Studies Of The Effect Of Hydrogen Sulfide On Myocardial Fibrosis In Diabetic Rats And The Related Mechanisms Involved

Diabetic cardiomyopathy（DCM） is one of the chronic complications of diabetes and it has severely impact on people’s health and quality of life. There are no specific treatment for DCM and its prevention and control is an important issue in the field of medical research at present. The occurrence and process of the DCM is complex, the existing research shows that the myocardial fibrosis is the important performance and progress process in it. Hydrogen sulfide（H₂S） has been found to be the third gaseous signal molecule and it has a variety of physiological and biological function in bodies, such as anti-atherosclerosis, anti-hypertensive, anti-fibrosis, analgesia, anti-Parkinson, and so on. The specific mechanism may be related with ER stress, oxidative stress, cell autophagy, protein kinase C（PKC） pathway and Janus Kinase（JAK） pathway. Cell autophagy is also called type II programmed cell death, a normal level of autophagy has an important role in regulating cell damage and aging, but excessive autophagy can lead to cell death. Researches found that cell autophagy was in a long-term activation state when myocardial infarction, hypertension and hyperglycemia occurred, it accelerated the cell death and development of these disease. Phosphatidylinositol 3-kinase/Serine/threonine kinase（PI3K/AKT） signal pathway is considered to be the most important survival pathways in cells and plays an important role in the regulation of cell autophagy. Research had shown that inhibition of the signal pathway could induce the occurrence of autophagy of macrophages and tumor cells. PKC can regulate cell metabolism, differentiation, proliferation, apoptosis and canceration. In diabetic rats, PKC induced myocardial hypertrophy, fibrosis, and then affected the effect of cardiac function. Heat shock protein 70（HSP70） is a kind of protection protein in a normal level, but has damage effect to bodies in an excessive expression. JAK/STAT signal pathway is widely involved in immunity, cell proliferation, differentiation, apoptosis, inflammation, tumor and other pathological physiological process, it widely exists in cells and tissues. Studies found that JAK/STAT signaling pathway played an important role in the happening and development of many fibrosis models. Research proved that H₂S could inhibit the diabetes mellitus（DM） myocardial fibrosis in rats. DM model was induced by streptozocin（STZ） in this article to observe the expression of cell autophagy, PI3K/AKT pathway, PKC/HSP70 pathway and JAK/STAT pathway in diabetic rats and to study the the relationship between hydrogen sulfide and these pathways and effects of hydrogen sulfide on diabetic rats myocardial fibrosis, and then further to know the molecular mechanism of hydrogen sulfide on myocardial fibrosis in diabetic rats.Part I: Improvement effect of hydrogen sulfide on myocardial fibrosis in diabetic rats Objective: To observe the effect of hydrogen sulfide on STZ-induced myocardial fibrosis in diabetic rats. Methods: 40 adult male Sprague-Dawley（SD）rats for experiment were randomly divided into 4 groups（n=10）: normal rats group（Control group）, Diabetic rats group（STZ group）, H₂S intervened in diabetic rats group（STZ+H₂S group）, H₂S intervened in normal rats group（H₂S group）. Rats in STZ group and STZ+H₂S group were treated with STZ（dissolved in sodium citrate buffer） intraperitoneally（40 mg/kg） to establish DM models while the rats in Control group and H₂S group treated with isodose saline solutiononly. 72 h after STZ injection, took the tail venous blood to test fasting blood glucose, the rats with blood glucose higher than 16.7 mmol/L were considered as successfully diabetic model rats. Then treated the rats in STZ+H₂S group and H₂S group with sodium hydrosulfide（Na HS, 100μmol/kg） dissolved in phosphate buffer saline（PBS） by intraperitoneal injection as a H₂S donor while the rats in the Control group and STZ group treated with isodose PBS solution. 