神经肽P物质对高体积分数氧暴露下大鼠Ⅱ型肺泡上皮细胞的调控作用

Regulative Effect of Neuropeptide Substance P on Type Ⅱ Alveolar Epithelial Cells in Rat Exposed to Hyperxia

目的探讨神经肽P物质(SP)在高体积分数氧(高氧)肺损伤中的调控机制及其与c-Jun氨基末端激酶(JNK)信号转导通路的关系。方法分离纯化原代早产鼠Ⅱ型肺泡上皮细胞(AECⅡ),随机分为空气暴露组(空气组)、高氧暴露组(高氧组)、SP干预空气暴露组(空气加SP组)及SP干预高氧暴露组(高氧加SP组)。空气组和高氧组分别置于氧体积分数为210 mL/L和950 mL/L密闭氧仓暴露48 h;空气加SP组及高氧加SP组于暴露前加入1×10-6mol/LSP,再分别置于氧体积分数为210 mL/L和950 mL/L密闭氧仓中暴露48 h。各组分别于12、24、48 h取样,常规脱水、包埋、切片,电镜下观察AECⅡ的形态变化;3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四唑法及流式细胞仪测定其增殖率和凋亡率;蛋白质免疫印迹法检测磷酸化JNK的动态变化。结果与空气组比较,高氧组暴露12、24、48 h后AECⅡ增殖率均明显降低(Pa<0.05),凋亡率均明显升高(Pa<0.05),高氧加SP组AECⅡ在暴露12、24、48 h后增殖率均明显升高(Pa<0.05),凋亡率明显下降(Pa<0.05),形态学损伤明显改善。高氧刺激可导致JNK的磷酸化激活,磷酸化JNK在高氧损伤的AECⅡ表达均明显增加(Pa<0.05),SP干预后,磷酸化JNK表达明显降低(Pa<0.05)。结论SP可促进高氧暴露下AECⅡ的增殖,并通过抑制JNK信号激活从而抑制AECⅡ的凋亡,对氧化应激状态下的AECⅡ细胞有保护作用。

Objective To explore the regulative mechanism of neuropeptide substance P（SP） in lungs injury induced by hyperxia and the relationship between SP and c - Jun N - terminal kinase （JNK） signal transduction pathway. Methods The type II alveolar epithelia] cells（ AEC H ） in primary premature rats were isolated and purified. They were randomly divided into 3 groups：air group, hyperxia group, air plus SP group and hyperxia plus SP group. Air group and hyperxia group were placed in the 210 mL/L and 950 mL/L closed oxygen chamber for 48 h, respectively. With regarded to air plus SP group and hyperxia plus SP group, 1×10^-6 mol/L SP was added before the exposure. As following, the group was exposed to 210 mL/L and 950 mL/L oxygen for 48 h, respectively. Samples of each group were obtained at 12,24 and 48 h and were dehydrated and embedded for morphologic change of AEC H was observed under electron microscope. 3 - （4,5 - dimethyhhiazol- 2 -yl） -2,5 -diphenyltetrazolium bromide and flow cytometry was employed to detect the growth rate and apoptosis rate ,respectively. Dynamic change of phosphorylated JNK（ p - JNK） was determined using Western blot. Results Growth rate of AEC Ⅱin hyperxia group decreased siguifcantly （ Pa 〈 0.05 ） and the apoptosis rate increased remarkably （ Pa 〈 0.05 ） compared with air group. The growth rate increased notablcly （ P, 〈 0.05 ） and the apoptosis rate decreased obviously （ P 〈 0.05 ） after the intervention of SP. Meanwhile, the morphalogic injure improved siguifcantly. Hyperxia stimulation could result in activation of JNK phosphorylation,and the expression of p - JNK increased remar- kably（ Pa 〈 0.05 ） in the impaired AEC H induced by hyperxia. Nevertheless, the expression of p -JNK decreased notablcly after the intervention of SP（Pa 〈 0.05）. Conclusions SP can promote the proliferation and inhibit the apoptosis of AEC 11 exposed to hyperxia via suppressing activation of JNK signal and then inhibiting the apoptosis,which have a protective effect on AEC H under oxidative stress.