Kuang Mei, Cen Yanyan, Qin Rongxin, Shang Shenglan, Zhai Zhaoxia, Liu Chao, Pan Xichun, Zhou Hong
Department of Pharmacology, College of Pharmacy, the Third Military Medical University, Chongqing, China.
Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, China.
Cell Physiol Biochem. 2018;47(2):475-488. doi: 10.1159/000489982. Epub 2018 May 22.
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.
An enzyme-linked immunosorbent assay was used to detect TNF-α 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), Beclin1, 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-Beclin1 and Beclin1-PI3KC3, and the ubiquitination of Beclin1.
AS inhibited TNF-α 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, Beclin1, and PI3KC3. Importantly, the IP results showed AS only inhibited K63-linked ubiquitylation not total ubiquitylation of Beclin1 by acting on TRAF6. This interrupted the TRAF6-Beclin1 interaction and subsequent the formation of Beclin1- PI3KC3 core complex of the PI3K-III complex.
AS inhibited LPS-induced cytokine release from macrophages by inhibiting autophagic activation. This effect was tightly related to blockade of the TRAF6-Beclin1-PI3KC3 pathway via decreasing K63-linked ubiquitination of Beclin1 and then interrupting the formation of Beclin1-PI3KC3 core complex of the PI3K-III complex. Our findings reveal the mechanism of AS's anti-inflammatory effect and is significant for future targeted investigations of sepsis treatment.
背景/目的：脂多糖（LPS）在脓毒症期间过度的炎性细胞因子产生中起关键作用。此前有报道称青蒿琥酯（AS）可通过减少LPS诱导的促炎细胞因子释放来保护脓毒症小鼠。在本研究中，进一步探究了AS抗炎作用的可能机制。
采用酶联免疫吸附测定法检测巨噬细胞中TNF-α和IL-6的释放。使用特异性小干扰RNA（siRNA）敲低靶基因的mRNA表达。采用透射电子显微镜和激光共聚焦显微镜观察自噬的变化。进行蛋白质印迹法检测肿瘤坏死因子受体相关因子6（TRAF6）、Beclin1、Ⅲ类磷脂酰肌醇3激酶（PI3KC3）、自噬相关蛋白5（ATG5）和聚集体蛋白1的蛋白水平。采用免疫沉淀（IP）和荧光共定位检测TRAF6-Beclin1和Beclin1-PI3KC3之间的相互作用以及Beclin1的泛素化。
AS抑制LPS诱导的RAW264.7细胞、小鼠骨髓来源单核细胞（BMDM）和腹腔巨噬细胞（PM）中TNF-α和IL-6的释放。然而，当使用3-甲基腺嘌呤、巴弗洛霉素A1或靶向Atg5基因的siRNA抑制自噬时，AS对LPS诱导的细胞因子释放的抑制作用减弱。值得注意的是，AS抑制LPS诱导的自噬激活而非降解。此外，在缺乏TLR4的巨噬细胞中AS的这些作用消失，在TRAF6下调的巨噬细胞中AS的作用减弱，表明AS通过TLR4-TRAF6信号通路抑制LPS诱导的细胞因子释放和自噬激活。蛋白质印迹结果显示AS可降低TRAF6、Beclin1和PI3KC3的水平。重要的是，IP结果显示AS仅通过作用于TRAF6抑制Beclin1的K63连接的泛素化而非总泛素化。这中断了TRAF6-Beclin1相互作用以及随后PI3K-III复合物的Beclin1-PI3KC3核心复合物的形成。
AS通过抑制自噬激活抑制LPS诱导的巨噬细胞细胞因子释放。这种作用与通过减少Beclin1的K63连接的泛素化进而中断PI3K-III复合物的Beclin1-PI3KC3核心复合物的形成来阻断TRAF6-Beclin1-PI3KC3途径密切相关。我们的研究结果揭示了AS抗炎作用的机制，对未来脓毒症治疗的靶向研究具有重要意义。
