A novel function of artesunate on inhibiting migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients
A novel function of artesunate on inhibiting migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients
复制标题
青蒿琥酯抑制类风湿性关节炎成纤维样滑膜细胞迁移和侵袭的新功能
DOI：
10.1186/s13075-019-1935-6
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发表时间：
2019-06-24
影响因子：
4.9
通讯作者：
Dai, Lie
中科院分区：
文献类型：
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作者：
Ma, Jian-Da;Jing, Jun;Dai, Lie
抗疟药青蒿琥酯能抑制胶原诱导的大鼠关节炎模型的炎症反应，并能防止软骨和骨的破坏，提示青蒿琥酯可能是治疗类风湿性关节炎（RA）的有效药物。本研究旨在探讨其对RA患者成纤维样滑膜细胞（FLS）侵袭性的影响。方法采用闭合式针吸活检法获取活动期RA患者滑膜组织，分离培养FLS。RA-FLS用不同浓度的青蒿琥酯治疗，而甲氨蝶呤或羟氯喹作为对照药物。CCK-8法、EdU法、Annexin V-FITC/PI法、transwell法、F-actin法分别检测RA-FLS的细胞活力、增殖、细胞周期、凋亡、迁移、侵袭和伪足形成。此外，通过Proteome profiler人类蛋白酶阵列分析表达的蛋白酶的相对变化，并通过定量实时PCR（qPCR）、蛋白质印迹和ELISA验证。通过qPCR和Western blot测量MAPK、NF-B、AP-1和PI 3 K/Akt通路的信号分子的表达。PDK-1敲低特异性抑制剂AR-12或siRNA转染用于验证青蒿琥酯对RA-FLS.ResultsArtesunate的药理学机制显着抑制RA-FLS的迁移和侵袭在剂量依赖性的方式与或没有TNF-刺激。这种作用是通过青蒿琥酯抑制MMP-2和MMP-9的产生来介导的，并且用外源性MMP-9预处理逆转了青蒿琥酯对RA-FLS侵袭的抑制作用。青蒿琥酯对RA-FLS迁移和侵袭的抑制作用强于羟氯喹，抗炎作用也强于羟氯喹。青蒿琥酯和甲氨蝶呤之间检测到类似的抑制作用，并且当联合时观察到协同作用。从机制上讲，青蒿琥酯显著抑制PDK-1表达以及Akt和RSK 2磷酸化，其方式与PDK-1特异性抑制剂AR-12或PDK-1通过siRNA转染敲低相似。这种抑制的结果在抑制RA-FLS迁移和侵袭以及降低MMP-2和MMP-9 expression.ConclusionsOur研究表明青蒿琥酯是能够抑制迁移和侵袭RA-FLS通过抑制PDK 1诱导的Akt和RSK 2磷酸化激活提示青蒿琥酯可能是一个潜在的疾病修饰抗风湿药物类风湿关节炎。
IntroductionAnti-malarial drug artesunate can suppress inflammation and prevent cartilage and bone destruction in collagen-induced arthritis model in ratssuggesting it may be a potent drug for rheumatoid arthritis (RA) therapy. We aimed to investigate its effect on the invasive property of fibroblast-like synoviocytes (FLS) from patients with RA.MethodsSynovial tissues were obtained by closed needle biopsy from active RA patients, and FLS were isolated and cultured in vitro. RA-FLS were treated with artesunate at various concentrations, while methotrexate or hydroxychloroquine was employed as comparator drugs. Cell viability, proliferation, cell cycle, apoptosis, migration, invasion, and pseudopodium formation of RA-FLS were assessed by CCK-8 assays, EdU staining, Annexin V-FITC/PI staining, transwell assays, or F-actin staining, respectively. Further, relative changes of expressed proteases were analyzed by Proteome profiler human protease array and verified by quantitative real-time PCR (qPCR), Western blot, and ELISA. The expression of signaling molecules of MAPK, NF-B, AP-1, and PI3K/Akt pathways were measured by qPCR and Western blot. PDK-1 knockdown by specific inhibitor AR-12 or siRNA transfection was used to verify the pharmacological mechanism of artesunate on RA-FLS.ResultsArtesunate significantly inhibited the migration and invasion of RA-FLS in a dose-dependent manner with or without TNF- stimulation. The effect was mediated through artesunate inhibition of MMP-2 and MMP-9 production, and pre-treatment with exogenous MMP-9 reversed the inhibitory effect of artesunate on RA-FLS invasion. Artesunate had a stronger inhibitory effect on migration and invasion of RA-FLS as well as greater anti-inflammatory effect than those of hydroxychloroquine. Similar inhibitory effect was detected between artesunate and methotrexate, and synergy was observed when combined. Mechanistically, artesunate significantly inhibited PDK-1 expression as well as Akt and RSK2 phosphorylationin a similar manner to PDK-1-specific inhibitor AR-12 or PDK-1 knockdown by siRNA transfection. This inhibition results in suppression of RA-FLS migration and invasion as well as decreased MMP-2 and MMP-9 expression.ConclusionsOur study demonstrates artesunate is capable of inhibiting migration and invasion of RA-FLS through suppression of PDK1-induced activation of Akt and RSK2 phosphorylationsuggesting that artesunate may be a potential disease-modifying anti-rheumatic drug for RA.