枸杞多糖抑制核因子κB(NF-κB)通路降低骨关节炎软骨细胞炎性细胞因子水平

Lycium barbarum polysaccharide inhibits NF-κB pathway to reduce the level of inflammatory cytokines in osteoarthritis chondrocytes

目的探讨枸杞多糖对骨关节炎(OA)软骨细胞增殖、细胞中免疫相关细胞因子水平的影响及其潜在机制。方法收集骨关节炎样本,分离培养OA软骨细胞。以(0、100、200、400、800)μg/m L枸杞多糖作用OA软骨细胞,噻唑蓝(MTT)法检测细胞增殖确定最佳枸杞多糖剂量。以400μg/m L枸杞多糖处理OA软骨细胞,实时荧光定量PCR检测细胞中诱导型一氧化氮合酶(i NOS)、生长转化因子β(TGF-β)、核因子κBp65(NF-κBp65)的mRNA水平,Western blot法检测细胞i NOS、TGF-β、NF-κBp65的蛋白水平,ELISA检测培养OA软骨细胞上清液中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平。结果当枸杞多糖剂量为(200、400、800)μg/m L时,OA软骨细胞活力明显低于0μg/m L组,且400μg/m L组、800μg/m L组细胞活力低于200μg/m L组,而400μg/m L组和800μg/m L组细胞活力无明显变化,选取400μg/m L为最适剂量。与对照组相比,400μg/m L枸杞多糖处理的OA软骨细胞上清液中IL-1β和TNF-α水平降低,细胞中i NOS在mRNA和蛋白水平上表达下调,而TGF-β表达上调,同时,NF-κBp65蛋白水平降低。结论枸杞多糖能够降低OA软骨细胞炎性细胞因子水平,抑制NF-κB信号通路,从而改善骨关节炎症损伤。

Objective To investigate the effect of Lycium barbarum polysaccharide( LBP) on the proliferation and immune-related cytokines in osteoarthritis( OA) chondrocytes. Methods We isolated chondrocytes from OA samples,andthen detected cell proliferation of OA chondrocytes treated with LBP at 0,100,200,400 and 800 μg/m L by MTT assay. OAchondrocytes were treated with 400 μg/m L LBP which was determined to be the optimal concentration by MTT assay.Real-time fluorescence quantitative PCR and Western blot analysis were performed to detect the expression of induced nitricoxide synthase( i NOS),growth transformation factor beta( TGF-β) and nuclear factor-κBp65( NF-κBp65). And the levels ofinterleukin 1 beta( IL-1β) and tumor necrosis factor alpha( TNF-α) in the supernatant were detected by ELISA. Results Cellviability of OA chondrocytes treated with LBP at 200,400 or 800 μg/m L was significantly lower than that at 0 μg/m L,and cellviability of 400 μg/m L group and 800 μg/m L group was lower than that of 200 μg/m L group. There was no significantdifference in the cell viability between the 400 μg/m L group and 800 μg/m L group. So 400 μg/m L LBP was used forsubsequent experiments. Compared with the control group, the levels of IL-1β and TNF-α in the supernatant of OAchondrocytes after treated with 400 μg/m L LBP decreased;the expression of i NOS mRNA and protein were down-regulated;theexpression of TGF-β was up-regulated;and the expression level of NF-κBp65 protein was reduced. Conclusion LBP couldreduce the levels of immune-related cytokines in OA chondrocytes and inhibit the NF-κB signaling pathway,which isinstrumental in improving the OA injury.