Integrating Network Pharmacology, Microbiomics, and Metabolomics to Uncover the Therapeutic Effect of Liubao Tea on Osteoarthritis

Background: Osteoarthritis (OA) is a debilitating joint disorder with no effective disease-modifying drugs. Liubao tea, a traditional Chinese dark tea, possesses various bioactivities, including anti-inflammatory and gut microbiota-regulating effects. However, its potential therapeutic role and mechanism of action in OA remain unexplored.Methods: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the chemical components of Liubao tea. Network pharmacology was employed to identify the active components and their potential targets in OA treatment. A destabilization of the medial meniscus (DMM)-induced mouse OA model was established and treated with low/high-dose Liubao tea extract. Micro-CT, histological staining (H&E, Safranin O-Fast Green), and ELISA were performed to evaluate joint structure, cartilage damage, and inflammatory cytokine levels. 16S rRNA sequencing, fecal microbiota transplantation (FMT), and untargeted serum metabolomics were conducted to explore gut microbiota and metabolic changes. Additionally, Brequinar, a de novo pyrimidine synthesis inhibitor, was used to verify the role of pyrimidine metabolism.Results: UPLC-MS/MS and network pharmacology analyses identified 1,989 metabolites in Liubao tea, including 273 bioactive components (e.g., flavonoids, lignans) that targeted 602 OA-related genes. Liubao tea treatment significantly ameliorated DMM-induced OA progression, as evidenced by improved subchondral bone microarchitecture (increased BV/TV, Tb.N, Tb.Th; decreased Tb.Sp), reduced cartilage erosion (lowered modified Mankin and OARSI scores), and suppressed systemic inflammation (decreased IL-6, IL-1beta, TNF-alpha levels). Liubao tea remodeled gut microbiota homeostasis (increased -diversity and altered bacterial taxa), and FMT from Liubao tea-treated mice recapitulated its anti-OA effects. Metabolomic analysis revealed that Liubao tea significantly downregulated the pyrimidine metabolism pathway, and Brequinar treatment mimicked its therapeutic benefits, confirming the role of pyrimidine metabolism suppression in OA alleviation.Conclusion: Liubao tea attenuates OA progression by modulating gut microbiota composition and inhibiting the pyrimidine metabolism pathway, highlighting its potential as a novel natural therapeutic strategy for osteoarthritis.

背景：骨关节炎（Osteoarthritis, OA）是一种致残性关节疾病，目前尚无有效的疾病修正治疗药物。六堡茶作为中国传统黑茶，具备多种生物活性，包括抗炎与调节肠道菌群的作用。但其在骨关节炎中的潜在治疗价值与作用机制仍未被探明。
方法：采用超高效液相色谱-串联质谱（Ultra-performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS）分析六堡茶的化学成分；通过网络药理学筛选其治疗骨关节炎的活性成分及潜在靶点。构建内侧半月板不稳（destabilization of the medial meniscus, DMM）诱导的小鼠骨关节炎模型，分别给予低、高剂量六堡茶提取物干预。通过Micro-CT、苏木精-伊红（Hematoxylin-Eosin, H&E）染色、番红O-固绿（Safranin O-Fast Green）染色及酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay, ELISA）评估关节结构、软骨损伤程度与炎症因子水平。采用16S rRNA测序、粪便菌群移植（fecal microbiota transplantation, FMT）及非靶向血清代谢组学探究肠道菌群与代谢变化；此外，使用布喹那（Brequinar）——一种嘧啶从头合成抑制剂，验证嘧啶代谢在骨关节炎进程中的作用。
结果：UPLC-MS/MS与网络药理学分析共鉴定出六堡茶中的1989种代谢物，其中273种为生物活性成分（如黄酮类、木脂素类），可靶向调控602个骨关节炎相关基因。六堡茶干预可显著缓解DMM诱导的骨关节炎进展：软骨下骨微结构得到改善（骨体积分数（Bone Volume/Tissue Volume, BV/TV）、骨小梁数量（Trabecular Number, Tb.N）、骨小梁厚度（Trabecular Thickness, Tb.Th）升高，骨小梁分离度（Trabecular Separation, Tb.Sp）降低），软骨侵蚀程度减轻（改良Mankin评分与OARSI评分降低），全身炎症反应得到抑制（白细胞介素-6（Interleukin-6, IL-6）、白细胞介素-1β（Interleukin-1β, IL-1β）、肿瘤坏死因子-α（Tumor Necrosis Factor-α, TNF-α）水平下调）。六堡茶可重塑肠道菌群稳态（提升菌群α多样性，改变菌群组成），将六堡茶干预小鼠的粪便菌群移植至模型小鼠体内可重现其抗骨关节炎作用。代谢组学分析显示，六堡茶可显著下调嘧啶代谢通路，而布喹那处理可模拟其治疗效果，证实抑制嘧啶代谢在骨关节炎缓解中发挥关键作用。
结论：本研究表明，六堡茶可通过调控肠道菌群组成并抑制嘧啶代谢通路延缓骨关节炎进展，为骨关节炎提供了一种新型天然治疗策略。