| Background: Cardiac arrest(CA)remains a difficult problem in clinical practice.Although international guidelines on cardiopulmonary resuscitation(CPR)have been constantly updated and the scientific research on resuscitation has made continuous progress,Post–resuscitation myocardial dysfunction is one of the main reasons for low in-hospital survival rate after CPR.Pathogenesis of post-resuscitation myocardial dysfunction is still controversial.Animal CA modle experiments suggested that apoptosis mediated by ER stress may be one of the main pathological mechanisms of post-resuscitation myocardial injury.Endoplasmic reticulum stress is often accompanied by autophagy.The role of autophagy in ischemia reperfusion injury is still controversial.It is generally showed that ischemia-induced autophagy is beneficial in the early stage of ischemia reperfusion,while overinduction is harmful in the reperfusion stage.However,some studies have proposed that increased autophagosome formation in the reperfusion stage is not due to overinduction,but due to impaired autophagosome clearance.Mild hypothermia therapy has been shown to improve survival and reduce the risk of neurological dysfunction after CA-CPR,but whether mild hypothermia has a protective effect on cardiac function after cardiopulmonary resuscitation and its mechanism remains unclear.Objective: To determine the protective effect of mild hypothermia on cardiac function after CPR,the effects of mild hypothermia on apoptosis of cardiomyocytes after resuscitation,endoplasmic reticulum stress and autophagy flux,the mechanism of autophagy flux and endoplasmic reticulum stress and their relationship in the protective effect of mild hypothermia on cardiac function after CPR,and the possible signal pathways involved.It provides a new target and a new idea for the prevention and treatment of mild hypothermia to improve post-resuscitation myocardial dysfunction.Methods: A total of 56 pigs were randomly divided into 7 groups: sham group,normothermia group,mild hypothermia group,autophagy inhibitor chloroquine(CQ)+ normothermia group,mild hypothermia +CQ group,normothermia + endoplasmic reticulum stress inducer chlamycin(TN)group,and mild hypothermia +TN group.A CA model of 8-minute ventricular fibrillation was established in pigs.Pigs in the mild hypothermia group received mild hypothermia treatment with a target temperature of33℃ after ROSC and were rewarmed after maintained for 12 h.Hemodynamic indexes were monitored in pigs at 0 h、0.5 h、6 h、12 h and 24 h after ROSC,and the level of myocardial marker c Tn I was detected at 0 h、0.5 h、6 h、12 h and 24 h after ROSC.The left ventricular ejection fraction of the heart at 0 h、0.5 h、6 h and 24 h after ROSC was detected by ultrasonic Doppler.All the animals were sacrificed 24 h after ROSC and the myocardial tissue was collected.The pathological changes of myocardial tissue in different groups were observed by transmission electron microscopy.The apoptosis of myocardial tissue was detected by TUNEL;the protein expression levels of apoptosis related proteins Caspase-3,Bax and Bcl-2,the protein expression levels of endoplasmic reticulum stress indicators GRP78,CHOP and Caspase-12,the protein expression levels of autophagy related indicators LC3-II,p62 and LAMP2 and their regulatory signaling pathways,and the protein expression levels of phosphorylated AMPK and phosphorylated S6 were detected using western blotting.Results: 1.All pigs in each group were resuscitated successfully without death;at 24 h after ROSC,and 7 pigs survived in the mild hypothermia group with a survival rate of87.5%,6 pigs survived in the mild hypothermia group with a survival rate of 75%.The survival rate in the mild hypothermia group was increased by 12.5% without statistical significance(P=0.60).2.Compared with normothermia group,pigs in the mild hypothermia group had a significantly lower level of troponin I and significantly increased cardiac ejection fraction 12 h after ROSC;morphological changes of myocardial ultrastructure were significantly improved after 24 h of ROSC.3.At 24 h after ROSC,apoptosis ratio of cardiomyocytes in the mild hypothermia group was significantly lower than that in the normal temperature group,whereas the protein levels of pro-apoptotic proteins Caspase-3 and Bax were significantly decreased,and the protein expression level of anti-apoptotic protein Bcl-2 was up-regulated.4.At 24 h after ROSC,the protein expression levels of ER stress indicators GRP78,CHOP and Caspase-12 in the mild hypothermia group were all decreased compared with the normal temperature group,whereas the proportion of cell apoptosis was increased,the protein levels of Caspase-3 and Bax were significantly decreased,and the protein expression level of Bcl-2 was up-regulated.Compared with the mild hypothermia +TN group,the protein expression levels of GRP78,CHP and Caspase-12 were all upregulated,and the proportion of cell apoptosis was increased,the protein levels of Caspase-3 and Bax were significantly decreased,and the protein expression level of Bcl-2 was up-regulated.5.At 24 h after ROSC,the protein expression levels of LC3-II and p62 were significantly decreased in the mild hypothermic group compared with the normal temperature group,whereas the protein expression level of LAMP2 was upregulated.Meanwhile,the proportion of cell apoptosis was decreased,the protein levels of Caspase-3 and Bax were significantly decreased,and the protein expression level of Bcl-2 was up-regulated.6.The protein expression levels of GRP78,CHOP and Caspase-12 in the mild hypothermia +CQ group were significantly increased compared with the mild hypothermia group at 24 h after ROSC.The protein levels of LC3-II in the mild hypothermia +TN group and normothermia +TN group were significantly increased compared with the mild hypothermia group and normothermia group,respectively.7.P-AMPK/AMPK was significantly increased while p-S6/S6 was significantly decreased in mild hypothermia group 24 h after ROSC compared with normothermia group.Conclusion: 1.Mild hypothermia can inhibit myocardial cell apoptosis after CPR,reduce myocardial injury,and improve myocardial dysfunction after resuscitation.2.Mild hypothermia can play a cardiac protective role by inhibiting endoplasmic reticulum stress-mediated apoptosis of cardiomyocytes after CPR.3.Autophagy flux is damaged and autophagosome clearance function is decreased after CPR.Mild hypothermia can inhibit myocardial apoptosis by repairing the damaged autophagy flux and play a cardiac protective role.4.Endoplasmic reticulum stress after CPR can induce autophagy,but does not damage autophagy flux.Autophagy has a negative feedback effect on ER stress,and part of the mechanism of mild hypothermia alleviating ER stress after CPR is related to the repair of damaged autophagy flux.5.Mild hypothermia can up-regulate AMPK signal transduction pathway after CPR,and the mechanism by which mild hypothermia improves post-resuscitation myocardial dysfunction may be related to the up-regulation of AMPK signal transduction pathway.The mechanism of mild hypothermia to repair damaged autophagy flux may be related to the activation of AMPK signaling pathway. |
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