Screening of a Panel of Low Molecular Weight Compounds That Inhibit Synovial Fibroblast Invasion in Rheumatoid Arthritis
Screening of a Panel of Low Molecular Weight Compounds That Inhibit Synovial Fibroblast Invasion in Rheumatoid Arthritis
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抑制类风湿性关节炎滑膜成纤维细胞侵袭的一组低分子量化合物的筛选
DOI：
10.4049/jimmunol.1901429
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发表时间：
2020-12-15
影响因子：
4.4
通讯作者：
Urano, Takeshi
中科院分区：
文献类型：
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作者：
Sugiura, Tomoko;Kamino, Hiroki;Urano, Takeshi
滑膜成纤维细胞侵袭增多及其参与软骨损伤是类风湿关节炎(RA)的特征表型。为了确定抑制滑膜成纤维细胞侵袭的低分子化合物，使用实时细胞分析系统对从RA患者手术标本中酶法分离的人滑膜成纤维细胞进行了初步筛选(n=330)。为了评估筛选中确定的抑制物的效果，用伤口愈合试验测量滑膜成纤维细胞的迁移，用免疫印迹法测定细胞内信号分子的磷酸化。在筛选中确定了几种候选抑制剂，包括针对血小板衍生生长因子受体(PDGFR)、Akt、PI3K和糖原激酶合成酶3(GSK-3)的抑制剂。这些抑制剂在72 h后强烈抑制滑膜成纤维细胞的迁移，在48 h时下调Akt的磷酸化(Ser(473))，当从培养条件中去除这些抑制剂时，迁移和磷酸化Akt(Ser(473))的水平都恢复了。此外，除PDGFR抑制剂IV外，所有类别的抑制剂都能抑制滑膜成纤维细胞的细胞增殖和IL-6的产生。有趣的是，GSK-3抑制剂增加了抗炎细胞因子IL-10的产生，但抑制了来自健康捐赠者的内毒素刺激的巨噬细胞产生IL-23。总之，阻断PDGFR、PI3K或GSK-3作为以滑膜成纤维细胞侵袭/迁移为靶点的RA治疗可能具有治疗价值。
Increased invasion of synovial fibroblasts and their involvement in cartilage damage are characteristic phenotypes of rheumatoid arthritis (RA). To identify low molecular weight compounds that suppress synovial fibroblast invasion, a panel of inhibitors (n = 330) was initially screened using a real-time cell analysis system for human synovial fibroblasts that were enzymatically isolated from surgical samples of RA patients. To evaluate the effects of the inhibitors identified in the screen, synovial fibroblast migration was measured using a wound-healing assay, and phosphorylation of intracellular signaling molecules was determined by immunoblots. Several candidate inhibitors were identified in the screen, including inhibitors against platelet-derived growth factor receptor (PDGFR), Akt, PI3K, and glycogen kinase synthetase 3 (GSK-3). These inhibitors strongly suppressed synovial fibroblast migration after 72 h and downregulated phosphorylation of Akt (Ser(473)) at 48 h. When the inhibitors were removed from the culture conditions, both migration and phosphorylated Akt (Ser(473)) levels were restored. Furthermore, all the categories of inhibitors except for PDGFR inhibitor IV decreased cell proliferation as well as IL-6 production in synovial fibroblasts. Interestingly, GSK-3 inhibitors increased anti-inflammatory cytokine IL-10 production but suppressed IL-23 production from LPS-primed macrophages obtained from healthy donors. In conclusion, blocking PDGFR, PI3K, or GSK-3 could have therapeutic value as an RA treatment that targets the invasion/migration of synovial fibroblasts.