Kuo et al. J Transl Med (2016) 14:78 DOI 10.1186/s12967-016-0831-y

RESEARCH

Hericium erinaceus mycelium and its isolated erinacine A protection from MPTP-induced neurotoxicity through the ER stress, triggering an apoptosis cascadeHsing‑Chun Kuo1,2, Chien‑Chang Lu3, Chien‑Heng Shen4, Shui‑Yi Tung4,5, Meng Chiao Hsieh6, Ko‑Chao Lee3, Li‑Ya Lee7, Chin‑Chu Chen7, Chih‑Chuan Teng1, Wen‑Shih Huang5,6, Te‑Chuan Chen8 and Kam‑Fai Lee9*

Abstract

Background: Hericium erinaceus is an edible mushroom; its various pharmacological effects which have been investi‑gated. This study aimed to demonstrate whether efficacy of oral administration of H. erinaceus mycelium (HEM) and its isolated diterpenoid derivative, erinacine A, can act as an anti‑neuroinflammatory agent to bring about neuroprotec‑tion using an MPTP (1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahydropyridine) mouse model of Parkinson’s disease, which results in motor disturbances, in addition to elucidating the mechanisms involved.

Methods: Mice were treated with and without HEM or erinacine A, after MPTP injection for brain injuries by the degeneration of dopaminergic nigrostriatal neurons. The efficacy of oral administration of HEM improved MPTP‑induced loss of tyrosine hydroxylase positive neurons and brain impairment in the substantia nigra pars compacta as measured by brain histological examination.

Results: Treatment with HEM reduced MPTP‑induced dopaminergic cell loss, apoptotic cell death induced by oxidative stress, as well as the level of glutathione, nitrotyrosine and 4‑hydroxy‑2‑nonenal (4‑HNE). Furthermore, HEM reversed MPTP‑associated motor deficits, as revealed by the analysis of rotarod assessment. Our results demonstrated that erinacine A decreases the impairment of MPP‑induced neuronal cell cytotoxicity and apoptosis, which were accompanied by ER stress‑sustained activation of the IRE1α/TRAF2, JNK1/2 and p38 MAPK pathways, the expression of C/EBP homologous protein (CHOP), IKB‑β and NF‑κB, as well as Fas and Bax.

Conclusion: These physiological and brain histological changes provide HEM neuron‑protective insights into the progression of Parkinson’s disease, and this protective effect seems to exist both in vivo and in vitro.

Keywords: Hericium erinaceus mycelium, Erinacine A, Parkinson’s disease, Endoplasmic reticulum stress

© 2016 Kuo et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

BackgroundHericium erinaceus (Lion’s mane or Yamabushitake) is an edible mushroom with medicinal properties; it grows on old or dead broadleaf trees. It is used as a food and herbal medicine in Japan and China without harmful effects [1]. The mushroom may be a good candidate for inducing

neuronal differentiation and promoting neuronal survival [2]. Both the mycelium (erinacines A-I) and the fruiting bodies (Hericenone C-H) are the source of many bioac-tive extracts with drug efficacy. Hericium erinaceus has been extensively documented and possesses a range of therapeutic properties, such as antioxidant activity [3], hypolipidemic activity [4], hemagglutinating activity [5], antimicrobial activity [6], antiaging activity [7], immune modulation and anticancer activities [8, 9]. Erinacine A has small molecular weight components that are the major active agents isolated from the cultured mycelium

Open Access

Journal of Translational Medicine

*Correspondence: lkf2758@gmail.com 9 Department of Pathology, Chang Gung Memorial Hospital, Chiayi, TaiwanFull list of author information is available at the end of the article

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of H. erinaceus. These diterpenoid compounds also play a role in varied functions, including neuroprotec-tion through nerve growth factor (NGF) synthesis [10]. Therefore, H. erinaceus is attracting attention as a novel resource, not only for medicinal drugs, but also for die-tary phytochemicals for disease prevention and health promotion through use of its biological properties [11]. Our previous study focused on exploring the biological agent of erinacine A from H. erinaceus mycelium and its structural elucidation by ethanol extraction and HPLC analysis techniques [12, 13]. However, the mechanism by which H. erinaceus mycelium and its isolated diterpenoid derivative, erinacine A, promote neuron cell survival and protection from MPTP-induced neurotoxicity remains poorly understood, as does the mechanism by which H. erinaceus mycelium and erinacine A initiate neuropro-tection against MPTP injury to the brain.

