To Explore The Effect Of Calcitonin Gene-related Peptide On The Autophagy Level Of H9c2 Cardiomyocytes After Hypoxia/reoxygenation And Its Mechanism

Objective:1.To explore the effect of calcitonin gene-related peptide on the autophagy level of H9c2cardiomyocytes after hypoxia/reoxygenation.2.To study the effect of calcitonin gene-related peptide on the related proteins in the PI3K/AKT/mTOR signaling pathway of hypoxia/reoxygenation H9c2 cardiomyocytes.Methods:Cultivate H9c2 cardiomyocytes to the logarithmic growth phase,simulate in vivo ischemia/reperfusion of cardiac cells to establish a hypoxia/reoxygenation model of H9c2cardiomyocytes.The H9c2 cardiomyocytes in the six-well plate were randomly divided into four groups,each with three wells,and divided into blank group,normal cultured cells according to different processing factors.Hypoxia/reoxygenation group（H/R group）,H9c2cardiomyocytes were hypoxia/reoxygenated for 3h and reoxygenated for 2h to prepare the hypoxia/reoxygenation model.H/R+CGRP（CGRP group）,add 1×10^-8mol/L CGRP to the culture well for pretreatment 20 minutes before H/R.H/R group+CGRP+CGRP_8-37 group(CGRP_8-37 group)was added 1×10^-7mol/L CGRP_8-37 30min before H/R for 10min,and CGRP was added for 20min.Use CCK-8 to detect cell viability;detect LDH activity in cell supernatant;use TUNEL method to detect cell apoptosis;JC-1 was used to detect the level of mitochondrial membrane potential;Using western blot method H9c2 cardiomyocytes LC3Ⅰ,LC3Ⅱ,Beclin 1,AKT,P-AKT,mTOR,P-mTOR expression level.Results:CCK-8 detection:Compared with the blank group,the survival rate of H9c2cardiomyocytes decreased after H/R treatment（P<0.01）,and the survival rate of cardiomyocytes increased after CGRP pretreatment（P<0.01）.This effect was affected by CGRP_8-37 reversed（P<0.01）.ELISA method to detect LDH activity:Compared with the blank group,H9c2 cardiomyocyte LDH activity increased after H/R treatment（P<0.01）,and LDH activity decreased after CGRP treatment（P<0.01）.This effect was antagonized by CGRP_8-37（P<0.01）.Detection by TUNEL method:The apoptotic rate of cardiomyocytes after H/R treatment was higher than that of the blank control group（P<0.01）,and the apoptotic rate of cardiomyocytes decreased after CGRP treatment（P<0.01）.This effect was antagonized by CGRP_8-37（P<0.01）.Fluorescence probe JC-1 detection:Compared with the blank group,the mitochondrial membrane potential of the H/R group was significantly reduced（all P<0.01）.Compared with the H/R group,the CGRP group significantly inhibited H/R-induced reduction of the mitochondrial membrane potential of H9c2 cardiomyocytes（all P<0.01）.Compared with the CGRP_8-37 group,the CGRP inhibitor CGRP_8-37 can reverse the effect of CGRP on H9c2 cardiomyocytes（P<0.01）.Western Blot detection:Compared with the blank group,the expression of Beclin-1 and the ratio of LC3Ⅱ/LC3Ⅰin cardiomyocytes of the H/R group increased（P<0.01）,and the ratio of P-AKT/AKT and P-mTOR/mTOR value decreased（P<0.01）.Compared with the H/R group,Beclin-1expression and LC3Ⅱ/LC3Ⅰratio in H/R+CGRP group decreased（P<0.01）,P-AKT/AKT ratio,P-mTOR/mTOR value increased（P<0.01）,CGRP_8-37 can reverse the effect of CGRP（P<0.01）.Conclusion:CGRP may inhibit excessive autophagy by activating the AKT/mTOR pathway,increase cell survival rate,reduce cell apoptosis,and protect H/R cardiomyocyte.