The Mechanism Of Limb Ischemic Postconditioning Induced Protective Autophagy In Cerebral Ischemia-reperfusion Injury

Sources of research work:Guangxi Natural Science Fund Project NO 2016GXNSFAA380310Objection and significance:Our previous study on cerebral ischemia-reperfusion injury found that the protective mechanism of limb ischemic postconditioning may be related to activation of P38MAPK signal transduction pathways.Autophagy is an important intracellular self-catabolic process that relies on the lysosomal pathway to degrade long half-life proteins and organelles.It is an important regulator of cell survival and death.However,it’s not clear whether limb ischemic postconditioning activating autophagy to reduce cerebral ischemia-reperfusion injury.And the specific mechanism is also unclear.In this study,we established the model of focal cerebral ischemia-reperfusion in rats to explore whether the limb ischemic postconditioning can induce autophagy to protect brain,and further clarify whether JNK and AMPK/mTOR signaling pathways are the upstream pathways of autophagy after limb ischemic postconditioning.It provide a theoretical basis for the application of limb ischemic postconditioning to clinical practice.MethodsPart1:The effects of autophagy after limb ischemic post conditioning in cerebral ischemia and reperfusion injury.In this experiment,rats were randomly divided into 5 groups,which were Sham group,I/R group,LPostC group,Rapa group and 3-MA group.The neuronal damage was observed by HE staining and TTC staining.The changes of Beclin 1,LC3Ⅱ and Cathepsin B were detected by immunohistochemistry and Western blotting.Patr2:The mechanisms of JNK signal pathway-dependent autophagy in limb ischemic postconditioning.In this experiment,rats were randomly divided into 4 groups,which were Sham group,I/R group,LPostC group and sp6 group.The changes of Beclin 1,LC3Ⅱ and p-JNK were detected by Western blotting.Part3:The mechanisms of AMPK/mTOR signal pathway-dependent autophagy in limb ischemic postconditioning.In this experiment,rats were randomly divided into 4 groups,which were Sham group,I/R group,LPostC group and compound C group.The changes of Beclin 1,LC3Ⅱ,p-AMPK and p-mTOR were detected by Western blotting.ResultsPart 1:1.HE staining showed that:In I/R group,the neurons were disordered,and many neurons appeared karyopycnosis and had vacuolar change.The pathological changes of LPostC group were lighter than those in I/R group group.The pathological changes of Rapa group were more lighter than those in LPostC group,and the pathological changes of 3-MA were the most sevetity.2.TTC staining showed that:Compared with I/R group,LPostC group had small infarct size(P<0.05).Compared with LPostC group,the infarct volume was more smaller than those in Rapa group(P<0.05)and the infarct volume was larger in 3-MA group(P<0.05).3.The immunohistochemistry showed that:Compared with Sham group,the expressions Beclin 1 and CathepsinB increased in I/R group(P<0.05).Compared with I/R group,the expressions of Beclin 1 and CathepsinB increased in LPostC group(P<0.05).Compared with LPostC group,the expression of Beclin 1 and Cathepsin B were further increased in Rapa group(P<0.05)and decreased in 3-MA group(P<0.05).4.The Western blotting showed that:Compared with Sham group,the level of LC3Ⅱ,Beclin 1 and CathepsinB increased in I/R group(P<0.05).Compared with I/R group,the level of LC3Ⅱ,Beclin 1 and CathepsinB increased in LPostC group(P<0.05).Compared with LPostC group,the level of LC3Ⅱ,Beclin 1 and Cathepsin B were further increased in Rapa group(P<0.05)and decreased in 3-MA group(P<0.05).Part2:1.The Western blotting showed that:Compared with Sham group,the protein level of Beclin 1 and LC3Ⅱ were increased in I/R group(P<0.05).Compared with I/R group,the protein level of Beclin 1 and LC3Ⅱ were fuurther increased in LPostC group(P<0.05).Compared with LPostC group,the protein level of Beclin 1 and LC3Ⅱ were decreased in sp6 group(P<0.05).2.The p-JNK Western blotting showed that:Compared with Sham group,the protein level of p-JNK was increased in I/R group(P<0.05).Compared with I/R group,the protein level of p-JNK was further increased in LPostC group(P<0.05).Compared with LPostC group,the protein level of p-JNK was decreased in sp6 group(P<0.05).Part 31.The Western blotting showed that:Compared with Sham group,the protein level of Beclin 1 and LC3Ⅱ were increased in I/R group(P<0.05).Compared with I/R group,the protein level of Beclin 1 and LC3Ⅱ were further increased in LPostC group(P<0.05).Compared with LPostC group,the protein level of Beclin 1 and LC3Ⅱ were decreased in compound C group(P<0.05).2.The p-AMPK Western blotting showed that:Compared with Sham group,the protein level of p-AMPK was increased in I/R group(P<0.05).Compared with I/R group,the protein level of p-AMPK was further increased in LPostC group(P<0.05).Compared with LPostC group,the protein level of p-AMPK was decreased in compound C group(P<0.05).3.The p-mTOR Western blotting showed that:Compared with Sham group,the protein level of p-mTOR was decreased in I/R group(P<0.05).Compared with I/R group,the protein level of p-mTOR was further decreased in LPostC group(P<0.05).Compared with LPostC group,the protein level of p-mTOR was increased in compound C group(P<0.05).Conclusion1.Limb ischemic postconditioning plays a protective role in cerebral ischemia-reperfusion injury by activating autophagy.2.JNK signaling pathway is a possible pathway to activate autophagy after limb ischemic postconditioning.3.AMPK/mTOR signaling pathway is a possible pathway to activate autophagy after limb ischemic postconditioning.