Parkinson’s disease (PD) involves a distinct sequence of events behind the selective neuronal death that occurs in PD, but these events are not fully understood [14–16]. Numerous diseases of the nervous system, such as Parkinson’s disease (PD) produce excessive free radi-cal generation (reactive oxygen species [ROS] and reac-tive nitrogen species [RNS]), which then cause oxidative damage. These include lipids, oxidative S-nitrosylation proteins and nucleic acids, which have been linked to apoptosis by the high levels of ROS in dopamine neurons due to dopamine metabolism. Various disease models for PD also show the involvement of the drug 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) [17, 18]. Furthermore, the MPTP animal model is useful for the study of neurodegeneration in PD. The neurotoxic effects of MPTP are thought to be mediated by its metabolite 1-methyl-4-phenylpyridinium ion (MPP+) and mono-amine oxidase-B (MAO-B) in neuron cells, leading to a number of deleterious effects on cellular function, such as impairing the dopaminergic nigrostriatal neurons, generating free radicals from the mitochondria and a neuroinflammatory response, similar to those seen in PD [19, 20]. Our previous investigation focused on explor-ing the biological agent of erinacine A from H. erinaceus mycelium, its structural elucidation by ethanol extrac-tion and HPLC analysis techniques [12, 13]. However, the mechanism by which H. erinaceus mycelium and its isolated diterpenoid derivative, erinacine A, are able to effectively improve the neuroprotective effects of the endoplasmic reticulum (ER) stress pathway and apopto-sis, as well as how the signal cascades become activated, remain poorly understood.

Numerous studies have demonstrated that the ER stress pathway might be crucial in various CNS degen-erative diseases [21]. In fact, ER stress may be related to neuronal death. In particular, the JNK/p38 MAPK/CHOP

pathways involved in ER-stress-induced apoptosis in the neurons are implicated in PD [22]. In addition, energy metabolism with cultured neuronal cells, including dopaminergic neurons, showed that MPP +  triggers ER stress and induces a number of genes [23]. Thus, extreme oxidant and peroxide levels from the neurotoxicity of MPTP suggest that inhibition of antioxidant defenses results in inflammatory effects and generation of ROS or RNS found in PD-related neuron damage [24, 25]. MPTP injury of the brain then induces oxidative stress, which leads to activate the multiple-cellular-signaling pathway, such as the IRE1α pathway. IRE1α binds TNF receptor-associated factor 2 (TRAF2), apoptosis signal-regulating kinase 1 (ASK1) and downstream kinases that further activate Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB), which has also been linked to PD [26, 27]. In the present study, we explore the biological agent of H. erinaceus mycelium that is associated with protec-tion against ER stress and loss of dopaminergic nigrostri-atal neurons.

In our previous study, we investigated the molecular mechanisms underlying H. erinaceus that inhibit global cerebral ischemic injury via inactivation of the iNOS/RNS and p38 MAPK/CHOP pathways, which may be among the possible pathways involved in stroke-related neuron injury [12, 13]. In the present study, we assess the neuroprotective effect of H. erinaceus mycelium and its isolated compound erinacine A, as well as its rele-vance to idiopathic PD in the MPTP mouse model. We were able to demonstrate that H. erinaceus mycelium, a known antioxidant, is able to protect against the endo-plasmic reticulum stress induced by the loss of dopamin-ergic neurons and disordered motor function by MPTP injury. This results in its isolated compound erinacine A promoting neuronal cell survival due to MPP+ -medi-ated induction expression of Fas and Bax via IRE1α/TRAF2 complex formation and phosphorylation of the JNK1/2, p38 and NF-kB pathways. Moreover, developing more effective dietary H. erinaceus mycelium for PD is an important goal.

MethodsHericium erinaceus extracts and analysis of erinacine AFresh mycelium of H. erinaceus was extracted with etha-nol. 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 elu-ent. 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 exe-cuted according to the previous study with minor modi-fications. The analytical column used was a COSMOSIL

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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 fol-lowing 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).

