The Experimental Study On The Neuroprotective Action Of Decoction Of Poxuehuayu On Neural Damage Model Induced By Oxygen-glucose Deprivation In Vitro

Purpose:By employing cortical neurons cultured in vitro,this experiment aims at studying the protective action of decoction of Poxue Huayu on Oxygen-Glucose deprivation injury to neurons,and exploring the best protective action by the best culturing method of neurons,mould condition as well as the dose.This will lay the foundation of further study and application.Method:Isolate and culture cerebral cortex cells from embryonic rat.By using serum medium promote cell adhesion and growth,and adding the neurobasal medium with B27 and L-glutamine and the primary culture of cerebral cortex neurons will be achieved.Then a considerable proportion of neurons will be harvested(the number of NSE positive cells are greater than 90%,identified with immunocytochemistry).The growth curve of cells is determinate by CCK-8 method;set up both negative and positive control group,and establish Oxygen-Glucose deprivation model with CCK-8 method.The cells what have been cultured with different drug concentration at the 7th day,establish normal oxygen control group,and detect the survival rate of the cells with CCK-8 method.Conduct T-test and anova to survival rate of the cells under the environment of Oxygen-Glucose deprivation with SPSS.17.0.Result:1.The primarily culture of cortical neurons of embryonic rat with neurobasal medium with B27 and L-glutamine,which grows stably from the sixth to eighth day and they present a tendency of decay at the ninth day.2.The model which under the Oxygen-Glucose Deprivation12 h and reoxygenation 12 h,the survival rate has declined,compared with the control group the survival rate is 60%.3.Adding the decoction of DI DANG of 2.5%,5% and 10% compared to the negative control group the survival rate of neurons model injured by OGD has increased,the discrepancy is statistically significant(P<0.05).The group of adding the decoction of DI DANG of 5% compared to the positive control group,the survival rate of neurons model has increased and the discrepancy is statistically significant(P<0.05).And there is no statistical significance(P>0.05)between the positive control group.4.The Oxygen-Glucose deprivation group with LY294002,what is a PI3K/AKT inhibitors,compared to the negative control group,the survival rate has decreased and the group at the same time adding the decoction of DI DANG of 5% the survival rate has increased and both discrepancy of the survival rate is statistically significant(P<0.05).Conclusion:1.With the primary culture method of neurons of 14-16 days with fetal bovine serum culture medium and neurobasal medium with B27 and L-glutamine a relatively high purity neurons can be cultured in vitro,which can be used for the study of their relative features.2.A sample and stable cortical neurons model can be build with the methods of Oxygen-Glucose deprivation 12 h and reoxygenation 12 h wih EBSS medium and Ischemia anoxic tank in vitro.3.The Decoction of DI DANG can protect the cortical neurons model from the Oxygen-Glucose Deprivation and dose positively correlated and the protective effect is better than the positive control group.4.The protective effect of the decoction of DIDANG is may be attributed to PI3K/AKT signaling pathways.