Protection Of Hydrogen Sulfide Against Myocardial Fibrosis As Well As Its Mechanism

Myocardial fibrosis is a common pathological process occurred at certain stage of multiple heart diseases, leading to increased myocardial stiffness, ventricular diastolic and systolic dysfunction, which finally results in heart failure. Hydrogen sulfide (H2S), the third gasotransmitter discovered after nitric oxide and carbon monoxide, plays key role in mediating cardiovascular disease development. H2S slow releasing agent GYY4137(morpholin-4-ium4 methoxyphenyl phosphinodithioate) is a pH-dependent H2S release agent that may bear potential value in clinics, however, with its effect on myocardial fibrosis to be demonstrated. This current study mainly aimed to resolve whether H2S slow releasing agent would protect against myocardial fibrosis, and if so, its underlying mechanism.Healthy male SHR rats,12 weeks old, were divided randomly into hypertensive control (SHR) and GYY4137-administrated group, the third group is non-hypertensive control group of WKY rats (WKY). The rats in GYY4137 group received intraperitoneal injection of GYY4137 with 3 dosages, respective of 10 mg/kg/day (GYY10),25 mg/kg/day (GYY25) and 50 mg/kg/day (GYY50). The SHR and WKY group were injected with saline of equal volume. The daily injection procedure lasted for 4 week. At the end of 4-week administration,the picric acid-sirius red staining indicated that the area of collagen existed interstitial of myocardial tissue, as well as perivascular collagen, reduced remarkably. The collagen was analyzed and found that total amount of collagen decreased and the cross-linking formation was suppressed, as well as type Ⅰ and Ⅲ collage expression.Cardiac fibroblasts from neonatal rats were pretreated with GYY4137 of different concentrations (12.5 μM,25 μM,50 μM) for 4 h, followed with angiotension Ⅱ (AngⅡ) of 10-7M for another 24 h. The cck-8 kit detection both indicated that the proliferation stimulated with AngⅡ can be alleviated with GYY4137 pretreatment. The increased total collagen in culture medium of cardiac fibroblasts stimulated with AngⅡ was suppressed by GYY4137. The cell cycle of fibroblasts was detected with flow cytometry by PI staining, and found that GYY4137 reduced the amount of fibroblasts in S phase, indicating that the cell proliferation was inhibited. The Real-time PCR analysis showed that mRNA level type Ⅰ and Ⅲ collagen were significantly ecreased after GYY4137 pretreatment. While western blot and immunofluorescence results showed that after AngⅡ stimulation, protein expression of type Ⅰ and Ⅲ increased significantly, which was declined after GYY4137 treatment, along with reactive oxygen species level within fibroblasts detected with DCFH-DA fluorescent probe. Further detection found that the transforming growth factor-β1(TGF-β1), smooth muscle actin(α-SMA) protein expression were significantly increased in AngⅡ stimulated fibroblasts and after giving GYY4137 pretreatment, TGF-β1 protein levels and α-SMA expressionwere significantly decreased.Conclusion:GYY4137 protects fibroblasts against myocardial fibrosis, which may be attributed to suppression of oxidative stress, down-regulated TGF-β1 and α-SMA expression.