摘要
目的 探讨毛兰素调节CC趋化因子配体2(CCL2)/CC趋化因子受体2(CCR2)轴对关节软骨细胞炎性损伤的影响.方法 将人关节软骨细胞分为control组(正常培养)、LPS组、毛兰素低(10 nmol/L)、中(25 nmol/L)、高(50 nmol/L)剂量组、pcDNA 组(转染 pcDNA3.1)、pcDNA-CCL2 组(转染 pcDNA3.1-CCL2),实时荧光定量 PCR(qRT-PCR)检测细胞中 CCL2、CCR2 mRNA表达水平;MTT法、平板克隆实验与流式细胞仪分别检测细胞增殖和凋亡情况;qRT-PCR和ELISA检测IL-6、IL-1β、TNF-α水平;Western blot法检测细胞中增殖细胞核抗原(PCNA)、细胞抗凋亡因子B细胞淋巴瘤蛋白2(Bcl-2)、Bcl-2相关X蛋白(Bax)及CCL2/CCR2通路蛋白表达.结果 与control组比较,LPS组细胞凋亡率、IL-6、IL-lβ、TNF-α mRNA表达水平、CCL2、CCR2 mRNA及Bax、CCL2、CCR2蛋白表达水平显著升高,OD490值、集落形成数、PCNA、Bcl-2蛋白表达水平降低(P＜0.05);与LPS组比较,毛兰素低、中、高剂量组细胞凋亡率、IL-6、1L-iβ、TNF-α mRNA表达水平、CCL2、CCR2 mRNA及Bax、CCL2、CCR2蛋白表达水平逐渐降低,OD490值、集落形成数、PCNA、Bcl-2蛋白表达水平逐渐升高(P＜0.05);过表达CCL2减弱了毛兰素对LPS诱导的关节软骨细胞的影响(P＜0.05).结论 毛兰素可抑制LPS诱导的关节软骨细胞凋亡和炎症反应,其机制可能与抑制CCL2/CCR2通路有关.
Abstract
Objective To investigate the effect of Erianin on inflammatory injury of articular chondrocytes by regulating the C-C motif chemokine ligand 2(CCL2)/C-C motif chemokine receptor 2(CCR2)axis.Methods Human articular chondrocytes were grouped into control group(normal culture),LPS group,low(l0nmol/L),medium(25nmol/L),and high(50nmol/L)dose groups,pcDNA group(transfected with pcDNA3.1),and pcDNA-CCL2 group(transfected with pcDNA3.1 CCL2).Real-time fluorescence quantitative PCR(qRT-PCR)was applied to detect the expression levels of CCL2 and CCR2 mRNA in cells.MTT assay,plate cloning assay,and flow cytometry were applied to detect cell proliferation and apoptosis,respectively.qRT-PCR and ELISA were applied to detect levels of IL-6,IL-1β,and TNF-α.Western blotting was applied to detect the expressions of proliferating cell nuclear antigen(PCNA),anti apoptotic factor B cell lymphoma protein 2(Bcl-2),Bcl-2 associated X protein(Bax),and CCL2/CCR2 pathway protein in cells.Results Compared to those in the control group,the cell apoptosis rate,IL-6,IL-1β,TNF-α mRNA expression levels and contents,CCL2,CCR2 mRNA,and Bax,CCL2,and CCR2 protein expression levels obvious increased in the LPS group,and the OD490 value,colony formation number,and PCNA and Bcl-2 protein expression levels decreased(P＜0.05).Compared to those in the LPS group,the cell apoptosis rate,IL-6,IL-1β,TNF-α mRNA expression levels and contents,CCL2,CCR2 mRNA,and Bax,CCL2,and CCR2 protein expression levels obvious decreased in the the low,medium,and high dose Erianin groups,and the OD490 value,colony formation number,and PCNA and Bcl-2 protein expression levels increased(P＜0.05).Overexpression of CCL2 weakened the effect of Erianin on LPS-induced articular chondrocytes(P＜0.05).Conclusion Erianin inhibits LPS-induced apoptosis and inflammatory response of articular chondrocytes.The mechanism may be related to the inhibition of CCL2/CCR2 pathway.
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