AnimalsC57BL/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 Institu-tional Animal Care and Use Committee of Chang Gung University of Science and Technology. Mice were oper-ated 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 ran-domly 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 (H2O) 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 antibodiesMouse monoclonal antibodies against tyrosine phos-pho-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 antibod-ies 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 deoxy-cholate, protease inhibitor cocktail was purchased from Sigma (St. Louis, MO, USA).

Behavioral testingBehavioral 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 rotat-ing rod and the time until they fell off was recorded. This

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

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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.

ImmunohistochemistryAfter 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 imme-diately fixed in 10  % formalin overnight. The brain sec-tions were then dehydrated with graded ethanol, passed through chloroform, and embedded in paraffin, which were assessed by hematoxylin and eosin (H and E) stain-ing. 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 neg-ative control. Using the slides, the presence of cytoplasm stained with brown was scored as positive. The protein expression were quantitatively evaluated using an Olym-pus 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 cultureThe 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 assayCell 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 spectrophoto-metrically 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 soft-ware. 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 analysisCells were lysed with a buffer containing 1  % NP-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS) and a protease inhibitor mixture (phenylmethylsul-fonyl fluoride, aprotinin and sodium orthovanadate). The total cell lysate (50 μg of protein) was separated by SDS–polyacrylamide gel electrophoresis (PAGE) (12  % run-ning, 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 analysesData were reported as the mean  ±  standard devia-tion (SD) of three independent experiments and were analyzed by one-way analysis of variance (ANOVA). The data were analyzed using the SAS software statisti-cal package “SigmaPlot,” version 9.0 (SAS Institute Inc., Cary, NC, USA) [33].

ResultsHericium erinaceus mycelium (HEM) inhibits the cytotoxic effect of neuron cells in the MPTP‑treatment animal modelOur previous study demonstrated that H. erinaceus mycelium and its structural analog erinacine A have nerve-growth properties, allowing them to aid in the pre-vention of ischemic injury to neurons in the central nerv-ous system of excessive oxidative stress subjects [12, 13]. Based on these studies, we assayed whether H. erinaceus mycelium provides substantial therapeutic advantages by suppressing brain impairment in a mouse model that resembles PD. Figure  1 shows the neuroprotection of HEM in an MPTP-treated animal model. Figure 2 shows the expressions of the results of TUNEL as well as tyros-ine hydroxylase (dopaminergic neurons). There was also a reduction in the number of positive TUNEL cells in the striatum and substantia nigra, a marker of apoptosis,

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when the MPTP mice treated with HEM were compared with the untreated MPTP mice (MPTP = 38 ± 5; 10.76 and 21.52  mg/day, HEM =  24 ±  3 and 7 ±  2, *  <0.05). Tyrosine hydroxylase (TH) is expressed exclusively in most dopaminergic neuronal cell types. TH is the enzyme responsible for the conversion of the amino acid l-tyros-ine to dopamine, while PD can be indicated as a TH-deficiency syndrome of the striatum. Immunoreactivity of phospho-Tyrosine hydroxylase was found to reside mostly in the brain striatum area in the MPTP group 24  h after administering HEM. Quantification of the dopaminergic neuron cells in terms of pathology showed that the dosage of MPTP administration was inclined to decrease the number of normal neurons compared to the control group. However, a significant increase was found between the MPTP and MPTP  +  HEM (10.76 and 21.52  mg/day) group (untreated MPTP =  32 ±  5; HEM treated MPTP = 72 ± 6 # <0.05 and 116 ± 13) (see Fig. 2c).

HEM treatment‑attenuated brain protein ROS oxidants and behavioral impairment in mice with MPTP intoxicationIn a previous study, an animal model of Parkinson’s disease showed the result of the presence of excessive free radicals after brain tissue damage; these have been reported to damage dopamine neurons due to dopamine formation for PD [23]. These findings demonstrated that HEM was able to inhibit oxidative nitrotyrosine proteins and lipid peroxidation 4-HNE, resulting in the dopa-minergic nigrostriatal neurons’ apoptosis. This suggests that the ER stress effectors of CHOP via p38 MAPK may contribute to the neuroprotective effects by MPTP administration (Fig. 3, p < 0.05). Figure 4 shows the neu-roprotective activities of HEM in an MPTP-treated ani-mal model. Motor function of MPTP injection of HEM was also determined by a rotary rod apparatus, in com-parison to an MPTP group. Five days after MPTP treat-ment, the mice showed significant motor function, as indicated by a decrease in the time periods on a rotary rod at 6  days, as compared to the control group MPTP mice (p  <  0.05, n  =  6). Significant differences were found between the HEM-treated and MPTP groups. The HEM group showed reduced motor dysfunction

