白藜芦醇通过NF-κB信号通路抑制软骨细胞炎症因子的表达

Resveratrol inhibits the expression of inflammatory cytokines in cartilage cells through NF-κB signaling pathway

背景:炎症因子如IL-1β、TNF-α等可通过一系列级联反应引起关节软骨破坏和炎症反应,在骨关节炎软骨病变中尤为重要。白藜芦醇(resveratrol,Res)具有抗炎、抗氧化等作用,研究Res对软骨细胞炎症因子表达的影响可为其预防、治疗骨关节炎提供参考依据。目的:探讨Res对脂多糖(lipopolysaccharides,LPS)诱导小鼠软骨细胞核因子-κB(nuclear factor-kappa B,NF-κB)活化及炎症因子基因表达的调节及其相关机制。方法:体外分离培养小鼠膝关节软骨细胞,Ⅱ型胶原免疫荧光检测进行软骨细胞鉴定。根据加入不同培养物分为3组:LPS组(1μg/ml LPS);Res组(100μmol/L Res+1μg/ml LPS);空白对照组。酶联免疫吸附试验(ELISA)法检测各组软骨细胞中NF-κB抑制蛋白α(inhibitor of NF-κB-α,I-κBα)的表达量,细胞免疫荧光染色观察NF-κB核转位情况,实时定量聚合酶链反应(RT-PCR)测IL-1β、TNF-αm RNA表达量。结果:荧光显微镜下可见软骨细胞核呈蓝色荧光,胞质呈红色荧光。ELISA检测:LPS组的I-κBα表达量低于空白对照组(P<0.05),Res组明显高于LPS组(P<0.01)。细胞免疫荧光染色检测:与空白对照组相比,LPS组中NF-κB发生明显的核转位,而Res组中NF-κB的核转位受到抑制。RT-PCR检测:LPS组中IL-1β和TNF-αm RNA的表达量与空白对照组相比明显上调,而Res组中IL-1β和TNF-αm RNA的表达量与LPS组相比明显下调,均有统计学差异(P<0.05)。结论:Res可通过NF-κB信号通路影响软骨细胞IL-1β、TNF-αm RNA的表达,进而降低软骨细胞炎症反应,延缓关节软骨细胞退变。

Background：Inflammatory cytokines such as IL-1 beta, TNF alpha can lead to cartilage destruction and inflam-matory reaction through a series of cascade reaction, especially in cartilage lesions. Resveratrol has anti-inflammatory and antioxidant function. It can provide reference for the prevention and treatment of osteoarthritis by researching the effect of resveratrol on inflammatory cytokines expression of cartilage cells. Objective：To investigate how resveratrol regulates the activation of nuclear factor-Kappa B （NF-κB） and the gene expression of inflammatory cytokines in lipopolysaccharide-in-duced cartilage cells and its related mechanism. Methods： Mouse knee cartilage cells were isolated and cultured in vitro, which were identified by typeⅡcollagen immunofluorescence. The experiment was divided into three groups according to different culture：LPS group （1μg/ml LPS）, resveratrol group （100μmol/L resveratrol＋1μg/ml LPS）, and blank control group. The expression level of inhibitor of NF-κB-α（I-κBα） were detected by using enzyme-linked immunosorbent assay （ELISA）. NF-κB nuclear translocation was observed by immunofluorescence staining. The expression of IL-1β、TNF-αmRNA was measured by real-time quantitative polymerase chain reaction （RT-PCR）. Results：The cell nucleus of chondro-cytes was blue and cytoplasm was red under the fluorescent microscope. ELISA detection：the expression of I-κBαin LPS group was significantly lower than that in control group （P〈0.05）, while the expression of I-κBαin resveratrol group was significantly higher than that in LPS group （P〈0.01）. Immunofluorescence staining：the nuclear translocation of NF-κB in LPS group significantly changed compared with the control group, while the nuclear translocation of NF-κB in resveratrol group was inhibited. RT-PCR test：as compared with the control group, the expression of IL-1βand TNF-αmRNA in LPS group were upregulated, while resveratrol group were downregulated compared to LPS group （P〈0.05）. Conclusions：Resve-ratrol can inhibit the expression of IL-1βand TNF-αmRNA in cartilage cells through NF-κB signal pathway, decrease the inflammatory reaction of chondrocytes, and then delay the degeneration of articular cartilage cells.