关键词: 机械牵张, 高迁移率族蛋白B1, 细胞外调节蛋白激酶, 免疫组织化学, 反转录-聚合酶链反应, 免疫印迹法, 肺泡上皮细胞

Abstract: Objective To investigate the role of extracellular regulated protein kinase (ERK) signal pathway in mechanical stretch induced high mobility group box 1 protein (HMGB1) expression on alveolar epithelial cells (A549). MethodsA549 cells were cultured and seeded at 1×10SUP>5 /SUP>cells/ml in 6-well Bioflex cell culture plates. Subsequently, the cells were exposed to cyclic mechanical stretch at 14% (group B) elongation for 4 hours using Flexercell 4000T cell stretching unit. In group C, cells were pretreated with PD98059 for 2 hours before mechanical stretch. Cells in group A without stretch were served as control. The expression of HMGB1 protein and mRNA in A549 cells were detected by immunocytochemisty staining and RT-PCR, respectively. ERK activity was measured by Western blotting method. Results Immunocytochemisty staining indicated that the expression of HMGB1 protein in A549 cells was increased obviously in group B (EM>P/EM><0.05) and decreased in group C (EM>P/EM><0.05). Polymerase chain reaction (RT-PCR) showed that the expression of HMGB1 mRNA was also significantly increased in group B (EM>P/EM><0.05) and decreased in group C (EM>P/EM><0.05). Western blotting analysis confirmed the activation of ERK in A549 cells by mechanical stretch (EM>P/EM><0.05). PD98059, an inhibitor of ERK, might significantly inhibit mechanical stretch induced HMGB1 protein and mRNA expression in A549 cells (P<0.05). Conclusion Mechanical stretch could regulate the expression of HMGB1 gene and protein in A549 cells through ERK signal pathway.

Key words: Mechanical stretch, High-mobility group box-1, ERK, Alveolar epithelial cell, Immunohistochemistry, RT-PCR, Western blotting