灵仙新苷对膝骨关节炎软骨细胞凋亡和自噬的影响

Effects of Clematichinenoside AR on Apoptosis and Autophagy of Knee Osteoarthritis Chondrocytes

目的观察灵仙新苷(C-AR)对体外培养的膝骨关节炎(KOA)软骨细胞凋亡及自噬的影响,探讨其改善KOA的作用机制。方法从接受全膝关节置换术的KOA患者软骨组织提取软骨细胞,通过甲苯胺蓝及Ⅱ型胶原免疫荧光染色鉴定软骨细胞。将细胞分为对照组、模型组和C-AR组,对照组不予干预,模型组和C-AR组采用白细胞介素(IL)-1β诱导24 h构建KOA模型,C-AR组造模前用30μmol/L C-AR预处理1 h。CCK8法检测细胞活力,ELISA检测细胞IL-6和IL-8含量,流式细胞仪检测活性氧(ROS)含量和细胞凋亡率,Western blot检测Bax、Caspase-3、Bcl-2、Beclin1、LC33B蛋白表达,qRT-PCR检测Bax、Caspase-3、Bcl-2 mRNA表达,免疫荧光检测LCB表达。结果与对照组比较,模型组软骨细胞活力显著降低(P<0.01),IL-6和IL-8含显著增加(P<0.01),ROS含量和细胞凋亡率显著升高(P<0.01),Bax、Caspase-3 mRNA和蛋白表达显著升高(P<0.01),Bcl-2 mRNA和蛋白表达显著降低(P<0.01),Beclin1、LC3B蛋白表达及LC3BⅡ/LC3BⅠ比值显著降低(P<0.01);与模型组比较,C-AR组软骨细胞活力显著升高(P<0.05),IL-6和IL-8含量显著减少(P<0.05),ROS含量和细胞凋亡率显著降低(P<0.01),Bax、Caspase-3 mRNA和蛋白表达显著降低(P<0.01),Bcl-2 mRNA和蛋白表达显著升高(P<0.01),Beclin1、LC3B蛋白表达及LC3BⅡ/LC3BⅠ比值显著升高(P<0.01)。结论C-AR可减轻体外培养的KOA软骨细胞炎症反应,降低细胞凋亡率,其机制可能与激活自噬有关。

Objective To observe the effects of clematichinenoside AR(C-AR)on apoptosis and autophagy of chondrocytes of knee osteoarthritis(KOA)in vitro;To explore its mechanism of improving KOA.Methods The chondrocytes were extracted from KOA patients undergoing total knee arthroplasty,and identified by toluidine blue and typeⅡcollagen immunofluorescence staining.Cells were divided into control group,model group and C-AR group.The control group was non-intervened,the model group and C-AR group were induced by interleukin(IL)-1βfor 24 h to establish KOA model.The C-AR group was pretreated with 30μmol/L C-AR for 1 h before modeling.The cell activity was detected by CCK8 method,the contents of IL-6 and IL-8 were detected by ELISA,the content of reactive oxygen species(ROS)and apoptosis rate were detected by flow cytometry,the expressions of Bax,Caspase-3,Bcl-2,Beclin1 and LC3B protein were detected by Western blot,the mRNA expressions of Bax,caspase-3 and Bcl-2were detected by qRT-PCR,the expression of LC3B was detected by immunofluorescence.Results Compared with the control group,the activity of chondrocytes in the model group significantly reduced(P<0.01),the contents of IL-6and IL-8 significantly increased(P<0.01),the content of ROS and apoptosis rate increased significantly(P<0.01),the expression of Bax and Caspase-3 mRNA and protein increased significantly(P<0.01),the expression of Bcl-2 mRNA and protein decreased significantly(P<0.01),and the expression of Beclin1 and LC3B protein the ratio of LC3BⅡ/LC3BⅠdecreased significantly(P<0.01).Compared with the model group,the activity of chondrocytes in C-AR group significantly increased(P<0.05),the contents of IL-6 and IL-8 significantly reduced(P<0.05),the content of ROS and apoptosis rate decreased significantly(P<0.01),the expression of Bax and Caspase-3 mRNA and protein decreased significantly(P<0.01),the expression of Bcl-2 mRNA and protein increased significantly(P<0.01),the expression of Beclin1 and LC3B protein and the ratio of LC3BⅡ/LC3BⅠincreased significantly(P<0.01).Conclusion C-AR can reduce the inflammatory reaction of KOA chondrocytes cultured in vitro and decrease the apoptosis rate,and its mechanism may be related to the activation of autophagy.