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Advances in Clinical Medicine
Vol. 14  No. 05 ( 2024 ), Article ID: 88000 , 5 pages
10.12677/acm.2024.1451680

转录因子GABPA对机械通气相关肺损伤中 调控紧密连接蛋白的影响

孔磊1，曹乐2,3，赵涛1,3*

1日照市人民医院麻醉科，山东 日照

2山东第二医科大学麻醉学院，山东 潍坊

3日照市麻醉与呼吸重症基础研究重点实验室，山东 日照

收稿日期：2024年4月29日；录用日期：2024年5月24日；发布日期：2024年5月31日

摘要

目的：探讨转录因子GABPA在机械通气相关肺损伤中对紧密连接蛋白Occludin表达的影响。方法：对小鼠肺上皮细胞(MLE-12)进行机械牵张，牵张时间为0 h、2 h、4 h，牵张幅度为20%。利用GABPA siRNA预处理MLE-12敲低GABPA基因48 h后，再对MLE-12细胞进行机械牵张，牵张时间为0 h、4 h，牵张幅度为20%。机械牵张后，提取蛋白后，利用蛋白质印迹法检测GABPA和Occludin的表达。结果：与CS (0 h)组比较，CS (2 h)组、CS (4 h)组中GABPA表达均升高(P < 0.05)、Occludin表达均降低(P < 0.05)。与CS (4 h) + si-Nc组比较，CS (4 h) + si-GABPA组Occludin表达升高(P < 0.05)。结论：机械牵张导致VILI中的转录因子GABPA表达升高，紧密连接蛋Occludin表达降低；GABPA在调控Occludin中起重要作用，是VILI潜在治疗靶点。

关键词

机械通气，急性肺损伤，GA结合蛋白转录因子α，紧密连接蛋白

Effect of GABPA on the Regulation of Tight Junction in Ventilation-Induced Lung Injury

Lei Kong1, Le Cao2,3, Tao Zhao1,3*

1Department of Anesthesiology, The People’s Hospital of Rizhao, Rizhao Shandong

2School of Anesthesiology, Shandong Second Medical University, Weifang Shandong

3Rizhao Key Laboratory of Basic Research on Anesthesia and Respiratory Intensive Care, Rizhao Shandong

Received: Apr. 29th, 2024; accepted: May 24th, 2024; published: May 31st, 2024

ABSTRACT

Objective: To investigate the effect of GABPA on the expression of Occludin in ventilator-induced lung injury. Methods: MLE-12 cells were treated with cyclic stretch with 20% amplitude for 0 h, 2 h, 4 h. MLE-12 cells were pre-treated with GABPA siRNA to knock down GABPA gene for 48 h, and then treated with cyclic stretch with 20% amplitude for 0 h and 4 h. After cyclic stretch, the protein of MLE-12 cells was extracted, and the expressions of GABPA and Occludin were detected by Western blotting. Results: Compared with CS (0 h) group, the expression of GABPA increased in CS (2 h) group and CS (4 h) group (P < 0.05), and the expression of Occludin decreased (P < 0.05). Compared with CS (4 h) + si-Nc group, the expression of Occludin increased in CS (4 h) + si-GABPA group (P < 0.05). Conclusion: Mechanical stretch increased the expression of GABPA and decreased the expression of Occludin in VILI. GABPA plays an important role in regulating Occludin and is a potential therapeutic target in VILI.

Keywords:Mechanical Ventilation, Acute Lung Injury, GA Binding Protein Transcription Factor α, Tight Junction Protein

Copyright © 2024 by author(s) and Hans Publishers Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).

