HIF-1α/GLUT1信号通路在中耳低氧致中耳渗液中的机制研究

Secretory otitis media（OME） is a common disease in department of ENT. Dysfunction of eustachian tube is the main cause for the treatment. Hypoxia of middle ear caused by dysfuntion of eustachian tube has been confirmed. Until now the mechanism of hypoxia in effusion of OME has not been studied. HIF-1 gene is currently the only and a highly specific nuclear transcription factor to hypoxia, which can play an important role in hypoxic conditions. Our preliminary experiment has confirmed the higher expression of HIF-1α in clinical middle ear mucosa sample with dysfuntion of eustachian tube and OME rat model caused by dysfunction of eustachian tube. So How HIF-1α activated in hypoxia situation induces effusion in middle ear ? Our further preliminary experiment has confirmed that HIF-1 can upregulation GLUT1, then promote glycolysis and cause the consumption of ATP, which will downregulation Na-K-ATP enzyme and Na-K-ATP ion channel function. Our study, therefore, aims to explore the activation status of HIF-1α/GLUT1 signaling and the function Na+-K+-ATPase both in animal model of OME caused by dysfunction of euatachian tube and in vitro. Hopefully to provide scientific theory for the development of new preventive measures and treatment measures of eustachian tube dilatation.

分泌性中耳炎（OME）是耳鼻喉科常见疾病，咽鼓管功能障碍是其治疗后容易复发的主要因素，而咽鼓管功能障碍所致中耳低氧已被证实。目前尚未有关中耳腔低氧参与OME中耳渗出机制研究的报道，我们前期预实验已证实在咽鼓管功能不良的临床中耳粘膜以及咽鼓管堵塞的OME大鼠中耳粘膜中存在HIF-1α的高表达，HIF-1α是目前已知的在低氧环境下高度特异的核转录因子，那么低氧状态下活化的HIF-1α是如何介导中耳渗出呢？我们进一步预实验显示，HIF-1α通过上调GLUT1促进糖酵解，消耗ATP，结果将导致Na-K-ATP酶活性及Na-K-ATP离子通道功能下调，最终引起中耳渗液。本课题拟通过体内外实验，采用形态学、分子生物学及电生理的方法，探讨中耳低氧诱导HIF-1α转录通过上调GLUT1促糖酵解增强，下调Na-K-ATP酶活性及离子通道，导致中耳渗出的新机制，为开发新的预防和治疗OME措施提供科学理论依据。

低氧环境下HIF-1转录后启动一系列细胞因子活性上调，包括介导GLUT1致糖酵解增加，ATP含量减少，导致Na -K -ATP酶活性下降及Na -K -ATP离子通道改变功能紊乱，最终继发引起水通道功能下调，离子和水分子介导的液体转运能力过于薄弱则会引起中耳腔积液过多，形成SOM。后者已被研究与中耳腔渗出、SOM的发生密切相关，而咽鼓管功能障碍引起中耳腔缺氧亦已被证实。本项目拟应用在体的动物模型以及离体细胞试验、应用听觉电生理学、形态学、分子生物学等方法检测咽鼓管功能障碍致中耳腔缺氧引起的SOM大鼠模型HIF/GLUT1信号活化状态、AQPs表达的改变以及Na -K -ATP酶功能的改变，阐明HIF/GLUT1信号通路诱导AQPs/Na -K -ATP酶功能下调致水通道、离子通道功能障碍在中耳炎发生机制中的作用。我们的实验已证实在咽鼓管功能障碍的SOM大鼠模型中耳粘膜HIF-1α /GLUT1的高表达。我们同时采用EASB上皮细胞在不同低氧环境下进行低氧培养，然后检测低氧培养下HIF-1α/GLUT1的表达，以及采用膜片钳进行低氧培养下上皮细胞Na -K -AT离子通道的检测。结果显示，不同氧浓度下的低氧状态培养，上皮细胞表达HIF-1α/GLUT1均呈现随着时间的推移先升高后下降的趋势，且Na 电流值随着低氧程度及低氧时间逐渐下降，后逐渐趋于稳定，证实在中耳低氧状态下HIF-1转录后介导GLUT1致糖酵解增加，ATP含量减少，导致Na -K -ATP酶活性下降及Na -K -ATP离子通道改变功能紊乱，最终继发引起水通道功能下调。同时我们发现在诱导中耳低氧，通过圆窗膜渗透导致淋巴液处于低氧状态，毛细胞受到低氧刺激而发生缺血缺氧改变，mir34a上调，并介导Bcl-2下调，导致内耳毛细胞凋亡增多，并随着缺氧时间的延长而出现听力损伤。揭露临床关于长期咽鼓管功能不良，中耳低氧导致渗液、上鼓室内陷袋等形态学改变后，逐渐出现感音神经性听力损失的可能分子生物学机制。