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目的:通过体外细胞模型，探讨蠲痹汤对骨关节炎（OA）的作用机制。方法:将ATDC5细胞分为4组：正常组、OA组、蠲痹汤组和Fer-1组。OA组加入10 ng/ml白细胞介素（IL）-1β诱导炎症杜尔伯科改良伊格尔（DMEM）培养基；蠲痹汤组加入IL-1β＋蠲痹汤诱导炎症DMEM培养基；Fer-1组加入IL-1β＋Fer-1诱导炎症DMEM培养基。进行蛋白质印迹法（Western blot）试验检测软骨细胞的肿瘤坏死因子-α（TNF-α）、诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2），Collagen Ⅱ、Aggrecan、基质金属蛋白酶13（MMP-13）、p53、ACSL4、SLC7A11和GPX-4的表达，使用细胞免疫荧光染色检测GPX-4的表达。利用化学发光法检测软骨细胞的代谢产物丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH）、Fe 2＋、线粒体内铁的含量。多组比较采用单因素方差分析。 结果:Fer-1组和蠲痹汤组的TNF-α（3.282±0.261、2.255±0.253）、iNOS（5.046±0.478、3.443±0.039）、COX-2（3.484±0.333、2.450±0.467）蛋白相对表达水平低于IL-1β组（4.384±0.601、6.863±0.637、4.812±0.496），差异有统计学意义（ t＝3.848、7.432、5.579、10.500、4.289、7.629， P<0.05）。Fer-1组和蠲痹汤组的MMP-13（2.738±0.224、4.276±0.663）、p53（3.364±0.898、4.289±0.936）、ACSL4（2.897±0.767、2.776±0.594）蛋白相对表达水平低于IL-1β组（5.779±0.018、7.413±1.635、6.077±0.847），差异有统计学意义（ t＝10.640、5.258、4.752、3.666、6.048、6.279， P<0.05）。Fer-1组和蠲痹汤组的Collagen Ⅱ（0.752±0.143、0.609±0.073）、Aggrecan（0.663±0.139、0.570±0.022）、SLC7A11（0.623±0.037、0.400±0.034）、GPX-4（0.686±0.041、0.537±0.065）蛋白相对表达水平高于IL-1β组（0.298±0.049、0.268±0.154、0.097±0.021、0.163±0.063），差异有统计学意义（ t＝4.661、3.895、4.631、3.538、23.700、13.660、12.890、9.226， P<0.05）。Fer-1组和蠲痹汤组的GPX-4（15.476±2.519、12.133±0.703）荧光强度高于IL-1β组（5.141±0.153），差异有统计学意义（ t＝8.117、5.491， P<0.05）。Fer-1组和蠲痹汤组的线粒体内铁（9.689±2.680、11.043±1.818）荧光强度低于IL-1β组（16.604±3.262），差异有统计学意义（ t＝3.675、2.955， P<0.05）。Fer-1组和蠲痹汤组的MDA[（8.755±0.390）、（10.915±0.379） nmol/ml]、Fe 2＋[（4.200±0.041）、（5.189±0.083） nmol]含量低IL-1β组[（13.130±0.430） nmol/ml、（7.109±0.071） nmol]，差异有统计学意义（ t＝14.960、7.573、59.230、39.090， P<0.05）。Fer-1组和蠲痹汤组的GSH[（7.215±0.382）、（6.988±1.246） μmol/g prot]含量高于IL-1β组[（4.826±0.426） μmol/g prot]，差异有统计学意义（ t＝4.240、3.838， P<0.05）。 结论:蠲痹汤通过调控SLC7A11-GSH-GPX4轴抑制软骨细胞铁死亡减轻炎性反应和细胞外基质降解。

Objective:The mechanism of action of osteoarthritis (OA) induced by Juanbi decoction is investigated in this test using an in vitro cell model.Methods:The ATDC5 cells were divided into four groups: the normal group, theinterleukin (IL) -1β group, the IL-1β+ Juanbi decoction group, and the IL-1β+ Fer-1 group. In the IL-1β group, Dulbecco’s modified Eagle’s medium (DMEM) was used to induce inflammation with 10 ng/ml IL-1β. The IL-1β+ Juanbi decoction group was treated with DMEM medium to induce inflammation. The IL-1β+ Fer-1 set was added to induce inflammation in the DMEM medium.The expression of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), CollagenⅡ, Aggrecan, matrix metalloproteinase-13 (MMP-13), p53, ACSL4, SLC7A11 and GPX-4 in chondrocytes was assessed via Western blotting. GPX-4 expression was evaluated through immunofluorescence staining. Chemiluminescence was utilized to measure the levels of malondialdehyde (MDA), glutathione peroxidase (GSH), Fe 2+, and mitochondrial iron in