Nrf2对三氯甲烷所致L02细胞氧化损伤的调控作用

职业与健康 ›› 2026, Vol. 42 ›› Issue (20): 2765-2771.

Nrf2对三氯甲烷所致L02细胞氧化损伤的调控作用

钟苑芳¹, 谭诗莹², 邱奕冰³, 胡黎明¹, 邹志辉⁴, 余日安⁴()

¹ 深圳市宝安区公共卫生服务中心，广东深圳 518100
² 广州市白云区人和镇卫生院，广东广州 510475
³ 广东潮州卫生健康职业学院，广东潮州 515632
⁴ 广东药科大学公共卫生学院，广东广州 510310

收稿日期:2025-04-07 修回日期:2025-10-17 出版日期:2026-10-15 发布日期:2026-09-14
通信作者: 余日安，E-mail：yurian.tj@163.com
作者简介:钟苑芳，女，副主任医师，主要从事环境与职业流行病学研究工作。
基金资助:
深圳市宝安区科技创新局2020年宝安区医疗卫生基础研究项目(2020JD113);深圳市宝安区医学重点学科建设经费资助

Regulatory effect of Nrf2 on oxidative damage induced by trichloromethane in L02 cells

ZHONG Yuanfang¹, TAN Shiying², QIU Yibing³, HU Liming¹, ZOU Zhihui⁴, YU Ri'an⁴()

¹ Shenzhen Bao'an District Public Health Service Center， Shenzhen， Guangdong 518100， China
² Guangzhou Baiyun District Renhe Town Health Center， Guangzhou， Guangdong 510475， China
³ Guangdong Chaozhou Health Vocational College， Chaozhou， Guangdong 515632， China
⁴ School of Public Health， Guangdong Pharmaceutical University，Guangzhou， Guangdong 510310， China

Received:2025-04-07 Revised:2025-10-17 Online:2026-10-15 Published:2026-09-14
Contact: YU Ri'an，E-mail：yurian.tj@163.com

摘要/Abstract

摘要：

目的了解核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）对三氯甲烷（trichloromethane，TCM）所致人正常肝细胞株L02细胞氧化损伤的调控作用。方法将处于对数生长期的L02细胞分为叔丁基对苯二酚（tert-butylhydroquinone，tBHQ）预处理组和单纯TCM染毒组。采用比色分析法检测超氧化物歧化酶（superoxide dismutase，SOD）、过氧化氢酶（catalase，CAT）、谷胱甘肽过氧化物酶（superoxide dismutase，GSH-Px）活力和丙二醛（malondialdehyde，MDA）水平；采用RT-PCR和Western blot法测定Nrf2、血红素加氧酶1（heme oxygenase-1，HO-1）、谷氨酸半胱氨酸连接酶催化亚基（glutamatecysteine ligase catalytic subunit，GCLC）及醌氧化还原酶1[NAD（P）H：quinone oxidoreductase 1，NQO1]mRNA以及蛋白水平的相对表达。结果与tBHQ对照组相比，tBHQ+TCM中剂量组L02细胞中CAT、SOD和GSH-PX活力明显增加，并随染毒剂量增加呈上升趋势，而MDA含量则显著上升（F=9.396、10.744、13.991、8.991，均P<0.05）；tBHQ+TCM低、中、高剂量组L02细胞中NQO1和HO-1 mRNA的表达均明显升高（F=32.724、110.290，均P<0.05）；当TCM剂量增大到12 mmol/L时L02细胞中Nrf2 mRNA和NQO1蛋白表达均显著升高（F=2.612、14.596，均P<0.05），而GCLC mRNA和蛋白的表达则逐渐下调（F=141.341、13.616，均P<0.05）。与同剂量单纯TCM染毒组比较，tBHQ+TCM低剂量组L02细胞中CAT、SOD活力下降，MDA含量升高，而tBHQ+TCM中、高剂量组L02细胞中CAT、SOD和GSH-PX活力上升，MDA含量下降（F=32.845、18.018、41.511、37.317，均P<0.05）。tBHQ+TCM低、中剂量组L02细胞Nrf2、HO-1、NQO1和GCLC mRNA表达均明显上调（F=35.567、191.428、161.252、1 314.869，均P<0.05）；而tBHQ+TCM高剂量组的L02细胞Nrf2 mRNA明显上调，但Nrf2蛋白表达则明显下调（F=4.150，P<0.05）；NQO1、GCLC蛋白表达均明显上调（F=31.398、5.048，均P<0.05），HO-1蛋白表达则明显下调（F=9.421，P<0.05）。结论 tBHQ具有诱导Nrf2信号因子增强表达的作用并进一步诱导其下游靶分子在mRNA转录和蛋白质合成两个层面上的相对表达均有所升高；Nrf2信号因子的表达增加可提高细胞的抗氧化能力，显著减弱TCM对L02细胞的氧化应激损伤。

