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

Abstract

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

CLC Number:

R114

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.

Figures/Tables 6

References 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