Adipose-derived Mesenchymal Stem Cells Ameliorate The Myocardial Injury Of Diabetic Cardiomyopathy By Promoting Macrophages’ Polarization

Objective: This research mainly aimed to study the effect and underlying mechanism of mesenchymal stem cells (MSCs) on diabetic cardiomyopathy (DCM)rats’ heart injury and function and macrophages’ polarization. The purpose is to find a new therapeutic target to prevent from the development and progression of DCM, and provide forceful experimental and theoretical support for MSCs therapy on chronic inflammation of diabetic cardiac complications in clinic.Methods:①Group the rats at random,and establish DCM rat model. Then we detected fasting blood glucose (FBG), serum total cholesterol (TC), serum triglyceride(TG) and fasting insulin (FINS) by blood biochemical autoanalyzer and ELISA kit.We performed H&E dyeing and used echocardiography to assess cardiac function and determine specific time to transplant MSCs to DCM rats. ②We separated, cultivated and identified rats’ adipose-derived MSCs, then transplanted cells to DCM rats via tail vein. After four times MSCs treatment,we contrast the differences of rats’ cardiac systolic and diastolic function through echocardiogram and blood biochemical indicators in different groups. After being executed, the rats’ hearts were programmed by hematoxylin and eosin staining and Masson dyeing. We detected the quantity of macrophages and the proportion of M1, M2 in different hearts by flow cytometry. We tested the concentration of several inflammatory cytokines in blood serum and cardiac tissues of different groups. ③ We separated, cultivated and identified peritoneal macrophages of rats. We used high glucose combined with chronic inflammation to induce injury in vitro to mimic the microenvironment of DCM hearts, then used intervention factors to specific groups, and co-cultured with MSCs in the Trans-well system for 48 hours. After that, we detected and analyzed the proportion of different macrophages phenotypes by flow cytometry, and the concentration of cytokines in cell supernatants by ELISA.Results:①Compared with normal control group,FBS,FINS,TC and TG of DCM rats were significantly increased (P<0.05), LVEF, FS and E/A declined obviously, DCM hearts presented cardiomyocyte hypertrophy and inflammatory cells infiltration by H&E staining.②After MSCs treatment, compared with normal group,besides the aggravation of above indicators, DCM hearts showed severe inflammation and fibrosis. Flow cytometry and ELISA showed that increased proportion of M1 macrophages and high concentration of IL-6、TNF-α in DCM hearts.Compared with DCM group, FBG, FINS, TC and TG of MSCs-treated rats were obviously decreased (P<0.05). The structure (the thickness of ventricular wall and ventricular chamber) and cardiac function (systolic and diastolic function) of MSCs-treated hearts ameliorated significantly. The pathological staining results showed that inflammation and fibrosis of MSCs-treated hearts alleviated significantly.The quantity of total macrophages, especially the proportion of type 2 macrophages(M2) obviously increased (P<0.05), on the contrary, the proportion of type 1 macrophages (Ml) decreased significantly (P<0.05) in the MSC-treated hearts by Flow cytometry. ELISA results showed that the concentration of IL-6, TNF-a in the serum and heart tissue of MSCs group obviously declined, the concentration of IL-10 obviously increased (P<0.05). ③ Macrophages stimulated by high glucose and chronic inflammation showed increased proportion of M1 macrophages(P<0.05).After co-cultured with MSCs for 48 hours, the proportion of M2 macrophages increased significantly, the proportion of M1 decreased significantly (P<0.05).Compared with MSCs co-cultured group, macrophages co-cultured with MSCs which was preconditioned by COX-2 inhibitor showed obviously increased proportion of M1 and decreased M2 (P<0.05). However, after adding PGE2 to COX-2 inhibitor-preconditioned MSCs, the proportion of M1 declined and M2 increased significantly (P<0.05). Changes of the concentration of IL-6,TNF-a were similar with M1, and IL-10 was same as M2.Conclusions:①Blood glucose and lipid metabolism of DCM rats were obviously abnormal, DCM hearts had obvious inflammation and fibrosis, systolic and diastolic function of DCM hearts were already decreased. The inflammation of DCM was mainly caused by increased M1 macrophages and high concentration of inflammatory cytokines.②MSCs infusion could ameliorate glucose and lipid metabolism,alleviate myocardial injury and improve cardiac function of DCM rats. Otherwise, MSCs could alleviate inflammation and fibrosis of DCM hearts, increase the proportion and quantity of M2 macrophages to repair the injured heart and remove the inflammation in DCM heart and body. ③High glucose combined with chronic inflammation induced macrophages to M1 macrophages which is a kind of proinflammatory phenotype, but MSCs could promote injured macrophages to polarize to M2 phenotype via COX-2-PGE2 pathway, and regulate inflammatory reaction.