淫羊藿苷促进软骨细胞增殖的机制研究

Study on the mechanism of icariin promoting the proliferation of chondrocytes

目的探讨淫羊藿苷(icariin,ICA)对软骨细胞增殖功能的影响及其作用机制。方法选取SD大鼠四肢长骨进行关节软骨细胞的分离与培养,将第二代细胞接种至96孔板或6孔板进行培养,并将其随机分为对照组、低剂量组、中剂量组和高剂量组,对照组软骨细胞采用常规培养基培养,低剂量组、中剂量组和高剂量组软骨细胞分别在常规培养基础上添加0.1、1、10 μmol/L的ICA,干预1、3、5 d后采用MTT法与流式细胞术分别检测四组软骨细胞的增殖和凋亡情况,并通过实时荧光定量聚合酶链反应分析ICA干预5 d后Wnt/β-catenin通路相关基因的表达水平。结果 ICA干预1 d后,四组软骨细胞在450 nm波长处的吸光度(optical density 450,OD450)与凋亡率比较差异均无统计学意义(均P > 0.05)。ICA干预3 d后,中剂量组、高剂量组软骨细胞的OD450均显著高于对照组(均P < 0.05),凋亡率均显著低于对照组(均P < 0.05),而低剂量组与对照组软骨细胞的OD450比较差异无统计学意义(P > 0.05)。ICA干预5 d后,低剂量组、中剂量组和高剂量组软骨细胞的OD450均显著高于对照组(均P < 0.05),凋亡率均显著低于对照组(均P < 0.05)。低剂量组、中剂量组和高剂量组软骨细胞的Wnt2、β-catenin、CCND1相对表达水平均显著高于对照组(均P < 0.05)。结论 ICA能通过促进Wnt/β-catenin信号通路而促进软骨细胞的增殖,抑制细胞凋亡,且呈剂量、时间依赖性。

Objective To investigate the mechanism of icariin (ICA) promoting the proliferation of chondrocytes. Method The articular chondrocytes were isolated and cultured from the long limb bones of SD rats. The second generation cells were inoculated into 96-well plate or 6-well plate for culture, and they were randomly divided into control group, low dose group, medium dose group and high dose group. Chondrocytesin in control group was cultured in conventional medium, while chondrocytes in low dose group, medium dose group and high dose group were given 0.1, 1 and 10 μmol/L respectively on the basis of conventional culture. MTT assay and flow cytometry were performed to detect the proliferation and apoptosis of cells after 1, 3 and 5 d of ICA intervention. The expression of Wnt/β-catenin pathway related genes was analyzed by quantitative reverse transcriptase-mediated polymerase chain reaction after 5 d of ICA intervention. Result At 1 day after ICA intervention, there were no significant differences in optical density 450 (OD450) and apoptotic rate among four groups (all P > 0.05). At 3 days after ICA intervention, the OD450 of chondrocyte in medium dose group and high dose group were significantly higher than that in control group (all P < 0.05), and the apoptotic rate were significantly lower than that in control group (all P < 0.05), but there was no significant difference in OD450 between low dose group and control group (all P > 0.05). At 5 days after ICA intervention, the OD450 of chondrocytes in low dose group, middle dose group and high dose group were significantly higher than that in control group (all P < 0.05), and the apoptotic rate were significantly lower than that in control group (all P < 0.05). The relative expression levels of Wnt2,β-catenin and CCND1 of chondrocytes in low dose group, middle dose group and high dose group were significantly higher than those in control group (all P < 0.05). Conclusion ICA could promote chondrocyte proliferation and inhibit cell apoptosis by promoting Wnt/β-catenin signaling pathway in a doseand time-dependent manner.