Showing papers in "Acta Pharmacologica Sinica in 2003"

Recent advances in traditional plant drugs and orchids.

Abstract: The main objective of this paper is to review recent advances in plant drug research and developments in orchid study, in an attempt to provide useful references for plant drug studies. Plants have been used as medicine for millennia. Out of estimated 250 000 to 350 000 plant species identified so far, about 35 000 are used worldwide for medicinal purposes. It has been confirmed by WHO that herbal medicines serve the health needs of about 80 percent of the world's population; especially for millions of people in the vast rural areas of developing countries. Meanwhile, consumers in developed countries are becoming disillusioned with modern healthcare and are seeking alternatives. The recent resurgence of plant remedies results from several factors: 1) the effectiveness of plant medicines; 2) the side effect of most modern drugs; and 3) the development of science and technology. It has been estimated that in the mid-1990s over 200 companies and research organizations worldwide are screening plant and animal compounds for medicinal properties. Actually, several important drugs used in modern medicine have come from medicinal plant studies, eg, taxol/paclitaxel, vinblastine, vincristine, topotecan, irinotecan, etoposide, teniposide, etc. As for drugs derived from orchids, some novel discoveries, both in phytochemical and pharmacological properties, were reported by some universities. However, studies on plants are very limited. Only about a third of the million or so species of higher plants have been identified and named by scientists. Of those named, only a tiny fraction has been studied. Nowadays the linking of the indigenous knowledge of medicinal plants to modern research activities provides a new approach, which makes the rate of discovery of drugs much more effective than with random collection.

Horizontal gene transfer-emerging multidrug resistance in hospital bacteria.

Abstract: The frequency and spectrum of antibiotic resistant infections have increased worldwide during the past few decades. This increase has been attributed to a combination of microbial characteristics, the selective pressure of antimicrobial use, and social and technical changes that enhance the transmission of resistant organisms. The resistance is acquired by mutational change or by the acquisition of resistance-encoding genetic material which is transferred from another bacteria. The spread of antibiotic resistance genes may be causally related to the overuse of antibiotics in human health care and in animal feeds, increased use of invasive devices and procedures, a greater number of susceptible hosts, and lapses in infection control practices leading to increased transmission of resistant organisms. The resistance gene sequences are integrated by recombination into several classes of naturally occurring gene expression cassettes and disseminated within the microbial population by horizontal gene transfer mechanisms: transformation, conjugation or transduction. In the hospital, widespread use of antimicrobials in the intensive care units (ICU) and for immunocompromised patients has resulted in the selection of multidrug-resistant organisms. Methicillin-resistant Staphylococci, vancomycin resistant Enterococci and extended-spectrum beta-lactamase (ESBL) producing Gram negative bacilli are identified as major problem in nosocomial infections. Recent surveillance studies have demonstrated trend towards more seriously ill patients suffering from multidrug-resistant nosocomial infections. Emergence of multiresistant bacteria and spread of resistance genes should enforce the application of strict prevention strategies, including changes in antibiotic treatment regimens, hygiene measures, infection prevention and control of horizontal nosocomial transmission of organisms.

Pharmacological actions of Uncaria alkaloids, rhynchophylline and isorhynchophylline.

Abstract: The pharmacological actions of Uncaria alkaloids, rhynchophylline and isorhynchophylline extracted from Uncaria rhynchophylla Miq Jacks were reviewed The alkaloids mainly act on cardiovascular system and central nervous system including the hypotension, brachycardia, antiarrhythmia, and protection of cerebral ischemia and sedation The active mechanisms were related to blocking of calcium channel, opening of potassium channel, and regulating of nerve transmitters transport and metabolism, etc

Antitumor effects of curcin from seeds of Jatropha curcas.

Abstract: AIM: To study the antitumor effects of curcin from Jatropha curcas. METHODS: Antitumor activity of curcin was tested by MTT assay. The N-glycosidase activity of curcin was determined by characterization of R-fragment in gel. A cell-free system, rabbit reticulocyte lysate, was introduced to quantify the inhibitory activity of curcin on protein biosynthesis. RESULTS: The curcin had a powerful inhibitory action upon protein synthesis in reticulocyte lysate with an IC50 (95 % confidence limits) value of 0.19 (0.11-0.27) nmol/L. The IC50 (95 % confidence limits) of curcin on SGC-7901, Sp2/0, and human hepatoma was 0.23 (0.15-0.32) mg/L, 0.66 (0.35-0.97) mg/L, 3.16 (2.74-3.58) mg/L, respectively. Curcin was found to have no toxic to Hela cells and normal cells (MRC). After the rRNA of ribosome was treated with curcin and aniline at acidic condition, a cleaved R-fragment of approximately 450 nt appeared, but this fragment did not occur after treatment with curcin only. A comparison of the amino acid sequences of curcin, ricin A-chain and trichosanthin revealed that there were relatively high similarities among them. The percentages of homology between curcin and ricin A chain, between curcin and trichosanthin were found to be 54 % and 57 % respectively. Especially, the conserved residues forming the active sites of the A chain of ricin and trichosanthin occurred in curcin. CONCLUSION: Curcin has an obvious antitumor effect and its mechanisms are related to the N-glycosidase activity.

