MKK6p38α信号通路在机械牵张诱导肺泡上皮细胞表达晚期糖基化终末产物受体中的作用

Effect of mitogen-activated protein kinase kinase 6-p38α signal pathway on receptor for advanced glycation end-product expression in alveolar epithelial cells induced by mechanical stretch

目的探讨p38α及其上游激酶丝裂素活化蛋白激酶6（MKK6）在机械牵张诱导肺泡上皮细胞（A549）表达晚期糖基化终末产物受体（RAGE）中的作用。方法应用阳离子脂质体DOTAP介导的基因转染方法将p38a显性无活性突变体p38α（AF）＋绿色荧光表达载体pGFP及MKK6b的组成性活性突变体MKK6b（E）＋pGFP分别导入A549细胞，以质粒pcDNA3＋pGFP作为阴性对照。以pGFP作为空白对照。应用蛋白质免疫印迹法（Westernblotting）检测转染基因。建立体外细胞牵张应变模型，观察各组细胞在施加20％牵张应变时p38激酶活性变化及RAGE的蛋白和mRNA表达变化。结果Western blotting检测结果显示，外源基因成功转染A549细胞，并在细胞内表达。在A549细胞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p38a（AF）转染细胞内p38激酶活性显著下降；MKK6b（E）基因转染可促进20％牵张应变诱导A549细胞RAGE的蛋白和mRNA表达，而p38α（AF）基因转染则抑制牵引应变诱导的RAGE蛋白和mRNA表达。结论牵张应变通过MKK6p38α信号通路调节A549细胞RAGE的表达。

Objective To investigate the role of mitogen-activated protein kinase kinase 6 （MKK6） p38α in mechanical stretch induced receptor for advanced glycation end-product （RAGE） expression in alveolar epithelial cells （A549）. Methods Recombinant plasmids were transfected into A549 ceils with liposome DOTAP. A549 cells transfected with p38α （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. The infection and expression of gene were assessed by Western blotting analysis. The activity of p38 kinase in A549 cells and the expression of RAGE protein and mRNA were observed. Results The transfection of MKK6b （E） led to a markedly increase in p38 kinase activity compared with control group. In contrast, the transfection of p38α（AF） significantly inhibited p38 kinase activity. Compared with control group, stretch markedly increased RAGE protein and mRNA expression in MKK6b （E） transfected cells, while it markedly decreased RAGE expression in p38α （AF） transfected cells. Conclusion MKK6 p38αsignaling pathway regulates the expression of RAGE induced by mechanical stretch in A549 cells.