巨噬细胞炎性蛋白-2在暴露于脂多糖的巨噬细胞高迁移率族蛋白B1分泌中的作用
Macrophage Inflammatory Protein-2 in High Mobility Group Box 1 Secretion of Macrophage Cells Exposed to Lipopolysaccharide.
作者信息
Chaochao Qin, Lou Guohua, Yang Ying, Liu Yanning, Hu Ying, Min Zheng, Chen Ping, He Jiliang, Chen Zhi
机构信息
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
Department of Environmental Medicine, School of Public Health, Zhejiang University, Hangzhou, China.
出版信息
Cell Physiol Biochem. 2017;42(3):913-928. doi: 10.1159/000478646. Epub 2017 Jun 26.
BACKGROUND/AIMS: Macrophage inflammatory protein-2 (MIP-2), a type of leukocyte chemokine, is primarily produced by macrophages, and levels increase significantly in early inflammation. However, the precise biological functions and mechanisms of MIP-2 in the development of inflammation remain unclear. The purposes of the present study were to investigate the role of MIP-2 in inflammation induced by lipopolysaccharide (LPS) in vitro and to determine the possibility of blocking the high mobility group box 1 (HMGB1) signalling pathway via MIP-2 inhibition.
METHODS
Macrophage cells (RAW264.7, U937 and THP-1 cells) were divided into control and treatments groups. Expression levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), HMGB1, chemokine (C-C motif) ligand-2 (Ccl-2), Toll-like receptor-4 (TLR-4), inducible nitric oxide synthase (iNOS), phosphorylated MAPKs (p38, ERKs, JNKs), PI3K/Akts, JAKs/STAT3, IκB, and cytoplasmic and nuclear NF-κB p65 in RAW264.7 cells were detected by qRT-PCR, enzyme-linked immunosorbent assay (ELISA) or western blot assays.
RESULTS
mip-2 siRNA and an anti-MIP-2 antibody significantly reduced the expression levels of Ccl-2, TLR-4, iNOS, IL-6, IL-1β, HMGB1, and TNF-α in RAW264.7 cells exposed to LPS (P<0.01). Additionally, mRNA expression levels of HMGB1 and TLR-4 in cells treated with LPS+mip-2 siRNA were significantly lower than those in cells treated with LPS alone (P<0.01 or P<0.05). The MIP-2 antibody significantly suppressed activation of p38-MAPK, p-STAT3, and p-Akts and translocation of NF-κB p65 from the cytoplasm to the nucleus in RAW264.7 exposed to LPS (P<0.01 or P<0.05).
CONCLUSION
mip-2 siRNA and the MIP-2 antibody can reduce the inflammatory effects induced by LPS in macrophage cells. The mechanisms may occur through down-regulation of p38-MAPK, STAT3 and Akts phosphorylation and translocation of NF-κB p65. MIP-2 plays an important role in inflammation induced by LPS.
背景/目的：巨噬细胞炎性蛋白-2（MIP-2）是一种白细胞趋化因子，主要由巨噬细胞产生，在炎症早期水平显著升高。然而，MIP-2在炎症发展过程中的确切生物学功能和机制仍不清楚。本研究的目的是探讨MIP-2在体外脂多糖（LPS）诱导的炎症中的作用，并确定通过抑制MIP-2阻断高迁移率族蛋白B1（HMGB1）信号通路的可能性。
方法
将巨噬细胞（RAW264.7、U937和THP-1细胞）分为对照组和处理组。通过qRT-PCR、酶联免疫吸附测定（ELISA）或蛋白质印迹法检测RAW264.7细胞中白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、HMGB1、趋化因子（C-C基序）配体-2（Ccl-2）、Toll样受体-4（TLR-4）、诱导型一氧化氮合酶（iNOS）、磷酸化丝裂原活化蛋白激酶（p38、细胞外信号调节激酶、c-Jun氨基末端激酶）、磷脂酰肌醇-3激酶/蛋白激酶B、Janus激酶/信号转导子和转录激活子3、IκB以及细胞质和细胞核中NF-κB p65的表达水平。
结果
mip-2小干扰RNA（siRNA）和抗MIP-2抗体显著降低了暴露于LPS的RAW264.7细胞中Ccl-2、TLR-4、iNOS、IL-6、IL-1β、HMGB1和TNF-α的表达水平（P<0.01）。此外，用LPS+mip-2 siRNA处理的细胞中HMGB1和TLR-4的mRNA表达水平显著低于单独用LPS处理的细胞（P<0.01或P<0.05）。MIP-2抗体显著抑制了暴露于LPS的RAW264.7细胞中p38丝裂原活化蛋白激酶、磷酸化信号转导子和转录激活子3以及磷酸化蛋白激酶B的激活，以及NF-κB p65从细胞质到细胞核的转位（P<0.01或P<0.05）。
结论
mip-2 siRNA和MIP-2抗体可降低LPS在巨噬细胞中诱导的炎症效应。其机制可能通过下调p38丝裂原活化蛋白激酶、信号转导子和转录激活子3以及蛋白激酶B的磷酸化和NF-κB p65的转位而发生。MIP-2在LPS诱导的炎症中起重要作用。
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