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目的探讨SP600125对周期牵张致A549细胞上调白细胞介素-8(IL-8)表达的影响。方法将A549细胞以密度为5×105种植于特制的硅胶树脂膜上,DMEM培养基培养48 h后,置于细胞牵张仪行周期性牵张。牵张组:牵张强度为细胞体积扩大15％,牵张周期:30个周期／min,牵张时间分别为0(未进行牵张)、15、30、60、120 min。预防处理组:将JNK抑制剂SP600125加入培养基,终浓度为50μmmol／L孵育30 min后再行上述处理,收集细胞和培养液,提取细胞中的总蛋白和RNA,用RT-PCR方法测定细胞IL-8 mRNA表达,用Western blot测定细胞JNK和磷酸化JNK(p-JNK)的水平,采用ELISA法测定细胞培养液中IL-8浓度。结果周期牵张30～120 min可导致牵张组A549细胞IL-8 mRNA、IL- 8浓度增加,且呈时间依赖性,周期牵张120 min时达高峰,p-JNK水平升高;SP600125可减弱周期牵张导致的上述改变(P<0．05)。结论周期牵张致A549细胞IL-8的上调与JNK的激活有关。
Objective To investigate the effect of SP600125 on the up-regulation of interleukin-8 (IL-8) expression in A549 cells induced by periodic stretch. Methods A549 cells were seeded onto a special silicone resin membrane at a density of 5 × 105. After cultured in DMEM for 48 h, cells were placed in a cell stretch apparatus for periodic stretch. Stretching group: Stretching intensity of 15% of the cell volume expansion, stretch cycle: 30 cycles / min, stretch time were 0 (no stretch), 15,30,60,120 min. Preventive treatment group: adding JNK inhibitor SP600125 into the culture medium and incubating at a final concentration of 50 μmmol / L for 30 min, then performing the above treatment, collecting the cells and the culture broth, extracting the total protein and RNA in the cells, measuring the cells by RT-PCR The expression of IL-8 and IL-8 mRNA were detected by Western blot. The levels of JNK and phosphorylated JNK (p-JNK) were detected by Western blot. The concentration of IL-8 in culture medium was determined by ELISA. Results The cyclic stretch of 30-120 min resulted in the increase of concentration of IL-8 mRNA and IL-8 in stretch group A549 cells in a time-dependent manner, reaching the peak at 120 min of stretch and increasing the level of p-JNK. Weakening the above changes caused by periodic stretch (P <0.05). Conclusion The up-regulation of IL-8 in A549 cells induced by periodic stretch is related to the activation of JNK.