Showing papers in "Physiology and Pharmacology in 2018"

Carbon nanotubes provide longer lasting gastroprotective effects for anandamide in stress-induced gastric ulcer in rat

Abstract: Introduction: Anandamide (AEA) has shown a wide spectrum of pharmacological activities including the effects against the peptic ulcer, meanwhile, the poor solubility or short half-life may negatively affect the effectiveness of this valuable cannabinoid. Based on the superior properties of carbon nanotubes (CNTs) for controlled drug delivery, we aimed to prepare AEA-CNTs complex and evaluate its therapeutic potential in an experimental model of gastric ulcer. Methods: Amino-functionalized multi-walled CNTs-AEA (MWCNTs-AEA) complex was prepared using COOH-MWCNTs and then characterized by Fourier transform infrared spectroscopy and transmission electron microscopy. Gastric ulcer was induced by water immersion and restrain stress (WRS) for 3.5 and 6 h in rats and the gastric lesion and oxidative stress were evaluated. Results: AEA at higher doses reduced the gastric ulcer area and malondialdehyde content and elevated glutathione level and superoxide dismutase and catalase activities after 3.5-h WRS but it was ineffective after 6-h WRS. MWCNTs-AEA complex showed therapeutic effects after both 3.5and 6-h WRS. Conclusion: Aminated MWCNTs are suitable carriers for AEA as they provide longer lasting effects for this cannabinoid. The antioxidant mechanism may be involved in the gastroprotective effects of MWCNTs-AEA complex. iD D ow nl oa de d fr om p hy ph a. ir at 1 5: 56 + 03 30 o n S at ur da y D ec em be r 29 th 2 01 8 39 | Physiol Pharmacol 22 (2018) 38-47 Hassanzadeh et al. degradation inhibitors or AEA uptake blockers has been the focus of intense research (Boger et al., 2000). According to the protective effects of the endocannabinoid system against the gastric lesions and its regulatory role in feeding behavior and inflammatory bowel disease (Di Sabatino et al., 2011; Orio et al., 2011; Shujaa et al., 2009), this ubiquitous signalling system may be considered as an emerging target for the therapeutic interventions against the gastrointestinal (GI) disorders. AEA with a wide spectrum of pharmacological activities including the effects against the psychological disorders, neurotoxicity and cancer (Adinolfi et al., 2013; Gobbi et al., 2005; Milton, 2002), has also shown therapeutic potential against the peptic ulcer in which the classical drugs have shown limited efficiency or numerous side-effects (Warzecha et al., 2011). Meanwhile, the poor solubility or short half-life of AEA (Jarho et al., 1996) may negatively affect its effectiveness. Over the last few decades, the outstanding breakthroughs in nanotechnology have resulted in the development of novel treatment strategies including the advanced nanovectors for delivery of compounds with poor solubility or short half-life. In this context, carbon nanotubes (CNTs) which may be used for the protection or targeted delivery of a wide variety of compounds, have been the focus of intense research. Indeed, CNTs have been represented as the attractive theranostic agents due to their improved biocompatibility and solubility, high thermoelectrical conductivities and superior mechanical properties (Cellot et al., 2009; Kam and Dai, 2005). These nanostructures may be used for biosensing, high-resolution imaging, tissue engineering and controlled release of drugs or growth factors (Bhirde et al., 2009; Fabbro et al., 2012; Mohammadi et al., 2009; Son et al., 2006). This background prompted us to prepare CNTs-AEA complex and evaluate its suitability to provide longerlasting effects for AEA in an experimental model of gastric ulcer. Materials and methods Preparation of CNTs-AEA complex Based on the improved dispersibility and reduced toxicity of amino functionalized multi-walled CNTs (MWCNTs) (Lee et al., 2011), we aimed to prepare aminated MWCNTs-AEA complex. Meanwhile, instead of the direct aminization of MWCNTs, we initially used acidified MWCNTs (COOH-MWCNTs) as the carboxylation of CNTs prior to the aminization enhances the reactivity of CNTs and facilitates further aminization (Hamdi et al., 2015). Aminefunctionalization of MWCNTs was performed as previously described (Chen et al., 2014; Hamdi et al., 2015; Lee et al., 2011) with some modifications. In brief, 500 mg of COOHMWCNTs (Plasmachem GmbH, Berlin, Germany) and 50 ml of 98% thionyl chloride (SOCl2, Sigma Aldrich, Germany) were sonicated using ultrasonic system (Tecna 6, TecnoGaz, Italy) at 70% amplitude for 40 min and stirred using a magnetic stirrer (IKA, Germany) at 25 °C for 48 h. Then, the suspension was filtered with 0.45 μm pore-sized microporous membrane (Sartorius, Germany), washed 5 times with tetrahydrofuran to remove the excess SOCl2 and vacuumed at 25 °C for 25 min. The residue was reacted with 50 ml of ethylenediamine (EDA) (Sigma Aldrich, Germany) and stirred for 10 h. Afterwards, the suspension was filtered, washed 5 times with tetrahydrofuran, vacuumed for 25 min, dialyzed in the deionized distilled water using a dialysis bag (MW cut-off 14 KD) for 72 h and vacuumed to obtain amine-modified MWCNTs. In order to prepare aminated MWCNTsAEA complex, AEA (N-arachidonoyl-ethanolamine, Tocris Bioscience, UK) was dissolved in Tween 80 (Sigma-Aldrich, Germany), 98% ethanol and phosphate-buffered saline (PBS) (1:2:18 v/v). Then, AEA (50 μM) was added to the mixture of aminated MWCNTs and PBS (0.25% w/v), stirred at 25 °C for 24 h and centrifuged by sigma-3k30 centrifuge (Sigma, Germany) at 10,000 rpm for 20 min. After the removal of supernatant, the sample was washed with PBS, re-centrifuged at 10,000 rpm for 20 min and dispersed in 10 ml of PBS. Characterization of MWCNTs Fourier transform infrared (FTIR) spectrophotometer (Shimadzu, Japan) was used to characterize the chemical structures of MWCNTs. The morphologies of MWCNTs were evaluated by transmission electron microscopy (TEM, Philips CM12). In vivo experiments Animals Male Wistar rats weighing 250-280 g were housed in pairs under the standard laboratory conditions D ow nl oa de d fr om p hy ph a. ir at 1 5: 56 + 03 30 o n S at ur da y D ec em be r 29 th 2 01 8 Carbon nanotube-anandamide complex and gastric ulcer Physiol Pharmacol 22 (2018) 38-47 | 40 (temperature: 22 ± 1 °C, humidity: 55-65%) on a 12-h light/dark cycle. Animals had unlimited access to water but were fasted for 18 h prior to the experiments. The maintenance and care of experimental animals complies with National Institutes of Health guidelines for the humane use of laboratory animals and has been approved by Institutional Ethics Committee. Animal groups and induction of gastric ulcer Water immersion restraint stress (WRS) is a wellestablished stress model which mimics the clinical acute gastric ulcerations and is suitable for evaluating the stress ulceration and demonstrating the mechanism of stress-induced gastric injury that might result in the development of novel therapeutic agents (Ernst et al., 1998). Animals were randomly assigned into the following groups; intact (n=6), vehicle-treated without exposure to WRS (n=6), exposed to WRS for 3.5 and 6 h (n=10/group), intraperitoneally (ip) treated with 0.05, 0.5 and 1 mg/kg of AEA (Dembinski et al., 2008; Warzecha et al., 2011) (Tocris Bioscience, UK) dissolved in ethanol, or 0.2, 2 and 4 mg/kg of MWCNTs-AEA complex (containing 0.05, 0.5 and 1 mg/kg of AEA, respectively), COOH-MWCNTs, EDAMWCNTs or vehicle 30 min before the exposure to WRS (n=10/group). Gastric ulcer was induced by WRS as previously described (Takagi et al., 1964) with some modifications. Animals were individually placed in a cage and immersed in water at 23 °C for 3.5 and 6 h. Evaluations of the gastric ulcer and histological alterations Following 3.5or 6-h WRS, each animal was sacrificed with 100 mg/kg of thiopental (Altana, Wesel, Germany), abdomen was incised, stomach was removed and opened along the greater curvature, washed by ice-cold normal saline, and the area of lesions of the oxyntic mucosa were determined using the computerized planimeter (Morphomat, Carl Zeiss, Germany). For the histological evaluations, gastric tissue samples were fixed in 10% buffered formalin for 24 h, embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin for further assessments by light microscope (Olympus Bx 10, Japan) equipped with a digital camera (Olympus DP12, Japan) (Dembinski et al., 2005). Biochemical assays Gastric tissue homogenates [10% w/v in ice-cold phosphate-buffered saline (0.1 M/L)] were provided using a homogenizer (Polytron, Heidolph RZR 1, Germany), centrifuged at 10,000 g at 4 °C for 15 min and then the pure supernatant was used for the measurement of malondialdehyde (MDA) and reduced glutathione (GSH) contents and superoxide dismutase (SOD) and catalase (CAT) activities according to the manufacturer’s instruction (Sigma Aldrich, Germany). In brief, the absorption of a pinkcoloured chromophore (due to the reaction of MDA with thiobarbituric acid) was determined at 532 nm using a spectrophotometer (UV-1601, Shimadzu, Japan) and MDA content was expressed as nM/mg tissue (Esterbauer et al., 1990). GSH content was measured based on its reaction with 5,5’-dithiobis (2nitrobenzoic acid) leading to the formation of a yellow-coloured product with an absorbance at 412 nm and expressed as nM/mg tissue (Jollow et al., 1974). CAT activity was assessed based on the rate of hydrogen peroxide degradation at 240 nm and expressed as U/mg tissue (Aebi, 1984). SOD activity was evaluated based on the extent of the inhibition of amino blue tetrazolium formazan formation in the mixture of nicotinamide adenine dinucleotide, phenazine methosulphate and nitroblue tetrazolium. The colour intensity was determined at 560 nm and the enzyme activity was expressed as U/mg tissue (Kakkar et al., 1984). Statistical analysis Three-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for data analysis. Data are presented as mean±SEM (standard error of the mean) and the level of significance was set at P<0.05.

Effects of crocin on cognitive and spatial memories in rats under chronic isolation stress

Abstract: Introduction: Certain types of chronic mental stress impair memory. On the other hand, crocin is introduced in the medical literature as an effective component of saffron with remedial effects on memory impairment. This study investigated the effects of crocin on spatial and cognitive memories, locomotor activity, novel recognition conditions and serum corticosterone levels in rats under chronic isolation stress. Methods: Male rats were randomly allocated to the five groups of control, sham, isolation stress (St.I), St.I-C30 and St.I-C60. The latter two groups were exposed to chronic isolation stress (6h/day) receiving two levels of crocin (30 and 60 mg/kg, respectively) over a period of 21 days. The object location and novel object recognition tests (OLT and NOR) were used to evaluate spatial and cognitive memories, respectively. Results: The OLT results revealed that chronic isolation stress led to significantly decreased locomotor activity in all the stressed groups; the NOR test, however, yielded similar results only in the St.I group. Moreover, isolation stress was found to lead significant declines in spatial and cognitive memories. Finally, crocin administration led to improvements in impaired memory in St.I-C30 and St.I-C60 groups. There were significant enhancements in serum corticosterone levels in the St.I and St.I-C30 groups as compared with the control group. Conclusion: Our findings indicate that spatial and cognitive memory impairments are strongly affected by isolation stress and crocin especially at its high dose of 60 mg/kg, exhibits better protective effects against cognitive memory deficit induced by chronic isolation stress. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 22 + 03 30 o n M on da y N ov em be r 4t h 20 19 255 | Physiol Pharmacol 22 (2018) 254-268 khani et al. throughout their lives (Izadi et al., 2018a; Izadi et al., 2018b; Rudramma et al., 2003; Sampath et al., 2014; Sandstrom and Hart, 2005; Shao et al., 2015; Zimmerberg et al., 2003; Zlatković et al., 2014). It has been observed that isolation of rats affects their brain development, brain neurochemical responses and subsequent adult behavior (Arango et al., 2001; Caldji et al., 2000; Lehmann and Feldon, 2000; Weiss and Feldon, 2001). It is, therefore, hypothesized that stress impairs both the activity of the hypothalamic–pituitary–adrenal (HPA) axis and cognition, suggesting potential alterations in brain functions (Sandstrom and Hart, 2005). Certain studies have investigated possible ways of alleviating the deleterious effects of mental stress on memory as a risk factor of mood disorders associated with the Alzheimer’s disease (Alkadhi, 2011). Crocin, as a carotenoid pigment, is the effective compound of saffron (Crocus sativus L) that has long been used as a drug in medicine (Abdullaev, 2002; Alavizadeh and Hosseinzadeh, 2014; Mohajeri et al., 2010). Previous studies indicated the beneficial anti-oxidant, anti-lipidemic and antiinflammatory effects of crocin (Abdullaev, 2002; Assimopoulou et al., 2005; He et al., 2005; Lee et al., 2005; Nam et al., 2010). The water maze test has shown crocin improves memory deficit induced by restraint stress (Ghadrdoost et al., 2011). Isolation stress is a kind of stress anyone is likely to be exposed to throughout their lives. Affected individuals may present memory deficits as a result of chronic isolation stress. However, few studies have been focused on the physiological aspects of isolation stress such as brain functioning. The present study was, therefore, designed and conducted to investigate the protective effects of crocin on serum corticosterone (CORT), locomotor activity and novel recognition. Moreover, the object location and novel object recognition (OLT and NOR) tests were used to explore spatial and cognitive memories, respectively, only in rats affected by chronic stress isolation. Materials and methods Experimental design The experiments were performed on forty adult male Wistar rats (Pasteur Institute, Tehran, Iran) weighing 250‒300g. The animals were maintained under controlled temperature (22±2°C) and humidity (50±5%) conditions over 12-h light/dark cycles with ad libitum access to food and water. All the experiments were performed in accordance with the standards set by the Committee on Ethical Standards of Isfahan University of Medical Sciences (IR.mui.rec.1394.3.934) and the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, 1996 Rev.). Rats were randomly assigned to the following five groups (n=8 in each group): control (Co), sham (Sh; receiving saline as vehicle daily for 21 days), chronic isolation stress (St.I; stressed in individual housing 6 h/day for 21 days) and two groups receiving daily crocin doses (30 and 60 mg/kg) accompanied by a period of 21 days of isolation stress. Finally, all rats were prepared on day 21 for the tests (Fig. 1). The animals were evaluated not only in terms of their locomotor activity and recognition of novel conditions but also for their spatial and cognitive memories as judged by the object location and novel object recognition tests, respectively. Serum corticosterone levels were also determined after decapitation. Drugs Crocin (Sigma Aldrich Co., USA) was purchased in powder form and dissolved in a saline to be injected Fig.1. Protocols for isolation stress treatments and crocin administration. D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 22 + 03 30 o n M on da y N ov em be r 4t h 20 19 Crocin and memory in chronic isolation stress Physiol Pharmacol 22 (2018) 254-268 | 256 intraperitoneally (IP) to the rats in the experimental groups at doses of 30 and 60 mg/kg/day for 21 consecutive days. The prevalently used and least effective doses of crocin administered to rodents in the experiments have been reported to be 30 and 60 mg/kg/day (Khalili and Hamzeh, 2010; Vakili et al., 2014). In addition, previous studies have shown no biochemical, hematological or histopathologic toxicity in rodents (mice and rats) due to chronic injection administration of 15-80 mg/kg, IP of crocin (Hosseinzadeh et al., 2010 ; Kianbakht and Hashem Dabaghian, 2015). However, higher doses administered over long periods (21 days) might be

Induction of traumatic brain and spinal cord injury models in rat using a modified impactor device

