Hydrogen Sulfide Improves Myocardial Fibrosis In Diabetic Rats By Decreasing Endoplasmic Reticulum Stress

Background and objective Studies have shown that Endoplasmic reticulum(ER) stress can induce cell apoptosis through various channels and participate in myocardial fibrosis of diabetes mellitus(DM), therefore,ER stress may be involved in the occurrence and development of diabetic cardiomyopathy(DC), and ultimately affect heart function. However, there is no special drug to prevent and treat DC. Hydrogen sulfide(H2S) was found to be the third gaseous signal molecule with a variety of physiological and biological function, including anti-ER stress effect. Previous studies have shown that H2 S acts as a potent inhibitor of fibrosis in the heart of diabetic rat. However, the mechanism of H2 S inhibiting myocardial fibrosis is unclear. This study is aimed to establish the DM model, and demonstrate whether ER stress is involved in the formation of the myocardial fibrosis, and whether H2 S can improve cardiac remodeling of diabetic rats by suppressing ER stress and reducing cell apoptosis.Methods Adult male Sprague-Dawley(SD) rats for experiment were randomly divided into 5 groups: Control group, STZ group, C1 group, C2 group, H2 S group. STZ group, C1 group and C2 group were treated with STZ intraperitoneally( 40 mg/kg) to establish DM models. 72 h after injection, we take the tail venous blood, the rats with blood glucose higher than 16.7 mmol/L were considered as successfully diabetes mellitus(DM) rats. Then treated C1 and C2 group with different concentrations of sodium hydrosulfide(Na HS) solution( 30 mol/kg, 100 mol/kg) by intraperitoneal injection as a H2 S donor. After 8 weeks of experiment, the collagen I and III in cardiac tissues were measured by Immunostaining; Masson staining was used to detect the myocardial collagen fibers; The expressions of hydroxyproline was detected by alkali hydrolysis method in myocardial tissue of each group; Hematoxylin eosin(HE) staining was used to observe the myocardial pathological changes; The expressions of ER stress related protein(GRP78, CHOP and Caspase-12), H2 S enzyme(cystathionine-gamma-lyase, CSE, mercaptopyruvate sulfurtransferase, MPST) as well as apoptosis related protein(Cleaved Caspase-3) in heart tissue of every group were detected by Western blotting; The morphology of ER and muscle fiber were observed by transmission electron microscope.Results 1.The rat model of DM was established successfully. Blood glucose was measured after STZ injection for 72 h, the results showed that blood glucose increased significantly, and the blood glucose of each rat were higher than 16.7 mmol/L. 2. Effect of H2 S on BW, HW and HW/BW of DM rats. At the end of the experiment, BW and HW of DM rats were significantly lighter than the control group, the HW/BW value was significantly higher than the control group, which suggesting that cardiac hypertrophy may exist in DM rats. While,H2 S has no effect on BW, HW and HW/BW of DM rats. 3.H2 S can improve myocardial fibrosisin of DM heart. Results of Immunostaining showed that the expression of collagen type I and III is significantly higher than the control group in DM heart, and the collagen fibers arranged in disorder. While, H2 S can decrease the expression of collagen type I and III of DM. Results of Masson stainingshowed that the area of myocardial collagen deposition was bigger than control group, and H2 S can decrease the deposition area of collagen. Results of alkali hydrolysis showed that the expression of hydroxyproline is significantly higher than the control group in heart tissues of DM. However, H2 S can decrease hydroxyproline expression in heart in DM. The results of HE staining showed that cardiac collagen of DM rats were arranged in disorder, and the gap between myocardial fibers become broad. While, H2 S can improve the change of myocardial pathological.Results of transmission electron microscope showed that myocardial fiber edema occurred, and myocardial fiber showed disordered arrangement and focal lytic necrosis. While, H2 S can reduce the myocardial fiber edema and necrosis. 4.H2 S can up-regulate the expression of H2 S enzyme in DM rats. Results ofwestern blotting show that the expression of H2 S enzyme, CSE and MPST, were decreased, while H2 S can increase the expression of CSE and MPST. 5. H2 S can down-regulate ER stress of myocardial in STZ induced DM rats. Results of western blotting show that the expression of ER stress marker protein, GRP78, CHOP and Caspase-12, in the DM rat myocardial increased significantly. While, H2 S can down-regulate the expression of GRP78, CHOPand Caspase-12.Results of transmission electron microscope showed that ER edema appeared in varying degrees, and H2 S can decrease alleviate ER edema. 6.H2 S can decrease myocardial apoptosisin STZ induced DMrats. Results of western blotting show that the expression of Cleaved Caspase-3, which is the apoptosis related protein, in the DM rat myocardial increased significantly. While, H2 S can decrease the expression of Cleaved Caspase-3.Conclusion 1. The pathogenesis of myocardial fibrosis in DM rat may be related to the the decrease of endogenic H2 S and up regulation of ER stress. 2. H2 S can inhibit cardiac fibrosis in DM rats, the mechanism may be related to the down regulating of ER stress.