从Dkk/Wnt信号通路探讨清热活血方药治疗类风湿关节炎的“护骨”机制

Rheumatoid arthritis (RA) is to control the synovial inflammation, and inhibit bone destruction for the treatment of the target. Heat, dampness, and blood stasis are important factors to the bone destruction oh active RA. Clinical research confirmed Qingrehuoxue Decoction could delay RA bone destruction, in addition the natural fund research showed that it could inhibit IL-17, cut RANKL expression, raised OPG express, which regulated the OPG/RANKL/RANK pathways, restrained osteoclast function, finally the bone destruction delayed. It has been initially proved the Qngrehuoxue Decoction "protect bone" scientific hypothesis. Study also found that, not only in the role of Qingrehuoxue Decoction to regulate osteoclast action and may also to regulate osteoblast action, and then correct into osteoclast and osteoblast imbalance. "Osteo immunology " consider that the imbalance of osteoclast and osteoblast is core mechanism of RA bone destruction, Wnt signal pathway is the core of osteoblast adjustment function, and Dkk-1 as its inhibitor, also is the RA disease activity and bone destruction biological marker. In this study, the bone marrow mesenchymal stem cells and PBMC trained technologies, from Dkk/Wnt signaling pathways of mRNA, genes, protein levels, multi-level Qingrehuoxue Decoction formulas balance of osteoblasts to the role of further interpret its "protect bone" theory scientific content.

类风湿关节炎(RA)以控制滑膜炎、抑制骨破坏为治疗目标。湿热瘀是活动期RA骨破坏重要因素,临床研究证实清热活血方药具有延缓RA骨破坏的作用，前期自然基金研究表明,清热活血方药通过抑制IL-17,下调RANKL表达,上调OPG表达,从而调节OPG/RANKL/RANK通路,抑制了破骨细胞功能,延缓了骨破坏,初步证实了清热活血方药"护骨"的科学假说。研究中还发现,清热活血方药不仅作用于破骨细胞,也可能对成骨细胞起作用,进而纠正成破骨细胞失衡。"骨免疫"学说认为成破骨细胞平衡被打破是RA骨破坏核心机制,Wnt信号通路是调节成骨细胞功能的核心,Dkk-1为其抑制剂,也是RA疾病活动和骨破坏的生物标志物。本研究采用骨髓间充质干细胞与PBMC共培养技术,从Dkk/Wnt信号通路入手,从基因、mRNA、蛋白水平多层次探讨清热活血方药在骨平衡中对成骨细胞所起的作用,进一步诠释其"护骨"理论的科学内涵。

本课题基于“骨免疫”学说，以临床应用有效之清热活血方药，采用 CIA 大鼠模型，（1）进行类风湿关节炎（RA）的病理形态学观察；（2）利用成纤维滑膜细胞与外周血单个核细胞共培养体系，观察骨保护素（OPG）、IL-17 拮抗对成骨细胞分化的作用；（3）运用 Wnt 双荧光素酶报告基因检测 PBMC 中 Wnt 信号途径的活性，采用实时定量逆转录-聚合酶链反应(RT-PCR)研究方法，检测 DKK－1 和β-catenin 等因子在分子及 mRNA水平的表达；（4）采用 westen-blot 检测， 采用 Laemmli 法进行 SDS-PAGE 电泳，检测血清β-catenin、 LRP 5 和 TCF 等含量。结果表明：清热活血方药通过调节 OPG、IL-17、β-catenin，从而调整了 DKK/Wnt信号通路，延缓了RA关节骨破坏。本研究结果诠释了清热活血方药治疗 RA的抗炎与减轻骨破坏作用的内在机制，揭示了其对 RA 活动期“护骨”作用途径和科学内涵，证实了湿热瘀血是 RA 骨破坏的病机关键、清热活血中药可以“护骨”的科学假说，使 RA中医治疗的基础理论有一定的突破，丰富了中医治疗RA理论，是中医治疗学在基础研究中的重要理论探索。