Background: Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive. Methods: The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3Ⅱ/I, TNF-α, IL-1β, etc., were detected under normal or Drp1 interference conditions.Results: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P<0.05). However, the high cytoplasmic LC3Ⅱ/I ratio and increased aggregation of p62 could be reversed by 44％ and 88％ by Drp1 short hairpin RNA (shRNA) (P<0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P<0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75％ to a low LC3Ⅱ/I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P<0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P>0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4–5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P<0.05). Furthermore, TNF-α and IL-1β increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen–glucose deprivation/reoxygenation (OGD/R) conditions (P<0.05). Conclusions: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.

Xue Zeng;Yun-Dong Zhang;Rui-Yan Ma;Yuan-Jing Chen;Xin-Ming Xiang;Dong-Yao Hou;Xue-Han Li;He Huang;Tao Li;Chen-Yang Duan

Department of Anaesthesiology,the Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China;Department of Neurology,the Third Affiliated Hospital of Chongqing Medical University,Chongqing 401120,China;Department of Cardiovascular Surgery,Xinqiao Hospital,Army Medical University,Chongqing 400037,China;State Key Laboratory of Trauma,Burns and Combined Injury,Department of Shock and Transfusion,Daping Hospital,Army Medical University,Chongqing 400042,China

军事医学研究（英文）

[1]Xue Zeng;Yun-Dong Zhang;Rui-Yan Ma;Yuan-Jing Chen;Xin-Ming Xiang;Dong-Yao Hou;Xue-Han Li;He Huang;Tao Li;Chen-Yang Duan-.Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis)[J].军事医学研究（英文）,2022(06):668-685

aggresome,cristae,Undegraded,extracellularly

Activated,Drp1,regulates,p62,mediated,autophagic,flux,aggravates,inflammation,cerebral,ischemia,reperfusion,via,ROS,RIP1,RIP3,axis,Background,Cerebral,injury,CIRI,refers,secondary,brain,that,can,occur,when,blood,supply,ischemic,tissue,restored,However,mechanism,underlying,such,remains,elusive,Methods,male,C57,mice,underwent,middle,artery,occlusion,MCAO,Among,them,were,further,treated,Mitochondrial,division,inhibitor,Mdivi,acetylcysteine,NAC,SH,SY5Y,cells,cultured,glucose,medium,hypoxic,conditions,then,transferred,normal,Then,flow,mitochondrial,structure,mtDNA,copy,number,intracellular,reactive,species,expression,profiles,cardiac,length,density,content,well,Ser616,LC3,etc,detected,interference,Results,levels,elevated,by,times,after,high,cytoplasmic,increased,aggregation,could,reversed,short,hairpin,shRNA,fluorescence,intensity,phosphorylation,induced,attenuated,scavenger,Necrostatin,enhanced,RFP,activation,In,addition,although,production,caused,considerable,accumulation,autophagosomes,packaging,secretion,exosomes,containing,which,decreased,Furthermore,derived,deprivation,reoxygenation,OGD,Conclusions,activated,accelerated,formation,while,inhibiting,transition,autolysosomes,pathway,secreted,leading,inflammatory,cascades,damaged,resulting,excessive,generation,blockage,degradation,triggering,vicious,cycle

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