Cytoprotective Effects of the Tiger's Milk Mushroom Lignosus rhinocerotis (Agaricomycetes) Sclerotia against Oxidative Stress in PC12 Cells

Abstract

Lignosus rhinocerotis (Cooke) Ryvarden has been reported to possess numerous pharmacological effects. However, little is known about its potential role in mitigating the detrimental effects of oxidative stress. The present study investigated the cytoprotective effects of L. rhinocerotis extracts against hydrogen peroxide (H2O2)-induced oxidative stress of rat pheochromocytoma (PC12) cells. In the pre-treatment model, PC12 cells were pre-treated with aqueous (LRAQ) or ethanolic (LRET) extracts of L. rhinocerotis for 24 h, followed by 30 μM of H2O2 for 24 h. In the co-treatment model, the cells were incubated with LRAQ or LRET and H2O2 for 2 or 24 h to induce oxidative stress. Cell viability, intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and apoptotic cells with activated caspase-3/7 were quantified. Additionally, LRET was separated into fractions by chromatographic methods prior to analysis by gas chromatography-mass spectrometry (GCMS). 320 μg/ml aqueous extract showed a significant cytoprotective effect of 70.0 ± 22.4% and 133.92 ± 8.8% in the pre-treatment and co-treatment models, respectively, compared to untreated H2O2-challenged cells. LRAQ also showed a reduction (p < 0.05) in the percentage of depolarized cells of 37.6 ± 0.6% at 640 ug/ml and 53.4 ± 4.5% at 320 ug/ml in the pre-treatment and co-treatment models, respectively, compared to untreated H2O2-challenged cells. LRAQ or LRET showed a reduction (p < 0.01) in caspase 3/7 activity compared to untreated H2O2-challenged cells in the co-treatment model. However, LRAQ or LRET did not reduce excessive ROS formation (p > 0.05). The cytoprotective effects could be attributed to the presence of fatty acids, phenols, phytosterols, and dicarboxylic acids. In conclusion, L. rhinocerotis extracts demonstrated cytoprotective effects against H2O2-induced oxidative stress in an in vitro model, contributing to the maintenance of cellular integrity through the regulation of mitochondrial function and apoptosis.