Showing papers by "Tao Chen published in 2016"

Crocin protects retinal ganglion cells against H2O2-induced damage through the mitochondrial pathway and activation of NF-κB

Abstract: Glaucoma is a degenerative nerve disorder that results in irreversible blindness. It has been reported that the apoptosis of retinal ganglion cells (RGCs) is a hallmark of glaucoma. Oxidative stress is one of the major factors that cause apoptosis of RGCs. Crocin has many beneficial effects, including antioxidant and anti-apoptotic actions. However, the mechanism by which crocin protects against oxidative stress‑induced damage to RGCs remains unclear. The present study aimed to investigate the mechanism by which crocin protects RGC-5 cells against H2O2-induced damage. H2O2 was used to establish a model of oxidative stress injury in RGC-5 cells to mimic the development of glaucoma in vitro. Different concentrations (0.1 and 1 µM) of crocin were added to test whether crocin was capable of protecting RGCs from H2O2-induced damage. WST-1, lactic dehydrogenase (LDH) release and Annexin V/FITC assays were then performed. Levels of reactive oxygen species (ROS) were detected using a ROS assay kit, mitochondrial membrane potential (ΔΨm) was analyzed by JC-1 staining, caspase-3 activity was examined using a Caspase-3 assay kit, and the protein levels of Bax, Bcl-1 and cytochrome c were measured using western blot analysis. In addition, the protein level of phosphorylated nuclear factor-κB (p-NF-κB) p65 was also evaluated using western blot analysis. The results showed that crocin protected RGC-5 cells from apoptosis, decreased LDH release and enhanced cell viability. Additional experiments demonstrated that crocin decreased ROS levels, increased ΔΨm, downregulated the protein expression of Bax and cytochrome c, promoted Bcl-2 protein expression and activated NF-κB. Taken together, the findings of this study indicate that crocin prevented H2O2‑induced damage to RGCs through the mitochondrial pathway and activation of NF-κB.

Crocin Upregulates CX3CR1 Expression by Suppressing NF-κB/YY1 Signaling and Inhibiting Lipopolysaccharide-Induced Microglial Activation

Abstract: Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Microglial activation has been shown to be deleterious to RGCs and may participate in the progression of glaucoma. Crocin, one of the major active ingredients in saffron, has been found to inhibit microglial activation. However, the mechanism remains unclear. The aim of this study was to investigate whether crocin can inhibit lipopolysaccharide (LPS)-induced microglial activation and to clarify the mechanisms involved. The influence of crocin on primary RGCs and LPS-stimulated BV2 microglial cells survival was determined by the MTT and lactate dehydrogenase assays, or by flow cytometry. BV2 cells were pretreated with various concentrations of crocin for 2 h followed by 1 μg/mL LPS stimulation. Microglial markers and pro-inflammatory mediators were assessed by real-time PCR, western blot and ELISA. Furthermore, CX3CR1 expression was detected and the underlying mechanism was examined. The concentrations of crocin ranged from 0.1 to 1 μM, and did not show any cytotoxicity in RGC and BV2 cells. After crocin pretreatment, the expression of microglial markers (CD11b and Iba-1) and pro-inflammatory mediators (iNOS, COX-2, IL-1β, and TNF-α) induced by LPS were significantly decreased in a dose-dependent manner. Additionally, CX3CR1 expression was remarkably increased by crocin via the suppression of NF-κB/Yin Yang 1 (YY1) signaling in BV2 cells. In conclusion, crocin effectively suppresses microglial activation and upregulates CX3CR1 expression by suppressing NF-κB/YY1 signaling.

Puerarin Attenuates N-Methyl-D-aspartic Acid-induced Apoptosis and Retinal Ganglion Cell Damage Through the JNK/p38 MAPK Pathway.

