Neuroprotective Effects of Erinacine A on an Experimental Model of Traumatic Optic Neuropathy.

Erinacine A (EA), a natural neuroprotectant, is isolated from a Chinese herbal medicine, Hericium erinaceus. The aim of this study was to investigate the neuroprotective effects of EA in a rat model of traumatic optic neuropathy. The optic nerves (ONs) of adult male Wistar rats were crushed using a standardized method and divided into three experimental groups: phosphate-buffered saline (PBS control)-treated group, standard EA dose-treated group (2.64 mg/kg in 0.5 mL of PBS), and double EA dose-treated group (5.28 mg/kg in 0.5 mL of PBS). After ON crush, each group was fed orally every day for 14 days before being euthanized. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined using flash visual-evoked potentials (fVEP) analysis, retrograde Fluoro-Gold labelling, and TdT-dUTP nick end-labelling (TUNEL) assay, respectively. Macrophage infiltration of ON was detected by immunostaining (immunohistochemistry) for ED1. The protein levels of phosphor-receptor-interacting serine/threonine-protein kinase1 (pRIP1), caspase 8 (Cas8), cleaved caspase 3 (cCas3), tumour necrosis factor (TNF)-α, tumour necrosis factor receptor1 (TNFR1), interleukin (IL)-1ß, inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), and superoxide dismutase 1 (SOD1) were evaluated by Western blotting. When comparing the standard EA dose-treated group and the double EA dose-treated group with the PBS-treated group, fVEP analysis showed that the amplitudes of P1−N2 in the standard EA dose group and the double EA dose-treated group were 1.8 and 2.4-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The density of RGC in the standard EA dose-treated group and the double EA dose-treated group were 2.3 and 3.7-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The TUNEL assay showed that the standard EA dose-treated group and the double EA dose-treated group had significantly reduced numbers of apoptotic RGC by 10.0 and 15.6-fold, respectively, compared with the PBS-treated group (p < 0.05). The numbers of macrophages on ON were reduced by 1.8 and 2.2-fold in the standard EA dose-treated group and the double EA dose-treated group, respectively (p < 0.01). On the retinal samples, the levels of pRIP, Cas8, cCas3, TNF-α, TNFR1, IL-1ß, and iNOS were decreased, whereas those of Nrf2, HO-1, and SOD1 were increased in both EA-treated groups compared to those in the PBS-treated group (p < 0.05). EA treatment has neuroprotective effects on an experimental model of traumatic optic neuropathy by suppressing apoptosis, neuroinflammation, and oxidative stress to protect the RGCs from death as well as preserving the visual function.

Apoptotic mechanisms of gastric cancer cells induced by isolated erinacine S through epigenetic histone H3 methylation of FasL and TRAIL.

Erinacine S, the new bioactive diterpenoid compound isolated from the ethanol extract of the mycelia of Hericium erinaceus, displays great health-promoting properties. However, the effects of erinacine S on inductive apoptosis in cancer cells such as gastric cancer and its molecular mechanisms remain unclear. Our results demonstrated that erinacine S treatment significantly induces cell apoptosis with increased ROS production in gastric cancer cells, but not in normal cells. Significantly, erinacine S also showed its inhibitory effects on tumor growth in an in vivo xenograft mouse model. Furthermore, immunohistochemical analyses revealed that erinacine S treatment significantly increases the FasL and TRAIL protein, whereas it decreases the levels of PCNA and cyclin D1 in the gastric cancer xenograft mice. Consistently, in AGS cells, erinacine S treatment not only triggers the activation of extrinsic apoptosis pathways (TRAIL, Fas-L and caspase-8, -9, -3), but it also suppresses the expression of the anti-apoptotic molecules Bcl-2 and Bcl-XL in a time-dependent manner. In addition, erinacine S also causes cell cycle G1 arrest by the inactivation of CDKs/cyclins. Moreover, our data revealed that activation of the ROS-derived and AKT/FAK/PAK1 pathways is involved in the erinacine S-mediated transcriptional activation of Fas-L and TRAIL through H3K4 trimethylation on their promoters. Together, this study sheds light on the anticancer effects of erinacine S on gastric cancer and its molecular mechanism in vitro and in vivo.

