DataSheet1_Network analysis combined with pharmacological evaluation strategy to reveal the mechanism of Tibetan medicine Wuwei Shexiang pills in treating rheumatoid arthritis.docx|藏药数据集|类风湿性关节炎治疗数据集

Tibetan medicine is an important part of traditional Chinese medicine and a significant representative of ethnic medicine in China. Tibetan medicine is gradually recognized by the world for its unique curative effects. Wuwei Shexiang pills (WPW) has been widely used to treat “Zhenbu” disease (Also known as rheumatoid arthritis) in Tibetan medicine, however, its potential bioactive ingredients and mechanism for RA treatment remain unclear. In this study, we used a combination of gas chromatography-mass spectrometry (GC-MS), ultra-performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC-Q-TOF/MS), network analysis and experimental validation to elucidate the potential pharmacodynamic substances and mechanisms of WPW in the treatment of rheumatoid arthritis (RA). The results showed that songoramine, cheilanthifoline, saussureanine C, acoric acid, arjunolic acid, peraksine, ellagic acid, arjungenin and other 11 components may be the main activities of WPW in the treatment of RA. PIK3CA, AKT, MAPK, IL-6, TNF, MMP1, MMP3, and CDK1 are considered as core targets. PI3K-AKT, MAPK, apoptosis, cell cycle, and other signaling pathways may be the key pathways for WPW to play a role in the treatment of RA. Furthermore, we validated the underlying molecular mechanism of WPW predicted by network analysis and demonstrated its possible mechanism through in vivo animal experiments. It was found that WPW could significantly improve the degree of paw swelling, and reduce ankle joint diameter and arthritis index. Further histomorphological analysis showed that WPW could reduce the degree of synovial tissue inflammation and ankle joint cartilage damage. Meanwhile, WPW could down-regulate the levels of IL-6, IL-1β, and IL-17, and increase the levels of IL-10 and IL-4 in the serum of AA rats. TUNEL staining confirmed that WPW could significantly promote the apoptosis of synovial cells. Moreover, the immunohistochemical results showed that WPW decreased the expression of PI3K, AKT, MAPK, MMP1, MMP3, CDK1, and Bcl-2, as well as increased the expression of Bax protein. In conclusion, we successfully combined GC-MS, UPLC-Q-TOF/MS, network analysis, and experimental validation strategies to elucidate the inhibition of inflammation by WPW in AA model rats via PI3K/AKT, MAPK, cell cycle and apoptotic pathways process. This not only provides new evidence for the study of potential pharmacodynamic substances and the mechanism of WPW in the treatment of RA, but also provides ideas for the study of other Tibetan medicine compound preparations.

藏药作为中医的重要组成部分，以及中国民族医药的杰出代表，日益受到世界的认可，其独特的治疗效果备受瞩目。乌维石香丸（WPW）在藏医药中广泛用于治疗“真布”病（亦称类风湿关节炎），然而，其潜在的生物活性成分及对类风湿关节炎的治疗机制尚不明确。本研究通过结合气相色谱-质谱联用（GC-MS）、超高效液相色谱串联四极杆飞行时间质谱（UPLC-Q-TOF/MS）、网络分析和实验验证等方法，旨在阐明WPW在治疗类风湿关节炎中的潜在药效物质和作用机制。研究结果表明，松果胺、鸡骨草素、草果胺C、熊果酸、阿胶酸、帕拉辛、没食子酸、阿胶苷等11种成分可能是WPW治疗类风湿关节炎的主要活性成分。PIK3CA、AKT、MAPK、IL-6、TNF、MMP1、MMP3、CDK1被认为是核心靶点。PI3K-AKT、MAPK、细胞凋亡、细胞周期等信号通路可能为WPW在治疗类风湿关节炎中发挥作用的关键途径。此外，我们验证了网络分析预测的WPW潜在分子机制，并通过体内动物实验展示了其可能的机制。研究发现，WPW可显著改善足肿胀程度，减少踝关节直径和关节炎指数。进一步的病理形态学分析表明，WPW可减轻滑膜组织炎症和踝关节软骨损伤。同时，WPW可下调AA大鼠血清中IL-6、IL-1β和IL-17的水平，并提高IL-10和IL-4的水平。TUNEL染色证实，WPW可显著促进滑膜细胞的凋亡。此外，免疫组化结果显示，WPW降低了PI3K、AKT、MAPK、MMP1、MMP3、CDK1和Bcl-2的表达，并增加了Bax蛋白的表达。综上所述，我们成功地将GC-MS、UPLC-Q-TOF/MS、网络分析和实验验证等策略相结合，阐明WPW通过PI3K/AKT、MAPK、细胞周期和凋亡途径抑制AA模型大鼠炎症的机制。这不仅为WPW潜在药效物质和治疗机制的研究提供了新的证据，也为其他藏药复方制剂的研究提供了新的思路。