桂枝加葛根汤通过SDF-1/CXCR4信号轴改善大鼠椎间盘终板软骨细胞退变的研究

Guizhi plus Gegen Decoction improves rat intervertebral disc endplate chondrocyte degeneration through SDF-1/CXCR4 signal axis

[目的]探讨桂枝加葛根汤在体外力学刺激诱导的大鼠终板软骨细胞退变中的作用与机制。[方法]分离培养大鼠的终板软骨细胞,采用Flexcell FX-5000^(TM)细胞应力加载系统建立终板软骨细胞退变模型,实验分为对照组、模型组、桂枝加葛根汤组(造模前200 mL/L桂枝加葛根汤含药血清处理细胞24 h)、桂枝加葛根汤+SDF-1α组(造模前200 mL/L桂枝加葛根汤含药血清和100μg/L SDF-1α处理细胞24 h)。CCK-8法和流式细胞术检测细胞增殖与凋亡,甲苯胺蓝染色与鬼笔环肽染色观察细胞形态变化,免疫荧光染色检测细胞中CXC趋化因子受体4(CXCR4)阳性表达,逆转录-聚合酶链式反应(qRT-PCR)和蛋白免疫印迹法(Western blot)分别检测细胞中Ⅱ型胶原纤维α1(COL2A1)、HMG盒超家族成员9(SOX9)mRNA与蛋白表达水平变化。[结果]与对照组比较,大鼠终板软骨细胞在力学刺激后细胞活性下降(P<0.05),细胞凋亡率增加(P<0.05),细胞出现退变,且形态由多边形变成纺锤形,细胞中COL2A1、SOX9 mRNA与蛋白的相对表达量均下降(P<0.05);与模型组比较,加入桂枝加葛根汤处理的终板软骨细胞活性升高(P<0.05),细胞凋亡率降低(P<0.05),细胞退变得到抑制,细胞形态改变有所减轻,同时,COL2A1、SOX9 mRNA与蛋白的相对表达量升高(P<0.05),而在桂枝加葛根汤与SDF-1α共培养的细胞中未发现改善作用。[结论]桂枝加葛根汤可体外干预SDF-1/CXCR4信号通路,阻断SDF-1与软骨细胞表面的CXCR4受体结合,减轻大鼠椎间盘终板软骨细胞退变。

[Objective]To investigate the effect and mechanism of Guizhi plus Gegen Decoction in the degeneration of rat endplate chondrocytes induced by mechanical stimulation in vitro.[Methods]Isolate and culture rat endplate chondrocytes,the Flexcell FX-5000TM cell stress loading system was used to establish an endplate chondrocyte degeneration model,the experiment was divided into control group,model group,Guizhi plus Gegen Decoction group(200 mL/L Guizhi plus Gegen Decoction-containing serum treated cells for 24 h before modeling),Guizhi plus Gegen Decoction+SDF-1αgroup(200 mL/L Guizhi plus Gegen Decoction and 100μg/L SDF-1αtreated cells for 24 h before modeling).CCK-8 method and flow cytometry detect cell proliferation and apoptosis,toluidine blue staining and phalloidin staining to observe cell morphology changes,immunofluorescence staining was used to detect the positive expression of CXC chemokine receptor 4(CXCR4)in the cells.qRT-PCR and Western blot were used to detect the changes in the expression levels of type II collagen fibersα1(COL2A1)and SOX9 mRNA and protein in the cells.[Results]Compared with the control group,the cell activity of rat endplate chondrocytes decreased after mechanical stimulation(P<0.05),and the apoptosis rate increased(P<0.05),and the cells degenerated,and the shape changed from polygon to spindle,and the relative expression of COL2A1 and SOX9 mRNA and protein in the cells decreased(P<0.05);compared with the model group,the activity of endplate chondrocytes treated with Guizhi plus Gegen Decoction increased(P<0.05),the apoptosis rate was decreased(P<0.05),the cell regression was inhibited,and the cell morphology changes were alleviated.At the same time,the relative expression of COL2A1 and SOX9 mRNA and protein increased(P<0.05),but no improvement was found in the cells co-cultured with Guizhi plus Gegen Decoction and SDF-1α.[Conclusion]Guizhi plus Gegen Decoction can intervene in the SDF-1/CXCR4 signaling pathway in vitro,block the binding of SDF-1 to the CXCR4 receptor on the surface of chondrocytes,and reduce the degeneration of chondrocytes in the endplate of rat intervertebral disc.