| Background and objective: Cardiac Arrest(CA)is one of the leading causes of death in patients worldwide,and cardiopulmonary resuscitation(CPR)is the most basic rescue technology and method for patients with cardiac arrest.Timely and effective cardiopulmonary resuscitation plays a vital role in saving patients’ lives.However,cerebral ischemia-reperfusion injury caused by cardiopulmonary resuscitation is a fundamental reason for poor prognosis and high mortality.Therefore,an in-depth study of the mechanism of brain injury after cardiopulmonary resuscitation and active exploration of brain protection drugs after resuscitation are fundamental problems that need to be resolved urgently in the clinic.Metformin is one of the primary drugs for type 2 diabetes,and more and more studies have shown neuroprotective function in recent years besides lowering blood sugar.This study explores the brain-protective effect and potential mechanism of metformin on rats after cardiopulmonary resuscitation and provides potential therapeutic targets for metformin to improve brain injury after cardiopulmonary resuscitation.Methods: In this study,electrical stimulation was used to induce cardiac arrest and cardiopulmonary resuscitation in rats.After 14 days of metformin pretreatment,the survival rate,the number of cases of recovery of autonomic circulation,the score of nerve function and the nerve injury of brain tissue were observed.The pro-inflammatory factors IL-6,TNF-a,IL-1β,and the expression of anti-inflammatory factors IL-10 and IL-4 in plasma and brain tissues in the sham group,CA/CPR group,and metformin treatment group were compared three days after cardiopulmonary resuscitation.At the same time,MDA content and SOD activity in brain tissues were detected.The TUNEL assay was used to detect the apoptosis of nerve cells in each group,and the levels of apoptosis-related proteins Bcl-2 and Bax were detected by Western blot.By detecting the level of JC-1 in the brain tissue,the changes in mitochondrial membrane potential in the brain tissue of rats in each group were reflected.Western blot and immunohistochemical methods were used to detect the levels of Grp78 and XBP-1 in the brain tissues of each group;The rats,after cardiopulmonary resuscitation,were treated with endoplasmic reticulum stress inhibitor combined with metformin.Western blot was used to detect Grp78 and CHOP protein expression,and the 7-day survival rate and NDS score were calculated.The rats in the CA/CPR group,metformin group,metformin group+ autophagy inhibitor group,and metformin +AMPK inhibitor group were treated after cardiopulmonary resuscitation.Western blot was used to detect the expression of autophagy protein LC3,P62,and AMPK signaling pathway,and the 7-day survival rate and NDS scores were calculated.Results: The rat models of cardiac arrest and cardiopulmonary resuscitation were successfully established by electrical stimulation.Compared with the CA/CPR group,the survival rate and the number of recovery cases of spontaneous circulation after cardiopulmonary resuscitation treated with metformin were significantly increased,and the neurological function score was significantly increased.In addition,the number of apoptotic and necrotic cells in brain tissues treated with metformin was significantly reduced,and the brain tissue injury was significantly inhibited.Compared with the CA/CPR group,the levels of pro-inflammatory factors IL-6,TNF-ɑ,and IL-1β in serum and brain tissues of rats treated with metformin were reduced after cardiopulmonary resuscitation,while the levels of anti-inflammatory factors IL-10 and IL-4 were increased.In addition,after metformin treatment,the production of MDA in brain tissues of the cardiopulmonary resuscitation rats was significantly decreased,while SOD activity was significantly increased.Compared with CA/CPR group,the percentage of apoptotic cells in the metformin group decreased,while the expression of Bcl-2 was significantly increased,and the expression of Bax decreased.After treatment with metformin,the mitochondrial membrane potential in rat brain decreased after cardiopulmonary resuscitation,indicating that metformin could inhibit mitochondrial damage after cardiopulmonary resuscitation.Compared with CA/CPR group,metformin could inhibit the expression of Grp78 and XBP-1 in rats after cardiopulmonary resuscitation.After treatment with an endoplasmic reticulum stress inhibitor combined with metformin,the expression changes of GRP78 and XBP-1 induced by metformin could be further downregulated.At the same time,compared with metformin alone,metformin combined with an endoplasmic reticulum stress inhibitor could significantly improve the survival rate and neurological function of rats.Compared with the control group,the expression level of autophagy-related gene LC3 II increased significantly after metformin treatment,while the level of P62 protein decreased,indicating that metformin treatment might promote autophagy after cardiopulmonary resuscitation.The treatment of autophagy inhibitors might inhibit the protective effect of metformin on the survival rate and neurological function of rats after cardiopulmonary resuscitation.Compared with the control group,metformin treatment could significantly activate the AMPK signaling pathway,and the addition of an AMPK signal inhibitor in the metformin treatment group could significantly inhibit the expression of autophagy-related genes.In addition,compared with the metformin alone,the survival rate and neurological function score of rats in the metformin combined with the AMPK inhibitor group markedly decreased,indicating that metformin could promote autophagy via the AMPK signaling pathway and has a protective effect on rat brain tissues after cardiopulmonary resuscitation.Conclusion: Metformin exerts a protective effect on the brain of rats after cardiopulmonary resuscitation.Metformin might inhibit brain injury after cardiopulmonary resuscitation by inhibiting inflammatory reaction,oxidative stress process,neuronal apoptosis,mitochondrial damage,endoplasmic reticulum stress,and activating the AMPK signal pathway to promote autophagy,providing a potential basis for metformin in the clinical treatment of brain injury after cardiopulmonary resuscitation. |
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