Abstract:

Objective To observe the improvement effect of total flavonoids of Sophora flavescens （SF） on the oxidative stress in the Leydig’s cells TM3 of the mice induced by lead acetate （LA） and their influences on the Keap1/Nuclear factor E2-related factor 2 （Nrf2） signaling pathway， and to discuss the potential mechanism of the improvement effect of SF on oxidative stress in the LA-induced TM3 cells. Methods The survival rates of the TM3 cells after treated with various concentrations of LA （0， 10， 20， 50， and 80 μmol·L^－1） and SF （0， 12.5， 25.0， 50.0， and 80.0 mg·L^－1） for 24 h were detected by MTT assay. The TM3 cells were randomly divided into blank control group， LA group， LA + low dose of SF group， LA+middle dose of SF group， and LA+high dose of SF group. Except for blank control group， the TM3 cells in the other groups were induced with 50 μmol·L^－1 LA for 24 h to establish the oxidative stress model. The TM3 cells in LA+low dose of SF group， LA+middle dose of SF group， and LA+ high dose of SF group were then treated with 12.5， 25.0， and 50.0 mg·L^－1 SF， respectively. The survival rate of the TM3 cells was detected by MTT assay； the activities of superoxide dismutase （SOD） and levels of malondialdehyde （MDA） in the TM3 cells in various groups were detected by WST-1 assay and thiobarbituric acid （TBA） method； the expression levels of Nrf2， Keap1， and Caspase-9 proteins in the TM3 cells in various groups were detected by detected by Western blotting method. Results The MTT assay resuts showed that compared with 0 μmol·L^－1 LA group，the survival rates of the TM3 cells in 10， 20， 50， and 80 μmol·L^－1 LA groups were decreased （P<0.01）； compared with 0 mg·L^－1 SF group，the survival rates of the TM3 cells in 12.5， 25.0， 50.0， and 80.0 mg·L^－1 SF groups were significantly decreased （P<0.05 or P<0.01）； compared with LA group， the survival rates of the TM3 cells in LA+low dose of SF group， LA+middle dose of SF group， and LA+high dose of SF group were significantly increased （P<0.01）. The WST-1 assay and TBA method results showed that compared with LA group，the SOD activities and MDA levels in the TM3 cells in LA+low dose of SF group， LA+middle dose of SF group， and LA+high dose of SF group were significantly decreased （P<0.01）.The Western blotting results showed that compared with LA group， the expression levels of Nrf2 protein in the TM3 cells in LA+low dose of SF group， LA+middle dose of SF group， and LA+high dose of SF group were increased（P<0.01），and the expression levels of Keap1 and Caspase-9 proteins were decreased （P<0.01）. Conclusion SF has certain improvement effect on the oxidative stress in the LA-induced TM3 cells and can reduce the expression level of Keap1 protein and increase the expression level of Nrf2 protein in the TM3 cells.

Key words: Sophoral flavones, Lead acetate, Leydig’s cell, Oxidative stress, Kelch-like ECH-associated protein 1, Nuclear factor erythroid 2-related factor 2