Abstract: Introduction: The use of standard rodent model, allows for the understanding of neuronal injury physiopathology and helping development of therapeutic strategies. Because of eliminating technical problems, we designed a modified impactor device with ability to induce different degrees according to kilodyne from very mild to very severe of spinal cord injury (SCI) and traumatic brain injury (TBI) models in rat. Methods: For standardization and determining of optimal performance of the device to induce varying injuries, 47 adult male Wistar rats were used, and 8 different forces were applied in spinal cord and brain tissues. Results: The hematoxylin and eosin and 2, 3, 5-triphenyltetrazolium chloride (TTC) results demonstrated that by increasing the level of forces, histological changes in the spinal cord and brain were significantly enhanced. Different injuries had significant effect on the Basso-Beattie-Brenham and elevated body swing test outcomes, and there were significant differences between groups in comparison with control group. Conclusion: Our results showed that the modified device could be valid to produce precise SCI and TBI models, goal to replicate SCI and TBI in humans as much as possible. However, it might be considered that aspects of SCI and TBI models are complicate and more examination is necessary. iD Induction of traumatic brain and spinal cord injury models in rat using a modified impactor device D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 14 + 03 30 o n M on da y N ov em be r 4t h 20 19 229 | Physiol Pharmacol 22 (2018) 228-239 Ghorbani et al. tissue devastation (Gaetz, 2004; Cernak, 2005; Masel and DeWitt, 2010). This primary stage is followed by secondary phase of SCI characterizing with cellular and molecular dysfunctions (Thompson et al., 2005; Marklund et al., 2006; Bramlett and Dietrich, 2007). Among different techniques, contusion method using New York University (NYU), the Ohio State University (OSU) (Stokes, 1992) and the Infinite horizon (IH) devices (Lee et al., 2012) are widely applied for induction of SCI in rodents (Iwanami et al., 2005; Khuyagbaatar et al., 2015). The SCI producing by the NYU device results from dropping of a 10-g rod from different heights on exposed special part of the spinal cord (Gruner, 1992). In the primary version, NYU impactor device couldn’t record digital parameters, but in the ultimate version the device is able to control some mechanical parameters to create different injuries, however, as a disadvantage, duration of impact cannot be exactly controlled (Iwanami et al., 2005). The OSU or electromagnetic spinal cord injury device (ESCID) can produce a range of spinal cord injuries by applying different displacement onto exposed spinal cord (Noyes, 1987; Stokes et al., 1992). The device improved in 2000 to investigate in SCI model in mouse (Jakeman et al., 2000) and cervical spine injury in the rat (Pearse et al., 2005). There is merely a limitation of the OSU impactor that discerning the zero point for the impactor sometimes is difficult (Cheriyan et al., 2014). Finally, the IH device is made for crating different severity of injuries using a force-controlled impact on the spinal cord tissue and graded contusion injury will generate by entering a predetermined force in specific software which calibrated according to mild (100kdyn), moderate (150kdyn) and sever (200kdyn) forces at different segments of spinal cord in the rat (Scheff et al., 2003). In this device, an apparent limitation revealed by some researchers relating to spinal cord holder which finally caused to create a custom type of clamping instrument to cope with that technical problem (Streijger et al., 2013). Considering, TBI models divided into four specific models as follow; fluid percussion injury (FPI) (Dixon et al., 1987), weight-drop impact injury (Marmarou et al., 1994), blast injury (Leung et al., 2008) and controlled cortical impactor (Lighthall, 1988; Dixon et al., 1991). The FPI model creates by a fluid rapid injection into the brain space following craniotomy and the different degrees of injury relies on the strength of the pressure (McIntosh et al., 1989). As a beneficial change in next version, FPI model improved by providing a microprocessor-controlled and contemplating adjustable biomechanical parameters (Kabadi et al., 2010). Weight-drop model, a weight releases straight onto the exposed dura matter from a desired height to induce a cortical injury (Feeney et al., 1981). Overtly, one remarkable disadvantage of the weight-drop models relates to high variation in creating different severities of injury; however, because of several reasons, including economic efficiency, easy to accomplish and similar TBI method in human, it should be considered as an applicable and common approach in experimental condition (Xiong et al., 2013). Blast brain injury causes by blast which commonly happens in various military personnel (Long et al., 2009). The controlled cortical impact (CCI), injury model induces by an air or electromagnetic device (Xiong et al., 2013). The advantage of CCI compared to other models is adjustable mechanical parameters, such as velocity, time and depth (Wang and Ma, 2010), and with this way, severity of injury can be appropriate for specific experiments ( Goodman et al., 1994). Therefore, all of these devices were generally employed and developed for traumatic brain and spinal cord injuries, separately, and each one has its specific feature of injury. In this study a modified device is produced inducing either SCI or TBI models in rats. To gain several targets effectively, we have designed a device with usability in the spinal cord and brain trauma models that could create more wide range of injury with slight variation and low heterogeneity in order to help researcher for understanding precise pathophysiological

The effect of saffron aqueous extract on oxidative stress parameters and important biochemical enzymes in the testis of streptozotocin-induced diabetic rats

Abstract: Introduction: Sexual dysfunction and infertility are frequently associated with diabetes in men and experimental animals. Oxidative stress and alteration in testis are responsible for complication in diabetes. Saffron has antidiabetic and antioxidant properties that improves the functions of various organs. Therefore, the aim of the present study was to investigate the effects of administration of saffron aqueous extract in testis tissues of diabetic rats. Methods: The fasted rats were injected by a single intraperitoneal (ip) injection of a freshly prepared solution of streptozocin (STZ, 65mg/kg) in 0.1 M cold citrate buffer (pH=4.5). Three days after STZ administration, the animals with fasting blood glucose concentrations of over 250mg/dl were considered to be diabetic and were used in the experimental groups as follows: normal control (1), diabetic control (2), saffron control (3) and saffron treated (4). The treatment was started on the 7th day after STZ injection with ip injection of saffron (200mg/kg), five doses and weekly to groups (3 and 4). At the end of the experimental period, fasting blood glucose levels and the activity of ALT, AST, ALP, LDH, SOD, CAT, GPx and MDA content were determined in testis tissues. Results: Results showed saffron administration decreased elevated biochemical enzymes levels in testis of diabetic rats. Also, saffron significantly increased CAT and GPx activities in testis of diabetic rats. MDA levels had no significant changes in all experimental groups. Conclusion: The results demonstrated that saffron administration improved antioxidant enzymes function against oxidative stress. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 22 + 03 30 o n M on da y N ov em be r 4t h 20 19 29 | Physiol Pharmacol 22 (2018) 28-37 Hasanpour et al. remains untreated, it will lead to serious damages in different vital organs including male reproductive system. A large number of studies in men with diabetes, and in animal models show that diabetes causes male infertility due to sexual dysfunction and hypogonadism. Diabetes affects male reproductive function at different levels including changes in the quality of sperm, spermatogenesis, testicular morphology, sertoli glucose metabolism, ejaculation disorder, reduction in testosterone and libido (Jain and Jangir, 2014). Testis are considered as the primary reproductive organ and are one of the major organs affected by diabetes (Baccetti et al., 2002; Koh, 2007). Diabetic rats exhibit decreased testicular weight, sperm count, sperm motility and testosterone levels, and increased frequency of abnormal spermatogenesis (Scarano et al., 2006). Changes in testicular tissue including testis apoptosis may be the major component of the infertility in diabetes (Roy et al, 2014). Therefore, consideration the abnormalities in testis could be really beneficial to treat or reduce serious damages and infertility. Over production of reactive oxygen species (ROS) and impaired antioxidant defenses are accompanied with diabetes. Evidence indicates that oxidative stress in diabetes people is the trigger for many alterations on sexual function. In this regard, reducing oxidative stress in patients suffering from diabetes could be really valuable (Amaral et al., 2008). There are number of medications used to treat people with diabetes; however, common medications for diabetes does not always prevent the complications of the disease. In recent years, attention has been focus on herbs with high antioxidant activity to prevent and protect oxidative damage caused by free radical species in diabetes (Stavic, 1994). Crocus sativus L. commonly known as saffron is a perennial stem less herb of the Iridaceae family. Saffron has been used in traditional medicine as an antispasmodic, eupeptic, gingival sedative, anti-catarrhal, nerve sedative, carminative, diaphoteric, expectorant, stimulant, stomachic and aphrodisiac agent. The pharmacological activities of saffron are attributed to many of its active constituents including safranal, crocetin, crocin and quercetin (Rios et al., 1996). Theses constituents of saffron exhibits high antioxidant activity which makes it an ideal candidate for treatment of disease associated with oxidative stress (Khajuria et al., 2010). Testicular dysfunction in diabetes is well established, whereas the impact of oxidative stress associated with diabetes on the testis remains elusive. Herein, the aim of the present study was to evaluate the effect of saffron aqueous extract on oxidative stress parameters including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and malondialdehyde (MDA) in testis of diabetes rats and to investigate the performance of saffron extract on the improvement of testicular tissue by determining the activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). Materials and methods Experimental animals Thirty-two adult healthy male albino Wistar rats (7-8 weeks) weighting 200–225 g were purchased from Shiraz University of Medical Science (Shiraz, Iran). The animals were housed in standard cages at room temperature (23±1 °C) with a 12:12-h light-dark cycle with free access to tap water and balanced diet (ad libitum). For adaptation, all animals were kept in this condition one week prior to the study.

The effect of curcumin against 6-hydroxydopamine induced cell death and Akt/GSK disruption in human neuroblastoma cells

Abstract: Introduction: Parkinson’s disease (PD) is the second most common neurodegenerative disease, characterized by the continuous deficit of dopaminergic neural cells in the substantia nigra pars compacta. The natural compounds from plant extracts, such as turmeric, have been proposed as alternative sources for anti-PD drugs. Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line used as an in vitro model for the study of dopaminergic cells. The neurotoxin 6hydroxydopamine (6-OHDA) has been known to induce cell death in dopaminergic neural cells. Curcumin, as the main ingredient of turmeric, has been shown to protect against some animal models of PD. The purpose of the present study was to assess the potential neuroprotective effect of curcumin against the 6-OHDA-induced cell death in SH-SY5Y cells and to delineate its effect on Akt/GSK-3β signaling. Methods: The cells were exposed to 6-OHDA with/without different doses of curcumin and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) and morphological observations. According to the MTT results, the protective doses of curcumin (2 and 2.5μM) were selected for further studies. Western blot assay was done to determine the phosphorylated and total amount of Akt and GSK-3β proteins. Results: 6-OHDA induced cell death and declined Akt/GSK-3β phosphorylation, while curcumin co-treatment partially restored these effects. Conclusion: Taken together, these findings suggest that curcumin protects the SHSY5Y cells from 6-OHDA-induced cell death and Akt/GSK-3β signaling alteration. Thus, our study indicates that curcumin has a partial cytoprotective effect in dopaminergic cell culture systems. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 :0 9 + 03 30 o n W ed ne sd ay O ct ob er 3 0t h 20 19 Curcumin reverses 6-OHDA toxicity in SH-SY5Y cells Physiol Pharmacol 22 (2018) 163-171 | 164 (Morgante et al., 2007). Then, there is an emerging interest in the use of novel therapeutic strategies especially natural molecules with neuroprotective properties. Curcumin, found in turmeric, is a yellow curry spice with a long history of use in cooking or medicine (Aggarwal et al., 2007). It has a wide pharmacological effects such as anti-inflammatory properties (Srimal and Dhawan, 1973), powerful antioxidant effects (Masuda et al., 1999), anti-protease activity (Sui et al., 1993) and cancer preventive effects (Kim et al., 1998). Recently, some studies have shown the neuroprotective effect of curcumin in experimental models of PD (Wang et al., 2017). In 6hydroxydopamine (6-OHDA) model of PD, rats pretreated with curcumin showed a protection of substantia nigra and dopamine levels in the striata (Zbarsky et al., 2005). Wang et al. (2009) reported that curcumin has the capability to restore mitochondrial membrane potential and cell viability in 6-OHDA-lesioned mouse embryonic stem cells. Similarly, Rajeswari and colleagues (2008) have shown an increment in striatal dopamine and DOPAC (3,4-Dihydroxyphenylacetic acid) levels after curcumin injection in MPTP (1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine) injected mice. Accordingly curcumin has been shown to prevent cell death in SH-SY5Y cells (Jaisin et al., 2011; Meesarapee et al., 2014), as the appropriate cellular model of dopaminergic neural cells (Song et al., 2012). 6OHDA has been shown to alter some signaling molecules; for instance, it affects Akt phosphorylation (Chen et al., 2004) and its downstream element glycogen synthesis kinase 3β (GSK-3β) (Amiri et al., 2016) which is negatively regulated by Akt-mediated phosphorylation at serine 9 (Stambolic and Woodgett, 1994). As Akt /GSK-3β signaling has been shown to take part in 6-OHDA induced cell death (Chen et al., 2004; Amiri et al., 2016), this study aimed to explore if the protective effect of curcumin against 6-OHDA induced toxicity is accompanied with Akt/GSK-3β signaling alteration. Materials and methods

Anti-cancer properties of the methanol extract of Boswellia serrata gum resin: Cell proliferation arrest and inhibition of angiogenesis and metastasis in BALB/c mice breast cancer model

Abstract: Introduction: Boswellia serrata is a medicinal plant with immense potential in combating cancer. Since many cancers therapeutics have their roots in natural products, we investigated the inhibitory effect of B. serrata gum resin alcoholic extract (BSE) on tumor growth, metastasis and angiogenesis in 4T1 breast cancer mouse

The rate of resistance to tetracyclines and distribution of tetA, tetB, tetC, tetD, tetE, tetG, tetJ and tetY genes in Enterobacteriaceae isolated from Azerbaijan, Iran during 2017

Abstract: Introduction: Enterobacteriaceae are the heterogeneous group of Gram-negative bacteria, which cause different infections. The incidence of resistance to antibiotics among the Enterobacteriaceae is growing. This study investigated antibiotic resistance features and tetracycline resistance genes distribution in Enterobacteriaceae isolates from Hospitals of Azerbaijan, Iran. Methods: The disc diffusion agar and agar dilution methods were used for assessment of antibiotics susceptibility patterns and minimum inhibitory concentration determination of tetracycline and minocycline. To detect eight tetracycline resistance genes (tetA, tetB, tetC, tetD, tetE, tetG, tetJ, and tetY), the PCR was performed in tetracycline-resistant isolates. Results: The resistance rate to tetracycline, minocycline, doxycycline, and tigecycline by the disc diffusion agar method were 58.8%, 24%, 43.6% and 0.4%, respectively. Fifty-one (20.4%) isolates were multiple drugs resistant. The minimum inhibitory concentration results showed 52% resistance to tetracycline and 22% for minocycline. The percentage of tet genes distribution was tetA (14.4%), tetB (18.4%), tetC (2%) and tetD (4.4%). However, tetE, tetG, tetJ and tetY genes were not detected in the present study. Conclusion: There is a moderate-high resistance rate to tetracycline among Enterobacteriaceae in Azerbaijan. The most effective antibiotic against Enterobacteriaceae was tigecycline followed by fosfomycin, imipenem and meropenem. The tet genes family especially tetA and tetB were prevalent among tetracycline-resistant isolates. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 05 + 03 30 o n M on da y N ov em be r 4t h 20 19 Resistance rate to tetracyclines Physiol Pharmacol 22 (2018) 205-212 | 206 most prominent pathogenic genera to human are included Escherichia coli, Klebsiella pneumoniae, Citrobacter spp., Proteus spp. and Morganella spp. (Stecher et al., 2012). E. coli and K. pneumoniae are more isolated from infections caused by Enterobacteriaceae in particular bloodstream infections (Kim et al., 2002). Common treatments for Gram-negative bacterial infections are aminoglycosides, beta-lactams, fluoroquinolones and tetracyclines (Hawkey and Finch, 2007). Tetracyclines are bacteriostatic antibiotics and can attach to the ribosome 30s subunit and prevent protein synthesis. They were discovered in the 1940s and were rigorously active against Gram-positive and Gram-negative bacteria and protozoan parasites (Chopra and Roberts, 2001). Amon the most wellknown tetracyclines, it can be referred to tetracycline, minocycline, doxycycline and tigecycline (Connell et al., 2003). Doxycycline as a second generation tetracycline has high absorbance rate and lipophilic properties (Sloan and Scheinfeld, 2008). Minocycline is a bacteriostatic agent as well. Its performance mechanism is similar to other tetracyclines (Ritchie and Garavaglia-Wilson, 2014). Tigecycline as a new tetracycline is obtained from the combination of 9-tbutylglycylamido side chain to minocycline due to a high affinity to ribosome (Deng et al., 2014). Tigecycline is more used against the Gram-negative bacteria (Livermore, 2005). The prescription range of tetracyclines is too wide including human and animals infections and even plants (Aminov et al., 2001). The price of tetracyclines is declining due to pharmaceutical advancement, so they are considered to be the most favorite and cost-effective antibiotics around the world. Tetracyclines alone or in part with other antimicrobial agents administrate to the treatment of infections caused by Enterobacteriaceae (Hirsch and Tam, 2010). The infections caused by E. coli have been treated with tetracycline, doxycycline and minocycline (Cunha, 2012). Before the mid-1950s, most bacteria were sensitive to tetracycline. The mechanisms of resistance to tetracycline include the decreased penetration, efflux pumps, ribosomal protection, target alternation and enzymatic modifications. Several plasmid genes contribute to resistance, which is known as otr and tet genes. Twenty-nine tet genes are known that to be named in the English alphabet and belong to a major facilitator superfamily which encodes the dependent membrane proteins. These proteins drove out the tetracyclines and protect ribosome (Chopra and Roberts, 2001). All detected tet genes are related to efflux pump and ribosomal protection mechanisms except the tetX gene, which belongs to the enzymatic alteration mechanism (Ng et al., 2001). Tetracyclines are not the first-line prescribed drugs for the treatment of Enterobacteriaceae infections, but increasing resistance to first-line drugs has turned them into an alternative treatment for these infections (Horcajada et al., 2014). In the present study, the antibiotic resistance features and the distribution of tetracycline resistance genes in Enterobacteriaceae isolated from Azerbaijan Hospitals were investigated. Materials and methods Bacterial isolation and identification A total of 250 Enterobacteriaceae isolates were gathered from clinical specimens. The organisms were identified by the microscopic feature and the differential tests such as indole production, urease, phenylalanine deaminase, glucose and lactose fermentation, motility test, methyl red, Voges Proskauer, citrate consumption and H2S production (Hansen et al., 2004). Finally, they were stored in tryptic soy broth medium including glycerol and preserved at -70°C freezers (Rohman et al., 2013). Informed consent was obtained from all human adult participants and from the parents or legal guardians of minors. The Ethic Committee of Tabriz University of Medical Sciences approved this study (Number: Ir.tbzmed.rec.1396.638). The disk diffusion agar method The disk diffusion susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (Jorgensen and Turnidge, 2015). Antimicrobial disks that were used in this study, purchased from “Mast” (UK) included ampicillin, piperacillin/tazobactam, imipenem, coamoxiclav, cefepime, ciprofloxacin, ceftazidime, nitrofurantoin, meropenem, aztreonam, cotrimoxazole, fosfomycin, gentamicin, amikacin, nalidixic acid, cefazolin, tetracycline, doxycycline, minocycline and tigecycline (Hudzicki, 2009). E. coli ATCC (American type culture collection) 25922 was used as a quality control strain. D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 05 + 03 30 o n M on da y N ov em be r 4t h 20 19 207 | Physiol Pharmacol 22 (2018) 205-212 Sheykhsaran et al. Minimum inhibitory concentration (MIC) determination In order to achieve MIC rate in Enterobacteriaceae isolates to tetracycline and minocycline, the agar dilution method was conducted. The results were interpreted according to the CLSI guidelines (Andrews, 2001). E. coli ATCC 25922 was used as a quality control strain. PCR To detect tetracycline resistance genes (tetA, tetB,tetC, tetD, tetE, tetG, tetJ, and tetY), DNA of organisms, which were resistant to tetracycline by the MIC method, were extracted by the boiling method (Zhang and Stewart, 2000). Then, the PCR was performed for screening of eight tet genes as previously described (Aminov et al., 2002). PCR products were evaluated by electrophoresis for 60 min on a 1.5% agarose gel at 85 Vand after staining with 0.5μg/ml ethidium bromide visualized under UV light (Akhi et al., 2017). Statistical analysis The data were evaluated by q2, Fisher exact test and qualitative statistics (percentages) using the SPSS software 24 (Washington, the USA), version 22.