关键词: 三氯甲烷, L02细胞, 叔丁基对苯二酚, Nrf2信号通路, 氧化损伤

Abstract:

Objective To investigate the regulatory effect of the nuclear factor erythroid 2-related factor 2（Nrf2） on trichloromethane（TCM）-induced oxidative damage in human normal liver cell line L02 cells. Methods L02 cells in logarithmic growth phase were divided into two groups：a tert-butylhydroquinone（tBHQ） pretreated group and a pure TCM exposed group.The activity of superoxide dismutase（SOD），catalase（CAT），superoxide dismutase（GSH-Px） and the level of malondialdehyde（MDA） were detected by colorimetric analysis. Relative expression levels of Nrf2，heme oxygenase-1（HO-1），glutamatecysteine ligase catalytic subunit（GCLC） and NAD（P）H：quinoneoxidoreductase1（NQO1） mRNA and protein were determined using RT-PCR and Western blot methods. Results Compared with the control group，the activities of CAT，SOD and GSH-PX in the tBHQ pre-treatment group were significantly increased，and showed an upward trend with increasing exposure dose，while the content of MDA was significantly increased（F=9.396，10.744，13.991，8.991，all P<0.05）. The expression of NQO1 and HO-1 mRNA in L02 cells treated with tBHQ+TCM low，medium，high dose were significantly increased（F=32.724，110.290，both P<0.05）. When the TCM dose was increased to 12 mmol/L，the expression of Nrf2 mRNA and NQO1 protein in L02 cells significantly increased（F=2.612，14.596，both P<0.05），while the expression of GCLC mRNA and protein gradually decreased（F=141.341，13.616，both P<0.05）. Compared with the same doses of pure TCM group，the tBHQ+low-dose TCM group showed a decrease in CAT and SOD activity and an increase in MDA content in L02 cells，while the tBHQ+ medium and high-dose TCM groups showed an increase in CAT，SOD，and GSH-PX activity and a decrease in MDA content in L02 cells（F=32.845，18.018，41.511，37.317，all P<0.05）. The mRNA expression of Nrf2，HO-1，NQO1 and GCLC in L02 cells showed significant increases in the tBHQ+lowand medium-dose TCM groups（F=35.567，191.428，161.252，1314.869，all P<0.05）；The mRNA expression of Nrf2 showed significantly increased in L02 cells in the tBHQ+high-dose TCM group，while the protein expression of Nrf2 was significantly decreased（F=4.150，P<0.05），the protein expression of NQO1 and GCLC were significantly increased（F=31.398，5.048，both P<0.05），and the expression of HO-1 protein was significantly decreased（F=9.421，P<0.05）. Conclusion tBHQ can induce the enhanced expression of Nrf2 signaling factors and further promote the upregulation of its downstream target molecules at both the mRNA transcription and protein synthesis levels.The upregulation of Nrf2 signaling factor can enhance the antioxidant capacity of cells and significantly reduce the oxidative stress damage caused by TCM on L02 cells.

Key words: Trichloromethane, L02 cells, Tert-butylhydroquinone, Nrf2 signaling pathway, Oxidative damage

中图分类号:

R114

钟苑芳, 谭诗莹, 邱奕冰, 胡黎明, 邹志辉, 余日安. Nrf2对三氯甲烷所致L02细胞氧化损伤的调控作用. [J]职业与健康, 2026, 42(20): 2765-2771.

ZHONG Yuanfang, TAN Shiying, QIU Yibing, HU Liming, ZOU Zhihui, YU Ri'an. Regulatory effect of Nrf2 on oxidative damage induced by trichloromethane in L02 cells. [J]OCCUPATION AND HEALTH, 2026, 42(20): 2765-2771.

