The Protective Effect Of Metformin On Myocardial Injury After Cardiac Arrest/Cardiopulmonary Resuscitation In Rats Through Activation Of AMPK Pathway

Objective: To investigate the protective effect of metformin(Met)preconditioning on myocardial injury after cardiac arrest/cardiopulmonary resuscitation(CA/CPR)and its possible mechanism.Methods: A total of 70 healthy adult male SD rats were divided into 5 groups according to random number table: Normal group(Sham group,n=10),Model group(CA group,n=25),Met group(n=25),Met+AMPK inhibitors Compound C(Met+CC group,n=5)and Met+Autophagy flow inhibitors chloroquine(Met+CQ group,n=5).The Met group,Met+CQ group and Met+CC group were gavaged with200mg/kg metformin once a day for 14 days,and the Sham group and the CA group were gavaged with the same amount of normal saline for consecutive 14 days.The Met+CC group and Met+CQ group were intraperitoneally injected with CC25mg/kg and CQ25mg/kg 1h before modeling,respectively.The control group was given the same amount of normal saline in the same way.The Sham group only received mechanical ventilation without CA/CPR induction,and the other 4 groups received mechanical ventilation and CA/CPR induction.24 h after ROSC,5 rats in each group were put to death.The contents of myocardial troponin I(c Tn I),creatine kinase isoenzyme MB(CK-MB)and lactate dehydrogenase(LDH)in plasma were determined by enzyme-linked immunosorbent assay(ELISA).The pathological changes of left ventricular tissue were observed by hematoxylin-eosin(HE)staining.The changes of cardiac tissue structure and the formation of autophagosomes were observed by transmission electron microscopy.The protein expressions of p62,LC3II/I,p-AMPK and AMPK in the myocardial texture of rats were determined by Western blotting.Results:(1)The cumulative survival rate at 7 days was 40%(8/20)in the CA group and 75%(15/20)in the Met group(P=0.025).(2)24h after ROSC,serum levels of c Tn I,CK-MB and LDH in CA group were significantly higher than those in Sham group(P<0.05),while those in Met group were lower than those in CA group(P<0.05).(3)After successful modeling,blood pressure was stable in the Sham group,and the mean arterial pressure(MAP)of the other groups at 0.5h,1h,2h,4h and 6h decreased compared with the Sham group,with statistical significance(P < 0.05).The blood pressure measured at 4h and 6h in the Met group was higher than that in the CA group,with statistical difference(P < 0.05).(4)There were statistical differences in LVEF,LVFS,LVDD,LVDS,LVEDV and LVESV in all groups compared with Sham group(P < 0.05).Compared with CA group,LVEF,LVFS,LVDS and LVESV in Met group were increased(P < 0.05),while LVDD and LVEDV were not significantly different(P > 0.05).(5)The myocardial tissue structure of the Sham group was complete,with no obvious swelling of myocardial cells and interstitium,and no obvious pathological changes;In the CA group,the myocardial tissue texture was blurred,the stroma was swollen,the myocardial cells had vacuolar changes,and the nuclear morphology was irregular and accompanied by the disappearance of some nuclei.The myocardial cell texture of Met group was still clear,and the degree of interstitial swelling and cell destruction was less than that of CA group.The myocardial tissue of Met+CC group and Met+CQ group had different degree of disorder,and the nuclear morphology was not regular.(6)The ultrastructure of the myocardium in the Sham group was normal,the moistening disc was clearly visible,the mitochondria were arranged neatly,no obvious swelling was observed,and the myofilaments were complete;The mitochondria of cardiomyocytes in CA group were obviously swollen and disordered,myofilaments dissolved and broken,and autophagosomes were also formed.The degree of mitochondrial swelling in Met group was less than that in CA group,and the formation of autophagosomes increased significantly.The above cells in Met+CC and Met+CQ groups were worse than those in Sham group.(7)LC3II/I protein expression in all groups was higher than that in Sham group(P < 0.05),and that in Met and Met+CC groups was higher than that in CA group(P < 0.05).The expression of p62 protein in CA group,Met group and Met+CC group was lower than that in Sham group(P < 0.05),Met group was lower than that in CA group,Met+ CQ group was higher than that in CA group(P < 0.05).The protein expression of p-AMPK/AMPK in all groups was higher than that in Sham group(P < 0.05),and that in Met group and Met+CC group were higher than that in CA group(P < 0.05).Conclusion: Metformin preconditioning has a protective effect on myocardial injury after CA/CPR,and this protective effect may be related to the activation of AMPK pathway mediated autophagy.