Anti-aging effect of astragalosides and its mechanism of action.

Abstract: AIM: To study the anti-aging effect of astragalosides (AST) and its mechanism of action. METHODS: Rotating rod test and step-down type passive avoidance test were performed to determine the effects of AST on motor and memory of D-galactose (D-gal)-induced senescent mice and the middle-aged mice. The proliferative response of splenocytes induced by Con A or LPS, IL-2 production of splenocytes induced by ConA of D-gal-treated mice and the middle-aged mice were also measured. RESULTS: AST (40 mg.kg(-1).d(-1), ig, for 10 weeks) was found to ameliorate age-related alternations in both motor response and memory, enhance the deteriorated cellular immunity in D-gal-treated mice and the pre-aged (17-month-old) mice. CONCLUSION: AST has an anti-aging effect on D-gal-induced senescent mice and has the effect of delaying senility of the middle-aged mice, which was related to its improvement of brain function and immunomodulatory effects.

Effects of chiral 3-n-butylphthalide on apoptosis induced by transient focal cerebral ischemia in rats.

Abstract: AIM: To investigate the effects of 3-n-butylphthalide (NBP) on apoptosis induced by transient focal cerebral ischemia in rats, compare the action potency of s-(-)-, r-(+)- and (+/-)-NBP, and clarify the enantiomer that played a main role. METHODS: DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay and gel electrophoresis. The expression of cytochrome c and caspase-3 protein was observed by Western blot analysis and immunohistochemistry. Middle cerebral artery was occluded for 2 h. RESULTS: Significant DNA fragmentation was detected at 24 h after reperfusion. This response was inhibited by s-(-)-NBP (5, 10 mg/kg i.p.). s-(-)-NBP 10 mg/kg almost completely inhibited DNA fragmentation, whereas r-(+)- NBP 10 mg/kg showed less effect. (+/-)-NBP (20 mg/kg) showed an inhibitory effect between that of s-(-)-NBP (10 mg/kg) and r-(+)-NBP (10 mg/kg). During the apoptotic process, cytochrome c was released into the cytosol and caspase-3 was activated. This effect was markedly inhibited by s-(-)-NBP, and the action potency of r-(+)- and (+/-)-NBP on the changes of cytochrome c and caspase-3 protein was similar to that on DNA fragmentation. CONCLUSION: NBP, especially its s-(-)-enantiomer, could potently reduce the release of cytochrome c, decrease the activation of caspase-3, and inhibit DNA fragmentation after transient focal cerebral ischemia. Our findings on the beneficial effects of NBP on cerebral ischemia-induced apoptosis might have important implications for the study and treatment of ischemic cerebrovascular diseases.

Effects of resveratrol on secondary damages after acute spinal cord injury in rats.

Abstract: AIM: To study the effects of resveratrol (Res) on secondary spinal cord edema, the activity of lactate dehydrogenase (LDH), Na+, K+-ATPase, and malondialdehyde (MDA) content in experimental spinal cord injured (SCI) rats. METHODS: The weight-dropping method was used to produce the experimental SCI in adult rats. Res ( 50, 100 mg/kg) and methylprednisolone (MPSS) 100 mg/kg were injected ip immediately after the induction of SCI. The effects of Res on edema, LDH, Na+, K+-ATPase, and MDA were determined at 1 h, 24 h, and 48 h after SCI compared with MPSS. The electron microscope was employed to investigate the ultrastructural effects of Res on axons, neurons, and subcellular organelles after SCI. RESULTS: Res obviously inhibited the secondary spinal cord edema with the most remarkable suppressing rate by 11.5 % at 48 h. Res significantly suppressed the activities of the lactate dehydrogenase with the highest suppressing rate > 40 % at 24 h. Res markedly improved the Na+, K+-ATPase activities that were promoted to the biggest extent of 60 % at 48 h. At the same time, Res (50 and 100 mg/kg) obviously reduced MDA production in the injured spinal cord tissue in comparison with the SCI model, the most remarkable effect of Res was detected at 48 h with the inhibitory rate >40 %. The ultrastructural findings suggested that SCI caused profound spinal cord damage, which could be protected or improved by injection of Res and MPSS. CONCLUSION: Both Res and MPSS effectively protected the spinal cord from secondary spinal cord injures. But the effects of Res 50 and 100 mg/kg were stronger in improving the energy metabolism system and inhibiting the lipid peroxidation in the local injured spinal cord after SCI than MPSS at the dose of 100 mg/kg. Res might have greatly potent therapeutic effects on SCI.

Genetic authentication of ginseng and other traditional Chinese medicine.

