目的 探讨芍药苷通过调控花生四烯酸5-脂氧合酶（arachidonate 5-lipoxygenase，ALOX5）表达介导铁死亡，从而缓解类风湿性关节炎（rheumatoid arthritis，RA）进展的作用机制。方法 通过GeneCards数据库获得RA靶点，CTD和SwissTargetPrediction数据库获得芍药苷靶点，收集GEO数据库GSE77298数据集中表达上调的差异表达基因，对RA靶点、表达上调的差异表达基因与芍药苷靶点取交集，获得交集靶点。通过String网站和Cytoscape软件获得蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络，根据度值算法筛选获得靶基因ALOX5。采用分子对接技术验证芍药苷与ALOX5的结合能力。将ALOX5的干扰RNA（siRNA）、ALOX5过表达质粒（oeALOX5）和阴性对照组（siNC/oeNC）转染至RA成纤维样滑膜细胞（RA-fibroblast like synovial cells，RA-FLS）中，设置siNC、siALOX5组、对照组、芍药苷（50 μg/mL）组、芍药苷＋oeNC组、芍药苷＋oeALOX5组。采用CCK-8、流式细胞术和Transwell实验检测细胞活力、凋亡、迁移及侵袭情况；采用ELISA检测炎症因子白细胞介素-6（interleukin-6，IL-6）、IL-1β和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平；采用试剂盒检测Fe²⁺和铁含量；采用Western blotting检测ALOX5和铁死亡相关蛋白谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）表达。结果 ALOX5在RA-FLS细胞中的表达显著高于人成纤维样滑膜细胞（P＜0.001）。干扰ALOX5抑制RA-FLS细胞迁移和侵袭（P＜0.001），并促进细胞凋亡（P＜0.001）。网络药理学分析发现ALOX5是芍药苷的靶基因。与对照组比较，芍药苷显著下调ALOX5的表达（P＜0.001），抑制细胞活力、迁移、侵袭和炎症反应（P＜0.001），促进细胞凋亡（P＜0.001），降低Fe²⁺和铁含量（P＜0.001），上调GPX4表达（P＜0.001）；过表达ALOX5显著逆转了芍药苷对RA-FLS细胞的作用（P＜0.05、0.01、0.001）。结论 芍药苷通过调控ALOX5表达介导铁死亡，从而缓解RA进展，为RA治疗提供了重要理论依据和新的方法。

Objective To explore the mechanism by which paeoniflorin alleviates the progression of rheumatoid arthritis (RA) by regulating the expression of arachidonate 5-lipoxygenase (ALOX5) and mediating ferroptosis. Methods RA targets were obtained from GeneCards database, paeoniflorin targets were obtained from CTD and SwissTargetPrediction databases, and up-regulated differentially expressed genes were collected from the GSE77298 dataset in GEO database. The intersection of RA targets, up-regulated differentially expressed genes and paeoniflorin targets was obtained. A protein-protein interaction (PPI) network was obtained through String website and Cytoscape software, and the target gene ALOX5 was screened based on the degree algorithm. Molecular docking technology was used to verify the binding ability of paeoniflorin with ALOX5. ALOX5 interfering RNA (siRNA), ALOX5 overexpression plasmid (oeALOX5) and negative control group (siNC/oeNC) were transfected into RA-fibroblast like synovial cells (RA-FLS), siNC, siALOX5 group, control group, paeoniflorin (50 μg/mL) group, paeoniflorin + oeNC group, and paeoniflorin + oeALOX5 group were set up. Cell viability, apoptosis, migration and invasion were detected using CCK-8, flow cytometry and Transwell assays; ELISA was used to detect the levels of inflammatory factors interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α); Kit was used to detect Fe²⁺ and iron contents; Western blotting was used to detect the expressions of ALOX5 and ferroptosis associated protein glutathione peroxidase 4 (GPX4). Results The expression of ALOX5 in RA-FLS cells was significantly higher than that in human fibroblast like synovial cells (P < 0.001). Interference with ALOX5 inhibited migration and invasion of RA-FLS cells (P < 0.001), and promoted cell apoptosis (P < 0.001). Network pharmacology analysis revealed that ALOX5 was the target gene of paeoniflorin. Compared with control group, paeoniflorin significantly down-regulated the expression of ALOX5 (P < 0.001), inhibited cell viability, migration, invasion and inflammatory response (P < 0.001), promoted cell apoptosis (P < 0.001), reduced Fe²⁺ and iron contents (P < 0.001), and up-regulated GPX4 expression (P < 0.001); Overexpression of ALOX5 significantly reversed the effect of paeoniflorin on RA-FLS cells (P < 0.05, 0.01, 0.001). Conclusion Paeoniflorin mediates ferroptosis by regulating ALOX5 expression, thereby alleviating the progression of RA and providing important theoretical basis and new methods for RA treatment.

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河南省科技攻关项目(242102310010);河南省医学科技攻关计划联合共建项目(LHGJ20230890);河南省高等学校重点科研项目(26B310003)