Upregulation of NF-E2-related factor-2-dependent glutathione by carnosol provokes a cytoprotective response and enhances cell survival

Aim: To explore whether glutathione (GSH) increased through Nrf-2 activation is involved in the cytoprotective effects of carnosol in HepG2 cells. Methods: Human hepatoma cell line HepG2 were exposed to rosemarry essential oil or carnosol. Cell viability was measured using an Alamar blue assay. The production of intracellular GSH was determined using monochlorobimane. The level of protein or mRNA was examined by Western blotting or RT-PCR, respectively. Results: Rosemarry essential oil (0.005%-0.02%) and carnosol (5 and 10 mol/L) increased the intracellular GSH levels and GSH synthesis enzyme subunit GCLC/GCLM expression. Rosemary essential oil and carnosol increased nuclear accumulation of Nrf2 and enhanced Nrf2-antioxidant responsive element (ARE)-reporter activity. Transfection of the treated cells with an Nrf2 siRNA construct blocks GCLC/GCLM induction. Furthermore, pretreatment of the HepG2 cells with essential oil and carnosol exerted significant cytoprotective effects against H2O2 or alcohol. In TNF alpha-treated cells, the nuclear translocation and transcriptional activity of NF-kappa B was abolished for 12 h following carnosol pretreatment. Cotreatment with GSH also suppressed NF-kappa B nuclear translocation, whereas cotreatment with BSO, a GSH synthesis blocker, blocked the inhibitory effects of carnosol. Conclusion: This study demonstrated that Nrf2 is involved in the cytoprotective effects by carnasol, which were at least partially mediated through increased GSH biosynthesis.