Abstract: The main objective of this paper is to review the chemical and genetic methods used in authentication of ginseng, especially the recent advances in microsatellite genotyping and its application to the authentication of other traditional Chinese medicines (TCM). The standardization and modernization of TCM hinge on the authentication of their botanical identities. Analysis of well-characterized marker compounds is now the most popular method for identifying the herbal materials and quality control of TCM, eg, ginsenoside profiling for authentication of Panax species. However, in many herbal species the chemical composition of the plant changes with the external environment and processing conditions, which lowers the reliability of these authentication methods. In the light of the advances in molecular biotechnology in the past few decades, genetic tools are now considered to provide more standardized and reliable methods for authentication of herbal materials at the DNA level. These genetic tools include random amplified polymorphic DNA (RAPD), DNA fingerprinting using multi-loci probes, restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and microsatellite marker technology. The practicality of these methods varies in terms of their sensitivity, reliability, reproducibility, and running cost. Using ginseng as an example, we reviewed the advantages and limitations of these molecular techniques in TCM authentication. We have developed a set of microsatellite markers from American ginseng that are able to differentiate Panax ginseng and Panax quinquetolius with the resolution down to farm level, ie, confirmation of its botanical identity and origin. Compared with other molecular techniques, microsatellite marker technology is more robust, accurate, reproducible, reliable, and sensitive. This is essential for large-scale TCM authentication centers.

A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening.

Abstract: AIM: To constructed a three-dimensional (3D) model for the 3C like (3CL) proteinase of SARS coronavirus (SARS_CoV), and to design inhibitors of the 3CL proteinase based on the 3D model. METHODS: Bioinformatics analyses were performed to search the homologous proteins of the SARS_CoV 3CL proteinase from the GenBank and PDB database. A 3D model of the proteinase was constructed by using homology modeling technique. Target- ing to the 3D model and its X-ray crystal structure of the main proteinase (M pro ) of transmissible gastroenteritis virus (TGEV), virtual screening was performed employing molecular docking method to identify possible 3CL proteinase inhibitors from small molecular databases. RESULTS: Sequence alignment indicated that the SARS_CoV 3CL proteinase was extremely homologous to TGEV M pro , especially the substrate-binding pocket (active site). Accordingly, a 3D model for the SARS_CoV 3CL proteinase was constructed based on the crystal structure of TGEV M pro . The 3D model adopts a similar fold of the TGEV M pro , its structure and binding pocket feature are almost as same as that of TGEV M pro . The tested virtual screening indicated that 73 available proteinase inhibitors in the MDDR database might dock into both the binding pockets of the TGEV M pro and the SARS_CoV 3CL proteinase. CONCLUSIONS: Either the 3D model of the SARS_CoV 3CL proteinase or the X-ray crystal struc- ture of the TGEV M pro may be used as a starting point for design anti-SARS drugs. Screening the known proteinase inhibitors may be an appreciated shortcut to discover anti-SARS drugs.

Spatiotemporal relationships among D-serine, serine racemase, and D-amino acid oxidase during mouse postnatal development.

Abstract: AIM: To elucidate the spatiotemporal relationships among D-serine, serine racemase, and D-amino acid oxidase (EC 1.4.3.3; DAO) in mouse cortex, striatum, cerebellum, heart, lung, liver, spleen, kidney, and skeletal muscle during mouse postnatal development. METHODS: The transcription levels of serine racemase and DAO were assayed by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of serine racemase were examined by Western blot. DAO activities were assayed by colorimetric method. D-serine was measured by HPLC. RESULTS: In cortex, striatum, and cerebellum, free D-serine increased drastically after birth and coincided well with the increase of serine racemase expression. However, among the 9 tissues examined, DAO activities were detected only in cerebellum and kidney. During the 3rd week, DAO activity in cerebellum and kidney increased dramatically, which concurred with the drastic decline of D-serine content in these tissues. On the other hand, while D-serine and serine racemase fall to trace level in cerebellum and kidney at the 3rd weekend, DAO activities in these tissues increased continuously. CONCLUSION: The free D-serine is mainly synthesized by serine racemase. However, novel mechanisms might be involved in D-serine deposition in mouse tissues with high level of D-serine and no detectable DAO activity such as cortex and striatum. DAO in cerebellum and kidney might have other physiological functions in addition to degrading D-amino acid.

Regulatory effect of Ganoderma lucidum polysaccharides on cytotoxic T-lymphocytes induced by dendritic cells in vitro.

Abstract: AIM: To study the regulatory effects of Ganoderma lucidum polysaccharides (Gl-PS) on cytotoxicity and mechanism of specific cytotoxic T-lymphocytes (CTL) induced by dendritic cells (DC) in vitro during the stage of antigen presentation. METHODS: Cultured murine bone marrow-derived DC were pulsed with P815 tumor cell lysates and co-incubated with or without various concentrations of Gl-PS (0.8, 3.2, or 12.8 mg/L) at the same time. P815 specific CTL were induced by spleen lymphocytes stimulated with mature DC. Non-adherent cells and culture supernatants were harvested on d 5 for analysis of specific cytotoxicity with lactate dehydrogenase (LDH) activity assay, mRNA expression of IFNgamma, granzyme B with RT-PCR assay, and protein expression of IFNgamma, granzyme B with ELISA or Western blot assay, respectively. RESULTS: Three concentrations of Gl-PS promoted LDH activities released into culture supernatants (P<0.01). It also increased mRNA expression of IFNgamma in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) and granzyme B in CTL (P<0.01). Protein production of IFNgamma in culture supernatants (P<0.05) and protein expression of granzyme B in CTL (Gl-PS 12.8 mg/L vs RPMI medium 1640, P<0.05) were also augmented by Gl-PS. CONCLUSION: Gl-PS is shown to promote the cytotoxicity of specific CTL induced by DC which were pulsed with P815 tumor antigen during the stage of antigen presentation, and the mechanism of cytotoxicity is believed to be going through IFNgamma and granzyme B pathways.

