活化蛋白C通过靶向VLA⁃3⁃中性粒细胞亚群来减轻结核杆菌诱导的人气道上皮细胞炎症反应线粒体能量代谢障碍

doi:10.3969/j.issn.1006⁃5725.2020.23.010

摘要/Abstract

摘要：

目的探究活化蛋白C 通过靶向VLA⁃3⁃中性粒细胞亚群来减轻结核杆菌诱导的人气道上皮细胞炎症反应线粒体能量代谢障碍的机制。方法人支气管上皮BEAS⁃2B细胞分为3组：BEAS⁃2B组（正常培养的细胞系），结核杆菌诱导组（用50个细菌/细胞的结合杆菌感染BEAS⁃2B细胞培养物），BEAS⁃2B转染组（携带活化蛋白C 的慢病毒载体与BEAS⁃2B 进行细胞转染）。通过可溶性整联蛋白结合测定活化蛋白C与VLA⁃3的结合度；逆转录定量PCR（RT⁃qPCR）实时分析IL⁃6、IL⁃8、MCP⁃1的mRNA表达；荧光探针JC⁃1 评估线粒体功能；通过Biosciences 测量线粒体膜电位；ELISA 和TUNEL 鉴定细胞凋亡及活力情况。结果 VLA⁃3较VLA⁃3和αVβ3结合程度升高（P < 0.05）。结核杆菌诱导组较BEAS⁃2B组IL⁃6、IL⁃8、MCP⁃1mRNA 表达升高（P < 0.05），BEAS⁃2B 转染组较结核杆菌诱导组IL⁃6、IL⁃8、MCP⁃1mRNA 表达降低（P <0.05）。BEAS⁃2B组较结核杆菌诱导组线粒体复合物Ⅰ、线粒体复合物Ⅳ活性升高（P < 0.05），结核杆菌诱导组较BEAS⁃2B转染组线粒体复合物Ⅰ、线粒体复合物Ⅳ活性降低（P < 0.05）。BEAS⁃2B组较结核杆菌诱导组膜电位升高（P < 0.05），结核杆菌诱导组较BEAS⁃2B转染组膜电位降低（P < 0.05）。BEAS⁃2B组较结核杆菌诱导组细胞凋亡率降低（P < 0.05），结核杆菌诱导组较BEAS⁃2B转染组细胞凋亡率升高（P < 0.05）。BEAS⁃2B组较结核杆菌诱导组细胞活力升高（P < 0.05），结核杆菌诱导组较BEAS⁃2B转染组细胞细胞活力降低（P < 0.05）。结论活化蛋白C 对整联蛋白VLA3 有更强的亲和力，可靶向作用VLA3 中性粒细胞亚群降低人气道上皮细胞的炎症反应，提高线粒体复合物Ⅰ、复合物Ⅳ活性，有效抑制结合杆菌诱导的细胞线粒体膜电位下降。

关键词: 活化蛋白C, 整联蛋白VLA?3, 细胞凋亡, 线粒体功能障碍

Abstract:

Objective To investigate the mechanism of activated protein C to alleviate the mitochondrialenergy metabolism disorder induced by Mycobacterium tuberculosis in human airway epithelial cells by targetingVLA⁃3⁃neutrophil subpopulation. Methods Human bronchial epithelial BEAS⁃2B cells were divided into threegroups：BEAS⁃2B group（normal culture cell line），Mycobacterium tuberculosis induction group（BEAS⁃2B cellculture was infected with 50 bacteria/cell conjugated bacteria），and BEAS⁃2B transfection group（BEAS⁃2B cellswere transfected with lentiviral vector carrying activated protein C）. The binding degree of activated protein C to vla⁃3 was determined by soluble integrin binding. RT qPCR was used to analyze the mRNA expression of IL⁃6，IL⁃8and MCP⁃1. The function of mitochondria was evaluated by fluorescence probe JC⁃1 and mitochondrial membranepotential was measured by biosciences. Cell apoptosis and viability were detected by ELISA and TUNEL. Results VLA⁃3 was more highly expressed than VLA⁃3 and αVβ3（P < 0.05）. The expression of IL⁃6，IL⁃8 and MCP⁃1mRNA in the induction group of Mycobacterium tuberculosis was higher than that in BEAS⁃2B group（P < 0.05）. TheBEAS⁃2B transfection group was more sensitive than IL⁃6，IL⁃8 and MCP in the induction group of Mycobacteriumtuberculosis. 1 mRNA expression was decreased（P < 0.05）. Compared with the Mycobacterium tuberculosis induc⁃tion group，the mitochondrial complex Ⅰ and mitochondrial complex Ⅳ activity were significantly increased in theBEAS⁃B group（P < 0.05），and the mitochondrial complex I and mitochondrial complex Ⅳ activity were decreasedin the Mycobacterium tuberculosis induction group compared with the BEAS⁃B transfection group（P < 0.05）. Themembrane potential of the BEAS⁃2B group was higher than that of the Mycobacterium tuberculosis induced group（P < 0.05），and the membrane potential of the tuberculosis induction group was lower than that of the BEAS⁃2Btransfection group（P < 0.05）. The apoptosis rate of BEAS⁃2B group was lower than that of Mycobacterium tubercu⁃losis induction group（P < 0.05），and the apoptosis rate of Mycobacterium tuberculosis induction group was higherthan that of BEAS⁃2B transfection group（P < 0.05）. The cell viability of the BEAS⁃2B group was higher than thatof the Mycobacterium tuberculosis induction group（P < 0.05），and the cell viability of the tuberculosis inductiongroup was lower than that of the BEAS⁃2B transfection group（P < 0.05）. Conclusion Activated protein C has astronger affinity for integrin VLA3，which can target VLA3 neutrophil subpopulation to reduce the inflammatoryresponse of human airway epithelial cells，improve mitochondrial complex Ⅰ，complex Ⅳ activity，and effectivelyinhibit binding. Bacillus⁃induced decrease in mitochondrial membrane potential.

Key words: activation protein C, integrin vla?3, apoptosis, mitochondrial dysfunction

陈焰, 王岗玲, 吴海明. 活化蛋白C通过靶向VLA⁃3⁃中性粒细胞亚群来减轻结核杆菌诱导的人气道上皮细胞炎症反应线粒体能量代谢障碍[J]. 实用医学杂志, 2020, 36(23): 3217-3221.

CHEN Yan, WANG Gangling, WU Haiming.

Activated protein C reduces mitochondrial energy metabolism disorder induced by Mycobacterium tuber⁃culosis in human airway epithelial cells by targeting VLA⁃3⁃neutrophil subpopulation [J]. The Journal of Practical Medicine, 2020, 36(23): 3217-3221.

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活化蛋白C通过靶向VLA⁃3⁃中性粒细胞亚群来减轻结核杆菌诱导的人气道上皮细胞炎症反应线粒体能量代谢障碍

Activated protein C reduces mitochondrial energy metabolism disorder induced by Mycobacterium tuber⁃culosis in human airway epithelial cells by targeting VLA⁃3⁃neutrophil subpopulation

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