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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 investigate the effect of calcitonin gene related peptide (CGRP) on the oxidative damage of fetal rat alveolar type Ⅱ epithelial cells (AEC Ⅱ) exposed to high volume fractional oxygen (hyperoxia) and whether its effect is regulated by extracellular signal regulated kinase (ERK) Mediated. Methods AECⅡ was isolated and cultured on embryonic day 21. The cells were divided into 6 groups: air group, air CGRP group, air CGRP antagonist group, hyperoxia group, hyperoxia group CGRP group, hyperoxia CGRP group Antagonist group. The air group and the hyperoxia group were cultured for 18 h in air with an oxygen volume fraction of 210 mL.L-1 or oxygen in 850 mL.L-1, respectively. CGRP group and CGRP antagonist group were added CGRP before air or hyperoxia exposure or both CGRP and its receptor antagonist CGRP8-37 at the final concentration of 10-8mol.L-1 and 10-7mol.L-1 . The levels of LDH, AKP and malondialdehyde (MDA) in culture medium were measured by immunoturbidimetric assay. The levels of reactive oxygen species (ROS) were measured by flow cytometry. The expression of cytoplasmic surfactant protein C (SP-C) Western blot was used to detect the expression of phosphorylated ERK1,2 (p-ERK1 / 2). Results The levels of MDA, LDH, AKP, ROS and p-ERK1 / 2 in hyperoxia group were significantly higher than those in the air group [(2.29 ± 0.10) μmol·L -1 vs 1.06 ± 0.14 μmol·L -1 5.01) UL-1 vs (25.92 ± 3.68) UL-1, (24.63 ± 2.92) UL-1 vs (10.34 ± 1.78) UL-1, 47.74 ± 3.35 vs 25.96 ± 5.04, 1.21 ± 0.06 vs 0.45 ± 0.05, Pa <0.01], while the expression of SP-C in cells was significantly lower than that in air group (22.75 ± 3.31 vs 43.50 ± 4.42). Compared with hyperoxia group and hyperoxia-CGRP antagonist group, MDA, LDH, ROS, AKP level was significantly decreased, while the expression of p-ERK1 / 2 and SP-C was significantly increased (Pa <0.01). There were no significant differences in the expression of MDA, LDH, ROS, AKP and SP-C between air group, air CGRP group and air CGRP antagonist group. The expression of p-ERK1 / 2 in air CGRP group was significantly higher than that in air group And air CGRP antagonist group (Pa <0.01). Conclusion CGRP can reduce the oxidative damage of AEC Ⅱ induced by hyperoxia. Its mechanism may be mediated by the activation of ERK.