| Experiment 1 Effect of Electroacupuncture Preconditioning on Apoptosis and Autophagy in Rats with Cerebral Ischemia Reperfusion InjuryObjective:To observe the effects of electroacupuncture preconditioning on motor function,pathological morphology,apoptosis and autophagy in ischemic penumbra of rats with cerebral ischemia-reperfusion injury.Methods:153 SPF male Sprague-Dawley rats were divided into sham operation group,model group and electroacupuncture preconditioning group according to the random number table method.36 rats in each group were divided into three subgroups:reperfusion 2h,24h and 72h,with 12 rats per subgroup.Cerebral ischemia was induced by middle cerebral artery occlusion(MCAO)for 2h,followed by reperfusion.At 2h,24h and 72h after reperfusion,neurobehavioral assessment was performed on all rats.HE staining was used to observe the degree of pathological damage in the ischemic penumbra.TUNEL was used to detect apoptosis in the ischemic penumbra of brain tissue.Western blotting was performedto identify expression of LC3 ?/? in the ischemic penumbra of brain tissue.Result:1.mNSS:Compared with the model group,mNSS in the electroacupunct-ure preconditioning group were significantly lower at each time point after surgery(P<0.05).2.HE staining:At each time point after operation,compared with the model group,the extent of pathological injury in the cerebral ischemia-reperfusion injury area was reduced in the electroacupuncture precondit ioning group(P<0.05).3.TUNEL:Compared with the model group,the number of apoptotic cells in the ischemia-reperfusion injury area was decreased in the electroa-cupuncture preconditioning group(P<0.05).4.Western blotting:Compared with the model group,the ratio of LC3 ?/?protein in the ischemia penumbra was decreased(P<0.05),and autophagy was reduced in the electroacupuncture preconditioning group.Conclusion:1.Electroacupuncture preconditioning can reduce mNSS scores in rats with cerebral ischemia-reperfusion injury and improve the degree of pathological damage in the ischemia penumbra;2.Electroacupuncture preconditioning can reduce the number of apopto-tic cells in the cerebral ischemic penumbraand inhibit apoptosis for promoti-ng brain protection;3.Electroacupuncture preconditioning can reduce the ratio of LC3 ?/? in ischemic penumbraand inhibit autophagy for promoting brain protection.Experiment 2 Effect of Electroacupuncture Preconditioning on p53/mTOR Pathway Related Protein in Rats with Cerebral Ischemia Reperfusion InjuryObjective:To observe the effect of electroacupuncture preconditioning on expressio-n of p53/mTOR pathway-related proteins in rats with cerebral ischemia-reperfusion injury.Methods:At 24h after ischemia/reperfusion,the expression of p53,p-p53,mTOR,and p-mTOR in ischemic brain tissue was detected by immunohistochemistric-al tests and Western blotting.The effects of electroacupuncture precondition-ning on p53/mTOR pathway related proteins were observed in rats of cerebral ischemia reperfusion injury.Result:1.Immunohistochemistry:After reperfusion for 24 hours,compared with the sham group,the p53,p-p53,mTOR,p-mTOR positive cells counts in the other group were significantly increased(P<0.05).Compared with the model group,the p53,p-p53,mTOR,p-mTOR positive cell counts in the electroacupuncture preconditioning group were significantly lower(P<0.05).2.Western blotting:After reperfusion for 24 hours,the expression of p53,p-p53,mTOR,and p-mTOR protein in the other group was significantly higher than that in sham group(P<0.05).Compared with the model group,the expression of p53,p-p53,mTOR,and p-mTOR protein was significantly lower in the electroacupuncture preconditioning group(P<0.05).Conclusion:Electroacupuncture preconditioning can down-regulate the expression of p53,p-p53,mTOR,and p-mTOR in ischemic penumbra after cerebral ischemia reperfusion via regulating the p53/mTOR signaling pathway.Experiment 3 The Mechanism of p53/mTOR Pathway on Electroacupuncture Preconditioning Reduces Cerebral Ischemia Reperfusion Injury in RatsObjective:To observe how electroacupuncture preconditioning regulates apoptosis and autophagy through the p53/mTOR pathway,thus exerting brain protect-ion.Methods:90 SPF male Sprague-Dawley rats were randomly divided into sham operation group,model group,mTOR inhibitor group,electroacupuncture preconditioning group,electroacupuncture preconditioning+mTOR agonist,and electroacupuncture preconditioning+vehicle,with 12 rats in each group.Cerebral ischemia was induced by middle