过刊浏览

P38 及上游激酶MKK6 对周期性牵张应变诱导的肺泡巨噬细胞高迁移率族蛋白B1 表达的调控

丁宁, 肖慧, 高巨, 许立新, 佘守章

广州市第一人民医院1 麻醉科； 门诊部，广州 510180； 广州中医药大学第二附属医院麻醉科，广州 510120

摘要

本文旨在探讨P38 及其上游激酶——丝裂原活化蛋白激酶激酶6 (mitogen-activated protein kinase kinase 6, MKK6)对 周期性牵张应变诱导的大鼠肺泡巨噬细胞高迁移率族蛋白B1 (high mobility group box 1 protein, HMGB1) 表达的调控作用。 应用DOTAP 脂质体介导的基因转染方法将P38α 的显性无活性突变体P38(AF) + pGFP 以及MKK6b 的组成性活性突变体 MKK6b(E) + pGFP 分别导入大鼠肺泡巨噬细胞，以质粒pcDNA3 + pGFP 作为阴性对照，以pGFP 作为空白对照。应用 荧光显微镜和Western blot 方法观察转染基因的表达情况。建立体外大鼠肺泡巨噬细胞周期性牵张应变模型，观察各组细 胞在施加20% 周期性牵张应变时P38 激酶活性变化，HMGB1 蛋白和mRNA 的表达变化情况。荧光染色及Western blot 检 测结果显示，外源基因成功转染大鼠肺泡巨噬细胞，并在细胞内表达。在大鼠肺泡巨噬细胞周期性牵张应变模型中观察 到，MKK6b(E)转染细胞内P38 激酶活性明显升高，而P38(AF)转染细胞内P38 激酶活性显著下降；MKK6b(E)基因转染 可促进牵张应变(应变幅度为20%)诱导的大鼠肺泡巨噬细胞HMGB1 蛋白和mRNA的表达，而p38(AF)基因转染则抑制牵引 应变诱导的HMGB1 蛋白和mRNA 表达。本研究表明，周期性牵张应变通过MKK6-P38α 信号通路调节大鼠肺泡巨噬细胞 HMGB1 的表达。

关键词： P3 8 丝裂原活化蛋白激酶; 机械应力; 肺泡巨噬细胞; 高迁移率族蛋白B1

分类号：R322.3; R329.2

[Regulation of P38 and MKK6 on HMGB1 expression in alveolar macrophages induced by cyclic mechanical stretch.] [Ariticle in Chinese]

DING Ning, XIAO Hui, GAO Ju, XU Li-Xin, SHE Shou-Zhang

Departments of Anesthesiology and Out-patient clinic, Guangzhou First Municipal People’s Hospital, Guangzhou 510180, China； Department of Anesthesiology, the Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510120, China

Abstract

The aim of the present study was to investigate the role of mitogen-activated protein kinase kinase 6 (MKK6)-P38 signaling pathway in cyclic mechanical stretch-induced high mobility group box 1 protein (HMGB1) expression in alveolar macrophages. In the study, Sprague-Dawley rats were anesthetized and then sacrificed by bloodletting. The lungs were lavaged six times with prechilled PBS. Alveolar macrophages were isolated from lavage samples. Recombinant plasmids were transfected into alveolar macrophages with liposome DOTAP. Alveolar macrophages transfected with P38(AF)/pGFP and MKK6b(E)/pGFP plasmids were taken as treated groups, while the groups that transfected with pcDNA3 plasmid and pGFP plasmid served as blank transfection group and control group, respectively. All the groups were then cultured in 6-well Bioflex cell culture plates and exposed to cyclic mechanical stretch at 20% elongation using Flexercell 4000T cell stretching unit. The results showed that the transfection of MKK6b(E) led to a marked increases in P38 kinase activity compared with control group. In contrast, the transfection of P38(AF) significantly inhibited P38 kinase activity. Compared with control group, HMGB1 protein and mRNA expression in MKK6b(E) transfected cells increased markedly, while HMGB1 expression in P38(AF) transfected cells decreased markedly. These results suggest that MKK6-P38 MAPK
signaling pathway regulates the expression of HMGB1 induced by cyclic mechanical stretch in alveolar macrophages.

Key words: p38 mitogen-activated protein kinase; mechanical stress; alveolar macrophage; high mobility group box 1

收稿日期：2008-10-17　　录用日期：2008-12-22

通讯作者：许立新　　E-mail: gzxulixin@163.com

引用本文：

丁宁, 肖慧, 高巨, 许立新, 佘守章. P38 及上游激酶MKK6 对周期性牵张应变诱导的肺泡巨噬细胞高迁移率族蛋白B1 表达的调控[J]. 生理学报 2009; 61 (1): 49-55.

DING Ning, XIAO Hui, GAO Ju, XU Li-Xin, SHE Shou-Zhang. [Regulation of P38 and MKK6 on HMGB1 expression in alveolar macrophages induced by cyclic mechanical stretch.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (1): 49-55 (in Chinese with English abstract).