| Background: Diabetic myocardial injury, which is one of the common complications of diabetes mellitus, carries a substantial risk for the subsequent development of heart failure and increased mortality. It is characterized by a variety of morphological changes, including myocyte hypertrophy, myofibril depletion, interstitial fibrosis, and intramyocardial microangiopathy. Previous studies have demonstrated that hyperglycemia, lipid accumulation, increased oxidative stress, impaired calcium handling, mitochondrial dysfunction, renin-angiotensin system activation, myocardial inflammation and apoptosis are probably involved in the pathogenesis of diabetic myocardial injury. Hydrogen sulfide(H2S), the third gaseous signaling molecule identified after nitric oxide and carbon monoxide, is endogenously generated from cysteine by the pyridoxal-5’-phosphate-dependent enzymes, including cystathionine β-synthase and cystathionine γ-lyase. In recent years, accumulating evidence has demonstrated that H2 S plays critical roles in the physiology and pathophysiology of cardiovascular system. H2 S attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function and reduces the morbidity and mortality associated with ischemia-induced heart failure. Decreased endogenous production of H2 S predisposes to vascular remodeling and early development of atherosclerosis. In the present study, we established a streptozocin-induced diabetic rat model to investigate the protective effects of H2 S against diabetes-induced myocardial injury and explore the potential mechanisms involved.Methods: The diabetic rat model was induced with a single intraperitoneal injection of streptozotocin(65 mg/kg). Rats were randomly devided into four groups: Na HS group(normal rats intraperitoneally administered with Na HS at a dose of 14 μmol/kg.d), DM group(diabetic rats intraperitoneally injected with an equivalent volume of physiological saline), DM+Na HS group(diabetic rats intraperitoneally administered with Na HS at a dose of 14 μmol/kg.d), and control group(normal rats intraperitoneally injected with an equivalent volume of physiological saline). After 16 weeks of streptozotocin injection, the plasma and myocardial levels of H2 S were determined by the Methylene Blue method. Cardiac structure and function were evaluated by echocardiography. Rat left ventricular tissue was stained with haematoxylin and eosin and Masson’s Trichrome. Myocyte cross-sectional area and collagen volume fraction were measured using an image analysis software. The expression of types I and III collagen in cardiac tissue was examined by immunohistochemistry. The levels of inflammatory markers in myocardial tissue, including tumour necrosis factor-α(TNF-α), interleukin(IL)-1β, IL-6 and IL-8, were determined using commercial ELISA kits. Oxidative stress was evaluated by detecting malondialdehyde(MDA) levels and superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) activities. Nrf2-ARE binding activity was evaluated by electrophoretic mobility shift assay. Cardiomyocyte apoptosis was detected by TUNEL staining and PI3 K activity was measured by ELISA. Western blotting was used to detect the expression of Nrf2, HO-1, NQO1, Bax, Bcl-2 and caspase-3 and the phosphorylation of JNK(Thr183/Tyr185), P38(Thr180/Tyr182), Akt(Ser473), Bad(Ser136) and caspase-9(Ser196). Myocardial cells were administered with 100 μmol/L Na HS for 30 min prior to exposure to 30 mmol/L glucose for 24 h. The generation of reactive oxygen species(ROS) was measured and cardiomyocyte apoptosis was determined by Annexin V/PI staining. To confirm whether H2 S suppresses high glucose-induced oxidative stress in an Nrf2-dependent manner, we transfected cardiomyocytes with Nrf2-specific si RNA and subjected them to Na HS and high glucose, and then nuclear Nrf2 expression and ROS production were detected. To determine whether H2 S reduces high glucose-induced cardiomyocyte apoptosis by inhibiting MAPK signalling and activating PI3K/Akt pathway, myocardial cells were pre-conditioned with JNK inhibitor SP600125(10 μmol/L), p38 inhibitor SB203580(20 μmol/L) or PI3 K inhibitor LY294002(10 μmol/L) for 60 min, followed by exposure to high glucose, and then phosphorylation of JNK, P38 and Akt was determined and cardiomyocyte apoptosis was detected.Results: Our findings showed that endogenous generation of H2 S was decreased in the diabetic rats, whereas exogenous administration of Na HS increased H2 S contents in both plasma and myocardial tissue. After 16 weeks of streptozotocin injection, the diabetic rats exhibited left ventricular enlargement, wall thickening, myocardial fibrosis and cardiac dysfunction. H2 S could attenuate myocardial hypertrophy and interstitial fibrosis and improve left ventricular function in diabetic rats. TNF-α, IL-1β, IL-6 and IL-8 levels were significantly elevated in the myocardium of diabetic rats, whereas H2 S was found to attenuate the inflammatory reaction induced by diabetes. There were marked increases in MDA levels and decreases in SOD and GSH-Px activities in the diabetic rats, whereas Na HS administration was associated with reduced oxidative stress in the diabetic myocardium. H2 S could increase the binding activity of Nrf2-ARE, induce the nuclear accumulation of Nrf2, and up-regulate the protein expression of HO-1 and NQO1, which consequently alleviated the oxidative injury in the myocardium of diabetic rats. Na HS pre-treatment could activate Nrf2 signalling and decrease the ROS generation in cardiomyocytes induced by high glucose, while Nrf2 si RNA-transfected cells which were pre-treated with Na HS and then exposed to high glucose did not exhibit a marked decline in ROS production, suggesting that H2 S could reduce high glucose-induced oxidative stress by activating Nrf2 signalling pathway. In the diabetic rats, the cardiomyocyte apoptotic index was higher, and Bax and caspase-3 expression was increased and Bcl-2 expression was decreased. Na HS administration was found to reduce cardiomyocyte apoptosis and the ratio of Bax/Bcl-2 in diabetic rats, suggesting that H2 S could reduce high glucose-induced cardiomyocyte apoptosis by inhibiting mitochondrial apoptotic pathway. The protein levels of phospho-JNK and phospho-p38 were elevated in the myocardium of diabetic rats, whereas H2 S was found to inhibit the phosphorylation of MAPKs in diabetic myocardium. High glucose could induce cardiomyocyte apoptosis. Na HS pre-treatment could inhibit JNK and p38 pathways and reduce cellular apoptosis. Myocardial cells pre-treated with SP600125 or SB203580 exhibited decreased apoptosis rate, which suggested that H2 S could reduce high glucose-induced apoptosis by inhibiting JNK and p38 pathways. The PI3 K activity and the expression of phospho-Akt, phospho-caspase-9 and phospho-Bad were decreased in the myocardium of diabetic rats. H2 S was found to activate PI3K/Akt pathway and promote the phosphorylation of apoptosis regulatory proteins Bad and caspase-9 in diabetic myocardium. Na HS pre-treatment could increase the activity of PI3K/Akt and reduce the apoptosis of cardiomyocytes exposed to high glucose, while myocardial cells which were pre-treated with LY294002 and Na HS and then exposed to high glucose did not exhibit a marked decrease in apoptosis rate, suggesting that H2 S could reduce high glucose-induced apoptosis by activating PI3K/Akt pathways.Conclusions: Our study demonstrates that H2 S alleviates diabetes-induced myocardial injury via attenuation of inflammation, oxidative stress and apoptosis. Moreover, H2 S may reduce high glucose-induced oxidative stress by activating Nrf2/ARE pathway and may exert anti-apoptotic effects in diabetic myocardium by inhibiting JNK and p38 pathways and activating PI3K/Akt signalling.
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