Zingiber officinale extract pre-treatment ameliorates astrocytes activation and enhances neuroprotection in pentylenetetrazol-induced kindling model of epilepsy in mice

Abstract: Introduction: Recently, herbal medicine is widely used as an alternative and complementary therapy in several neurological disorders such as epilepsy. The anti-inflammatory and neuroprotective effects of Zingiber officinale or ginger have been well-documented. The present study was designed to evaluate the effects of ginger extract pre-treatment on seizures behavior, neuronal density and astrocytes activation in pentylenetetrazol (PTZ)induced kindling model. Methods: Kindling model was induced in mice by repetitive administration of PTZ at sub convulsive dose. Hydroalcoholic extract of ginger at doses of 25, 50 or 100 mg/kg were daily injected 10 days before PTZ injections and intraperitoneal administration of extract was continued 1h before each PTZ injection. Immunostaining against NeuN and GFAP as neuronal and astrocyte markers, respectively, was carried out on brain tissue sections. Results: Our data showed that ginger extract pre-treatment, especially at dose of 100 mg/kg, reduced the seizures behavior in PTZ receiving animals. Immunostaining against NeuN biomarker demonstrated that neuronal death was alleviated in animals under treatment of ginger extract. Furthermore, application of ginger extract attenuated the number of GFAP expressing cells in hippocampus of fully-kindled animals. Conclusion: Overall, our data suggest that ginger pre-treatment exerts significant neuroprotective effect by attenuation of astrocytes activation in PTZ-induced kindling model. It can be concluded that ginger might be used as effective supplementary agent in epileptic patients. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 02 + 03 30 o n M on da y N ov em be r 4t h 20 19 93 | Physiol Pharmacol 22 (2018) 92-102 Naeimi et al. hyperexcitability possesses major role in epileptogenesis (Devinsky et al., 2013). Glial cells including astrocytes, microglia and oligodendrocytes are regarded as the most abundant cells in the central nervous system (Pelvig et al., 2008). Astrocytes and microglia are involve in various physiological functions and play important roles in homeostasis of the nervous system (Devinsky et al., 2013). Upon the injury, the proliferation of glial cells increases and activated cells migrate to the damage area. Activated astrocytes and microglia effectively inhibit the spread of injury to the surrounding region of lesion site. Despite the beneficial effect of glial cells, activated astrocytes and microglia ultimately form glial scar which leads to neuronal dysfunction and axonal loss (Guo et al., 2014). Gliosis is considered as common hallmark of brain injuries such as stroke (Barreto et al., 2011) and neurodegenerative disorders including Alzheimer's disease (AD) (Guo et al., 2014) and epilepsy (Lehrmann et al., 2008). It has been well-understood that changes in morphology, molecular composition and proliferation of astrocytes occur in epileptic foci (Devinsky et al., 2013). Additionally, astrocytes activation has been found in experimental models of epilepsy and brain tissues of epileptic patients (Devinsky et al., 2013). It has been shown that activated glial cells can promote epileptogenesis through enhancement of excitability and inflammation. Activated glial cells induce neuronal hyperexcitability by disruption of ions, water and neurotransmitters regulation (Devinsky et al., 2013). Furthermore, astrocytes and microglia release several inflammatory factors which in turn facilitate the epileptogenesis process (Vezzani and Granata, 2005; Vezzani et al., 2008; Vezzani et al., 2011; Vezzani et al., 2013). Accumulating body of evidences demonstrated that some anti-inflammatory drugs can effectively reduce the seizures behavior (Ikonomidou-Turski et al., 1988; Marchi et al., 2011). Despite the emergence of numerous anti-epileptic drugs, the current therapeutic approaches are effective only in 40% of epileptic patients (Schmidt, 2011). Therefore, a demand for producing of new types of anti-epileptic drugs continuously exists. Currently, herbal medicine has been introduced as complementary and alternative strategy in treatment of epilepsy. Ginger is derived from the rhizome of the Zingiber officinale Roscoe and it is widely being used as spice and food additive in worldwide (Hosseini and Mirazi, 2014). Ginger or its effective compounds such as gingerol or shogaol have been shown to exhibit beneficial effects in treatment of several diseases such as rheumatoid arthritis, neuropathy disorders, gingivitis and stroke (Lantz et al., 2007). Several evidences have demonstrated that Z. officinale has antioxidant (Masuda et al., 2004; Mashhadi et al., 2013), anti-inflammatory (Grzanna et al., 2005; Lantz et al., 2007) and neuroprotective effects both in vitro and in vivo (El-Akabawy and El-Kholy, 2014). It has been well documented that anti-inflammatory effects of shogaol is partly mediated by inhibiting the production of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), and proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and by down-regulating inducible nitric oxide syntheses, p38 mitogenactivated protein kinase and nuclear factor kappa B (NF-κB) expression (Ha et al., 2012; Shim et al., 2011). [6]-shogaol treatment also remarkably increases the level of histone H3 acetylation and heat-shock protein 70 and suppresses the expression of histone deacetylase 1 (Shim et al., 2011). In addition, it has been shown that ginger treatment increases the neuroprotection by augmentation of brain anti-oxidant defense mechanisms (Shanmugam et al., 2011). Previous studies suggested the beneficial effects of ginger in various neurological disorders such as AD (Grzanna et al., 2004; Oboh et al., 2012). In recent years, the effect of acute and chronic administration of ginger extract on seizure threshold was evaluated in acute model of seizure (Hosseini and Mirazi, 2014; Hosseini and Mirazi, 2015). To our knowledge, there is no study that examined the anti-convulsant mechanism of ginger extract on kindling model of epilepsy. Pentylenetetrazol (PTZ) as GABAA receptor antagonist is extensively used to evaluate the epileptogenesis process as well as testing of novel antiepileptic drugs (Dhir, 2012). For development of PTZ-induced chemical kindling model of epilepsy, PTZ is repetitively administrated at subconvulsive dose. The neuronal loss and glial activation in hippocampus of PTZ receiving animals has been well-addressed (Anissian et al., 2018; Gol et al., 2017; Kaur et al., 2015). The present study was designed to examine the D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 02 + 03 30 o n M on da y N ov em be r 4t h 20 19 Ginger extract, neuronal loss and glial activation Physiol Pharmacol 22 (2018) 92-102 | 94 effect of ginger extract pre-treatment on seizures behavior in PTZ-induced kindling model. Furthermore, the level of astrocytes activation and neuronal density was evaluated in fully-kindled animals which have received the ginger hydroalcoholic extract. Materials and methods Drugs PTZ and sodium valproate (VPA) were obtained from Sigma-Aldrich (St.Louis, Mo USA). Preparation of ginger hydroalcoholic extract The rhizomes of Z. officinale were purchased from local market and authenticated by a botanist (herbarium number: 94302). Three hundred grams of dried ginger rhizomes were mechanically powdered and mixed with 500 ml of ethanol. The prepared mixture was shaken for 72 hours in 25°C using shaker-incubator (labnet. 311DS. USA). The supernatant solvent was filtered through a whatman filter paper. Solvent evaporation was carried out by rotary evaporator under vacuum condition (Moghadamnia et al., In press). Prepared extract was dissolved in sterile saline solution containing 0.1% Tween-80 and was injected at appropriate dose based on body weight of animals.

Effect of Syzygium aromaticum (clove) extract on morphine withdrawal side effect in male reproductive system should be addressed

Abstract: Introduction: To study the effect of withdrawal syndrome in morphine-dependent

Interactive effect of aqueous-alcoholic extract of ginger as well as GABAA receptor agonist and antagonist on pain sensitivity in male rats

Abstract: Pain, at all forms including mental, emotional and physical is one of the most frequent complains of patients and it is almost always a sign of imbalance in one or more parts of the body (Vachon-Presseau et al., 2016). Accordingly, the design of therapeutic interventions to reduce pain is perhaps the physician’s greatest challenge (Bonakdar and Sukiennik, 2016). Previously, it has been shown that Zingiber officinale (ginger) owns a number of beneficial effects for attenuating pain. Ginger is widely used as a spice in Physiology and

Evaluation of the GABAA receptor on pain sensitivity in male rat pretreated with Valeriana officinalis extract using formalin test

Abstract: Introduction: : It seems that Valeriana officinalis (valerian) extract through gammaamino-butyric acid A (GABAA) receptor possesses analgesic effect. The aim of the present study was to investigate the effect of muscimol and picrotoxin on pain sensitivity in male rats pretreated with valerian extract using the formalin test. Methods: Thirty-five male rats weighing 200-250g in standard temperature 20±2 °C and light cycle of 12/12h used. Animals were randomly divided to 7 groups: sham 1 (injection of saline); sham 2 (pretreated with valerian + ICV injection of artificial cerebro spinal fluid); experimental1 (injection of valerian extract); experimental 2 or 3 (pretreated with valerian extract + ICV injection of muscimol 250 and 500 ng/rat); experimental 4 or 5 (pretreated with valerian extract + ICV injection of picrotoxin 250 and 500 ng/rat). Valerian extract 400 mg/kg was administrated by intraperitoneal injection. Pain evaluation was done by the formalin test. Lateral ventricles cannulated unilaterally by the stereotaxic procedure. Results: Data showed that valerian extract significantly decreased pain sensitivity in the late phase of the formalin test in comparison to sham 1 group. Muscimol in both doses significantly decreased pain in comparison to sham 1, while at the dose of 500 ng/rat significantly increased pain sensitivity in comparison to sham 2 at late phases of formalin test. Picrotoxin at both doses significantly decreased pain sensitivity in comparison to sham 1, while significantly increased pain sensitivity in comparison to the sham 2 at late phases of formalin test. Conclusion: According to present results, valerian extract had analgesic effect through the GABAA receptor. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 25 + 03 30 o n M on da y N ov em be r 4t h 20 19 119 | Physiol Pharmacol 22 (2018) 118-123 Taherianfard et al. in medicine in different countries (Benke et al., 2009; Khayat Nouri and Namvaran Abbas Abad, 2011). Several studies have reported anticonvulsant effects of valerian extract and they have mentioned its effects mediated through the GABA system (Khayat Nouri and Namvaran Abbas Abad, 2011; TorresHernandez et al., 2015). Furthermore, valerian extract possesses sedative, hypnotic and anxiolytic properties (Dietz et al., 2005; Fernandez et al., 2004; Heidari and Razban, 2004; Solati and Sanaguye Motlagh, 2008). Phytochemical studies have shown valerian contains valepotriates, alkaloids, sesquiterpenes, alcohols, volatile oils, aromatic compounds and other polar and non-polar organic compounds. These are responsible for mentioned effects of valerian extract (Fernandez et al., 2004; Torres-Hernandez et al., 2015). Several potential mechanisms have proposed for valerian effects. Antianxiety properties of valerian relates to the GABA system function (Khayat Nouri and Namvaran Abbas Abad, 2011; Yuan et al., 2004; Murphy et al., 2010). GABA is a main inhibitory neurotransmitter in central nervous system of mammals (Froestl, 2011). Activation of GABAA receptor opens chloride channel and makes intracellular hyperpolarization (Farrant and Nusser, 2005). Muscimol is an agonist of GABAA receptor and causes pain reduction (Reis et al., 2007). Studies have shown that administration of muscimol reduced pain in different pain tests (Gilbert and Franklin, 2001; Mahmoudi and Zarrindast, 2002). Picrotoxin is a competitive antagonist of GABAA receptor and changes pain response in the formalin test (Heidari et al., 1996). There are many investigations on GABAA receptor and pain, on the other hand, relation between valerian and pain was investigated; but the analgesic effect of these two factor simultaneously was not evaluated. Therefore, the aim of the present investigation was to evaluate analgesic effect of muscimol (GABAA agonist) and picrotoxin (GABAA antagonist) in adult male rats pretreated with valerian extract using formalin test. Materials and methods Animals Thirty-five adults’ male Sprague Dawley rats weighing 200-250g were used. Animals housed in standard condition and maintained under controlledtemperature 20±2 °C and light-dark cycle as 12/12h. Rats were fed standard food and water ad libitum. The animals randomly divided into 7 groups (5 rats per group): sham 1, intraperitoneal (ip) injection of saline; sham 2, pretreated with ip injection of valerian 400 mg/kg + intracerebroventricular (ICV) injection of artificial cerebro spinal fluid (ACSF); experimental 1, ip injection of valerian 400 mg/kg; experimental 2 and 3, pretreated with ip injection of valerian 400 mg/kg + ICV injection of muscimol 250 or 500 ng/rat and experimental 4 and 5; pretreated with ip injection of valerian 400 mg/kg + ICV injection of picrotoxin 250 or 500 ng/rat. Intraperitoneal injection of valerian 400 mg/kg at single dose and ICV injection of ACSF, muscimol and picrotoxin performed 15 and 30 minutes before formalin test, respectively. Preparation of valerian extract To prepare the valerian extract, 500g of dried valerian rhizome was ground and then added to the solvent 70% ethanol. Every 100 grams of powdered rhizome dissolved in 400ml of the above solution. The solution stirred for one hour on shaker to obtain almost uniform solution then placed at room temperature for 48 hours (25°C). After 48 hours, the solution filtered through whatman filter paper. Evaporator and lyophilized devices used for condensing of the final solution. After this process, the dried powder well mixed, in a fit ratio with distilled water on shaker until being dissolved completely and ip injectable solution (400mg/kg; LD50= 3300 mg/kg) was prepared (Patočka and Jakl, 2010). Stereotaxic procedure Animals anesthetized with ip injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Rats fixed in the stereotaxic apparatus using blunt ear bars. The skull was carefully exposed and stainless steel guide cannula (23-gauge needle), were inserted bilaterally 3.5 mm above the lateral ventricle. The coordinates for lateral ventricle were 0.5 mm anterior to Bregma and 1.5 mm lateral to midline. The guide cannula fixed to the skull via dental acrylic cement and two tiny stainless steel screws. At the end, animals were given a 7-day recovery period.

The effect of AT2 and Mas receptors antagonists on renal hemodynamic and excretory disorders induced by ischemia/reperfusion in male and female rats

