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.
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.
基金项目
国家自然科学基金(82274490)
安徽省高等学校科研重点项目(自然科学类)(2022AH050449)
安徽省第"115"创新团队项目(第十二批)(皖人才办[2019]1号)
安徽省名中医刘健工作室建设项目(中医药发展秘[2018]11号)
安徽省中医药领军人才项目(中医药发展秘[2018]23号)
国家科技期刊平台
NSTL
维普
万方数据