Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation

Abstract

In recent years, the medicinal mushroom Antrodia cinnamomea, known as "niu-chang chih" has received much attention with regard to its possible health benefits; especially its hepatoprotective effects against various drugs, toxins, and alcohol induced liver diseases. However, the molecular mechanism underlying this protective effect of Antrodia cinnamomea and its active compound antroquinonol was poorly understood. In the present study we evaluated to understand the hepatoprotective efficacy of antroquinonol and ethanolic extracts of mycelia of Antrodia cinnamomea (EMAC) in vitro and in vivo. The protective mechanism of antroquinonol and EMAC against ethanol-induced oxidative stress was investigated in cultured human hepatoma HepG2 cells and ICR mice model, respectively. HepG2 cells were pretreated with antroquinonol (1-20μM) and oxidative stress was induced by ethanol (100mM). Meanwhile, male ICR mice were pretreated with EMAC for 10 days and hepatotoxicity was generated by the addition of ethanol (5g/kg). Hepatic enzymes, cytokines and chemokines were determined using commercially available assay kits. Western blotting and real-time PCR were subjected to analyze HO-1 and Nr-2 expression. EMSA was performed to monitor Nrf-2 ARE binding activity. Possible changes in hepatic lesion were observed using histopathological analysis. Antroquinonol pretreatment significantly inhibited ethanol-induced AST, ALT, ROS, NO, MDA production and GSH depletion in HepG2 cells. Western blot and RT-PCR analysis showed that antroquinonol enhanced Nrf-2 activation and its downstream antioxidant gene HO-1 via MAPK pathway. This mechanism was then confirmed in vivo in an acute ethanol intoxicated mouse model: serum ALT and AST production, hepatocellular lipid peroxidation and GSH depletion was prevented by EMAC in a dose-dependent manner. EMAC significantly enhanced HO-1 and Nrf-2 activation via MAPKs consistent with in vitro studies. Ethanol-induced hepatic swelling and hydropic degeneration of hepatocytes was significantly inhibited by EMAC in a dose-dependent manner. These results provide a scientific basis for the hepatoprotective effects of Antrodia cinnamomea. Data also imply that antroquinonol, a potent bioactive compound may be responsible for the hepatoprotective activity of Antrodia cinnamomea. Moreover, the present study highly supported our traditional knowledge that Antrodia cinnamomea as a potential candidate for the treatment of alcoholic liver diseases.