Table_6_Synergistic Effects of Erzhi Pill Combined With Methotrexate on Osteoblasts Mediated via the Wnt1/LRP5/β-Catenin Signaling Pathway in Collagen-Induced Arthritis Rats.docx

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by chronic synovitis, bone erosion, and bone loss. Erzhi Pill (EZP), a classic Chinese patent medicine, is often used to treat osteoporosis and shows a capacity for bone metabolism regulation. Methotrexate (MTX), an essential drug for RA treatment, has been reported to inhibit generalized bone loss in RA patients. However, the combined therapeutic effects and mechanism of EZP and MTX in RA have not been fully elucidated. The aim of this study was to investigate the synergistic effect of EZP and MTX on RA and to explore the underlying mechanism through network pharmacological prediction and experimental verification. Chemical compounds of EZP, human target proteins of EZP and MTX, and RA-related human genes were identified in the Encyclopedia of Traditional Chinese Medicine database, PubChem database, and NCBI database, respectively. The molecular network of EZP and MTX in RA was generated and analyzed with Ingenuity Pathway Analysis software according to the datasets. Then, MTX monotherapy, EZP monotherapy, and combined MTX and EZP therapy were administered to collagen-induced arthritis rats, followed by assessment of pathological score, bone damage, bone alkaline phosphatases (BALP), and tartrate-resistant acid phosphatase (TRACP), and of gene levels related to the Wnt1/LRP5/β-catenin pathway according to network pharmacological analysis. Finally, serum samples from MTX-, EZP- and MTX+EZP-treated rats were used to treat the rat osteoblast (OB)-like UMR-106 cell line to evaluate gene levels related to Wnt1/LRP5/β-catenin. Network pharmacological analysis showed that the Wnt/β-catenin signaling pathway was the top signaling pathway shared among MTX, EZP, and RA. The results from in vivo experiments indicated that EZP combined with MTX reduced arthritis severity, alleviated ankle bone damage, increased BALP and decreased TRACP serum levels, and regulated the mRNA expression of Wnt1, LRP5, β-catenin, Runx2, BALP, and BGP in the ankles. In vitro experiments showed that EZP combined with MTX could also improve the expression of genes related to the Wnt1/LRP5/β-catenin pathway. This study demonstrated that EZP in combination with MTX played a synergistic role in regulating OBs in RA, which was connected to the modulatory effect of EZP and MTX on the Wnt1/LRP5/β-catenin signaling pathway.

类风湿关节炎（Rheumatoid arthritis, RA）是一类以慢性滑膜炎、骨侵蚀及骨丢失为核心特征的慢性全身性自身免疫性疾病。二至丸（Erzhi Pill, EZP）作为经典中成药，临床常用于治疗骨质疏松，且具备调节骨代谢的功效。甲氨蝶呤（Methotrexate, MTX）是类风湿关节炎治疗的核心用药，已有研究证实其可抑制RA患者的全身性骨丢失。然而，二至丸与甲氨蝶呤联合用于RA治疗的协同效应及潜在作用机制尚未完全阐明。本研究旨在探讨二至丸与甲氨蝶呤对RA的协同治疗作用，并通过网络药理学预测结合实验验证，解析二者联合用药的分子机制。
研究人员分别从中医百科数据库（Encyclopedia of Traditional Chinese Medicine）、PubChem数据库及NCBI数据库中，筛选得到二至丸的化学成分、二至丸与甲氨蝶呤的人类靶蛋白以及RA相关人类基因。基于上述数据集，采用Ingenuity Pathway Analysis软件构建并分析二至丸与甲氨蝶呤干预RA的分子调控网络。
随后，将胶原诱导性关节炎大鼠随机分为甲氨蝶呤单药组、二至丸单药组及联合给药组，通过大鼠踝关节病理评分、骨损伤程度评估、血清骨碱性磷酸酶（bone alkaline phosphatases, BALP）与抗酒石酸酸性磷酸酶（tartrate-resistant acid phosphatase, TRACP）水平检测，结合网络药理学分析结果，检测踝关节组织中Wnt1/LRP5/β-连环蛋白通路相关基因的mRNA表达水平。此外，收集三组大鼠的血清样本，体外培养大鼠成骨细胞样UMR-106细胞系，以此评估Wnt1/LRP5/β-连环蛋白通路相关基因的表达变化。
网络药理学分析结果显示，Wnt/β-连环蛋白信号通路是甲氨蝶呤、二至丸与RA三者共享的核心信号通路。体内实验结果表明，二至丸联合甲氨蝶呤可显著减轻关节炎严重程度，缓解踝关节骨损伤，升高血清BALP水平并降低TRACP含量，同时调控踝关节组织中Wnt1、LRP5、β-连环蛋白、Runx2、BALP及BGP的mRNA表达。体外实验进一步证实，二至丸与甲氨蝶呤联合给药可有效上调Wnt1/LRP5/β-连环蛋白通路相关基因的表达水平。
本研究证实，二至丸与甲氨蝶呤联合可通过调控Wnt1/LRP5/β-连环蛋白信号通路，对RA模型中的成骨细胞发挥协同调控作用，为二者联合治疗RA提供了实验依据与理论支撑。