The Effect And Mechanism Of Dapagliflozin In Improving Angiotensin Ⅱ-induced Myocardial Fibrosis

Background:Heart failure is the terminal state of various cardiac diseases,and cardiac remodeling is an important risk factor for heart failure.At present,the efficacy of drugs for heart remodeling is limited.It is of great significance to find new drugs for the prevention and treatment of heart failure.Sodium-glucose cotransporter 2（SGLT2）inhibitor,the important new drug for the treatment of patients with type 2 diabetes mellitus,reduces the risk of the first hospitalization for heart failure,possibly through glucose-independent mechanisms,but the underlying mechanisms remain largely unknown.Objective:1.To observe the effects of DAPA on Ang Ⅱ-induced cardiac remodeling and cardiac dysfunction in non-diabetic rats,and explore the effects of DAPA on the expression of TGF-β/Smad pathway and NHE-1.2.To observe the effect of DAPA on Ang Ⅱ-induced cardiac fibroblasts（CFs）fibrosis,and research the effects and mechanism of DAPA on the intracellular Ca²⁺concentration of CFs.Methods:Animal experiment:24 Sprague-Dawley（SD）rats,chronic infusion of Ang Ⅱ at a dose of 520 ng/kg per minute for 4 weeks with ALZET^?mini-osmotic pumps,were treated with either SGLT2inhibitor DAPA 5mg/kg or vehicle alone.Rats were randomly divided into 4 groups:（1）Control group（CTL,n=6）;（2）Control+dapagliflozin group（CTL+DAPA,n=6）;（3）AngiotensinⅡgroup（AngⅡ,n=6）;（4）AngiotensinⅡ+dapagliflozin group（AngⅡ+DAPA 5mg/kg,n=6）.Echocardiography was performed to determine cardiac structure and function.Hematoxylin-eosin staining was used to observe the structure of the heart.Picric Acid-Sirius Red staining and Masson staining were used to observe the collagen deposition in the heart.Immunohistochemical staining was used to test the expression of Collagen I、III、TGF-β1 and the markers of fibroblast-to-myofibroblast transformationα-SMA.Western blot was used to detect the expression of Collagen I、III、α-SMA、TGF-β/Smad pathway and NHE-1.Cell experiments:Cardiac fibroblasts（CFs）were treated with Ang Ⅱ（1μM）with the indicated concentration of dapagliflozin（0,0.5,1,10μM）.The Ca²⁺fluorescent indicator probe Fluo-3AM was used to label free calcium ions in the cytoplasm,and the intracellular Ca²⁺concentration was evaluated by flow cytometry and immunofluorescence.Western blot was used to detect the expression of Collagen I、III、α-SMA、TGF-β/Smad pathway and NHE-1.Results:Animal experiment:1.The results of echocardiography showed that compared with the CTL group,the End-diastole interventricular septum thickness,diastolic left ventricular posterior wall thickness,left ventricular fractional shortening and the left ventricular ejection fraction were increased in the Ang Ⅱ group and the left ventricular end-diastolic diameter,left ventricular end-systolic diameter,end-diastolic volume,end-systolic volume were decreased in the Ang Ⅱ group.After DAPA treatment,these indicators were significantly improved.The blood pressure,the plasma glucose levels,blood lipids and biochemical indexes of rats in each group did not change significantly.2.Compared with the CTL group,the heart weight of rats and the cross-sectional area of cardiomyocytes,ANP,BNP,Ang Ⅱ in the Ang Ⅱ group increased significantly,and these changes were significantly reduced after DAPA treatment.3.The results of Picric Acid-Sirius Red staining and Masson staining showed that the positive area of the Ang Ⅱ group was larger than that of the CTL group,obviously flakes and nets,while the positive area of the Ang Ⅱ+DAPA group was significantly reduced.The results of immunohistochemistry and Western blot showed that the expression of Collagen I,Collagen III andα-SMA in AngⅡgroup increased significantly,while the expression of AngⅡ+DAPA group was significantly decreased.4.The results of Western blot showed that the expression of TGF-β1 and the ratio of p-Smad2/Smad2 and p-Smad3/Smad3 in the myocardial tissue of the Ang Ⅱ group increased significantly,the expression of the inhibitory molecule Smad7 of the TGFβ/Smad pathway decreased,while in the AngⅡ+DAPA group,the expression of the TGF-β1 and the ratio of p-Smad2/Smad2 and p-Smad3/Smad3 decreased,and the expression of Smad7 increased.In addition,in the Ang Ⅱ group,the expression of NHE-1 in the rat myocardial tissue was significantly increased,while after DAPA treatment,the expression of NHE-1 was significantly reduced.Cell experiment:1.In the in vitro experiments,compared with the CTL group,Ang Ⅱ increased the expression of Collagen I,Collagen III,TGF-β1,andα-SMA of CFs,while DAPA intervention reduced the expression of these fibrosis-related proteins in a dose-dependent manner.2.Ang Ⅱ increased the expression of NHE-1 protein,while DAPA significantly decreased the expression of NHE-1.In addition,the results of flow cytometry and immunofluorescence showed that AngⅡsignificantly increased the intracellular Ca²⁺concentration;while DAPA could reduce the intracellular Ca²⁺concentration.These results indicated that DAPA might reduce fibrosis by affectting intracellular Ca2+concentration through NHE-1.Conclusion:1.DAPA could improve Ang Ⅱ-induced cardiac remodeling and cardiac dysfunction in non-diabetic rats,and its mechanism may be related to the inhibition of TGF-β/Smad pathway and the expression of NHE-1.2.DAPA could reduce the Ang Ⅱ-induced conversion of CFs into myofibroblasts and the production of ECM protein,the mechanism of which may be related to the reduction of intracellular Ca²⁺concentration through the inhibition of NHE-1.