橄榄苦苷对 IL－1β诱导的大鼠软骨细胞损伤的作用及机制研究

摘要

AIM:To investigate the effect of oleuropein on interleukin-1β( IL-1β)-induced SD rat articular chondrocytes .METHODS:The SD rat articular chondrocytes were isolated by 2 step enzyme digestions .The chondrocytes were cultured in vitro.Inverted microscopic observation was performed during the culture .Alcian blue staining and type II collagen immunohistochemical staining were used to identify the chondrocytes .The effects of oleuropein on the viability of chondrocytes were determined by CCK-8 assay.The cells in 3rd passage were pretreated with oleuropein at 10, 50 or 100 μmol/L and subsequently stimulated with IL-1βat 10 μg/L for 24 h.Production of prostaglandin E 2 ( PGE2 ) and ni-tric oxide (NO) were evaluated by the Griess reaction and an enzyme linked immunosorbent assay (ELISA).The mRNA expression of matrix metalloproteinase ( MMP)-1 and MMP-13 was measured by real-time PCR.The protein levels of in-ducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-κB) were detected by Western blotting .RESULTS:The cell viability of chondrocytes was not significantly impaired by treating with oleuropein at concentration of 10, 50 or 100μmol/L for 24 h compared with control group .Pretreatment with oleuropein significantly in-hibited the production of PGE 2 and NO induced by IL-1β.Oleuropein also significantly decreased the IL-1β-stimulated MMP-1 and MMP-13 mRNA expression in articular chondrocytes .Pretreatment with oleuropein inhibited the IL-1β-media-ted activation of NF-κB by suppressing the degradation of its inhibitory protein IκBαin the cytoplasm .CONCLUSION:Oleuropein inhibits IL-1β-induced inflammatory gene expression by suppressing NF-κB activation at the transcriptional le-vel, suggesting a new mechanism for the anti-inflammatory effects of oleuropein as a novel agent on treating with osteoarthri-tis.%目的：研究橄榄苦苷对白细胞介素1β（ IL－1β）诱导的SD大鼠关节软骨细胞的影响。方法：采用酶两步顺序消化法消化SD大鼠关节软骨分离细胞，体外培养软骨细胞，光学显微镜观察细胞形态，阿尔新蓝染色及Ⅱ型胶原免疫组化法对软骨细胞进行鉴定。橄榄苦苷对软骨细胞的细胞毒性采用CCK－8实验进行评估。取第3代软骨细胞，分别用浓度为10、50或100μmol／L的橄榄苦苷预先处理，随后用IL－1β刺激细胞24 h，所生成的NO和前列腺素E2（ PGE2）的量分别用格里斯重氮化反应和酶联反应吸附实验进行评估。基质金属蛋白酶（ MMP）－1和MMP－13 mRNA的表达利用实时荧光定量PCR进行定量检测。采用免疫印迹实验分析诱导型一氧化氮合酶（iNOS）、环氧化酶2（COX－2）和活化NF－κB的蛋白表达。结果：软骨细胞在不同浓度橄榄苦苷培养24 h后，其生存能力与对照组相比无明显差异。橄榄苦苷可以显著降低IL－1β刺激下软骨细胞MMP－1和MMP－13 mRNA的表达及NO、PGE2的生成。橄榄苦苷通过减少IκB蛋白的降解从而抑制IL－1β介导的NF－κB通路的活化。结论：橄榄苦苷可通过NF－κB信号转导通路调控炎症的发生发展，进一步明确了橄榄苦苷对关节炎防治作用的分子机制，为骨关节炎的免疫治疗提供了重要的基础理论依据。