in a dose-dependent manner compared to the MPTP group (p  <  0.01, n =  6; Fig.  3). Based on these studies, we tested whether HEM administration provided a pro-tection of oxidative stress on densitometric analysis of neurotrophic factors in the brain striatum. As shown in Table 1, infusion of MPTP had a pro-inflammatory effect on brain injury, as shown through enzymatic analyses of the MPTP treatment group compared to a healthy con-trol group. Dopamine, NGF and glutathione (GSH) levels were significantly lowered in the MPTP treatment group (MPTP infusion vs. normal saline infusion, p  <  0.05, n = 6). Neuron GSH levels have recently been established as an indicator of oxidative stress in various diseases. The administration of the HEM groups significantly (HEM treatment vs. MPTP infusion, *  <0.01, n =  6) elevated dopamine, NGF and the GSH level in the MPTP group.

Inhibition effects of erinacine A treatment on MPP + ‑induced ROS‑related apoptosis and the ER stress‑signaling mechanismsOur previous findings demonstrated the effects of the biological agent of erinacine A from HEM fractiona-tion and its effect on ischemia reperfusion injury. This suggests that the inactivation of the iNOS/RNS and p38 MAPK/CHOP pathways may contribute to the neu-roprotective effects for both cortical and subcortical infarctions. MPTP exerts its neurotoxic effect through biological activation by MPP+, leading to mitochon-drial energy loss, generation of toxic ROS and neu-ron cell death [34]. To clarify whether erinacine A has an inhibitory effect on the MPP+ model of neuronal injury that is cytotoxic to N2a cells and the molecular mechanisms underlying the MPP+ -mediated activa-tion of IRE1a-inducing Jun N-terminal kinase (JNK1/2) and p38 MAP Kinase (MAPK), we induced N2a cells to MPP+ for 24 h, performed MTT assays and examined the protein levels of the ER stress-signaling pathway. Significantly decreased cell death was found in the eri-nacine A group compared to the MPP+ group (Fig. 5a). As shown in Fig.  5b, the extent of apoptosis of MPP+ induction was quantified as a percentage of annexin V-positive cells and shown as 20  %. Erinacine A treat-ment of N2a cells also resulted in increased neuron

(See figure on previous page.) Fig. 2 Effect of Hericium erinaceus mycelium (HEM) on brain histological examination in MPTP treated animal model. MPTP animal models (MPTP injection and brains harvested) were performed on mice described in “Methods” section. a Histological examination of brain were revealed to stria‑tum and substantia nigra zones as indicated by immunohistochemical staining of TUNEL. Representative brain sections stained as saline infusion control group (I); Mice with MPTP injection (II); HEM treatment (10.76 and 21.52 mg/day) (III, IV); Apoptotic cells were measured under microscopy as described in “Methods” section. The MPTP group exhibited dopamine deficiency syndrome of the striatum, as indicated by apoptotic cells (filled arrow). Magnification, ×100. b Histological examination of brain were revealed in brain, as indicated by immunohistochemical staining of tyrosine Hydroxylase p‑Ser 31. Magnification, ×200. The cells were counted from 10 fields (200× magification) of each brain sample. c The results from statistical analysis are the means of cells and were calculated per microscope field from six animals per group. Data are expressed as mean ± SD of independent experiments. *p < 0.05, MPTP group versus MPTP + HEM group. #P < 0.05, MPTP group versus MPTP + HEM group

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survival by 6 and 3  %, respectively. Erinacine A might have neuroprotection effects on neuron injury caused by oxidative stress of MPP+.

