白藜芦醇对肥胖者骨性关节炎软骨细胞的作用及对TLR4、NF-<em>κ</em>BmRNA表达的影响-东南大学学报医学版

白藜芦醇对肥胖者骨性关节炎软骨细胞的作用及对TLR4、NF-κBmRNA表达的影响

单位：1. 中国医科大学公共卫生学院营养与食品卫生教研室, 辽宁沈阳 110122;
2. 内蒙古自治区地方病防治中心, 内蒙古呼和浩特 010030;
3. 中国医科大学附属盛京医院骨科, 辽宁沈阳 110004

分类号：R151.2

出版年·卷·期（页码）：2015·34·第三期（342-346）

摘要：

目的:探讨白藜芦醇是否可通过抑制TLR4/NF-κB信号通路发挥抗骨性关节炎的作用.方法:采用两步酶消化法分离培养肥胖者骨性关节炎软骨细胞.免疫细胞化学染色及甲苯胺蓝染色对细胞进行表型鉴定.应用MTT法测定白藜芦醇对软骨细胞增殖活力的影响.ELISA法检测细胞培养基上清TNF-α的水平.RT-PCR法检测TLR4、NF-κBmRNA表达水平.结果:软骨细胞Ⅱ型胶原免疫细胞化学染色及蛋白多糖甲苯胺蓝染色呈阳性.白藜芦醇浓度在6.25~100 μmol·L^-1时对细胞增殖均无抑制作用,其中低浓度的白藜芦醇(6.25~12.5 μmol·L^-1)能促进细胞增殖(P <0.05).IL-1β(10 ng·ml^-1)能抑制细胞增殖(P <0.05).白藜芦醇浓度在6.25~12.5 μmol·L^-1时,对IL-1β诱导的细胞活力降低有显著抑制作用.相比对照组,IL-1β诱导组的培养基上清中TNF-α水平及软骨细胞TLR4、NF-κBmRNA表达均明显增加(P <0.01),而加入白藜芦醇(6.25~12.5μmol·L^-1)处理后,TNF-α水平及TLR4、NF-κBmRNA表达均显著降低(P <0.01).结论:白藜芦醇可能通过抑制TLR4/NF-κB信号通路发挥抗骨性关节炎效应.

Objective: To investigate whether resveratrol can exert anti-osteoarthritic effect via the TLR4/NF-κB signaling pathway. Methods: Human articular chondrocytes were isolated and cultured by two-step eny matic digestion. The chondrocytes were identified by immunocytochemistry staining and toluidine blue staining. The cell viability was determined by the MTT assay. The levels of TNF-α in cell culture supernatant were measured by ELISA. The expression of TLR4 and NF-κB mRNA were examined by RT-PCR. Results: Cultured chondrocytes presented positive cells by Type Ⅱ collagen immunocytochemistry staining and toluidine blue staining. MTT assay showed that resveratrol (6.25-100 μmol·L^-1) had no discernable inhibition effects on chondrocytes cultured in the presence or absence of IL-1β. At 6.25 and 12.5 μmol·L^-1, resveratrol significantly stimulated chondrocyte proliferation. In addition, viability of cells exposed to IL-1β (10 ng·ml^-1) was significantly impaired but not in the presence of resveratrol (6.25-12.5 μmol·L^-1) (P <0.05). The level of TNF-α and the expression of TLR4 and NF-κB mRNA were significantly upregulated in the presence of IL-1β (10 ng·ml^-1) (P <0.01) compared with control group, while resveratrol (6.25-100 μmol·L^-1) treatment could dramatically downregulate TNF-α levels and TLR4 and NF-κB mRNA expression (P <0.01). Conclusion: The anti-inflammatory effect of resveratrol on human articular chondrocyte may at least partly via the inhibition of the TLR4/NF-κB signaling pathway.

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