淫羊藿苷对强直性脊柱炎成纤维细胞向成骨型分化的影响

Effect and Mechanism of Osteogenic Differentiation of Fibroblasts in Ankylosing Spondylitis under Intervention of Icariin

目的异位成骨是强直性脊柱炎病理过程的核心环节。成纤维细胞是AS异位成骨的靶细胞,BMP/Smad信号传导通路是骨形成过程中的一条重要通路。该研究通过观察淫羊藿苷对体外培养强直性脊柱炎成纤维细胞增殖和凋亡的作用,同期观察淫羊藿苷对体外培养AS成纤维细胞成骨表型表达及BMP/Smad信号通路蛋白表达的影响,初步探讨淫羊藿苷干预强直性脊柱炎异位成骨的分子机制。方法采用组织学原代培养法,建立人髋关节囊成纤维细胞系,应用免疫组化法鉴定细胞来源。以第3代对数生长期的AS成纤维细胞为研究对象,将实验分为AS空白对照组与淫羊藿苷小剂量组(25μg/mL)、中剂量组(50μg/mL)与大剂量组(100μg/mL)。MTT法检测淫羊藿苷干预下AS成纤维细胞的增殖情况;Annexin V法流式细胞术检测淫羊霍苷对AS成纤维细胞凋亡的影响。检测淫羊藿苷作用下AS成纤维细胞成骨表型的表达状况,观察其ALP活性、胶原合成及OC分泌量。Western blotting技术检测在不同剂量淫羊藿苷干预下,AS成纤维细胞BMP/Smad信号通路中Smad1、Smad5、Smad4及成骨标志基因Cbfa-1蛋白的表达情况。结果淫羊藿苷100μg/mL可明显抑制AS成纤维细胞增殖(P<0.05),且无细胞毒产生,各组细胞不同时间点增殖情况无显著差异;淫羊藿苷各剂量组对AS成纤维细胞凋亡均无明显作用。大剂量淫羊藿苷对AS成纤维细胞ALP活性、胶原合成及OC分泌均有明显抑制作用。与AS对照组相比,淫羊藿苷作用后AS成纤维细胞BMP/Smad信号通路Smad1、Smad4、pSmad1以及Cbfa-1蛋白表达受明显抑制(P<0.05)。结论淫羊藿苷能有效抑制AS成纤维细胞增殖,且抑制AS成纤维细胞成骨表型的表达,其分子机制可能是淫羊藿苷通过干预BMP/Smad信号通路活化,从而调节成骨标志基因Cbfa-1表达,抑制AS成纤维细胞向成骨型分化,可能是淫羊藿苷干预AS异位成骨的机制之一。

Objective Ectopic osteogenesis is the core of the pathological process of ankylosing spondylitis.Fibroblasts are the target cells of ectopic osteogenesis.The BMP/Smad signal transduction pathway is an important pathway in the process of bone formation.We observed the effect of icariin on proliferation and apoptosis of cultured ankylosing spondylitis fibroblasts in vitro,and the influence of the expression of osteoblastic phenotype of fibroblasts and the protein of BMP/Smad signaling pathway.It preliminarily discussed the molecular mechanism of icariin in the treatment of ectopic ossification of ankylosing spondylitis.Methods By using histological method of primary culture,hip capsule fibroblast cell line of human was established.Immunohistochemistry was used to identify the origin of the cell.The third generation of AS fibroblasts in logarithmic growth phase was used as the research object.The experiment was divided into AS blank control group and icariin small dose group(25 μg/mL),middle dose group(50 μg/mL) and large dose group(100 μg/mL).MTT assay was used to detect the proliferation of AS fibroblasts under the intervention of icariin.Annexin V flow cytometry was used to detect the effect of icariin on the apoptosis of fibroblasts.The expression of osteogenic phenotype of AS fibroblasts was observed.The activity of alkaline phosphatase(ALP),collagen synthesis and osteocalcin(OC) secretory volume in clear supernatant liquid were tested.The expressions of Smad1,Smad5,Smad4 and bone marker gene Cbfa-1 protein in the BMP/Smad signaling pathway of AS fibroblasts under the intervention of icariin were tested by Western blotting.Results Icariin with a dose of 100 μg/mL could significantly inhibit the proliferation of AS fibroblasts(P<0.05),and there was no cytotoxicity or significant difference in cell proliferation at different time points in each group.Icariin had no significant effect on apoptosis of AS fibroblasts in all dose groups.Large doses of icariin inhibited the activity of ALP,collagen synthesis and OC secretion in AS fibroblasts.Compared with the AS control group,the expressions of Smad1,Smad5,Smad4 and bone marker gene Cbfa-1 protein in the BMP/Smad signaling pathway of AS fibroblasts under the intervention of icariin were significantly inhibited(P<0.05).Conclusion Icariin can effectively inhibit the proliferation of AS fibroblasts and reduce the osteoblastic phenotype expression of AS fibroblasts.The molecular mechanism may be that icariin can regulate the expression of Cbfa-1 gene and inhibit the differentiation of AS fibroblasts into osteoblasts by inhibiting the activation of BMP/Smad signaling pathway,which may be one of the mechanisms of icariin intervening AS heterotopic ossification.