氧化应激在石英粉尘致肺泡巨噬细胞内质网应激性凋亡中的作用机制

Role of oxidative stress in endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust

目的 探讨氧化应激在石英粉尘致肺泡巨噬细胞内质网应激性凋亡途径中的作用机制.方法 健康成年雄性Wistar大鼠72只,随机分为对照组、染尘组、染尘＋N-乙酰半胱氨酸（NAC）组及NAC组,每组18只.采用非暴露式气管内注入法染尘,染尘组及染尘＋NAC组一次性注入含5％超细石英粉尘的生理盐水1 ml,对照组及NAC组注入等量的无菌生理盐水,染尘＋NAC组及NAC组在大鼠染尘后第2天开始,以灌胃的方式接受每天NAC 100 mg/kg抗氧化治疗,每周连续给药5d,直至实验结束.每组分别于染尘结束后14、28、56 d随机选取6只大鼠,股动脉放血处死、取肺,进行支气管肺泡灌洗,收集支气管肺泡灌洗液,分离纯化巨噬细胞,流式细胞术检测肺泡巨噬细胞凋亡率及巨噬细胞活性氧（ROS）含量,紫外比色法检测巨噬细胞蛋白羰基含量,蛋白免疫印迹（Western blot）法检测肺泡巨噬细胞caspase-12的蛋白表达水平.结果 与对照组比较,染尘组大鼠在染尘后第14天ROS含量、蛋白羰基含量、caspase-12蛋白表达水平及细胞凋亡率开始升高,染毒后第28、56天上述指标持续升高,差异有统计学意义（P＜0.05）.与染尘组比较,染尘＋NAC组大鼠在染尘后第14、28及56天巨噬细胞ROS含量均降低,差异有统计学意义（P＜0.05）.与染尘组比较,染尘＋NAC组大鼠染尘后第28、56天巨噬细胞羰基含量均降低,差异有统计学意义（P＜0.05）.与染尘组比较,染尘＋NAC组大鼠caspase-12蛋白表达量和凋亡率在第14、28及56天均降低,差异有统计学意义（P＜0.05）.结论 氧化应激可能是启动粉尘致肺泡巨噬细胞内质网应激性凋亡的原因之一,同时氧化应激引发的内质网内蛋白的氧化损伤可能是其中间环节之一.

Objective To investigate the role of oxidative stress in the endoplasmic reticulum stressinduced apoptosis of alveolar macrophages triggered by quartz dust.Methods Seventy-two healthy adult Wistar rats were randomly divided into control group,quartz dust group,quartz dust plus N-acetyl cysteine （NAC） group,and NAC group,with 18 rats in each group.One milliliter of sterile saline （for the control and NAC groups） or l ml of saline with 5% ultrafine quartz dust （for dust group and dust plus NAC group） was given to each rat by non-exposed endotracheal infusion.From the second day after dust infusion,rats in dust plus NAC group and NAC group received intragastric administration of NAC （100 mg/kg）.In each week,the treatment with NAC lasted for 5 consecutive days,followed by 2 days＆#39; interval.For each group,6 rats were randomly selected on the 14th,28th,or 56th day after dust exposure; they were sacrificed by bloodletting from the femoral artery,and the lungs were collected.Bronchoalveolar lavage fluid was collected to separate macrophages.The protein expression of caspase-12 in alveolar macrophages,the apoptosis rate and reactive oxygen species （ROS） content of alveolar macrophages,and the protein carbonyl content of alveolar macrophages were determined by Western blot,flow cytometry,and colorimetry,respectively.Results Increased protein expression of caspase-12,apoptosis rate,and content of ROS and protein carbonyl were discovered on the 14th day in the dust group,in comparison with the control group （P〈0.05）,and the increase lasted till the 28th and 56th days.（P〈0.05）.Compared with the dust group,the dust plus NAC group showed significant decreases in the content of ROS on the 14th,28th,and 56th days （P〈0.05）,significant decreases in the content of protein carbonyl on the 28th and 56th days （P〈0.05）,and significant decreases in the protein expression of caspase-12 and apoptosis rate （P〈0.05）.Conclusion Oxidative stress is potentially involved in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust.Oxidative damage of protein in the endoplasmic reticulum may play an important role in the process.