Protection Of Carnosic Acid Against Oxidative Damage By Inducing Phase 2 Enzymes Mediated By PI3K/Akt And Nrf2 In HepG2 Cells

Objective:To investigated whether Carnosic acid (CA) could induce antioxidant and phase 2 enzymes and confer protection against oxidative damage in HepG2 cells.Methods:HepG2 cells were randomly divided into four groups (n=3): control group; carnosic acid (CA) group (5μM,10μM,20μM). The activity of NAD(P)H:quinone oxidore-ductase (NQO1) in HepG2 cells were measured by 2,6-dichloroindophenol reduction method. The activities of Glutathione Reductase (GSH) were determined with the method of chemical colorimetry. The protein expressions of nuclear factor E2-related factor2 (Nrf2), NQO1 and Heme oxygenase-1 (HO-1) in HepG2 cells were detected by Western blotting. HepG2 cells were randomly divided into five groups (n=3):control group; 20μM CA group; 20μM CA+inhibitor group (LY294002; Dicoumarol; ZNPPIX). The protein expressions of Nrf2, NQO1 and HO-1 in HepG2 cells were detected by Western blotting.HepG2 cells were randomly divided into four groups (n=3):control group; PI3K inhibitor group; 20μM CA group; 20μM CA group+PI3K inhibitor group. The protein expressions of Akt and P-Akt in HepG2 cells were detected by Western blotting. HepG2 cells were randomly divided into five groups and damaged by H2O2 (n=4):control group; H2O2 groups; CA treated group (5μM,10μM,20μM). MTT method was used to examine cell survival rate. The activities of lactate dehydrogenase (LDH) were determined with the method of chemical colorimetry. Then HepG2 cells were randomly divided into six groups (n=4):control group; 20μM CA treated group; 20μM CA group+inhibitor (LY294002, Dicoumarol, ZNPPIX) treated group. MTT method was used to examine cell survival rate. The activities of lactate dehydrogenase (LDH) were determined with the method of chemical colorimetry.Results:The results showed that CA could induce Heme Oxygenasel (HO-1), quinone oxidoreductase 1 (NQO1) and glutathione reductase (GSH) expression in HepG2 cells in a time-and dose-dependent manner and protect them against hydrogen peroxide caused oxidative damage. The induction of HO-1 and NQO1 by CA was accompanied with the accumulation of nuclear factor-E2-related factor-2 (Nrf2) in nucleus and the activation of phosphatidylinositol 3-kinase PI3K/Akt signal pathway. Additionally, the specific inhibitor of PI3K/Akt could obviously decrease not only the induced expression of HO-1, NQO1 and GSH but also the antioxidant effect of CA. In conclusion, this study proved that CA exerts antioxidant effect by inducing HO-1, NQO1 and GSH expression mediated by PI3K/Akt and Nrf2.Conclusion:The result indicated that CA increased the antioxidant and phase 2 enzymes through an activation of the PI3K-Akt/Nrf2 pathway. This may be a primary mechanism of antioxidative action of CA concerned with its therapeutic effectiveness in HepG2 cells.