Artesunate Attenuates Pro-Inflammatory Cytokine Release from Macrophages by Inhibiting TLR4-Mediated Autophagic Activation via the TRAF6-Beclinl-PI3KC3 Pathway
Artesunate Attenuates Pro-Inflammatory Cytokine Release from Macrophages by Inhibiting TLR4-Mediated Autophagic Activation via the TRAF6-Beclinl-PI3KC3 Pathway
复制标题
青蒿琥酯通过 TRAF6-Beclinl-PI3KC3 途径抑制 TLR4 介导的自噬激活，从而减弱巨噬细胞促炎性细胞因子的释放
DOI：
10.1159/000489982
复制
发表时间：
2018-01-01
影响因子：
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通讯作者：
Zhou, Hong
中科院分区：
文献类型：
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作者：
Kuang, Mei;Cen, Yanyan;Zhou, Hong
背景/目的：脂多糖（LPS）在脓毒症期间过度炎症细胞因子产生中起关键作用。以前，青蒿琥酯（AS）的报告，以保护脓毒症小鼠通过减少LPS诱导的促炎细胞因子的释放。本研究进一步探讨了AS抗炎作用的可能机制。方法：采用酶联免疫吸附法检测巨噬细胞释放TNF-α和IL-6的情况。特异性小干扰RNA（siRNA）用于敲低靶基因的mRNA表达。透射电镜和激光共聚焦显微镜观察自噬的变化。Western blotting检测肿瘤坏死因子受体相关因子6（TRAF 6）、Beclinl、磷脂酰肌醇3-激酶III（PI 3 KC 3）、自噬相关蛋白5（ATG 5）和螯合体1的蛋白水平。免疫共沉淀（IP）和荧光共定位检测TRAF 6-Beclinl和Beclinl-PI 3 KC 3之间的相互作用以及Beclinl的泛素化。结果如下：AS抑制LPS诱导的RAW264.7细胞、小鼠骨髓源性单核细胞（BMDM）和腹腔巨噬细胞（PM）释放TNF-α和IL-6。然而，当使用3-MA、巴弗洛霉素A1或靶向Atg 5基因的siRNA抑制自噬时，AS对LPS诱导的细胞因子释放的抑制降低。值得注意的是，AS显示出抑制LPS诱导的自噬激活而不是降解。而AS的这些作用在缺乏TLR 4的巨噬细胞中丧失，并且在TRAF 6下调的巨噬细胞中降低，表明AS通过TLR 4-TRAF 6信号传导抑制LPS诱导的细胞因子释放和自噬激活。Western blotting结果显示AS可降低TRAF 6、Beclinl和PI 3 KC 3的水平。重要的是，IP结果显示AS仅通过作用于TRAF 6抑制K63连接的泛素化，而不是Beclini的全部泛素化。这中断了TRAF 6-Beclini相互作用，随后形成了PI 3 K-III复合物的Beclini-PI 3 KC 3核心复合物。结论：AS通过抑制自噬激活抑制LPS诱导的巨噬细胞释放细胞因子。这种作用与TRAF 6-Beclinl-PI 3 KC 3途径的阻断密切相关，其通过降低Beclini的K63连接的泛素化，然后中断PI 3 K-HI复合物的Beclinl-PI 3 KC 3核心复合物的形成。我们的研究结果揭示了AS的抗炎作用的机制，并为未来有针对性的研究脓毒症的治疗具有重要意义。(C)2018作者（S）由S发布。Karger AG，巴塞尔
Background/Aims: Lipopolysaccharide (LPS) plays a critical role in excessive inflammatory cytokine production during sepsis. Previously, artesunate (AS) was reported to protect septic mice by reducing LPS-induced pro-inflammatory cytokine release. In the present study, the possible mechanism of the anti-inflammatory effect of AS was further investigated. Methods: An enzyme-linked immunosorbent assay was used to detect TNF-alpha and IL-6 release from macrophages. Specific small interfering RNAs (siRNAs) were used to knockdown the mRNA expression of target genes. Transmission electron microscopy and laser confocal microscopy were used to observe changes in autophagy. Western blotting was performed to detect the protein levels of tumor necrosis factor receptor-associated factor6 (TRAF6), Beclinl, phosphatidylinositol 3-kinase class III (PI3KC3), autophagy-related protein 5 (ATG5), and sequestosome 1. Immunoprecipitation (IP) and fluorescent co-localization were used to detect the interactions between TRAF6-Beclinl and Beclinl-PI3KC3, and the ubiquitination of Beclinl. Results: AS inhibited TNF-alpha and IL-6 release from RAW264.7 cells, mouse bone marrow-derived monocytes (BMDMs) and peritoneal macrophages (PMs) induced by LPS. However, the inhibition by AS of LPS-induced cytokine release decreased when autophagy was inhibited using 3-MA, bafilomycin A1, or a siRNA targeting the Atg5 gene. Notably, AS showed an inhibition of LPS-induced autophagic activation not degradation. Whereas, these effects of AS were lost in macrophages lacking TLR4 and decreased in macrophages with down-regulated TRAF6, indicating that AS inhibited LPS-induced cytokine release and autophagic activation via TLR4-TRAF6 signaling. Western blotting results showed AS could reduce the levels of TRAF6, Beclinl, and PI3KC3. Importantly, the IP results showed AS only inhibited K63-linked ubiquitylation not total ubiquitylation of Beclini by acting on TRAF6. This interrupted the TRAF6-Beclini interaction and subsequent the formation of Beclini- PI3KC3 core complex of the PI3K-III complex. Conclusion: AS inhibited LPS-induced cytokine release from macrophages by inhibiting autophagic activation. This effect was tightly related to blockade of the TRAF6-Beclinl-PI3KC3 pathway via decreasing K63-linked ubiquitination of Beclini and then interrupting the formation of Beclinl-PI3KC3 core complex of the PI3K-HI complex. Our findings reveal the mechanism of AS's anti-inflammatory effect and is significant for future targeted investigations of sepsis treatment. (C) 2018 The Author(s) Published by S. Karger AG, Basel