图/表 6

参考文献 26

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引物	引物序列（5′→3′）	长度（bp）
ACTB	上游：TCTGGCGGCACCACCATGTAC	183
ACTB	下游：CTGCTTGCTGATCCACATCTGCTG	183
Nrf2	上游：TGGGCCCATTGATGTTTCTG	124
Nrf2	下游：TGCCACACTGGGACTTGTGTTTA	124
HO-1	上游：TTGCCAGTGCCACCAAGTTC	150
HO-1	下游：TCAGCAGCTCCTGCAACTCC	150
GCLC	上游：GTCCACAAATTGGCAGACAATGA	147
GCLC	下游：ACTCTGGTGAGCAGTACCACAAACA	147
NQO1	上游：GGATTGGACCGAGCTGGAA	100
NQO1	下游：GAAACACCCAGCCGTCAGCTA	100

引物	引物序列（5′→3′）	长度（bp）
ACTB	上游：TCTGGCGGCACCACCATGTAC	183
ACTB	下游：CTGCTTGCTGATCCACATCTGCTG	183
Nrf2	上游：TGGGCCCATTGATGTTTCTG	124
Nrf2	下游：TGCCACACTGGGACTTGTGTTTA	124
HO-1	上游：TTGCCAGTGCCACCAAGTTC	150
HO-1	下游：TCAGCAGCTCCTGCAACTCC	150
GCLC	上游：GTCCACAAATTGGCAGACAATGA	147
GCLC	下游：ACTCTGGTGAGCAGTACCACAAACA	147
NQO1	上游：GGATTGGACCGAGCTGGAA	100
NQO1	下游：GAAACACCCAGCCGTCAGCTA	100

TCM剂量（mmol/L）	CAT（U/mg prot）		SOD（U/mg prot）		GSH-Px（U/mg prot）		MDA（nmol/mg prot）
TCM剂量（mmol/L）	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组
12	2.05±0.10^abc	2.43±0.07^abd	60.67±1.25^abc	68.80±2.33^bcd	24.56±0.16^abc	34.49±1.07^abd	0.47±0.02^abc	0.25±0.04^abd
8	2.76±0.13^a	2.23±0.12^bd	65.16±0.48^ab	72.36±2.15^abd	27.02±0.68^ab	33.75±1.34^abd	0.36±0.03^ab	0.24±0.05^abd
4	2.94±0.21	1.88±0.16^d	67.48±2.08^a	64.78±0.91^d	30.07±0.46^a	31.22±0.91^ad	0.26±0.00^a	0.32±0.02^ad
0	3.06±0.03	2.10±0.15^d	71.15±0.98	66.92±0.83^d	35.67±1.53	28.92±1.33^d	0.17±0.01	0.17±0.02

TCM剂量（mmol/L）	CAT（U/mg prot）		SOD（U/mg prot）		GSH-Px（U/mg prot）		MDA（nmol/mg prot）
TCM剂量（mmol/L）	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组
12	2.05±0.10^abc	2.43±0.07^abd	60.67±1.25^abc	68.80±2.33^bcd	24.56±0.16^abc	34.49±1.07^abd	0.47±0.02^abc	0.25±0.04^abd
8	2.76±0.13^a	2.23±0.12^bd	65.16±0.48^ab	72.36±2.15^abd	27.02±0.68^ab	33.75±1.34^abd	0.36±0.03^ab	0.24±0.05^abd
4	2.94±0.21	1.88±0.16^d	67.48±2.08^a	64.78±0.91^d	30.07±0.46^a	31.22±0.91^ad	0.26±0.00^a	0.32±0.02^ad
0	3.06±0.03	2.10±0.15^d	71.15±0.98	66.92±0.83^d	35.67±1.53	28.92±1.33^d	0.17±0.01	0.17±0.02

TCM剂量（mmol/L）	HO-1 mRNA		NQO1 mRNA		GCLC mRNA		Nrf2 mRNA
TCM剂量（mmol/L）	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组	TCM组	tBHQ+TCM组
12	1.40±0.03^abc	1.58±0.03^abc	1.77±0.04^abc	3.09±0.07^abcd	1.25±0.01^ac	2.30±0.06^abcd	1.28±0.03^abc	2.30±0.21^bd
8	1.71±0.03^ab	2.03±0.08^abd	1.56±0.02^ab	2.59±0.18^abd	1.38±0.02^ab	3.09±0.05^abd	1.09±0.01^ab	2.09±0.35^d
4	1.21±0.01^a	1.47±0.02^ad	1.38±0.04^a	2.34±0.16^ad	1.24±0.02^a	3.27±0.02^ad	0.92±0.02^a	1.82±0.08^d
0	1.00±0.01	1.28±0.06^d	1.00±0.01	2.09±0.07^d	1.00±0.01	2.85±0.10^d	1.00±0.03	1.97±0.16^d