Abstract: Introduction: Renal ischemia-reperfusion (RIR) may disturb renin-angiotensin system components. In this study, the effects of Mas receptor (A779) and AT2 receptor (PD123319) antagonists were examined in RIR rats. Methods: Total 60 male and female Wistar rats were assigned into 10 groups (n=6 in each group), including sham-operated group, RIR groups treated with the vehicle, A779, PD123319, or A779+PD123319. The rats were subjected to 30 minutes renal ischemia followed by 75 minutes reperfusion and the vehicle/antagonists were started to infuse 15 minutes after beginning of reperfusion for 60 min. Mean arterial pressure (MAP) and renal perfusion pressure responses to antagonists were assessed. Measurements for kidney function parameters also were performed. All the measurements were made at the end of 60 min vehicle/antagonist infusion. Results: MAP has altered significantly during RIR times (P=0.004), but no significant difference was observed between two genders. The RIR itself in injured rats (compared to sham operated rats) decreased urine flow (UF), creatinine clearance (Ccr), filtrate load of sodium (FNa) and sodium excretion rate (ENa) significantly in both genders (P<0.05). The antagonists infusion caused significant decrease in Ccr and FNa in male and female rats subjected to RIR when compared with vehicle (P<0.05), but the UF decreased significantly (P<0.05) only in PD123319 treated groups; however, there was no significant difference in ENa between the RIR groups in both genders. Conclusion: Our findings showed the importance role of Mas receptor and AT2 receptor on renal function after kidney ischemia/reperfusion in RIR rat model. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 23 + 03 30 o n M on da y N ov em be r 4t h 20 19 AT2 and Mas receptors antagonists in RIR Physiol Pharmacol 22 (2018) 133-140 | 134 following RIR (Kontogiannis and Burns, 1998) while RAS has been considered as an important regulatory system of renal function (Jalal Hassanshahi et al., 2017; Rice et al., 2004). Furthermore, female is more resistant than male to RIR induced kidney injury (Fekete et al., 2004) and the RAS functions are different between two genders (Hilliard et al., 2011). Moreover, it has been made clear that the renal function reduces slowly more in female subjected to RIR than male rats (Neugarten et al., 2000; Silbiger and Neugarten, 1995). Generally RAS has two pivotal opposite roles in vascular system. In one hand vasoconstriction by angiotensin II (Ang II) through Ang II type 1 receptor and on the other hand vasodilation by Ang II and angiotensin 1-7 (Ang 1-7) through Ang II type 2 receptor (AT2R) and Mas receptor (MasR) respectively (Hassanshahi and Nematbakhsh, 2018; Kaschina et al., 2017; Yousif et al., 2017). Hence there are two main RAS vasodepressor pathways (including angiotensinconverting enzyme [ACE]-AT2RAng II and ACE2MasR-Ang1-7 arms) that regulates the functions of RAS classical pathway (ACEAT1 receptor-Ang II arm) (Dilauro and Burns, 2009; Ferrario and Varagic, 2010). In this regard, it is reported that the renal AT2R expression increased early after RIR and it can attenuate the RIR induced renal injury (Matavelli et al., 2011). Furthermore, it has been shown that Ang 1-7 via MasR has renoprotective effect in RIR model (da Silveira et al., 2010; Maleki et al., 2018) and MasR genetic deficiency can lead to glomerular hyperfiltration and renal fibrosis (Pinheiro et al., 2009). Also, it is known that angiotensin antagonist's administration can exert significant effects on the kidney subjected to RIR (Rabie et al., 2012). According to this result, we hypothesized that in RIR model, AT2R and MasR can have an important role in modifying the renal injury. In order to assess this hypothesis, renal functions were evaluated while AT2R and MasR were blocked by PD123319 and A779 respectively in male and female RIR rat models. Materials and methods Animal The total of 60 male and female Wistar rats (male: 202.67±0.84 g, n=30 and female: 178.13±0.83 g, n=30) were housed in a temperature of 23-25°C with a 12 h light/dark cycle and were fed with rat chow and had free access to tap water ad libitum. The maintenance and care of experimental animals comply with National Institutes of Health guidelines for the humane use of laboratory animals, and has been confirmed by the National Institute of Medical Research (NIMAD, # 943759). Male or female rats were divided into 5 groups. In summary, the designed groups were as follows (n=6 rats in each group): group 1, sham-operated male rats treated with vehicle (saline); group 2, bilateral RIR male rats treated with vehicle (control group); group 3, bilateral RIR male rats treated with MasR antagonist (A779); group 4, bilateral RIR male rats treated with AT2R antagonist (PD123319) and group 5, bilateral RIR male rats treated with both PD123319 and A779. The groups 6-10 were included female rats that received the same regimen as groups 1-5. Surgical preparation Rats were anesthetized with 1.7g kg -1 bodyweight urethane (Sigma St. Louis USA). The trachea was isolated to insert an air ventilation tube in order to facilitate breathing. The left jugular vein was cannulated with polyethylene tubing (PE 9658, Microtube Extrusions, North Rocks NSW, Australia) for vehicle/antagonists infusion. Also, catheters were implanted into the left carotid and femoral arteries connected to a pressure transducer and a bridge amplifier (Scientific Concepts, Vic., Melbourne, Australia) for measuring mean arterial pressure (MAP) and renal perfusion pressure (RPP) respectively. MAP and RPP were monitored during the experiment continuously. The bladder also was cannulated to drain urine during the experiment. Finally, under general anesthesia, surgery was performed through an incision on left and right quadrant of the abdomen under sterile conditions and the renal vessels were prepared to induce ischemia by vessels clamping. During the entire period of surgical procedure rectal temperature was maintained at 37±1°C. Experimental protocol: baseline measurement and RIR induction The animals were allowed to stabilize for 30-45 minutes as equilibrium time for baseline measurements. The baseline data for MAP and RPP were obtained over the last 5 minutes of equilibrium D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 23 + 03 30 o n M on da y N ov em be r 4t h 20 19 135 | Physiol Pharmacol 22 (2018) 133-140 Samimiat & khosravi et al. time. Then in RIR groups, the left and right renal arteries were ligated with micro bulldog clamp and kidneys were subjected to ischemia for 30 minutes and they followed by reperfusion for 75 minutes. MAP and RPP were monitored during the RIR period and data for MAP and RPP were obtained over the last 5 minutes of 30 min renal ischemia time and between 10 and 15 minutes after the reperfusion period as reperfusion time. The experimental protocol was done in all animals but in sham-operated groups was done without RIR induction. ntagonist responses Based on groups specified, the animals were subjected to receive either vehicle (saline), MasR antagonist (A779, Bachem, King of Prussia, MO, USA) or AT2R antagonist (PD123319, Sigma, St. Louis, MO, USA). The A779 and PD123319 were dissolved in 0.9% w/v saline and at the 15 minutes after the beginning of reperfusion the vehicle or antagonist were infused. A779 was administered via jugular vein tube as a bolus dose of 50 μg kg -1 followed by continuous infusion at 50μg kg -1 h -1 using microsyringe pumps (New Era Pump System Inc. Farmingdale, NY, USA). PD123319 was administered with bolus doses of 1 mg kg -1 followed by continuous infusion at 1 mg kg -1 h -1 using microsyringe pumps. The 60 minutes post vehicle or antagonist infusion was considered as antagonist effect time for the measurement. MAP and RPP were determined over the last 5 minutes period of antagonists’ effect time. Urine sample also was obtained after the 60 min of vehicle/antagonist infusions. Finally, blood samples were obtained via heart puncture and the animal was sacrificed humanly. Then the serum and urine creatinine (Cr) levels were determined by commercial kits (Pars Azmoon, Tehran, Iran) using RA-1000 system. Also, the serum and urine Na + levels were obtained using flame photometer assay. Statistical analysis Data analyzing was done by SPSS 20 software and presented as the mean±SEM. ANOVA for repeated measure data and two ways ANOVA were applied using Tukey test as post hoc test. P≤0.05 was considered to be significant.

High and low temperatures affect rat hippocampal synaptosome’s viability and functions

Abstract: Introduction: Synaptosomes are sealed particles that contain mitochondria, cytoskeleton and vesicles which are necessary to synaptic events like neurotransmitter release and uptake in the nervous system. However, the effect of high and low temperatures on synaptosome membrane integrity and function during a time course after its extraction is less known. The purpose of this study was to assess synaptosome viability and function at 37, 4°C and room temperature (RT) during 6 hours after its extraction. Methods: Hippocampi of 40 male Wistar rats were used for synaptosome preparation. To ensure synaptosome membrane integrity and function, lactate dehydrogenase activity (LDH) and GABA uptake were assessed during 6 successive hours after their extraction at 37, 4°C and RT. Results: Our results showed that at 37°C, synaptosome membrane integrity was reduced 3 hours but at 4°C and RT, it occurred 5 hours following their extraction. The results of synaptosome function analysis coincide with LDH enzyme assay data, meaning that GABA uptake faced a 50% reduction from the initial value at 37°C after 3 hours and at RT after 5 hours. We also found that GABA uptake was reduced at 4°C in the first hour after extraction because the low temperature inhibits GABA transporters. Conclusion: Synaptosomes preserved their viability and function at RT, 37 and 4°C at least for 3 hours after extraction and reduced over time. For long term application of synaptosomes, it is better to keep them at 4°C. iD Physiol Pharmacol 22 (2018) 73-81 D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 19 + 03 30 o n M on da y N ov em be r 4t h 20 19 Synaptosome viability and function Physiol Pharmacol 22 (2018) 73-81 | 74 by Whittaker in the late 1950s and detected as detached synapses by electron microscopy (Whittaker, 1993; Whittaker and Gray, 1962). Synaptosomes are acquired by homogenization of fresh brain tissue in an appropriate isotonic solution which causes the nerve terminals to be separated from their axon stalks. Then, the membrane of presynaptic terminals is resealed, which contains cytoplasm, cytoskeleton, mitochondria and synaptic vesicles with the presynaptic and occasionally postsynaptic membranes (Dunkley et al., 2008; Whittaker et al., 1964). Synaptosomes have widely been applied as an in vitro model to investigate the molecular mechanisms of brain synapses specially storage, release, and uptake of neurotransmitters. As a result, our knowledge about the function of synapse ending at a physiological, cellular and molecular level was enhanced (Breukel et al., 1997; Dunkley et al., 1988; Dunkley et al., 2008; Whittaker, 1993). Different procedures have been developed to isolate synaptosomes including ultrafiltration, electrophoresis, sucrose and percoll gradients; however, a median-speed centrifuge technique seems to be more appropriate (Dunkley et al., 1988; Dunkley et al., 2008; Enriquez et al.,1990; Kamat et al., 2014; Stadler and Tashiro, 1979). Synaptosomes can be obtained from any part of the brain tissues. In addition, the nerves of the non-neurological tissue are also involved in the synaptosomes preparation (Jonakait et al.,1979). If a chemical active ingredient is found abundantly in synaptosomes, it can be assumed as a neurotransmitter or coneurotransmitter. Moreover, mixed fraction synaptosomes can be applied to assess the mechanisms of neurotransmitters release (Breukel et al., 1997). Exposure of adult albino rats to the high ambient temperature at 35 °C for 2-12 hours or at 45 °C for 12 hours increased the acetylcholinesterase activity of their brain synaptosomes (Mukhopadhyay and Poddar, 1990). Several studies have investigated the effects of organophosphate compound on the GABA uptake of synaptosomes in cerebral cortex, cerebellum and hippocampus, suggesting that the GABA uptake was optimal one hour after synaptosome extraction (Ghasemi et al., 2007; Pourabdolhossein et al., 2009; Shahroukhi, et al., 2007). Furthermore, Hosseini et al. reported 35 minute is the optimal time for GABA release by rat cerebral synaptosomes (Hosseini et al., 2004). Incubation of synaptosomes at room temperature (22-25 °C) gradually increases the percentage of microtubule containing synaptosomes (41-47%) while its stabilization at low temperature leads to a significant reduction in the number of synaptosomes (Hajos et al., 1979). Despite the significance of synaptosomes in pharmaceutical, structural and functional studies, there is no empirical data on synaptosomes viability and function at different temperatures and time points after their extraction. Thus, the main ambition behind this study was to examine the synaptosomes survival and function at different temperatures and time points by measuring lactate dehydrogenase enzyme (LDH) and GABA uptake. We used hippocampal synaptosome due to its high synaptic contacts ratio compared to the other brain regions (Cragg, 1975). Materials and methods

Effect of Plantago major extract on doxorubicin-induced nephropathy in rat

Abstract: Introduction: Nephropathy is defined as rational loss of renal function related with glomerulosclerosis and declining glomerular filtration rate. Inflammation and oxidative stress play a critical role in nephropathy. Plantago major has antioxidant effects. The aim of present study is the investigation of the effect of Plantago major hydroalcoholic extract on the oxidative stress and renal function in kidney of rat. Methods: Rats were divided into five groups: control (Co), doxorubicin (DOX), doxorubicin+ vitamin E (DOX+ Vit E), 600mg/kg Plantago major (PM)+ doxorubicin (PM600+ DOX), 1200mg/kg Plantago major (PM)+ doxorubicin (PM1200+ DOX). DOX (5mg/kg, IV), Vit E and PM extract (600 and 1200mg/kg, PO) were administrated for 35 days. Finally, urine, blood samples and renal tissue were collected to measurement of redox markers, functional parameters and renal index percentage. Results: The renal superoxide dismutase (SOD) activity, total thiol and functional parameters significantly reduced and malondialdehyde (MDA) concentration increased in DOX group in comparison with control group. The renal SOD, catalase activities and total thiol content were significantly increased and MDA level decreased in PM treated groups along with DOX group in comparison with DOX group. The functional parameters significantly enhanced in treated groups with PM in comparison with the DOX group. The extract did not relive enhanced renal index induced by DOX. Conclusion: Hydro-alcoholic extracts of PM, specially at its high dose led to an improvement in DOX-induced renal function and oxidative stress.

Diabetes mellitus linked Alzheimer’s disease – A review on sporadic form of Alzheimer’s disease

Abstract: This review mainly deals with scientific data related to sporadic Alzheimer’s disease (AD) particularly related to diabetes mellitus (DM). AD is divided into sporadic AD and familial AD. It is known to be the most common cause of dementia. Sporadic form of AD results from multiple etiologic factors including metabolic, environmental and genetic factors. DM linked AD is known to be one of major challenges to health care system in these days. Both type 1 and type 2 DM is strongly related to cognitive impairment and known to be a major risk factor in the development of probable Alzheimer’s disease. In this review, the various mechanisms involved in the development of neuronal degeneration associated with chronic hyperglycaemia are discussed. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 06 + 03 30 o n M on da y N ov em be r 4t h 20 19 Diabetes mellitus linked Alzheimer’s disease Physiol Pharmacol 22 (2018) 141-145 | 142 plaques. The accumulation of senile plaque leads to activation of microglial cells and proinflammatory cytokines which are responsible for inflammatory responses in neocortex and hippocampus. Imbalance between kinases and phosphatases causes hyperphosphorylation of tau protein that results in neuronal loss and degeneration. The hyperphosphorylated or pathologically modified tau protein aggregate into neurofibrillary tangles in brain and contributes to neuronal loss and degeneration (Ghanemi, 2015; Saido, 2013; Belluti et al., 2013; Thota et al., 2007). Diabetic encephalopathy is a condition triggered by DM, which is characterized by electrophysiological, structural and neurochemical changes in the brain leading to cognitive impairments. The most common altered cognitive function in early onset of DM is memory impairment (Liu et al., 2008). The prominent common pathological feature in DM and AD is extracellular aggregation of insoluble amyloid beta proteins. Common pathological features between diabetes mellitus and Alzheimer’s disease Amyloidogenesis DM and AD are known to be amyloid forming diseases. The pathological hallmark of AD is the extracellular amyloid plaque. The primary component of this amyloid plaque is the Aβ peptide 1–42. Aβ is derived from the amyloid precursor protein (APP) by proteolytic cleavage. APP is the base of molecular pathology of which impairs glucose metabolism and tolerance in brain (Bigl et al., 2003). The deposition of amyloidogenic peptide is also seen in the islets of Langerhans in DM in the same manner. The islet amyloid deposit is known as Islet Amyloid Polypeptide (IAPP) or amylin which is a 37 amino acid peptide. IAPP is co secreted with insulin. Together with the insulin and glucagon IAPP acts as a satiety and adiposity signal in regulation of food intake and body weight. The human Aβ1-42 and IAPP are highly susceptible for aggregation and thus they can easily lead to the formation of polymers (Yang and Song, 2013; Sun et al., 2012). Aggregates of Aβ oligomers and also their early intermediate assemblies provoke toxicity to neurons. Similarly, aggregates IAPP produces toxicity to βcells of islets of Langerhans (Porat et al., 2003). IAPP oligomers reacts same way toward cultured β cells as how Aβ oligomers react to neurons (Zhao and Townsend, 2009). Impairment in energy metabolism Glucose is the main source of energy for the neurons in the brain. Thus any impairment in the utilization and glucose metabolism forms the pathological basis of DM (Hoyer, 1990). In total body glucose, 18-30% is consumed by brain. Disruption in the supply, transport or utilization of glucose can lead to neuronal damage and functional deficits in brain. Neuronal death always accompanies insulin resistance in developing human brains which may even cause permanent brain damage (Dunne et al., 2004; Strachan et al., 1997; Tun et al., 1990; Richardson, 1990). This gives a clear indication that cognitive function is affected by disruption in glucose metabolism. Clinical studies have revealed that AD patients had severely impaired glucose metabolism in their cerebral cortex. In early stages, the hypometabolism was found to be prominent in parietotemporal and the posterior cingulate regions, as the disease progresses it is observed spread to the prefrontal cortex (Mosconi et al., 2008; Small et al., 2000). AD genetic risk factor is strongly correlated to abnormal glucose metabolism. Metabolic reduction of glucose in posterior cingulate regions of AD transgenic animal model was reported by Valla et al. (2008). The association between impaired glucose transport and neurodegeneration links the major glucose GLUT1 which is selectively expressed in the endothelium of the blood brain barrier. Significant reduction of GLUT1 is seen in aged humans and AD transgenic mice model coinciding with hippocampal atrophy (Hooijmans et al., 2007) Oxidative stress and high level of advanced glycation end products (AGEs) Non-enzymatic glycation and oxidation of cellular proteins, nucleic acids and lipids by reducing sugars leads to accumulation of AGEs in chronic hyperglycemia. Accumulated AGEs binds to the receptor “RAGE” expressed on various cells such as microglia, vascular smooth muscle cells, mononuclear phagocytes and endothelial cells. AGEs interact with RAGE and triggers signalling cascade D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 06 + 03 30 o n M on da y N ov em be r 4t h 20 19 143 | Physiol Pharmacol 22 (2018) 141-145 Sathiya et al. leading to increased oxidative stress by up-regulating transcription factor NF-κB. This leads to increased secretion or production of inflammatory cytokines such as TNF-α and IL-6. Thus the interaction of RAGE-AGE is depicted as a major source of oxidative stress and inflammation which is correlated with diabetic complications as well as neurodegenerative diseases. AGE is known to be an accelerating factor for Aβ aggregation (Reddy et al., 2002). Evidence suggest that the neurofibrillary tangles are stabilized by glycation of tau protein which actually results from accumulated AGE products. Thus AGE contributes to amyloidosis and neurofibrillary tangle formation in brain. Abnormally hyperphosphorylated tau protein localised in neuronal axons is known to be the major component in neurofibrillary tangles (Zhao and Townsend, 2009; Smith et al., 1995). Role of insulin receptor and insulin receptor substrates Modulation of synaptic plasticity and cognition are the major functions of insulin receptor in brain (Zhao and Townsend, 2009). The abundant distribution of insulin receptors in cerebral cortex, amygdala and hippocampus are the evidences for their involvement in cognitive process and synaptic activity. Studies have shown that animals with increased amount of insulin receptor and insulin receptor substarte-1 in the hippocampal synaptic membranes show better spatial memory compared to the diabetic animals which showed reduction in hippocampal insulin receptor. Accumulated Aβ competes with insulin to bind to the insulin receptor and interferes with insulin signalling. Impaired insulin signalling is reported to be a main cause of reduced clearance of Aβ oligomers (Yang and Song, 2013).