Abstract: Purpose To explore the protective effect of puerarin on N-methyl-D-aspartic acid (NMDA)-induced retinal ganglion cells (RGCs) injury and its underlying mechanism. Materials and methods Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. NMDA was used to mimic the glutamate activation, cell apoptosis, reactive oxygen species (ROS), malondialdehyde levels, SOD and NO production, nNOS and iNOS expression, as well as caspase-3 activity, Bcl-2, and Bax expression in the RGCs were analyzed by ELISA, RT-PCR, and Western blotting. A rat model of retinal injury was used to detect the protective effect of puerarin. Results Puerarin protected against NMDA-induced RGCs injury in a dose-dependent manner. Compared with the NMDA-treated group, puerarin pretreatment significantly reduced ROS and malondialdehyde levels, promoted SOD and NO production, and downregulated nNOS and iNOS expression in the RGCs. Mechanism analysis showed that pretreatment with puerarin could effectively offset the increase of Bax expression and caspase-3 activity brought by NMDA, and promote Bcl-2 expression in the RGCs. Puerarin pretreatment also effectively inhibited NMDA-induced JNK and p38 phosphorylation in the RGCs, whereas pretreatment with either JNK agonist anisomycin or p38 agonist P79350 could significantly compensate the effects caused by puerarin. Furthermore, puerarin prevented RGCs loss in the retinal injury induced by intravitreal NMDA in a rat model. Conclusions The present results of this study demonstrated that puerarin protected against NMDA-induced apoptosis and RGCs damage through the JNK/p38 MAPK pathway.

Mechanism Underlying the Analgesic Effect Exerted by Endomorphin-1 in the rat Ventrolateral Periaqueductal Gray.

Abstract: The ventrolateral periaqueductal gray (vlPAG) is an important brain area, in which 5-HTergic neurons play key roles in descending pain modulation. It has been proposed that opioid peptides within the vlPAG can excite the 5-HTergic neurons by alleviating tonic inhibition from GABAergic neurons, the so-called disinhibitory effect. However, no direct morphological evidence has been observed for the micro-circuitry among the opioid peptide-, GABA-, and 5-HT-immunoreactive (ir) profiles nor for the functional involvement of the opioid peptides in the intrinsic properties of GABAergic and 5-HTergic neurons. In the present study, through microscopic observation of triple-immunofluorescence, we firstly identified the circuitry among the endomorphin-1 (EM1, an endogenous ligand for the μ-opioid receptor)-ir terminals and GABA-ir and 5-HT-ir neurons within the rat vlPAG. The synaptic connections of these neurons were further confirmed by electron microscopy. Through the in vitro whole-cell patch-clamp method, we showed that EM1 has strong inhibitory effects on the spiking of GABAergic neurons. However, although the resting membrane potential was hyperpolarized, EM1 actually increased the firing of 5-HTergic neurons. More interestingly, EM1 strongly inhibited the excitatory input to GABAergic neurons, as well as the inhibitory input to 5-HTergic neurons. Finally, behavioral results showed that pretreatment with a GABA(A) receptor antagonist potentiated the analgesic effect of EM1, while treatment with a GABA(A) receptor agonist blocked its analgesic effect. In summary, by utilizing morphological and functional methods, we found that the analgesic effect of EM1 is largely dependent on its potent inhibition on the inhibitory inputs to 5-HTergic neurons, which overwhelms EM1's direct inhibitory effect on 5-HTergic neurons.

Decreased Endomorphin-2 and μ-Opioid Receptor in the Spinal Cord Are Associated with Painful Diabetic Neuropathy

