Zhu Lang-Jing, Yang Tie-Cheng, Wu Qiang, Yuan Li-Ping, Chen Zhen-Wei, Luo Min-Hong, Zeng Hai-Ou, He Dong-Ling, Mo Cai-Ju
Department of Nephrology, The Eighth Affiliated Hospital, Sun Yat-Sen University, Shenzhen 518033, PR China; Department of Rheumatology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, PR China.
Department of Nephrology, The Eighth Affiliated Hospital, Sun Yat-Sen University, Shenzhen 518033, PR China.
Cytokine. 2017 May;93:26-33. doi: 10.1016/j.cyto.2017.05.001. Epub 2017 May 12.
Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) play a crucial role in RA through producing inflammatory cytokines and proteases which could cause cartilage destruction. We showed previously that elevated expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) in RA synovium correlated significantly with the severity of synovitis and the number of infiltrated inflammatory cells. The aims of this study are to detect the roles of TRAF6 in RA-FLSs.
Synovium were collected by closed needle biopsy from inflamed knees of active RA patients, and FLSs were isolated by modified tissue culture method. Expression of TRAF6 and CD55 in RA synivium was tested by double immunofluorescence (IF) staining. TRAF6 in RA-FLSs was inhibited using Lentiviral-TRAF6-shRNA transfection. Real-time PCR and ELISA were used to detect the mRNA expression and secretion of matrix metalloproteinase (MMP) and pro-inflammatory cytokines. Cell Counting Kit-8 was used to detect cell proliferation, flow cytometry was used to detect cell cycle, and Annexin V assay was used to detect cell apoptosis.
We showed that in the intimal and subintimal area of RA synovium, TRAF6 was expressed obviously not only in CD55+ cells, but also in some other CD55- cells. TRAF6 expression in RA-FLSs was suppressed effectively by Lentiviral-TRAF6-shRNA transfection. Inhibition of TRAF6 in RA-FLSs mitigated the mRNA levels and secretion of pro-inflammatory cytokines and MMPs, such as IL-1β, IL-8, IL-6, TNF-α, MMP-13, and MMP-3. In addition, it decreased the proliferation of RA-FLSs, blocked RA-FLSs in G0/G1-phase, and inhibited the cells to go into S-phase and G2/M-phase, but not facilitated apoptosis of RA-FLSs.
TRAF6 plays direct roles in the pro-inflammatory effects and proliferation of RA-FLSs. TRAF6 may serve as a potential treatment target in RA.
类风湿关节炎（RA）成纤维样滑膜细胞（FLS）通过产生可导致软骨破坏的炎性细胞因子和蛋白酶，在RA中发挥关键作用。我们之前表明，RA滑膜中肿瘤坏死因子受体相关因子6（TRAF6）表达升高与滑膜炎严重程度及炎性细胞浸润数量显著相关。本研究旨在检测TRAF6在RA - FLS中的作用。
通过闭合针吸活检从活动期RA患者发炎的膝关节收集滑膜，并采用改良组织培养方法分离FLS。通过双重免疫荧光（IF）染色检测RA滑膜中TRAF6和CD55的表达。使用慢病毒 - TRAF6 - shRNA转染抑制RA - FLS中的TRAF6。采用实时PCR和ELISA检测基质金属蛋白酶（MMP）和促炎细胞因子的mRNA表达及分泌。使用细胞计数试剂盒 - 8检测细胞增殖，流式细胞术检测细胞周期，Annexin V检测法检测细胞凋亡。
我们发现，在RA滑膜的内膜和内膜下区域，TRAF6不仅在CD55 + 细胞中明显表达，在其他一些CD55 - 细胞中也有表达。慢病毒 - TRAF6 - shRNA转染有效抑制了RA - FLS中TRAF6的表达。抑制RA - FLS中的TRAF6可减轻促炎细胞因子和MMPs（如IL - 1β、IL - 8、IL - 6、TNF - α、MMP - 13和MMP - 3）的mRNA水平和分泌。此外，它降低了RA - FLS的增殖，将RA - FLS阻滞在G0/G1期，并抑制细胞进入S期和G2/M期，但未促进RA - FLS的凋亡。
TRAF6在RA - FLS的促炎作用和增殖中起直接作用。TRAF6可能是RA的一个潜在治疗靶点。