Protective effects of omega-3, atorvastatin, vitamin E and vitamin C against doxorubicin-induced cardiotoxicity

Abstract: Introduction: The stress-oxidative is involved in doxorubicin (DOX)-induced cardiotoxicity. Due to the potential and previous reported for antioxidant properties of atorvastatin, omega-3, vitamin E and vitamin C, their efficacy to prevention of DOX-induced cardiotoxicity was investigated in this study Methods: Fifty-six male rats were divided into 8 groups which received omega-3, atorvastatin, vitamin E, vitamin C, normal saline and dimethyl sulfoxide (DMSO) via gavage for 14 days then a single dose of DOX (20 mg/kg) was injected intraperitoneally except two last groups that received only normal saline or DMSO. The level of oxidative stress parameters like ferric reducing ability of plasma (FRAP) before and after DOX injection and malondialdehyde (MDA) of heart were estimated. Also the histopathologic assessments were done on heart sample at the end of experimental period. Results: The results showed that compared to other agents, omega-3 could emerge as the most protection against DOX. Its pretreatment led to one of the most FRAP changing percent meanwhile less MDA value and cardio pathologic indexes almost close to control groups compared to that of other agents (P<0.01). Conclusion: Omega-3 may have a promising protective effect against DOX-induced

Urban traffic noise pollution disturbs spatial learning and memory and increases anxiety-like behavior in adult male rats

Abstract: Introduction: Noise pollution is an unwanted inevitable distribution of the modern and industrialized life of mankind. With the expansion of urban life, humans are daily exposed to noise pollution which can cause anxiety and disorders in cognitive activities. The present study was aimed to investigate the impact of sub-chronic urban traffic noise pollution on learning, memory and anxiety-like behavior in adult male rats. Methods: Thirty two adult male Wistar rats (weighing 275-300g) were used in the present experimental study. The animals were divided into two groups: the control and the noiseexposed. The rats in the test group were exposed to a 90dB noise recorded from a crowded street traffic for 6h/10 days. Control rats were intact. Morris water maze (MWM) and an elevated plus maze (EPM) were used to assess spatial learning and memory and anxiety-like behavior in rats. Results: The findings displayed that both control and noise-exposed group improved their maze steering over 4 days of experiment in MWM; however, noise-exposed group had more latency and traveled-distance in MWM to find the hidden platform in probe trial compared to those of control (P<0.05). Moreover, noise-exposed group showed a significant increase in weight gain compared to the control group (P<0.05). In addition, the spent time in open arm of the EPM was significantly decreased compared to controls (P<0.05). Conclusion: Urban traffic noise pollution for a short-term period causes a meaningful increase on weight gain, disorders in retrieval memory and increase in anxiety-like behavior in

Effectiveness of intravenous promethazine and diazepam in the treatment of peripheral vertigo in emergency department visits

Abstract: Introduction: To improve our understanding of dizziness, its assessment, as well as its management to identify the appropriate treatment in order to reduce the costs and to increase the patients’ quality of life. Methods: A single blind parallel group randomized controlled trial was conducted on 164 participants with dizziness. One group received 25mg/ml promethazine and another received 5mg/ml diazepam. To assess the severity of dizziness, Visual Analog Scale was used prior to and two hours after treatment. Results: Both promethazine and diazepam had significant effects on decreasing the severity of dizziness, i.e. the severity score decreased from 9.31 to 1.81 in promethazine versus 9.33 to 4.50 in diazepam (P<0.001). Conclusion: A single IV dose of promethazine is more effective in reducing vertigo as compared with diazepam. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 6 :4 7 + 03 30 o n W ed ne sd ay M ar ch 1 1t h 20 20 Promethazine, diazepam and peripheral vertigo Physiol Pharmacol 22 (2018) 213-218 | 214 (Halmagyi and Baloh, 1996). Traditionally, it has been divided into four main subtypes (Drachman and Hart, 1972): (i) vertigo, where patients express illusory sensation of self-motion, which is often the result of a vestibular system disorder; (ii) pre-syncopal dizziness, a light-headed sensation associated with cerebral hypo perfusion; (iii) psychogenic dizziness, associated with a mental health issue, such as generalized anxiety and (iv) disequilibrium and nonspecific dizziness, often associated with neuromuscular causes. In older people, dizziness may have a multifactorial etiology, with many dizzy older patients fulfilling criteria for two or more of the subtypes described above (Kroenke et al., 1992; Tinetti et al., 2000a; Sloane et al., 2001). Treatment of peripheral vertigo is symptomatic and mainly includes anti-nausea and inhibitors of the ear balance system but there is no confirmed standard guideline to select the type and duration of treatment (Strickland et al., 2003; Roceanu et al., 2016). The benzodiazepines, anticholinergic and phenothiazine are some common medications used in the treatment of vertigo. The efficacy of corticosteroids has also previously been reported in some studies (Strupp et al., 2004; Amini et al., 2015; Roceanu et al., 2016; Shahrami et al., 2016). Currently due to the variety of vertigo treatments, the lack of a single protocol in dealing with dizziness, complications of medical and surgical treatments as well as limitation of exercise and physical therapy to resolve dizziness, there are limitations on how to deal with vertigo. For this reason, we decided to compare the effectiveness of intravenous promethazine and diazepam, two drugs that have the least side effects and maximum effect on reducing dizziness, in the treatment of peripheral vertigo in patients referring to the emergency department. The current randomized control trial was carried out to improve the understanding of dizziness, its assessment as well as its management to identify the appropriate treatment so as to reduce the costs and to increase patients’ quality of life. A single blind parallel group randomized controlled trial was conducted after receiving the ethics approval and patient informed consent from 164 participants randomly selected from among individuals with dizziness who had referred to Baqiyatallah Hospital, Tehran, in 2015. The Ethics Committee at Baqiyatallah University of Medical Sciences approved the study. Also, the present study was reviewed and approved by Iranian Registry of Clinical Trials (IRCT 20171221037984N1). Dizziness was diagnosed by a Emergency Medicine with more than 10 years of experience. Patients were randomized into two groups using a computer generated randomization list. One group received 25mg/ml promethazine intravenously and the other group received 5mg/ml diazepam intravenously. If the patients did not respond to the treatment initially, they would be assigned to the other group, and still if they did not respond to the other treatment protocol, they would undergo other more aggressive treatments. To assess the severity of dizziness, Visual Analog Scale (VAS) was used prior to and two hours after the treatment in setting position. This way, patients were asked to score the severity of their vertigo from 1 to 10. To evaluate the reliability, test–retest reliability proved to be good, but higher among literate (r: 0.94, P<0.001) compared with illiterate patients (r: 0.71, P<0.001) prior to and after their visit to the clinic. The inclusion criteria were age over 18 and diagnosis of peripheral vertigo. Potential participants were excluded if they; (i) had a degenerative neurological condition, (ii) were currently (in the past 24 hours) receiving treatment for their dizziness, (iii) had a cognitive impairment (a General Practitioner Assessment of Cognition (GPCOG) of <5) (Brodaty et al., 2002) and/or, (iv) were unable to walk 20m without difficulty using a walking aid, and had evidences of developing drug-induced vertigo and head trauma. Participants were asked to sign an informed consent form before answering the questionnaire. All personal information remained anonymous. Data were analyzed using Statistical Package for Social Sciences (SPSS), version 19 (SPSS Inc. Chicago, IL). Sample size was calculated using sample size formula. We considered α=0.05 and power=90%. Normal distribution variables (approved by one-sample Kolmogorov–Smirnov test) were compared using independent sample t-test between the groups and paired sample t-test within the groups. In addition, Chi square test was run to compare categorical variables between the two groups. Analysis of the simultaneous effect of variables was carried out running logistic regression. Furthermore, repeated measures two-way ANOVA was employed to compare dizziness score and D ow nl oa de d fr om p pj .p hy ph a. ir at 6 :4 7 + 03 30 o n W ed ne sd ay M ar ch 1 1t h 20 20 215 | Physiol Pharmacol 22 (2018) 213-218 Fayyaz et al. duration between groups during time. P-values <0.05 were considered as statistically significant. The average ages of the patients who consumed diazepam and promethazine were 45±11 and 47±12, respectively. Of the patients who were taking diazepam, 24.3% (n=17) were male and 75.5% (n=53) were female and 32.9% (n=23) reported a positive history of an underlying disease. In addition, among patients who took promethazine, 43.6% (n=41) were male and 56.4% (n=53) were female and 37.2% (n=35) were with a positive history of an underlying disease. Both promethazine and diazepam had significant effects on decreasing severity of dizziness; the severity score decreased from 9.31 to 1.81 in promethazine group versus 9.33 to 4.50 in diazepam group (P<0.001), but comparison of the means showed that promethazine was more effective (Table 1). Furthermore, the influence of the drugs was not found to be significantly associated with any factor, including age, gender, duration of vertigo, etc. According to Table 2, there is no significant relationship between the mean age (P=0.729) as well as gender (P=0.266) and the effects of the drug; however, promethazine had better effects in males than in females. The means of dizziness duration in patients who consumed diazepam and promethazine were 7±2 hours and 8±2 hours, respectively. According to the results of repeated measures two-way ANOVA, the statistical relationship between dizziness duration as well as scores and the effect of drugs were not observed to be significant after treatments (P>0.05). Furthermore, no significant relationship was found between underlying disease and the drug effect (P=0.833) (Table1). In addition, no significant relationship was observed between cause of dizziness and effect of the drugs (P=0.947); however, the effects of promethazine was better in patients who had dizziness due to Meniere’s disease (Table 2). Vertigo is a common complaint in the emergency departments that influences daily activities and increases medical consultations (Irving et al., 2002). Although many pharmacological agents appear to be clinically useful, insufficient investigations have been carried out to find the most effective anti-vertigo medication with the least adverse effects. Therefore, we conducted the current randomized, double-blind trial to compare the effects of two common intravenously administered medications, i.e. promethazine and diazepam. To treat vertigo, several medications from different pharmacologic groups have been employed, including antihistamines, anticholinergics, benzodiazepines, calcium channel blockers, diuretics, neuroleptics, psychotherapeutic agents and corticosteroids. It seems that these medications influence on the level of neurotransmitters involved in the transmission of impulses through vestibular neurons and in maintenance of tone in the vestibular nuclei (Kerber et al., 2008). Promethazine and diazepam are among the drugs used in emergency departments to treat vertigo. Promethazine, a component of phenothiazine drugs, acts as an antiemetic agent with dopamine, histamine Table 1: Comparison of mean dizziness score in diazepam and promethazine groups prior and two hours after

Mespilus germanica leaves flavonoids improve passive avoidance memory and apoptosis in a rat model of amyloid-β neurotoxicity

Abstract: Introduction: Alzheimer’s disease (AD) is a common progressive, neurodegenerative disorder with no preventive or curative therapy until now. Use of natural products as an important source of neuroprotective flavonoids against AD has been considered recently. In this study, the effect of Mespilus germanica leaves (MGL) flavonoids treatment on memory dysfunction and apoptosis in the amyloid beta (Aβ)-treated rat was investigated. Methods: Forty-eight male Wistar rats (220-250g) were divided into 6 groups (n=8): saline, Aβ, treatment (5, 7.5 and 10 mg/kg MGL flavonoids) and positive control group. Step through the passive avoidance test was performed on the 22nd day to examine learning and memory. Immediately afterward, the animals were killed and their brains were removed to measure the levels of cytochrome c in brain homogenate. Results: Our results showed significant improvement in passive avoidance task as flavonoid (10mg/kg) increased step-through latency (P=0.003) and decreased the time spent in dark compartment (P=0.001) significantly. In addition, the levels of cytochrome c which was significantly increased in the Aβ-injected group was reduced remarkably in the flavonoid treatment group (P=0.029). Conclusion: Therefore, MGL flavonoid can improve Aβ1-42 induced memory dysfunction in rats and its effect might be partially due to their role in decreasing apoptosis. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 18 + 03 30 o n M on da y N ov em be r 4t h 20 19 Mespilus g. flavonoids and memory improvement Physiol Pharmacol 22 (2018) 219-227 | 220 toxins and harmful factors (Newman et al., 2007), blocks synaptic plasticity (Nakamura et al., 2001), loses functional neurons (Nakamura et al., 2001) and finally, declines memory and cognition (Lambert et al., 1998). A line of research in AD has concentrated on apoptosis. It has been reported that apoptosis is a major cause of neuronal death in AD (Youle and Strasser, 2008). Both intrinsic and extrinsic pathways of apoptosis are involved in AD (Devi et al., 2006). In the intrinsic pathway, mitochondria release cytochrome c. It binds with APAF-1 (apoptotic protease activating factor-1) to make a complex with procaspase-9 leading to activation of caspase-9 and then caspase-3 (Asadi et al., 2015). Cytochrome C is an important component of the electron transport chain and shuttles electron between mitochondrial complexes III and IV. In addition, it plays a critical role during apoptosis. It is widely believed that the release of cytochrome c from mitochondria is one of the early stages of apoptotic cascades and is also considered as a point of no return in cell death (Jazvinšćak Jembrek et al., 2015). Although there are five FDA approved drugs for management of AD, none of them cures or prevent disease and just offer symptomatic benefits. Over the last decade, growing evidence has been found on the potential of dietary flavonoids for helping prevention of cognitive function. Flavonoids are a large family of polyphenolic compounds synthesized by plants and provide much of the flavor and color of fruits and vegetables. They have the capacity to cross the blood-brain barrier (BBB) and have been detected in the rat brain in areas related to learning and memory shortly after oral administration (Krishnaveni, 2012). The effect of flavonoids on Aβ-induced memory impairment has been assessed in many studies (Balouchnejadmojarad, 2009; Ejaz Ahmed et al., 2013). Although the exact mechanisms are not clear, it has been suggested that flavonoids can enhance cognitive function via their neuroprotective properties and also, they can stimulate neurogenesis, therefore, enhance neuronal function (Spencer, 2007; Spencer, 2008). One of the extremely rich sources of flavonoids is Medlar. It is the fruit of Mespilus germanica in the family of Rosaceae (Gülçin et al., 2011). The medlar is an edible fruit and modern medicine has recognized its healing properties in the treatment of some diseases (Ansari et al., 2006; Ahmady et al., 2013; Ramezani et al., 2016). However, the effects of MGL flavonoids on impaired learning and memory induced by Aβ1-42 in rats has not been reported. Based on these findings as previously reported, this study examined the possible effects of MGL flavonoids on memory dysfunction and brain cytochrome c levels in a rat model of amyloid-β neurotoxicity. Materials and methods

The effects of subchronic social and isolation stresses on learning and memory trend in male rats

