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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 kinase6,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。
This study was designed to investigate the effects of P38 and its upstream mitogen-activated protein kinase kinase 6 (MKK6) on the cyclic stretch-induced rat high-mobility group protein B1 high mobility group box 1 protein, HMGB1). The DOTAP liposome-mediated gene transfection method was used to introduce the dominant negative mutant P38 (AF) + pGFP of P38α and the constitutively active mutant MKK6b (E) + pGFP of MKK6b into rat alveolar macrophages, Plasmid pcDNA3 + pGFP as a negative control, pGFP as a blank control. The expression of transfected gene was observed by fluorescence microscope and Western blot. Periodontal stretch model of alveolar macrophages in vitro was established. The changes of P38 kinase activity and the expression of HMGB1 protein and mRNA in the cells were observed under 20% cyclic strain. Fluorescent staining and Western blot results showed that the exogenous gene was successfully transfected into rat alveolar macrophages and expressed in the cells. P38 kinase activity was significantly increased in MKK6b (E) -transfected cells and P38 kinase activity was significantly decreased in P38 (AF) -transfected cells in a model of periodic alveolar macrophage stretch-induced strain; MKK6b (E) ) Gene transfection promoted the expression of HMGB1 protein and mRNA in rat alveolar macrophages induced by stretch strain (strain amplitude 20%), whereas p38 (AF) gene transfection inhibited the traction induced strain HMGB1 protein and mRNA expression . This study shows that periodic stretch distorting regulates the expression of HMGB1 in rat alveolar macrophages through the MKK6-P38α signaling pathway.