The Mechanism Of Madecassoside On Microglia During Oxygen Glucose Deprivation And Reintroduction

Cerebral ischemia is a major diseases to human health and survival and one of the leading causes of human disability and mortality in the world. However, even after the restoration of blood flow, reperfusion itself causes tissue damage beyond that caused by ischemia. ischemia-reperfusion(I/R) injury result in a series of tissue damage and cell death process, its pathogenesis is not fully understood,there is no specific medicines. The hall mark of brain inflammation is the activation of microglia. Upon activation,the microglia can produce proinflammatory cytokines which plays an important role in the development of cerebral ischemia. Thus, drugs with the effects to inhibit microglia activation would provide neuroprotective effects. Recent reports strongly suggest that regulating microglia function may be a promising therapeutic approach for treating cerebral ischemia. Targeting at the prevention of the activation and generation of inflammatory cytokines of the microglia has become the focus of the development of treatment drugs.Madecassoside(MA) extracts exhibit a wide range of pharmacological activities, including anti-inflammatory, antioxidant, wound healing. We have confirmed that MA exerts a neuroprotective effect against cerebral I/R injury in rats, we found that MA reduced cortical infarct size and neurological deficit scores, and the protective effect may be related to the inhibition inflammation of CNS, but the definite mechanism remains to be further explored. In this study, we established oxygen and glucose deprivation and reintroduction(OGD/R) model in vitro, to further study the protective effect of MA on activation of microglia, TLR4/MyD88 signaling pathway and its downstream molecule NF-κB-mediated inflammatory. To launch the study as order "signal pathway- microglia- neuroinflammation" to clarify the anti-inflammatory mechanisms of madecassoside against cerebral ischemia and provide a scientific basis for drug development and clinical application.Objective: To study the protective effect madecassoside(MA) on BV2 microglia OGD/R injury, and the anti-inflammatory effect of MA on LPS-induced injury, To confirm further the roles of TLR4/myeloid differentiation protein88(MyD88)/nuclear factor-kappa B(NF-κB) pathway and inflammation reaction in the neuroprotective effects of madecassoside.Methods: We established oxygen and glucose deprivation and reintroduction(OGD/R) model of BV2 microglia in vitro, grouping into OGD/R+MA(50μM), OGD/R+MA(100μM), OGD/R+MA(200μM), OGD/R and Control group. We established LPS-induced inflammation model of BV2 microglia, experimental groups divided LPS+MA(200μM), LPS and Control group. Cell viability was determined by inverted microscope and the MTT assay. mRNA level of proinflammatory cytokines(IL1β, IL1 R, IL6, TNFα, TLR4, MyD88 and NF-κB)was determined with real-time PCR. Level of IL1β, IL6 and TNFα in culture medium was determined with enzyme-linked immunosorbent assay. TLR4 and its downstream signaling molecules, MyD88 and NF-κB expressions were detected by immunofluorescense and Western blot.Conclusion: MA has a protective effect for the OGD/R injury of BV2 microglia, the mechanism may be associated with reduced expression of TLR4 and its downstream MyD88-dependent signaling pathway, inhibits the expression of the downstream nuclear transcription factor NF-κB, thereby inhibiting inflammatory cytokines regulated by NF-κB to reduce microglia-mediated inflammatory damage.