Study On The Neuroprotective Effect And Mechanism Of Cynomorium Songaricum Ethyl Acetate Extract Based On GPR30 Recepto

Background:It has been generally provided that estrogen played a critical role in central nerve system,regulate synaptic elasticity,anti formation of Aβ and free radical.Recent research found that estrogen protect nerve cells from oxidative damage induced by H₂O₂ by binding with GPR30 associated with stimulate multi signal pathways,speculate that GPR30 is a potential therapeutic target for neuroprotection.Objective:Previous study have shown that EtOAc extract of Cynomorium Songaricum（ECS）can improve both E2 level and the ability of learning and memory in ovariectomized rat,protect sk-nsh cells from Aβ injury.To contribute a better understanding of the effect of ECS in protect nerve system,we use Neuro2a cells as model and different estrogen antagonist blocked the effects of ECS,then detect expression of synaptophysin.Based on this,using H₂O₂ induced cell oxidative damage model to detect the anti-oxidative effect of ECS.Methods:Part one Take Neuro2a as model,use CCK8 method to detect the effect of ECS on different cells,and observe the influence of ECS on cells morphology.Using Western-blot method,giving ER blocker ICI182780 and GPR30 blocker G15 to detect the effect of ECS on syn,p-CREB and p-erk expression.Using MAPK antagonist PD98059 to detect the influence on p-CREB and p-erk expression and immunofluorescence detect the location and fluorescence intensity of synaptophysin.Part two Establishment of H₂O₂ induced cell oxidative injuried model.Using CCK8 method to detect the cell viability rate ECS on H₂O₂ damaged cells in different times.Part three Research on the mechanism of H₂O₂ damaged cells and protective effect of ECS.Using Western blot method to detect the expression of synaptophysin and PSD95 of H₂O₂ damged cells,Giving GPR30 antagonist G15 to detect the effect of ECS on H₂O₂damged cells and expression of synaptophysin and PSD95.And detect the expression of p-ERK and pp38 in H₂O₂ damaged cells.Giving both ERK and P38 antagonist PD98059 and SB203580 to detect the anti-oxidative effect of ECS.Results:1.In the condition of complete medium,100 mg/L ECS can significantly improve cell viability at 24h,and has no significant effect on primary nerve cell viability.2.The results of western blot has shown that ECS improve the expression of synaptophysin and p-CREB,p-ERK.GPR30 antagonist G15 attenuated the effect of ECS,but ER antagonist ICI182780 didn’t.MAPK antagonist PD98059 and GPR30 antagonist G15 blocked the effect of ECS.Immunofluorescence results has shown that the synaptophysin located in cytodendrite.ECS increased the immunoreactivity products.3.In the condition of complete medium,200μM H₂O₂ significantly decrease the cell viability at 2 hours,the viability rate is 50%.100mg/L ECS significantly reduce the oxidative effects of H₂O₂.at 24h.4.The results of western blot has shown that H₂O₂ significantly reduced the expression of synaptophysin and PSD95,while ECS has an protective effect,but GPR30 antagonist G15 attenuate the protective effect of ECS.ECS attenuate the effect of H₂O₂ in reducing the expression of p-p38 and p-ERK,while PD980059 and SB203580 blocked the effect of ECS.Conclusion:ECS has shown a neuroprotection effect.We suppose that ECS activate GPR30 to regulate the expression of p-erk and p-CREB to regulate gene transcription and translation,cause functional synaptic elasticity protein syn increased.H₂O₂ decreased cell viability and the expression of syn and PSD95 and p-erk,increased the expression of p-p38.ECS protects cells from H₂O₂ induced oxidative-damage.ECS activates GPR30 receptor to increase expression of syn and PSD95,and increased the expression of MAPK p-ERK and dereased the expression of p-p38 to exert neuroprotection effects.