丹参酮ⅡA通过影响BMP/Smad信号对强直性脊柱炎异位成骨的干预作用

Mechanism of Tanshinone ⅡA on Ectopic Osteogenesis of Ankylosing Spondylitis by Affecting BMP/Smad Signal

目的成纤维细胞向成骨细胞的分化是AS异位成骨的关键。该研究通过观察丹参酮ⅡA对强直性脊柱炎成纤维细胞增殖及BMP/Smad信号通路蛋白表达的影响,探讨其抑制强直性脊柱炎成纤维细胞向成骨型分化的分子机制。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空白对照组与丹参酮ⅡA不同浓度组(12.5μg/mL、25μg/mL、50μg/mL、75μg/mL、100μg/mL)。MTT法检测丹参酮ⅡA培养24 h、48 h、72 h后,AS成纤维细胞的增殖情况。Western blotting技术检测在丹参酮ⅡA 25μg/mL、50μg/mL、75μg/mL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。结果丹参酮ⅡA 25μg/mL、50μg/mL、75μg/mL对AS成纤维细胞增殖有明显抑制作用,且在24 h最明显;与AS对照组相比,丹参酮ⅡA作用后AS成纤维细胞BMP/Smad信号通路Smad1、Smad4、pSmad5以及Cbfa-1蛋白表达受明显抑制(P<0.05)。结论丹参酮ⅡA能有效抑制AS成纤维细胞增殖,并通过抑制BMP/Smad信号通路蛋白及成骨标志基因Cbfa-1表达抑制AS成纤维细胞向成骨型分化,可能是丹参酮ⅡA干预AS异位成骨的机制之一。

Objective The differentiation of fibroblastsinto osteoblasts is the key to AS ectopic osteogenesis.To observe the effect of tanshinone ⅡA on the proliferation and the expression of BMP/Smad signaling protein in fibroblasts of ankylosing spondylitis and investigate the molecular mechanism of its inhibition on osteogenic differentiation of ankylosing spondylitis fibroblasts.Methods Using histological method of primary culture, hip capsule fibroblast cell line of human was set up and immunohistochemistry was used to identify the cell source.Based on the third generation of the logarithmic phase of AS fibroblasts as the research object, the experiment was divided into AS controlgroup and different concentrations of tanshinoneIIA groups(12.5 μg/mL,25 μg/mL,50 μg/mL).We observed the proliferation of AS fibroblasts under the culture of the tanshinoneIIA after 24 h, 48 h and 72 h by MTT methodand the expressions of Smad1,Smad5,Smad4 and bone marker gene Cbfa-1 protein in the BMP/Smad signaling pathway of AS fibroblasts by Western blotting.Results Tanshinone ⅡA groups(12.5 μg/mL,25 μg/mL,50 μg/mL) inhibited the cell proliferation of AS,and it was the most obviousin 2 h.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 tanshinoneIIA was significantly inhibited(P<0.05).Conclusion TanshinoneIIA can inhibit AS proliferation of fibroblasts, and by inhibiting the expressions of BMP/Smad signaling pathway and osteogenic marker genes Cbfa-1 to decrease differentiation of fibroblast cells to osteoblast, which may be one of the mechanisms of tanshinone IIA against AS ossification.