神经肽P物质对高氧暴露下Ⅱ型肺泡上皮细胞的影响及其与细胞外信号调节激酶信号转导通路关系研究

Effect of neuropeptide substance P on the type Ⅱ alveolar epithelial cells exposed to hyperxia and relationship with extracellular signal-regulated protein kinase signal transduction pathway.

目的探讨感觉神经肽P物质(SP)对高氧暴露下Ⅱ型肺泡上皮细胞(AECⅡ)的影响及其与细胞外信号调节激酶(ERK)信号转导机制的关系。方法分离纯化原代早产鼠AECⅡ,随机分为空气暴露组、高氧暴露组、SP干预空气暴露组、SP干预高氧暴露组,空气暴露组氧体积分数为0.21(21%),高氧暴露组氧体积分数为0.95(95%),SP干预组于暴露前加入SP1×10-6mol/L,在置于氧体积分数为0.21(21%)和0.95(95%)中各组分别暴露12、24和48h,电镜观察AECⅡ的形态变化;MTT法及流式细胞仪测定其增殖率和凋亡率;蛋白质免疫印迹法(Western blot)检测磷酸化ERK的动态变化。结果与空气暴露组比较,高氧组暴露12、24、48h后AECⅡ增殖率明显降低,凋亡率明显增加,而SP干预后其增殖率明显增加,凋亡率明显下降,形态学的损伤也有明显的改善。高氧刺激可导致ERK的磷酸化激活,磷酸化ERK在高氧损伤的AECⅡ表达明显增加,SP干预后,磷酸化ERK表达更明显。结论SP可促进高氧暴露AECⅡ的增殖并抑制其凋亡,其通过促进ERK信号激活对氧化应激状态下的AECⅡ细胞可起到保护作用。

Objective To investigate the regulatory mechanism of neuropeptide substance P（SP） in lung injury induced by hyperxia and the relationship between SP and extracellular signal-regulated protein kinase （ERK）signal transduction pathway. Methods The primary premature rats type II alveolar epithelial cells （AEC Ⅱ ） were isolated and purified. They were randomly divided into 3 groups, including air group, hyperxia group and SP intervention group. Air（21% oxygen） group and hyperxia（95% oxygen） group were placed in the closed oxygen chamber for 48 h respectively. With regard to SP intervention group, SP（ 1×10^-6mol/L） was added before the exposure.As following, the group was exposed to 21% and 95% oxygen for 48 h respectively. Sample of each group was obtained at 12,24 and 48 h and morphologic change of AEC Ⅱ was observed under electron microscope.MTT and flow cytometry was employed to detect the growth rate and apoptosis rate respectively.Dynamic change of phosphorylated ERK was determined using Western blot. Results Growth rate of AEC Ⅱ in hyperxia group decreased siguifcantly and the apoptosis rate increased remarkbly compared to air group.The growth rate increased notablely and the apoptosis rate decreased obviously after the intervention of SP.Meanwhile, the morphologic injure improved signifcantly.Hyperxia stimulation could result in activation of ERK phosphorylation, and the expression of phosphorylated ERK increased remarkbly in the impaired AEC Ⅱ induced by hyperxia.Nevertheless, the expression of ERK increased notably after the intervention of SP. Conclusion SP could promote the proliferation and inhibit the apoptosis of AEC Ⅱ exposed to hyperxia via suppressing and then inhibiting the activation of ERK signal apoptosis, which has a protective effect on AEC Ⅱ under oxidative stress.