1、1 0 2CARCINOGENESIS，TERATOGENESIS&MUTAGENESIS论著癌变畸变突变Vol.35No.2Mar.2023收稿日期：2022-12-05；修订日期：2023-03-06基金项目：国家自然科学基金青年基金(31900892)作者信息：王钊，E-mail：。*通信作者，孔德钦，E-mail：；刘 江 正，E-mail：糜烂性毒剂氮芥染毒对肝细胞线粒体结构和功能的影响王钊1，刘思佳1，2，刘建豪1，2，马丞飞1，2，赵昱舜1，2，徐安琦1，2，艾多1，2，戚羽佳3，孔德钦1，*，刘江正1，*(1空军军医大学军事预防医学系军事毒理学与防化医学教研室，陕西省自由基生物
2、学与医学重点实验室，教育部特殊作业环境危害评估与防治重点实验室，陕西西安710032；2空军军医大学基础医学院学员二大队，陕西西安710032；3南京中医药大学翰林学院资产与实验室管理处，江苏泰州225300)Effects of blister agentnitrogen mustard exposureon the structure and functionof mitochondria in hepatocytesWANG Zhao1,LIU Sijia1,2,LIU Jianhao1,2,MA Chengfei1,2,ZHAO Yushun1,2,XU Anqi1,2,AI Duo1
3、,2,QI Yujia3,KONG Deqin1,*,LIU Jiangzheng1,*(1.Department of Military Toxicology and Chemical Defense Medicine,School of Military Preventive Medicine,Air Force Medical University;Key Laboratory of Free Radical Biology and Medicine of Shaanxi Province;Key Laboratory of Environmental Hazard Assessment
4、 and Preventionof Special Operations of Ministry of Education,Xian 710032;2.TheSecond Brigade of Basic Medical College,Air Force Medical University,Xian 710032,Shaanxi;3.Assets and Laboratory ManagementDivison,Hanlin College,Nanjing Traditional ChineseMedicine University,Taizhou 225300,Jiangsu,China
5、)【摘要】目的：探讨体外和体内模型中糜烂性毒剂氮芥(HN2)染毒对肝细胞线粒体结构和功能的影响。方法：体外模型中，HepG2细胞分为正常对照组和HN2染毒组，正常对照组给予含0.1%DMSO的无血清培养基处理36 h，HN2染毒组给予8 mol/L氮芥染毒36 h，分别采用CCK-8法检测细胞活性，JC-1探针法检测线粒体膜电位，NADPH速率法检测细胞匀浆ATP含量，Seahorse细胞能量代谢分析仪检测线粒体功能；体内模型中，20只C57BL/6小鼠分为正常对照组和HN2染毒组，HN2染毒组按2 mg/kg腹腔单次注射盐酸氮芥，正常对照组注射生理盐水，染毒后3 d后采集血清和肝组织，测定血
6、清谷丙转氨酶(ALT)和谷草转氨酶(AST)活性，进行肝组织HE染色病理检测和电镜观察，并测定肝组织ATP含量和线粒体酶复合物I、II和III的活性。结果：在体外模型中，与正常对照组相比，HN2染毒组细胞活性降低了约16.2%(P0.05)，线粒体膜电位下降了约33.2%(P0.05)，线粒体能量代谢明显异常。在体内模型中，与正常对照组比较，HN2染毒后，血清ALT和AST活性显著升高(P0.05)，同时肝组织存在大量炎症细胞浸润并伴有肝实质细胞损伤，肝细胞内线粒体形态发生显著异常，表现为数量增多、体积缩小、线粒体嵴缺失或断裂，线粒体酶复合体I、II和III的活性均显著降低(P0.05)。结论
7、：HN2染毒能够导致肝细胞发生线粒体结构损伤和功能异常，线粒体可能是HN2诱导肝损伤的主要毒作用靶点之一，本研究结果为糜烂性毒剂诱导急性肝损伤的防治策略提供了新思路。【关键词】氮芥；糜烂性毒剂；线粒体；急性肝损伤；中毒中图分类号：R827.15文献标志码：A文章编号：1004-616X(2023)02-0102-08doi：10.3969/j.issn.1004-616x.2023.02.004【ABSTRACT】OBJECTIVE：Toinvestigateeffectsoftheblisteragent，nitrogenmustard(HN2)，onmitochondrial struct
8、ure and function of hepatocytes，based on in vitro and in vivo models.METHODS：In thein vitro model，HepG2 cells were divided into normal control and HN2 exposure groups.The normal controlgroup was treated with serum-free medium containing DMSO(0.1%)for 36 h，and the HN2 exposure groupwas treated with n
