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Inhibition of HMGB1/RAGE-mediated endocytosis by HMGB1 antagonist box A, anti-HMGB1 antibodies, and cholinergic agonists suppresses inflammation
Molecular Medicine ( IF 6.0 ) Pub Date : 2019-04-11 , DOI: 10.1186/s10020-019-0081-6
Huan Yang 1 , Hui Liu 1 , Qiong Zeng 1 , Gavin H Imperato 1 , Meghan E Addorisio 1 , Jianhua Li 1 , Mingzhu He 2 , Kai Fan Cheng 2 , Yousef Al-Abed 2, 3, 4, 5 , Helena E Harris 6 , Sangeeta S Chavan 1, 3, 4, 5 , Ulf Andersson 7 , Kevin J Tracey 1, 3, 4, 5
Affiliation  

BackgroundExtracellular high mobility group box 1 protein (HMGB1) serves a central role in inflammation as a transporter protein, which binds other immune-activating molecules that are endocytosed via the receptor for advanced glycation end-products (RAGE). These pro-inflammatory complexes are targeted to the endolysosomal compartment, where HMGB1 permeabilizes the lysosomes. This enables HMGB1-partner molecules to avoid degradation, to leak into the cytosol, and to reach cognate immune-activating sensors. Lipopolysaccharide (LPS) requires this pathway to generate pyroptosis by accessing its key cytosolic receptors, murine caspase 11, or the human caspases 4 and 5. This lytic, pro-inflammatory cell death plays a fundamental pathogenic role in gram-negative sepsis. The aim of the study was to identify molecules inhibiting HMGB1 or HMGB1/LPS cellular internalization.MethodsEndocytosis was studied in cultured macrophages using Alexa Fluor-labeled HMGB1 or complexes of HMGB1 and Alexa Fluor-labeled LPS in the presence of an anti-HMGB1 monoclonal antibody (mAb), recombinant HMGB1 box A protein, acetylcholine, the nicotinic acetylcholine receptor subtype alpha 7 (α7 nAChR) agonist GTS-21, or a dynamin-specific inhibitor of endocytosis. Images were obtained by fluorescence microscopy and quantified by the ImageJ processing program (NIH). Data were analyzed using student’s t test or one-way ANOVA followed by the least significant difference or Tukey’s tests.ResultsAnti-HMGB1 mAb, recombinant HMGB1 antagonist box A protein, acetylcholine, GTS-21, and the dynamin-specific inhibitor of endocytosis inhibited internalization of HMGB1 or HMGB1-LPS complexes in cultured macrophages. These agents prevented macrophage activation in response to HMGB1 and/or HMGB1-LPS complexes.ConclusionThese results demonstrate that therapies based on HMGB1 antagonists and the cholinergic anti-inflammatory pathway share a previously unrecognized molecular mechanism of substantial clinical relevance.

中文翻译:


通过 HMGB1 拮抗剂盒 A、抗 HMGB1 抗体和胆碱能激动剂抑制 HMGB1/RAGE 介导的内吞作用,从而抑制炎症



背景细胞外高迁移率族蛋白 1 (HMGB1) 作为转运蛋白在炎症中发挥核心作用,它与通过晚期糖基化终产物 (RAGE) 受体内吞的其他免疫激活分子结合。这些促炎复合物靶向内溶酶体区室,其中 HMGB1 使溶酶体通透。这使得 HMGB1 伙伴分子能够避免降解、渗入细胞质并到达同源的免疫激活传感器。脂多糖 (LPS) 需要该途径通过访问其关键的胞质受体、小鼠 caspase 11 或人类 caspase 4 和 5 来产生细胞焦亡。这种溶解性、促炎性细胞死亡在革兰氏阴性脓毒症中起着基本的致病作用。本研究的目的是鉴定抑制 HMGB1 或 HMGB1/LPS 细胞内化的分子。方法在抗 HMGB1 单克隆抗体存在下,使用 Alexa Fluor 标记的 HMGB1 或 HMGB1 和 Alexa Fluor 标记的 LPS 复合物在培养的巨噬细胞中研究内吞作用(mAb)、重组 HMGB1 盒 A 蛋白、乙酰胆碱、烟碱性乙酰胆碱受体亚型 α 7 (α7 nAChR) 激动剂 GTS-21 或内吞作用的动力特异性抑制剂。通过荧光显微镜获得图像并通过 ImageJ 处理程序 (NIH) 进行量化。使用学生 t 检验或单向方差分析,然后进行最小显着性差异或 Tukey 检验来分析数据。 结果抗 HMGB1 mAb、重组 HMGB1 拮抗剂 A 蛋白、乙酰胆碱、GTS-21 和内吞作用的动力特异性抑制剂抑制内化培养的巨噬细胞中的 HMGB1 或 HMGB1-LPS 复合物。这些药物阻止巨噬细胞响应 HMGB1 和/或 HMGB1-LPS 复合物而激活。结论这些结果表明,基于 HMGB1 拮抗剂和胆碱能抗炎途径的治疗具有先前未被认识的具有重要临床意义的分子机制。
更新日期:2019-04-11
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