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降钙素基因相关肽通过增加自噬对抗新生大鼠氧化应激性肺损伤

邹振庄^1,2, 王少华², 黄元露¹, 冯伟^1,*

¹南华大学附属第二医院，衡阳 421000；²深圳市福田区妇幼保健院，深圳 518000

摘要

本研究组前期研究显示，降钙素基因相关肽(calcitonin gene-related peptide, CGRP)对高氧诱导的氧化应激性肺损伤具有保护作用。本研究旨在探讨细胞自噬是否参与CGRP对新生大鼠氧化应激性肺损伤的保护作用。新生Sprague-Dawley (SD)大鼠随机分为5组：空气对照组(Control组)、高氧损伤模型组(Model组)、CGRP干预组(Model + CGRP组)、Model + CGRP + 自噬激动剂组(Model + CGRP + Rapamycin组)和Model + CGRP+自噬抑制剂组(Model + CGRP + LY294002组)。采用新生SD大鼠持续吸氧(吸入氧浓度百分比FiO2为90%~95%)14天的方法制备氧化应激性肺损伤模型。苏木精-伊红(HE)染色观察肺组织病理变化并测定肺泡平均线性截距(mean linear intercept, MLI)。透射电镜观察肺泡II型上皮细胞(type II alveolar epithelial cell, AECII)内自噬囊泡数量改变。Western blot检测肺组织裂解液中Caspase-3、Bcl-2、mTOR和Beclin-1蛋白的表达水平。结果表明，与Model组相同时间点相比，CGRP处理显著增加AECII内自噬囊泡数量和肺组织中Beclin-1蛋白表达水平，降低肺组织mTOR蛋白表达水平，同时减轻病理变化，降低肺组织MLI值和Caspase-3蛋白表达水平，提高Bcl-2蛋白表达水平。此外，加入自噬激动剂Rapamycin可增强CGRP的上述保护作用，而自噬抑制剂LY294002可以逆转CGRP的上述保护作用。以上结果提示，CGRP可以通过增强自噬减轻高氧诱导的新生大鼠肺损伤。

关键词： 支气管肺发育不良; 降钙素基因相关肽; 细胞自噬; 新生大鼠

Calcitonin gene-related peptides protect against oxidative stress-induced lung injury via increasing autophagy in neonatal rats 

ZOU Zhen-Zhuang^1,2, WANG Shao-Hua², HUANG Yuan-Lu¹, FENG Wei^1,*

¹The Second Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang 421000, China；²Women & Children Health Institute Futian Shenzhen, Shenzhen 518000, China

Abstract

Our previous studies have shown that calcitonin gene-related peptide (CGRP) exerts protective effects on the acute lung injury induced by oxidative stress. This study was aimed to investigate whether autophagy was involved in the protection of CGRP against oxidative stress-induced lung injury in neonatal rats. Newborn Sprague-Dawley (SD) rats were randomly divided into five groups: Control group, oxidative stress model group (Model group), Model + CGRP group,  Model + CGRP + Rapamycin (an autophagy agonist) group, and Model + CGRP + LY294002 (an autophagy inhibitor) group. The model of hyperoxia-induced lung injury was established by continuous inhalation of oxygen (FiO2 = 90%–95%) for 14 days in neonatal SD rats. Pathological changes of lung tissue were observed by hematoxylin and eosin (HE) staining, and mean linear intercept (MLI) was measured. The quantitative changes of autophagic vesicles (AV) in type II alveolar epithelial cells (AECII) were measured under the transmission electron microscope. The protein expressions of Caspase-3, Bcl-2, mTOR, and Beclin-1 in lung tissue lysates were detected by Western blot. The results showed that, compared to the Model group at the same time point, the number of AV in AECII and the expression level of Beclin-1 protein of the lung tissue were increased, while the expression level of mTOR protein was decreased, with alleviated pathological changes, reduced MLI value and Caspase-3 protein expression level, increased Bcl-2 protein expression level in the lung tissue of Model + CGRP group. In addition, we found that the protective effect of CGRP on hyperoxia-induced lung injury could be enhanced by autophagy activator Rapamycin and abolished by autophagy inhibitor LY294002. Together, these findings indicate that CGRP could attenuate hyperoxia-induced lung injury in neonatal rats by enhancing autophagy.

Key words: bronchopulmonary dysplasia; calcitonin gene-related peptide; autophagy; neonatal rats

收稿日期：　　录用日期：

通讯作者：冯伟　　E-mail: 13873409212@163.com

DOI: 10.13294/j.aps.2022.0057

引用本文：

邹振庄, 王少华, 黄元露, 冯伟. 降钙素基因相关肽通过增加自噬对抗新生大鼠氧化应激性肺损伤[J]. 生理学报 2022; 74 (4): 548-554.

ZOU Zhen-Zhuang, WANG Shao-Hua, HUANG Yuan-Lu, FENG Wei. Calcitonin gene-related peptides protect against oxidative stress-induced lung injury via increasing autophagy in neonatal rats . Acta Physiol Sin 2022; 74 (4): 548-554 (in Chinese with English abstract).