活性氧介导的肺组织细胞线粒体凋亡在染矽尘大鼠肺纤维化中的作用

On the effect of reactive oxygen species induced pneumonocyte mitochondrial apoptosis on pulmonary fibrosis with rats exposed to silica dust

目的 通过抑制大鼠肺组织细胞内活性氧（ROS）活性,探讨ROS在肺组织细胞线粒体凋亡介导肺纤维化中的作用及矽肺的发病机制。方法 24只SD成年雄性大鼠,体质量为180~220g,随机分为生理盐水对照组、矽肺模型组和N-乙酰基-L-半胱氨酸（N-Acetyl-L-cysteine,NAC）干预组,每组8只。矽肺模型组和NAC干预组大鼠采用非气管暴露法一次性肺内注入50g/L二氧化硅悬浊液1ml,对照组注入等体积生理盐水,NAC干预组自造模当天起每天以灌胃方式给予600mg/kg NAC 1ml,各组动物均可自由进食和饮水。造模28d后统一处死所有实验动物,收集左侧肺灌洗回收液,分离纯化肺组织细胞,流式细胞仪检测肺组织细胞内ROS含量和线粒体膜电位（mitochondrial transmembrane potential,MTP）的变化水平;另取灌洗侧肺组织,Trizol固定,检测I型和III型前胶原mRNA表达水平;将灌洗侧部分肺组织裂解,免疫印迹法（Western blotting）检测肺组织内细胞色素C（CytC）,天冬半胱氨酸特异性蛋白-9（cysteinyl aspartate specific protease-9,Caspase-9）和Caspase-3的表达水平;右侧肺组织用多聚甲醛固定,苏木精-伊红（HE）染色,制作病理标本,观察肺组织病理变化及肺纤维化程度。结果 NAC干预组大鼠ROS含量和线粒体去极化率均较矽肺模型组低,但高于生理盐水对照组（P〈0.05）;矽肺模型组大鼠肺组织内CytC、Caspase-9、Caspase-3及I型和III型前胶原mRNA的表达水平最高,NAC干预组各指标均高于生理盐水对照组（P〈0.05）;NAC干预组大鼠肺组织纤维化病变较矽肺模型组减轻,但仍高于生理盐水对照组。结论 被NAC干预的染矽尘大鼠肺组织细胞内ROS活性被抑制后,线粒体凋亡信号转导通路被阻断,肺组织纤维化程度减轻。因此,ROS可能在肺组织细胞线粒体凋亡致肺纤维化中发挥重要的作用。

Objective To explore the role of reactive oxygen species （ROS） in pneumonocyte mitochondrial apoptosis pathway and the pathogenesis of silicosis. Methods 24 adult male Sprague--Dawley （SD） rats weighing 180--220g were randomly divided into control group, silicosis model group and N-- Acetyl--L--cysteine （NAC） intervention group with 8 rats in each group. The silicosis model group and the NAC group were adopted non--tracheal exposure method of disposable intrapulmonary injection 50g/L silica suspension lml to establish animal silicosis model. The control group were injected with same volume physiological saline, the NAC intervention group was treated with 600mg/kg NAC 1ml by gavage from theright day of establishing silicosis model, each animal could freely eat and drink. They were all sacrificed after 28days. We collected one side lung lavage fluid, separated and purified pneumonocyte. The ROS contents and mitochondrial transmembrane potential （MTP） changes were detected by flow cytometry, the lavage side lung tissue was partly fixed by Trizot reagent to detect type I and type III proeollagen mRNA; Another section of lavage side lung tissue was extracted for protein measurement of cytochrome C （CytC）, cysteinyl aspartate specific protease-9（caspase-9） and cysteinyl aspartate specific protease-3 （easpase-3）. The other side of the lung tissue was fixed with paraformaldehyde and stained with hematoxylin-eosin （HE）. The pathological change and the severity of pulmonary fibrosis was observed under a microscope. Results ROS contents and MTP changes were lower in the NAC group compared with the silicosis model group. CytC, easpase-9, easpase-3, type I and type Ill procollagen mRNA expression levels were lower in the NAC treatment group than the model group, but higher than that of the control group, the severity of lung fibrotie lesions observed from the pathological slices showed the same trend. Conclusions After the activity of ROS in pneumonoeyte from rats exposed to silica dust was inhibited by NAC, the mitochondrial apoptosis can be suppressed and the severity of pulmonary fibrosis disease reduced. Therefore, ROS may be a critical factor in the mitochondriat apoptosis induced pulmonary fibrosis.