Mechanism Of Liraglutide Promoting Autophagy To Alleviate Myocardial Ischemia-reperfusion Injury In Diabetic Mice

Objective:To investigate the protective effect and molecular mechanism of liraglutide on alleviating myocardial ischemia-reperfusion injury in in diabetic mice.Methods:1.C57BL/6 mice were randomly divided into four groups,control group（Con）,diabetes mellitus group（DM）,diabetic mellitus ischemia-reperfusion group（DM+I/R）,liraglutide intervention group（DM+I/R+Lir）.Mice diabetes model was established by intraperitoneal injection of 30mg/kg streptozotocin combined with high-fat diet.200μg·kg^-1 liraglutide was injected subcutaneously.After 4 weeks intervention,myocardial ischemia-reperfusion model was established,mice body weight and fasting blood glucose were measured.Cardiac function was measured by animal ultrasound imaging system.The myocardial infarction area was detected by Evans blue and TTC staining.Myocardial cell morphology was observed by HE staining.Western blot and immunofluorescence were used to detect the expressions of autophagy related proteins LC3 A/B and p62 in myocardial tissue,and Western blot was used to detect the expressions and phosphorylation of AMPK and m TOR in AMPK/m TOR signaling pathway.2.In vitro experiment,rat cardiomyocytes（H9C2）were divided into 6 groups,control group（Con）,high glucose group（HG）,high glucose hypoxia/reoxygenation group（HG+H/R）,Liraglutide intervention group（HG+H/R+Lir）and liraglutide+Compound C group（HG+H/R+Lir+CC）and Liraglutide+rapamycin group（HG+H/R+Lir+Rap）.H9C2 cells were cultured in 25m M high-glucose medium to establish a high-glucose model.Cells were treated with AMPK inhibitor Compound C or m TOR inhibitor rapamycin,respectively.200n M liraglutide was added.Cell hypoxia/reoxygenation injury model was established by cell hypoxia apparatus.CCK-8 kit was used to detect the viability of H9C2 cells.Western blot and immunofluorescence were used to detect the expressions of autophagy related proteins LC3A/B and p62 in H9C2cells.Western blot was used to detect the expressions and phosphorylation of AMPK and m TOR proteins in AMPK/m TOR pathway.Results:1.The effects of liraglutide on blood glucose and body weight in mice and its protective effect on myocardial ischemia-reperfusion injury.（1）Compared with the Con group,the blood glucose of mice in the DM group was increased[（7.05±0.20）vs（9.21±0.42）,P<0.05],indicating that the diabetes model of mice was successfully established.After liraglutide intervention,compared with DM+I/R group,DM+I/R+Lir group decreased blood glucose[（9.93±0.16）vs（8.56±0.20）,P<0.05]and body weight[（32.48±0.75）vs（28.89±0.61）,P<0.05].The results showed that liraglutide could reduce blood glucose and body weight in diabetic ischemia-reperfusion mice.（2）The results of mice cardiac function test showed that compared with DM+I/R group,LVEF[（33.71±3.48）vs（52.20±3.30）,P<0.05]in liraglutide intervention group was increased.LVESD[（3.42±0.13）vs（2.55±0.12）,P<0.05]and LVESV[（48.11±1.96）vs（23.73±2.71）,P<0.05]decreased,indicating that liraglutide can improve the cardiac function of mice with diabetic ischemia-reperfusion injury.（3）Myocardial infarction size test results showed compared with DM+I/R group,the myocardial infarction area in DM+I/R+Lir group was significantly decreased[（67.18±3.81）%vs（32.63±14.92）%,P<0.05].The results showed that liraglutide could reduce myocardial infarction size in mice with diabetic ischemia-reperfusion injury.（4）Western Blot analysis showed that compared with DM+I/R+Lir group,LC3Ⅱ/Ⅰratio increased and p62 protein expression decreased in DM+I/R+Lir group than those in DM+I/R group,indicating that liraglutide could promote myocardial autophagy in mice with ischemia-reperfusion injury.The p-AMPK/AMPK ratio increased and p-m TOR/m TOR ratio decreased（P<0.05）,indicating that liraglutide can activate AMPK/m TOR signaling pathway in myocardial ischemia-reperfusion injury mice.2.Effects of liraglutide on autophagy and AMPK/m TOR signaling pathway in myocardial cells injured by high glucose hypoxia/reoxygenation injury.（1）Compared with HG+H/R group,H9C2 cells morphology was improved and cell viability increased in HG+H/R+Lir group[（0.48±0.04）vs（0.73±0.08）,P<0.05]by observing cell morphology and CCK-8 detection.These results indicated that liraglutide could reduce H9C2 cells high glucose hypoxia/reoxygenation injury and increase cell viability.（2）Western Blot analysis of autophagy and AMPK/m TOR signaling pathway related proteins showed that compared with HG+H/R group,LC3Ⅱ/Ⅰratio increased and p62protein expression decreased in HG+H/R+Lir group（P<0.05）.The results indicated that liraglutide could promote autophagy in H9C2 cells high glucose hypoxia/reoxygenation injury.p-AMPK/AMPK ratio increased,while p-m TOR/m TOR ratio decreased（P<0.05）,indicating that liraglutide could activate AMPK/m TOR signaling pathway in H9C2cells high glucose hypoxia/reoxygenation injury.These effects were partially reversed after treatment with AMPK inhibitor Compound C,while the m TOR inhibitor rapamycin promoted these effects,suggesting that liraglutide acts on H9C2 cells high glucose hypoxia/reoxygenation injury via the AMPK/m TOR signaling pathway.Conclusion:Liraglutide has a protective effect on myocardial ischemia-reperfusion injury in diabetic mice,which promotes autophagy by activating AMPK/m TOR signaling pathway,providing a theoretical basis for the prevention and treatment of ischemia-reperfusion injury in clinical diabetic conditions and further improving the prognosis and survival rate of patients.