Metformin Alleviate The Apoptosis Of Cardiomyocytes And Promote Regeneration Through Activating AMPK/Hedgehog Signaling Pathway

Background:Myocardial infarction（MI）is mainly caused by necrosis of myocardium whose blood and oxygen supply are blocked on the condition that spasm,stenosis or occlusion existed in coronary artery.The probable mechanism of this process is related to the rupture of coronary artery atherosclerosis plaque,collagen are exposed to bloodstream after vascular endothelial cells get injured.The platelets adhere to vessel wall,which result in hemodynamic changes and then promote the formation of thrombus in a hypercoagulable blood condition,finally ischemia and hypoxia occurred when blockage appears in blood flow.Recently,the morbidity of MI in our country has been rising by years,age of incidence presents a younger tendency according to analyzing the epidemiological characteristics.Cardiomyocytes suffer a lot of loss because of the increasing cell apoptosis rate after MI happened,and the proliferation of fibroblast will lead to adverse ventricle remodeling.Thus,it is vital to alleviate cardiac structure and function for mitigating apoptosis or promoting cell regeneration and angiopoiesis.Metformin is the clinical first-line drugs of Type 2 Diabetes,it can decrease the blood glucose and weaken insulin resistance.Present reports indicated that metformin plays a crucial role in cardiac vascular system by activating AMPK or PI3K/Akt signals.It is confirmed that metformin can alleviate ischemia reperfusion injury and ventricle adverse remodeling in vivo.Also,it can mitigate apoptosis of cardiac cells in vitro.However,no research have been made on that metformin is able to alleviate apoptosis and promote proliferation of H9C2 myocardial cells through Hedgehog pathway,and there isn’t no report about the regulation of cell proliferation that metformin works on certain components of ubiqutin proteasome system such as UBE2C.Objective（1）To investigate the effect and mechanism of metformin apply to rat H9C2cardiomyocytes’hypoxia-reoxygenation injury induced by CoCl₂.（2）To explore the function and probable mechanism that metformin exerts on cardiac cells.Methods Divided the myocardial cells into 5 groups:control group（NC）,treated with cobalt chloride（CoCl₂）,treated with metformin（MET）,combined CoCl₂withmetformin（CM）,combined CM with Compound C,an AMPK inhibitor（CMCC）.（1）CCK-8 assay was done to detect the influences of metformin and CoCl₂ exert on cell viability.（2）Clone formation assay was performed to explain cell regeneration and forming sphere after exposing to drugs.（3）Western blot was used to detect the expression level of related target proteins.（4）Application of Flow Cytometry was to ascertain the apoptosis index after treating with drugs.Results（1）The level of HIF-1α,P-AMPK and Cleaved-caspase3 had risen in the group treated with CoCl₂ while Bcl-2,an anti-apoptotic protein,which expression was reduced.Treated cells with metformin,by contrast,could obvious promote phosphorylation of AMPK,protein level of PTEN and Bcl-2 were also improved,Cleaved-caspase3’s expression was decreased.However,those changes could be blocked by AMPK inhibitor,named Compound C.（2）The results of Flow Cytometry indicated that each group of cardiomyocytes’apoptotis rate was2.22%（NC）,31.65%（CoCl₂）,3.69%（MET）,15.08%（CM）,respectively.（3）Compared with NC group,the molecules belong to Hedgehog signaling,such as PTCH1,SMO,Gli-1,that protein levels had shown a remarkable rising trend after adding different concentrations of metformin into medium for different periods of time.Besides,the expression of ubiquitin conjugating enzyme UBE2C,cell cycle-related protein such as CDC20 and CyclinA were increased,what’s more,both of them exerted an dose-dependent relationship and time-dependent relationship.Conclusion CoCl₂ could induce cell into a state of hypoxia,ultimately led to apoptosis.Metformin could mitigate hypoxia and ameliorate the apoptosis of myocardial cells.Besides,metformin can improve the protein expression of UBE2C,CDC20 and CyclinA,facilitated the process of cell proliferation.The mechanism may be related to PTEN/AMPK and Hedgehog signaling pathway.