Anti-inflammatory and anti-platelet aggregation activity of human placental extract.

Abstract: AIM: To find the anti-inflammatory and anti-platelet aggregatory activity of human placental extract (HPE, Placentrex). METHODS: The HPE was studied for anti-inflammatory effect in Wistar rats on carrageenin, serotonin (5-HT), and prostaglandin E1 (PGE1) induced edema in acute model and cotton pellet induced granuloma on sub-acute model. Anti-platelet aggregation was studied against protection of adinosine diphosphate (ADP)-induced aggregation of human platelet through in vitro study. RESULTS: HPE showed positive results both in acute and sub-acute models of inflammation. Highly significant (P<0.01) results were obtained against 5-HT induced acute inflammation and cotton pellet induced sub-acute inflammation in comparison with standard (diclofenac sodium) and control (normal saline) drugs. The anti-inflammatory property of HPE in animal model was well supported with clinical study of platelet aggregation. There was highly significant (P<0.01) inhibition of platelet aggregation with HPE at different doses against ADP. CONCLUSION: Our data suggest that human placental extract may be useful in suppressing inflammation and platelet aggregation.

Effect of ghrelin on septic shock in rats.

Abstract: AIM: To study the role of ghrelin in the late stage of septic shock in rats. METHODS: The rat model of septic shock was made by caecal ligation and perforation. At the time of operation ghrelin 10 nmol/kg was infused through femoral vein followed by a sc injection at 8 h after operation. Hemodynamic parameters including heart rate (HR), mean arterial blood pressure (MABP), LVdp/dtmax, and left ventricular end-diastolic pressure (LVEDP) in survival rats were measured at 18 h after surgery. Plasma glucose and lactate concentrations, plasma ghrelin level and myocardial ATP content were assayed. The mortality rate in rats with septic shock was also observed. RESULTS: Compared to that of septic shock group, MABP of rats in ghrelin-treated group increased by 33 % (P <0.01). The values of +LVdp/dtmax and -LVdp/dtmax increased by 27 % and 33 %, respectively (P <0.01), but LVEDP decreased by 33 % (P < 0.01). The plasma glucose concentration and myocardial ATP content increased by 53 % and 22 %, respectively, but plasma lactate concentration decreased by 40 % in ghrelin-treated rats (P < 0.01). The plasma ghrelin level in rats with septic shock was 51 % higher than that of rats in sham group, and was negatively correlated with MABP and blood glucose concentration (r=-0.721 and -0.811, respectively, P <0.01). The mortality rates were 47 % (9/19) in rats with septic shock and 25 % (3/12) in rats of ghrelin-treated group, respectively. CONCLUSION: Treatment with ghrelin could correct partly the abnormalities of hemodynamics and metabolic disturbance in septic shock of rats.

Multiple signalling options for prostacyclin

Abstract: The fate of a cell following stimulation by the prostanoid prostacyclin is cell specific, depending not only on the ability of prostacyclin to activate the cell surface prostacyclin (IP) receptor and regulate its coupling to various G proteins, but also on its ability to act intracellularly via the nuclear peroxisome proliferator-activated receptor family (PPAR). This review will highlight the different signalling options available to prostacyclin, and discuss the consequences for cell responses.

Antioxidants attenuate reperfusion injury after global brain ischemia through inhibiting nuclear factor-kappa B activity in rats

Abstract: AIM: To investigate the effects of two antioxidants on the alterations of nuclear factor kappaB (NF-kappaB) activity and p65, p50 protein expression and phosphorylation of IkappaBalpha in rat hippocampus following global brain ischemia. METHODS: Using a 4-vessel occlusion (4-VO) as brain ischemia model, NF-kappaB protein (p65 or p50 subunit) expression was examined by Western blot analysis, and NF-kappaB activity was assayed by electrophoretic mobility shift assay (EMSA), and neuronal loss was observed by histology. RESULTS: NF-kappaB activity displayed a time-dependent manner, and p65, p50 proteins showed their peak levels after ischemia/reperfusion 6 h. NF-kappaB inductions (p65: 4.79+/-0.78, p50: 5.50+/-0.33, sham control=1) and activity (4.93+/-0.95) after 6 h of reperfusion were markedly reduced by pretreatment with antioxidants pyrrolidine dithiocarbamate (PDTC, 200 mg/kg) (p65: 1.11+/-0.74, p50: 1.38+/-0.98, activity: 2.20+/-0.86, respectively) or N-acetylcysteine (NAC, 300 mg/kg) (p65: 0.64+/-0.39, p50: 1.89+/-0.87, activity: 0.61+/-0.65), and histological observations of the pyramidal layer of CA1 also showed a reduction of neuronal loss in rat hippocampus (70 %+/-5 % or 92 %+/-4 % cells are survival, respectively). Furthermore, PDTC and NAC prevented the decrease (from 0.50+/-0.10 to 0.80+/-0.20 or 1.20+/-0.24, respectively) and phosphorylation (from 2.00+/-0.15 to 0.46+/-0.10 or 0.41+/-0.10, respectively) of IkappaBalpha protein in the cytoplasm. CONCLUSION: The protective effects of antioxidants against ischemia/reperfusion-induced injury may be mediated by down-regulation of NF-kappaB activity. NF-kappaB activation and deactivation are controlled mainly through phosphorylation and degradation of IkappaBalpha following brain ischemia.

