玻璃酸钠上调miR-92a-3p表达保护IL-1β诱导的软骨细胞损伤

Sodium Hyaluronate Upregulates the Expression of miR-92a-3p and Protects Chondrocytes from IL-1β-induced Injury

目的:探讨玻璃酸钠(HA)对白细胞介素1β(IL-1β)诱导的软骨细胞损伤的影响及作用机制。方法:取人软骨细胞第3代对数生长期的细胞进行试验。部分软骨细胞分别转染miR-92a-3p抑制剂(inhibitor)、miR-92a-3p模拟物(mimics)和相应对照(inhibitor-NC、mimics-NC)至48 h后,将细胞分为Control组(用PBS处理未经转染的细胞24 h)、IL-1β组(用0.1μL10 ng/ml的IL-1β诱导未经转染的细胞24 h)、IL-1βmimics-NC组和IL-1βmiR-92a-3p mimics组(用0.1μL10 ng/ml的IL-1β分别诱导经mimics-NC、miR-92a-3pmimics转染的细胞24 h)、IL-1β+HA组(用0.1μL 10 ng/ml的IL-1β诱导未经转染的细胞24 h后,加入0.1μL 100μg/ml HA培养24 h)、IL-1β+HA inhibitor-NC组和IL-1β+HA miR-92a-3p inhibitor组(用0.1μL 10 ng/ml的IL-1β分别诱导经inhibitor-NC、miR-92a-3p inhibitor转染的细胞24 h后,分别加入0.1μL 100μg/ml HA培养24 h)。用实时荧光定量PCR检测各组细胞中miR-92a-3p的表达,用四甲基偶氮唑盐法(MTT)法检测细胞活性,用流式细胞仪检测细胞凋亡,用Western Blot法检测基质金属蛋白酶3(MMP-3),基质金属蛋白酶13(MMP-13)、和Ⅱ型胶原纤维α1基因(COL2A1)蛋白水平的表达。结果:与Control相比,miR-92a-3p在IL-1β组中表达显著降低(P<0.01)。当转染miR-92a-3p mimics后,IL-1β诱导的软骨细胞miR-92a-3p表达升高、细胞活力增加、凋亡率降低、细胞中MMP-3及MMP-13蛋白水平降低、COL2A1蛋白水平升高(P<0.01)。HA可增加IL-1β诱导的软骨细胞中miR-92a-3p的表达、增加细胞活性、降低细胞凋亡率、降低细胞中MMP-3和MMP-13、升高COL2A1蛋白水平(P<0.05,P<0.01)。结论:HA可通过增加miR-92a-3p表达抑制IL-1β诱导的软骨细胞凋亡,增加细胞活性,保护IL-1β诱导的软骨细胞损伤。

Objective To explore the effect of hyaluronic acid sodium(HA)on IL-1β-induced chondrocyte injury and its mechanism.Methods The third-generation logarithmic growth cells of human chondrocytes were tested.Some chondrocytes were transfected with miR-92 a-3 p inhibitor,miR-92 a-3 p mimics and corresponding controls(inhibitor-NC and mimics-NC)for 48 hours.The cells were then randomly divided into a control group(with non-transfected cells treated with PBS for 24 hours),IL-1βgroup(with un-transfected cells induced with 0.1μL 10 ng/ml IL-1βfor 24 h),IL-1βmimics-NC group and IL-1βmiR-92 a-3 p mimics group(The cells transfected with mimics-NC and mir-92 a-3 p mimics were induced by IL-1βof 0.1μL 10 ng/ml for 24 h respectively),IL-1β+HA group(with untransfected cells were induced with IL-1βof 0.1μL 10 ng/m L for 24 h,and then cultured with HA of 0.1μL 100μg/mL for 24 h),IL-1β+HA inhibitor-NC group and IL-1β+HA miR-92 a-3 pinhibitor group(cells transfected with interactivator NC and miR-92 a-3 p inhibitor were induced with IL-1βof 0.1μL 10 ng/mL for 24 h,and HA of 0.1μL 100μg/m L was added for 24 h).The expression of miR-92 a-3 p and cell activity was detected using the real-time fluorescence quantitative PCR and methyl thiazolyl tetrazolium(MTT)assay.Moreover,the cell apoptosis was detected using the flow cytometry,and protein levels of matrix metalloproteinase-3(MMP-3),matrix metalloproteinase-13(MMP-13)and COL2 A1 were measured using Western Blotting.Results The average expression of Mir-92 A-3 p of the IL-1βgroup decreased significantly compared with the control group(P<0.01).After being transfected with miR-92 a-3 p mimics,the average expression of miR-92 a-3 p,cell viability and COL2 A1 protein level increased,while the apoptosis rate MMP-3 and MMP-13 protein levels decreased significantly(P<0.01).HA could increase the expression of miR-92 a-3 p,cell activity and protein level of COL2 A1,but decrease the apoptosis rate,as well as the expression of MMP-3 and MMP-13(P<0.05,P<0.01).Conclusion HA can inhibit IL-1β-induced chondrocyte apoptosis,increase cell activity and protect IL-1β-induced chondrocyte injury by increasing the expression of miR-92 a-3 p.