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

doi:10.3969/j.issn.1004-616x.2024.01.001

癌变·畸变·突变 ›› 2024, Vol. 36 ›› Issue (1): 1-8.doi: 10.3969/j.issn.1004-616x.2024.01.001

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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

孔德钦¹, 刘思佳^1,2, 刘建豪^1,2, 马耀^1,2, 马丞飞^1,2, 赵昱舜^1,2, 周嘉恒³, 师敏婕¹, 李嘉³, 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

收稿日期:2023-06-30 修回日期:2023-10-19 出版日期:2024-02-19 发布日期:2024-02-19
通讯作者: 李嘉, 刘江正
作者简介:孔德钦,E-mail:1084789645@qq.com。
基金资助:
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）

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

KONG Deqin¹, LIU Sijia^1,2, LIU Jianhao^1,2, MA Yao^1,2, MA Chengfei^1,2, ZHAO Yushun^1,2, ZHOU Jiaheng³, SHI Minjie¹, LI Jia³, 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 and Prevention of Special Operations of Ministry of Education, Xi'an 710032;
2. The Second Brigade of Basic Medical College, Air Force Medical University, Xi'an 710032;
3. Department of Recuperation and Rehabilitation for Flight Personnel, School of Aerospace Medicine, Air Force Medical University, Xi'an 710032, Shaanxi, China

Received:2023-06-30 Revised:2023-10-19 Online:2024-02-19 Published:2024-02-19

摘要/Abstract

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。

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

Abstract: 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 toxicity.

Key words: blister agent, 2-chloroethyl ethyl sulfide, hepatocyte nuclear factor-1b, BEAS-2B cells, oxidative stress

中图分类号:

R994.7

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. 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[J]. 癌变·畸变·突变, 2024, 36(1): 1-8.

KONG Deqin, LIU Sijia, LIU Jianhao, MA Yao, MA Chengfei, ZHAO Yushun, ZHOU Jiaheng, SHI Minjie, LI Jia, LIU Jiangzheng. Involvement of hepatocyte nuclear factor-1b in lung bronchial epithelial cell injury induced by 2-chloroethyl ethyl sulfide[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2024, 36(1): 1-8.

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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

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