8 weeks later, Hematoxylin eosin（HE） staining and Van Gieson（VG） staining were used to analyze the pathomorphologycal changes of myocardial fibers. The expressions of type I and III collagen, MMP-8, MMP-13, MMP-14, TIMP-1, TIMP-2 proteins in heart tissue of every group were detected by Western blotting.Results: HE staining demonstrated that, in the Control group, the myocardial cells were orderly arranged and had normal intercellular space and no interstitial fibrosis. Compared with Control group, relatively disorganized myocardial cells, broadened intercellular space and obvious interstitial fibrosis were observed in STZ group, while the H₂S group had no such changes. Compared with STZ group, the myocardial cells were arranged orderly relatively, intercellular space was generally normal and there was a little interstitial fibrosis in STZ+H₂S group. VG staining showed that the myocardial fibers arranged compactly and orderly and there was little collagen deposition around the myocardial tissue and muscle clearance in Control group. While the STZ group rats myocardial fiber arranged evacuation and disorder, collagen deposition around myocardial tissue increased significantly. Compared with STZ group, the myocardial fibers arranged orderly and the collagen deposition around the myocardial tissue was decreased significantly in STZ+H₂S group. Western blotting method results showed: compared to control group, the expression level of type I and III collagen, MMP-8, MMP-13, MMP-14, TIMP-1 and TIMP-2 proteins of STZ group was significantly increased in the heart tissue of rats. And these changes were reversed by treating with H₂S. Conclusion: Hydrogen sulfide can improve STZ-induced myocardial fibrosis in diabetic rats.Part II: Improvement effect of hydrogen sulfide on cardiomyocyte autophagy and expression of PI3K/AKT pathway in diabetic rats Objective: To observe the expression level of cell autophagy in diabetic rats myocardial, and discusses the effect and mechanism of hydrogen sulfide on it. Methods: The steps of rats to be put to death were the same with the methodology in part I. Transmission electron microscope（TEM） was adopted to observe autophagosomes. The expressions of autophagy related proteins such as Beclin-1, Atg3, Atg5, Atg16, PI3 K and AKT1 proteins in heart tissue of every group were detected by Western blotting. Results: TEM results showed that autophagosome in myocardial tissue was not found in the Control group, STZ+H₂S and H₂S group, but the myocardial tissue of STZ group had. Western blotting method results showed that, compared with the Control group, the expression level of Beclin-1, Atg3, Atg5 and Atg16 were significantly increased in STZ group, while the expression of PI3 K and AKT1 obviously down-regulated. After the H₂S intervention, the expression level of Beclin-1, Atg3, Atg5 and Atg16 in STZ+H₂S group was significantly lower than those in STZ group, at the same time, the expression of PI3 K and AKT1 increased significantly in STZ+H₂S group compared with STZ group. Conclusion: Decreased expression of PI3K/AKT pathway and over expression of cell autophagy may be involved in the formation and progress of the myocardial fibrosis in diabetic rats. The protection mechanism of hydrogen sulphide on myocardial fibrosis in diabetic rats may be related with its function of down-regulating cell autophagy and up-regulating PI3K/AKT1 expression. Part III: The study of other mechanism of the protection effect of hydrogen sulfide on myocardial fibrosis in diabetic rats Objective: To explore the other possible mechanisms of the effect of hydrogen sulfide on myocardial fibrosis in diabetic rats. Methods: The steps of rats to be put to death were the same with the methodology in part I. The expressions of PKC, HSP70, JAK-1, STAT-1, STAT-3, STAT-5, STAT- 6 proteins in heart tissue of every group were detected by Western blotting. Results: Western blotting method results showed that: compared with Control group, the expression level of PKC, HSP70, JAK-1, STAT-1, STAT-3, STAT-5, STAT-6 proteins was increased significantly in the myocardial tissue of STZ group rats. After the intervention by H₂S, compared with STZ group, the expression level of these proteins was decreased significantly in the myocardial tissue of STZ+H₂S group rats. Conclusion: The expression of PKC/HSP70 and JAK/STAT pathway related proteins increases significantly in myocardial tissue of diabetic rats, which may be involved in the occurrence and progress of myocardial fibrosis in diabetic rats. The mechanism that H₂S improve myocardial fibrosis in diabetic rats may be related to PKC/HSP70 signal pathway and JAK/STAT signal pathway.