Evaluation Of Influence Of Wenyang Huoxue Tongluo Medicine On Expression Of TGF-β1, MMP-9 And MCP-1 In Mice And The Skin Fibroblasts With Bleomvcin-incuced Scleroderma

Objective: Evaluate the influence of Wenyang Huoxue Tongluo(WHT) medicine on expression of TGF-β1, MMP-9 and MCP-1 in mice with bleomycin-induced scleroderma(BIS) and Cultured primary skin fibroblasts.Methods: 50 female Balb/c mice were used in this study. Bleomycin was injected into the subcutaneous tissue of 45 mice to establish BIS models, as well as 5 as normal controls. The BIS mice were fed with WHT, prednisone, WHT plus prednisone or water only as control for 28 days. The pathological changes in the skin and lung tissue of mice were evaluated with HE staining and Masson staining. The level of TGF-β1, MMP-9 and MCP-1 in serum was detected with ELISA, as well as which in the skin tissue was detected with immunohistochemistry. The skin fibroblasts from BIS mice were cultured in medium with or without C3 f, and then WHT was added with different dose and duration. The influence of WHT on proliferation of skin fibroblasts was evaluated by MTT assay. The level of TGF-β1,MMP-9 and MCP-1 in supernatant of cultured cells was detected with ELISA.Results:(1)The microscopic changes in BIS skin such as the increased total skin thickness, accumulated type I collagen, thickened vascular wall and narrowed vessels,as well as induced skin appendages under the dermis, accompanied with dominant inflammatory cells infiltration, were observed by HE and Masson staining. The pathological changes in pulmonary tissue were also observed in BIS mice. Alveolar interval became significantly thickened with lots of inflammatory cell infiltration and collagen deposition. Thickened small blood vessel wall made the vessel narrowed and even obstructed. However, the pathological changes in BIS mice treated with WHT or prednisone were more mild than in that ones without treatment(P<0.05,respectively).(2)The location and amount of TGF- β1,MCP-1 and MMP-9 in skin tissue were different in BIS mice. Expression of TGF-β1 and MCP-1 in BIC mice skin tissue were significantly decreased in WHT or WHT+prednisone treated groups compared to no-treated ones(P<0.05, respectively). Contrastly, MMP-9 level in skin tissues was increased in treated groups, especially in WHT+prednisone one.(3) In serum of BIS mice, TGF-β1 and MCP-1 increased significantly than in normal control(P <0.01). And after treated with WHT or WHT+prednisone, the level of TGF-β1and MCP-1 were significantly decreased(P <0.05), compared with no-treated one.Like influence of WHT on expression of MMP-9 in skin tissue, which also interestingly increased in WHT treated groups.(4)The levels of TGF-β1 and MCP-1in supernatant of skin fibroblasts co-cultured with C3 f were significantly higher than which without C3f(P < 0.05). However, no change was found about the level of MMP-9 with or without C3f(P> 0.05). WHT also decreased the level of TGF-β1 and MCP-1, while MMP-9 increased, which dependent on the dose.(P <0.05).Conclusion: WHT can improve the pathological changes in skin and pulmonary tissue of BIS mice, which might partially result from that WHT down-regulate TGF-β1 and MCP-1 as well as up-regulate MMP-9 in skin fibroblasts from BIS mice.