The Protective Effect Of Liraglutide On Cardiac Function In Diabetic Cardiomyopathy Rats And The Pathological Model Of Atherosclerosis In ApoE^-/- Mice Induced By High-fat Diet

Part 1 Protective effect of liraglutide on cardiac function in diabetic cardiomyopathy ratsBackground and objective:As a specific cardiomyopathy,diabetic cardiomyopathy(DCM),has a complicated pathogenesis and is easy to progress to heart failure.It is one of the main complications that cause death in diabetic patients.The new-type hypoglycemic drug liraglutide has been confirmed by clinical studies to have cardiovascular protection,but its mechanism of action is still fully clarified.This study aims to explore the protective effect of liraglutide on the cardioprotection of DCM rat models,and to provide a theoretical basis for revealing the mechanism of action of liraglutide in clinical treatment of DCM to improve its cardiac function.Methods:100 SD rats were randomly divided into control group and type 2 diabetes(type 2 diebetes mellitus,T2DM)model;The rats in the control group were fed with ordinary diet;Rats in the model were fed with high-sugar and high-fat diet,after 4 weeks,They were given intraperitoneal injection of 30 mg/kg of streptozotocin(STZ)diluted with citrate buffer solution,and the control group was given the same dose of citrate buffer solution intraperitoneally.The blood glucose was measured at 72 h and one week later,if the blood glucose was≥16.7 mmol/L in both times,the T2DM model was successfully established.After the T2DM rats were fed with high-sugar and high-fat diet for 4 weeks,the heart function was tested by echocardiography,and the standard DCM rat model was established successfully.After successful modeling,they were randomly divided into DCM group,low-dose liraglutide group,and high-dose liraglutide group;rats were injected subcutaneously with liraglutide(100/200 μg/kg)or equivalent normal saline,daily once for 12 consecutive weeks.After 12 weeks of treatment,echocardiography was used to detect cardiac function;18F-FDG PET/CT gated myocardial metabolism imaging;The four items of blood glucose and blood lipids were measured;heart weight index(HWI)was measured;H&E staining,Masson staining,TUNEL staining and WGA immunofluorescence were used to detect rat cardiomyopathy;Metabolomics was used to detect the expression of myocardial metabolites in rats.Results:Compared with the control group,The fasting blood glucose,total cholesterol(total cholesterol,TC),total triglyceride(total triglycerides,TG),low density lipoprotein cholesterol(low density lipoprotein cholesterol,LDL-C)levels of DCM group were increased significantly;Ejection fraction(EF)and fractional shortening(FS)were significantly reduced,and the ratio of the maximum blood flow in the early diastole of the left ventricle to the maximum blood flow in the mitral atrial systole(E’/A’)was significantly increased;Myocardial fiber breakage,fibrous tissue proliferation,apoptotic cells increase,and myocardial cell hypertrophy;The myocardial 18F-FDG uptake value increased significantly;The levels of metabolites such as phosphatidylcholine and phosphatidylethanolamine increased significantly and the choline metabolic pathway is most affected.Conclusions:Liraglutide may change the expression of phosphatidylcholine,phosphatidylethanolamine and other metabolites through the choline metabolism pathway,thereby reducing the myocardial damage of DCM rats and improving the cardiac function of DCM rats.Part 2 Pathological model of atherosclerosis induced by high fat diet in ApoE-/-miceBackground and objective:The formation process of atherosclerosis(AS)is complex,and its pathogenesis involves various pathophysiological processes,such as inflammation,lipid metabolism disorders and oxidative stress.ApoE-/-mice are commonly used animal models for the study of atherosclerosis,after fed western diet,their blood lipid levels are increased significantly,and the aorta presents typical pathological features of atherosclerotic plaque.Current studies have shown that oxidized low density lipoprotein(oxLDL)and proprotein convertase subtilisin/kexin type 9,PCSK9 in circulating blood play an important role in the pathogenesis of AS and is closely related to the degree of aortic valve calcification.This study aims to investigate the pathological characteristics of ApoE-/-mice atherosclerosis model induced by high-fat diet,and to clarify the relationship between lipid component deposition,especially oxLDL,and the degree of aortic valve calcification,and the relationship between PCSK9 and the degree of renal calcification.Methods:10 8-week-old male C57BL/6J mice were taken as the normal group,and 20 8week-old male ApoE-/-mice were randomly divided into control group and model group;The normal group and control group were given ordinary diet,and the model group was given high fat diet,continuous feeding for 24 weeks.Mice in each group were tested by echocardiography;After the experiment,the mice were sacrificed and samples were taken to test the levels of TC,TG,LDL-C,HDL-C,oxLDL and PCSK9;Oil red O staining was used to detect mice aortic plaque and liver fat deposits;H&E staining was used to detect the pathological changes of the aortic valve,liver,and kidney structures.Masson staining was used to detect the degree of fibrosis of liver and kidney;Von kossa staining was used to detect the degree of aortic valve calcification;Alizarin red staining was used to detect the degree of kidney tissue calcification;Immunohistochemistry was used to detect the expression level of oxLDL in mice aortic valve and PCSK9,BMP2 and Osteopontin in kidney;Western blot was used to detect the expression of BMP2 and Osteopontin protein in kidney tissue.Results:Compared with the normal group,the level of serum TC,TG,LDL-C,oxLDL and PCSK9 in the control group were increased significantly,while the HDL-C level was decreased;The area of aortic atherosclerotic plaque was increased significantly;The peak velocity of transvalvular blood flow of the aortic valve was increased,and the thickness and degree of calcification of the aortic valve were significantly increased;The expression of oxLDL in the aortic valve was increased significantly;Fatty degeneration of the liver,fibrosis and lipid deposition were increase significantly;The kidneys have glomerular shrinkage,cell proliferation,renal interstitial fibrosis,and glomerular calcification were significantly increased;The expression of BMP2 and Osteopontin in kidney tissue was significantly increased.Compared with the control group,the above-mentioned changes in the model group were more significant,and the expression level of PCSK9 in the kidney tissue was significantly increased.Conclusions:High-fat diet accelerates the AS process in ApoE-/-mice,induces aortic valve calcification,liver lipid deposition,and kidney calcification,and significantly increases the expression of aortic valve oxLDL and the protein expression levels of PCSK9,BMP2 and Osteopontin in the kidney,suggesting that PCSK9,BMP2 and Osteopontin may be involved in the process of kidney calcification.