治尪汤通过成纤维样滑膜细胞Wnt/β-catenin通路抑制类风湿关节炎骨破坏的机制研究

Rheumatoid arthritis (RA) is a disease with high rate of disability which is lack of effective treatments to inhibit the progression of bone destruction. In TCM, the pathogenesis of RA bone destruction is a deficiency of the liver and kidney, with the evil invasion of wind, cold, dampness and heat. Bushen Quhan Zhiwang Decoction (briefly as Zhi Wang Decoction) with the function of invigorating liver and kidney, expelling cold, dampness and stasis, can effectively alleviate the symptoms of RA, delay the progress of bone destruction. It had been demonstrated that the mechanism may be related to its effect of inhibiting inflammatory of fibroblast-like synoviocytes and destructing factors. Thus we put forward a scientific hypothesis: Zhi Wang Decoction plays a role of anti-bone destruction based on regulating Wnt/β-catenin pathway in FLS cells in RA. Animal and cell experiments will be applied in this study to observe the effect of Zhi Wang Decoction on anti- bone destruction and the effect on the Wnt/ beta -catenin pathway，which to confirm the mechanism of the decoction regulating Wnt/β-catenin pathway in FLS. The effect on the upstream activator Wnt3a, upstream key inhibitors of DKK-1 and sFRP3 will be detected to reveal the possible mechanism of drugs regulating Wnt/ beta-catenin pathway. This study provides the experimental basis for the traditional Chinese medicine in the treatment of RA bone damage and it plays an important role in optimizing prescription and improving the curative effect of traditional Chinese medicine.

类风湿关节炎（RA）致残率高，目前治疗方法不能有效抑制骨破坏进展。RA骨破坏的病机为肝肾亏虚、风寒湿热诸邪外侵，具有补益肝肾、散寒除湿、祛瘀通络作用的补肾祛寒治尪汤（简称治尪汤）可有效缓解RA症状、延缓骨破坏进展，前期研究提示，其机制可能与抑制成纤维化滑膜细胞（FLS）炎症与骨破坏因子有关，据此提出科学假说：治尪汤调节RA的FLS细胞Wnt/β-catenin通路发挥抗骨破坏作用。本研究首先在体内和体外研究治尪汤抗骨破坏的作用，以及对Wnt/β-catenin通路的影响，证实治尪汤调节FLS的Wnt/β-catenin通路抗骨破坏的作用机制，在此基础上，研究药物对通路上游激动剂Wnt3a、上游关键抑制剂DKK-1和sFRP3的影响，揭示药物调节Wnt/β-catenin通路的可能作用途径。本研究为中药治疗RA骨破坏的作用提供实验依据，对于进一步优化处方、提高中药疗效具有重要作用。

动物实验运用胶原诱导关节炎（collagen-induced arthritis, CIA）大鼠模型，分为正常组、模型组、治尪汤组和甲氨蝶呤组，在初次免疫同时给药干预，治疗42天，结果显示，治尪汤显著抑制CIA模型大鼠炎症和骨破坏，抗炎起效时间比甲氨蝶呤快，能抑制大鼠膝关节TNF-α、IL-6、MMP13和β-catenin的mRNA和蛋白的表达，研究表明，RA存在wnt/β-catenin通路的活化，这与炎症和骨破坏可能有关。进而采用SW982细胞，分别用Wnt经典通路激动剂Wnt3a和促炎因子TNFα、ILIL-6、LPS刺激，以治尪汤含药血清干预。结果显示，Wnt3a刺激导致wnt5a、DKK1、RANKL、β-catenin mRNA表达均显著升高，β-catenin蛋白表达升高，治尪汤含药血清干预能减少DKK1、RANKL、β-catenin mRNA表达，减少β-catenin蛋白表达，但对MMP3、TNF-a、IL-6、RANK、RANKL、Wnt5a蛋白表达无作用，结果提示wnt/β-catenin通路激活可导致骨破坏因子和促炎因子的升高，治尪汤对Wnt/β-catenin的活化有抑制作用；促炎因子 TNF-a刺激可引起RANKL、IL-6和TNF-a、IL-8 mRNA表达的增高，但wnt3a、wnt5a、β-catenin的蛋白和mRNA表达无改变，治尪汤含药血清干预后，RANKL、IL-6和TNF-a、IL-8 mRNA表达显著降低，结果提示治尪汤对促炎因子激活的骨破坏因子RANKL和炎症因子IL-6和TNF-a、IL-8 有很好的抑制作用，细胞实验表明， 治尪汤可能通过抑制Wnt/β-catenin的活化对骨破坏因子起抑制作用。本研究表明：Wnt/β-catenin通路异常与RA炎症和骨破坏有关，治尪汤通过抑制Wnt/β-catenin通路活化，发挥抗炎抗骨破坏的作用。本研究对认识RA发病机制、改善RA骨破坏的治疗现状，具有一定作用。