补肾痹通方联合骨髓间充质干细胞对损伤软骨细胞的保护机制及SOX9、MMP-13表达的影响

The protective mechanism of Bushen Bitong recipe combined with bone marrow mesenchymal stem cells on injured chondrocytes and its effects on the expression of SOX9 and MMP-13

目的:探讨补肾痹通方(BSBT)联合骨髓间充质干细胞(BMSCs)对白介素(IL)-1β诱导的损伤软骨细胞的保护机制及性别决定区Y框蛋白9(SOX9)、基质金属蛋白酶13(MMP-13)表达的影响。方法:使用10 ng/ml的IL-1β建立损伤软骨细胞模型,实验分为对照组、模型组(IL-1β)、中药组(IL-1β+BSBT)、干细胞组(IL-1β+BMSCs)和联合组(IL-1β+BSBT+BMSCs);CCK-8法检测各组软骨细胞增殖情况;RT-qPCR法和Western blot法分别检测软骨细胞内SOX9、MMP-13、Ⅱ型胶原α1重组蛋白(COL2A1)、IL-10的基因和蛋白表达;ELISA检测各组细胞培养上清中肿瘤坏死因子-α(TNF-α)、IL-6、胰岛素样生长因子1(IGF-1)、碱性成纤维细胞生长因子(bFGF)的水平。结果:与模型组比较,各组软骨细胞活性显著增强(P<0.01),软骨细胞中的SOX9、COL2A1、IL-10的基因和蛋白水平显著升高(P<0.01),MMP-13的基因和蛋白水平明显降低(P<0.01),软骨细胞培养上清中TNF-α、IL-6的含量显著降低(P<0.01),IGF-1、bFGF的水平升高(P<0.01),其中联合组变化最明显(P<0.05)。结论:BSBT联合BMSCs可有效保护损伤软骨细胞,其机制可能是通过上调SOX9,下调MMP-13,抑制炎症,改善软骨细胞微环境,促进关节软骨的修复与再生。

Objective:To explore the protective mechanism of Bushen Bitong recipe(BSBT)combined with bone marrow mesenchymal stem cells(BMSCs)on IL-1β-induced injured chondrocytes,as well as the effects on the expression of SOX9 and MMP-13.Methods:An injured chondrocyte model was established using 10 ng/ml of IL-1β,and the experiments were divided into control group,model group(IL-1β),traditional Chinese medicine group(IL-1β+BSBT),stem cell group(IL-1β+BMSCs),and combined group(IL-1β+BSBT+BMSCs).CCK-8 assay for chondrocyte proliferation in each group.And gene and protein expression of SOX9,MMP-13,and IL-10 in chondrocytes by RT-qPCR and Western blot,respectively.The levels of TNF-α,IL-6,IGF-1,and bFGF in the cell culture supernatants of each group were detected by ELISA.Results:Compared with the model group,the chondrocyte activity was highly significantly enhanced in all groups(P<0.01),the gene and protein levels of SOX9,COL2A1 and IL-10 in chondrocytes were highly significantly elevated(P<0.01),and the gene and protein levels of MMP-13 were highly significantly reduced(P<0.01),and the content levels of TNF-αand IL-6 in the supernatants of chondrocyte culture were highly significantly decreased(P<0.01),and the content levels of IGF-1 and bFGF were highly significantly increased(P<0.01),with the best performance in the combined group(P<0.05).Conclusion:BSBT combined with BMSCs effectively protected injured chondrocytes,possibly by up-regulating SOX9 and down-regulating MMP-13,inhibiting inflammation,improving the microenvironment of chondrocytes,and promoting the repair and regeneration of articular cartilage.