Abstract: Introduction: Psychological stresses influence brain functions such as learning and memory. Environmental factors like types and durations of stress affect brain responsiveness. This study investigated the effects of two subchronic social and isolation stresses on learning, memory, adrenal glands weight and corticosterone levels in the hippocampus and frontal cortex. Methods: Eighteen male rats were randomly allocated into three experimental groups: control, social stress and isolation stress groups. Rats were under stresses for 7 days. Latency of entrance into the dark room was evaluated as brain function, using the passive avoidance test before inducing of electrical shock (as initial latency) and on days 1, 3, 5 and 7 after foot shock. In addition, corticosterone levels were measured in the homogenized hippocampus and frontal cortex. Results: The latencies of days 1, 3 and 5 were significantly lower in an isolation stress group than the control group. The latency of day 7 significantly decreased in social and isolation stress groups, compared to the control group. The adrenal glands weight showed significant enhancements in social and isolation stress groups, compared to the control group. Although, the weight of the adrenal glands significantly increased in an isolation stress group, compared to the social stress group. There was a significant enhancement in the corticosterone levels in the hippocampus, but not frontal cortex in isolation stress group. Conclusion: It was concluded that subchronic isolation stress severely deteriorated brain functions (learning and memory) compared to the subchronic social stress. In addition, isolation stress affected corticosterone levels in the hippocampus more than frontal cortex. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 20 + 03 30 o n M on da y N ov em be r 4t h 20 19 83 | Physiol Pharmacol 22 (2018) 82-91 Izadi et al. duration (Jaggi et al., 2011; Radahmadi et al., 2017a; Radahmadi et al., 2017b; Ranjbar et al., 2015). According to the type of stress, there are social, isolation, immobility and many other types of stress that each influences the physiological system of the body through different neural circuits (Campos et al., 2013). For instance, cat-induced stress causes memory impairments (Sandi et al., 2005), heat stress caused cognitive disorders (Lee et al., 2015) and restraint stress accelerated memory deficits via oxidative damage, the corticosterone (CORT) levels and other biochemical stress markers in the hippocampus and frontal cortex (Azadbakht et al., 2015; Dastgerdi et al., 2017; Eidelkhani et al., 2015; Huang et al., 2015; Radahmadi et al., 2017b). On the other hand, based on the stress duration category, a variety of acute, subchronic and chronic stress exists (Bali et al., 2014; Jaggi et al., 2011; Radahmadi et al., 2017a; Ranjbar et al., 2015; Ranjbar et al., 2017). Previous studies indicated that acute stress improved memory (Henckens et al., 2009; Zheng et al., 2008), whereas chronic stress leads to impairment (Radahmadi et al., 2017a; Ranjbar et al., 2015). In addition, subchronic exposure to noise stress impaired memory and cognition with reduction of locomotor activity in open field test (Naqvi et al., 2012). Cognitive deficits also were caused along with chronic mild stress using the novel object recognition test (Papp et al., 2017). Therefore, it seems that stress studies present paradoxical results on brain functions and memory. On the other hand, in the current study hippocampus (as the main region of memory) and frontal cortex (as other region of memory) were selected for measuring CORT levels, because they are involved in both memory processing and stress pathway (McEwen et al., 2016). In addition, both regions send excitatory projections to the paraventricular nucleus of the hypothalamus for activating hypothalamushypophysis adrenal axis in stress (Kinlein and Karatsoreos, 2015). In addition, these regions have abundant CORT receptor (McKlveen et al., 2015; Raineki et al., 2018). Despite a vast amount of researches about the stress on brain functions, none of the studies directly determined which one of the sub chronic stress types was more harmful on learning and memory. Therefore, the present study was designed to investigate the effects of two subchronic psychological stresses (social and isolation stress) on learning, memory trend, the weight of the adrenal glands (as one of the stress indexes), as well as hippocampal and frontal cortex corticosterone levels in the same laboratory conditions. Materials and methods Experimental animals Experiments were performed on eighteen adult male Wistar rats weighting 200-250 g. The animals were maintained under 12 h light/dark cycles at controlled temperature (22±2°C) and humidity (50±5%) conditions with ad libitum access to food and water. All the experiments were performed in accordance with the standards set by the Ethics Committee of Isfahan University of Medical Sciences and the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80– 23, 1996 Rev). All behavioral experiments (learning and memory) were carried out between 14:00 and 16:00 pm. Memory function was evaluated by the passive avoidance test at different intervals (1, 3, 5 and 7 days) after foot electrical shock. Rats were randomly assigned to the following three groups (n=6 in each group): control, social stress and isolation stress groups. Rats were under social and isolation stresses for seven consecutive days. In addition, at the last day of the protocol, the adrenal glands were carefully dissected and immediately weighed (fresh tissue) as a stress index (Radahmadi et al., 2017a; Ranjbar et al., 2017; Ulrich-Lai et al., 2006). In addition, hemi-hippocampus and hemifrontal cortex were instantly dissected to be kept on dry ice, for evaluation of CORT level (Dastgerdi et al., 2017). Experimental procedures Stress paradigm For induction of social stress, rats were transferred to the new cage with new neighbors for every 24 hours, as one kind of psychological stress (Grippo et al., 2007). In addition, for induction of isolation stress, rats were kept in individual cages without any other neighbors (Grippo et al., 2007; Kalshetti et al., 2015). Since, rats are social creatures, isolation stress is considered as a psychological stress condition (Grippo et al., 2007). Stress was inducted for seven consecutive days, as subchronic stress and/or mid D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 20 + 03 30 o n M on da y N ov em be r 4t h 20 19 Subchronic stress and memory trend Physiol Pharmacol 22 (2018) 82-91 | 84 stress (7 days) was the strongest stress condition, with respect to other timing of stress (Forsberg et al., 2015; Patki et al., 2013; Ranjbar et al., 2015; Ranjbar et al., 2016; Ranjbar et al., 2017; Sahin et al., 2015). Behavioral paradigms The passive avoidance apparatus (64 cm × 25 cm × 35 cm) divided into two compartments of identical size (32 cm × 25 cm × 35 cm) with sliding guillotine doors and grid floors. On the first day, rats were placed in the light compartment and were allowed to explore the whole apparatus (sliding door open) without the electrical shock over a period of 300s. The day after, an acquisition trial was performed, rats were placed individually in the light compartment for the 60s and then the sliding door was raised. When the rat entered the dark compartment, the door was closed and an inescapable scrambled single foot electric shock (0.5mA, 2s; once) was delivered through the grid floor by a shock unit (Dastgerdi et al., 2017). The initial latency of entrance into the dark room was recorded before inducing of electrical shock. In probe trials (1, 3, 5 and 7 days after foot shock), the rat placed in the light compartment again for 60s, then the sliding door was raised and rat accessed to the dark compartment without any shock. The time to enter the dark compartment (up to a maximum of 300s) was recorded. If an animal did not enter the dark compartment within 300s, the trial was terminated. The absence of entry to the dark compartment or a longer duration in the light compartment indicated as a positive response, because the passive avoidance task determines the ability of a rat to remember a foot shock delivered. In addition, latencies of the initial and probe trial on day 1 (before and after foot shock, respectively) indicated learning. Meanwhile, memory changes have been shown by the comparison of probe trial latencies (Hosseini et al., 2014; Radahmadi et al., 2013; Radahmadi et al., 2015a). Measurement of adrenal glands weight At the end of the experimental period, adrenal glands weight were measured for each rat. Assessment of CORT levels in the hippocampus and frontal cortex At the end of the experiments, the animals were sacrificed at 12:00–14:00 pm by decapitation on day 8. Following decapitation, the brain of each animal was immediately dissected from the skull and the hemi hippocampus and hemi frontal cortex were instantly dissected to be kept on dry ice, which were subsequently immersed in ProblockTM 50, EDTA free (Gold Bio Co., USA) and in a phosphate buffer solution (0.01M, pH 7.4), separately. Indeed, this solution contained a complete protease inhibitor cocktail (Radahmadi et al., 2015a). The hippocampus and frontal cortex were homogenized and centrifuged in a cooled centrifuge (4°C, 10,000g) for 20 min. The supernatant was collected and stored at −80 °C, until further assessment. The commercial Enzyme Linked Immuno Sorbent Assay (ELISA) kit (Zellbio Co., Marburg, Germany) was used to assess the CORT level in the hippocampus and frontal cortex. The amount of CORT was determined in a given volume of the supernatant (Dastgerdi et al., 2017; Radahmadi et al., 2015a; Radahmadi et al., 2015b). Statistical analysis Biochemical and behavioral data on days 1, 3, 5 and 7 were analyzed using the one-way ANOVA followed by LSD post-hoc t

Dynamic changes of hemodynamic parameters and cardiac transcription of sirtuins in adaptive and mal-adaptive phases of pressure overload-induced hypertrophy in rats

Abstract: Introduction: The aim of the study was to investigate the structural and hemodynamic changes as well as cardiac transcriptional profile of the key regulatory proteins, sirtuins family (SIRT1-7), in adaptive and mal-adaptive phases of left ventricular hypertrophy (LVH). Methods: LVH was induced in male Wistar rats (190±20g) by abdominal aortic banding. The third and sixteenth weeks post-surgery were considered as adaptive and mal-adaptive phases of hypertrophy (H3w and H16w groups, respectively). Blood pressure (BP) was recorded through the carotid artery catheter. Cell area and fibrosis were assessed using haematoxylin/eosin and Masson trichrome staining, respectively. The sirtuins mRNA levels were quantified using quantitative RT–PCR technique. Results: H3w rats had a higher systolic and diastolic BP, cardiomyocytes area and heart to body weight ratio (HW/BW) compared with control (intact animals). Although cell size and HW/BW increased in the H16w group, systolic and diastolic BP did not change significantly in comparison with control. In H3w group, SIRT1/3/6/7 mRNAs levels increased significantly. In H16w group, SIRT1/3/5/6/7 mRNAs levels declined in comparison with H3w group. SIRT2 and SIRT4 mRNA levels did not change significantly among the experimental groups. Conclusion: During progression of cardiac hypertrophy transcriptional profile of sirtuins changes in parallel to structural and hemodynamic parameters. Therefore, it can validate sirtuins as the pharmacological targets for the treatment of pathological LVH. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 15 + 03 30 o n M on da y N ov em be r 4t h 20 19 Sirtuins transcription in cardiac hypertrophy Physiol Pharmacol 22 (2018) 279-291 | 280 12.8% of all death worldwide, has been known as the most common cause of LVH, as prevalence of LVH in hypertensive patient is about 40-60% (Martinez et al., 2003; Lazzeroni et al., 2016). Following uncontrolled hypertension, high peripheral resistance load placed on the left ventricle results in hypertrophy response in the heart. During the early phase of LVH, cardiac performance increases to adapt the heart with the increased workload. However, sustained LVH can develop into decompensating or mal-adaptive stage with prolonged volume or pressure stress. The consequences of this persistent hypertrophy include activation of pro-apoptotic and pro-fibrotic pathways, abnormal calcium handling, progressive inflammation, imbalance between the myocardial oxygen supply and consumption and ion current disturbance. Therefore, prolonged LVH will ultimately result in deterioration of contractile performance of the heart and arrhythmia and eventually heart failure and sudden death (Kehat and Molkentin, 2010 ). Pressure overload-induced LVH progression is accompanied by alteration in heart geometry includes increase of heart weight, LV weight, LV thickness and interventricular septum thickness (concentric hypertrophy). In the late phase of LVH, the eccentric phenotype of hypertrophy is appeared in the heart which is characterized by dilatation of the left ventricular chamber and weakened heart. In compensated phase of hypertrophy, cardiac hypercontractility preserves the systolic function with higher ejection fraction. However, while LVH progresses to decompensated phase, ejection fraction progressively reduced (Lazzeroni et al., 2016). During progression of LVH, excessive deposition of extracellular matrix proteins contributes in myocyte misalignment, distorted heart architecture and reduced tissue compliance which ultimately leads to cardiac failure. The negative correlation between ejection fraction and interstitial fibrosis has been shown in patient with pressure overload-induced LVH (Hein et al., 2003). Despite the significant progress in reducing LVH-induced heart failure, the prevalence of this disease and the consequent mortality rate is still high worldwide. Considering the disastrous complication of LVH progression, new therapeutic approaches for treatment of this disease are highly needed. A hallmark of hypertrophy progression from adaptive (early) to mal-adaptive (late) phase is the genetic reprogramming of several cellular signaling in the cardiomyocytes. Identification of genes involved in cardiac hypertrophy progression improves our overall understanding of the molecular differences between adaptive and mal-adaptive phases of cardiac hypertrophy leading to discovery of new therapeutic approaches (Lai et al., 2013). Emerging studies have revealed that histone/protein deacetylases (sirtuins) contribute to the regulation of physiological processes such as metabolism, cell growth, cell survival and stress response in the heart (Matsushima and Sadoshima 2015; Katsi et al., 2016). Seven sirtuins (SIRT1-7) have been identified in mammalian cells. The first one, SIRT1, which is a nucleocytoplasmic protein, regulates distinct metabolic and cell survival pathways in the heart. SIRT1 plays an important role in the regulation of physiological and pathological cardiac cell growth, but the underlying mechanism is poorly understood (Planavila et al., 2011; Lai et al., 2014) . The role of SIRT2 in cardiovascular system has been investigated in recent studies. SIRT2 deacetylases histones, tubulin and a broad range of transcription factors, thereby contributing to the control of genomic stability, metabolism and inflammation (Inoue et al., 2007). It has been shown that SIRT2 is involved in microtubule stabilization in diabetic cardiomyopathy (Yuan et al., 2015) and hypertension-induced vascular remodeling (Hashimoto-Komatsu et al., 2011). Recently, it has been shown that SIRT2 is a negative regulator of pathological hypertrophy through deacetylation and inactivation of the nuclear factor of activated T-cells (NFAT) transcription factor (Sarikhani et al., 2018) . Mitochondrial sirtuin, SIRT3, has been shown to protect the heart against oxidative stress by suppressing cellular levels of reactive oxygen species (ROS) and improvement of mitochondrial function (Chen et al., 2015a). The cardioprotective effects of another mitochondrial sirtuin, SIRT4, are currently under debate. For example, SIRT4 ameliorated hypoxia-induced apoptosis in cardiomyoblast by influencing caspase activity (Liu et al., 2013a); however, promoted pathological hypertrophy and fibrosis by inhibition of manganese superoxide dismutase activity (Luo et al., 2017). The crucial role of SIRT5 in regulation of cardiac D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 15 + 03 30 o n M on da y N ov em be r 4t h 20 19 281 | Physiol Pharmacol 22 (2018) 279-291 Hosseinnezhad et al. function has been shown in recent studies (Sadhukhan et al., 2016). It has been demonstrated that SIRT5 was down-regulated in cardiomyocytes following oxidative stress and SIRT5 knocking down led to decreased cell viability and increased apoptosis (Liu et al., 2013b; Sadhukhan et al., 2016; Hershberger et al., 2017). SIRT5 knockout mice had reduced fatty acid and glucose oxidation as well as increased mortality when undergoing transverse aortic constriction to induce cardiac hypertrophy (Hershberger et al., 2017). Studies have revealed that the nuclear protein SIRT6 exerts cardioprotective effects. Sundaresan et al. investigated the role of SIRT6 in cardiac hypertrophy progression in human hearts and animal models of LVH. They showed that SIRT6 deficient mice exhibited left ventricular hypertrophy and cardiac failure. Whereas, SIRT6 over-expression protected the hearts against hypertrophy, as characterized by decrease of HW/BW, cardiomyocyte size and fibrosis. Furthermore, cardiac expression of SIRT6 reduced in human failing hearts in comparison with normal hearts (Sundaresan et al., 2012). The association between genetic variant in the SIRT6 gene and atherosclerotic plaque has also been shown (Dong et al., 2011). There is less information about the cardiovascular effects of the last sirtuin, SIRT7, which is localized in the nucleoli. Some novel cardioprotective effects of SIRT7 have been shown in recent studies. Following myocardial ischemia, SIRT7-deficient mice showed lower survival rate because of the cardiac rupture (Araki et al., 2015). Vakhrusheva et al. (2008) showed SIRT7 deficient mice developed cardiac hypertrophy which was accompanied by myocardial fibrosis, inflammation and increased apoptosis. Decrease of cardiac transcription of SIRT7 has also been reported in aged hearts (Wronska et al., 2016). In general, disturbances in the sirtuins expression or activity is strictly linked to the pathological processes in the heart. However, each of sirtuins has a distinct regulatory pathway and proposing the same expressional profile for all of the situins in different heart diseases is difficult. Consequently, understanding the transcriptional and expressional alteration of these proteins in cardiovascular diseases such as hypertension and LVH could allow for targeting these proteins as the novel paradigm in anti-hypertrophic drug discovery. Despite the importance of cardiovascular effects of sirtuins, there is no report on the comparison of sirtuins transcriptional profiles between early and progressive phases of LVH. Therefore, the aim of the present study is to investigate the cardiomyocytes size, fibrosis and hemodynamic parameters (systolic and diastolic blood pressure, heart rate) as well as cardiac transcriptional levels of sirtuin family in adaptive and mal-adaptive phases of pressure overload-induce hypertrophy in rats. Materials and methods Animal care and experimental protocol All experimental procedures in this study were performed in accordance with the ethical guidelines for animal research approved by the Ethics Committee for Animal Experiments of Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Thirty two, 8week-old male Wistar rats (190±20g) were purchased from the animal house of Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Animals were housed u

Inhibition of Candida albicans yeast– hyphal transition by combination of fluconazole with amphotericin B

