The Study On The Mechanism Of Empagliflozin On Diabetic Myocardial Oxidative Stress And Fibrosis

Objective:1.The aim of this study was to investigate the effect of empagliflozin(EM)on myocardium injury and the potential mechanism in T2 DM KK-Ay mice.2.To explore the effect of EM on glycemic control in patients with diabetic cardiomyopathy.3.To explore the effect of EM on cardiac function in patients with diabetic cardiomyopathy and its influence on blood lipids,blood pressure,fibrosis index,oxidative stress related indicators and inflammatory factor levels.Method:1.Thirty 8-week old KK-Ay mice(genetic T2 DM model)were randomized into two groups(15 per group).The groups were as follows:(1)DM group: T2 DM KK-Ay mice with no treatment;(2)DM+EM group: T2 DM KK-Ay mice treated with EM(10mg/kg/day).Fifteen 8-week old healthy C57BL/6J mice with no treatment were enrolled ioto control group.All the animals were raised for 8 weeks.Cardiac structures and functions in all mice were assessed by echocardiography after 8 weeks of treatment.The body weight and right tibia length of all mice were monitored.Plasma lipid levels,blood glucose levels,insulin levels,glycosylated hemoglobin?LPO level?glutathione peroxidase(GSH-Px)?superoxide dismutase(SOD)and malondialdehyde(MDA)levels were detected.The extent of myocardial fibrosis was assessed by immunohisto-chemistry and Masson staining.Western blot was used to detect Nrf2,HO-1,TGF-?1,type I collagen,type III collagen,p-Smad2,Smad2,p-Smad3,Smad3,Smad7,?-SMA(?-actin)and NOX4 expression levels to analyze cardiac oxidative stress and fibrosis indicators.2.A total of 48 patients with diabetic cardiomyopathy admitted to the Metabolic Disease Hospital of Tianjin Medical University from January 2018 to December 2018,who receive mono-or multi-drug combination therapy with DPP4 inhibitors,glycosidase inhibitors and sulfonylureas,were randomly divided into EM treatment group and control group(non-EM group),and 24 patients in each group.Patients in the EM group were given an oral dose of 10 mg/day on the original basis and continued treatment for 12 weeks.The control group was treated with thehypoglycemic regimen for 12 weeks in the above four types of hypoglycemic drugs.Baseline data were recorded.LVEF,LVEDD and E/A were measured before and after treatment by cardiac Doppler ultrasonography.Blood glucose-related indicators,blood lipid-related indicators,hsCRP and NT-proBNP levels,fibrosis-related indicators,Inflammatory-related factor levels,oxidative stress indicators were measured and compared.Result:1.The body weight gain of mice in the DM group was significantly higher than that of the control group.EM treatment affected the body weight gain in diabetic mice,and the body weight gain of DM+EM group is significantly lower than that of DM group.The heart weight/tibial length ratio in the DM+EM group had no significant difference with that in the DM group(P<0.05).Fasting and non-fasting blood glucose levels of mice in the DM+EM group were significantly decreased after8 weeks of EM treatment compared with the DM group,but it was still higher than the blood glucose in the control group.The parameter for long-term glycaemic conditions,HbA1 c,was dramatically increased in DM mice and was significantly decreased by the use of EM.At the end of drug treatment,the DM+EM group significantly reduced insulin levels compared to the DM group,but was still higher than that of the control group.While statistical differences in TC,HDL-C,LDL-C,and TG were not detected among the DM and DM+EM groups,the above four parameters were significantly increased in the DM and DM+EM groups compared with the control group(P<0.05).The cardiac parameters were measured by M-mode and Doppler echocardiographies.The HR of DM mice was slightly lower than that of the control and DM+EM groups(P<0.05)and there was no significant difference between the control and DM+EM groups.LV mass was decreased in all of the DM group compared with the control group and DM+EM group.Although the values of LVIDs and IVSs were not significantly different in the three groups,the LV myocardial mass/body mass of the DM group was significantly lower than the other two groups(P<0.05).Treatment with EM could inhibit the reduction of LVIDd.There was obvious impairment in EF,FS,FAC and E/A ratio in DM mice compared