Cytoprotective effect is one of common action pathways for antidepressants.

Abstract: AIM: To explore the possible common action mechanism of antidepressants. METHODS: The cell viability was detected by MTT assay. The intracellular Ca2+ concentration ([Ca2+]i) was measured by Fura 2-AM fluorescence labeling assay. Using RT-PCR, the mRNA level of nerve growth factor (NGF) was also detected. RESULTS: High concentration of corticosterone (0.2 mmol/L) was incubated with PC12 cells to simulate the lesion state of brain neurons in depressive illness. Three main kinds of antidepressants used in clinic [(1) tricyclic antidepressants (TCAs), such as desipramine (DIM) 0.625-10 micromol/L; (2) selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine (FLU) 0.625-10 micromol/L; (3) monoamine oxidase inhibitors (MAOIs), such as moclobemide (MOC) 2.5-40 micromol/L] protected cells from the lesion induced by corticosterone. While antipsychotic drug chlorpromazine or anxiolytic agent diazepam 0.4-50 micromol/L had no such effect. Moreover, DIM 1, 5 micromol/L or FLU 1, 5 micromol/L attenuated the [Ca2+]i overload induced by corticosterone 0.1 mmol/L for 48 h in PC12 cells. Furthermore, treatment with DIM or FLU 10 micromol/L for 48 h elevated the NGF mRNA expression in PC12 cells. CONCLUSION: Despite a remarkable structural diversity, the cytoprotective effect can be viewed as the common action pathway of the antidepressants. Moreover, attenuation of the intracellular Ca2+ overload and elevation of neurotrophic factor (such as NGF) expression is one of the mechanisms of cytoprotective effect of antidepressants.

Differentiation of endothelial progenitor cells from human umbilical cord blood CD 34+ cells in vitro.

Abstract: AIM: To study the time course of the expression of stem cell marker and endothelial cell markers on human cord blood CD34+ cells during in vitro differentiation process of endothelial progenitor cells (EPC). METHODS: CD34+ cells were selected and enriched from human cord blood by magnetically activated cell sorting (MACS), and cultured in dishes coated with or without fibronectin (Fn). Endothelial cells were identified by staining the cells with anti Flk-1 and vWF antibodies. The percentage of AC133+ cells in adherent CD34+ cell population was analyzed by fluorescence-activated cell sorting (FACS). RESULTS: The expression of Flk-1 and vWF on adherent CD34+ cells increased during the culture time, with 27.0 % positive for Flk-1 and negative for vWF at d 3, and 100 % positive for both Flk-1 and vWF at d 7. When cells were cultured in Fn-treated dishes, the percentages of Flk-1 and vWF positive cells increased to 34 % and 47 %, respectively at d 3, and 100 % at d 7. In contrast, the percentages of AC133+ cells among the adherent cell population decreased rapidly, and similar changes occurred in cells cultured in the presence of Fn. CONCLUSION: The gradual appearance of endothelial cell markers and the disappearance of stem cell marker characterized the in vitro differentiation of endothelial progenitor cells. Fibronectin accelerated the differentiation process of EPC.

Neuroprotective effect of ONO-1078, a leukotriene receptor antagonist, on transient global cerebral ischemia in rats.

Abstract: AIM: To determine whether ONO-1078 {pranlukast, 4-oxo-8-[p-(4-phenylbutyloxy)benzoyl-amono]-2-(tetrazol5-yl)-4H-1-benzopyran hemihydrate}, a potent leukotriene receptor antagonist, possesses a neuroprotective effect on global cerebral ischemia in rats, and to explore its possible mechanism of action. METHODS: Transient global cerebral ischemia was induced by four-vessel occlusion for 10 min and followed by 72-h reperfusion. ONO-1078 (0.03-0.3 mg/kg) and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one, a neuroprotective agent) 10 mg/kg were ip injected 30 min before ischemia and 1 h after reperfusion, and once a day afterward. Neurological outcome was evaluated before ischemia and 24, 48, 72 h after reperfusion. Neuron density, the expressions of N-methyl-Daspartate (NMDA) receptor subunit proteins (NR1, NR2A, NA2B) and vascular cell adhesion molecule 1 (VCAM-1) in the cerebral cortex and hippocampus were measured at 72 h after reperfusion. RESULTS: ONO-1078 (0.1, 0. 3 mg/kg) and edaravone (10 mg/kg) improved ischemia-induced neurological deficiency and reduced neuron death. ONO-1078 (0.1, 0.3 mg/kg) significantly inhibited the enhanced expression of NMDA receptor subunit protein NR2A in the cortex and VCAM-1 in the hippocampus of ischemic rats. CONCLUSION: ONO-1078 possesses a neuroprotective effect on global cerebral ischemia in rats, and its mechanism may be partly related to the inhibition of the upregulation of NR2A and VCAM-1 in different regions of the brain.

