Neuroprotective Effects of Extracts from Tiger Milk Mushroom Lignosus rhinocerus Against Glutamate-Induced Toxicity in HT22 Hippocampal Neuronal Cells and Neurodegenerative Diseases in Caenorhabditis elegans

Abstract

Despite the Tiger Milk Mushroom Lignosus rhinocerus (LR) having been used as a traditional medicine, little is known about the neuroprotective effects of LR extracts. This study aims to investigate the neuroprotective effect of three extracts of LR against glutamate-induced oxidative stress in mouse hippocampal (HT22) cells as well as to determine their effect in Caenorhabditis elegans. In vitro, we assessed the toxicity of three LR extracts (ethanol extract (LRE), cold-water extract (LRC) and hot-water extract (LRH)) and their protective activity by MTT assay, Annexin V-FITC/propidium iodide staining, Mitochondrial Membrane Potential (MMP) and intracellular ROS accumulation. Furthermore, we determined the expression of antioxidant genes (catalase (CAT), superoxide dismutase (SOD1 and SOD2) and glutathione peroxidase (GPx)) by qRT-PCR. In vivo, we investigated the neuroprotective effect of LRE, not only against an Aβ-induced deficit in chemotaxis behavior (Alzheimer model) but also against PolyQ40 formation (model for Morbus Huntington) in transgenic C. elegans. Only LRE significantly reduced both apoptosis and intracellular ROS levels and significantly increased the expression of antioxidant genes after glutamate-induced oxidative stress in HT22 cells. In addition, LRE significantly improved the Chemotaxis Index (CI) in C. elegans and significantly decreased PolyQ40 aggregation. Altogether, the LRE exhibited neuroprotective properties both in vitro and in vivo.

Keywords: Caenorhabditis elegans; HT22; Lignosus rhinocerus; PolyQ40; chemotaxis; glutamate toxicity; neuroprotection; oxidative stress.

Figure 1

The protective effect of different concentrations of Lignosus rhinocerus (LR) extracts against glutamate-induced toxicity in HT22 cells. Viability of untreated control cells was set at 100%. (a) Treatment of cells with an LR ethanol extract (LRE) and LRE plus glutamate; (b) Treatment of cells with LRC and LRC plus glutamate; (c) Treatment of cells with LRH and LRH plus glutamate. Values are mean ± SEM of at least three independent runs. Significant differences: ^### p < 0.001 vs. control; *** p < 0.001 vs. glutamate alone.

Figure 2

Quantitative flow cytometric analysis of the abundance of apoptotic HT22 cells using Annexin V-FITC/PI staining. Annexin V+/PI– cells are seen in the lower right are in the early stage and annexin V+/PI+ in the upper right quadrant are late stage. (a) Treatment of cells with glutamate, NAC and LRE alone or in combination with 5 mM glutamate; (b) Treatment of cells with glutamate, NAC and LRC and LRH alone or in combination with 5 mM glutamate; (c) Representative scatter plots of the distribution of annexin V and PI-stained cells. Values are mean ± SEM of at least three independent runs. ^### p < 0.001 vs. control; *** p < 0.001 vs. glutamate alone.

Figure 3

Protective effect of different concentrations of LR extracts on Mitochondrial Membrane Potential (MMP) in HT22 cells. (a) Treatment of cells with LRE, N-acetylcysteine (NAC), carbonylcyanide 3-chlorophenylhydrazone (CCCP) and LRE, NAC plus glutamate; (b) Treatment of cells with LRH, LRC and LRH, LRC plus glutamate; (c–h) Representative fluorescence micrographs with TMRE staining using fluorescence microscopy; (c) control group; (d) DMSO group; (e) 5 mM glutamate group; (f) 100 µg/mL LRE co-treatment group; (g) 200 µg/mL; (h) 0.25 NAC group. Values are mean ± SEM of at least three independent runs. ^## p < 0.01 vs. control; * p < 0.05; ** p < 0.01 vs. glutamate alone.

Figure 4

The effect of different concentrations of LR extracts on intracellular ROS accumulation in HT22 cells. ROS levels of untreated control cells were set at 100%. (a) Treatment of cells with glutamate, NAC and LRE alone or in combination with 5 mM glutamate; (b) Treatment of cells with glutamate, NAC and LRC and LRH alone or in combination with 5 mM glutamate; (c) Representative fluorescence micrographs of DCFH-DA stained control cells; (d) DMSO group; (e) 5 mM glutamate group; (f) 100 µg/mL LRE and (g) 200 µg/mL of LRE co-treatment groups; (h) 0.25 NAC group. Values are mean ± SEM of at least three independent experiments. ^# p < 0.05, ^### p < 0.001 vs. control; * p < 0.05; *** p < 0.001 vs. glutamate alone.

Figure 5

The effect of different concentrations of LRE extracts on the expression of antioxidant genes (catalase (CAT), superoxide dismutase (SOD1 and SOD2) and glutathione peroxidase (GPx)) in HT22 cells given alone or in combination with 5 mM glutamate. (a) CAT; (b) DOD1; (c) SOD2; (d) GPx. Values are mean ± SEM of at least three independent runs. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001 vs. control; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. glutamate alone.

Figure 6

The effect of different concentrations of LRE extracts against Aβ-induced reduction of chemotaxis behavior in C. elegans. (a) treatment of CL2355 worms (Aβ+) with LRE and EGCG (positive control); (b) treatment of CL2122 worms (Aβ-) with LRE and EGCG (positive control). Values are mean ± SEM of at least three independent runs. ** p < 0.01; *** p < 0.001 vs. DMSO.

Figure 7

The effect of different concentrations of LRE extracts against PolyQ40 aggregation in C. elegans. (a) PolyQ40 formation after treatment with of different concentration of LRE; (b) Visualization of PolyQ40 formation by fluorescence microscopy: (b) untreated worms; (c) DMSO-treated worms; (d–f) after treatment with 50, 100 and 200 μg/mL of LRE; (g) worms treated with 50 μg/mL of EGCG. Values are mean ± SEM of at least three independent runs. *** p < 0.001 vs. DMSO.

Figure 7

The effect of different concentrations of LRE extracts against PolyQ40 aggregation in C. elegans. (a) PolyQ40 formation after treatment with of different concentration of LRE; (b) Visualization of PolyQ40 formation by fluorescence microscopy: (b) untreated worms; (c) DMSO-treated worms; (d–f) after treatment with 50, 100 and 200 μg/mL of LRE; (g) worms treated with 50 μg/mL of EGCG. Values are mean ± SEM of at least three independent runs. *** p < 0.001 vs. DMSO.