TNF-α在构建大鼠骨性关节炎模型中对内质网应激的影响探讨

摘要

目的 观察不同剂量肿瘤坏死因子-α(tumor necrosis factor,TNF-α)在构建大鼠骨性关节炎(osteoarthritis,OA)模型中对内质网应激的影响,初步探讨大鼠骨性关节炎病理过程中内质网应激的作用.方法 将20只SPF级SD雄性大鼠随机分为健康对照组、TNF-α1 ng/mL干预组、TNF-α5ng/mL干预组和TNF-α10ng/mL干预组,分别给予不同剂量TNF-α处理,4周后取材,培养提取的各组原代软骨细胞,采用MTT法检测不同剂量TNF-α对软骨细胞活力的影响;ELISA法检测内皮细胞粘附分子-1(endothelial cell adhesion molecule-1,sICAM-1)含量变化,TUNEL法检测各组软骨细胞凋亡率,蛋白印迹法(Western blot)检测炎症因子MMP9、ICAM1的表达以及内质网应激指标IRE1、p-IRE1、GRP78的水平变化.结果 MTT法检测提示加入不同剂量的TNF-α后软骨细胞活力未见明显改变;TUNEL法检测结果提示,与对照组相比,TNF-α干预组软骨细胞凋亡率较高(P＜0.01),并呈浓度依赖性促进细胞凋亡;Western blot结果表明,与对照组相比,软骨细胞的p-IRE1、IRE1、GRP78、MMP9及ICAM1的表达水平呈TNF-α浓度依赖性升高(P＜0.01).结论 在TNF-α构建大鼠骨性关节炎模型中,炎症因子与内质网应激指标均发生显著改变,内质网应激可能在大鼠骨性关节炎的发生发展过程中发挥重要影响,且当TNF-α浓度为10 ng/mL时,其对炎性因子及内质网指标的作用效果最为显著.%Objective To explore the influence of TNF-α on endoplasmic reticulum stress responses in the rat model of osteoarthritis.Method A total of 20 male SD mice were randomly divided into the following groups:① Control group;②TNF-α1 ng/mL group;③TNF-α 5ng/mL group;④TNF-α 10ng/mL group.Firstly,after 4 weeks of treatment,we extracted the primary chondrocytes from each group.Secondly,the effects of different doses of TNF-α on chondrocyte viability were detected using MTT,the endothelial cell adhesion molecule-1 (slCAM-1) was detected using ELISA,the apoptosis of chondrocytes was detected using the TUNEL method and the expressions of inflammatory markers MMP9 and ICAM1 and markers of endoplasmic reticulum stress response including IRE1,p-IRE1 and GRP78 were detected using Western blot.Results The MTT assay showed that chondrocyte viability did not change significantly after adding different doses of TNF-α.TUNEL assay showed that the apoptotic rate of chondrocytes was higher in the TNF-α group than in the control group (P ＜ 0.01),and the apoptosis rate increased in a dosedependent manner.The Western blot result showed that compared with the control group,the expression of p-IRE1,IRE1,GRP78,MMP9 and ICAM1 in chondrocytes increased in a dose-dependent manner (P ＜ 0.01).Conclusion In the rat model of osteoarthritis induced by TNF-α,the inflammatory factors and endoplasmic reticulum stress responses changed significantly,and ERS might play an important role in the development and progression of osteoarthritis in rats.When TNF-α concentration was 10ng/mL,the effect on inflammatory factors and endoplasmic reticulum parameters was the most significant.