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线粒体ATP 敏感钾通道调控哮喘大鼠气道平滑肌细胞增殖

赵建平^*, 高敏, 叶永军, 胡伟华, 周志刚, 胡红玲

华中科技大学同济医学院附属同济医院呼吸疾病研究所，武汉 430030

摘要

本文旨在探讨线粒体ATP 敏感钾(mitochondrial ATP-sensitive K+, MitoKATP)通道对哮喘大鼠气道平滑肌细胞(airway smooth muscle cells, ASMCs)增殖的影响及其调控机制。36 只Sprague-Dawley (SD)大鼠随机分为哮喘组(n=18) 和正常组 (n=18)，哮喘组大鼠采用卵清蛋白(ovalbumin, OVA)致敏及激发的方法制备哮喘模型，正常组大鼠吸入等量生理盐水。取 各组大鼠肺组织，分离出ASMCs 进行培养，将样本分6 组分别进行干预：(1)正常组；(2)正常 + MitoKATP 通道开放剂 diazoxide 组(diazoxide 组)；(3)正常 + MitoKATP 通道阻断剂5-HD 组(5-HD 组)；(4)哮喘(asthma)组；(5) asthma + diazoxide 组； (6) asthma + 5-HD 组。利用罗丹明(Rhodamine123, R-123)荧光染色、激光共聚焦显微镜成像检测线粒体膜电位(ΔΨm)， DCFH-DA荧光染色法检测细胞内活性氧(reactive oxygen species, ROS)含量，RT-PCR 检测核因子κB (nuclear factor-κB, NF- κB) mRNA 的表达，流式细胞仪检测细胞凋亡和MTT 法检测细胞增殖情况。结果显示：(1) Diazoxide 组与正常组相比， R-123 荧光强度增强，ΔΨm 去极化，ROS 及NF-κB 的表达增高，细胞增殖增多、凋亡减少(P<0.05)；而5-HD 组与正常 组相比，上述指标均无显著差异；(2) 与正常组相比，Asthma 组R-123 荧光强度增强，ΔΨm 去极化，ROS 及NF-κB 的 表达增高，细胞增殖增多、凋亡减少(P<0.05)；Asthma + diazoxide 组与Asthma 组相比，R-123 荧光强度和ΔΨm 去极化 增强，ROS 及NF-κB 表达增高，细胞增殖增多、凋亡减少(P<0.05)；Asthma + 5-HD 组与Asthma 组相比，R-123 荧光 强度和ΔΨm 去极化减弱，ROS 及NF-κB 表达降低，细胞增殖减少、凋亡增多(P<0.05)；(3)各组大鼠ASMCs 的R-123 荧 光强度，NF-κB mRNA表达量与ROS 含量均呈正相关；MTT 的吸光度值与NF-κB mRNA表达量呈正相关，凋亡细胞百 分比与NF-κB mRNA表达量呈负相关。以上结果提示，哮喘可引起大鼠ASMCs 的MitoKATP 通道开放及ΔΨm 去极化，ΔΨm 去极化促进ROS 的表达，ROS 可能作为信号分子刺激NF-κB 表达增加，进而促进ASMCs 增殖，参与气道重构。

关键词： 哮喘; 线粒体膜电位; 线粒体ATP 敏感钾通道; 活性氧; 核因子κ B ; 平滑肌

分类号：R332；R562.2+5

[Regulation of rat airway smooth muscle cell proliferation by mitochondrial ATP-sensitive K+ channel in asthmic rats.] [Ariticle in Chinese]

ZHAO Jian-Ping^*, GAO Min, YE Yong-Jun, HU Wei-Hua, ZHOU Zhi-Gang, HU Hong-Ling

Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Abstract

The objective of this paper was to investigate the effect and mechanism of mitochondrial ATP-sensitive K+ (MitoKATP) channel on the proliferation of airway smooth muscle cells (ASMCs) in asthmic rats. Thirty-six Sprague-Dawley (SD) rats were randomly assigned into 2 groups (18 in each): (1) Asthma group: the asthmic rat model was established by ovalbumin (OVA) sensitization and excitation; (2) Normal group: rats were subjected to inhalation of equal amount of normal saline. The rat ASMCs were isolated from fresh lung tissues and cultured respectively as follows: (1) Control group: normal ASMCs were cultured under normoxia for 24 h; (2) Diazoxide group: normal ASMCs were cultured under normoxia for 24 h with diazoxide (an opener of MitoKATP channel); (3) 5-HD group: normal ASMCs were cultured under normoxia for 24 h with 5-hydroxydecanoate (5-HD) (an antagonist of MitoKATP channel); (4) Asthma group: Asthmic ASMCs were cultured under normoxia for 24 h; (5) Asthma + diazoxide group: Asthmic ASMCs were cultured under normoxia with diazoxide for 24 h; (6) Asthma + 5-HD group: Asthmic ASMCs were cultured under normoxia with 5-HD for 24 h. The mitochondrial membrane potential (ΔΨm) was detected using Rhodamine 123 (R-123). The level of reactive oxygen species (ROS) was detected by DCF fluorescence. The expression of nuclear factor-kappa B (NF-κB) mRNA was examined by RTPCR. The proliferation and apoptosis of rat ASMCs were examined respectively by MTT colorimetric assay and cell cycle analysis. The results were as follows. (1) After exposure to diazoxide for 24 h, the R-123 fluorescence intensity, the ROS level, NF-κB mRNA expression and the MTT absorbance value (A value) in normal ASMCs were significantly increased, and the apoptosis of rat ASMCs was significantly decreased compared to the control group (P<0.05). However, there was no significant changes in those indices after the normal ASMCs had been exposed to 5-HD for 24 h. (2) In Asthma and Asthma + diazoxide groups, the R-123 fluorescence intensity, ROS level and the MTT A value were markedly increased, and the apoptosis was markedly decreased compared to control group (P<0.05). These changes were more obvious in Asthma + diazoxide group than those in Asthma group (P<0.05). 5-HD partly weakened the effect of asthma on the R-123 fluorescence intensity, ROS level and the MTT A value and the apoptosis of rat ASMCs (P<0.05). R-123 fluorescence intensity and NF-κB mRNA expression were positively correlated with ROS level. NF-κB mRNA expression was positively correlated with the MTT A value and negatively correlated with the apoptosis of rat ASMCs. All the results suggest that the opening of MitoKATP channel followed by a depolarization of ΔΨm contributes to the increase in ROS level and NF-κB mRNA expression in rat ASMCs and to the unbalance between cell proliferation and apoptosis of ASMCs induced by asthma. This might be a mechanism of the development of airway remodeling in asthma.

Key words: Asthma; mitochondrial membrane potential; mitochondrial ATP-sensitive K+ channel; reactive oxygen species; nuclear factor-κB; smooth muscle

收稿日期：2008-10-06　　录用日期：2008-12-11

通讯作者：赵建平　　E-mail: zhaojp@tjh.tjmu.edu.cn

引用本文：

赵建平, 高敏, 叶永军, 胡伟华, 周志刚, 胡红玲. 线粒体ATP 敏感钾通道调控哮喘大鼠气道平滑肌细胞增殖[J]. 生理学报 2009; 61 (1): 65-71.

ZHAO Jian-Ping, GAO Min, YE Yong-Jun, HU Wei-Hua, ZHOU Zhi-Gang, HU Hong-Ling. [Regulation of rat airway smooth muscle cell proliferation by mitochondrial ATP-sensitive K+ channel in asthmic rats.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (1): 65-71 (in Chinese with English abstract).