The Effect Of Astragalus Injection On JNK3 After Hypoxia/Hypoglycemia And Reoxygenation In Hippocampal Neurons Of Rats

Ischemic cerebrovascular disease has become common ailment in clinic, and reperfusion injury is one of the its significant mechanisms,moreover it become a important factor which restricts curative effect of ischemic cerebrovascular disease,thus it is essential that probe into pathophysiological mechanism of cerebral ischemia-reperfusion and enhance prevention and therapy.Prospective researchs about ischemia reperfusion show generation of free radical,intracellular Ca²⁺ over loading,cell apoptosis and so on in exterior and interior.Neuron apoptosis is serious link of pathophysiological program about cerebral ischemia-reperfusion,however it is not clear that how the stress and mediators releasing which ischemia-reperfusion originates mediate neuron apoptosis by signal pathway.JNK3 in mitogen activated protein kinase family has relative to signal transduction of cell apoptosis,but it is reported rarely that whether JNK3 signal pathway mediate neuron apoptosis after ischemia-reperfusion or not.Astragalus injection is frequent drug which cure cerebral vessel diseases,however it has not been illustrated that inhibiting cell apoptosis mechanism is by which signal transduction executes it.We can probe into the molecular mechanism which astragalus injection protects brain through investigate the effect of astragalus injection on the expression and activity of JNK3 interrelated by apoptosis after hypoxia/ hypoglycemia and reoxygenation in hippocampal neurons of rats. The first partThe effect of Astragalus injection on the expression and activity of JNK3 after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of ratsAIM:To investigate the effect of astragalus injection on the expression and activity of JNK3(c-jun N terminal kinase) interrelated by apoptosis after hypoxia/ hypoglycemia and reoxygenation in hippocampal neurons of rats.METHODS:Divided the hippocampal neurons cultured for eight days into four groups:normal control group,original astragalus injection group, hypoxia/hypoglycemia and reoxygenation group,astragalus injection group. hypoxia/hypoglycemia and reoxygenation group,astragalus injection group and original astragalus injection group were treated with hypoglycemia and reoxygenation after deprived of oxygen and glucose for 30 minutes.Methods of immunohistochemistry and western blotting were used respectively to measure the expression of JNK3 after hypoxia/ hypoglycemia and reoxygenation 0h,0.5h,2h,6h,24h,72h,120h;Methods of ELISA were used respectively to measure the OD value of JNK3 after hypoxia/ hypoglycemia and reoxygenation 0h,0.5h,2h,6h,24h,72h,120hRESULTS:It can be observed in normal control group that hippocampal neuronal nucleolus was integrated,cellular tuber was distinct and cytokinesis was dyed by yellow a lot;It can be observed in hypoxia/ hypoglycemia and reoxygenation group that hippocampal neuronal nucleolus was crimple, cellular tuber was shrinked,large numbers of cytokinesis was dyed by yellow and there are yellow granule.Compared with normal control group,the numbers of JNK3 protein positive neuronal cells every time points increased obviously in the hypoxia/ hypoglycemia and reoxygenation group(P＜0.05); The change of neuronal configuration and the numbers of JNK3 positive neuronal cells in original astragalus injection group accorded with hypoxia/ hypoglycemia and reoxygenation group(P＞0.05);It can be observed in astragalus injection group that hippocampal neuronal nucleolus was crimple slightly and segmental cytokinesis was dyed by yellow.Compared with hypoxia/ hypoglycemia and reoxygenation group,the numbers of JNK3 protein positive neuronal cells every time points were less obviously in the astragalus injection group besides 120h(P＜0.05).Compared with normal control group,the mean optic density(MOD) of expression of JNK3 protein in hippocampal neurons of rats every,time points increased obviously in hypoxia/hypoglycemia and reoxygenation group(P＜0.05)besides 120h, Compared with normal control group,the mean optic density(MOD) of expression of JNK3 protein in hippocampal neurons of rats in 120h decreased obviously in hypoxia/hypoglycemia and reoxygenation group(P＜0.05); Compared with hypoxia/ hypoglycemia and reoxygenation group,the mean optic density(MOD)of expression of JNK3 protein every time points in original astragalus injection group had no