| Background : Acute myocardial infarction(AMI)is the leading cause of death worldwide.For patients with acute myocardial infarction,early revascularization can reduce myocardial injury and infarct area.But reperfusion itself will lead to further aggravation of myocardial injury,Which is called myocardial ischemia/reperfusion injury(MI/RI).Many studies have shown that glucagon-like peptide-1 receptor agonists Exendin-4 and autophagy play important roles in reducing myocardial ischemia/reperfusion injury,but the relationship between them is still unclear.Objective:To explore the relationship between autophagy,Exendin-4 and hypoxia/reoxygenation injuryMethods:H9C2 cells were cultured in vitro.The cell viability was detected by CCK8 method,and three groups of pre-experiments were carried out.Firstly,the experimental model of hypoxia / reoxygenation was established,in which the H9C2 cells were treated with hypoxia for 6 hours,and the reoxygenation time was determined by detecting the cell viability at different times of reoxygenation(0 h,1 h,2 h,4 h).Then,the effects of different drug concentrations of Exendin-4(12.5n M,25 n M,50 n M,100 n M,200 n M)on normal cardiomyocytes were detected,and whether Exendin-4 was toxic to normal H9C2 cells was observed.Finally,the best concentration of Exendin-4to reduce the damage of H/R was detected(12.5n M,25 n M,50 n M,100 n M,200 n M).After pre-experiment,Western blot was used to detect the effect of Exendin-4 on the expression of autophagy related protein LC3 II in different times of reoxygenation(0h,1h,2h,4h)to preliminary verify the relationship between Exendin-4 and autophagy.Then autophagy inhibitor chloroquine(CQ)was added,and the cells were divided into normal control group,H/ R group,H/R+ Ex-4 group and H/R+Ex-4+CQ group.The expression of autophagy related proteins P62,LC3 II and LAMP-2 were detected by Western blot to investigate whether Exendin-4 regulated autophagy flux.Finally,intracellular reactive oxygen species(ROS)level was detected by flow cytometry,cell viability was detected by CCK8,LDH release level was detected by LDH kit,the expression of apoptosis-related proteins Bcl-2 and Bax were detected by Western blot to verify whether Exendin-4 postconditioning plays a protective role in regulating autophagy flux in cardiomyocyte hypoxia / reoxygenation injury.Results:(1)Compared with the normal control group,the cell viability was significantly decreased in H/R group,and further decreased with the extension of reoxygenation time(P < 0.05).(2)Compared with the normal control group,there were no significant differences in the viability of cardiomyocytes treated with different concentrations of Exendin-4 for 4hours(P > 0.05).(3)Compared with the normal control group,the cell viability of H/R group was decreased(P < 0.05).There was no significant difference in cell viability between H/R+12.5n M Ex-4 group and H/ R group(P > 0.05).With the increase of dose,the cell viability increased gradually and reached the peak in H/R+50n M Ex-4 group(P <0.05).However,with the further increase of the dose,the cell viability did not increase(P > 0.05).(4)After cardiomyocytes were treated with Exendin-4 at different periods of reoxygenation,the expression of autophagy-related protein LC3 II increased at first and then decreased.(5)Compared with the normal control group,the expression of autophagy-related proteins LC3 II,P62 were increased and the expression of LAMP-2 protein was decreased in H/R group(P < 0.05).Compared with H/R group,the expression of LC3 II,P62 in H/R+Ex-4 group were decreased and the expression of LAMP-2 was increased(P < 0.05).There were no significant differences in the expression of autophagy related proteins between H/R+Ex-4+CQ group and H/R group(P > 0.05).(6)Compared with the normal control group,the ROS level was increased,cell viability was decreased and LDH release level was increased(P < 0.05),the expression of pro-apoptotic protein Bax was increased,and the expression of anti-apoptotic protein Bcl-2 was decreased in H/R group(P < 0.05).Compared with H/R group,the ROS level was decreased,cell viability was increased and LDH release level was decreased(P < 0.05),the expression of Bax was decreased,and Bcl-2 was increased in H/R+Ex-4 group(P < 0.05).There were no significant differences in ROS level,cell viability,LDH release level and the expression of apoptosis-related proteins between H/R+Ex-4+CQ group and H/R group(P > 0.05).Conclusion :(1)Autophagy was involved in the effect of Exendin-4 on hypoxia /reoxygenation injury of cardiomyocytes.(2)Exendin-4 not only promoted the formation of autophagosome,but also promoted the fusion of autophagosome and lysosome,repaired the damaged autophagy flux,and reduced the hypoxia /reoxygenation injury of myocardial cells. |
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