TOM7 Silencing Exacerbates Focal Cerebral Ischemia Injury In Rat By Targeting PINK1/Beclin-1-Mediated Autophagy

Background: TOM7 is a component of the protein translocase outer mitochondrial membrane(TOM)complex.TOM complex is essential for cell survival and forms the main entry gate into mitochondria for most mitochondrial proteins.TOM7 may destabilize the interactions of the TOM complex and the TOM receptors,which may be affect the insertion of proteins into the outer membrane.However,an understanding of how TOM7 affects cerebral ischemia injury is limited.PINK1-mediated autophagy plays an important role in preventing cerebral ischemia injury.Objective: To determine whether TOM7 affects cerebral ischemia injury by regulating autophagy through the PINK1/Beclin-1 signaling pathway.Methods:(1)Focal cerebral ischemia was induced by photothrombotic cerebral ischemic model in rats.The rats were successively perfused transcardially with 0.9% sodium chloride and decapitated at 1d,3d,5d,7d,and the total protein was extracted from peri-infarct cortical tissues.Western blot was used to detect the expression of TOM7 after PT.(2)Three TOM7 siRNAs(si-TOM7-1,si-TOM7-2,si-TOM7-3)and one scramble siRNA were designed and chemically synthesized.TOM7 siRNAs were injected into the right lateral cerebral ventricle 48 hours before the photothrombotic stroke model.Western blot was used to detect transfection efficiency.(3)With TOM7 interference,neurological deficits,infarct volume,and histological assessment were measured to detect the cerebral injury.(4)With TOM7 interference,autophagosome and the expression of autophagy related proteins Beclin-1,p62 and LC3 after cerebral ischemia in rats were detected.(5)The PT model with PINK1 activated after TOM7 interference was set up.Recombinant protein of PINK1 was injected into the right lateral cerebral ventricle 24 hours before building the models.Then,the expression of autophagy related proteins Beclin-1,p62 and LC3 after cerebral ischemia in rats were detected.Results:(1)The expression of TOM7 was increased after PT stroke as compared to the sham group,and peaked around 3 days(p < 0.001).(2)The TOM7 expression level was significantly decreased by TOM7 siRNA and the most effective siRNA of TOM7 was siRNA-3(P <0.001).(3)Compared with the PT group and scramble group,the scores in the group with the TOM7 siRNA showed an increase,the cerebral infarction volume increased obviously and the nerve cell injury was aggravated.(4)After cerebral ischemia,following TOM7 knockdown,autophagosomes were observed in the PT and scramble groups,but not observed in the TOM7 siRNA group.Compared with the PT group and scramble group,Beclin1 and LC3?/? ratio were decreased and p62 was increased in TOM7 siRNA.(5)Compared with the TOM7 siRNA group,the expression of Beclin1 and LC3?/? ratio were increased and p62 was decreased after the recombinant protein of PINK1 administrated.Conclusions:(1)The expression of TOM7 was increased after PT stroke and may exhibit neuroprotective effects against cerebral ischemia injury.(2)TOM7 affects cerebral ischemia injury by regulating autophagy through the PINK1/Beclin-1 signaling pathway.