A Novel Phytochemical, DIM, Inhibits Proliferation, Migration, Invasion and TNF-α Induced Inflammatory Cytokine Production of Synovial Fibroblasts From Rheumatoid Arthritis Patients by Targeting MAPK and AKT/mTOR Signal Pathway
A Novel Phytochemical, DIM, Inhibits Proliferation, Migration, Invasion and TNF-α Induced Inflammatory Cytokine Production of Synovial Fibroblasts From Rheumatoid Arthritis Patients by Targeting MAPK and AKT/mTOR Signal Pathway
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一种新型植物化学物质 DIM，通过靶向 MAPK 和 AKT/mTOR 信号通路，抑制类风湿关节炎患者滑膜成纤维细胞的增殖、迁移、侵袭和 TNF-α 诱导的炎症细胞因子的产生
DOI：
10.3389/fimmu.2019.01620
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发表时间：
2019-07-23
影响因子：
7.3
通讯作者：
Jie, Ligang
中科院分区：
文献类型：
--
作者：
Du, Hongyan;Zhang, Xi;Jie, Ligang
在类风湿关节炎(RA)的发病机制中，活化的RA成纤维样滑膜细胞(RA-Fls)表现出与肿瘤细胞相似的增殖特征，继而对软骨的侵蚀最终会导致关节的破坏。因此，寻找能够有效抑制RA-FLSS异常激活、延缓RA进展的化合物势在必行。3‘3-二吲哚甲烷(DIM)是十字花科蔬菜中吲哚-3-甲醇酸催化齐聚的主要产物，在某些实体肿瘤中具有抑制迁移、侵袭和致癌的功能。本研究旨在探讨DIM对RA-FLSS的抗增殖、抗转移和抗炎作用及其分子机制。为了做到这一点，我们从RA患者和动物模型中分离出原代RA-Fls。CCK-8法、Scratch法和Transwell法分别检测细胞增殖、迁移和侵袭能力。采用实时荧光定量聚合酶链式反应和免疫印迹方法，定量检测DIM对RA-FLS中基质金属蛋白酶(MMPs)、炎性因子、肿瘤坏死因子-α(TNF-a)异常激活信号通路中某些炎性因子的表达及关键分子的影响。此外，在C57BL/6小鼠体内评价了DIM对佐剂性关节炎(AIA)模型的影响。结果表明，DIM在体外可抑制RA-FLS的增殖、迁移和侵袭。同时，DIM显著抑制肿瘤坏死因子-α诱导的基质金属蛋白酶-2、基质金属蛋白酶-3、基质金属蛋白酶-8和基质金属蛋白酶-9的表达水平，以及促炎因子IL-6、IL-8和IL-1β的表达水平。机制研究表明，DIM不仅能抑制MAPK通路中p38、JNK的磷酸化活化，而且还能抑制AKT、mTOR及其下游分子在AKT/mTOR通路中的磷酸化活化。此外，DIM治疗降低了血清中促炎症细胞因子的表达水平，并减轻了AIA小鼠膝关节关节炎的严重程度。综上所述，我们的研究结果表明，DIM在体外可以通过阻断MAPK和AKT/mTOR通路，抑制RA-FLSS的增殖、迁移和侵袭，减少由TNF-α诱导的促炎因子，并在体内防止炎症和膝关节破坏，提示DIM可能对RA具有治疗潜力。
In rheumatoid arthritis(RA) pathogenesis, activated RA fibroblast-like synoviocytes (RA-FLSs) exhibit similar proliferative features as tumor cells and subsequent erosion to cartilage will eventually lead to joint destruction. Therefore, it is imperative to search for compounds, which can effectively inhibit the abnormal activation of RA-FLSs, and retard RA progression. 3'3-Diindolylmethane (DIM), the major product of the acid-catalyzed oligomerization of indole-3-carbinol from cruciferous vegetables, has been reported to be functionally relevant to inhibition of migration, invasion and carcinogenesis in some solid tumors. In this study, we explored the anti-proliferation, anti-metastasis and anti-inflammation effects of DIM on RA-FLSs as well as the underlying molecular mechanisms. To do this, primary RA-FLSs were isolated from RA patients and an animal model. Cell proliferation, migration and invasion were measured using CCK-8, scratch, and Transwell assays, respectively. The effects of DIM on Matrix metalloproteinases (MMPs) and some inflammatory factors mRNA and key molecules such as some inflammatory factors and those involved in aberrantly-activated signaling pathway in response to tumor necrosis factor a (TNF-a), a typical characteristic mediator in RA-FLS, were quantitatively measured by real-time PCR and western blotting. Moreover, the effect of DIM on adjuvant induced arthritis(AIA) models was evaluated with C57BL/6 mice in vivo. The results showed that DIM inhibited proliferation, migration and invasion of RA-FLS in vitro. Meanwhile, DIM dramatically suppressed TNF-alpha-induced increases in the mRNA levels of MMP-2, MMP-3, MMP-8, and MMP-9; as well as the proinflammatory factors IL-6, IL-8, and IL-1 beta. Mechanistic studies revealed that DIM is able to suppress phosphorylated activation not only of p38, JNK in MAPK pathway but of AKT, mTOR and downstream molecules in the AKT/mTOR pathway. Moreover, DIM treatment decreased expression levels of proinflammatory cytokines in the serum and alleviated arthritis severity in the knee joints of AIA mice. Taken together, our findings demonstrate that DIM could inhibit proliferation, migration and invasion of RA-FLSs and reduce proinflammatory factors induced by TNF-alpha in vitro by blocking MAPK and AKT/mTOR pathway and prevent inflammation and knee joint destruction in vivo, which suggests that DIM might have therapeutic potential for RA.