Abstract: Painful diabetic neuropathy (PDN) is one of the most common complications in the early stage of diabetes mellitus (DM). Endomorphin-2 (EM2) selectively activates the opioid receptor (MOR) and subsequently induces antinociceptive effects in the spinal dorsal horn. However, the effects of EM2-MOR in PDN have not yet been clarified in the spinal dorsal horn. Therefore, we aimed to explore the role of EM2-MOR in the pathogenesis of PDN. The main findings were the following: (1) streptozotocin (STZ)-induced diabetic rats exhibited hyperglycemia, body weight loss and mechanical allodynia; (2) in the spinal dorsal horn, the expression levels of EM2 and MOR decreased in diabetic rats; (3) EM2 protein concentrations decreased in the brain, lumbar spinal cord and CSF in diabetic rats but were unchanged in the plasma; (4) the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was significantly higher in diabetic rats than in control rats; and (5) intrathecal injection of EM2 for 14 days in the early stage of PDN partially alleviated mechanical allodynia and reduced MOR expression in diabetic rats. Our results demonstrate that the EM2-MOR signal may be involved in the early stage of PDN.

Characterization of the anterior cingulate cortex in adult tree shrew.

Abstract: The anterior cingulate cortex (ACC) is a key brain region for the perception of pain and emotion. Cellular and molecular mechanisms of the ACC are usually investigated in rodents such as mice and rats. Studies of synaptic mechanisms in primates are limited. To facilitate the translation of basic results from rodents to humans, it is critical to use a primate-like animal model for the investigation of the ACC. The tree shrew presents a great opportunity for this as they have similar genome sequences to primates and are considered to have many similarities to primates. In the present study, by combining anatomy, immunostaining and micro-optical sectioning tomography methods, we examined the morphological properties of the ACC in the tree shrew and compared them with the mouse and rat. We found that the ACC in the tree shrew is significantly larger than those found in the mouse and rat. The sizes of cell bodies of ACC pyramidal cells in tree shrew are also larger than that found in the mouse or rat. Furthermore, there are significantly more apical/basal dendritic branches and apical dendritic spines of ACC pyramidal neurons in tree shrew. These results demonstrate that pyramidal cells of the ACC in tree shrews are more advanced than those found in rodents (mice and rats), indicating that the tree shrew can be used as a useful animal model for studying the cellular mechanism for ACC-related physiological and pathological changes in humans.

A disparate subset of double-negative T cells contributes to the outcome of murine fulminant viral hepatitis via effector molecule fibrinogen-like protein 2.

Abstract: The underlying immune-mediated mechanisms involved in virus-induced severe hepatitis have not been well elucidated. In this study, we investigated the role of CD3(+)CD4(-)CD8(-) double-negative T (DN T) cells in the pathogenesis of fulminant viral hepatitis (FVH) induced by murine hepatitis virus strain 3 (MHV-3). After MHV-3 infection, the proportions of DN T cells increased significantly in BALB/cJ mice, and splenic DN T cells expressing high levels of CD69 were recruited by MHV-3-infected hepatocytes to the liver. Serum levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin increased, accompanied by massive hepatocyte necrosis. These DN T cells were predominantly consisted of a TCRαβ(+) subset expressing high levels of CD44 and did not produce cytokine except IL-2. Adoptive transfer of this subset of DN T cells to the MHV-3-infected mice resulted in an increase in murine fibrinogen-like protein 2 (mfgl2) expressions in association with massive fibrin deposition in the liver. Following MHV-3 infection, membrane mfgl2 expression and functional procoagulant activity increased remarkably in the DN T cells. Introduction of a recombinant adenovirus which encoded a microRNA specifically targeting mfgl2 gene (Ad-mfgl2-miRNA) in vivo significantly inhibited the hepatic expression of mfgl2 and improved survival in mice. However, under this condition, adoptive transfer of the DN T cells accelerated the disease progression and reversed the benefit from mfgl2 gene silence, leading to a 100 % death rate. Our results demonstrate that DN T cells contribute to the outcome of MHV-3-induced FVH via an important effector molecule mfgl2.

Expression of 58-kD Microspherule Protein (MSP58) is Highly Correlated with PET Imaging of Tumor Malignancy and Cell Proliferation in Glioma Patients.

