目的 探讨川芎嗪对高湿环境骨关节炎（high humidity osteoarthritis，HHOA）模型大鼠的治疗作用及潜在机制。方法 建立HHOA大鼠模型，给予川芎嗪干预8周后，通过番红O-固绿染色观察软骨组织形态学变化；检测血清中基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9）、基质金属蛋白酶抑制因子-1（tissue inhibitors of metalloproteinase-1，TIMP-1）、前列腺素内过氧化物合酶2（prostaglandin-endoperoxide synthase 2，PTGS2）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）和超氧化物歧化酶（superoxide dismutase，SOD）的水平；通过普鲁士蓝染色检测软骨组织的铁沉积情况；免疫组化法检测软骨组织中GPX4和长链脂酰辅酶A合成酶4（acyl-CoA synthetase long-chain family member 4，ACSL4）蛋白表达；结合血清非靶向代谢组学分析代谢物变化及其富集的通路。体外构建软骨细胞铁死亡模型，给予主要代谢物N-乙酰基血清素干预后，测定GSH/氧化型谷胱甘肽（oxidized glutathione，GSSG）值、MDA和SOD水平，采用Western blotting检测GPX4和ACSL4蛋白的表达。结果 与HHOA组比较，川芎嗪组大鼠血清中MMP-9、PTGS2和MDA水平明显降低（P＜0.05、0.01），血清中TIMP-1、GPX4、SOD和GSH水平明显升高（P＜0.05、0.01），关节软骨的病理损伤得到明显改善，关节软骨中铁离子沉积减少，GPX4蛋白表达增加，ACSL4蛋白表达减少。代谢组学分析结果显示，HHOA组与假手术组之间共筛选出2 695个差异代谢物，其中下调代谢物数量显著多于上调代谢物；川芎嗪高剂量组与HHOA组之间共筛选出2 846个差异代谢物，以上调代谢物为主。KEGG通路富集分析结果显示，HHOA组与假手术组差异代谢物主要富集于碳代谢和氨基酸代谢等多条代谢通路，川芎嗪与HHOA组差异代谢物主要富集于与氨基酸代谢及物质转运密切相关的多条通路。与假手术组比较，HHOA组大鼠血清中γ-谷氨酰亮氨酸、3-吲哚丙酸和N-乙酰基血清素水平显著降低（P＜0.05）；与HHOA组比较，川芎嗪高剂量组γ-谷氨酰亮氨酸、3-吲哚丙酸和N-乙酰基血清素水平显著升高（P＜0.05）。体外实验结果显示，主要代谢物N-乙酰基血清素显著提高软骨细胞铁死亡模型GSH/GSSG值和SOD活力（P＜0.05），降低MDA水平（P＜0.05），上调GPX4蛋白和下调ACSL4蛋白的表达（P＜0.05、0.01）。结论 川芎嗪能通过改善HHOA大鼠的代谢组学谱，调节以N-乙酰基血清素为代表的色氨酸代谢途径，缓解GPX4/ACSL4轴介导的脂质过氧化及铁离子沉积，从而减轻软骨细胞铁死亡并延缓HHOA的进展。

Objective To investigate the therapeutic effects and potential mechanisms of tetramethylpyrazine (TMP) in a high humidity osteoarthritis (HHOA) rat model. Methods The HHOA rat model was established, and rats were given TMP for eight weeks. Safranin O-fast green staining was used to observe morphological changes in cartilage tissue. The levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1), prostaglandin-endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4), glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured. Prussian blue staining was performed to detect iron deposition in cartilage. Immunohistochemistry was used to assess the expressions of GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) in cartilage. Serum untargeted metabolomics was performed to analyze changes in metabolites and their enriched pathways. In vitro, a ferroptosis model of chondrocytes was established and treated with main metabolite N-acetylserotonin. GSH/oxidized glutathione (GSSG) value, MDA and SOD levels were measured, the expressions of GPX4 and ACSL4 were measured by Western blotting. Results Compared with HHOA group, levels of MMP-9, PTGS2 and MDA in serum of rats in TMP group were significantly reduced (P < 0.05, 0.01), while the levels of TIMP-1, GPX4, SOD and GSH in serum were significantly increased (P < 0.05, 0.01). The pathological damage to the articular cartilage was significantly improved, and the deposition of iron ions in articular cartilage was reduced, GPX4 protein expression was increased, and ACSL4 protein expression was reduced. Metabolomics analysis showed that 2 695 differential metabolites were screened between HHOA group and sham group, with significantly more down-regulated metabolites than up-regulated metabolites; A total of 2 846 differential metabolites were screened between TMP high-dose group and HHOA group, with up-regulated metabolites being the main ones. KEGG pathway enrichment analysis results showed that the differential metabolites between HHOA group and sham group were mainly enriched in multiple metabolic pathways such as carbon metabolism and amino acid metabolism, the differential metabolites between TMP group and HHOA group were mainly enriched in multiple pathways closely related to amino acid metabolism and substance transport. Compared with sham group, the levels of γ-glutamylleucine, 3-indoolepropionic acid and N-acetylseletonin in serum of HHOA group rats were significantly reduced (P < 0.05). Compared with HHOA group, the levels of γ-glutamylleucine, 3-indoolepropionic acid and N-acetylseletonin in TMP high-dose group were significantly increased (P < 0.05). The in vitro experimental results showed that the main metabolite N-acetylserotonin significantly increased the GSH/GSSG value and SOD activity in ferroptosis model of chondrocytes (P < 0.05), decreased MDA level (P < 0.05), up-regulated GPX4 protein and down-regulated ACSL4 protein expressions (P < 0.05, 0.01). Conclusion TMP could improve the metabolomic profile of HHOA rats, regulate the tryptophan metabolism pathway represented by N-acetylserotonin, alleviate GPX4/ASCL4 axis mediated lipid peroxidation and iron ion deposition, thereby reducing ferroptosis in chondrocytes and delaying the progression of HHOA.

R285.5

国家自然科学基金资助项目（82305263）;国家自然科学基金资助项目（82575098）;国家自然科学基金资助项目（82205147）;广东省基础与应用基础研究基金资助项目（2025A1515010393，2023A1515012626）;广州市中医药重大科技项目（2025CX002）;广州中医药大学校院联合科技创新基金资助项目（GZYSE2024U01，GZYFT2024G08）;广东省中医药防治难治性慢病重点实验室开放课题（KF2023MB03）;国家中医药管理局郭程湘全国名老中医药专家传承工作室[国中医药人教发（2016）41]