降钙素基因相关肽对氧化应激肺损伤保护作用的Wnt7b/β-catenin信号机制

A study on the protective effects of calcitonin gene related peptide against oxidative stress in premature lung via Wnt7b/β-catenin pathway

目的探讨高氧暴露后早产鼠肺组织Wnt7b和β-连环蛋白(β-catenin)水平变化,高氧、降钙素基因相关肽(CGRP)干预后Wnt通路下游基因转录调控因子T细胞因子(TCF)、靶基因c-myc mRNA的表达,了解Wnt信号转导途径是否参与了氧化应激导致的肺泡Ⅱ型内皮细胞(AECⅡ)损伤死亡和肺发育障碍,以及其与CGRP肺保护作用的关系。方法 western blot检测95%高氧暴露早产鼠肺组织及高氧、CGRP干预后原代AECⅡWnt7b、β-catenin蛋白水平,RT-PCR测定TCF及c-myc mRNA表达。结果高氧暴露后3天肺组织Wnt7b及β-catenin蛋白表达即较空气对照组显著增强(P<0.05),7天时蛋白水平达到峰值,14天时表达有所下降,但仍明显高于空气对照组(P<0.05)。与空气对照组比较,高氧组AECⅡTCF及c-myc mRNA表达明显增强(P<0.01),高氧+CGRP组较单纯高氧组TCF、c-myc mRNA表达水平升高更为显著(P<0.01),空气+CGRP组TCF、c-myc mRNA表达与空气对照组差异无统计学意义(P>0.05)。结论高氧暴露后Wnt7b/β-catenin信号通路激活,启动下游增殖相关基因转录,可能是氧化应激性肺损伤时AECⅡ自我修复的机制之一。CGRP可促进Wnt7b/β-catenin活化,Wnt7b/β-catenin信号转导途径可能参与了CGRP的肺保护作用。

Objective To study the expression of Wnt7 b /β-catenin in the lungs of premature rat after hyperoxia exposure and the expression of downstream gene transcription regulator T-cell factor（ TCF） and target gene c-myc after CGRP and hyperoxia intervention. To understand the possible role of Wnt pathway in oxidative stress induced type Ⅱ alveolar epithelial cell（ AEC Ⅱ） injury and disturbances of lung development. Methods Premature rats and AEC Ⅱ were exposed to hyperoxia（ 95% O2） and CGRP treatment. The activation of Wnt7 b /β-catenin was assessed using Western blot and expression of TCF and c-myc mRNA using RT-PCR assay. Results The Wnt7 b and β-catenin protein levels were remarkably increased 3 d after hyperoxia exposure（ P 〈0. 05） and peaked at 7 d,then tapered but still higher than air control at 14 d（ P 〈0. 05）. Expression of TCF and c-myc mRNA were elevated in hyperoxia group than air control group（ P 〈0. 01）,also elevated in hyperoxia ＋ CGRP group than hyperoxia group（ P 〈0. 01）,and no differences between air ＋ CGRP group and air control group（ P 〉0. 05）. Conclusions Hyperoxia exposure can activate Wnt7 b /β-catenin pathway and downstream proliferation-associated gene transcription. CGRP may be a protective regulator via Wnt7 b / β-catenin pathway in premature lungs after hyperoxia-induced injury.