Erinacine A prevented cell death and was involved in the modulation of IRE1α/ΤRΑF2 complex levels, as well as ER‑associated protein expression by MPP+ in neuron cellsOur results clearly showed that MPP+ resulted in neuron cell death and that oxidative damage was relative to the expression of Fas, Bax and CHOP, as well as to activation of the ER stress-signaling pathway, such as the p-JNK1/2, p38 MAPK and NF-κB triggering pathways, which may function downstream of the ER stress to induce apopto-sis in response to neuronal damage, as an index of exces-sive oxidative damage with Western blotting (Fig.  6a). Conversely, treatment with erinacine A also resulted in a significant inhibition of JNK1/2 and p38 phosphorylation and ER-associated protein expression for 24  h. In addi-tion, erinacine A was also found to induce effects on the IRE1α/ΤRΑF2 association (Fig. 6). In order to verify the effects of oral administration of HEM and erinacine A on the relationships between neuroprotection, inflamma-tion and ER stress in the brain, Fas protein was selected for further examination using immunohistochemistry assays in  vivo after MPTP injury and HEM treatment. As shown in Fig.  7, mice with MPTP impairment dem-onstrated increased expression of Fas (CD95) in the brain tissue samples. In particular, HEM and relative erinacine A treatment resulted in a significant reduction of Fas expression. These results are consistent with the results in vitro, indicating that HEM is applicable when protecting MPTP-induced brain neuron injury. Taken together, the results and the data showed that H. erina-ceus mycelium and relative erinacine A administration in neuron cells is essential to involvement in oxidative

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Fig. 4 Effect of Hericium erinaceus mycelium (HEM) treatment on motor function by the rocking rotarod assessment of coordina‑tion. Mean duration (in seconds) of the time spent on the rocking rotarod as a function of increasing motor function for MPTP and HEM administration mice. At 5 days after the MPTP injection, the mice exhibited a significant motor dysfunction as indicated by a decrease in the time period on rotarod. The mice with MPTP treated with HEM(III) and assessed by rotarod apparatus. Data were expressed as mean ± SD (n = 6/group). *p < 0.05, 4 days, control group versus MPTP and MPTP + HEM groups. #P < 0.05, 5 days, MPTP group versus MPTP + HEM groups. & P < 0.05, 5 days, MPTP group versus MPTP + HEM groups

Table 1 Biochemical effects of MPTP administration animal model by Hericium erinaceus mycelium

* P < 0.05, when compared with MPTP group

Neurotoxic effects of the striatum

Control (n = 6) MPTP (n = 6) MPTP + HEM5.38 mg/day (n = 6)

MPTP + HEM10.76 mg/day (n = 6)

MPTP + HEM21.52 mg/day (n = 6)

Dopamine (ng/ml) 3.6 ± 0.15 1.7 ± 0.1 3.1 ± 0.1* 3.8 ± 0.1* 4.1 ± 0.1*

NGF (ng/ml) 2.8 ± 0.5 1.2 ± 0.1 1.9 ± 0.3* 2.3 ± 0.4* 2.7 ± 0.3*

GSH/mg 140 ± 11 62 ± 6 94 ± 8 134 ± 10 138 ± 8*

(See figure on previous page.) Fig. 3 Effect of Hericium erinaceus mycelium (HEM) on brain histological nitrotyrosine and 4‑HNE protein expression in MPTP animal model. Histological examination of brain were revealed to somata of dopamine neurons by immunohistochemical staining. a Mice were sacrificed and the brains were separated. Representative brain sections stained control group (I); Mice treated with MPTP at 6 days (I); HEM administration [10.76 (III) and 21.52 (IV) mg/day] staining of nitrotyrosine (a) and 4‑HNE (b) protein in brain tissue. c Evaluation of nuclear protein expression and nitrotyros‑ine and 4‑HNE expression were quantitative in the striatum. The area of dopamine neurons positive stained area from the SN was evaluated from three randomly selected observation fields of each brain section. Quantitative of immunohistochemical proteins by average integrated optical density (AIOD). Positive stained area was evaluated from three randomly selected observation fields of each liver section. Data were expressed as mean ± SD (n = 6/group). *p < 0.05, nitrotyrosine expression, MPTP group versus MPTP + HEM group. #P < 0.05, 4‑HNE expression, MPTP group versus MPTP + HEM group

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Fig. 5 Effect of MPTP on assessment of cell viability in N2a cells for 24 h. a after 24 h their viability was measured using an MTT assay. b After an indicated treatment for 24 h, the cells were stained with FITC‑conjugated Annexin‑V and PI for flow cytometry analysis as described in “Methods” section. The percentages presented in each frame depicted the apoptotic cells. *p < 0.05, indicates significant difference compared to an untreated group of N2a

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stress-signaling MPP+ -mediated cell death along both IRE1α and JNK1/2/NF-κB-related pathways.

