杜鹃素上调靶向基因表达抑制白细胞介素-1β诱导的软骨细胞损伤

Study of Farrerol on the Inhibition of IL-1β-induced Chondrocyte Injury by Upregulating miR-499a-5p Expression

目的:探讨杜鹃素对白细胞介素-1β(IL-1β)诱导的软骨细胞损伤的影响及其可能作用机制。方法:IL-1β诱导人正常软骨细胞建立细胞损伤模型,不同浓度的杜鹃素处理软骨细胞,miR-NC、miR-499a-5p mimics分别转染至软骨细胞后加入10 ng/mL IL-1β处理24 h,anti-miR-NC、anti-miR-499a-5p分别转染至软骨细胞后加入90μmol/L杜鹃素与10 ng/mL IL-1β共同处理24 h;ELISA法检测IL-6、TNF-α、IFN-γ的水平;流式细胞术检测细胞凋亡率;qRT-PCR法检测miR-499a-5p的表达量;Western Blot检测凋亡相关蛋白表达量。结果:杜鹃素处理后IL-1β诱导的软骨细胞中IL-6、TNF-α、IFN-γ的水平降低(P<0.05),凋亡率、Cleaved-caspase3、Cleaved-caspase9蛋白水平降低(P<0.05),miR-499a-5p的表达量升高(P<0.05),且呈剂量依赖性;转染miR-499a-5p mimics后IL-6、TNF-α、IFN-γ的水平降低(P<0.05),凋亡率、Cleaved-caspase3、Cleaved-caspase9蛋白水平降低(P<0.05);转染anti-miR-499a-5p可降低杜鹃素对IL-1β诱导的软骨细胞炎症反应和凋亡的影响。结论:杜鹃素可通过增加miR-499a-5p表达而抑制炎症反应及细胞凋亡从而减轻IL-1β诱导的软骨细胞损伤。

Objective:To explore the efficacy of farrerol on interleukin-1β(IL-1β)-induced chondrocyte damage and its possible mechanism.Methods:Human normal chondrocyte was induced by IL-1βto establish a cell injury model.Chondrocyte was treated by different concentrations of farrerol.The miR-NC and miR-499a-5p mimics were transfected into chondrocytes and treated with 10 ng/mL IL-1βfor 24 h.The anti-miR-NC and anti-miR-499a-5p were transfected into chondrocytes and treated with 90μmol/L farrerol and 10 ng/mL IL-1βfor 24 h.ELISA method was used to detect the levels of IL-6,TNF-α,IFN-γ.Flow cytometry was used to detect the rate of apoptosis;qRT-PCR was used to detect the expression of miR-499a-5p.Western Blot was used to detect the protein expression of cleaved-caspase3 and cleaved-caspase9.Results:After farrerol treatment,the levels of IL-6,TNF-αand IFN-γin chondrocytes induced by IL-1βwere decreased(P<0.05),and the apoptosis rate and the protein levels of cleaved-caspase3,cleaved-caspase9 were decreased(P<0.05),while the expression of miR-499a-5p was increased(P<0.05),and was dose-dependent.After transfection of miR-499a-5p mimics,the levels of IL-6,TNF-α,and IFN-γwere decreased(P<0.05),and the rate of cell apoptosis and the protein levels of cleaved-caspase3,cleaved-caspase9 were decreased(P<0.05).Transfection of anti-miR-499a-5p could reduce the effect of farrerol on IL-1β-induced inflammation and apoptosis of chondrocytes.Conclusion:Farrerol could inhibit the inflammation and apoptosis by up-regulating the expression of miR-499a-5p,thereby reducing IL-1β-induced chondrocyte injury.