Mechanisms of irbesartan in prevention of renal lesion in streptozotocin-induced diabetic rats

Abstract: AIM: To investigate the mechanisms of angiotensin II receptor antagonist irbesartan (Irb) in prevention of renal lesion in streptozotocin (STZ)-induced diabetic rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: normal control (group N), diabetic nephropathy (group DN), and diabetic nephropathy treated with Irb (group DNI). Diabetes was induced by injection of STZ ip after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), urinary albumin excretion (Ualb), and 24-h proteinuria (24hUpro) were observed in the rats at week 4, 8, and 12, respectively. Creatinine clearance (Ccr), the kidney weight (KW), profile of kidney hypertrophy (KW/BW), renal tissue protein contents (RTP), glomerular area (AG), glomerular volume (VG), and width of glomerular basement membrane (GBM) were determined after the rats were sacrificed at week 12. Renal expression of connective tissue growth factor (CTGF) and transforming growth factor-beta1 (TGF-beta1) were determined by immunohistochemistry. RESULTS: There was no significant difference in BG between group DN and DNI (P >0.05). Irb prevented the increasing of Ualb excretion, 24 hUpro, and Ccr in diabetic rats ( P < 0.01). Furthermore, Irb markedly inhibited the increasing of KW, KW/BW, RTP, AG, and VG shown in diabetic rats (P <0.05, P <0.01, respectively). Irb prevented the thickening of GBM and immunostaining of CTGF (P <0.01). The extent of CTGF expression was positively correlated with the glomerular immunostaining for TGF-beta1 and size of VG (P <0.01). CONCLUSION: Irb exerts an early renal protective role to diabetic nephropathy, possibly through inhibition of renal hypertrophy and expression of CTGF.

Microbial transformation of naproxen by Cunninghamella species.

Abstract: AIM: The metabolites of naproxen produced by Cunninghamella species were isolated and identified, and further to compare the similarities between microbial transformation and mammalian metabolism. METHODS: Naproxen was transformed by three strains of Cunninghammella species (Cunninghamella blakeslesna AS 3.153, Cunninghamella echinulata AS 3.2004, and Cunninghamella elegans AS 3.156). The metabolites of naproxen were separated and assayed by liquid chromatography-mass spectrometry method. Semi-preparative HPLC was used to isolate the major metabolite, and the structure was identified by nuclear magnetic resonance (NMR) and mass spectrometry. RESULTS: Naproxen was transformed into 2 metabolites, desmethylnaproxen and desmethylnaproxen-6-O-sulfate, both were the known mammalian metabolites. The conjugated metabolite was newly detected in microbial transformation samples. CONCLUSION: The microbial transformation of naproxen has some similarities with the metabolism of naproxen in mammals. The fungi belonging to Cunninghamella species could be used as complementary in vitro models for drug metabolism to predict and produce the metabolites of drugs in mammals.

A biphasic opioid function modulator: agmatine.

Abstract: Recently it has been revealed that some agents that are not able to interact with opioid receptors play an important role in regulating the pharmacological actions of opioids Especially, some of them show biphasic modulation on opioid functions, which enhance opioid analgesia, but inhibit tolerance to and substance dependence on opioids We would like to call these agents which do not interact with opioid receptors, but do have biphasic modulation on opioid functions as biphasic opioid function modulator (BOFM) Mainly based on our results, agmatine is a typical BOFM Agmatine itself was a weak analgesic which enhanced analgesic action of morphine and inhibited tolerance to and dependence on opioid The main mechanisms of agmatine were related to inhibition of the adaptation of opioid receptor signal transduction induced by chronic treatment of opioid

P-glycoprotein restricted transport of nimodipine across blood-brain barrier.

Abstract: AIM: To examine whether the transport of nimodipine (NMD) from the circulating blood into the brain is restricted by P-glycoprotein (P-gp) in rat brain capillary endothelial cells (BCEC). METHODS: When cells reached confluence, a time course of NMD uptake was recorded by incubation with a medium containing NMD 10 mg/L at 37 degrees C. Effects of various agents in the steady-state uptake of NMD were tested by co-administration with NMD and each compound to cells at 37 degrees C for 90 min. The uptake of NMD was measured for 90 min. Effects of P-gp inhibitors on the efflux of NMD from primary cultured BCEC were studied by administration of erythromycin, clarithromycin, cyclosporin A (CsA), and Hanks' solution after the accumulation of NMD by BCEC at 37 degrees C for 90 min. RESULTS: The uptake of NMD by primary cultured rat BCEC was time-dependent, and the steady-state uptake of NMD was increased in the presence of several substrates of P-gp in BCEC. The steady-state uptake was also significantly increased (P CONCLUSION: The permeability of NMD into the brain is restricted by P-gp and increased by co-administration with P-gp inhibitors.