Effects of Hericium erinaceus Mycelium Extracts on the Functional Activity of Purinoceptors and Neuropathic Pain in Mice with L5 Spinal Nerve Ligation.

Neuropathic pain is a serious clinical problem that is difficult to treat. Purinoceptors (P2Rs) transduce pain perception from the peripheral to the central nervous system and play an important role in the transmission of neuropathic pain signals. We previously found that the crude extracts of Hericium erinaceus mycelium (HE-CE) inhibited P2R-mediated signaling in cells and reduced heat-induced pain in mice. The present study explored the effects of HE-CE on neuropathic pain. We used adenosine triphosphate (ATP) as a P2R agonist to generate Ca2+ signaling and neuronal damage in a cell line. We also established a neuropathic mouse model of L5 spinal nerve ligation (L5-SNL) to examine neuropathic pain and neuroinflammation. Neuropathic pain was recorded using the von Frey test. Neuroinflammation was evaluated based on immunohistofluorescence observation of glial fibrillary acidic protein (GFAP) levels in astrocytes, ionized calcium-binding adaptor molecule1 (iba1) levels in microglia, and IL-6 levels in plasma. The results show that HE-CE and erinacine-S, but not erinacine-A, totally counteracted Ca2+ signaling and cytotoxic effects upon P2R stimulation by ATP in human osteosarcoma HOS cells and human neuroblastoma SH-SY5Y cells, respectively. SNL induced a decrease in the withdrawal pressure of the ipsilateral hind paw, indicating neuropathic pain. It also raised the GFAP level in astrocytes, the iba1 level in microglia, and the IL-6 level in plasma, indicating neuroinflammation. HE-CE significantly counteracted the SNL-induced decrease in withdrawal pressure, illustrating that it could relieve neuropathic pain. It also reduced SNL-induced increases in astrocyte GFAP levels, microglial iba1 levels, and plasma IL-6 levels, suggesting that HE-CE reduces neuroinflammation. Erinacine-S relieved neuropathic pain better than HE-CE. The present study demonstrated that HE inhibits P2R and, thus, that it can relieve neuropathic pain and neuroinflammation.

Post-Treatment with Erinacine A, a Derived Diterpenoid of H. erinaceus, Attenuates Neurotoxicity in MPTP Model of Parkinson's Disease.

Hericium erinaceus, a valuable pharmaceutical and edible mushroom, contains potent bioactive compounds such as H. erinaceus mycelium (HEM) and its derived ethanol extraction of erinacine A, which have been found to regulate physiological functions in our previous study. However, HEM or erinacine A with post-treatment regimens also shows effects on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity, but its mechanisms remain unknown. By using annexin-V-fluorescein-isothiocyanate (FITC)/propidium iodide staining and a 2',7' -dichlorofluorescin diacetate (DCFDA) staining assay, the cell death, cell viability, and reactive oxygen species (ROS) of 1-methyl-4-phenylpyridinium (MMP+)-treated Neuro-2a (N2a) cells with or without erinacine A addition were measured, respectively. Furthermore, signaling molecules for regulating the p21/GADD45 cell death pathways and PAKalpha, p21 (RAC1) activated kinase 1 (PAK1) survival pathways were also detected in the cells treated with MPP+ and erinacine A by Western blots. In neurotoxic animal models of MPTP induction, the effects of HEM or erinacine A and its mechanism in vivo were determined by measuring the TH-positive cell numbers and the protein level of the substantia nigra through a brain histological examination. Our results demonstrated that post-treatment with erinacine A was capable of preventing the cytotoxicity of neuronal cells and the production of ROS in vitro and in vivo through the neuroprotective mechanism for erinacine A to rescue the neurotoxicity through the disruption of the IRE1α/TRAF2 interaction and the reduction of p21 and GADD45 expression. In addition, erinacine A treatment activated the conserved signaling pathways for neuronal survival via the phosphorylation of PAK1, AKT, LIM domain kinase 2 (LIMK2), extracellular signal-regulated kinases (ERK), and Cofilin. Similar changes in the signal molecules also were found in the substantia nigra of the MPTP, which caused TH+ neuron damage after being treated with erinacine A in the post-treatment regimens in a dose-dependent manner. Taken together, our data indicated a novel mechanism for post-treatment with erinacine A to protect from neurotoxicity through regulating neuronal survival and cell death pathways.