9、itrogen mustard(8 mol/L).After HN2 treatment for 36 h，cell viability was detected byCCK-8 method，mitochondrial membrane potential by JC-1 probe method，ATP content in cell homogenateand liver tissue by NADPH rate method，and mitochondrial function by Seahorse cell energy metabolismanalyzer.Twenty C57B
10、L/6 mice were divided into normal control group and HN2 exposure group.The HN2exposure group was given a single intraperitoneal injection of nitrogen mustard hydrochloride(2 mg/kg)，andthe normal control group was injected with normal saline.Serum and liver tissues were used to measure论著癌变畸变突变1 0 3CA
11、RCINOGENESIS，TERATOGENESIS&MUTAGENESIS2023 年 3 月第 35 卷第 2 期activitiesofserumalanineaminotransferase(ALT)andaspartateaminotransferase(AST)，andpathologicalexaminationoflivertissuesandelectronmicroscopeobservationswereperformed.ContentsofATPandmitochondrial enzyme complexes I，II and III activities in l
12、iver tissues were determined by corresponding kits.RESULTS：In the in vitro model，compared with the normal control group，HN2 exposure decreased cellactivitybyabout16.2%(P0.05)，themitochondrialmembranepotentialbyabout33.2%(P0.05)，andmitochondrial energy metabolism was significantly abnormal.In the in
13、vivo model，compared with the normalcontrol group，HN2 exposure caused significant increased activities of serum ALT and AST.At the same time，there was large number of inflammatory cell infiltrations into liver tissues accompanied by liver parenchymalcell damage.Mitochondrial morphology was significan
14、tly abnormal：manifested as increased number，decreasedsize，loss or fragmentation of mitochondrial cristae，and significantly decreased activity of mitochondrial enzymecomplexesI，IIandIII(P0.05).CONCLUSION：HN2exposureinducedmitochondrialdamageinhepatocytes and this may be one of the main toxic targets
15、of HN2 poisoning.Results from our study providenew ideas for the prevention and treatment of acute liver injury induced by blister agents such as HN2.【KEY WORDS】nitrogen mustard；blister agent；mitochondria；acute liver injury；poisoning糜烂性毒剂(硫芥、氮芥和路易氏剂等)作为致死性化学战剂，曾在战争历史上广泛使用1。糜烂性毒剂是目前日本遗留在我国化学武器的主要毒剂类型
16、，该类毒剂导致的意外中毒事件迄今仍时有发生2。由于糜烂性毒剂的化学结构简单和化学性质稳定，其合成和释放相对容易，可能在化学恐怖袭击中被大量使用，对国家安全和人民群众健康构成了潜在的现实威胁3。糜烂性毒剂经皮肤或呼吸道中毒后，能够快速吸收入血，进而广泛的分布全身，导致机体重要器官的严重损伤1。实验研究和临床研究均表明，大剂量糜烂性毒剂中毒后会导致急性肝损伤，表现为血清谷丙转氨酶(alanine aminotransferase，ALT)和谷草转氨酶(aspartate aminotransferase，AST)持续升高和肝组织病理结构异常，其机制可能与氧化应激和炎症密切相关4。线粒体是细胞内重要的能量代谢细胞器，主要用于合成三磷酸腺苷(adenosine triphosphate，ATP)，被称为“细胞能量工厂”5。线粒体还广泛参与了凋亡调控和细胞信号转导等生命过程6。大量文献表明，线粒体是活性氧(ROS)生成的重要场所，由于其富含不饱和脂肪酸也极容易遭受氧化应激的损伤6。线粒体在调控氧化还原平衡方面发挥了重要的功能7。线粒体的结构和功能异常还参与了炎症反应的发生和发展8。目前，糜烂性毒