Effects of scutellarin on liver function after brain ischemia/reperfusion in rats.

Abstract: AIM: To investigate the effects of scutellarin (Scu) on liver function after brain ischemia/reperfusion in Wistar rats. METHODS: Rats were pretreated with Scu for 7 d and then subjected to a brain ischemia/reperfusion injury induced by a middle cerebral artery occlusion. The levels of nitric oxide (NO), xanthine oxidase (XOD), alanine transaminase (ALT), and aspartate aminotransferase (AST) in serum or liver tissues and the activities of antioxidant enzymes and cytochrome P450-dependent monooxygenases (CYPs) in liver tissues after brain ischemia/reperfusion were determined. RESULTS: In vehicle-treated rats, XOD, ALT, and AST activities (P CONCLUSION: These results demonstrated that pretreatment with Scu could attenuate hepatocellular damage elicited by brain ischemia/reperfusion in rats and this protection is in major part by its antioxidant activity.

Induction of hemopoiesis by saenghyuldan, a mixture of Ginseng radix, Paeoniae radix alba, and Hominis placenta extracts.

Abstract: AIM: To examine the efficacy of saenghyuldan and its components, Ginseng Radix, Paeoniae Radix Alba, and Hominis Placenta extracts (SHD, GR, PRA, and HP, respectively) on the hemopoiesis in a myelosuppression model system. METHODS: Susceptibility to cyclophosphamide (CP) and S180 carcinoma was determined in SHD, GR, PRA, and HP-treated mice. Analysis of peripheral blood and bone marrow cells was demonstrated by changes in cell types and histopathologic examination. The expression of cytokine mRNAs involved in hemopoiesis was examined by RT-PCR. RESULTS: SHD and its separated components (GR, HP, and PRA, respectively) significantly increased the survival in CP- and S180-treated mice. The hematology data demonstrated that all the agents augmented monocyte and leucocyte counts in the peripheral blood and increased bone marrow density and the ratio of leukocyte to erythrocyte in the bone marrow. These findings were positively correlated with the up-regulation of cytokine mRNA expression such as granulocyte colony-stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin (TPO), stem cell factor (SCF), and c-Kit. CONCLUSION: SHD is an effective remedy for the bone marrow failure and myelosuppression occurring during chemotherapy.

Small envelope protein e of sars: cloning, expression, purification, cd determination, and bioinformatics analysis

Abstract: AIM: To obtain the pure sample of SARS small envelope E protein (SARS E protein), study its properties and analyze its possible functions. METHODS: The plasmid of SARS E protein was constructed by the polymerase chain reaction (PCR), and the protein was expressed in the E coli strain. The secondary structure feature of the protein was determined by circular dichroism (CD) technique. The possible functions of this protein were annotated by bioinformatics methods, and its possible three-dimensional model was constructed by molecular modeling. RESULTS: The pure sample of SARS E protein was obtained. The secondary structure feature derived from CD determination is similar to that from the secondary structure prediction. Bioinformatics analysis indicated that the key residues of SARS E protein were much conserved compared to the E proteins of other coronaviruses. In particular, the primary amino acid sequence of SARS E protein is much more similar to that of murine hepatitis virus (MHV) and other mammal coronaviruses. The transmembrane (TM) segment of the SARS E protein is relatively more conserved in the whole protein than other regions. CONCLUSION: The success of expressing the SARS E protein is a good starting point for investigating the structure and functions of this protein and SARS coronavirus itself as well. The SARS E protein may fold in water solution in a similar way as it in membrane-water mixed environment. It is possible that beta-sheet I of the SARS E protein interacts with the membrane surface via hydrogen bonding, this beta-sheet may uncoil to a random structure in water solution.

Agmatine blocked voltage-gated calcium channel in cultured rat hippocampal neurons.

Abstract: AIM: To investigate the mechanism of agmatine by observing the effect of agmatine on the voltage-gated channels in rat hippocampal neurons. METHODS: The whole-cell patch recording technique was performed to record the voltage-gated potassium, sodium, and calcium currents in cultured rat hippocampus. Agmatine was applied directly to the single neuron using a pressure injector with microtubules. RESULTS: Agmatine (500 micromol/L) had no significant effect on the voltage-gated potassium and sodium channels. Agmatine reversibly blocked the voltage-gated calcium channel and the blockade was enhanced with the increasing concentration of agmatine. The inhibitory rates were 21%+/-4%, 35%+/-6%, 49%+/-6%, 67%+/-4%, 69%+/-6%, 86%+/-8%, and 87%+/- 9%, at the concentration of 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100 micromol/L, respectively. IC50 was (1.2+/-0.4) micromol/L. Two-way ANOVA revealed that change of membrane potential displayed a significant interaction with the blockade by agmatine. CONCLUSION: Agmatine reversibly blocked the voltage-gated calcium channel in rat hippocampal neurons in a concentration- and voltage-dependent way. Agmatine might perform its physiological and pharmacological effects partially by blocking the calcium channel.

Effects of endothelin and nitric oxide on organ injury, mesenteric ischemia, and survival in experimental models of septic shock.