DiscussionER stress-induced apoptosis is implicated in various pathological conditions, but the mechanisms of H. eri-naceus mycelium on the suppression of oxidative stress

and JNK/p38 MAPK as well as Fas, Bax and CHOP pro-tein expression, which confer neuroprotection in PD involving ER stress–mediated signaling to neuron apop-totic pathways, remain unclear. It has been previously reported that a number of H. erinaceus extracts, used as a medicinal mushroom, can improve sleep quality, ameliorate depression and neurodegenerative disease, and improve cognitive impairment [35]. Moreover, it was reported that polysaccharides purified from the liq-uid culture broth of HEM and erinacine A enhanced the growth of mice adrenal nerve cells, improved the exten-sion of neurite extension and expanded catecholamine in the brain of mice. Recently, studies were conducted on the synergistic effects of HEM extracts and exog-enous NGF on neurite outgrowth in a glioma cell line. These studies demonstrate that the HEM extracts contain certain neuroactive components that induce NGF syn-thesis and promote neurite outgrowth, which has been shown to be a mycelium- growth-associated metabolite that stimulates NGF synthesis in cultured astrocytes [7]. Using an in  vivo MPTP animal model and mouse neu-ron cell culture MPP+ -induced apoptosis (Fig.  1), our in vivo data demonstrated that the dopaminergic lesions and oxidative stress in the striatum and substantia nigra, as well as motor disorder, were significantly decreased after treatment with HEM (Figs. 2, 3, 4). In addition, we deployed several neuron cell death assays to confirm that the MPP+ damage, as measured by brain neuron death, is significantly decreased with erinacine A treatment at the concentration of 5 μM for 24 h in N2a cells (Fig. 5).

Studies have shown that ER stress- and inflammation-induced apoptosis is a key pathogenic event in disease processes as divergent as metabolism disease, cancer, hepatitis, heart disease and neurological diseases [5, 8, 22, 36]. The overall objective of this study made it neces-sary to develop more effective drugs from natural com-pounds of edible mushrooms to prevent neuronal death at high MPP+ exposures related to the mechanism of ER stress-induced apoptosis. The specific goal was link-ing upstream ER stress-mediated events to downstream apoptosis execution pathways in a model involving the JNK1/2, p38, NF-kB and CHOP pathways. Of current interest is the concept that prolonged CHOP expression leads to the release of ER calcium stores resulting from ER stress-induced expression of the Fas death receptor and Bax mitochondrial pathways of apoptosis through a pathway involving JNK and p38. Figure  6 shows that treatment of N2a cells with erinacine A increased the level of phosphorylated JNK and p38 MAPK as a result of an upregulation of CHOP and NFκB at 24  h, as well as a decrease in the Fas/Bax expression and IRE1α/TRAF2 complex interaction as early as 3  h. Our results showed that erinacine A inhibited MPP+ -induced

p-p38 21 kDa

N

H246 3 246 3 246MPP MPP+EA

p-JNK 1/2 48 kDa

β-actin42 kDa

NF-kB p65 65 kDa

IP:TRAF2WB: IRE1α120 kDa

Fas33 kDaBax19 kDa

CHOP 19 kDa

Histone H1

p-IKB-β65 kDa

p-p38 21 kDa

N

H246 3 246 3 246MPP MPP+EA

p-JNK 1/2 48 kDa

β-actin42 kDa

NF-kB p65 65 kDa

IP:TRAF2WB: IRE1α120 kDa

Fas33 kDaBax19 kDa

CHOP 19 kDa

Histone H1

p-IKB-β65 kDa

p-JNK1/2 CHOP IRE1αp-p38

Pro

tein

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l(F

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l)