obvious change,however the mean optic density(MOD)of expression of JNK3 protein in hippocampal neurons of rats every time points decreased obviously in the astragalus injection group besides 120h(P＜0.05).Compared with normal control group,the OD value and concentration of JNK3 kinase in hippocamal neurons of rats every time points increased obviously in hypoxia/hypoglycemia and reoxygenation group(P＜0.05) besides 120h,Compared with normal control group,the OD value and concentration of JNK3 kinase in hippocampal neurons of rats in 120h decreased obviously in hypoxia /hypoglycemia and reoxygenation group(P＜0.05);Compared with hypoxia/ hypoglycemia and reoxygenation group,the OD value of and concentration JNK3 kinase every time points in original astragalus injection group had no obvious change,however the OD value and concentration of JNK3 kinase in hippocampal neurons of rats every time points decreased obviously in astragalus injection group besides 120h (P＜0.05).CONCLUSION:astragalus injection could inhibit the expression of JNK3 protein after hypoxia/hypoglycemia and reoxygenation,meanwhile the activity of expressed JNK3 redused. The second partAstragalus injection inhibits the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of ratsAIM:To investigate the effect of astragalus injection on the expression of JNK3(c-jun N terminal kinase) mRNA interrelated by apoptosis after hypoxia/ hypoglycemia and reoxygenation in hippocampal neurons of rats.METHODS:Divided the hippocampal neurons cultured for eight days into four groups:normal control group,original astragalus injection group, hypoxia/hypoglycemia and reoxygenation group,astragalus injection group. hypoxia/hypoglycemia and reoxygenation group,astragalus injection group and original astragalus injection group were treated with hypoglycemia and reoxygenation after deprived of oxygen and glucose for 30 minutes.Methods of In situ hybridization and RT-PCR were used respectively to measure the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation 0h,0.5h,2h,6h,24h,72h,120h.RESULTS:It can be observed in normal control group that the volume of hippocampal neuronal nucleolus was accretion,cellular tuber was distinct and cytokinesis was dyed by yellow a lot; It can be observed in hypoxia/hypoglycemia and reoxygenation group that hippocampal neuronal nucleolus was crimple,cellular tuber was shrinked, large numbers of cytokinesis was dyed by yellow and there are yellow granule.Compared with normal control group,the numbers of JNK3 mRNA positive neuronal cells every time points increased obviously in the hypoxia/ hypoglycemia and reoxygenation group(P＜0.05);The change of neuronal configuration and the numbers of JNK3 mRNA positive neuronal cells in original astragalus injection group accorded with hypoxia/hypoglycemia and reoxygenation group(P＞0.05);It can be observed in astragalus injection group that hippocampal neuronal nucleolus was crimple slightly and segmental cytokinesis was dyed by yellow.Compared with hypoxia/hypoglycemia and reoxygenation group,the numbers of JNK3 mRNA positive neuronal cells every time points were less obviously in the astragalus injection group besides 120h(P＜0.05).Compared with normal control group,the mean optic density(MOD) of expression of JNK3 mRNA in hippocampal neurons of rats every time points increased obviously in hypoxia/hypoglycemia and reoxygenation group(P＜0.05);Compared with hypoxia/ hypoglycemia and reoxygenation group,the mean optic density(MOD)of expression of JNK3 mRNA every time points in original astragalus injection group had no obvious change(P＞0.05),however the mean optic density(MOD)of expression of JNK3 mRNA in hippocampal neurons of rats every time points decreased obviously in astragalus injection group besides 120h(P＜0.05).CONCLUSION:Astragalus injection could inhibit the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation,accordingly inhibited hippocampal neuronal apoptosisCONCLUSIONS1 Astragalus injection could inhibit the expression of JNK3 protein after hypoxia/hypoglycemia and reoxygenation,and inhibit the activity of JNK3 protein after hypoxia/hypoglycemia and reoxygenation.2 Astragalus injection could inhibit the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation.3 Astragalus injection could inhibit hippocampal neuronal apoptosis by inhibiting the expression of JNK3 interrelated by apoptosis after hypoxia/ hypoglycemia and reoxygenation in hippocampal neurons of rats.