Hericium erinaceus extracts and analysis of erinacine A

Fresh mycelium of H. erinaceus was extracted with ethanol. The extract was concentrated and fractionated by solvent partition between ethylacetate and water. The ethylacetate fraction was subjected to silica gel column chromatography using n-Hexane–ethylacetate as the eluent. The Hexane–acetone eluate was subjected to silica gel column chromatography according to the previous study [9, 12, 13]; HPLC analysis of erinacine A was executed according to the previous study with minor modifications. The analytical column used was a COSMOSIL 5C18-AR-II (250 × 4.6 mm; particle size 5 μm, Nacalai USA, Inc., Kyoto, Japan). Separation was performed at 40 °C using two different gradients for the mobile phase, which consisted of two solvents, methanol (A) and 2.0 % acetic acid in water (B). The gradient elution had the following profile: 0–20 min, 60–90 % (A); 20–25 min, 90 % (A). The retention time of erinacine A was approximately ~17 min at a flow rate of 1.0 mL/min with a scanning UV wavelength at 340 nm. The 3 mg/g erinacine A in the H. erinaceus extracted with 85 % ethanol was confirmed and quantified by HPLC as shown in Fig. 1 [12, 13]. Chemical compounds studied in this article Erinacine A (PubChem CID: 10410568).

Fig. 1 Simplistic flow chart of neuroprotective activities of Hericium erinaceus mycelium (HEM) in a MPTP treated animal model Full size image

Animals

C57BL/6 mice (8–10 weeks old, 20–28 g) were kept individually in a 12-h light/dark cycle cage and had free access to water and food. Animal care and the general protocols for animal use were approved by the Institutional Animal Care and Use Committee of Chang Gung University of Science and Technology. Mice were operated on according to the modified MPTP-induced PD’s model, can be induced by the intraperitoneal injections of MPTP-HCl (30 mg/kg; Sigma, St. Louis, MO) or saline in 5 day. Four groups (six mice in each group) were randomly assigned to a sham control group, a MPTP group, three H. erinaceus wet mycelia (HEM) groups (5.38, 10.76 and 21.52 mg) and erinacine A groups (1 mg/kg). HEM was dissolved in water (H 2 O) and mice received HEM oral administration indicated during the 25 days before the onset of MPTP induction and HEM starting after the first MPTP injection and continuing through 5 additional days. Erinacine A was dissolved in dimethyl sulfoxide (DMSO) and administered intraperitoneally for 5 days before the MPTP induction. The sham-operated group animals received an equivalent volume of saline. Mice were killed 5 days after MPTP injection and brains were harvested, sectioned, and processed [28].

Chemical reagents and antibodies

Mouse monoclonal antibodies against tyrosine phospho-Hydroxylase (Ser31), GAPDH, β-actin, 4-Hydroxy-2-nonenal (4-HNE), Nitro-tyrosine, CHOP, Fas, Bax, NFkB p65, Histone H1, TRAF2, IRE1α, and phospho-IKB-β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal antibodies against phospho-p38 MAPK (Thr180/Tyr182) and phospho-JNK1/2 (Thr183/Tyr185) were purchased from Cell Signaling Technology (Beverly, MA, USA). The TdT-mediated dUTP Nick End Labeling (TUNEL) kits were purchased from Roche (Germany). MPP+ (1-methyl-4-phenylpyridinium), SDS, NP-40, while sodium deoxycholate, protease inhibitor cocktail was purchased from Sigma (St. Louis, MO, USA).

Behavioral testing

Behavioral assessments on mice were made 1–6 days after MPTP injection. Motor performance was assessed with a rotary rod apparatus using a protocol similar to that described [29]. For the rotarod tests, the paradigms were used: rocking—direction of rotation with each full turn of the rod at 10 rpm for 3 min to a level just below the bottom of the rod. The mice were placed on the rotating rod and the time until they fell off was recorded. This was repeated six times until the total time on the rod for the control group was 3 min. Both the total time spent on the rotating rod for each mice, five groups (Control, MPTP, HEM), were recorded.

Immunohistochemistry

After the administration of a large dose of chloral hydrate, the mice were killed by decapitation 8 days after MPTP treatment. The brains were quickly removed and placed in ice-cold saline for 10 min. Next, the brains were cut into seven 4 μm-thickness slides transversely from neuron impairment area using a mouse brain matrix (Harvard Apparatus, MA, USA) and then immediately fixed in 10 % formalin overnight. The brain sections were then dehydrated with graded ethanol, passed through chloroform, and embedded in paraffin, which were assessed by hematoxylin and eosin (H and E) staining. Paraffin sections of the striatum and substantia nigra were used for immunohistochemistry (IHC). Staining was performed using a biotinylated secondary antibody (Vectastain Universal Elite ABC Kit). Monoclonal rabbit antibodies against tyrosine Hydroxylase, nitro-tyrosine, 4-HNE and Fas (CD95) were diluted in a ratio of 1:100. The omission of primary antibodies was used as the negative control. Using the slides, the presence of cytoplasm stained with brown was scored as positive. The protein expression were quantitatively evaluated using an Olympus Cx31 microscope with an Image-pro Plus medical image analysis system. The digital images were captured using a digital camera (Canon A640). The positive area and optical density of the positive cells were determined by measuring three randomly selected microscopic fields (100×, 200× magnification) on each slide. The IHC index was defined as average integral optical density (AIOD) (AIOD = positive area × optical density/total area) [30].

Cell culture

The mouse N2a (Neuro-2a) cells were purchased from the American Tissue Culture Collection (ATCC, USA). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10 % fetal calf serum (Gibco), non-essential amino acids, 1 mM sodium pyruvate and 1 % antibiotics (100 units/mL of penicillin and 100 μg/mL of streptomycin). All experiments were performed in plastic tissue culture flasks, dishes or in microplates (Nunc, Naperville, Denmark). Incubation was carried out at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air [31].

Assessment of cell viability and apoptosis assay

Cell viability, as previously reported by MTT quantitative colorimetric assay, was capable of detecting viable cells. The cells were seeded at 2 × 104 cells/ml density and incubated with MPP + for 24 h. Thereafter the medium was changed and incubated with MTT (0.5 mg/ml) for 4 h. The viable cell number is directly proportional to the production of formazan following solubilization with isopropanol, which can be measured spectrophotometrically at 563 nm [31]. Annexin V/propidium iodide (Biosource International, USA) was used to quantify the percentage of apoptosis cells. Flow cytometric analysis was performed with a FACSCaliber using CellQuest software. Data were analyzed with CellQuest and WinMDI software. The apoptotic cells (V+/PI-) were measured by the fluorescence-activated cell sorter analysis in a FACS analyzer (Becton–Dickinson). The data represented three independent experiments [32].

Preparation of total cell extracts and immunoblotting analysis

Cells were lysed with a buffer containing 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS) and a protease inhibitor mixture (phenylmethylsulfonyl fluoride, aprotinin and sodium orthovanadate). The total cell lysate (50 μg of protein) was separated by SDS–polyacrylamide gel electrophoresis (PAGE) (12 % running, 4 % stacking) and analyzed by using the designated antibodies and the Western-Light chemiluminescent detection system (Bio-Rad, Hercules, CA), as previously described [31].

Statistical analyses

Data were reported as the mean ± standard deviation (SD) of three independent experiments and were analyzed by one-way analysis of variance (ANOVA). The data were analyzed using the SAS software statistical package “SigmaPlot,” version 9.0 (SAS Institute Inc., Cary, NC, USA) [33].