with thecontrol group.EM treatment could largely restore EF,FS,FAC and E/A ratio in DM mice,which were similar to those in the control group.LPO concentration and MDA level were significantly higher in DM mice than in control and DM + EM groups(P<0.05),whereas the levels of SOD and GSH-Px were significantly lower in DM mice compared with the other two groups(P<0.05).We found that the NOX4 was greatly elevated in DM mice,and NOX4 expression in the DM+EM group was significantly decreased compared with DM group(P<0.05).Immunohistochemical staining of TGF-?1 showed that brown-stained positive cells of TGF-?1 increased significantly and were distributed in the myocardial tissue in the DM group.Compared with the DM group,EM markedly reduced the expression of TGF-?1(P<0.05).In addition,the positive percentage of collagen I and collagen III decreased dramatically in the DM + EM group compared with the DM group(both P<0.05).The analysis of the Masson's trichrome stain pictures revealed that there was a significant difference in the median cardiac connective tissue fraction among the three groups(P<0.05).The DM group had the highest connective tissue fraction when compared with the control group and the DM+EM group(all P<0.05),However,there was no significant difference between the control and DM+EM groups.Nrf2 and HO-1 levels of total protein or nuclear protein in the DM+EM group increased dramatically compared with those in the DM group(P<0.05).TGF-?/Smad signalling was highly activated in the heart tissue of diabetic mice,which can upregulate the levels of TGF-?1,p-Smad2,p-Smad3,collagen I,collagen III and?-SMA.Treatment with EM significantly suppressed experssion of TGF-?1,p-Smad2,p-Smad3,collagen I,collagen III and ?-SMA in the heart tissue of diabetic mice.Smad7 was decreased in the DM group and greatly increased with EM treatment.2.There were no significant differences in baseline data between the EM group and non-EM group(P>0.05).The data were comparable.There were no significant differences in fasting blood glucose,postprandial 2h blood glucose,glycosylated hemoglobin between the two groups(P>0.05).After 12 weeks of treatment,the two groups of patients had significant improvement in fasting blood glucose,postprandial2 h blood glucose,glycosylated hemoglobin(P<0.05),but there was no significant difference between the two groups after treatment(P>0.05).BMI was significantlylower in the EM group than the control group(P<0.05).The HOMA-IR indexes were significantly improved after treatment(P<0.05),but the above indicators of EM group were better than the control group(P<0.05).The level of AGEs in the EM group was also lower than that in the control group(P<0.05).After 12 weeks of treatment,the serum levels of TC,TG,LDL-C,hsCRP and NT-proBNP were significantly improved in the two groups(P<0.05),but the above indicators in the EM group were better than the control group(P<0.05).The serum levels of PIIINP and TGF-?1 were significantly decreased after treatment in both groups(P<0.05),but the above indicators were better in the EM group than the control group(P<0.05).The levels of GSH-Px,SOD and IL-10 increased after treatment with EM group,while the levels of MDA and IL-6 decreased(P<0.05).The LVEF,E/A and LVEDD levels in the EM group were significantly improved compared with the control group.The systolic blood pressure of patients in the EM group was significantly decreased after treatment(P<0.05),but there was no significant difference in diastolic blood pressure compared to pre-treatment(P>0.05).Conclusion:1.Empagliflozin improved diabetic myocardial structure and function,decreased myocardial oxidative stress and ameliorated myocardial fibrosis.Further study indicated that empagliflozin suppressed oxidative stress and fibrosis through inhibition of the TGF-?/Smad pathway and activation of Nrf2/ARE signalling.In conclusion,these results indicate that empagliflozin is a promising drug for the prevention and treatment of diabetic cardiomyopathy.2.Empagliflozin can effectively control blood glucose levels in patients with diabetic cardiomyopathy.Empagliflozin can effectively improve oxidative stress and inflammation in patients with diabetic cardiomyopathy.Empagliflozin can effectively improve the level of fibrosis and the heart function in patients with diabetic cardiomyopathy.