肝细胞核因子-1b在糜烂性毒剂2-氯乙基乙基硫醚诱导急性肺支气管上皮细胞损伤中的作用及其机制  

Involvement of hepatocyte nuclear factor-1b in lung bronchial epithelial cell injury induced by 2-chloroethyl ethyl sulfide

作　　者：孔德钦 刘思佳 刘建豪 马耀 马丞飞 赵昱舜 周嘉恒 师敏婕 李嘉 刘江正 KONG Deqin;LIU Sijia;LIU Jianhao;MA Yao;MA Chengfei;ZHAO Yushun;ZHOU Jiaheng;SHI Minjie;LI Jia;LIU Jiangzheng(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 and Prevention of Special Operations of Ministry of Education,Xi'an 710032;The Second Brigade of Basic Medical College,Air Force Medical University,Xi'an 710032;Department of Recuperation and Rehabilitation for Flight Personnel,School of Aerospace Medicine,Air Force Medical University,Xi'an 710032,Shaanxi,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/陕西省自由基生物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]空军军医大学航空航天医学系飞行人员康复与疗养教研室,陕西西安710032

出　　处：《癌变．畸变．突变》2024年第1期1-8,共8页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金青年基金(32100996,31900892);陕西省重点研发计划项目(2023-YBSF-297);陕西省自然科学基础研究计划项目(2021JQ-343)。

摘　　要：目的:探讨肝细胞核因子-1b(HNF-1b)在糜烂性毒剂2-氯乙基乙基硫醚(CEES)诱导人源性肺支气管上皮细胞(BEAS-2B)损伤中的作用及其机制。方法:用不同浓度(0、0.4、0.6、0.8、1.0和1.2 mmol/L)的CEES染毒BEAS-2B细胞24 h,使用CCK-8法检测细胞活力,光镜下观察细胞形态,分别采用DCFH-DA和MitoSOX荧光探针检测细胞总活性氧(ROS)和线粒体ROS水平。Western blot检测BEAS-2B细胞中HNF-1b蛋白的表达。再利用慢病毒感染技术构建过表达HNF-1b的BEAS-2B细胞系,用1 mmol/L CESS处理24 h后,分别采用CCK-8法测定细胞活力,Annexin V-FITC/PI双染法检测细胞凋亡率,MitoSOX和DHE荧光探针检测线粒体ROS和细胞总ROS水平,JC-1染色法检测线粒体膜电位。结果:与对照组比较,0.6~1.2 mmol/L CEES染毒后细胞活力均降低(P<0.01);细胞形态发生损伤性改变;0.8~1.2 mmol/L CEES染毒后细胞总ROS和线粒体ROS水平均增加(P<0.01);CEES染毒组细胞HNF-1b蛋白表达水平显著下调(P<0.01)。慢病毒转染后,与对照组正常细胞相比,CEES染毒组HNF-1b过表达细胞的细胞活力显著增加(P<0.01),细胞凋亡率降低(P<0.01),线粒体膜电位的损伤缓解,且线粒体ROS和细胞内总ROS水平显著降低(P<0.01)。结论:糜烂性毒剂CEES能够导致肺支气管上皮细胞中HNF-1b表达降低,过表达HNF-1b能够减轻CEES诱导的细胞损伤和凋亡,抑制线粒体功能障碍,其机制可能与抗氧化有关。上述结果提示HNF-1b可能是糜烂性毒剂导致肺损伤的新靶点,激活HNF-1b可能是糜烂性毒剂毒性靶点治疗的新策略。OBJECTIVE:To investigate the involvement of hepatocyte nuclear factor-1b(HNF-1b)in human lung bronchial epithelial cell injury induced by a blister agent,2-chloroethyl ethyl sulfide(CEES).METHODS:The human BEAS-2B cells were treated in culture with various concentrations(0,0.4,0.6,0.8,1.0 and 1.2 mmol/L)of CEES for 24 h.The CCK-8 method was used to detect cell viability,and cell morphology was observed under light microscope.The DCFH-DA and MitoSOX fluorescent probes were used to detect total reactive oxygen species(ROS)and mitochondrial ROS levels,respectively.The protein expression of HNF-1b was assessed by Western blot.A BEAS-2B cell line which overexpresses HNF-1b was constructed by lentiviral infection.After exposure to 1 mmol/L CEES for 24 h,cell viability was determined by the CCK-8 method;apoptosis rate was detected by Annexin V-FITC/PI double staining method;mitochondrial ROS and whole-cell ROS levels were measured by MitoSOX and DHE probes,and mitochondrial membrane potential was detected by JC-1 staining.RESULTS:After exposure of BEAS-2B to 0.6-1.2 mmol/L CEES and compared to the controls,cell viability was reduced(P<0.01),cell morphology showed damage,the levels of total ROS and mitochondrial ROS were increased(P<0.01),and HNF-1b protein expression was significantly down-regulated(P<0.01).After exposure of the cells with over-expressed HNF-1b,the cell viability was significantly increased(P<0.01),apoptosis rate was decreased(P<0.01),mitochondrial membrane potential damage was relieved,and the levels of mitochondrial ROS and total whole-cell ROS were significantly decreased(P<0.01).CONCLUSION:Exposure of the regular BEAS-2B cells to CEES reduced the expression of HNF-1b and caused extensive damage.However,overexpression of HNF-1b in the BEAS-2B cells reduced the CEES-induced cellular damage,apoptosis and mitochondrial dysfunction.These results suggest that the protective effect of HNF-1b may mediated by antioxidation,and activation of HNF-1b may be a new strategy for the treatment of blister agent toxi

关 键 词：糜烂性毒剂 2-氯乙基乙基硫醚 肝细胞核因子-1b BEAS-2B细胞 氧化应激 

分 类 号：R994.7[医药卫生—毒理学]