cerebral artery occlusion(MCAO)for 24h,followed by reperfusion.Nissl staining was used to observe the degree of neuronal damage in cerebral ischemic reperfusion injury.Immunohistochemistrical test was used to observe the expression of Beclin-1 and LC3 protein in ischemic brain tissue.Immunofluorescence test was used to observe the nuclear metastasis of p53.Western blotting was used to detect the nuclear metastasis of p-p53,nuclear p53,p-mTOR,Beclinl,Bcl-2 and Bax in the ischemia-reperfusion of rats.The mechanism of electroacupuncture preconditioning forreducing cerebral ischemia-reperfusion injury in rats through the p53/mTOR pathway was investigated.Result:1.Nissl's staining:After reperfusion for 24h,there was no significant neuronal damage in the sham group.The degree of neuron damage in the other groups was significantly higher than that in the sham group.Compared with the model group,the degree of neuronal damage was lower in the inhibitor group,electroacupuncture preconditioning group and the electroacupuncture preconditioning+vehicle group.And the degree of neuronal damage in the electroacupuncture preconditioning+agonist group was similar to that of the model group.The degree of neuronal damage in the inhibitor group and the electroacupuncture preconditioning+ vehicle group was similar to that in the electroacupuncture preconditioningroup,while the degree of neuron damage was more severe in the electroacupuncture preconditioning+agonist group than the electroacupuncture preconditioning group.2.Immunohistochemistry:After 24 hours of reperfusion,compared with the sham group,the counts of Becline-1 and LC3 positive cells were significantly increased in the other groups(P<0.05).While compared with the model group,significant differences were found in the inhibitor group,the electroacupuncture preconditioning group and the electroacupuncture preconditioning+vehicle group(P<0.05),but there was no significant difference in the electroacupuncture preconditioning+agonist group(P>0.05).Compare with the electroacupuncture preconditioning group,t no significant difference was found in the inhibitor group and the electroacupuncture preconditioning+vehicle group(P>0.05),while there was a significant difference in the electroacupuncture preconditioning+agonist group(P<0.05).3.Immunofluorescence:After 24 hours of reperfusion,compared with the sham group,the number of p53 nuclear positive cells in the model group was significantly increased(P<0.05).Compared with the model group,the number of nuclear positive cells in the inhibitor group,the electroacupuncture preconditioning group and the electroacupuncture preconditioning+vehicle group was significantly reduced(P<0.05),while there was no significant difference in the electroacupuncture preconditioning+agonist group(P>0.05).Compared with the electroacupuncture preconditioning group,the number of nucleus positive cells in the electroacupuncture preconditioning+agonists group was significantlu increased(P<0.05).4.Western blotting:Compared with the sham group,results of western blotting showed that the expression of p-p53,nuclear p53,p-mTOR,Becline-1,Bc1-2 and Bax was significantly higher in the other groups(P<0.05).Compared with the model group,the expression was significantly reduced in the inhibitor group,the electroacupuncture preconditioning group,and the electroacupuncture preconditioning+vehicle group(P<0.05),while there was no significant difference in the electroacupuncture preconditioning+agonist group(P>0.05).Compared with the electroacupuncture precondition-ing group,no significant difference was found in the inhibitor group and the electroacupuncture preconditioning +vehicle group(P>0.05),while there was significant difference in the electroacupuncture preconditioning+agonist group(P<0.05)..Conclusion:1.Electroacupuncture preconditioning can reduce neuronal damage and improve neurological function in rats with cerebral ischemia-reperfusion injury;2.Electroacupuncture preconditioning can increase the level of autop-hagy initiating protein Becline-1,down-regulate the level of autophagy marker protein LC3,affect autophagy and regulate cell apoptosis through nuclear p53;3.Electroacupuncture preconditioning can down-regulate the expression of p-p53 and p-mTOR in the ischemic penumbra,thereby increasing the apoptosis markers Bcl-2/Bax ratio,and ultimately inhibiting apoptosis and autophagy during reperfusion injuryfor promoting brain protection. |
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