mTOR/4EBP1/HIF-1α/VEGF信号通路在哮喘小鼠肺组织中的表达及意义

Expression and significance of mTOR/4EBP1/HIF-1α/VEGF signaling pathway in lung tissues of asthmatic mice

摘要:

目的 探讨哺乳动物雷帕霉素靶蛋白（mTOR）/真核生物始动因子4E结合蛋白1（4EBP1）/缺氧诱导因子-1α（HIF-1α）/血管内皮生长因子（VEGF）信号通路在哮喘小鼠中的表达及意义。方法 40只SPF级6~8周龄雌性Balb/c小鼠随机分为对照组、哮喘组、布地奈德干预组及mTOR抑制剂（雷帕霉素）干预组，每组10只。卵清蛋白致敏激发建立哮喘小鼠模型，各干预组分别在激发前30 min给予雷帕霉素3 mg/kg腹腔注射或布地奈德混悬液1 mg雾化吸入，对照组和哮喘组以生理盐水代替。于末次激发24 h后处死小鼠，收集肺泡灌洗液（BALF），采用ELISA法测定HIF-1α、VEGF水平；取肺组织行苏木精-伊红（HE）染色观察其病理变化；免疫组化染色和Western blot法测定肺组织磷酸化的mTOR及4EBP1（p-mTOR及p-4EBP1）蛋白表达水平。Pearson法分析p-mTOR、p-4EBP1、HIF-1α、VEGF表达的相关性。结果 与对照组相比，哮喘组气管及其周围炎性细胞浸润明显，分泌物增多；BALF中HIF-1α、VEGF水平显著升高（P < 0.01）；肺组织 p-mTOR、p-4EBP1 表达显著增加（P < 0.01）。与哮喘组相比，各干预组气道炎症浸润明显减轻，分泌物减少；BALF中HIF-1α、VEGF水平明显下降（P < 0.01）；肺组织p-mTOR、p-4EBP1表达显著降低（P < 0.01）。对照组及两干预组间相比上述指标变化差异均无统计学意义（P > 0.05）。哮喘组小鼠p-mTOR、p-4EBP1、HIF-1α、VEGF表达水平两两互呈正相关（P < 0.05），而对照组及两干预组各指标间无相关性（P > 0.05）。结论 哮喘发生时p-mTOR、p-4EBP1、HIF-1α、VEGF可能协同参与了哮喘的发病过程。雷帕霉素能阻断这一过程，可能作为治疗哮喘的新靶点。

Abstract:

Objective To study the expression and significance of the mammalian target of rapamycin (mTOR)/eukaryote initiating factor 4E binding protein 1 (4EBP1)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway in asthmatic mice. Methods Forty SPF level 6-8 week-old female Balb/C mice were randomly divided into control, asthma, budesonide and mTOR inhibitor (rapamycin) intervention groups (n=10 each). The asthmatic mouse model was prepared via OVA induction and challenge test. The intervention groups were administered with rapamycin at the dosage of 3 mg/kg by an intraperitoneal injection or budesonide suspension at the dosage of l mg by aerosol inhalation respectively 30 minutes before the OVA challenge. The control and asthma groups were treated with normal saline instead. The concentrations of HIF-1α and VEGF in bronchoalveolar lavage fluid (BALF) were examined using ELISA 24 hours after the last challenge. The pathological changes of lung tissue were observed by hematoxylin-eosin (HE) staining. The p-mTOR and p-4EBP1 from the lung tissues were detected by immunohistochemistry and Western blot. Pearson analysis was used to study the correlation between p-mTOR, p-4EBP1, HIF-1α, and VEGF expression. Results Compared with the control group, inflammatory cell infiltration and secretions in the trachea increased in the asthma group. The levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues also increased (P < 0.01). Compared with the asthma group, inflammatory cell infiltration and secretions in the trachea were reduced in the two intervention groups, and the levels of HIF-1α and VEGF in BALF and p-mTOR and p-4EBP1 expression in lung tissues were also reduced (P < 0.01). There were no significant differences in the above changes between the two intervention groups and control group (P > 0.05). In the asthma group, there was a pairwise positive correlation between lung p-mTOR and p-4EBP1 expression and HIF-1α and VEGF levels in BALF (P < 0.05). However, there were no correlations in the above indexes in the intervention groups and control group. Conclusions p-mTOR, p-4EBP1, HIF-1α and VEGF together are involved in the pathogenesis of asthma. Rapamycin treatment can block this signaling pathway, suggesting that this pathway can be used as a novel target for asthma treatment.