The Therapetic Effects And Related Mechanism Of Isosteviol Sodium On Diabetic Cardiomyopathy

Diabetes mellitus?DM?,a serious disease threatening human health,is a chronic disease caused by insulin deficiency or insulin resistance.According to the World Health Organization?WHO?,DM will be the seventh leading cause of death in 2030.More serious than DM itself is its variety of macrovascular and microvascular complications including heart disease,which has become the leading cause of death in diabetic patients.In addition to causing hypertension,coronary heart disease,atherosclerosis and other diseases,hyperglycemia also directly induces damages to the heart,causes significant changes in the cardiac structures and functions,which is defined as diabetic cardiomyopathy?DCM?.The main characterization of cardiac structural changes caused by DCM is that myocardial hypertrophy as increasing in left ventricular mass,thickening of the ventricular wall,enlargement of the volume of the cavity,as well as significant collagen deposition and interstitial fibrosis,leading to arterial stiffness and fibrotic remodeling.The functional changes are firstly characterized by diastolic dysfunction,as the disease progresses to the advanced stages,systolic function may also declines.Meanwhile,cardiac output?CO?is greatly reduced and unable to meet the wholebody's demands of oxygen,nutrients and energy,ultimately leading to heart failure and even death.At present,there is still no specific drugs for DCM therapies,and the existing treatments cannot satisfy the clinical needs,which brings great risks to diabetic patients.This study was dedicated to demonstrate the therapeutic efficacy and potential mechanism of isosteviol sodium?STVNa?on DCM,using marketed trimetazidine?TMZ?as a control drug.For the commonality of type 1 diabetes and type 2 diabetes-induced heart disease,most studies using streptozotocin?STZ?-induced type 1 diabetes for studying the pathophysiology of DCM and for drug develpment because of its ease of operation.Therefore,this study selected type 1diabetes to study the dynamic development process of DCM,and demonstrate the effects of STVNa on the disease,as well as the underlying mechanisms of action and the differences between STVNa and TMZ.Firstly,streptozotocin?STZ?was used to induce type 1 diabetes in male Wistar rats.Different time points of 4,8,12,and 16 weeks were used to analyze the dynamic changes of hypertrophy,fibrosis,diastolic function and systolic function in DCM progresses.The results showed that after successful induction of diabetes,myocardial hypertrophy and fibrosis were evident from the 8^th week,dysfunction of the diastolic function?8 weeks?preceded systolic dysfunction?12 weeks?.However,increased expression levels of hypertrophy and fibrosis biomarkers?BNP and TGF-?1?were observed earlier(from the 4^th week),indicating that changes in molecular levels precede changes in structures and functions.Subsequently,the rats successfully induced diabetes were randomly grouped after 1 week of STZ injection,and then treated with solvent control,STVNa or TMZ for 11 weeks,or 11weeks treatment following no treatment of 4 weeks.The cardiac function and pathological changes and potential mechanisms were investigated.Diabetes causes cardiac hypertrophy and fibrosis,characterized by increased cardiomyocyte cross-sectional area,heart weight to body weight ratio?HW/BW?,interstitial fibrosis,type?and type?collagen production,as well as significantly elevated hypertrophy and fibrosis related markers.At the same time,the levels of reactive oxygen species?ROS?in myocardial tissue were significantly increased,and the total antioxidant capacity?T-AOC?and total reduced glutathione?GSH?were significantly decreased,together with superoxide dismutase?SOD?activity showed a trend of decline.Malondialdehyde?MDA?,the lipid peroxidation product,and the ROS marker 8-OHdG content were significantly increased.Moreover,the pro-inflammatory cytokines were greatly enhanced,including tumor necrosis factor-??TNF-??and interleukin-6?IL-6?in plasma and heart tissues were elevated,as well as the expression of related inflammatory genes,such as IL-1?,MCP-1,ICAM-1 and VCAM-1 were significantly increased.Both STVNa and TMZ significantly improved symptoms in almost all aspects of DCM,even after 4 weeks of discontinuation of treatment.Overall,STVNa works better than TMZ.Moreover,diabetes induced the formation of glycation end products?AGE?in blood and myocardial tissue,promoted RAGE expression in the heart,activated ERK and NF-?B signaling pathways.However,AMPK signaling pathway was inhibited.Both STVNa and TMZ had no significant effects on blood glucose,insulin or AGE-RAGE axis.STVNa inhibited ERK and NF-?B signaling,but had no significant effect on AMPK.However,TMZ further enhanced phosphorylation of ERK,stimulated AMPK signaling pathway,whereas inhibited NF-?B signaling pathway.Finally,in vitro studies were carried out to analyze the effects of STVNa or TMZ on H9c2cardiomyocytes injuries induced by high glucose?HG?.The potential signal transduction pathways were investigated.HG induced significant damages to cells,which was manifested by decreased cell viability and elevated hypertrophy and fibrosis symptoms.Both STVNa and TMZ could reduce HG-induced cell damage and inhibit various abnormal processes.Meanwhile,HG increased the expression of both RAGE and oxidative stress-associated Nox-2 and TRPC3,and the ERK and NF-?B signaling pathways were activated,while the AMPK and Akt signaling pathways were inhibited.STVNa inhibited ERK,NF-?B and Nox-2signaling and activated phosphorylation of Akt,but had no significant effect on AMPK or TRPC3.However,TMZ further enhanced phosphorylation of ERK,stimulated AMPK and Akt signaling pathways,whereas inhibited NF-?B signaling pathway as well as expression os Nox-2 and TRPC3.Consistent with animal experiments,STVNa and TMZ had no significant effects on AGE-RAGE axis.In addition,in vitro cell experiments confirmed that HG inhibit the glycolysis of H9c2 cells.STVNa significantly promoted glucose glycolysis,restored hexokinase?HK?and phosphofructokinase?PFK?activity,and did not affect pyruvate kinase?PK?activity.Interestingly,TMZ promoted HK activity but not glycolysis process and had no significant effects on PFK and PK.Oligomycin inhibits oxidative phosphorylation and decreases the viability of H9c2 cells,which could not be promoted by STVNa or TMZ.This study can provide some information for the pathological mechanism of diabetic cardiomyopathy and drug developments.At present,different studies of DCM with different time periods lead to different results about cardiac function,cardiac hypertrophy or fibrosis.This study analyzed the dynamic process of DCM development by setting different time points.The specific time of changes in cardiac structure and function will be critical for analysing results of similar researches.STVNa is a typical representative of natural plant extracts,and TMZ is the most commonly used metabolically-regulated anti-heart failure drug,both of which are expected to be effective approaches against complex chronic diseases induced by multiple factors such as DCM.This study confirmed the therapeutic effects of STVNa on DCM and preliminarily explained its mechanisms of action,which is different form TMZ.In the future,we will use gene knockout,overexpression and other means to deeply analyze the precise regulatory targets of drug action,clarify the mechanism of STVNa to provide effective method for the treatment of DCM.