Abstract: Introduction: Candidiasis represents a major threat to the life and health in immune-compromised individuals. The number of antifungal drugs is limited for the treatment of candidiasis. Combination therapy is one of the most frequently used techniques to alleviate this problem. Methods: Clinical isolates of Candida albicans were obtained from the immunecompromised patients. Antifungal susceptibilities to fluconazole and amphotericin B alone and in combination were performed by broth microdilution method. Eventually direct microscopic observation, time-kill kinetic assay, biomass and metabolic activity of the hypha, Sap enzyme activity and expression of SAP3 gene were carried out in C. albicans. Results: Combination of fluconazole with amphotericin B demonstrated synergistic and partial synergistic effects with fractional inhibitory concentration index ranged from 0.031 to 0.75. The data indicated that combination of fluconazole with amphotericin B exerted antifungal effects through reducing time-kill kinetic, yeast– hyphal transition, biomass and metabolic activity of the hypha and Sap enzyme activity in C. albicans. Additionally, the expression levels of the SAP3 gene were significantly down regulated (P<0.001) in C. albicans treated with combination of fluconazole with amphotericin B. Conclusion: Taken together, these events may confirm the potential uses of combination of fluconazole with amphotericin B against C. albicans. The results suggest that SAP3 gene could be probable target of synergistic interaction of fluconazole and amphotericin B in C. albicans. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 0 :5 3 + 03 30 o n W ed ne sd ay O ct ob er 3 0t h 20 19 Inhibition of Candida albicans hyphal transition Physiol Pharmacol 22 (2018) 195-204 | 196 acquisition. The secreted aspartyl proteinases (SAPs), degrades many human proteins on the lesion site, contributing to tissue penetration and tissue invasion in addition to evading immune responses. The proteolytic activity is attributed to a multigene SAP family of C. albicans with at least 10 members, SAP1– 10. C. albicans SAP1– 8 are secreted and released into the surrounding medium, whereas SAP9 and SAP10 remain bound to the cell surface (Chin et al., 2016; Dalle et al., 2010; Mayer et al., 2013). Research studies have demonstrated that there is differential expression pattern of the SAPs at various stages of the infection process. SAP1–3 appear to be essential for adherence of C. albicans to the host cell surface and tissue damage of localized infection, whereas SAP4–6 are involved in systemic disease. Little is known about SAP7 and SAP8 expression pattern in C. albicans pathogenesis. The SAP9 and SAP10 which are cellsurface associated proteases with function in cell surface integrity, cell separation and also indirectly contribute to adhesion (Borelli et al., 2007; Staniszewska et al., 2012). The oldest class of antifungal drugs is the polyenes, of which amphotericin B is highly fungicidal and directly bind ergosterol and disrupt the fungal cell membrane (Gray et al., 2012). Fluconazole is the most widely-used yeast-active azole that inhibit ergosterol biosynthesis and, in general, are fungistatic (Lass-Flörl, 2011). Nowadays, increasing resistance of C. albicans to antifungal drugs is a major problem. There is important to find a powerful tool to treat infections with resistant C. albicans. Use of combination the antifungal drug is a cornerstone for treatment of resistant Candida infections (Pianalto and Alspaugh, 2016). Given the synergistic combination of fluconazole with amphotericin B against C. albicans, we hypothesized that it could augment the efficacy on inhibition of yeast– hyphal transition. To that end, we aimed to determine the effects of fluconazole in combination with amphotericin B, against C. albicans hyphae and conducted a series of follow-up studies to investigate on their antifungal activity. Materials and methods Candida albicans isolates C. albicans ATCC 14053 was purchased from the Iranian Research Organization for Science and Technology. Ten clinical isolates of C. albicans were identified using morphological, biochemical and molecular techniques from immune-compromised patients who admitted in Shahid Beheshti hospital affiliated to Yasooj University of Medical Sciences. Written informed consent was obtained from patients for the use of the samples in research. All isolates were kept at −80°C in sterile 20% (v/v) glycerol stocks. This study was approved by Research Ethics Committee of our institute (Ethics No. 95-6). The study protocol conformed to the ethical guidelines of the 1995 Declaration of Helsinki. Clinical isolates of C. albicans were freshly subcultured onto Sabouraud dextrose agar (SDA, Difco Laboratories, Detroit, Michigan) supplemented with 300μg/ml of chloramphenicol. C. albicans cells were passaged at least twice to ensure viability and purity. Subsequently, the turbidity equivalent to 0.5 McFarland standards prepared from a fungal suspension and quantified by spectrophotometer at 530nm wavelengths. The suspension was adjusted to a concentration of 1–5×106 yeast cells/ml and diluted the cells to a final concentration of 0.5×103 –5×103 cells/ml. For each of the fungal isolates the viability of the yeast was >97% by viable pour plate counting method (Alizadeh et al., 2017; Harmal et al., 2012; Wayne, 2008). Antifungal drugs Fluconazole and amphotericin B powder were purchased from the Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Fluconazole and amphotericin B were dissolved in dimethylsulfoxide (DMSO). The fluconazole and amphotericin B are mixed in 1:1 ratio (Khodavandi et al., 2010; Wayne, 2008). Antifungal drug assays The concentration of fluconazole and amphotericin B were determined by broth microdilution antifungal susceptibility assay developed by the CLSI document (Wayne, 2008). The broth microdilution susceptibility test was performed using 96-well U-bottom tissue culture microplates containing 100μl/well of RPMI1640 with L-glutamine (Sigma-Aldrich). The 96-well U-bottom microplates were prepared with 100μl/well of the twofold dilution of fluconazole ranged from 0.03 to 64μg/ml and amphotericin B ranged from 0.03 to 16μg/ml, alone or in combination in RPMI-1640 with L-glutamine buffered to pH 7.0 with 0.165M D ow nl oa de d fr om p pj .p hy ph a. ir at 0 :5 3 + 03 30 o n W ed ne sd ay O ct ob er 3 0t h 20 19 197 | Physiol Pharmacol 22 (2018) 195-204 Khodavandi et al. morpholinophosphonyl sulfate. The microplates, including drugs and C. albicans cells, were kept at 4°C for 2h and incubated at 35°C. MICs were measured at 530nm using a Stat Fax 303 Reader (Awareness Technology, Inc., USA) after 24h (Mardani et al., 2018; Sharifynia et al., 2017; Shokohi et al., 2016; Wayne, 2008). Drug interaction was regulated as synergistic, additive, indifferent or antagonistic on the basis of the fractional inhibitory concentration (FIC) index using the results of MICs determined with the antifungal drugs (Khodavandi et al., 2010). Time-kill kinetic assay Yeast cells at a density of 1×106 cells/ml were treated with the 1×MIC of each antifungal drugs alone or in combination. Samples were incubated at 35°C. After different time intervals (0, 2, 4, 6, 8, 10, 12, 24 and 48h), the fungicidal activity of antifungal drugs or combination was measured by viable pour plate counting method. Fungistatic and fungicidal activity were considered to be achieved when the number of CFU/ml were <99.9% and ≥99.9% compared with the starting inoculum size, respectively (Klepser et al., 1998). Effect of antifungal drugs on Candida albicans yeast– hyphal transition The yeast– hyphal transition was performed in 96well U-bottom microplates. Briefly, the C. albicans ATCC 14053 cell suspension was provide in RPMI1640 medium with a starting cell density of 1×106 cells/ml and dispensed into the wells of a microplates at 100μl/well. Fluconazole and amphotericin B alone and in combination with the different MIC concentrations (2×MIC, 1×MIC, 1⁄2×MIC and 1⁄4×MIC) were added (100μl/well). Similarly, 100μl of RPMI1640 medium containing 5% DMSO without antifungal drugs was added into the selected wells for control. Microplates were incubated at 35°C for 90min. Subsequently, the mixture was incubated at 35°C for 16h with gentle shaking. The metabolic activity of hypha was quantitatively measured by colorimetric XTT [2,3-bis (2methoxy4nitro5 sulfophenyl)5[(phenylamino) carbonyl 2 – [Htetrazolium hydroxide] reduction and also crystal violet assays (Peeters et al., 2008). Colorimetric XTT reduction assay Subsequent to the appropriate incubation of the microplates, medium was aspirated from the wells and unattached cells were removed by washing with sterile phosphate buffer saline (PBS). Colorimetric change in the XTT reduction assay of hypha was performed as previously reported (Peeters et al., 2008). Briefly, 100μl of the XTT-menadione (SigmaAldrich) solution was added into each well containing prewashed hypha and incubated in the dark at 37°C for 5h. Following incubation, colorimetric XTT reduction was measured in a microplate reader at 490nm. Crystal violet assay C. albicans yeast– hyphal transition was also measured by crystal violet assay. The 100μl of 99% methanol was added to the prewashed hypha. The wells were dried and stained with 100μl of crystal violet solution (Sigma-Aldrich) for 20min. Afterwards, each well was washed with tap water and immediately destained with 150μl of acetic acid 33% (Sigma-Aldrich). The absorbance values measured in a microplate reader at 590nm (Peeters et al., 2008). Effect of antifungal drugs on Candida albicans yeast– hyphal transition via light microscopy Effect of fluconazole and amphotericin B alone and in combination based on different concentrations of MIC (2×MIC, 1×MIC, 1⁄2×MIC and 1⁄4×MIC) on C. albicans ATCC 14053 yeast– hyphal transition was evaluated by growing the cells (1×106 cells/ml) in RPMI-1640 using cel

Gastroprotective effect of sodium hydrosulfide against indomethacin-induced gastric ulcer in diabetic rats

Abstract: Introduction: The incidence rate of gastric erosions and ulcers in diabetic patients are higher due to failure of mucosal antioxidant defense and maintain enough blood flow. The present study evaluated the gastro-protective effect of sodium hydrosulfide (NaHS) against indomethacin-induced gastric lesions in diabetic rats. Methods: In order to test anti-ulcer activity of NaHS against indomethacin, four diabetic groups of rats including diabetic control and 3 NaHS-treated groups received a single dose of physiologic saline or NaHS at 320, 640 and 1280 μg/kg respectively, 30 min before ulcer induction by indomethacin. Five hours later, the animals were killed and their stomachs were removed for macroscopically and microscopically evaluations. In order to evaluate the antacid effect of NaHS, 4 groups of diabetic rats received physiologic saline or NaHS at 320, 640 and 1280 μg/kg and 30 min later anesthetized, underwent a midline laparotomy and then their pylorus ligated. Five hours later, the animals were killed, their stomachs were removed and pH of gastric effluents were measured. Results: Indomethacin induced gastric lesions in glandular part of the stomach. NaHS at 640 and 1280 μg/kg significantly decreased the indomethacin-induced gastric lesions in diabetic rats. The pH of gastric effluents and mucus content increased by NaHS at doses of 640 and 1280 μg/kg. Macroscopic and microscopic observations showed that mucosal erosions induced by indomethacin were significantly inhibited by NaHS. Conclusion: results suggest NaHS through decreasing the rate of gastric acid output and increasing the mucus production, protected the gastric mucosa against indomethacin-induced gastric lesions in diabetic rats. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 19 + 03 30 o n M on da y N ov em be r 4t h 20 19 49 | Physiol Pharmacol 22 (2018) 48-53 Nazariani et al. 2014), promoting the production of mucous secretion and increasing the mRNA expression level of prostaglandin E2 receptors (Mard et al., 2017) contributed in mucosal defense barrier. It has been shown that the gastric mucosa in diabetic rats has a more susceptibility to gastric irritants (Takeuchi et al., 1994; Tashima et al., 1998). A study showed that the prevalence of gastric erosions and gastric ulcers in diabetic patients is more than 45% (Boehme et al., 2007). Moreover, according to twenty-four pH studies, the prevalence of asymptomatic reflux in insulin-dependent diabetic patients were 28% higher than in healthy population (Lluch et al., 1999). There are many reasons for decreased ability of gastric mucosa against irritants such as inability of the gastric mucosa to deliver the enough amount of calcitonin-gene related peptide, impairment of gastric mucosal blood flow and decrement the efficiency of antioxidant defense system in the gastric mucosal layer (Goldin et al., 1997; Tashima et al., 1998). Therefore, the present study was designed to evaluate the gastro-protective effect of NaHS against indomethacin-induced gastric lesions in diabetic rats. Materials and methods

Protective effects of rosuvastatin against hyperglycemia-induced oxidative damage in the pancreas of streptozotocin-induced diabetic rats

Abstract: Introduction: According to the powerful antioxidant effects of rosuvastatin, the present study aimed to examine the protective effects of rosuvastatin against oxidative damage of diabetic pancreas by potentiation of the antioxidant capacity in streptozotocin-induced diabetic rats. Methods: Experiment was performed in four groups of male Wistar rats (n=6 in each group): normal, diabetic and two treatment groups (normal and diabetic rats treated with rosuvastatin). Rats were made diabetic by a single intravenous injection of streptozotocin (40 mg/kg) at the beginning of study. Treatment groups received orally rosuvastatin at dose of 10 mg/kg/day. After eight weeks, the pancreas tissues were removed under deep anesthesia. After tissue homogenization, the contents of glutathione and malondialdehyde (MDA) as well as superoxide dismutase (SOD) activity were assessed by biochemical methods. Results: Blood glucose of diabetic rats was above 350 mg/dl. The MDA content of the homogenized pancreas significantly increased in diabetic rats by 92%. Diabetes also decreased the content of glutathione (32%) as well as SOD activity (68%) of pancreas tissues. Treatment with rosuvastatin noticeably decreased the MDA levels of diabetic pancreas (90%). Moreover, rosuvastatin significantly increased the glutathione content (21%) and SOD activity (67%) of pancreas tissues in treated diabetic rats. Conclusion: Our findings reveal that rosuvastatin is able to attenuate the uncontrolled hyperglycemia-induced oxidative damage of pancreas through potentiation of the antioxidant defense system. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 9 :5 8 + 03 30 o n M on da y N ov em be r 4t h 20 19 Rosuvastatin and diabetic pancreas Physiol Pharmacol 22 (2018) 19-27 | 20 insulin resistance (type 2 diabetes) are the main reasons of diabetes (Poitout, 2008; Yoon and Jun, 1999). The glucose toxicity in the β-cell of pancreatic islets is more common in people with type 2 diabetes because most patients with type 2 diabetes have abnormally elevated postprandial glucose concentrations that continually damage the remaining β-cells (Robertson and Harmon, 2006). Hyperglycemia is the main symptom of diabetes and it has been reported a direct correlation between chronic hyperglycemia and generation of reactive oxygen species (ROS), (Maritim et al., 2003). Exposure to high glucose levels has been reported that enhances intra-islet levels of peroxide (Tanaka et al., 2002). According to measurements of the oxidative stress markers in patients with type 2 diabetes, diabetic patients show elevated markers for oxidative tissue damage and oxidants in the tissues, such as peroxides and oxidation of DNA bases (Reus et al., 2016; Yang et al., 2011). There are several pathways, including polyol and hexosamine pathways and also activation of protein kinase C and NADPHoxidase, through which chronic hyperglycemia can lead to the ROS generation within the islets of pancreas (Robertson and Harmon, 2006). Moreover, weakening of the antioxidant defense system of pancreatic islets is occurred under hyperglycemic conditions (Kakkar et al., 1998). It has been reported that diabetes substantially decreases the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) as well as the mRNAs and proteins levels of these antioxidant enzymes in pancreatic islets (Kakkar et al., 1998; Robertson and Harmon, 2006; Tanaka et al., 2002). Statins, 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, such as rosuvastatin have the pleiotropic effects independent of lipid-lowering actions in several pathological states (Arboix et al., 2010; Castro et al., 2008). Previously, improved endothelial function and reduced oxidative stress by rosuvastatin have been reported in streptozotocin-induced diabetic mice through a cholesterol-lowering independent mechanism (Giunti et al., 2010). Deng et al. (2015) showed that rosuvastatin decreases contrast-induced nephropathy through modulation of nitric oxide, oxidative stress, apoptosis and inflammatory responses in diabetic rats. Rosuvastatin also induces vasoprotection through increasing of nitric oxide bioavailability, which is possibly achieved by its inhibition on ROS production (Tian et al., 2011). In another study Calkin et al. (2008) indicated the anti-atherosclerotic effects of rosuvastatin in a model of atherosclerosis accelerated by diabetes that are independent of its lipid lowering effects. They also reported that treatment with rosuvastatin was associated with reduced accumulation of advanced glycation endproducts (AGE) and AGE receptor (RAGE) in plaques. Besides the beneficial effects of statins, dual effects of rosuvastatin on glucose homeostasis have been reported during diabetes. It has been demonstrated that rosuvastatin improves insulin sensitivity but impairs β-cells function (Salunkhe et al., 2016). Also, association between increased risk of diabetes with statins and impaired insulin sensitivity and insulin secretion has been reported, previously (Laakso and Kuusisto, 2017). According to the previous reporters, weakening of the antioxidant defense system of pancreatic islets and consequent oxidative damage to the pancreatic βcells has been occurred during diabetes (Coskun et al., 2005). Hence, because of the beneficial pleiotropic effects of rosuvastatin, the present study aimed to evaluate whether rosuvastatin attenuates the oxidative stress of pancreas and potentiates the antioxidant defense system of pancreas in streptozotocin-induced diabetic rats. Materials and methods Animals All procedures of the study were approved by the Institutional Care and Use of Animals Committee of the University of Baqiyatallah Medical Sciences, and were performed in accordance with accepted standards of animals use and care. The ethical code for the present research was IR.BMSU.REC.1396.170. We used male Wistar rats, weighing 280-320 g, obtained from the animal house facility center of Baqiyatallah University of Medical Sciences. Before procedures, the rats were allowed to acclimatize to the new situation with controlled temperature (22-24 °C), humidity (40-60%), light period (07.00-19.00) and also free access to the rat chow and water. Induction of diabetes An intravenous injection of streptozotocin (Sigma D ow nl oa de d fr om p pj .p hy ph a. ir at 9 :5 8 + 03 30 o n M on da y N ov em be r 4t h 20 19 21 | Physiol Pharmacol 22 (2018) 19-27 Yazdanimehr et al. Aldrich, USA) was used for induction of type 1 of diabetes. In brief, under light anesthesia using ethyl ether, diabetes was induced by an intravenous injection of 40 mg streptozotocin per kg body weight of rats in lateral tail vein. Five days after injection of streptozotocin, blood glucose levels were tested to confirm diabetes state and the rats with blood glucose levels over 350 mg/dl were considered as diabetic animals (Mohammadi et al., 2014; Pirmoradi et al., 2014). Blood glucose levels at the beginning and termination of experiment were measured using a commercial kit (Pars Azmoon Company, Tehran, Iran) by an enzymatic colorimetric method according to the manufacture protocols. Rosuvastatin treatment Rosuvastatin (AstraZeneca, UK) was orally administered at a dose of 10 mg/kg/day by oral gavage for eight consecutive weeks. The dose of rosuvastatin was decided according to the study of Deng et al. (2015). Rosuvastatin was dissolved in distilled water. Experimental protocols To perform the study, the rats were randomly divided into four groups of equal numbers (six rats in each group) as follows: normal group (normal healthy rats that used as normal control), rosuvastatin-treated normal rats (normal healthy rats that received orally rosuvastatin at a dose of 10 mg/kg/day for eight weeks), diabetic control (diabetic rats that used as diabetic control) and rosuvastatin-treated diabetic rats (diabetic rats that received orally rosuvastatin at a dose of 10 mg/kg/day for eight weeks). The rats of normal and diabetic control groups were administered orally distilled water as vehicle at same volume which the rosuvastatin-treated normal and diabetic rats received rosuvastatin solution.

