| Cerebrovascular disease is clinically common disease,is one of the threemajor killer diseases,serious harm to human health and life.With populationgrowth and increasing the degree of aging,its incidence, mortality andmorbidity were increasing.Ischemic cerebrovascular disease accounts for alarge proportion.The treatment of cerebral ischemia is the key to rapidrecovery of cerebral blood perfusion,and maintain bloodflow.However,perfusion recovery is still likely to further increase braindamage,that is cerebral ischemia-reperfusion injury.Ischemia reperfusioninjury is that subjected to certain time of ischemic to restore blood cause tissueinjury aggravated the pathological phenomenon.Cerebral ischemia-reperfusioninjury has great harm,the damage mechanisms involve many complex aspectsand factors,many year scientists have been the focus of attention.Themechanism of cerebral ischemical injury with increasing depth study,apoptosiscause for concern in the role of cerebral ischemia and reperfusion.Studies haveshown that neuronal apoptosis is an important form of cell death in ischemicbrain injury.Apoptosis by a variety of related genes and proteins,includingcysteine aspartic protease(Caspase).Caspase has most closely relation withapoptosis.Astragalus is ischemia-reperfusion brain disease commonly useddrugs for the clinical treatment of ischemia-reperfusion brain disease,but themechanism that through which special signal transduction pathway inhibitapoptosis has not been fully elucidated. Our group pre-test confirmed thatAstragalus injection injection inhibited from cultured hypoxia/hypoglycemiaand reoxygenation rat hippocampal neuronal apoptosis,and confirmed to playthe role of Astragalus injection by inhibiting the JNK pathway.However, Astragalus injection whether inhibit the JNK pathway in the in vivo situationof neuronal apoptosis is still unknown.By observing the astragalus on theexpression of Caspase-3after cerebral ischemia and reperfusion in rathippocampal neurons,to investigate the inhibitory of astragalus injection oncerebral ischemia and reperfusion of hippocampal neurons,and application ofagonists and inhibitor of the JNK investigate whether Astragalus injection ofanti-apoptotic effects by inhiting the JNK pathway.The first partThe effect of astragalus injection on the morphology of cerebralischemia and reperfusion in hippocampal CA1neurons.Aim:Observation of astragalus injection on cerebral ischemia andreperfusion the morphology of hippocampal CA1neurons.Methods:The adult male SD were randomly divided into4groups: sham operationgroup,model group(cerebral ischemia-reperfusion group),astragalus injectiongroup,original astragalus injection group.Prepare the rat model of cerebralischemia-reperfusion by four-vessel occlusion.Astragalus injectin group andoriginal astragalus injection were given astragalus and sterile deionizedwater(6ml/kg)before30min ischemia.With HEand TUNELstaining to observethe pathological changes of24h after perfusion.Results:HE staining:the model group rat hippocampus occur apparentpathological changes,hippocampal neurons arranged due to the rules,unclearboundaries,the normal cell structure disappeared,nuclei stain area,appearedcoagulation necrosis,cell obvious dropout,compared with sham-operationgroup the number of hippocampal CA1neurons was significantlyreduced(P<0.05). Compared with model group,the hippocampal CA1neuronssignificantly improved in the astragalus injection group(P<0.05),while theoriginal astragalus injection group did not change significantly(P>0.05).TUNEL staining results show that:compare withsham-operation group, apoptotic cells in model group were significantlyincreced(P<0.05);compared with model group,Astragalus injection group ofapoptotic cells were significantly reduced,the difference was statisticallysignificant(P<0.05), but original astragalus injection group has no significantdifference (P>0.05).Conclusion:Astragalus injection can reduce the loss of cerebralischemia-reperfusion damage in hippocampal CA1neurons,reducing apoptsis.The second partThe effect of astragalus injection on Caspase-3expression followingcerebral ischemia-reperfusion in rat hippocampal neurons.Aim:To investigate the effect of astragalus injection on the expression ofCaspase-3protein and mRNA after cerebral ischemia-reperfusion in rathippocampal neurons.Methods:Selection of adult male SD were randomly divided into4groups:sham-operation group, model group(cerebral ischemia-reperfusiongroup),astragalus injection group,original astragalus injection group. Modelingapproach with the first part, in addition to the sham-operation group,eachgroup were carried out cerebral ischemia0.5h.After reperfusion,respetivelyselect0h,0.5h,2h,6h,24h,72h,and120h using tissue immunochemistry,westernblotting to detect the expression