Fas

*

**

*

*

**

*

*

*

*

*

*

*

*

**

*

0

1

2

3

4ControlMPPMPP+EA

NFkBp65

* **

*

*

*

** * * * *

Fig. 6 Erinacine A mediated and ER‑associated proteins expres‑sion between untreated (control) and treated (MPP+) in N2a cells. a Nuclear and whole cell lysate proteins were prepared and ana‑lyzed by Immunoblotting, using antisera to detect Fas, Bax, p‑JNK, p‑p38,CHOP, NF‑kB p65, p‑IKB‑b and IRE1a (b‑actin and Histone H1 served as the internal control). The association of TRAF2 with IRE1‑a was determined by immunoprecipitation with TRAF2 followed by western blot with anti‑IRE1‑a antibody. b Protein levels were quanti‑fied by densitometric analysis with the control being set at 100 %. Data presented in western blot are derived from a representative study, and comparisons of protein expression are calculated from three independent experiments. *P < 0.05, when compared with control untreated group

Page 11 of 14Kuo et al. J Transl Med (2016) 14:78

neuron damage by inactivating oxidative stress-depend-ent CHOP expression. We also showed that both the Fas and Bax pathways, through induction of the Fas receptor itself and the mitochondrial pathway by MPP+ , and then specifically erinacine A, reduced the production of the ER stress-signaling pathway in dose-relative inhibition. Moreover, our histopathological and immunohistochem-ical assays provide unique evidence suggesting that HEM

is able to suppress neuron impairment in MPTP mice at doses below the safe starting-dose range (Conversion of Animal Doses), and that this involves the significant inhibition of neuron death and Fas expression (Fig.  5). These findings provide an integrated, unique mecha-nism linking the ER stress to apoptosis, and suggest that it is feasible to determine whether H. erinaceus and relative erinacine A are able to effectively improve the

II III

IV V VI

II III

IV V VI

I

*

0

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20

30

40

50

60

70

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rage

inte

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opt

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(AIO

D, %

)

Control MPTP II HEM III HEM IV HEM V EA VI

*

**

*

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rage

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)

Control MPTP II HEM III HEM IV HEM V EA VI

*

**

Fig. 7 Effect of Hericium erinaceus mycelium (HEM) on brain histological Fas protein expression in MPTP treated animal model. Histological exami‑nation of brain were revealed to striatum and substantia nigra zones as indicated by immunohistochemical staining. Representative brain sections stained as saline infusion control group (I); Mice with MPTP injection (II); HEM treatment (5.38, 10.76 and 21.52 mg/day) (III, IV, V); Erinacine A (i.p. VI); The results from statistical analysis are the means of cells and were calculated per microscope field from six animals per group. Data are expressed as mean ± SD of independent experiments. *p < 0.05, MPTP group versus MPTP + HEM group and MPTP + EA group

Page 12 of 14Kuo et al. J Transl Med (2016) 14:78

neuroprotective effects for intervention directed against PD’s ER stress-induced apoptosis.

Studies have shown that natural phytochemicals from certain plants have the capability to exert neuroprotec-tive effects in various experimental models of neuro-logical disorders, and also to have cytotoxic effects on cancer cells [13, 37]. Furthermore, reducing oxidative stress-induced free radicals via an antioxidant effect has been shown to protect against the neuronal damage caused by neurotrophin deficiencies and toxin-induced degenerative diseases in response to chemopreven-tive drugs, such as dietary phytochemicals, phenols, alkaloids, flavonoids and mushrooms [38]. Moreo-ver, finding a suitable neuroprotective agent is needed since this is a very important property with regard to its ability to cross the blood–brain barrier (BBB). To validate these findings, further study of H. erinaceus is needed to determine whether there are mediated actions to reach the target sites of the CNS in mice. However, phytochemicals that regulate neurodegenera-tive disease by targeting neurotrophins might provide a promising future. Both NGF and BDNF are expressed in dopaminergic neurons, suggesting that these act as a survival factor for the trophic support of neurons. Phy-tochemicals that potentiate neurotrophins and activate Trk receptors may serve to prohibit the onset of neu-rodegenerative PD [39]. On the other hand, it has been shown that JNK and p38 MAPK stress protein kinases are involved in inflammation and oxidative stress in neurodegenerative neuronal death in PD [36]. Our results showed that the upregulation of NGF protein and dopamine affected the striatum neuron cell with an increase of the antioxidative indicator GSH; these resulted in inhibitors targeting JNK and p38MAPK, preceded by the administration of H. erinaceus myce-lium (Table 1; Fig. 6).

