南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (1): 108-118.doi: 10.12122/j.issn.1673-4254.2024.01.13

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新风胶囊抑制软骨细胞炎症和细胞外基质降解：基于调控miR-502-5p/TRAF2/NF-κB轴

周 巧，刘 健，万 磊，朱 艳，齐亚军，胡月迪   

  1. 安徽中医药大学第二附属医院，安徽 合肥 230061；安徽中医药大学第一附属医院，安徽 合肥 230031；安徽中医药大学，安徽 合肥 230012
  • 发布日期:2024-01-24

Xinfeng Capsule alleviates interleukin-1β-induced chondrocyte inflammation and extracellular matrix degradation by regulating the miR-502-5p/TRAF2/NF-κB axis

ZHOU Qiao, LIU Jian, WAN lei, ZHU Yan, QI Yajun, HU Yuedi   

  1. Second Affiliated Hospital, Anhui University of Chinese Medicine, Hefei 230061, China; First Affiliated Hospital, Anhui University of Chinese Medicine, Hefei 230031, China; Anhui University of Chinese Medicine, Hefei 230012, China
  • Published:2024-01-24

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摘要： 目的 探讨新风胶囊（XFC）对白介素（IL）-1β诱导的软骨细胞功能的影响及作用机制。方法 构建IL-1β诱导的软骨细胞炎症模型；SD大鼠灌胃制备XFC含药血清。细胞增殖实验（CCK-8）和流式细胞术筛选最佳XFC含药血清浓度；双荧光素酶报告分析miR-502-5p和TRAF2的靶向关系。构建miR-502-5p抑制表达质粒（inhibitor）及阴性对照组，转染至软骨细胞中。分为对照组（NC），IL-1β 组，XFC组，IL-1β+NC-inhibitor，IL-1β+miR-502-5p inhibitor，IL-1β+miR-502-5p inhibitor+XFC最佳含药血清组，酶联免疫吸附试验（ELASA）检测IL-1β、肿瘤坏死因子-α（TNF-α）、IL-4、IL-10的水平。逆转录PCR（RT-PCR）、蛋白质免疫印迹实验（WB）、免疫荧光法检测Ⅱ型胶原α1（COL2A1）、基质金属蛋白酶13（MMP13）、软骨蛋白聚糖抗体（ADAMTS5）和miR-502-5p/TRAF2/NF-κB基因的表达。结果 与NC组相比，IL-1β组中软骨细胞活力下降，细胞凋亡率升高，且miR-502-5p、IL-4、IL-10和COL2A1表达下降，IL-1β、TNF-α和ADAMTS5、MMP13、TRAF2、NF-κB p65表达升高（P<0.05）。筛选得出20%XFC 为最佳含药血清浓度。与 IL-1β组相比，XFC 含药血清干预后，软骨细胞活力升高，细胞凋亡率下降，IL-1β、TNF-α、ADAMTS5、MMP13、TRAF2、NF-κB p65表达下降，miR-502-5p、IL-4、IL-10和COL2A1表达升高（P<0.05）。与NC-inhibitor 相比，转染miR-502-5p inhibitor后，IL-1β、TNF-α、ADAMTS5、MMP13、TRAF2、NF-κB p65表达升高，miR-502-5p、IL-4、IL-10和COL2A1表达下降；与miR-502-5p inhibitor相比，将miR-502-5p inhibitor转染的软骨细胞和最佳XFC含药血清共培养后，XFC含药血清能逆转miR-502-5p抑制状态对软骨细胞炎症和ECM的影响。结论 XFC抑制软骨细胞炎症、细胞外基质降解，减轻IL-1β诱导的软骨细胞损伤，其机制可能是通过调节miR-502-5p/TRAF2/NF-κB轴。

关键词: miR-502-5p；骨关节炎；新风胶囊；炎症；细胞外基质

Abstract: Objective To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule (XFC) on interleukin (IL)-1β-induced impairment of chondrocytes. Methods XFC-medicated serum was collected from SD rats with XFC gavage, and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry. Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2. In cultured human chondrocytes induced with IL-1β, the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum, alone or in combination, on expression levels of IL-1β, tumor necrosis factor-α (TNF-α), IL-4, and IL-10 were examined with ELISA, and the changes in the expressions of collagen type II alpha 1 (COL2A1), matrix metalloproteinase 13 (MMP13), adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR, Western blotting, and immunofluorescence assay. Results In cultured human chondrocytes, treatment with IL-1β significantly decreased the cell viability, increased cell apoptosis rate, lowered miR-502-5p, IL-4, IL-10, and COL2A1 expressions, and enhanced IL-1β, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 expressions (P<0.05), and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20% (P<0.05). Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1β, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 and lowered expressions of miR-502- 5p, IL-4, IL-10, and COL2A1, and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor. Conclusion XFC can inhibit IL-1β-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Key words: miR-502-5p; osteoarthritis; Xinfeng Capsule; inflammation; extracellular matrix