The Role And Mechanism Of Endogenous Hydrogen Sulfide In Fibrosis In Systematic Scleroderma

Background:Systematic scleroderma（systematic sclerosis,SSc）is a autoimmune disease with the core pathologic change of progressive fibrosis in both skin and inner organs.The activated fibroblast is the most important effector cell driving the fibrotic phenotype,but the regulating mechanism is yet unknown.Hydrogen sulfide（H₂S）is a newly discovered gas signaling molecular,playing important roles in many physiological process such as vascular tension regulation,immune system regulation and protection against oxidative stress.The clinical potential of H₂S has already been studied in cardiovascular and inflammatory disases.Cystathionine-γ-lyase（CSE）is one of the dominating enzymes in endogenous H₂S protection in skin.As is shown in recent studies,the down regulation of endogenous H₂S pathway may promote the occurrence and development in the hepatic and pulmonary fibrosis.However,there is yet no study in whether the endogenous H₂S/CSE is involved in the SSc fibrosis.From three aspects,cells,animal models and patients,we explore the possible roles and mechanism of endogenous H₂S pathway in systemic scleroderma fibrosis,which is of great significance for further understanding of the pathogenesis of SSc and finding new therapeutic targets.Objective:This study intended to investigate the potential role and mechanism of the endogenous H₂S pathway,in order to comprehending the SSc mechanism AND finding new therapeutic targets.Method:1.The serum and the skin fibroblasts of SSc patients and normal persons were collected,cultured and purified,and the serum H₂S level and CSE expression level of fibroblasts were measured by fluorescence probe,RT-PCR and Western Blot.2.SSc fibroblasts were treated with exogenous H₂S donor Na HS and GYY4137 for 72h to detect the m RNA and protein’s expression level of COL1A1 andα-SMA.SSc fibroblasts were treated with exogenous H₂S donor and CSE inhibitor PAG respectively,and their effects on fibroblast migration were detected by cell scratch test.CSE inhibitors PAG and CSE-si RNA were used to knock down the expression of CSE in normal fibroblasts,and the m RNA and protein expression levels of COL1A1 and a-SMA in fibroblasts were measured by Rt-PCR and Western Blot.3.SSc fibroblasts were treated with H₂S donor GYY4137 and NAHS for 72 hours,and the change of SMAD2/3 phosphorylation level induced by TGF-Βwere detected by Western Blot.4.Balb/C mouse were subcutaneously injected with bleomycin（BLM）for 4 weeks to induce skin fibrosis,and a blank control control group,a BLM control group,a Na HS treatment group and a PAG intervention group were set up.Skin samples were collected for HE staining and Masson staining,and the skin thickness and collagen accumulation were observed.Results:1.Compared with the normal control group,the content of H₂S in serum of patients with SSc was significantly decreased and negatively correlated with skin sclerosis index(n_SSc=25,n_nc=28,p<0.01)and the expression of m RNA and protein of CSE in fibroblasts of SSc patients was also significantly down-regulated（n=5,p<0.05）.2.After SSc fibroblasts were treated with H₂S donor Na HS and GYY4137,the migration of SSc fibroblasts was decreased,and the m RNA expression of COL1A1 andα-SMA was also decreased,while PAG,an inhibitor of CSE,promoted the migration of SSc fibroblasts（n=3,p<0.05）.When PAG and CSE-si RNA were used to Inhibit the action and expression of CSE in normal fibroblasts,the function of normal fibroblasts increased and the m RNA and protein expression of COL1A1increased significantly.3.After SSc fibroblasts were treated with H₂S donor Na HS and GYY4137,the level of SMAD2/3 phosphorylation induced by TGF-βdecreased.4.Compared with the control group,the back skin of the BLM model group was significantly thickened and the collagen fibers also increased and thickened.In the model group,after Na HS intraperitoneal injection,the dermis became thinner and collagen fibers decreased significantly,while after injection of CSE inhibitor PAG,the skin thickened and collagen fibers increased and thickened.Conclusion:Endogenous H₂S/CSE pathway is significantly down-regulated in systemic scleroderma.H₂S donors may inhibit fibroblast migration and collagen production by inhibiting SMAD3 phosphorylation in vivo and in vitro,thus playing an important anti-fibrosis role in SSc fibrosis.Endogenous H₂S pathway may be a new target for the treatment of SSc fibrosis.