补骨颗粒含药血清对大鼠软骨细胞凋亡及Trx2信号通路的影响

Effects of Bugu Granule drug-containing serum on rat chondrocyte apoptosis and Trx2 signaling pathway

目的观察补骨颗粒含药血清对体外培养的大鼠膝关节软骨细胞凋亡及对Trx2、ASK1及Caspase3表达的影响,从而探讨补骨颗粒预防骨性关节炎发生发展的作用机制。方法采用两步酶消化法分离培养大鼠软骨细胞,并进行传代培养。应用膜联蛋白V-FITC/PI染色后经流式细胞仪检测软骨细胞的凋亡情况。同时,通过电泳分离蛋白并通过蛋白质印迹分析Trx2、ASK1及Caspase3的表达情况。结果软骨细胞培养15 d左右铺满80%~90%的培养皿,大部分细胞呈梭形。流式细胞检测结果显示空白血清组的细胞凋亡率为22.80%,明显高于含药血清组(P<0.05),而20%含药血清组(15.91%)与30%含药血清组(17.93%)的细胞凋亡率又明显低于10%含药血清组(21.58%),各组间比较差异具有统计学意义(P<0.05)。蛋白质印迹分析结果显示含药血清组Trx2的表达量都明显增多,以10%含药血清组的表达量最多;空白组与10%含药血清组的ASK1与Caspase3的表达量比20%与30%含药血清组多。结论补骨颗粒可以通过激活Trx2信号通路而抑制软骨细胞的凋亡,从而起到预防骨性关节炎发生发展的作用。

Objective To observe the effects of Bugu Granule drug-containing serum on apoptosis of rat knee articular chondrocytes cultured in vitro,and the expression of Trx2,ASK1 and Caspase 3.Furthermore,the mechanism of Bugu Granule in preventing the development of osteoarthritis was discussed.Methods Rat chondrocytes were isolated and cultured by two-step enzymatic digestion,and subcultured afterwards.Apoptosis of chondrocytes was detected by flow cytometry after annexin V-FITC/PI staining.At the same time,proteins were separated by electrophoresis and the expression of Trx2,ASK1 and Caspase3 was analyzed by Western blot.Results The culture dishes were 80%-90%covered with chondrocytes that were cultured for about 15 days,and most of chondrocytes were fusiform.Flow cytometry result showed that the apoptosis rate of the blank serogroup(22.80%)was higher than drug-containing serum group(P<0.05).However,the apoptosis rate of 20%drug-containing serum group(15.91%)and 30%drug-containing serum group(17.93%)was significantly lower than the 10%drug-containing serum group(21.58%),and there were statistically significant differences between the groups(P<0.05).Western blot analysis showed that the expression level of Trx2 in the drug-containing serum group increased significantly,which was expressed most in the 10%drug-containing serum group.The expression levels of ASK1 and Caspase3 in the blank group and the 10%drug-containing serum group were higher than those in the 20%and 30%drug-containing serum groups.Conclusion Trx2 signaling pathway could be activated by Bugu Granule,and chondrocyte apoptosis could be inhibited,which plays a role in preventing the development of osteoarthritis.