独活寄生汤含药血清对大鼠退变软骨细胞线粒体凋亡通路的影响

摘要

目的：观察独活寄生汤含药血清对大鼠退变软骨细胞线粒体凋亡通路的影响，探讨独活寄生汤治疗骨关节炎的作用机制。方法：将36只健康清洁级2月龄SD大鼠随机分为生理盐水组（空白血清组）9只和独活寄生汤临床等效剂量组（含药血清组）27只。采用1 h、2 h、3 h不同采血时间点独活寄生汤含药血清和空白血清，将10%、15%、20%、30%不同浓度两种血清作用于体外培养第三代软骨细胞，确定含药血清最佳干预条件；再用含药血清最佳干预条件处理退变软骨细胞，分别干预24 h、48 h、72 h后，收集软骨细胞，进行相应指标的检测。采用Annexin V-FITC/PI染色流式检测退变软骨细胞凋亡情况，JC-1染色流式检测退变软骨细胞线粒体膜电位的改变，分光光度法检测退变软骨细胞中caspase-9和caspase-3活性。结果：浓度为10%的2 h采血时间点含药血清的促增殖作用最明显；血清干预后，退变软骨细胞的凋亡率逐渐降低，具有时间依赖性；线粒体膜电位逐渐减低；随着干预时间的延长， caspase-9、caspase-3活性均逐渐降低，与空白血清组相比，含药血清组降低更显著。结论：独活寄生汤可通过调控线粒体凋亡通路相关调节因子caspase-9、caspase-3表达，有效抑制软骨细胞凋亡。%Objective:To observe the effects of serum medicated with Duhuo Jisheng Decoction on apoptosis pathway of degenerating mitochondria of rat chondrocytes and probe the mechanism of Duhuo Jisheng Decoction in the treatment of osteoarthritis.Methods:36 healthy clean grade 2 months old SD rats were randomly divided into saline group(blank serum group)9 and Duhuo Jisheng Decoction clinical equivalent dose group(drug containing serum group) 27.The third generation of cartilage cells was cultured in vitro using different time points of sample collection such as 1 h,2 h and 3 h,serums medicated with 10%,15%,20% and 30% concentrations of Duhuo Jisheng Decoction and blank serums.The best intervention time of medicated serum was determined,which was then used to deal with the degenerating chondrocytes.After intervention for 24 h,48 h and 72 h,the chondrocytes were collected and tested for corresponding index.Annexin V-FITC/PI was used to detectthe apoptosis of degenerating chondrocytes. JC-1 staining lfow was used to test membrane potential change of degenerating chondrocyte mitochondria.Spectrophotometric detection was used to detect the activity of caspase-9 and caspase-3 in degenerating chondrocytes. Results:Medicated serum collected after 2 h and with 10% of the concentration had the most obvious effect on promoting proliferation.After serum intervention,the apoptosis rate of degenerating chondrocytes decreased gradually,dependent on time.After serum intervention,mitochondrial membrane potential decreased gradually.With prolonging of intervention time,the activity of caspase-9 and caspase-3 was gradually reduced,more signiifcantly of the medicated group than that of the blank serum group.Conclusion:Duhuo Jisheng Decoction can effectively inhibit chondrocyte apoptosis through the regulation of the expression of caspase-9 and caspase-3 related with mitochondrial apoptosis pathway.