Mrf2 Pathway Mediated Protection Against Oxidative Stress In Human HepG2 Cells

Objective: To investigate the effects of Salvianolic acid A (SAA) on phase 2 enzymes induction and Nrf2 activation in oxidative damaged HepG2 cells.Methods: (1) The HepG2 cells were divided into four groups(n=3): control group and Salvianolic acid A (SAA) treatment groups(0.2μM, 2μM, 20μM). The activities of Reduced Glutathione (GSH) were determined with the method of chemical colorimetry. The protein expressions of NAD(P)H: quinone oxidoreductase l (NQO1), Heme Oxygenase-1 (HO-1), Glutathione S-transferase (GST), NF-E2-related factor 2 (Nrf2) and Kelch-like ECH associated protein1 (Keap1) were determined by western blotting. The RT-PCR analysis was employed to detect the mRNA expression levels of HO-1 and Nrf2. (2) The HepG2 cells were randomly divided to five groups and damaged by H₂O₂ (n=5): control group, H₂O₂ group (3.5mM) and SAA pretreatment groups (0.2μM, 2μM, 20μM) plus H₂O₂. MTT method was used to detect cell survival rate. The content of lactate dehydrogenase (LDH) was examined by chemical colorimetry method. (3) The HepG2 cells were randomly divided into four groups (n=3): control group, SAA group (20μM), H₂O₂ group (3.5mM) and SAA pretreatment group (20μM) plus H₂O₂. The protein expressions of NQO1,HO-1,GST and Nrf2 were measured by western blotting. The RT-PCR analysis was employed to detect the mRNA expression levels of HO-1 and Nrf2. (4) The HepG2 cells were divided to four groups (n=3): negative control group (NC), siRNA (Nrf2) group, negative control plus SAA (20μM) group, siRNA (Nrf2) plus SAA (20μM) group. The protein expression of Nrf2 was measured by western blotting.Results: In present study, we demonstrated that Salvianolic acid A (SAA) upregulate the NQO1, HO-1 and GST expression in HepG2 cells in a time- and dose-dependent manner. In addition, SAA increased the expression and nuclear translocation of a basic leucine zipper transcription factor Nrf2, which is known to be involved in the regulation of numerous antioxidant enzymes. Specific silence of Nrf2 by siRNA down-regulated SAA-induced Nrf2 activation. At the same time, SAA markedly reduced the level of Keap1 protein in a dose-dependent way. Additionally, the increase of GSH content and HO-1, NQO1, GST expression were accompanied with the accumulation of Nrf2 in nucleus. Thus we concluded that the activation of Nrf2 signal pathway may be one of the important reasons to provide protection against oxidative damage in HepG2 cells.Conclusions: Administration of SAA could enhance the antioxidant capacity of normal HepG2 cells by increase of a series of phase 2 enzymes and provide protection against the oxidative damage caused by H₂O₂. The results indicate that SAA attenuate oxidative stress by activating Nrf2-mediated phase 2 enzymes induction.