Materials and chemicals

The H. erinaceus (KLU-M 1232) and G. lucidum (KLU-M 1233) basidiocarps were obtained from Ganofarm in Tanjung Sepat, Selangor. Ganoderma neo-japonicum (KLU-M 1231) basidiocarps were collected from a forest in Ulu Grik, Perak and G. frondosa basidiocarps (KLU-M 1229) were purchased from a hypermarket in Selangor, Malaysia. The mushrooms were identified and authenticated by experts in the Mushroom Research Centre, University of Malaya. Voucher specimens are deposited in the University of Malaya herbarium (KLU-M). Rat pheochromocytoma (PC-12Adh) cell line was purchased from American Type Culture Collection (ATCC; Rockville, MD, USA; Catalogue Number: CRL-1721.1). Kaighn’s Modification of Ham’s F-12 Medium (F-12 K medium), NGF-7S from murine submaxillary gland, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), MEK inhibitor (U0126, PD98059), PI3K inhibitor (LY294002), anti-neurofilament 200 (NF-200) antibody produced in rabbit and Anti-Rabbit IgG-Fluorescein isothiocyanate (FITC) antibody produced in sheep were obtained from Sigma Co. (St. Louis, MO, USA). ProLong® Gold Antifade Reagent with DAPI (4-6-Diamidino-2-phenylindole) was purchased from Life Technologies Corporation (California, USA). Fetal bovine serum (FBS) and horse serum (HS) were purchased from PAA Laboratories (Cölbe, Germany).

Preparation of aqueous extracts

The aqueous extracts were prepared according to Eik et al. [15]. Briefly, the fresh basidiocarps of H. erinaceus and G. frondosa were sliced, weighed and freeze-dried while G. lucidum and G. neo-japonicum were air dried. The dried basidiocarps were then ground into powder by a Waring commercial blender. The powder was then soaked in distilled water at a ratio of 1:20 (w/v) and 150 rpm at room temperature. After 24 h, the mixture was double boiled in a water bath for 30 min and after cooling was filtered through Whatman no. 4 filter paper. The resulting aqueous extracts were freeze-dried and kept at −20°C prior to use.

In vitro cell culture

The rat pheochromocytoma (PC-12Adh) cells were sustained in ATCC formulated F-12 K medium and supplemented with 15% (v/v) of heat-inactivated HS and 2.5% (v/v) of heat-inactivated FBS with final pH 6.8 - 7.2. The cells were subcultured every 2 to 3 days and incubated at 37 ± 2°C in a 5% CO 2 -humidified incubator.

Cell viability and cytotoxicity assay

Cell viability was assessed by the mitochondrial-dependent reduction of MTT to purple formazan. PC-12 cells were plated in 96-well plates at a density of 5 × 103 cells/well and incubated overnight at 37°C in a 5% CO 2 -humidified incubator. Then, the aqueous extracts (0–2500 μg/ml) were added into the cells. After 48 h of incubation, 20 μl of MTT (5 mg/ml) in PBS buffer (pH 7.4) was added into each well and incubated at 37°C for 4 h. Subsequently, the supernatant was carefully discarded by aspiration, and 100 μl of DMSO was then added into each well to dissolve the MTT formazan crystals, mixed thoroughly and incubated for 15 min. The extent of the reduction of MTT was determined by measurement of the absorbance at 540 nm with 690 nm as background absorbance with an ELISA microplate reader (Sunrise, Tecan, Austria). The complete F-12 K medium was the blank, and cells incubated in the medium only were denoted as the negative control.

Neurite outgrowth stimulation assay

Cells were plated in 12-well plates at a density of 5 × 103 cells per well in complete F-12 K medium. The cells were treated with freshly prepared aqueous extracts at various concentrations ranged from 25 to 100 μg/ml (w/v). Eik et al. [15] reported that 50 ng/ml (w/v) of NGF-7S from murine submaxillary gland was the optimum concentration for neuritogenesis in PC-12 cells. In the present study, cells treated with 50 ng/ml (w/v) of NGF or 50 μg/ml (w/v) of H. erinaceus served as positive controls. Cells in complete F-12 K medium without treatment served as a negative control. Assay plates were incubated for 48 h at 37 ± 2°C in a 5% CO 2 -humidified incubator.

Quantification of neurite outgrowth

The cell morphology was assessed under an inverted microscope (Nikon Eclipse TS100). Neurite extension of PC-12 cells was regarded as an index of neuritogenesis. Neurite that was double or more the length of the cell body diameter was scored positive for a neurite-bearing cell [14]. The images were captured with a QImaging Go-3 color CMOS Camera (QImaging, Canada) and by the image processor system, Image-Pro Insight (MediaCybernetics, MD). The percentage of differentiated cells was evaluated by scoring the proportion of neurite-positive cells to total cells in randomly 10 selected microscopic fields per well, with an average of 200–300 cells per well.

Treatment with specific inhibitors of signaling pathways

The MEK/ERK1/2 inhibitors (U0126, PD98059) and PI3K/Akt inhibitor (LY294002) were used in this study. Stock solutions (10 mM) of inhibitors were prepared in DMSO and stored at −20°C in the dark. Final concentrations of 10 μM of U0126, 30 μM of LY294002 and 40 μM of PD98059 were prepared by diluting in complete F-12 K medium just before use [16]. Cells were pre-incubated either with or without the inhibitor for 1 h at 37 ± 2°C in a 5% CO 2 -humidified incubator, respectively before the treatment with 50 ng/ml (w/v) of NGF or the optimum concentration of each aqueous extract resulting in the neurite outgrowth stimulation assay. Cells were then incubated for 48 h prior to scoring the neurite-bearing cells.

Immunofluorescence staining of neurofilament

Immunofluorescence assay was carried out according to Schimmelpfeng et al. [21] with some modifications. Briefly, cells were seeded in 12-well micro-chamber (ibidi, Martinsried, Germany) at a density of 5 × 103 cells per well in complete F-12 K medium. Then, the cells were pre-incubated either with or without the treatment of inhibitors. After 1 h, the cells were treated with the optimum concentration of each aqueous extract result in the neurite outgrowth stimulation assay for 48 h at 37 ± 2°C in a 5% CO 2 -humidified incubator. Subsequently, the cells were fixed with 4% formalin (v/v) at room temperature for 20 min. After three washings with PBS, the cells were incubated with anti-NF-200 antibody produced in rabbit (1:80 dilution in blocking buffer) at room temperature for 1 h. Then, the cells were incubated with fluorophore-conjugated secondary antibody, anti-Rabbit IgG-FITC antibody produced in sheep (1:80 dilution in blocking buffer) at room temperature for 1 h in the dark. Cells were mounted with aqueous mounting medium, ProLong® Gold Antifade Reagent with DAPI. Slides were observed under fluorescence illumination using FITC and DAPI filters and images were captured with Nikon’s Imaging Software, NIS-Elements.

Statistical analysis

All the experimental data were expressed as the mean ± standard deviation (SD). Statistical differences between groups were performed using one-way analysis of variance (ANOVA) of a minimum of three independent experiments and Duncan's multiple range tests (DMRT) P < 0.05 was considered to be significant.