Objective Autophagy was prominently activated by cerebral ischaemia.This study was to investigate the exact role of autophagy in ischaemic stroke.Methods Two rat models of transient middle cerebral artery occlusion(tM...Objective Autophagy was prominently activated by cerebral ischaemia.This study was to investigate the exact role of autophagy in ischaemic stroke.Methods Two rat models of transient middle cerebral artery occlusion(tMCAO)and permanent MCAO(pMCAO)were prepared.The brain tissues in the penumbra were obtained to observe the dynamic variations of autophagy activity with Beclin1 and LC3 antibodies by Western blotting.At the characteristic time points,when autophagy activity was markedly elevated or reduced,the autophagy activation signaling was intervened with rapamycin and 3-methyladenine,respectively.Thereafter,key proteins in the autopahgic/lysosomal pathway were detected with the antibodies of LC3,p62,ubiquitin,LAMP-1 and cathepsin B.Meanwhile,TTC staining,neurological score and immunofluorescence were performed to evaluate brain infarct volume,neurological deficit and neuron survival,respectively.Results Both Beclin1 and LC3 expression levels were remarkably altered at 6 h,12 h,2 days and 7 days after tMCAO.Interestingly,the dynamic changes of autophagy activity following pMCAO were identical to those after tMCAO.Neither autophagy induction nor autophagy inhibition was able to ameliorate the pMCAO-induced neurological injury due to lysosomal dysfunction,as indicated by low levels of LAMP-1 and cathepsin B,accompanied with the accumulation of LC3-II,ubiquitin and insoluble p62.Comparatively,autophagy induction elicited overt neuroprotection at 2 and 7 days after tMCAO,and this neuroprotection might be elicited by the enhancement of autophagy flux.Conclusion Our study suggests that autophagy confers neuroprotection at the subacute phase of tMCAO but has few effects on neurological outcomes after pMCAO.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.81960418 and No.81860411)the Yunnan Ten Thousand Talents Plan Young&Elite Talents Project(No.YNWR-QNBJ-2018-034)+2 种基金the Yunnan Applied Basic Research Projects Fund of Yunnan Provincial Department of Science&Technology(No.2019FB113 and No.202001AT070049)the Science Research Fund of Yunnan Provincial Department of Education(No.2018JS016 and No.2020J0066)the Doctoral Foundation of Kunming University of Science and Technology(No.KKSY201960010).
文摘Objective Autophagy was prominently activated by cerebral ischaemia.This study was to investigate the exact role of autophagy in ischaemic stroke.Methods Two rat models of transient middle cerebral artery occlusion(tMCAO)and permanent MCAO(pMCAO)were prepared.The brain tissues in the penumbra were obtained to observe the dynamic variations of autophagy activity with Beclin1 and LC3 antibodies by Western blotting.At the characteristic time points,when autophagy activity was markedly elevated or reduced,the autophagy activation signaling was intervened with rapamycin and 3-methyladenine,respectively.Thereafter,key proteins in the autopahgic/lysosomal pathway were detected with the antibodies of LC3,p62,ubiquitin,LAMP-1 and cathepsin B.Meanwhile,TTC staining,neurological score and immunofluorescence were performed to evaluate brain infarct volume,neurological deficit and neuron survival,respectively.Results Both Beclin1 and LC3 expression levels were remarkably altered at 6 h,12 h,2 days and 7 days after tMCAO.Interestingly,the dynamic changes of autophagy activity following pMCAO were identical to those after tMCAO.Neither autophagy induction nor autophagy inhibition was able to ameliorate the pMCAO-induced neurological injury due to lysosomal dysfunction,as indicated by low levels of LAMP-1 and cathepsin B,accompanied with the accumulation of LC3-II,ubiquitin and insoluble p62.Comparatively,autophagy induction elicited overt neuroprotection at 2 and 7 days after tMCAO,and this neuroprotection might be elicited by the enhancement of autophagy flux.Conclusion Our study suggests that autophagy confers neuroprotection at the subacute phase of tMCAO but has few effects on neurological outcomes after pMCAO.