chondrocytes. One-way analysis of variance was employed to compare multiple groups. Results:The relative protein expression levels of TNF-α (3.282±0.261, 2.255±0.253), iNOS (5.046±0.478, 3.443±0.039), and COX-2 (3.484±0.333, 2.450±0.467) in the IL-1β+ Fer-1 group and IL-1β+ Juanbi decoction group were significantly lower compared to the IL-1β group (4.384±0.601, 6.863±0.637, 4.812±0.496). The differences were statistically significant with t-values of 3.848, 7.432, 5.579, 10.5, 4.289, and 7.629 respectively ( P<0.05). The relative protein expression levels of MMP-13 (2.738±0.224, 4.276±0.663), p53 (3.364±0.898, 4.289±0.936), and ACSL4 (2.897±0.767, 2.776±0.594) in the IL-1β+ Fer-1 group and IL-1β+ Juanbi decoction group were significantly lower compared to the IL-1β group (5.779±0.018, 7.413±1.635, 6.077±0.847). The differences were statistically significant with t-values of 10.64, 5.258, 4.752, 3.666, 6.048, and 6.279 respectively ( P<0.05). The relative protein expression levels of CollagenⅡ (0.752±0.143, 0.609±0.073), Aggrecan (0.663±0.139, 0.570±0.022), SLC7A11 (0.623±0.037, 0.400±0.034), and GPX-4 (0.686±0.041, 0.537±0.065) in the IL-1β+ Fer-1 group and IL-1β+ Juanbi decoction group were significantly higher compared to the IL-1β group (0.298±0.049, 0.268±0.154, 0.097±0.021, 0.163±0.063). The differences were statistically significant with t-values of 4.661, 3.895, 4.631, 3.538, 23.7, 13.66, 12.89 and 9.226 respectively ( P<0.05). The fluorescence intensity of GPX-4 (15.476±2.519, 12.133±0.703) in the IL-1β+ Fer-1 group and IL-1β+ Juanbi decoction group exhibited a statistically significant increase compared to that in the IL-1β group (5.141±0.153, t=8.117, 5.491, P<0.05). The fluorescence intensity of mitochondria iron (9.689±2.680, 11.043±1.818) in the IL-1β+ Fer-1 group and IL-1β+ Juanbi decoction group exhibited a statistically significant decline compared to that in the IL-1β group (16.604±3.262, t=3.675, 2.955, P<0.05). The levels of MDA [ (8.755±0.390), (10.915±0.379) nmol/ml] and Fe 2+ [ (4.200±0.041), (5.189±0.083) nmol] in the IL-1β+ Fer-1 group and IL-1β+ Juanbi decoction group were significantly lower compared to the IL-1β group [ (4.200±0.041), (5.189±0.083) nmol]. The differences were statistically significant with t-values of 14.96, 7.573, 59.230 and 39.090 respectively ( P<0.05). The level of GSH [ (7.215±0.382), (6.988±1.246) μmol/g prot] in the IL-1β+ Fer-1 group and IL-1β+ Juanbi decoction group were significantly higher compared to the IL-1β group [ (4.826±0.426) μmol/g prot]. The differences were statistically significant with t-values of 4.24 and 3.838 respectively ( P<0.05) . Conclusion:The Juanbi decoction exerts inhibitory effects on chondrocyte ferroptosis and attenuates inflammation and extracellular matrix degradation by modulating the SLC7A11-GSH-GPX4 axis.