Thirteen-Week Oral Toxicity Evaluation of Erinacine AEnriched Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), Mycelia in Sprague-Dawley Rats.

Recently, erinacine A-enriched Hericium erinaceus (EAHE) mycelia have demonstrated therapeutic efficacy in animal models of neurodegenerative disease, including Alzheimer and Parkinson disease. Despite promising results from animal models, there have been no reports on its toxicity after long-term consumption. Hence, the present study was designed to evaluate the safety of EAHE mycelia through a 13-week subchronic rodent feeding study. Following 13 weeks of EAHE mycelia feeding at dosages of 0, 875, 1750, and 2625 mg/kg body weight in both male and female Sprague-Dawley rats, findings revealed neither any mortalities nor noticeable toxicological effects in all the rats during the investigation period. Physiological parameters including body weight and feed consumption patterns were unaffected by EAHE mycelia administration. The hematological and biochemical parameters as well as histopathological studies revealed no significant differences between the treatment and control groups. Conclusively, the obtained results suggested that EAHE mycelia could be relatively unharmful when used over an extended period, supporting its safe use in food preparation.

The Cyanthin Diterpenoid and Sesterterpene Constituents of Hericium erinaceus Mycelium Ameliorate Alzheimer's Disease-Related Pathologies in APP/PS1 Transgenic Mice.

Hericium erinaceus was used in traditional Chinese medicine for physiologically beneficial medicines. Recently, it has become a candidate in causing positive brain health-related activities. We previously reported that Hericium erinaceus mycelium ameliorates Alzheimer's disease (AD)-related pathologies. To reveal the role of the cyanthin diterpenoid and sesterterpene constituents on this effects, erinacine A and S were isolated and their effects on attenuating AD-related pathology in APPswe/PS1dE9 transgenic mice were investigated. A 30 day short-term administration of erinacine A and S were performed to explore the effect of each erinacine on AD-related pathology including amyloid ß production and degradation, plaque formation, plaque growth, glial activation and neurogenesis deterioration. Our results indicated the benefit effects of both erinacine A and S in cerebrum of APPswe/PS1dE9 mice, including: (1) attenuating cerebral plaque loading by inhibiting plaque growth; (2) diminishing the activation of glial cells; (3) raising the level of insulin degrading enzyme; and (4) promoting hippocampal neurogenesis. Moreover, erinacine A reduced the level of insoluble amyloid ß and C-terminal fragment of amyloid precursor protein which was not mediated by erinacine S. We further performed a long term administration of erinacine A and found that erinacine A recovered the impairment in the tasks including burrowing, nesting, and Morris water maze. Our data pointed out that although both erinacine A and S reduce AD pathology via reducing amyloid deposition and promoting neurogenesis, erinacine A can also inhibit amyloid ß production and is worth to be further developed for AD therapeutic use.

Genotoxicity profile of erinacine A-enriched Hericium erinaceus mycelium.

Hericium erinaceus (H. erinaceus) has a long history of usage in traditional Chinese medicine for the treatment of gastric disorders. Recently, it has become a well-established candidate in causing positive brain and nerve health-related activities by inducing nerve growth factor (NGF) from its bioactive ingredient, erinacine A. This active compound, which exists only in fermented mycelium but not in its fruiting body, increases NGF levels in astroglial cells in vitro as well as catecholamine and NGF levels in vivo. With increasing recognition of erinacine A in H. erinaceus (EAHE) mycelium improving neurodegenerative diseases, numerous products are being marketed based on these functional claims. To our knowledge, there have been no reports on the mutagenicity of EAHE prior to this paper. Hence, the present study was undertaken to determine the mutagenicity and genotoxicity effects of EAHE mycelium conducted in three standard battery of tests (reverse mutation, chromosomal aberration, and micronuclei tests) according to the latest guidelines in order to meet all international regulatory requirements and provide information on the safety of this new and promising natural remedy. Our results have indicated that EAHE mycelium did not significantly increase the number of revertant colonies in the bacterial reverse mutation test nor induce higher frequency of aberrations in the chromosome aberration test. Moreover, no statistically significant EAHE mycelium-related increase was observed in the incidence of reticulocytes per 1000 red blood cells and micronucleated reticulocytes per 1000 reticulocytes. In conclusion, the three standard battery of tests suggested that EAHE mycelium was devoid of mutagenicity and genotoxicity in the tested doses and experimental conditions.

