Role Of Circadian Rhythm Gene Clock After Cerebral Ischemia-reperfusion Injury In Rats

Objective: Ischemic stroke is a central nervous system disease at high risk of death and disability.The pathogenesis of ischemic stroke is complicated,and vascular recanalization in the acute phase of ischemic stroke often causes cerebral ischemiareperfusion injury.Clinical studies have shown that the incidence and prognosis of ischemic stroke at different time points are significantly different,which is closely related to the circadian rhythm.The molecular biological basis for generation and maintenance of circadian rhythm in living organisms is the circadian gene.Circadian genes are involved in the physiological and pathological processes of the body,and regulate endoplasmic reticulum stress,cell apoptosis,and cell autophagy,affecting the occurrence and development of central nervous system diseases(Alzheimer’s disease,stroke,and Parkinson’s disease).Studies have shown that the role of circadian rhythm in ischemic cerebrovascular disease is related to the regulation of circadian genes,but the data of its specific mechanisms are still scarce.This study intends to observe the effects of the circadian Clock(Circadian Locomotor Output Cycles Kaput)after focal cerebral ischemia-reperfusion in rats,and explore the effect of rhythm factors on the function of ischemic nerve cells via the endoplasmic reticulum stress pathway from the perspective of proteomics and apoptosis,thus provide a new evidence for the pathogenesis of cerebral ischemia-reperfusion injury.Methods: The rat middle cerebral artery occlusion/reperfusion model was constructed at four different Zeitgeber time points(ZT0,ZT6,ZT12,ZT18;6:00,12:00,18:00,24:00)to simulate focal cerebral ischemia-reperfusion injury conditions in vivo.The expression level of Clock gene was observed by immunoblotting;the infarct volume was assessed by using TTC staining;neurological deficits were evaluated by using Longa score;neuronal apoptosis was evaluated by using TUNELDAPI staining;differential proteome expression at different times was detected by using two-dimensional electrophoresis gel(2D-PAGE)combined mass spectrometry(MALDI TOF/TOF MS);prediction of possible downstream signaling pathway was analyzed by bioinformatics;the expression of downstream endoplasmic reticulum stress and mitochondrial apoptotic pathway-related protein were detected by immunoblot.Results: After the cerebral ischemia-reperfusion injury,Clock expression in the ischemic cortex was decreased at ZT0(6:00)and increase at ZT12(18:00)(p <0.05).Compared with ZT0,infarct size was decreased at ZT12(p <0.01).Compared with ZT0,the neurological deficit score was decreased at ZT18(p <0.05).Compared with ZT6,the number of intact neurons was increased at ZT18(p <0.05).Compared with ZT0,DNA fragments were reduced at ZT12(p <0.0001)and ZT18(p <0.001).Compared with ZT0,the endoplasmic reticulum stress protein ATF4 was decreased at ZT12(p<0.05)and ZT18(p <0.05).A total of 749 differentially expressed proteins were detected by 2D-PAGE combined with MALDI TOF / TOF MS between ZT0 and ZT18(p <0.05),and 8 of them were successfully identified.Compared with ZT0,PP1 B,TTHY,FAHD1 increased,and VDAC1,Q5M7T6,Q5XIJ3,VATB2,A0A0G2JWJ4 decreased at ZT18.Their signaling pathways involve duct acid secretion,metabolic pathways,synaptic vesicle circulation,and endoplasmic reticulum stress pathway.Conclusion: The severity of the cerebral ischemia-reperfusion injury exhibits a time-dependent pattern,which possible mechanism is that the circadian gene Clock affects the severity of apoptosis of ischemic neurons via endoplasmic reticulum stress pathway after cerebral ischemia-reperfusion.