ConclusionIn this study, we evaluated the molecular mechanisms underlying the roles of H. erinaceus against MPTP-induced neurotoxicity. Consequently, based on MPTP-treated mice, our results imply the possibility of a toxic effect of MPTP, a food provided by the mushroom com-ponents of H. erinaceus, on N2a cells and enable the evaluation of the protective ability and the underlying mechanisms against MPP+ cytotoxicity. In  vivo study demonstrated that H. erinaceus mycelium decreased the brain dopamine neuronal loss and motor function. Our study showed that erinacine A inhibited the neu-ron cell apoptosis as a result of oxidative stress signal-ing and the JNK/p38/NF-κB/CHOP/Fas/Bax pathways (Fig.  8). These results provide insights into the neu-roprotective activity of erinacine A of the Hericium

erinaceus mycelium, which thus may be promising can-didates for the treatment of neurodegenerative diseases such as PD.

Authors’ contributionsK‑FL Provision of study material, collection and assembly of data and histopathological evaluation, C‑CL Design, collection, assembly of data and manuscript writing, M‑CH Conception, collection, and assembly of data, C‑HS Provision of study material or animals, K‑CL Provision of study material or ani‑mals, C‑CT Provision of study material, collection, and assembly of data, K‑CL Administrative support, collection, and assembly of data (flow cytometry), L‑YL, L‑YL and C‑CC Provision of study material or animals, W‑SH and H‑CK Conception and design, financial support, administrative support, manuscript writing, final approval of manuscript. All authors read and approved the final manuscript.

Author details1 Department of Nursing, Chang Gung University of Science and Technology, Chiayi, Taiwan. 2 Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan. 3 Division of Colorectal Surgery, Department of Surgery, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Tai‑wan. 4 Department of Hepato‑Gastroenterology, Chang Gung Memorial Hos‑pital, Chiayi, Taiwan. 5 Chang Gung University College of Medicine, Taoyuan, Taiwan. 6 Division of Colon and Rectal Surgery, Department of Surgery, Chang Gung Memorial Hospital, Chiayi, Taiwan. 7 Grape King Biotechnology Inc., Zhong‑Li, Taiwan. 8 Division of Nephrology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan. 9 Department of Pathology, Chang Gung Memorial Hospital, Chiayi, Taiwan.

AcknowledgementsFunding for this study was provided in part by research Grants from the Tomorrow Medical Foundation, Taiwan. This study was supported by grants BMRPD42, CLRPG8D0112, CMRPG6E0181, CMRPF6E0011, CMRPF6E0041, CMRPF6E0021, CMRPG8C1261, CMRPG8C1262, CMRPG6C0013,

Fig. 8 Schematic presentation of the ER Stress‑signaling pathways involved in Hericium erinaceus mycelium and erinacine A suppressed MPP‑induced apoptosis in N2a cells. Hericium erinaceus mycelium has neuroprotective potential through blocking oxidative stress MPTP‑induced neuron damage and that erinacine A contribute to the protective action. The inhibition of effect of MPP on the ER stress triggering production of ROS activates IRE1a/TRAF2 complex forma‑tion and phosphorylation of JNK1/2, p38 and NF‑kB pathways, which inhibits the Fas and Bax up‑regulation and decreases an apoptosis cascade

Page 13 of 14Kuo et al. J Transl Med (2016) 14:78

CMRPG6C0302, and CMRPF6D0072 from Chang Gung Memorial Hospital‑Kaohsiung Medical Center, Chang Gung Memorial Hospital, and Chang Gung University of Science and Technology, Chia‑Yi Campus, Taiwan and by the Ministry of Science and Technology, Taiwan (MOST 103‑2622‑B‑255‑001‑CC3 and MOST 103‑2313‑B‑255‑001 and MOST 104‑2320‑B‑255 ‑003 ‑MY3).

Competing interestsThe authors declare that they have no competing interests.

Received: 5 January 2016 Accepted: 5 March 2016

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