A new drug design targeting the adenosinergic system for Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The expanded CAG repeats are translated into polyglutamine (polyQ), causing aberrant functions as well as aggregate formation of mutant Htt. Effective treatments for HD are yet to be developed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a novel dual-function compound, N(6)-(4-hydroxybenzyl)adenine riboside (designated T1-11) which activates the A(2A)R and a major adenosine transporter (ENT1). T1-11 was originally isolated from a Chinese medicinal herb. Molecular modeling analyses showed that T1-11 binds to the adenosine pockets of the A(2A)R and ENT1. Introduction of T1-11 into the striatum significantly enhanced the level of striatal adenosine as determined by a microdialysis technique, demonstrating that T1-11 inhibited adenosine uptake in vivo. A single intraperitoneal injection of T1-11 in wildtype mice, but not in A(2A)R knockout mice, increased cAMP level in the brain. Thus, T1-11 enters the brain and elevates cAMP via activation of the A(2A)R in vivo. Most importantly, addition of T1-11 (0.05 mg/ml) to the drinking water of a transgenic mouse model of HD (R6/2) ameliorated the progressive deterioration in motor coordination, reduced the formation of striatal Htt aggregates, elevated proteasome activity, and increased the level of an important neurotrophic factor (brain derived neurotrophic factor) in the brain. These results demonstrate the therapeutic potential of T1-11 for treating HD. CONCLUSIONS/SIGNIFICANCE: The dual functions of T1-11 enable T1-11 to effectively activate the adenosinergic system and subsequently delay the progression of HD. This is a novel therapeutic strategy for HD. Similar dual-function drugs aimed at a particular neurotransmitter system as proposed herein may be applicable to other neurotransmitter systems (e.g., the dopamine receptor/dopamine transporter and the serotonin receptor/serotonin transporter) and may facilitate the development of new drugs for other neurodegenerative diseases.

Neuroprotective principles from Gastrodia elata.

Serum deprivation-induced neuronal-like PC12 cell apoptosis was used as an ischemic/hypoxic model to screen neuroprotective compounds from the rhizomes of Gastrodia elata, a traditional Chinese medicine. Two active compounds, bis(4-hydroxybenzyl)sulfide (1) and N6-(4-hydroxybenzyl)adenine riboside (2), together with 15 known compounds were obtained from the active fraction. Compound 2 was further elucidated by chemical synthesis. Compounds 1 and 2 potently prevented PC12 cell apoptosis in concentration-dependent manners with EC50 values of 7.20 microM and 3.7 x 10-8 M, respectively, and IC50 values of 42.90 microM (Ki 24.10 microM) and 4.660 microM (Ki 2.620 microM), respectively, in an adenosine A2A receptor binding assay.

Dihydrophenanthrenes from Bletilla formosana.

Three new dihydrophenanthrenes, 4-methoxy-9,10-dihydrophenanthrene-1,2,7-triol (1), 1-(4-hydroxybenzyl)-4,7-dimethoxy-9,10-dihydrophenanthrene-2-ol (2), and 1,3,6-tri(4-hydroxybenzyl)-4-methoxydihydrophenanthrene-2,7-diol (3) together with seven known phenanthrene derivatives, six known flavonoids, a bibenzyl and three phenolic compounds were isolated from the whole plant of Bletilla formosana. Their structures were elucidated by spectroscopic, mainly 2D NMR spectrometry and chemical methods.