Abstract: The development of potent drugs to treat cardiopulmonary failure in sepsis, such as antibiotics and new immunomodulatory therapeutic approaches have not prevented sepsis from being a major health problem. Dysfunction of the vascular endothelium is an early event in septic shock. The recognition of endothelium-derived substances, such as nitric oxide and endothelin, important mediators of systemic inflammatory response syndrome, led to the proposal that pharmacological inhibition of nitric oxide and endothelin production could represent a useful strategy in the treatment of septic shock. Splanchnic ischemia and translocation of endotoxin from the gut to the circulation contributes significantly to the high mortality rate in sepsis-related syndromes. This vasoconstriction in the splanchnic circulation can be partially blocked by inducible nitric oxide synthase inhibitor aminoguanidine or endothelin receptor antagonist bosentan in experimental models of septic shock. It can be suggested that endothelin and nitric oxide may affect survival. Although septic shock is a highly complex pathophysiological state, the course of septic shock has different phases with different characteristics which need different (special) treatment strategy. The inhibition of nitric oxide production during hyperdynamic, earlier phase of sepsis combined with the blockade of endothelin receptors at a later stage during the hypodynamic, late phase appears to be a novel promising strategy for the therapy of septic shock. The aim of this review is to discuss the role of nitric oxide and endothelin in sepsis and the potential therapeutic implications of blockade of nitric oxide and endothelin as a target in treatment of human septic shock. Briefly the importance of timing of intervention is also emphasized.

Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors

Abstract: AIM: To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similar experimental systems. METHODS: Human lung tryptase and human skin chymase were purified by a similar procedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacryl gel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined by enzyme assays. RESULTS: The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native protease inhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain, benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsin inhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymatic activity of chymase. Native protease inhibitors alpha 1-antitrypsin and secretory leukocyte protease inhibitor (SLPI) inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymatic activity. All the 3 inhibitors had weak inhibitory actions on tryptase. CONCLUSION: The protease inhibitors tested had relatively good selectivity to either tryptase or chymase.

Glucosides of Chaenomeles speciosa remit rat adjuvant arthritis by inhibiting synoviocyte activities

Abstract: AIM: To investigate the effects of Glucosides of Chaenomeles speciosa (GCS) on rat adjuvant arthritis (AA) and to clarify the role of synoviocytes in this process. METHODS: Complete Freund's adjuvant was used to induce AA in rats. Secondary paw swelling of AA rats was measured with MK-550 volume meter. The pain response and polyarthritis index were scored. Synoviocytes were separated by incubation of collagenase and trypsin, and morphological changes of synoviocytes were observed by transmission electron microscope. Interleukin-1 (IL-1) production was measured by thymocyte proliferation assay. Tumor necrosis factor (TNFalpha) and prostaglandin E2 (PGE2) production were determined by radioimmunoassay. RESULTS: There were significant secondary inflammatory reactions in AA rats. The morphology of synoviocytes from AA rats was changed, companying the elevation of the level of IL-1,TNFalpha, and PGE2 produced by synoviocytes from AA rats. GCS (60 and 120 mg/kg, ig, 8 d) suppressed secondary inflammatory paw swelling, pain response, and polyarthritis index. It also improved ultrastructural changes of synoviocytes and inhibited IL-1,TNFalpha, and PGE2 production in AA rats. The inhibitory effect of GCS 120 mg/kg was more evident than that of Actarit 60 mg/kg. CONCLUSION: GCS reduced the secondary inflammatory in AA rats, which is associated with prevention of ultrastructural changes of synoviocytes and inhibition of secretion of proimflammatory cytokines.

Protective effects of scutellarin on superoxide-induced oxidative stress in rat cortical synaptosomes.

Abstract: AIM: To evaluate the effects of scutellarin on superoxide-induced oxidative stress in rat cortical synaptosomes. METHODS: Oxidative damage model was established by incubation with xanthine (0.3 mmol/L ) and xanthine oxidase (0.02U) at 37 oC for 30 min. The extent of membrane oxidation was assessed by malondialdehyde (MDA). Membrane fluidity was measured by fluorescence anisotropy (polarization) of the lipophilic probe 1,6-diphenyl-1,3, 5-hexatriene (DPH). Intracellular Ca 2+ concentration([Ca 2+ ]i) was measured by fluorescent spectrophotometry. Fura 2-AM was used as an indicator for [Ca 2+ ]i. Na + /K + -ATPase activity assay was based on the amount of inorganic phosphate (Pi) released during an enzymatic hydrolysis of ATP. RESULTS: Synaptosomes exposed to superoxide significantly elevated the levels of malondialdehyde (MDA) and [Ca 2+ ]i compared with those in normal group. These changes were accompanied by the decrease in membrane fluidity and Na + /K + -ATPase activity. Pretreatment with scutellarin (25-100 µmol/L) significantly ameliorated the oxidative damage of synaptosomes by reducing MDA levels and [Ca 2+ ]i, up-regulating membrane fluidity and restoring Na + /K + -ATPase activity. CONCLUSION: Scutellarin exerts a potent protective effect against oxidative damage in synaptosomes induced by superoxide.