of Caspase-3protein in rat hippocampalneurons.With Real-Time PCR method detect the expression of Caspase-3mRNA in rat hippocampal neurons.Results:Immunocytochemistry showed that:sham-operation group ratshippocampal CA1neurons nuclear regularity,there is a little yellow stainedcytoplasm.Model of hippocampal CA1neurons nuclear shrinkage,chromatin margination,a large number of yellow stained cytoplasm,while nucleus has alittle yellow particles；In addition to0h,0.5h,the expression of Caspase-3protein at each time point than the sham-operation group was significantlyincreased(P<0.05). With the model group original astragalus injection groupthe morphology and expression of Caspase-3in hippocampal CA1has nochange(P>0.05). Astragalus rat hippocampal CA1neurons has slight nuclearshrinkage, While part of the neurons pulp yellow dye;in addition to0h,0.5htime point,the hippocampal neurons expression of Caspase-3protein in thecorresponding time points significantly lower than model group(P<0.05). Asreperfusion time, the model group,original astragalus injection group andastragalus injection group can be seen the cytoplasm of positive neuronsgradually increased,reached a peak at24h.Western blotting and Real-TimePCR results showed that:compared with the sham-operation group,in additionto0h and0.5h,the model group, Caspase-3protein and mRNA expression wassignificantly increased(P<0.05),reperfusion after24h the expression ofCaspase-3protein and mRNA reached a peak.Compared with modelgroup,astragalus group except0h and0.5h each time point the expression ofCaspase-3protein and mRNA were significantly lower(P<0.05).But wihmodel group original astragalus injection group Caspase-3protein and mRNAexpression had no significant change(P>0.05).Conclusion：Astragalus can inhibit the expression of Caspase-3protein and mRNA inrat hippocampal neurons after cerebral ischemia-reperfusion,therebyinhibiting apoptosis in rat hippocampal neurons induced by cerebralischemia-reperfusion.The third partThe effect of JNK inhibitor and agonists on cerebral ischemia andreperfusion in rat hippocampal neurons Caspase-3expression.Aim:Observe the JNK inhibitor and agonists on cerebral ischemia and reperfusion in rat hippocampal Caspase-3expression.Methods:Selection of adult male SD rats were randomly divided into7groups:sham-operation group,model group(cerebral ischemia-reperfusion),original astragalus injection group,astragalus injection group,JNK inhibitorgroup(SP600125group),JNK agonist group (Anisomysin group),and DMSOgroup(drug solvent control group). Modeling approach with the first part,after24h reperfusion using tissue immunochemistry,western blotting andReal-Time PCR method to detect the expression of Caspase-3in hippocampalneurons.Results:Immunocytochemistry showed that:the sham-operation rat hippocampalneurons membrane integrity,1-2nucleoli,slight stained cytoplasm.Model ofhippocampal neurons has nucleus shrinkage, chromatin margination, a largenumber of cytoplasm stained yellow,the nucleus of a little yellowgranules,compared with the sham-operation group hippocampal neuronsCaspase-3protein was significantly increased(P<0.05).Compared with modelgroup, original astragalus injection group and DMSO group the expression ofCaspase-3protein in rat hippocampal neurons without significantchange(P>0.05).While astragalus injection group and SP600125group ofCaspase-3protein expression were significantly lower(P<0.05),but the twogroup was no significant difference(P>0.05).While Anisomysin group ofCaspase-3protein expression was significantly increased(P<0.05).Westernblotting and Real-Time PCR results showed that:compared with thesham-operation group,the expression of Caspase-3protein and mRNA inmodel group rat was significantly increased(P<0.05).Compared with modelgroup,original astragalus injection group,DMSO group the expression ofCaspase-3protein and mRNA was not significantly changed(P<0.05).whileastragalus injection group,SP600125group of Caspase-3protein and mRNAexpression was significantly lower (P<0.05),but this was no significantdifference between the two groups(P>0.05).While Anisomycin group of Caspase-3protein and mRNA expression was significantly increased(P<0.05).Conclusion:Astragalus can inhibit the activation of JNK pathway to reduced theexpression of Caspase-3,thus inhibiting apoptosis of hippocampal neuronsafter cerebral ischemia-reperfusion.Conclusions:1Astragalus injection can reduce the loss of cerebralischemia-reperfusion damage in hippocampal CA1neurons,reducing apoptsis.2Astragalus injection inhibit the expression of Caspase-3protein andmRNA after cerebral ischemia-reperfusion hippocampal neurons byinhibiting the activation of JNK signaling pahway,thereby inhibiting apoptosisin hippocampal neurons. |
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