http://creativecommons.org/licenses/by/4.0/

1. 引言

机械通气相关肺损伤(Ventilator-induced lung injury, VILI)常发生于临床全身麻醉机械通气患者和重症监护室呼吸机治疗患者中 [1] [2] 。由于呼吸机设置不当，如吸氧浓度过高或过低，或者呼出气体的压力过大等，均可能导致肺泡过度膨胀或萎陷，引起的跨肺压、剪应力增大，引起的肺组织、间质性结构和肺泡膜损伤，肺泡通透性增加，导致肺损伤 [3] [4] 。Occludin是紧密连接蛋白，通过紧密连接形成肺泡膜屏障，维持细胞通透性 [5] 。研究发现 [6] ，大潮气量机械通气或病理性机械牵张导致紧密连接蛋白Occludin表达降低，肺泡通透性增加。在VILI中，调控Occludin的机制尚未完全研究清楚。GA结合蛋白转录因子α (GA Binding Protein Transcription Factor α, GABPA)是一种重组蛋白，与DNA结合的转录因子，在生物学中扮演着重要的角色，调控细胞连接蛋白 [7] 。本研究拟探讨转录因子GABPA在VILI中调控细胞连接蛋白的机制。

2. 材料和方法

2.1. 实验细胞

小鼠肺上皮细胞(MLE-12)购自上海富衡生物科技有限公司。

2.2. 细胞培养和实验分组

完全培养基：DMEM/F-12培养基、10%胎牛血清、青霉素(100 U/ml)和链霉素(100 mg/ml)。将密度为5 × 105细胞/mL的MLE-12细胞接种于BioFlex六孔板(胶原I涂层)，培养箱的温度为37℃，CO2的浓度为5%。GABPA siRNA (si-GABPA)和阴性对照siRNA (si-Nc)由吉玛公司构建和合成。在MLE-12细胞生长至50%，将si-GABPA和si-Nc利用Lipofectamine 3000转染试剂转染至MLE-12细胞，转染48 h后进行机械牵张。使用FX-5000T Flexercell Tension Plus系统进行机械牵张，参数如下：牵张幅度为20%，频率为0.5 HZ，牵张和放松的比例为1:1，牵张时间为0 h、2 h、4 h。实验分组：CS (0 h)组、CS (2 h)组、CS (4 h)组、si-Nc组、si-GABPA组、CS (4 h) + si-Nc组、CS (4 h) + si-GABPA组。CS (0 h)组、CS (4 h)组、si-Nc组作为不同研究的对照组。每个实验至少进行了三个实验。

2.3. 蛋白质免疫印迹

在MLE-12细胞经过siRNA或机械牵张预处理后，提取MLE-12细胞蛋白。倒掉六孔板中的完全培养基后，利用4℃预冷的PBS进行洗涤，1 min/次，3次；配制细胞裂解液，RIPA:PMSF = 100:1，六孔板每孔加入细胞裂解液100 μl，冰上裂解30 min，裂解完后将细胞碎片和裂解液移至1.5 ml离心管中，于4℃下12,000 rpm离心20 min，取上清液即为MLE-12细胞蛋白。取5×loading buffer与细胞蛋白进行变性，95℃金属浴10 min。根据不同蛋白的分子量，选择合适的凝胶浓度与电泳条件能够让目的蛋白区域最大限度的分离开。采用SDS-PAGE电泳，每孔上样等体积总蛋白，加marker后开始电泳，80 V电压30 min，120 V电压60 min。PVDF膜进行转膜，转膜时间为60 min。5%脱脂奶粉室温封闭2小时，一抗4℃孵育过夜(Occludin, 1:1000; GABPA, 1:200; GAPDH, 1:1000)，二抗室温孵育2小时(1:5000)。通过FluorChem E检测蛋白条带，AlphaView软件分析Occludin和GABPA的相对表达量。

2.4. 统计学分析

使用SPSS 26.0软件行统计分析，所有数据均采用均值±标准差。采用单因素方差分析(ANOVA)评估显著差异，认为P < 0.05为差异具有统计学意义。

3. 结果

3.1. 机械牵张导致GABPA激活以及Occludin表达降低

20%的牵张幅度对MLE-12细胞进行机械牵张，时间为0 h、2 h和4 h。与CS (0 h)组比较，CS (2 h)组、CS (4 h)组的GABPA的表达均升高(P < 0.05)，Occludin表达量均降低(P < 0.05)；与CS (2 h)组，CS (4 h)组的GABPA的表达均升高(P < 0.05)，Occludin表达量均降低(P < 0.05) (见表1)。

