Effects And Mechanism Of Remote Ischemic Post-conditioning On Autophagy And Apoptosis Of Hippocampal Neurons After Cardiopulmonary Resuscitation In Rats

Background:Cardiac arrest(CA)refers to the sudden stop of blood pumping function,which is one of the critical illnesses that seriously endanger the health and survival of people.According to statistics,more than 500,000 people in each year in China.Post-CA brain injury is an important cause of death in patients after ROSC.Autophagy refers to the process in which long-lived proteins in cells and damaged organelles are degraded by the lysosomal pathway.They are widely involved in physiological processes such as growth and development,immunity,metabolism and aging,and are ubiquitous in eukaryotic cells.Recent studies have shown that excessive activation of autophagy can lead to cell death.Apoptosis is the main pathological change in the brain after cardiopulmonary resuscitation.Remote ischemic treatment is the treatment of transient,non-fatal ischemia and reperfusion in distal organs or sites.Studies have shown that remote ischemic postconditioning can improve neurological dysfunction after cardiopulmonary resuscitation,and the relationship between its protective mechanism and autophagy needs further study.objective:To investigate the Effects and mechanism of remote ischemic post－conditioning on autophagy and apoptosis of hippocampal neurons after cardiopulmonary resuscitation in rats.Methods:①Male Sprague-Dawley rats were used to establish a model of cerebral injury after cardiopulmonary resuscitation by inducing asphyxia.②Thirty male SD rats were randomly divided into sham operation group(Sham group,n=6)and cardiac arrest cardiopulmonary resuscitation group(CA/CPR group,n=24).The hippocampus of the rats in the CA/CPR group were sacrificed at 3h,6h,24h and 72h after spontaneous circulation recovery.The LC3II/I,Beclin-1 protein and mitochondrial autophagy activity related to autophagy activity were detected by Western blot.The expression of Parkin protein was detected by immunofluorescence,and the localization of LC3 in neuronal cells was observed by transmission electron microscopy.The formation of autophagosomes and ultrastructure of neurons were observed by transmission electron microscopy.③60 male Sprague-Dawley rats were divided into sham operation group(Sham group),cardiac arrest cardiopulmonary resuscitation group(CA/CPR group),and remote ischemic postconditioning group(RIPostC group).The experimental end point was 24 h after ROSC.Long-distance ischemic postconditioning refers to the clamping of the left femoral artery with arterial clips for 5 min immediately after successful resuscitation,release for 5 min,and repeat 3 cycles.Neurological deficit scores(NDS)were performed at different time points after spontaneous circulation recovery(ROSC).LC3II/I,Beclin-1 protein expression,Parkin and P53 mitochondrial translocation were detected by Western blot,and TUNEL method was used.The apoptosis of hippocampal neurons was detected.The localization of LC3 in neurons was detected by immunofluorescence.The expression of mitochondrial P53 was detected by immunofluorescence double-labeling.The formation of autophagosomes and ultrastructure of neuronal cells were observed by transrmission electron microscopy.Results:① The expressions of LC3Ⅱ/Ⅰ,Beclin-1 and mitochondrial Parkin in hippocampal neurons at 6h,24h and 72h after cardiac arrest in rats with cardiac arrest were significantly higher than those in Sham group(P<0.05),24h after ROSC.The protein expression level was at the peak,and the immunofluorescence absorbance of brain tissue after resuscitation was higher than that of Sham group(P<0.05).②Compared with the CA/CPR group,the RIPostC group had an increased NDS score after resuscitation,decreased hippocampal neuronal apoptosis,decreased LC3Ⅱ/Ⅰ and Beclin-1 protein expression,decreased mitochondrial Parkin and P53 translocation,and neuronal cells.Autophagic vacuoles are reduced and mitochondrial ultrastructural damage is reduced.Conclusion:The results of this study indicate that cardiopulmonary resuscitation can be established 5 minutes after cardiac arrest in rats to establish a stable model of brain injury after resuscitation.The autophagy activity of hippocampal neurons increased 6 h after ROSC,and the level of autphagy(including mitochondrial autophagy)increased first and then decreased,peaked at 24h after recovery,and was still activated 72 h after resuscitation.Remote ischemic post-conditioning improves neurological function after cardiopulmonary resuscitation in rats,which may be related to inhibition of excessive autophagy in neuronal cells(including mitochondrial autophagy)and apoptosis induced by P53 mitochondrial translocation.