低氧和IL-1β联合刺激对类风湿关节炎成纤维样滑膜细胞基质金属蛋白酶表达的影响及机制研究

Effect and mechanism of hypoxia and IL-1βco-stimulation on the expressions of matrix metalloproteinases in fibroblastlike synoviocytes from patients with rheumatoid arthritis

目的探讨低氧和IL-1β联合刺激对类风湿关节炎(RA)成纤维样滑膜细胞(FLS)基质金属蛋白酶(MMP)-1、MMP-3、MMP-9水平的影响,并分析缺氧诱导因子1α(HIF-1α)通路和NF-κB通路在其中的调控作用。方法应用组织块法从临床RA患者滑膜组织分离原代FLS,一阶段实验中,细胞分为常氧组、低氧组、IL-1β组、IL-1β+低氧组;二阶段实验中,细胞分为IL-1β+低氧组、YC组、BAY组、YC+BAY组。采用ELISA法和Real-time PCR法分别检测MMP-1、MMP-3、MMP-9水平及MMP-1、MMP-3、MMP-9 mRNA表达水平,采用Western blot法检测HIF-1α和磷酸化NF-κB p65(p-p65)的表达,采用免疫荧光法检测p-p65的表达及核移位。结果与常氧组比较,IL-1β组MMP-1、MMP-3、MMP-9水平均升高(均P<0.05);而与IL-1β组比较,IL-1β+低氧组MMP-1、MMP-3、MMP-9水平均升高(均P<0.05)。与常氧组比较,低氧组、IL-1β组MMP-1、MMP-3、MMP-9 mRNA表达水平均升高(均P<0.05);而与IL-1β组、低氧组比较,IL-1β和IL-1β+低氧组MMP-1、MMP-3、MMP-9水平均升高(均P<0.05)。与常氧组比较,低氧组、IL-1β组HIF-1α和p-p65核移位明显增加,而IL-1β+低氧组HIF-1α和pp65蛋白表达水平及核移位进一步增加。YC或BAY预处理细胞后,IL-1β+低氧组MMP-1、MMP-3、MMP-9水平和mRNA表达水平均下降(均P<0.05)。结论低氧和IL-1β能协同促进RA关节滑膜组织中FLS生成MMP,而HIF-1α和NF-κB通路在RA炎症缺氧微环境病变机制中起重要作用。

Objective To investigate the effect of hypoxia and IL-1βco-stimulation on the expression of MMP-1,MMP-3 and MMP-9 in fibroblast-like synoviocytes(FLSs)from patients with rheumatoid arthritis(RA)and related mechanisms.Methods FLSs were isolated from synovial tissue of RA patients by tissue block method.The primarily cultured FLSs were divided into normoxic group,hypoxic group,and IL-1βgroup,IL-1β+hypoxia group.The FLSs were treated with HIF-1αinhibitors YC-1 or NF-κB inhibitor BAY,then the cells were divided into IL-1β+hypoxia group,YC group,BAY group,YC+BAY group.The protein and mRNA levels of MMP-1,MMP-3 and MMP-9 were measured by ELISA and RT-PCR,respectively.The expression of HIF-1αand phosphorylated NF-κB p65(p-p65)were determined by Western blot.Nucleus shift of p-p65 was observed by cellular immunofluorescence assay.Results The protein expression levels of MMP-1,MMP-3 and MMP-9 were significantly higher in IL-1βgroup than those in the normoxia group(all P<0.05);the protein levels of MMP-1,MMP-3 and MMP-9 were significantly higher in the IL-1β+hypoxia group than those in the IL-1βgroup(all P<0.05).The mRNA expression levels of MMP-1,MMP-3 and MMP-9 were significantly higher in hypoxia group and IL-1βgroup than those in the normoxia group(all P<0.05).Compared with IL-1βgroup or hypoxia group,the expression levels of MMP-1,MMP-3 and MMP-9 in IL-1β+hypoxia group were significantly elevated(all P<0.05).Compared with normoxia group,the expression of HIF-1αand p-p65 protein and nuclear translocation in IL-1βgroup or hypoxia group were significantly increased,which were further increased in IL-1β+hypoxia group.Both HIF-1αinhibitors YC-1 and NF-κB inhibitor BAY significantly decreased the protein and mRNA levels of MMP-1,MMP-3 and MMP-9 in presence of hypoxia and IL-1βco-stimulation(all P<0.05).Conclusion Hypoxia and IL-1βcan synergistically promote the generation of MMPs by RA FLSs,which indicates that HIF-1αand NF-κB pathways plays an important role in the pathogenesis of RA inflammation and hypoxic microenvironment.