Table 1. The relative expressions of GABPA and Occludin with cyclic stretch of MLE-12

表1. 机械牵张MLE-12后GABPA和Occludin相对表达量

注：与CS (0 h)组比较，aP < 0.05；与CS (2 h)组比较，bP< 0.05。

3.2. 在VILI中，GABPA敲低后逆转Occludin表达的降低

与si-Nc组比较，si-GABPA组中GABPA的表达降低(P < 0.05)；与si-Nc组比较，CS (4 h) + si-Nc组中GABPA表达升高(P < 0.05)，Occludin表达降低(P < 0.05)；与CS (4 h) + si-Nc组比较，CS (4 h) + si-GABPA组中GABPA的表达降低(P < 0.05)，Occludin表达量升高(P < 0.05) (见表2)。

Table 2. The relative expressions of GABPA and Occludin with GABPA knockdown in VILI

表2. 在VILI中，GABPA敲低后，GABPA和Occludin相对表达量

注：与si-Nc组比较，aP < 0.05；与CS(4 h) + si-Nc比较，bP < 0.05。

4. 讨论

由于机械通气过程中肺泡过度扩张或肺内压过高可导致肺组织及间质结构的破坏和肺泡膜损伤，表现为肺水肿、肺顺应性降低和氧合功能障碍，严重可导致呼吸窘迫综合征，甚至出现多器官功能障碍综合征 [8] 。本研究采用机械牵张模拟临床上的机械通气，利用牵张幅度20%牵张4 h模拟机械通气相关肺损伤 [9] 。

相邻肺泡上皮细胞以紧密连接蛋白相连，用于维持肺泡的通透性，肺泡上皮细胞间紧密连接蛋白变化和再分布可导致肺泡通透性变化 [10] [11] 。Occludin是紧密连接蛋白中最具代表性的蛋白，主要负责密封细胞间连接、维持细胞通透性、参与维持肺泡上皮的完整性 [12] 。因此，本文采用Occludin作为研究肺泡上皮通透性的关键蛋白。研究发现 [13] ，在大潮气量机械通气过程中，Occludin表达下调，本文研究结果与之一致，在20%机械牵张幅度下，Occludin表达降低。我们需要对其调控机制进行研究。

GA结合蛋白转录因子α (GA Binding Protein Transcription Factor Alpha, GABPA)是CNC转录因子家族成员中获利最强的转录因子，主要表达于肝、肾、肺、消化道等各种细胞 [14] 。通过6个高度保守的环氧氯丙烷相关蛋白同源结构域与其他靶基因的启动子结合，启动目标基因转录 [15] 。本研究发现，在机械通气相关肺损伤中，GABPA表达升高，但是否调控紧密连接蛋白Occludin的表达仍需进一步研究。我们利用siRNA将GABPA敲低后，机械牵张后发现，Occludin表达较未敲低GABPA的MLE-12细胞表达升高，提示GABPA可调控紧密连接蛋白Occludin的表达。

本研究仅从体外细胞层面进行验证，尚缺乏体内动物实验验证，在以后的机械通气相关肺损伤的研究需要进行体内动物实验的研究，通过研究屏障通透性的调控机制来预防和治疗VILI。

5. 结论

综上所述，机械牵张导致VILI中的紧密连接蛋白Occludin表达降低。GABPA通过调控紧密连接蛋白Occludin的表达影响通透性。

基金项目

山东省自然科学基金(ZR2020QH004)，中国博士后科学基金第74批面上资助(2023M741864)；泰山学者青年专家(tsqn202211380)；日照市重点研发计划项目(2021ZDYF020207)。

文章引用

孔 磊,曹 乐,赵 涛. 转录因子GABPA对机械通气相关肺损伤中调控紧密连接蛋白的影响
Effect of GABPA on the Regulation of Tight Junction in Ventilation-Induced Lung Injury[J]. 临床医学进展, 2024, 14(05): 2265-2269. https://doi.org/10.12677/acm.2024.1451680

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    16. NOTES

    *通讯作者。

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