麝香酮通过调控微小RNA-223表达减轻白细胞介素-1β诱导的软骨细胞损伤

Muscone reduces interleukin-1β-induced chondrocyte injury by regulating microRNA-223 expression

目的探讨麝香酮对白细胞介素(IL)-1β诱导的软骨细胞损伤的影响及分子机制。方法将人软骨细胞分为对照组、IL-1β组、IL-1β+麝香酮低、中、高剂量组、IL-1β+anti-微小RNA(miR)-NC组、IL-1β+anti-miR-223组、IL-1β+麝香酮+miR-NC组、IL-1β+麝香酮+miR-223组。检测IL-6、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)水平;流式细胞术检测细胞凋亡;蛋白质印迹法(Western blot)检测B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X(bax)蛋白表达;实时荧光定量聚合酶链反应(RT-qPCR)检测miR-223表达水平。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果 IL-1β+麝香酮低、中、高剂量组软骨细胞IL-6[(13.24±1.18)、(8.34±0.83)、(4.71±0.43) ng/L]、TNF-α[(36.24±3.78)、(23.47±2.47)、(10.95±1.17) ng/L]、IFN-γ[(56.47±5.59)、(41.34±5.56)、(26.88±2.12) ng/L]水平低于IL-1β组,且呈剂量依赖性,差异有统计学意义(F=367.902、390.005、181.019,P<0.05)。IL-1β+麝香酮低、中、高剂量组软骨细胞凋亡率[(26.39±2.35)%、(18.71±1.25)%、(11.06±1.03)%]和bax表达水平(0.71±0.06、0.57±0.04、0.39±0.03)低于IL-1β组,bcl-2表达水平(0.35±0.03、0.47±0.04、0.61±0.05)高于IL-1β组,且呈剂量依赖性,差异有统计学意义(F=293.382、193.076、201.190,P<0.05)。IL-1β+麝香酮低、中、高剂量组软骨细胞中miR-223表达水平低于IL-1β组,且呈浓度依赖性,差异有统计学意义(2.69±0.22、2.03±0.21、1.41±0.13比3.35±0.24,F=122.850,P<0.05)。IL-1β+麝香酮+miR-223组miR-223、IL-6、TNF-α、IFN-γ、细胞凋亡率和bax表达高于IL-1β+麝香酮+miR-NC组,bcl-2表达水平低于IL-1β+麝香酮+miR-NC组,差异有统计学意义(t=21.226、18.782、21.394、16.912、18.254、16.100、18.522,P<0.05)。结论麝香酮可能通过下调miR-223减轻IL-1β诱导的软骨细胞损伤。

Objective To explore the effect and molecular mechanism of muscone on interleukin(IL)-1β-induced chondrocyte injury.Methods Human chondrocytes were divided into control group,IL-1βgroup,IL-1β+muscone low,medium and high dose group,IL-1β+anti-microRNA(miR)-NC group,IL-1β+anti-miR-223 group,IL-1β+muscone+miR-NC group,IL-1β+muscone+miR-223 group.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of IL-6,tumor necrosis factor-α(TNF-α)and interferonγ(IFN-γ).Flow cytometry was used to detect apoptosis.Western blotting was used to detect the expression of B-cell lymphoma/leukemia-2(bcl-2)and bcl-2 related X(bax)protein.The real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-223.Single-factor variance analysis was used and LSD-t tests were used for statistical analysis.Results The IL-6[(13.24±1.18),(8.34±0.83),(4.71±0.43)ng/L],TNF-α[(36.24±3.78),(23.47±2.47),(10.95±1.17)ng/L]and IFN-γ[(56.47±5.59),(41.34±5.56),(26.88±2.12)ng/L]in low,median,high dosage-muscone+IL-1βgroup were significantly lower than in the IL-1βgroup(F=367.902,390.005,181.019,P<0.05).The apoptosis rate[(26.39±2.35)%,(18.71±1.25)%,(11.06±1.03)%],and bax expression(0.71±0.06,0.57±0.04,0.39±0.03)in low,median,high dosage muscone+IL-1βgroup were significantly lower than in the IL-1βgroup,but the bcl-2 expression was significantly higher(F=293.382,193.076,201.190,P<0.05).The miR-223(2.59±0.21 vs.1.00±0.08),IL-6[(12.12±1.11)ng/L vs.(4.69±0.42)ng/L],TNF-α[(42.97±4.42)ng/L vs.(10.47±1.11)ng/L],IFN-γ[(58.72±5.64)ng/L vs.(24.34±2.32)ng/L],apoptosis rate[(25.96±2.25)%vs.(10.80±1.07)%]and bax expression(0.74±0.06 vs.0.38±0.03)in IL-1β+muscone+miR-223 group were significantly higher than in the IL-1β+muscone+miR-NC group,while bcl-2 expression(0.28±0.03 vs.0.64±0.05)was significantly lower(t=21.226,18.782,21.394,16.912,18.254,16.100,18.522,P<0.05).Conclusion Muscone may reduce IL-1β-induced chondrocyte injury by down-regulating miR-223.