Astragaloside Ⅳ Alleviates Myocardial Damage In Diabetic Cardiomyopathy By Regulating Autophagy Via AMPK-ULK1 Pathway

Objective: Diabetic cardiomyopathy(DCM),a common cardiovascular complication of diabetes mellitus(DM),can lead to severe heart failure and,even,death.Astragaloside Ⅳ(AS-Ⅳ),the major bioactive ingredient of Huang Qi,has been proved to alleviate cardiac damage and dysfunction induced by DM.However,mechanisms underlying the cardioprotective effect have not been fully clarified.This study aimed to investigate and examine the beneficial roles and potential mechanisms of AS-Ⅳ against DCM by experiments in vivo and vitro.Methods: db/db mice(male,8 weeks of age)and db/+ mice(male,8weeks of age)were selected for in vivo experiment.db/+ mice were set as a control group(Con group).db/db mice were randomly assigned to four groups(n = 6):(1)DCM group: vehicle-treated group,(2)AS-Ⅳ(L)group: low-dosage AS-Ⅳ-treated(25 mg/kg/d,i.g.)group,(3)AS-Ⅳ(H)group: high-doage AS-Ⅳ-treated(50 mg/kg/d,i.g.)group,and(4)Met group: metformin-treated(150 mg/kg/d,i.g.)group.Met group was considered the positive control group.db/db mice were treated with or without AS-Ⅳ or metformin challenge for 10 weeks.Cardiac function were evaluated with echocardiography and serum BNP level.The extent of myocardial injury was assessed by measuring the levels of myocardial injury markers.Hematoxylin-eosin(HE),Wheat germ agglutinin(WGA)and Masson stains were used to assess the structural changes in mice hearts,cardiomyocyte cross-sectional area,and the extent of myocardial fibrosis.The expression levels of autophagy-related and AMPK-ULK1 pathway proteins in the heart of mice were examined using Western blotting and immunohistochemical analyses.H9c2 cardiomyocytes were used for in vitro experiments and categorized into four groups:(1)Control group: low concentration of glucose(5.5 mmol/L),(2)HG group: high concentration of glucose(33.3mmol/L),(3)AS-Ⅳ group: high glucose concentration plus AS-Ⅳ(100μmol/L),and(4)compound C group: high glucose concentration plus ASⅣ with compound C(50 μmol/L).The CCK-8 assay was used to detect the viability of H9c2 cardiomyocytes.The expression levels of autophagy-related and AMPK-ULK1 pathway proteins in H9c2 cells were examined using Western blotting.Results:1.Compared with Control group,the levels of fasting blood glucose(FBG)and blood lipid were significantly higher in DCM group.After treatment with AS-Ⅳ,the levels of FBG,triglyceride and low-density lipoprotein cholesterol were significantly decreased.2.Compared with Control group,mice in DCM group exhibit obvious decreases in the ratio of peak velocity of early diastolic to late diastolic flow,left ventricular ejection fraction,and left ventricular fractional shortening.And the serum levels of brain natriuretic peptide,creatine kinase-MB isoenzyme,cardiac troponin and lactate dehydrogenase were increased.However,AS-Ⅳ treatment clearly improved the above indicators in db/db mice.3.In contrast to the Control group,the arrangement of cardiomyocytes was disordered,the cross-sectional area of was increased,and collagenous fibers in myocardial interstitium was increased in DCM group.AS-Ⅳ treatment clearly improved myocardial remodeling and attenuated cardiac fibrosis.4.Compared with Control group,the cell viability of H9c2 cells in HG group was decreased.AS-Ⅳ pretreatment inhibited the reduction in cell viability caused by high glucose concentrations,while Compound C pretreatment inhibited the effect of AS-Ⅳ pretreatment on H9c2 cells.5.In the cardiac tissue of db/db mice and high-glucose-treated H9c2 cells,the expression levels of the autophagy-related proteins Beclin-1 and LC3 were decreased,the expression level of P62 was increased,and the phosphorylation levels of AMPK and ULK1 were decreased.AS-Ⅳ treatment or pretreatment increased the expression levels of Beclin-1 and LC3,and decreased the expression level of P62,as well as increased the phosphorylation levels of AMPK and ULK1.However,in high-glucosetreated H9c2 cells,Compound C pretreatment inhibited the effect of ASⅣ pretreatment on the expression of relevant proteins in H9c2 cells.Conclusion:1.AS-Ⅳ treatment can alleviate cardiac fibrosis,improved cardiac dysfunction and myocardial remodeling in db / db mice.2.AS-Ⅳ can protect H9c2 cells against high glucose damage.3.AS-Ⅳ alleviates myocardial damage in DCM by restoring autophagy via AMPK-ULK1 pathway.