糜烂性毒剂氮芥染毒诱导急性肝损伤中自噬的变化及作用  

Changes and Role of Autophagy in Acute Liver Injury Induced by Blister Agent Nitrogen Mustard

作　　者：李佳威 刘梦慧 刘思佳 刘建豪 马丞飞 赵昱舜 徐安琦 艾多 孔德钦 李文丽 刘江正 LI Jiawei;LIU Menghui;LIU Sijia;LIU Jianhao;MA Chengfei;ZHAO Yushun;XU Anqi;AI Duo;KONG Deqin;LI Wenli;LIU Jiangzheng(Department of Military Toxicology and Chemical Defense Medicine,School of Military Preventive Medicine,Air Force Medical University,Xi'an Shaanri 710032,China)

机构地区：[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,陕西西安710032 [2]西安交通大学第二附属医院神经外科

出　　处：《联勤军事医学》2023年第11期903-910,共8页Military Medicine of Joint Logistics

基　　金：国家自然科学基金青年基金项目(31900892);陕西省重点研发计划一般项目(2023-YBSF-297);军事口腔医学国家重点实验室开放课题(2021KB04);空军军医大学军事医学与航空医学重大问题科技攻关课题(2022ZZXM042)。

摘　　要：目的建立糜烂性毒剂氮芥(nitrogenmustard,HN2)诱导小鼠急性肝损伤模型和HN2染毒HepG2细胞(人源性肝癌细胞系)损伤模型,探讨自噬在糜烂性毒剂致急性肝损伤中的作用及机制。方法体内模型中,将20只C57BL/6雄性小鼠随机分为正常对照组和HN2染毒组;HN2染毒组小鼠给予腹腔注射盐酸氮芥(2mg/kg),正常对照组小鼠给予腹腔注射等体积的生理盐水。染毒3天后收集血清和肝组织,测定血清天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)和丙氨酸氨基转移酶(alanineaminotransferase,ALT)活性,进行肝组织苏木精-伊红(hematoxy-lin-eosin,HE)染色病理检测,电镜观察线粒体等细胞器形态,Westernblot检测肝组织自噬相关蛋白LC3-Ⅱ/Ⅰ比值和p62的蛋白表达。体外模型中,HepG2细胞分为正常对照组和HN2染毒组;正常对照组细胞给予含二甲基亚砜(dime-thylsulfoxide,DMSO)(0.1%)的无血清培养基处理36h。HN2染毒组细胞给予盐酸氮芥(8μmol/L)处理36h。采用细胞计数试剂盒(cellcountingkit8,CCK-8)检测细胞活性,单丹磺酰尸胺(monodansylcadaverine,MDC)探针法检测线粒体自噬改变,自噬双标腺病毒(mRFP-GFP-LC3)检测肝细胞自噬流的改变;在细胞模型中,给予自噬激动剂雷帕霉素(rapamycin,RAPA)和自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)预处理干预,检测细胞活力水平。结果在体内模型中,与正常对照组比较,HN2染毒组小鼠血清ALT和AST活性显著升高(P均<0.05),同时肝组织存在大量炎细胞浸润并伴有肝实质细胞损伤;肝实质细胞内出现大量自噬体,肝组织LC3-Ⅱ/Ⅰ比值升高(P<0.05),同时p62的蛋白表达显著下调(P<0.05)。在体外模型中,与正常对照组相比,HN2染毒组细胞活性显著降低(P<0.05),MDC荧光强度显著升高,自噬体数量增加(P均<0.05)。在HN2染毒细胞模型中,给予自噬激动剂RAPA预处理后细胞活力显著升高(P<0.05),而给予自噬抑制剂3-MA预处理后细胞活力显�Objective To establish a mouse model of acute liver injury induced by blister agent nitrogen mustard(HN2)and the injury model of HepG2 cells(human hepatocellular carcinoma cell lines)exposed to HN2,and to explore the role and mechanism of autophagy in acute liver injury induced by blister agent.Methods In in vivo model,20 C57BL/6 male mice were randomly divided into the normal control group and the HN2 exposed group;the mice in the HN2 exposed group were intraperitoneally injected with nitrogen mustard hydrochloride(2 mg/kg)once time,and the mice in the normal control group were intraperitoneally injected with the same volume of saline.Three days after the exposure,serum and hepatic tissue were collected,the activities of serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)were measured,hematoxylin-eosin(HE)staining was performed for pathological examination of hepatic tissue,the morphology of organelle such as mitochondria was observed under electron microscope,and the autophagy related protein LC3-Ⅱ/Ⅰratio,protein expression of p62 in hepatic tissue were detected by Western blot.In the in vitro model,HepG2 cells were divided into a normal control group and HN2 exposed group;the cells in normal control group were treated with serum-free medium containing dimethyl sulfoxide(DMSO)(0.1%)for 36 h,while the cells in HN2 exposed group were treated with 8μmol/L nitrogen mustard hydrochloride of treatment after 36 h.Cell viability was detected by cell counting kit 8(CCK-8),the changes of mitophagy were detected by monodansylcadaverine(MDC)probe method,the changes in liver cell autophagy flow were detected by double labeled adenovirus(mRFP-GFP-LC3);in the cell model,pretreatment intervention with autophagy agonist rapamycin(RAPA)and autophagy inhibitor 3-methyladenine(3-MA)was administered to detect the cell viability level.Results In the in vivo model,compared with the normal control group,the serum ALT and AST viability of mice in HN2 exposed group significantly increased(all P<0.05),at the same

关 键 词：氮芥 糜烂性毒剂 自噬 急性肝损伤 中毒 

分 类 号：R827[医药卫生—临床医学]