鬼针草总黄酮联合甲氨蝶呤调控RANKL/RANK/OPG通路减缓类风湿关节炎骨破坏的作用机制研究

Rheumatoid arthritis(RA) is a systemic autoimmune disease with high prevalence in China. It can lead to severe disability mainly by destructing the bone. The aim of treatment is reducing or blocking bone destruction in RA patients. Methrotrexate (MTX)-based combined therapy has become consensus which can reduce bone destruction but the application in clinical practice is limited because of its liver toxicity. It’s urgent to find another effective and economic drug which has effect-enhancing and toxicity-reducing effect. Our previous studies found that TFB has anti-inflammatory and liver protective effects in CIA rats. Previous studies have demonstrated that RANK signal pathway plays a key role in bone destruction of RA. Hence, we propose a hypothesis: TFB combined with MTX could reduce bone destruction of RA through RANK signal pathway, ameliorate liver toxicity by inhibiting liver cell apoptosis, achieving a synergistic effect. This study aims to establish a rat model of CIA and culture osteoclasts in vitro, using multiple techniques including molecular biology, cell biology, pathology and imaging technology, to discuss the influence of inflammatory factor and key molecules in RANK pathway in synovial tissue and serum after intervention of TFB combined with MTX in CIA. Furthermore, we investigate the mechanism of TFB combined with MTX on RA bone destruction and liver protective effect, to provide the experimental basis for new combined therapy in treating RA.

我国类风湿关节炎（RA）罹患人群基数大，致残率高，骨破坏是其致残的主因，减轻或阻断骨破坏是RA的治疗目标。以甲氨蝶呤(MTX)为主的联合治疗方案已成共识，可减缓骨破坏，但因肝毒性而限制其临床应用。亟需寻找一种经济有效能与MTX有协同增效减毒的药物。本课题组前期研究发现鬼针草总黄酮(TFB)对胶原诱导关节炎大鼠（CIA）有抗炎及肝脏保护作用，既往已证实RANK信号通路在RA骨破坏中起关键作用。因此，我们提出假说：TFB联合MTX通过调控RANK通路减缓RA骨破坏，通过抑制肝细胞凋亡减轻肝毒性，发挥协同增效作用。本研究拟建立CIA大鼠模型并体外培养破骨细胞，采用分子生物学、细胞生物学、病理学和影像学等技术，研究TFB联合MTX干预CIA大鼠后其滑膜组织和血清中炎性因子、RANK通路关键分子及肝脏病理的影响。探讨TFB联合MTX对RA骨破坏及肝保护作用的机制，为RA联合治疗新方案提供实验依据。

类风湿关节炎(Rheumatoid arthritis RA)主要病理特征是滑膜慢性炎症及血管翳形成导致关节软骨及骨的破坏，减缓或阻止关节骨破坏的进程已成为类风湿关节炎（RA）治疗的重要目标之一。骨保护素(osteoprotegerin,OPG)、细胞核因子-κB受体活化因子配体(receptor activator of nuclear factor kappa B ligand, RANKL)、细胞核因子-κB受体活化因子(receptor activator of nuclear factor kappa B，RANK)三者互相作用，骨组织的局部骨形成和骨吸收处于动态平衡中，RANKL/OPG的比例是维持这个平衡的关键。鬼针草总黄酮（total flavonoids of Bidens bipinnata L.,TFB）具有广泛的生物活性，在本研究中我们通过建立胶原诱导性关节炎（CIA）大鼠的动物模型，予TFB进行干预，观察不同给药组CIA大鼠一般情况、关节肿胀、血清学、影像学、组织病理的改变，观察TFB对CIA大鼠的治疗效果。实验中CIA大鼠造模成功率达90%，结果提示TFB可减轻CIA大鼠关节肿胀度、关节炎评分、降低血清及关节组织中TNF，IL-1β和IL-17，减少RANKL增加OPG，使RANKL/OPG的比例下调，减轻CIA大鼠关节滑膜细胞增生、炎性细胞浸润及关节软骨破坏，降低X线影像学评分。实验结果证明：TFB可改善CIA大鼠关节炎症及骨破坏，通过降低TNF-α、IL-1β、IL-17等炎症因子的水平，降低RANKL，上调OPG的表达，通过调控RANKL/RANK/OPG信号通路减轻CIA大鼠的骨破坏。中药单体成分的药理作用及机制研究是目前中药研究领域的一个重要分支，因此我们在后续的研究中使用鬼针草总黄酮中有效的成分之一金丝桃苷的单体，对CIA大鼠滑膜成纤维细胞增殖及NF-KB信号通路的影响进行研究。细胞实验结果：金丝桃苷可抑制CIA大鼠滑膜成纤维细胞增殖，呈时间与浓度依赖性。金丝桃苷可以促进OPG表达，减少RANKL表达，抑制IL-6及TNF-a炎症细胞因子的产生，下调NF-KB、TAF6基因和蛋白的表达，其作用机制可能是通过抑制NF-KB信号通路的异常活化，从而抑制了类风湿关节炎滑膜细胞增生，减轻滑膜炎症。通过细胞实验证明金丝桃苷的药效作用。