Abstract: Background/Aims: The nucleolar 58-kDa microspherule protein (MSP58) has important transcriptional regulation functions and plays a crucial role in the tumorigenesis and progression of cancers. 3'-deoxy-3'-[18F]fluorothymidine (FLT) has emerged as a promising positron emission tomography (PET) tracer for evaluating tumor malignancy and cell proliferation. Methods: In the present study, the expression of MSP58 was evaluated by immunohistochemistry and the corresponding PET image was examined using FLT-PET in 55 patients with various grades of gliomas. Results: The immunoreactivity score (IRS) of MSP58 increased with tumor grade with grade IV gliomas exhibiting the highest expression and showed a highly significant positive correlation with the Ki-67 index (r = 0.65, P r = 0.61, P r = 0.59, P Conclusion: These results indicate that MSP58 plays an important role in cell proliferation and will be one of the potential candidates of molecular therapy targeting proliferation. FLT-PET might be used as an early measure of treatment response in the proliferation-targeted therapy.

A comparative study on protective effect of optical nerve between crocin and erigeron breviscapus in chronic ocular hypertensive rats

Abstract: Background The primary cause of glaucomatous optic nerve damage is apoptosis of nerve cell and disorder of retinal circulation.Erigeron breviscapus is confirmed to have a protective effect on retinal ganglion cells (RGCs) in rats with chronic ocular hypertension, and crocin has the effect of anti-inflammation and anti-apoptosis.However, whether crocin can protect RGCs against ocular hypertension damage is unclear. Objective This study was to investigate the protective effect of crocin on the optic nerve in chronic ocular hypertension. Methods Thirty-two SD rats were randomized into the sham operation group, model control group, erigeron group and crocin group, 8 rats for each group.The right eyes served as experimental eyes.Chronic ocular hypertensive models were established by episcleral vein cauterization in the rats of the model control group, erigeron group and crocin group, and only conjunctiva was cut off in the sham operation group.Erigeron (150 mg/kg) and crocin (20 mg/kg) was intraperitoneally injected in the erigeron group and crocin group respectively 30 minutes before operation and once daily after operation for 4 weeks, and 0.5 ml normal saline was used in the sham operation group and model control group.Intraocular pressure (IOP) was measured before surgery and 1 day, 3 days, 1 week, 2 weeks, 3 weeks and 4 weeks after surgery.The samples of eyeballs and optic nerve in the rats were prepared at 4 weeks after surgery.Retinal thickness was measured by hematoxylin and eosin staining.The apoptosis of RGCs was detected by TUNEL assay.The ultrastructure change of optic nerve was examined under the transmission electron microscope, and the expression of bcl-2 and bax proteins in retinal homogenates was analyzed by Western blot.The use and care of the animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee. Results The IOPs were significantly elevated in the model control group, erigeron group and crocin group in comparison with the sham operation group, and the IOPs was significantly higher in various time points after surgery than that before surgery (Fgroup=169.079, P=0.000; Ftime=50.505, P=0.000). The retinal thickness was (192.72±4.28), (165.15±3.89), (177.75±3.35) and (182.48±4.12)μm in the sham operation group, model control group, erigeron group and crocin group, and rat retinal thickness in the crocin group was significantly lower than that in the sham operation group and higher than that in model control group and erigeron group (all at P<0.05). The percentage of apoptotic RGCs in the sham operation group, model control group, erigeron group and crocin group, showed significant reduction in comparison with the model control group and erigeron group (all at P<0.05). The number of myelinated nerve fibers and bcl-2/bax values were significantly increased in the crocin group compared with the model control group and erigeron group (all at P<0.05). Conclusions Crocin affords a optic nerve by inhibiting RGC apoptosis and optic nerve degeneration in SD rats with chronic ocular hypertension, and the protecting effect of crocin is more prominent than that of erigeron breviscapus. Key words: Crocus/chemistry; Herbal medicine; Plant extracts/pharmacology; Ocular hypertension; Apoptosis; Retinal ganglion cells; Erigeron/chemistry; Optic nerve protection