Clinical Institute of Molecular Biology, People's Hospital, Peking University, Beijing, China.
Inflammation. 2012 Feb;35(1):377-87. doi: 10.1007/s10753-011-9365-x.
The purpose of this research is to study the effect of genistein on cytokines or growth factor-induced proliferation and transformation phenotype of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were primarily cultured. With respective stimulation of IL-1β, TNF-α, and EGF, genistein was applied to elucidate its effect on synoviocytes' growth number, cell proliferation assay, cell cycle using cell counts, (3)H-TdR incorporation and flow cytometry, the colony numbers under anchorage-independent condition, and the expression of MMP-2 and MMP-9 in synovial fibroblasts. EGF, IL-1β, and TNF-α increased (3)H incorporation in RA-FLS, respectively. EGF augmented clone numbers of RA-FLS under anchorage-independent condition and not IL-1β and TNF-α. Genistein had an inhibitory role on cell number and (3)H-TdR incorporation of RA-FLS stimulated with IL-1β, TNF-α and EGF; genistein arrested the cell cycle at G(1) restriction point; genistein decreased colony numbers under anchorage-independent condition stimulated by EGF in serum condition. IL-1β or TNF-α increased expression of MMP-9 and MMP-2 in rheumatoid synoviocytes; EGF stimulated expression of MMP-9 but not of MMP-2; genistein suppressed production of MMP-9 more than MMP-2 induced by IL-1β or TNF-α; rMMP-9, rMMP-2, or their inhibitors had no effect on the (3)H-TdR incorporation of synovial cells. Erk1/2 inhibitor (PD098 059) had obvious inhibitory effect on the (3)H incorporation induced by TNF-α or IL-1β; inhibitors of JNK (SP600 125) had no significant effect on the (3)H incorporation. While pretreatment with PD098059 had no marked inhibitory effect on MMP-9 expression induced by TNF-α or IL-1β, SP600125 decreased significantly the MMP-9 expression induced by TNF-α or IL-1β. Neither PD098059 nor SP600 125 could inhibit the MMP-2 expression induced by TNF-α or IL-1β. Genistein inhibited IL-1β, TNF-α or EGF-induced proliferation and MMP-9 expression in fibroblast-like synoviocytes of rheumatoid arthritis; the proliferation of RA-FLS was mediated by Erk1/2 but not JNK activation, while JNK activation was involved in the signal transduction pathway leading to MMP-9 expression in rheumatoid synoviocytes.
本研究旨在探讨金雀异黄素对细胞因子或生长因子诱导的类风湿关节炎成纤维样滑膜细胞（RA-FLS）增殖和转化表型的影响。首先培养 RA-FLS，分别用 IL-1β、TNF-α 和 EGF 刺激，应用金雀异黄素阐明其对滑膜细胞生长数量、细胞增殖试验、细胞周期的影响，采用细胞计数、（3）H-TdR 掺入和流式细胞术，在无锚定条件下的集落数，以及 MMP-2 和 MMP-9 在滑膜成纤维细胞中的表达。EGF、IL-1β 和 TNF-α 分别增加 RA-FLS 中的（3）H 掺入。EGF 增加 RA-FLS 在无锚定条件下的克隆数，而不是 IL-1β 和 TNF-α。金雀异黄素对 IL-1β、TNF-α 和 EGF 刺激的 RA-FLS 的细胞数和（3）H-TdR 掺入有抑制作用；金雀异黄素将细胞周期阻滞在 G1 限制点；金雀异黄素减少血清条件下 EGF 刺激的无锚定条件下集落数。IL-1β 或 TNF-α 增加类风湿滑膜细胞中 MMP-9 和 MMP-2 的表达；EGF 刺激 MMP-9 的表达，但不刺激 MMP-2 的表达；金雀异黄素抑制由 IL-1β 或 TNF-α 诱导的 MMP-9 的产生多于 MMP-2；rMMP-9、rMMP-2 或其抑制剂对滑膜细胞的（3）H-TdR 掺入无影响。Erk1/2 抑制剂（PD098059）对 TNF-α 或 IL-1β 诱导的（3）H 掺入有明显的抑制作用；JNK 抑制剂（SP600125）对（3）H 掺入无显著影响。虽然 PD098059 预处理对 TNF-α 或 IL-1β 诱导的 MMP-9 表达无明显抑制作用，但 SP600125 显著降低 TNF-α 或 IL-1β 诱导的 MMP-9 表达。PD098059 或 SP600125 均不能抑制 TNF-α 或 IL-1β 诱导的 MMP-2 表达。金雀异黄素抑制 IL-1β、TNF-α 或 EGF 诱导的类风湿关节炎成纤维样滑膜细胞的增殖和 MMP-9 表达；RA-FLS 的增殖是通过 Erk1/2 介导的，而不是 JNK 激活，而 JNK 激活参与了导致类风湿滑膜细胞中 MMP-9 表达的信号转导途径。
