| Objective: Diabetic cardiomyopathy(DCM)is a complex microvascular complication of diabetes.Its incidence is high,the pathogenesis is complex and unclear,and the treatment effect is poor.In recent years,Exosomes derived from human umbilical cord mesenchymal stemcells(HUCMSC-EXO)as a self-non-cellular system,Its role in the repair of myocardial cells,the occurrence and development of DCM,and the protection of cardiac function and cardiac structure have been gradually recognized.In our study,we investigated the role of autophagy in the development of diabetic cardiomyopathy and the therapeutic effect and potential molecular mechanism of HUCMSC-EXO on DCM,which may provide a new method for the treatment of DCM.Methods: In our investigation,30 male rats were fed and randomly assigned to one of three groups: the control group,the HUCMSC-EXO treatment group,or the DCM group.DCM group was induced by a combination of high-fat and high-carbohydrate diet and Streptozotocin(STZ)intraperitoneal injection,and then treated with HUCMSC-EXO via tail vein.The morphology and diameter of HUCMSC-EXO were observed by Transmission electron microscope(TEM).The exosome marker proteins CD9,CD81,and TSG101 were identified by Western blot(WB).Fasting blood glucose(FBG)was measured every week and the differences in blood glucose levels among the groups were statistically analyzed.Left ventricular internal dimension at end-systole(LVIDs)、left ventricular internal dimension at end-diastole(LVIDd)、left ventricular diastolic wall thickness(LVPWD)、 Interventricular septal thickness at diastole(IVSd)was measured by echocardiography.The Left ventricular ejection fraction(LVEF)and Left ventricular fractional shortening(LVFS)were calculated.The ratio of blood flow velocity during early and late diastole(E/A).Enzyme linked immunosorbent assay(ELISA)was used to test Myosin heavy chain beta(MHC-beta)、atrial natriuretic peptide(ANP)and B-type natriuretic peptide(BNP)the expression in rat tissues.The morphological alterations of cardiac tissue were examined using Hematoxylin-Eosin(HE),Masson,and Wheat Germ Agglutinin(WGA)staining.TEM was used to look into the arrangement of cardiomyocytes and the structure of mitochondria.Autophagy-related protein was discovered using immunohistochemistry(IHC).In the cardiac tissue of rats,there are microtubule-associated protein 1 light chain 3(LC3)and Beclin-1,a protein that resembles coiled-coil myosin.The autophagy-related proteins LC3,Beclin-1,and P62,as well as AMPK and UNC-51-like kinase 1(ULK1)in the autophagy signaling pathway,were all identified using Western blot(WB)in cardiac tissue.Results:1.HUCMSC-EXO were spherical vesicles with a diameter of 30-150 nm,and the specific exosome marker proteins CD9,CD81,and TSG101 were expressed.There was no significant reduction in fasting blood glucose in DCM rats after treatment with HUCMSC-EXO(P>0.05).2.Compared with the control group,LVIDs and LVIDd were significantly increased,LVPWD and IVSd were significantly thickened,LVEF,LVFS and E/A were decreased,and serum BNP level was increased in the DCM group.Compared with the model group,LVIDs,LVIDd,LVPWD,IVSd were decreased,LVEF,LVFS,E/A were increased,and serum BNP level was significantly decreased after treatment with HUCMSC-EXO(all P<0.05).3.Compared with the control group,the DCM group had an increased Heart weight/Tibial length(HW/TL)ratio,HE staining showed hypertrophy of cardiomyocytes,disordered arrangement of myocardial fibers,and breakage and dissolution of myofilaments.The morphology and fiber arrangement of cardiomyocytes were greatly enhanced after treatment with HUCMSC-EXO,and HW/TL decreased.In DCM rats,the extent of myocardial fibrosis has been demonstrated to be significantly increased by Masson staining,and WGA immunofluorescence demonstrated that DCM animals had considerably higher CSA of cardiac cells.After tail vein treatment with HUCMSC-EXO,the CSA of cardiomyocytes was reduced and the degree of fibrosis was improved(P<0.05).Compared with CON group,the levels of MHC-β and ANP,the markers of myocardial hypertrophy,were increased in DCM group,and decreased after intervention with HUCMSC-EXO.4.Transmission electron microscopy of the myocardial tissue revealed that the DCM group’s mitochondria were significantly more swollen and vacuolated than the control group’s,their mitochondrial cristlines had disappeared,their Z-lines were lighter,their myofilaments were disorganized,and their autophagolysosomes were significantly more numerous.The rats in the DCM group received therapy with HUCMSCEXO,which resulted in decreased mitochondrial swelling,a negligible quantity of mitochondrial cristolysis,a more ordered arrangement of myofilaments,and a miniscule amount of autophagolysosomes.According to WB data,the DCM group’s expression of the autophagy-related proteins LC3 and Beclin-1 rose in comparison to the control group,but P62 decreased.The expression of P62 rose while that of LC3 and Beclin-1reduced following treatment with HUCMSC-EXO.IHC data demonstrated that the DCM group’s cardiac tissue had higher levels of LC3 and Beclin-1 expression compared to the control group,and that these levels had decreased following treatment with HUCMSC-EXO(all P<0.05).5.WB findings revealed that after receiving treatment from HUCMSC-EXO,the expression of p-AMPK and p-ULK1 in the DCM group dropped and increased as compared to the control group(all P<0.05).Conclusion:1.HUCMSC-EXO can alleviate myocardial hypertrophy and improve cardiac function in DCM rats induced by STZ combined with high fat and high glucose.2.HUCMSC-EXO can reduce myocardial hypertrophy and improve cardiac function by activating AMPK/ULK1 signaling pathway in rats induced by STZ combined with high fat and high glucose. |
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