CGRP减轻高氧诱导的早产鼠肺泡Ⅱ型细胞损伤及对Gli1表达的影响

Calcitonin gene-related peptide alleviates type Ⅱ alveolar epithelial cells injury induced by hyperoxia and influences Gli1 expression

目的探讨降钙素基因相关肽(CGRP)对高氧致早产鼠肺泡Ⅱ型细胞(AECⅡ)损伤的保护作用及对Sonichedgehog(Shh)通路Gli1蛋白表达的影响和意义。方法分离纯化早产鼠AECⅡ,分为空气、空气+CGRP、空气+CGRP+CGRP拮抗剂(CGRP8-37)、高氧(95%O2)、高氧+CGRP及高氧+CGRP+CGRP8-37组。培养24 h后观察AECⅡ形态,流式细胞术检测凋亡率,荧光分子探针法检测细胞内活性氧(ROS),分光光度法检测丙二醛(MDA)和超氧化物歧化酶(SOD);RT-qPCR检测表面活性蛋白C(SPC)mRNA,Western blot检测Gli1蛋白表达。结果95%O2诱导24 h后,AECⅡ凋亡率比空气对照增加3.6倍,ROS水平增加1.5倍,MDA增加65%,SOD降低近一半,SPC mRNA和Gli1蛋白表达显著降低(均P<0.05);CGRP干预后可显著减轻上述变化(P<0.05)。结论CGRP可减轻高氧诱导的AECⅡ损伤,其机制可能与Shh信号通路的激活有关。

Objective To explore the protection of calcitonin gene-related peptide（CGRP） on typeⅡ alveolar epithelial cells（AECⅡ） injury induced by 95% oxygen,and influence on Gli1 protein in Sonic hedgehog（Shh） signal pathway.Methods AECⅡs were isolated from premature rats and exposed to air,air＋CGRP,air＋CGRP＋CGRP antagonists（CGRP8-37）,hyperoxia（95% oxygen）,hyperoxia＋CGRP and hyperoxia＋CGRP＋CGRP8-37 for 24 h respectively.Morphologic changes of AECⅡs were observed under electron microscope.The apoptosis was detected by flow cytometry.The level of cellular reactive oxygen species（ROS） was measured by fluorescence molecular probes,malondialdehyde（MDA） and superoxide dismutase（SOD） in AECⅡs were determined by spectrophotography.The expression of surfactant protein C（SPC） mRNA and protein of Gli11 in AECⅡs were detected by real-time quantitative polymerase chain reaction（RT-qPCR） or Western blot.Results Under the 95% oxygen condition,the apoptosis ratio of AECⅡs was increased 3.6-fold,the level of ROS increased 1.5-fold,and the activation of MDA and SOD also up-regulated sharply,SPC mRNA and Gli1 protein decreased significantly.Administration of CGRP before hyperoxia could attenuate these changes induced by 95% hyperoxia.Conclusions CGRP can alleviate AECⅡ injuries induced by 95% oxygen,which might be associated with the activation of Gli1 in Shh signal pathway.