1.Role of NRF2 signaling pathway in trichloromethane-induced oxidative stress in L02 cells
TAN Shi ying ZHONG Yuan fang QIU Yi bing ZOU Zhi hui YU ri an
China Occupational Medicine 2022;49(05):530-
Objective - ( )
To investigate the effects of nuclear factor erythroid 2 related factor 2 NRF2 on the oxidative stress
( ) Methods ) ,,
induced by trichloromethane TCM in human normal hepatocyte L02 cells. i L02 cells were stimulated with 1 2
, , , ( ),
4 8 12 16 and 20 mmol/L TCM solution dissolved in dimethyl sulfoxide and the control group and blank group were set
, - ,
up. After culturing for 24 hours the cell viability was detected by CCK 8 colorimetric method and the concentration of TCM
) -, -
stimulation was screened. ii L02 cells in logarithmic growth phase were randomly divided into control group and low medium
- , , ,
and high dose groups. After 24 hours of exposure to 0 4 8 and 12 mmol/L TCM the cells were collected. The activity of
( ), ( ), ( - ) ( )
superoxide dismutase SOD catalase CAT glutathione peroxidase GSH Px and the level of malondialdehyde MDA
NRF2, - (HO-1),
were detected by colorimetric analysis. The mRNA expression levels of heme oxygenase 1 glutamate cysteine
(GCLC) () (NQO1) -
ligase catalytic subunit and NAD P H quinone dehydrogenase 1 were detected by real time fluorescence
, - ,
polymerase chain reaction. The protein levels of NRF2 HO 1 GCLC and NQO1 were detected by Western blotting.Results ) , , , ,
i When the concentration of TCM was 4 8 12 16 and 20 mmol/L the survival rate of L02 cells decreased
( P ) , ,
significantly compared with the control group all <0.05 . The concentration of 0 4 8 and 12 mmol/L were selected as the
) , -
stimulation doses for subsequent experiments. ii Compared with the control group the activities of SOD and GSH Px in L02
( P ) ( P ), -
cells in the three doses groups decreased all <0.05 and the levels of MAD increased all <0.05 with a dose effect
- (P ),
relationship. The CAT activity of L02 cells in the medium dose group was lower than that in the control group <0.05 and the
- ( P )
CAT activity of L02 cells in the high dose group was lower than that in the others three groups all <0.05 . Compared with the
, NRF2 - (P ),NRF2
control group the relative expression levels of mRNA in L02 cells in the low dose group decreased <0.05
- (P ), NRF2
mRNA in L02 cells in the medium dose group increased <0.05 mRNA and NRF2 protein expression in L02 cells in
( P ) HO-1,GCLC, NQO1 ,
the highdose group increased both <0.05 . The relative expression level of mRNA and GCLC NQO1
( P )
protein expression in L02 cells in the three doses groups increased compared with the control group all <0.05 . The relative
NRF2 - - -
expression level of mRNA in L02 cells in the high dose group was higher than that in the low and medium dose groups
( P ), - (P ),
both <0.05 and the relative expression of NRF2 protein was higher than that in the low dose group <0.05 but the
HO-1 GCLC - - (
relative expression levels of and mRNA and HO 1 protein level were lower than those in the medium dose group all
P )Conclusion -
<0.05 . TCM exposure can inhibit the proliferation of L02 cells by inducing oxidative stress with a dose effect
,
relationship. In this process the antioxidant mechanism mediated by NRF2 was activated. The expression of antioxidant defense
, - ,
and detoxification related target genes downstream of NRF2 signaling pathway was activated and the expression of HO 1
-
GCLC and NQO1 was up regulated to alleviate the oxidative damage caused by TCM.