| Objective:To determine whether?-LA promotes Nrf2 and p-Nrf2 in HepG2 cells,investigate how?-LA replenishes rGSH through Nrf2 signaling pathway in HepG2 cells and explore the effect of?-LA antagonized cadmium-induced HepG2 cells damages in Nrf2 signaling pathwayMethods:The HepG2 cells were divided into control group,50?M?-LA treated group,25?M cadmium chloride treated group,50?M?-LA and 25?M cadmium chloride co-treated group,50?M?-LA+25?M cadmium chloride+5×10-44 mg/ml brusatol co-treated group,5×10-44 mg/ml brusatol treated group.For?-LA group,the cells were treated with 50?M?-LA for 16h.For cadmium group,the cells were treated with 25?M cadmium for 16h.For brusatol group,the cells were treated with 5×10-4mg/ml brusatol for 16h.For?-LA and cadmium co-treatment group,the cells were firstly pretreated with50?M?-LA for 8h,then treated with 25?M cadmium and 50?M?-LA for 16h.For?-LA+cadmium+brusatol co-treatment,the cells were pretreated with 50?M?-LA and brusatol for 8h,followed by exposure to 25?M cadmium for 16h in the presence of?-LA and brusatol.The protein of Nrf2,p-Nrf2,GCLC,GCLM and GR were measured by western blotting The mRNA levels of GCLC,GCLM and GR were measured by Real Time PCR.The intracellular concentrations of rGSH and GSSG were measured with the rGSH and GSSG assay kit.The cell viability was determined by MTT assay.Results:Resultes showed that cadmium significantly reduced the protein levels of Nrf2 and p-Nrf2?p<0.01,n=3?compared with untreated cells,.At the same time,cadmium decreased the mRNA and the protein levels of GCLC,GCLM and GR in HepG2 cells,the rGSH contents and rGSH/GSSG ratio in HepG2 cells?p<0.01,n=3?,and reduced the viability of HepG2 cells to 74%?p<0.01,n=3?.Compared with cadmium group,the protein levels of Nrf2 and p-Nrf2 in the HepG2 cells co-treated with?-LA and cadmium were significantly elevated?p<0.01,n=3?.At the same time,the mRNA and the protein levels of GCLC,GCLM and GR in the HepG2 cells co-treated with?-LA?50?M?and cadmium?25?M?,the rGSH contents,rGSH/GSSG ratio and the cell viability were significantly elevated?p<0.01,n=3?.However,inhibiting Nrf2 with brusatol significantly decreased the protein levels of Nrf2,p-Nrf2GCLC,GCLM and GRin the cells co-treated with?-LA and cadmium?p<0.01,n=3?.At the same time,inhibiting Nrf2 with brusatol significantly decreased the mRNA and the protein expressions of GCLC,GCLM and GR,the rGSH levels and rGSH/GSSG ratio,the cell viability in the cells.Conclusion:?-LA can promote the expression of Nrf2 and p-Nrf2 protein and the expression of GCLC,GCLM and GR,promote the increase of GCLC,GCLM and GR protein expression,and then elevate the content of rGSH and the ratio of rGSH/GSSG,and finally antagonize cadmium-induced cytotoxicity through Nrf2 signaling pathway in HepG2 cells. |
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