The Role Of Beclin 1/Bcl-2 Complex-dependent Autophagy In Brain Ischemic Tolerance Induced By Limb Ischemic Preconditioning

Background:It was found that remote ischemic preconditioning can counteract ischemia/reperfusion injury（I/R）.Previous studies in our laboratory have confirmed that limb ischemic preconditioning（LIP）can reduce I/R-induced neuronal damage in the CA1 region of the rat hippocampus through up-regulation of autophagy,but the exact mechanisms of the protective effect of LIP on brain ischemia/reperfusion injury have not been fully elucidated.Autophagy is the process by which intracellular components are transported to lysosomes or vesicles for degradation and recirculation and can promote tolerance to biotic/abiotic stresses in organisms.Autophagy eliminates damaged or aged cells,enables cells to maintain homeostasis and survive in vivo under a wide range of stressful conditions.In this study,we used the Wistar rat four-vessel occlusion whole brain ischemia/reperfusion model to investigate the role and mechanism of autophagy in LIP-induced brain ischemic tolerance.Bcl-2 acts as an apoptosis suppressor,which promotes cell survival and inhibits cell death.Bcl-2 binds to the evolutionarily conserved autophagy protein Beclin 1 to form the Beclin 1/Bcl-2 complex,thereby inhibiting Beclin1-dependent autophagy.Objective:The present study intends to investigate whether LIP can induce brain ischemic tolerance in rats by mediating the interaction between Bcl-2 and Beclin 1 and to explore the possible mechanisms.Methods:1.The role of autophagy in LIP-induced ischemic tolerance in rat brainAnimal groups and methods:Sixty SPF grade healthy male eight-week-old Wistar rats weighing 280～320 g.The animals were randomly divided into six groups of ten animals each after two days after recovery of permanently coagulated bilateral vertebral arteries:Sham BI group,BI group,Sham LIP+BI group,LIP+BI group,PBS+LIP+BI group and 3-MA（Autophagy Inhibitor）+LIP+BI group.Five animals were taken 7 days after the last surgery and observed for neuronal cell death in the CA1 region of the hippocampus using thionin staining.Five animals were sampled 24 h after the last surgery and the expression of autophagy-related protein LC3 and Beclin 1in the CA1 region of hippocampus was detected by applying Western blot technique.2.Effect of inhibition of Bcl-2 phosphorylation on LIP-induced ischemic tolerance in rat brainAnimal groups and methods:Sixty SPF grade healthy male eight-week-old Wistar rats weighing 280～320 g.The animals were randomly divided into six groups of ten animals each after two days after recovery of permanently coagulated bilateral vertebral arteries:Sham BI group,BI group,Sham LIP+BI group,LIP+BI group,DMSO+LIP+BI group and SP 600125（JNK inhibitor）+LIP+BI group.Five animals were taken 7 days after the last surgery and observed for neuronal cell death in the CA1 region of the hippocampus using thionin staining.Five animals were sampled 24 h after the last surgery,and the expression of Bcl-2^Ser70,Bcl-2 and autophagy-related proteins LC3 and Beclin 1 in the hippocampal CA1 region was detected by applying Western blot technique.Results:1.The role of autophagy in LIP-induced ischemic tolerance in rat brainThe results of thionin staining showed that the hippocampal CA1 area in the Sham BI group had neat and dense arrangement of pyramidal cells,with2～3 layers,complete cell morphology,full nuclei,deep-stained and clear nucleoli,and no obvious delayed neuronal death（DND）.In the CA1 region of BI group,there were scattered or patchy dead neuronal cells,and neuronal density（ND）value was significantly lower（P<0.05）.The number of dead neuronal cells was significantly reduced in the LIP+BI group compared with the BI group,which showed an increase in ND（P<0.05）.Compared with the LIP+BI group,3-MA+LIP+BI group had a reduced number of surviving pyramidal neurons,severe pyramidal neuron damage,and significantly lower ND values（P<0.05）.It was shown that inhibition of autophagy blocked the protective effect of LIP on brain injury due to whole brain ischemia.Western blot showed that the expression of autophagy-associated protein Beclin 1 and LC3 in the hippocampal CA1 region of Sham BI group was low.However,the expression of Beclin 1 and LC3 was obviously increased in the BI group compared with the Sham BI group（P<0.05）.The expression of the above two proteins was further increased in the LIP+BI group compared with the BI group,which was statistically significant（P<0.05）.Pretreatment with3-MA before LIP significantly attenuated the up-regulation of Beclin 1 and LC3 by LIP.The above results suggest that LIP exerts an anti-whole brain ischemic brain injury effect through up-regulation of autophagy.2.Effect of inhibition of Bcl-2 phosphorylation on LIP-induced ischemic tolerance in rat brainThe results of thionin staining showed that there was no obvious DND in the hippocampal CA1 region pyramidal cells in the Sham BI group,neuronal cells in the hippocampal CA1 area were neatly arranged and dense,with intact cell morphology,full nuclei,darkly stained and clear nucleoli.In the CA1region of the BI group,scattered,patchy dead neuronal cells were seen,and the ND value was significantly reduced compared with that of the Sham BI group（P<0.05）.Compared with BI group,the number of dead neurons became less and the ND value increased in LIP+BI group（P<0.05）.In the SP 600125+LIP+BI group,significant histological damage was seen in the pyramidal neurons in the CA1 region,with altered cell morphology,patchy death or absence,and significant DND（P<0.05）.It was shown that inhibition of the JNK pathway blocked Bcl-2 phosphorylation,thereby reversing the protective effect of LIP on brain injury due to global brain ischemia.Western blot showed that there were small amounts of Beclin 1,LC3,Bcl-2 and Bcl-2^Ser70proteins expressed in the hippocampal CA1 region of Sham rats.Compared with the Sham BI group,the expression of Bcl-2^Ser70/Bcl-2,Beclin 1,and LC3 was significantly enhanced and Bcl-2expression was reduced in the BI group（P<0.05）.Moreover,the expression of Bcl-2^Ser70/Bcl-2,Beclin 1,LC3,and Bcl-2 was enhanced in the LIP+BI group compared with the BI group.The expression of Bcl-2^Ser70/Bcl-2,Beclin1,LC3,and Bcl-2 was enhanced in the LIP+BI group compared with the BI group.However,the above effects of LIP were attenuated in the SP 600125+LIP+BI group.These experimental results suggested that LIP can play a brain protective role by up-regulating the phosphorylation of anti-apoptotic protein Bcl-2 at the Ser-70 site and thus up-regulating autophagy.Conclusions:1.Autophagy inhibitor 3-MA partially blocked the brain protective effect of LIP,while affecting the expression of autophagy-related proteins Beclin 1and LC3,suggesting that LIP induced brain ischemic tolerance in rats through up-regulation of autophagy;2.SP 600125 blocked the phosphorylation of Bcl-2 via the JNK pathway,thereby antagonizing the brain protective effect of LIP,and affected the expression of autophagy-related proteins Beclin 1,LC3 and Bcl-2^Ser70/Bcl-2,suggesting that LIP induces brain ischemic tolerance by up-regulating autophagy through up-regulation of Bcl-2 phosphorylation at the Ser-70 site.