降钙素基因相关肽调控细胞外信号调节激酶减轻高体积分数氧对胎鼠肺泡Ⅱ型上皮细胞的损伤作用

Damage-Reduced Effects of Calcitonin Gene-Related Peptide on Hyperoxia-Exposed TypeⅡ Alveolar Epithelial Cell Mediated by Extracellular Signal-Regulated Kinase

目的探讨降钙素基因相关肽(CGRP)对高体积分数氧(高氧)暴露下胎鼠肺泡Ⅱ型上皮细胞(AECⅡ)氧化损伤的影响,及其作用是否由细胞外信号调节激酶(ERK)所介导。方法原代分离培养孕21 d胎鼠AECⅡ,培养12 h待细胞贴壁后分为6组:空气组、空气CGRP组、空气CGRP拮抗剂组、高氧组、高氧CGRP组、高氧CGRP拮抗剂组。空气组和高氧组分别在氧体积分数为210 mL.L-1的空气或850 mL.L-1的氧气中培养18 h。CGRP组和CGRP拮抗剂组分别在空气或高氧暴露前加入CGRP或同时加入CGRP和其受体拮抗剂CGRP8-37,终浓度分别为10-8mol.L-1和10-7mol.L-1。用免疫比浊法测定培养液LDH、AKP和丙二醛(MDA)水平,流式细胞术测定细胞内活性氧(ROS)水平,荧光显微镜检测胞质表面活性蛋白C(SP-C)的表达情况,Western blot检测磷酸化ERK1,2(p-ERK1/2)的表达水平。结果高氧组MDA、LDH、AKP、ROS及p-ERK1/2水平均明显高于空气组[(2.29±0.10)μmol.L-1 vs(1.06±0.14)μmol.L-1,(58.79±5.01)U.L-1 vs(25.92±3.68)U.L-1,(24.63±2.92)U.L-1 vs(10.34±1.78)U.L-1,47.74±3.35 vs 25.96±5.04,1.21±0.06 vs 0.45±0.05,Pa<0.01],而细胞内SP-C表达则明显低于空气组(22.75±3.31 vs 43.50±4.42);与高氧组及高氧CGRP拮抗剂组比较,高氧CGRP组MDA、LDH、ROS及AKP水平显著降低,而p-ERK1/2及SP-C的表达水平则明显增高(Pa<0.01)。空气组、空气CGRP组、空气CGRP拮抗剂组组间MDA、LDH、ROS、AKP及SP-C表达水平比较差异均无统计学意义;空气CGRP组p-ERK1/2表达水平明显高于空气组及空气CGRP拮抗剂组(Pa<0.01)。结论CGRP可减轻高氧对AECⅡ的氧化损伤,其作用机制可能是通过ERK的活化来介导。

Objective To explore the effects of calcitonin gene-related peptide（CGRP） on typeⅡ alveolar epithelial cell（AECⅡ） exposed to hyperoxia and whether the mechanism is mediated by extracellular signal-regulated kinase（ERK） pathway. Methods AECⅡ were isolated from 21 d fetal rat lung and grew for 12 h to attach.Then AECⅡ were randomly divided into six groups：air group,CGRP/air group,CGRP8-37/air group,hyperoxia group,CGRP/O2 and CGRP8-37/O2 group.Air or hyperoxia environment was achieved by exposing AECⅡ into 210 mL·L-1 oxygen or 850 mL·L-1 oxygen for 18 h.CGRP group or CGRP8-37 group was carried out by adding 10-8 mol·L-1 CGRP or both CGRP and CGRP8-37（10-7 mol·L-1）,a receptor antagonist against CGRP,into medium before cultured in air or 850 mL·L-1 oxygen.Lactate dehydrogenase（LDH）,alkaline phosphatase（AKP） and malondialdehyde（MDA） were measured by immune turbidimetry and reactive oxygen species（ROS） by flow cytometry.Immunofluorescence microscopy was used to analyze the expression of surfactant protein C（SP-C） and Western blot was taken to detect the content of p-ERK1/2. Results The levels of MDA,LDH,AKP,ROS and p-ERK1/2 were markedly increased in hyperoxia group than those in air group[（2.29±0.10） μmol·L-1 vs（1.06±0.14） μmol·L-1,（58.79±5.01） U·L-1 vs（25.92±3.68） U·L-1,（24.63±2.92） U·L-1 vs（10.34±1.78） U·L-1,47.74±3.35 vs 25.96±5.04,1.21±0.06 vs 0.45±0.05,Pa0.01],whereas expression of SP-C was decreased in hyperoxia group compared with air group（22.75±3.31 vs43.50±4.42）.Levels of MDA,LDH,AKP and ROS were reduced with an elevated expression of p-ERK1/2 and SP-C in CGRP/O2 group compared with those in hyperoxia group and CGRP8-37/O2 group（Pa0.01）.There were no significant differences about the levels of MDA,LDH,AKP,ROS and SP-C among three groups cultured in air condition.The expression of p-ERK1/2 in CGRP/air group was also higher than that in air group or CGRP8-37/air group（Pa0.01）. Conclusion CGRP can protect lung epithelium cells against hyperoxic insult and its effects can mediate by ERK pathway.