Modulatory effect of ventromedial hypothalamic D2 receptors on leptin and glucose concentration

Abstract: Introduction: A specific role of dopamine D2 receptor signaling of midbrain and hypothalamic dopaminergic systems has not been yet identified in energy homeostasis. Here, we investigated effects of intra-ventromedial hypothalamus (VMH) administration of the D2 receptor agonist (quinpirole) and antagonist (sulpiride) on plasma leptin and glucose levels in fasted rats. Methods: A guide cannula was stereotaxically implanted in the VMH of male Wistar rats (n=6/group). In experiment day, the fasted rats (20-24h) received a recommended dose of D2 receptor agonist (quinpilroe: 0.5μg), antagonist (sulpiride: 0.005μg) and saline (0.5μl) injected into the VMH. Blood samples were collected at 0, 30 and 60 min after injection, and plasma leptin and glucose were measured by Eliza kit and glucose oxidase method, respectively. Results: Plasma leptin significantly increased in a time dependent manner in quinpirole group compared to control (P<0.01), while sulpiride markedly suppressed it (P<0.001). Increase in glucose levels was time dependently robust in quinpirole group (P<0.00). A significant reduction was observed in glucose levels in sulpiride compared to control group (P<0.05). There was also a negative correlation between glucose and leptin plasma levels in drug-treated rats. Conclusion: These data suggest that altered the VMH D2-mediated neurotransmission might contribute to an alteration in the metabolic phenotype (leptin secretion and plasma glucose level) of rats. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 09 + 03 30 o n M on da y N ov em be r 4t h 20 19 125 | Physiol Pharmacol 22 (2018) 124-132 Ghasemi et al. food, but they cannot consume food enough to survive (Zhou and Palmiter, 1995), suggesting an important role of DA signaling in physiological function of appetite centers. The importance of the DA signaling in the energy homeostasis is also confirmed by studies that indicating DA receptors are co-localized with the leptin receptors, where dopamine inhibits leptin signaling in the hypothalamus (Billes et al., 2012; Kim et al., 2010; Nogueiras and Seeley, 2012). Furthermore, D2R knock-out mice have an increased hypothalamic leptin sensitivity (Kim et al., 2010). Results obtained from previous studies suggest that leptin release is controlled by the dopaminergic neurons and that dopamine executes its physiological role on energy balance in partly via regulation of plasma level of leptin or other peripheral homeostatic signals (Doknic et al., 2002; Dunn et al., 2017; Kim et al., 2010; Kok et al., 2006b; Mastronardi et al., 2001). For example, D2R knock-out mice show a 75% reduction in the plasma leptin concentration compared to wild type mice (Kim et al., 2010). Conversely, previous observations provide the inhibitory effect of dopaminergic neurotransmission (systemic administration) on leptin secretion. In particular, it has been reported that treatment with bromocriptine, a dopamine D2 receptor agonist, significantly lowers the plasma leptin concentration in a single blood sample of humans with prolactinoma, even without affecting body weight (Doknic M et al., 2002). Furthermore, a single intravenous bolus injection of bromocriptine significantly reduced both basal and lipopolysaccharide-induced leptin release in rats, and short-term treatment with bromocriptine lowered circulating leptin levels in obese women, which suggests that dopaminergic neurotransmission is involved in the control of leptin release in human and animals (Dunn et al., 2017; Kok et al., 2006b; Mastronardi et al., 2001). Nevertheless, these studies have not identified a specific role for midbrain or appetite DA systems in the control of leptin release. In this regard, the role of hypothalamic dopaminergic neurotransmission on homeostasis system is unknown. Since D2R expression is relatively abundant in ventromedial hypothalamus, VMH (Bina and Cincotta, 2000; Clifton et al., 1991; Cooper and Al-Naser, 2006; Kuo, 2002; Meguid et al., 1997; Meguid et al., 2000; Volkow et al., 2011), hence, we investigated the role of local VMH D2R activation on regulation of plasma leptin concentration in rats. Furthermore, a significant correlation has been shown between plasma glucose levels and cerebrospinal fluid concentrations of the dopamine (Blum et al., 2014; Dunn et al., 2012; Umhau et al., 2003), raising the possibility that leptin may affect plasma glucose levels. Therefore, we also evaluated correlation between plasma glucose and leptin concentrations. Materials and methods

The effect of intracerebroventricular administration of insulin on memory impairment-induced by scopolamine in male rats

Abstract: Introduction: Cholinergic neuronal deficiency is one of the main causes of Alzheimer's pathology, which leads to learning and memory impairment. Scopolamine is a muscarinic cholinergic antagonist commonly used to induce Alzheimer's disease (AD). Insulin also regulates learning and memory function. Thus, the aim of this study was to determine the effect of central administration of insulin on passive avoidance learning. Methods: In this experiment, fifty-nine rats were divided into 6 groups: (1) intact, (2) sham, (3) scopolamine-saline, (4) scopolamine-insulin4, (5) scopolamine-insulin8 and (6) scopolamine-insulin16. In addition, scopolamine (70nmol/2μl) was injected into the right lateral ventricle, before the retrieval test of the inhibitory avoidance task. Then the effects of three doses of insulin (4, 8 or 16 mU/2μl) were investigated on the passive avoidance learning in an amnestic model induced by scopolamine. Results: Our results indicate that the retrieval of passive avoidance memory was significantly improved by intracerebroventricular administration of insulin in 4 and 8 mU/2μl doses but not in 16 mU/2μl. Conclusion: These results confirmed that insulin could improve the retrieval phase of passive avoidance memory that was impaired by scopolamine. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 22 + 03 30 o n M on da y N ov em be r 4t h 20 19 241 | Physiol Pharmacol 22 (2018) 240-246 Ehsani et al. acts as an in vivo model of Alzheimer's (Hoyer and Lannert, 1999). According to this concept, clinical studies in the past indicated that induced hyperinsulinemia with euglycemia preservation in the periphery can ameliorate memory in Alzheimer's patients and normal adults (Craft et al., 2003). Regarding the growing proofs of several studies, these outcomes confirm the association between peripheral (Gasparini et al., 2002) and central (Park et al., 2000) insulin effects on cognitive disorder. Insulin had an effective role in improving verbal memory in groups of patients with AD and mild cognitive impairments. However, insulin therapy, among those patients who possess the APOE (epsilon4+) genotype (a strong predictor of AD), had different effects than patients who do not carry this allele (Reger et al., 2006). Moreover, it has been shown that intranasal insulin administration had different effects on memory impairments, given the APOE genotype (Reger et al., 2008). Intranasal insulin has also demonstrated the efficacy and preventing effects on some symptoms and pathology of Alzheimer’s. A large body of experiments have shown that intranasal insulin can improve cognition (Craft et al., 2012; Rosenbloom et al., 2014; Salameh et al., 2015). Animal studies indicated that insulin in the nose is able to cross the cribriform plate, that easily spreads across the brain and is able to improve learning and memory impairments (Salameh et al., 2015). Most importantly, a clinical study has shown that memory impairment progression was delayed in AD patients with poor cognitive impairment, following intranasal insulin administration (Craft et al., 2012). Further research showed that due to glucose uptake enhancement following the increase in the amount of insulin in the frontal and parietotemporal cortex, the attentional state and general performance of these patients were boosted (Craft et al., 2012). Despite the effectiveness of insulin to improve memory impairment in AD, some ambiguities still exist. In addition, the underlying mechanisms of both insulin and cholinergic activity interaction, in passive avoidance (PA) learning are not fully understood. This study investigated how these mechanisms are created to prevent AD development. Thus, there may be alternative mechanisms that can be useful in lightening the way. Amnestic effects were induced in the animals using central infusion of a muscarinic cholinergic antagonist, scopolamine. Subsequently, the effects of three doses of insulin on retrieval were evaluated in a PA learning task. Materials and methods Animals Male Wistar rats (200–250g) that were provided from our own breeding colony, were used in this study. All experimental animals had free access to food and tap water. The environmental conditions consisted of constant temperature (25±2°C) and controlled humidity with a 12h light/dark cycle. The procedures in the passive avoidance tests were performed in accordance with the international principles for the experimental use of animals (Olfert et al., 1993). In this study, the experiments were done in a manner conforming to internationally accepted principles for the use of experimental animals (National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011) and were in accordance with the guidelines of the ethical committee at Mazandaran University of Medical Sciences. Surgical method and drug microinjection One week prior to the behavioral testing, the animals were anesthetized by a mixture of ketamine and xylazine (100 and 2.5mg/kg intraperitoneal, respectively). They were then placed in stereotaxic device (Stoelting, USA) and a guide cannula was fixed exactly above the right lateral ventricle of each rat, based on Paxinos and Watson atlas (AP: −0.8 mm from bregma; ML: 1.5 mm from midline; DV: −2.6 mm from the surface of skull). Dental acrylic cement was used for cannula fixation. Behavioral tests were performed a week after the recovery. To deliver the drugs, an injection needle (27 gauge), joined at one end to the polyethylene tube and at the other end to a 5μl Hamilton micro syringe was used. Treatment infusion was done through the 21-gauge guide cannula. When the injection needle was inserted correctly, a final volume of 2μl of the saline, scopolamine, 4, 8, or 16mU of insulin was delivered. The needle remained in place for at least 1 minute, to deliver the medication and avoid leaking towards the outside of the cannula. The injection time lasted 3 to 4 minutes. The doses of insulin were chosen based on previous studies that examined the enhanced D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 22 + 03 30 o n M on da y N ov em be r 4t h 20 19 Insulin effect on memory Physiol Pharmacol 22 (2018) 240-246 | 242 insulin-induced cognitive activity (Park et al., 2000; Grillo et al., 2009). The 70nmol scopolamine hydrobromide was dissolved in 2μl of sterile 0.9% saline based on earlier literature which investigated the effect of scopolamine-induced learning deficit (Albiston et al., 2004). Sterile 0.9% saline with the same volume was used as a solvent. Experimental design To investigate the effect of insulin, 6 experimental groups were used in this study. These six groups were divided into: (1) intact (n=5); (2) sham (sal-sal, n=10); (3) scopolamine-saline (sco-sal, n=11); (4) scopolamineinsulin4 (sco-Ins4, n=11); (5) scopolamineinsulin8 (sco-Ins8, n=11) and (6) scopolamineinsulin16 (sco-Ins16, n=11). Memory impairment was induced by scopolamine administration (70nmol, ICV) 35min before the retrieval test. Drugs infusions of insulin (4, 8 and 16mU, ICV), or saline were delivered 10min before the retrieval test. A graphical design for this study is shown in Figure 1. Passive avoidance apparatus The PA device was made up of two light and dark chambers with the same size (20×20×40 cm) and a rectangular guillotine door in the middle (8×8cm) that connected the two parts. The stainless steel rods were embedded on the floor of both parts spaced 1cm apart. From the floor of the dark portion, an electric current could pass through an electrical stimulator. The behavioral test was performed in standard conditions in a sound-isolated room. A 100W lamp was placed at 40cm above the light chamber (Ardeshiri et al., 2017). Behavioral testing PA training All animals were habituated to the device in two trials. For these sessions, the animals were put on the light chamber and after 10s the guillotine door was opened. The rats entered the dark part of the device due to their inherent tendency to darkness. After placing the rat in a dark chamber, the door was closed and after 30s, the rat was moved from the dark chamber to its cage. The habituation trial was repeated after 30min. After a similar period of time, the acquisition trial was conducted. The delay in entering the dark chamber in the acquisition trial (step-through latency, STLa) was recorded when the animal had entered the dark chamber completely. In the training session, immediately after the animals entered the dark chamber, the door was released and an electric shock (50-Hz, 1mA for 1.5s) was used. After 20s, the rats were returned into their home cage. The procedure was repeated after 2min. An electrical shock was delivered every time that the rat re-entered the dark chamber. Whenever the rat remained in the light chamber for 120 consecutive seconds, the training session was finished.

Preventive effect of natural dietary supplement -Flavin7- on the onset of spontaneous diabetes mellitus in bio-breeding diabetes prone rats

Abstract: Introduction: The aim of the present study was to evaluate the preventive effects of Flavin7 in prediabetic bio-breeding diabetes prone (BB-DP) rats. Methods: Foutthy rats were divided into 2 equal groups: group C (untreated control group) and group F7 with Flavin7 (natural dietary supplement F7 with bioflavonoids, 0.2 mg/l) in drinking water from 21 st day after birth to 171 st day of their life, respectively. Blood glucose, superoxide dismutase, glutathione peroxidase, catalase, total antioxidant capacity, glutathione, body weight, food intake, water intake and urine output were determined. Results: The age of diabetes onset was significantly higher for group F7 compared to group C (P<0.05). The incidence of diabetes was lower in group F7 than in group C. Blood glucose at the diabetes onset was higher in group C than in F7 group (P<0.05). Decrease of antioxidant status parameters, at the treatment onset as well as immediately after its termination showed a drop in the F7 group firstly, but increased progressively later, until the end of the experiment. Conclusion: F7 delayed the development of diabetes in BB-DP rats and prevented its onset. The severity of diabetes mellitus was milder in rats treated with F7 than in control group. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 21 + 03 30 o n M on da y N ov em be r 4t h 20 19 Flavin7 and onset of diabetes in BB-DP rats Physiol Pharmacol 22 (2018) 11-18 | 12 (Lukačínová et al., 2008). However, there are contradictory views in the literature that consider antioxidants as compounds with no detectable benefits in autoimmune diabetes in NOD mice (Li et al., 2006). To avoid the ethical and logistical constraints resulting from the type 1 diabetes studies in the outbred human population exposed to chemical and microbiological agents (triggers) accidentaly, we have to rely on animal models that can easily be used for examination, biopsy and autopsy. These models are eligible for the study of heredity (including genetically modified animals) and for testing of their responses to environmental factors (Lukačínová et al., 2013). Therefore, bio-breeding (BB) rats are the most appropriate model for these studies (Mordes et al., 2004). Antioxidants used in systemic therapy delay the onset of type 1 diabetes (Asmat et al., 2016; Hoffman, 2014; Kaneto et al., 2007). Despite many studies, type 1 diabetes remains resistant to prevention (Skyler et al., 2002), except of unacceptable toxic immunosuppression (Parving et al., 1999). Prevention of diabetes mellitus is an urgent issue not only for medicine, but also for whole human society at the beginning of this millennium. Epidemic growth, devastating complications, enormous expenses on a healthcare related and other arguments confirm the necessity of prevention. Many studies regarding hypoglycaemic and hypolipidemic effects of various flavonoids have been reported with administration of bioflavonoids to diabetic animals (Aarland et al., 2017; Cazarolli et al., 2006; Roslan et al., 2017), but also to humans (Geng et al., 2007; Williamson, 2017). The basic mechanism of antidiabetic effect of flavonoids is the suppression of oxidative stress (Ghosh and Konishi, 2007; Mehta et al., 2006). Increased oxidative stress and impaired nitric oxide synthesis are mechanism likely to play a major role in the pathogenesis of vascular complications of diabetes (Sudnikovich et al., 2007). Various natural dietary bioflavonoid-based supplement are used in order to improve health support and to increase a quality of life in many countries currently. One of these natural-compound supplements is Flavin7 (F7) a bioflavonoid-containing dietary supplement with potential antioxidant activity, composed of the extracts from seven different fruits in which a suspected influence on glucose homeostasis regulation, chronic diseases, mainly oncologic is hypothesized (Kello et al., 2017). Besides that, F7 has also antiinflammatory and antivirotic effects. Therefore, the aim of the present study was to verify the F7 preventative effect on autoimmune diabetes mellitus in pre-diabetic bio-breeding diabetes prone (BB-DP) rats, which is very similar to human type 1 diabetes. Materials and methods

Changes in the levels of hippocampal BDNF expression are accompanied with inflammatory dental pain-induced learning and memory impairment

Abstract: Introduction: Learning and memory requires a brain-derived neurotrophic factor (BDNF)-dependent phase in the hippocampus. It has been reported that chronic pain decreases hippocampal BDNF levels. We have also previously reported that noxious stimulation of the rat tooth pulp impairs learning and memory. Therefore, we decided to find the changes in the hippocampal BDNF expression which are associated with tooth pain and learning and memory impairment. Methods: Dental pulp nociception was induced by intradental injection of capsaicin (100μg) in male Wistar rats. BDNF expression levels were determined by semiquantitative RT-PCR and western blotting. Results: The data indicated that capsaicin elicited pain behaviors and impaired learning and memory in Morris water maze test. The protein and mRNA levels of BDNF were significantly (P<0.05) decreased in capsaicin-treated rats as compared with control animals. Furthermore, iboprofen (120mg/kg, ip) treatment caused a significant (P<0.05) up-regulation of the BDNF protein and mRNA in the hippocampus of capsaicin-injected animals. Conclusion: These findings suggest that inflammatory dental pain induces hippocampal function impairments by decreasing in BDNF expression. iD D ow nl oa de d fr om p pj .p hy ph a. ir at 0 :5 1 + 03 30 o n W ed ne sd ay O ct ob er 3 0t h 20 19 Dental pain-induced learning and memory impairment Physiol Pharmacol 22 (2018) 155-162 | 156 factors and their receptors (Kozlovskiy et al., 2012), elevated pro-inflammatory cytokines (Khairova et al., 2009) and alterations in cannabinoid receptor function (Chevaleyre et al., 2006). The hippocampus is an essential site for learning and spatial memory processing, and it is one of the few brain regions to display adult neurogenesis. It has been hypothesized that adult hippocampal neurogenesis is also involved in hippocampal-related learning and memory (Leuner et al., 2006). Damage to the hippocampal structures has been also associated with learning and memory impairments (Squire, 1992). It is likely that hippocampal vulnerability may result from cellular components involved in hippocampal apoptosis and neurogenesis (Kuhajda et al., 2002). We have previously reported that apoptotic factors are over-expressed in the hippocampus of adult male rats suffering from inflammatory pulpal pain (Raoof et al., 2015). However, the exact cellular mechanisms underlying the vulnerability of hippocampus remain to be elucidated. The involvement of neurotrophic growth factors in the underlying mechanism of hippocampal neurogenesis has been much reported recently (Lee and Son, 2009; Leal and Yassa, 2015). Adult hippocampal neurogenesis is positively affected by neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin3. Among these, BDNF has been intensively shown to be involved in learning, memory and synaptic plasticity (Poo, 2001; Leal and Yassa, 2015). Surprisingly, spatial learning and memory is impaired in a BNDF-deficient animal model (Mu et al., 1999; Petzold et al., 2015). Since the role of BDNF on the decreased hippocampal function following orofacial pain has not been fully elucidated, the present study was designed to analyze the expression level of BDNF in the hippocampus of dental pain suffering and dental paininduced cognitive impaired rats. Materials and methods