基于LXRs/NF-κB通路探讨痹痿同治的壮骨健膝方干预KOA滑膜炎的机制

Knee osteoarthritis(KOA) has the pathogenesis feature of "coexistence of impediment and wilting". The synovial inflammation and bone hyperplasia belong to "impediment syndrome" in Traditional Chinese Medicine(TCM); The cartilage degeneration and soft tissue atrophy belong to "wilting syndrome" in TCM. Zhuanggu Jianxi Decoction plays an crucial role in "Co-treatment of impediment and wilting", which can tonify the liver and kidney, strengthen the bones and tendons in the treatment of wilting syndrome and eliminate the wind and dampness, dredge the meridian in the treatment of impediment syndrome. Our previous projects of National Natural Science Foundation has shown that the decoction, from the perspective of curing the wilting syndrome, can protect the articular cartilage by many ways. So, how does it inhibit synovitis? The studies have shown that liver X receptors (LXRs) can regulate the NF-κB pathway and affect the release of inflammatory factors in type A synoviolytes, which have become the potential target for screening KOA synovitis control drugs. Now we put forward the hypothesis, Zhuanggu Jianxi Decoction may inhibit KOA synovitis by affecting the LXRs/NF-κB pathway, interfering with the expression of IL-1β, TNF-α and other cytokines. To verify the hypothesis, our reasearch group will use the multi-mode KOA synovitis models in rabbits and humans to study the intervention effect of the decoction and its effect on the expression of the related proteins and genes in the upstream and downstream of LXRs/NF-κB pathway by adopting the multiple node blocking control method from cell, tissue to integer, further clarify the targets and mechanism of its effect, and enrich the scientific connotation of the therapeutic principle of "Co-treatment of impediment and wilting" and lay a foundation for the promotion of the decoction.

膝骨关节炎（KOA）具有“痹痿同病”的病机特点，滑膜炎症、骨端增生等属“痹”，软骨退化、软组织萎缩等属“痿”。壮骨健膝方具有“痹痿同治”功效，以补肝肾强筋骨治痿，祛风湿通经络治痹，疗效显著。我们前期国家自然科学基金项目从治痿角度研究表明该方可多途径保护关节软骨。那么该方如何抑制滑膜炎呢？研究表明，A型滑膜细胞上的肝X受体（LXRs）能调节NF-κB通路，影响炎症因子释放，已成为筛选KOA滑膜炎防治药物潜在的靶标。现我们提出假说：壮骨健膝方可能通过影响LXRs/NF-κB通路，干预IL-1β、TNF-α等表达而抑制KOA滑膜炎。为验证假说，本研究拟借助兔与人多模式KOA滑膜炎模型，从整体、组织、细胞等层面，采用多节点阻滞对照观察方法，多角度研究该方对LXRs/NF-κB通路上下游相关蛋白及基因表达的影响，进一步明确其作用的靶点与环节，丰富“痹痿同治”治则的科学内涵，并为该方推广应用奠定基础。

本研究借助兔与人多模式膝骨关节炎（KOA）滑膜炎模型，从整体、组织、细胞等层面，采用多节点阻滞对照观察方法，多角度研究该方对LXRs/NF-κB通路上下游相关蛋白及基因表达的影响，进一步丰富该方针对KOA痹痿同治中“除痹”功效的科学内涵。首先从动物层面，通过观察壮骨健膝方对KOA滑膜炎模型兔一般状态，膝关节液中IL-1β、TNF-α、MMP-3、MMP-13的含量，膝关节滑膜组织形态，滑膜组织中LXRα、N-CoR、P50、P65蛋白及mRNA表达的影响，结果提示：该方能通过调控肝X受体/核因子κB信号通路的传导，减少下游炎症因子分泌，达到抑制KOA滑膜炎症反应的作用。接着从组织层面，通过观察该方含药血清对KOA滑膜炎兔膝滑膜及KOA滑膜炎患者膝滑膜LXRs/NF-κB通路LXRα、N-CoR、P50、P65蛋白表达及下游IL-1β、TNF-α、MMP3、MMP-13含量的影响，结果提示：该方可改善滑膜内衬层细胞增生及排列紊乱、炎性细胞浸润及纤维组织增生等病理表现，减少组织中炎症相关指标的含量，从而减轻兔及人KOA滑膜组织炎症反应，这可能与其上调滑膜组织中滑膜细胞LXRs/NF-κB通路上LXRα、N-CoR蛋白表达，从而下调P50、P65蛋白，降低通路的活性，进而减少IL-1β、TNF-α、MMP-3、MMP-13等下游产物有关。最后通过细胞层面，观察该方对脂多糖(Lipopolysaccharide，LPS)刺激的兔及人膝A型滑膜细胞LXRs/NF-κB信号通路中关键节点蛋白与mRNA表达及细胞上清液中IL-1β、TNF-α、MMP-3及MMP-13含量的影响，结果提示：该方可通过上调LXRα和N-CoR蛋白及mRNA表达，下调P50、P65蛋白及mRNA表达，从而抑制LXRs/NF-κB信号通路的传导，减少下游炎症因子的分泌