杯苋甾酮通过上调miR-204表达抑制IL-1β诱导的软骨细胞凋亡的实验研究

Cyasterone Inhibits IL-1βInduced Chondrocyte Apoptosis by Up-regulating MiR-204 Expression

目的:探讨杯苋甾酮对微小RNA(miR)-204表达及白细胞介素(IL)-1β诱导的软骨细胞凋亡的影响。方法:体外培养人正常软骨细胞C28/I2,设置①对照组(不做处理)、IL-1β组(50mg/L IL-1β)、低剂量杯苋甾酮组(2.5mg/L杯苋甾酮+50mg/L IL-1β)、中剂量杯苋甾酮组(5mg/L杯苋甾酮+50mg/L IL-1β)和高剂量杯苋甾酮组(10mg/L杯苋甾酮+50mg/L IL-1β);②正常对照组(不转染)、阴性对照组(转染NC-siRNA)和miR-204 siRNA组(转染miR-204 siRNA),三组均加入50mg/L IL-1β、10mg/L杯苋甾酮处理;流式细胞仪检测各组C28/I2细胞凋亡情况;酶联免疫(ELISA)法检测各组C28/I2细胞上清液中炎症因子IL-6、IL-8、肿瘤坏死因子α(TNF-α)水平;实时荧光定量PCR(qRT-PCR)法检测各组C28/I2细胞中miR-204表达情况;蛋白印迹(WB)法检测各组C28/I2细胞中细胞色素C(Cytochrome C)、Bcl-2相关X蛋白(Bax)、半胱天冬酶-3(Caspase-3)蛋白表达情况。结果:与对照组相比,IL-1β组C28/I2细胞活力、miR-204表达水平显著降低(P<0.05),细胞凋亡率、IL-6、IL-8、TNF-α表达水平及Cytochrome C、Bax、Caspase-3蛋白表达水平显著升高(P<0.05);与IL-1β组相比,随着杯苋甾酮剂量的增加,C28/I2细胞活力、miR-204表达水平逐次升高(P<0.05),细胞凋亡率、IL-6、IL-8、TNF-α表达水平及Cytochrome C、Bax、Caspase-3蛋白表达水平逐次降低(P<0.05);与阴性对照组相比,miR-204 siRNA组相比凋亡率显著升高(P<0.05)。结论:杯苋甾酮可能通过上调miR-204表达对IL-1β诱导的软骨细胞凋亡具有抑制作用。

Objective:To investigate the effects of Cyasterone on the expression of microRNA(miR)-204 and the apoptosis of chondrocytes induced by interleukin(IL)-1β.Methods:Human normal chondrocytes C28/I2 were cultured in vitro,and set up①control group(no treatment),IL-1βgroup(50 mg/L IL-1β),low-dose Cyasterone group(2.5 mg/L Cyasterone+50 mg/L IL-1β),medium dose Cyasterone group(5 mg/L Cyasterone+50 mg/L IL-1β)and high dose Cyasterone group(10 mg/L Cyasterone+50 mg/L IL-1β);②normal control group(no transfection),negative control group(transfect NC-siRNA)and miR-204 siRNA group(transfect miR-204 siRNA),three groups were adminstered 50 mg/L IL-1βand 10mg/L Cyasterone.The proliferation of C28/I2 cells was detected by CCK-8 method;the apoptosis of C28/I2 cells was detected by flow cytometry;the levels of IL-6,IL-8 and tumor necrosis factor-α(TNF-α)in the supeRNAtant of C28/I2 cells were detected by enzyme-linked immunosorbent assay(ELISA);the expression of miR-204 in C28/I2 cells was detected by real-time fluorescent quantitative PCR(qRT-PCR);in addition,the protein expressions of Cytochrome C,Bcl-2-related X protein(Bax)and Caspase-3 in C28/I2 cells were detected by Western blot(WB).Results:Compared with those in control group,the cell viability and miR-204 expression level of C28/I2 cells in IL-1βgroup were significantly lower(P<0.05),and the apoptosis rate,the expression levels of IL-6,IL-8 and TNF-α,the protein expression levels of Cytochrome C,Bax and Caspase-3 were significantly higher(P<0.05);moreover,compared with those in the IL-1βgroup,with the addition of Cyasterone and the increase of its dosage,the cell viability and the expression level of miR-204 in C28/I2 cells increased gradually(P<0.05),and the apoptosis rate,the expression levels of IL-6,IL-8 and TNF-α,the protein expression levels of Cytochrome C,Bax and Caspase-3 decreased gradually(P<0.05).Compared with negative control group,the apoptosis rate of miR-204 siRNA group were significantly increased(P<0.05).Conclusion:Cyasterone may inhibit the IL-1β-induced apoptosis of chondrocytes by up-regulating the expression of miR-204.