Ethanol Extract of Ganoderma lucidum Augments Cellular Anti-oxidant Defense through Activation of Nrf2/HO-1.
OBJECTIVE
The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL) against oxidative stress (hydrogen peroxide, H2O2) and elucidated the underlying mechanisms in a C2C12 myoblast cell line.


METHODS
Oxidative stress markers were determined by using the comet assay to measure reactive oxygen species (ROS) generation and deoxyribonucleic acid (DNA) damage. Cell viability and Western blotting analyses were employed to evaluate the cellular response to EGL and H2O2 in C2C12 cells. Transfection with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific small interfering ribonucleic acid (siRNA) was conducted to understand the relationship between Nrf2 expression and H2O2-induced growth inhibition.


RESULTS
The results showed that EGL effectively inhibited H2O2-induced growth and the generation of ROS. EGL markedly suppressed H2O2-induced comet-like DNA formation and phosphorylation of histone H2AX at serine 139 (p-γH2AX), a widely used marker of DNA damage, suggesting that EGL prevented H2O2-induced DNA damage. Furthermore, the EGL treatment effectively induced the expression of Nrf2, as well as heme oxygenase-1 (HO-1), with parallel phosphorylation and nuclear translocation of Nrf2 in the C2C12 myoblasts. However, zinc protoporphyrin IX, a HO-1 inhibitor, significantly abolished the protective effects of EGL against H2O2-induced accumulation of ROS and reduced cell growth. Notably, transient transfection with Nrf2-specific siRNA attenuated the cytoprotective effects and HO-1 induction by EGL, indicating that EGL induced the expression of HO-1 in an Nrf2-dependent manner.


CONCLUSIONS
Collectively, these results demonstrate that EGL augments the cellular anti-oxidant defense capacity through activation of Nrf2/HO-1, thereby protecting C2C12 myoblasts from H2O2-induced oxidative cytotoxicity.
Departments of Acupuncture and Moxibustion, Dong-Eui University College of Korean Medicine, Busan, Korea.
Yoo-Hwan
YH
Lee
Departments of Acupuncture and Moxibustion, Dong-Eui University College of Korean Medicine, Busan, Korea.
Jung-Hee
JH
Kim
Departments of Acupuncture and Moxibustion, Dong-Eui University College of Korean Medicine, Busan, Korea.
Choon-Ho
CH
Song
Departments of Acupuncture and Moxibustion, Dong-Eui University College of Korean Medicine, Busan, Korea.
Kyung-Jeon
KJ
Jang
Departments of Acupuncture and Moxibustion, Dong-Eui University College of Korean Medicine, Busan, Korea.
Cheol-Hong
CH
Kim
Anti-Aging Research Center, Dong-Eui University, Busan, Korea.
Ji-Sook
JS
Kang
Anti-Aging Research Center, Dong-Eui University, Busan, Korea; Department of Acupuncture and Moxibustion, Dong-Eui University College of Korean Medicine, Busan, Korea.
Yung-Hyun
YH
Choi
Departments of Acupuncture and Moxibustion, Dong-Eui University College of Korean Medicine, Busan, Korea.
Hyun-Min
0000-0003-3645-6109
HM
Yoon
Ganoderma lucidum; Nrf2; anti-oxidant; heme oxygenase-1; reactive oxygen species