Department of Neurosurgery, The Second Affiliated Hospital of the University of Traditional Chinese Medicine in Guizhou, Guiyang, Guizhou.
Department of Neurosurgery, The First Affiliated Hospital of Wannan Medical College, Wuhu, Anhui.
Neuroreport. 2022 Apr 6;33(6):243-251. doi: 10.1097/WNR.0000000000001550.
Microglia are the main effectors in the inflammatory process of the central nervous system. Once overactivated, microglia may release pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-18, etc.) and accelerate neurodegeneration. Here, we aimed to explore the mechanism of how m6A methyltransferase METTL3 affects the inflammatory response of microglia, appropriately inhibiting the overactivation of microglia.
Lipopolysaccharide (LPS) was used to construct a cellular inflammation model in vitro. To evaluate the expression of METTL3 and inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-18) in cells, RT-PCR and ELISA were carried out. The related protein (TRAF6, NF-κB and I-κB) expression was examined adopting Western blot. Dot blot experiment was used to assess the effect of regulating METTL3 on the m6A level. Methylated RNA immunoprecipitation reaction was used to measure the effect of METTL3 on the m6A level of TRAF6 mRNA 3'-UTR. The co-immunoprecipitation experiment (IP) proved that METTL3 combines with TRAF6.
In LPS-mediated microglial inflammation, METTL3 expression was increased, and the expression of inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-18) and inflammatory proteins (TRAF6 and NF-κB) were upregulated. METTL3 level was positively correlated with TRAF6, and the two proteins could bind to each other. Overexpression of METTL3 promoted the activation of the TRAF6-NF-κB pathway in an m6A-dependent manner, and inhibiting NF-κB attenuated METTL3-mediated microglial activation.
METTL3 promotes LPS-induced microglial inflammation by activating the TRAF6-NF-κB pathway.
小胶质细胞是中枢神经系统炎症过程中的主要效应细胞。一旦过度激活，小胶质细胞可能会释放促炎细胞因子（IL-1β、IL-6、TNF-α和 IL-18 等）并加速神经退行性变。在这里，我们旨在探讨 m6A 甲基转移酶 METTL3 如何影响小胶质细胞的炎症反应，适当抑制小胶质细胞的过度激活。
采用脂多糖（LPS）构建体外细胞炎症模型。采用 RT-PCR 和 ELISA 检测细胞中 METTL3 和炎症细胞因子（IL-1β、IL-6、TNF-α和 IL-18）的表达。采用 Western blot 检测相关蛋白（TRAF6、NF-κB 和 I-κB）的表达。点印迹实验用于评估调节 METTL3 对 m6A 水平的影响。甲基化 RNA 免疫沉淀反应用于测量 METTL3 对 TRAF6 mRNA 3'-UTR 的 m6A 水平的影响。共免疫沉淀实验（IP）证明 METTL3 与 TRAF6 结合。
在 LPS 介导的小胶质细胞炎症中，METTL3 表达增加，炎症细胞因子（IL-1β、IL-6、TNF-α和 IL-18）和炎症蛋白（TRAF6 和 NF-κB）的表达上调。METTL3 水平与 TRAF6 呈正相关，两者蛋白可以相互结合。METTL3 的过表达以 m6A 依赖的方式促进 TRAF6-NF-κB 通路的激活，抑制 NF-κB 减弱了 METTL3 介导的小胶质细胞激活。
METTL3 通过激活 TRAF6-NF-κB 通路促进 LPS 诱导的小胶质细胞炎症。
