Effect of Royal Sun Medicinal Mushroom Agaricus brasiliensis KA21 (Agaricomycetes) Cultivated Outdoors on Wound Healing and Maintaining Skin Elasticity.

Royal sun medicinal mushroom Agaricus brasiliensis KA21 has various beneficial pharmacological effects; however, these effects are influenced by its cultivation conditions. A. brasiliensis KA21 is rich in ß-glucan, which promotes wound healing. This study evaluated the wound healing-promoting effects of A. brasiliensis KA21 and the influence of cultivation conditions on these effects. Upon evaluation of its effect on wound healing, the outdoor-cultivated A. brasiliensis KA21 (KAOD) promoted wound healing in mice, and the effect of KAOD was stronger than that of the indoor-cultivated one (KAID). In addition, A. brasiliensis KA21 promoted the synthesis of collagen I and III, which are involved in promoting wound healing; KAOD strongly induced collagen III production. Furthermore, KAOD suppressed the decrease in skin elasticity after acetone application in our mouse model, which was not observed for KAID. These results show that KAOD is useful as a supplement in surgery and injury healing for humans and animals, improving immunity against pathogens in wound areas and promoting wound healing. In addition, KAOD may be useful in the field of skin aesthetics for repairing skin damage and maintaining skin elasticity.

Addition of Mucoadhesive Agent to Enzymatically Polymerized Caffeic Acid-Based Nasal Vaccine Formulation Attenuates Antigen-Specific Antibody Responses in Mice.

Mucosal vaccination is a promising strategy for combating infectious diseases caused by pathogenic microbes, as it can generate antigen-specific immune responses in both systemic and mucosal compartments. In our recent study, we developed a nasal vaccine system for Streptococcus pneumoniae infections in mice using enzymatically polymerized polyphenols such as caffeic acid. However, the efficacy of this mucosal vaccine system is approximately 70%, indicating a need for improvement. To address this issue, we hypothesized that incorporating a mucoadhesive agent that enhances mucosal absorption into a polyphenol-based mucosal vaccine system would improve vaccine efficacy. Contrary to our expectations, we found that adding a mucoadhesive agent, hydrophobically modified hydroxypropylmethylcellulose, to the vaccine system reduced the stimulation of antigen-specific antibody responses in both the mucosal (more than 90% reduction; P < 0.05) and systemic compartments (more than 80% reduction; P < 0.05). Although the addition of the mucoadhesive agent may have interfered with the interaction between the mucosal epithelium and the vaccine system, the underlying mechanism remains unclear, and further research is needed to fully understand the mechanisms involved.

Polymeric Caffeic Acid Acts as an Antigen Delivery Carrier for Mucosal Vaccine Formulation by Forming a Complex with an Antigenic Protein.

The development of mucosal vaccines, which can generate antigen-specific immune responses in both the systemic and mucosal compartments, has been recognized as an effective strategy for combating infectious diseases caused by pathogenic microbes. Our recent research has focused on creating a nasal vaccine system in mice using enzymatically polymerized caffeic acid (pCA). However, we do not yet understand the molecular mechanisms by which pCA stimulates antigen-specific mucosal immune responses. In this study, we hypothesized that pCA might activate mucosal immunity at the site of administration based on our previous findings that pCA possesses immune-activating properties. However, contrary to our initial hypothesis, the intranasal administration of pCA did not enhance the expression of various genes involved in mucosal immune responses, including the enhancement of IgA responses. Therefore, we investigated whether pCA forms a complex with antigenic proteins and enhances antigen delivery to mucosal dendritic cells located in the lamina propria beneath the mucosal epithelial layer. Data from gel filtration chromatography indicated that pCA forms a complex with the antigenic protein ovalbumin (OVA). Furthermore, we examined the promotion of OVA delivery to nasal mucosal dendritic cells (mDCs) after the intranasal administration of pCA in combination with OVA and found that OVA uptake by mDCs was increased. Therefore, the data from gel filtration chromatography and flow cytometry imply that pCA enhances antigen-specific antibody production in both mucosal and systemic compartments by serving as an antigen-delivery vehicle.

Refinement and complete solution NMR analysis of the structure of a 6-branched 1,3-ß-D-glucan (OL-2) isolate from Omphalialapidescens.

OL-2 is a water-soluble ß-glucan produced by Omphalia lapidescens. This versatile glucan has potential applications in various industries, including food, cosmetics, and pharmaceuticals. In addition, OL-2 is known for its promising applications as a biomaterial and drug, owing to its reported antitumor and antiseptic properties. Although the biological activities of ß-glucans vary depending on their primary structure, holistic clarification of OL-2 via solution NMR spectroscopy to ascertain its complete and unambiguous structure has not yet been achieved. In this study, a chain of solution NMR techniques, such as correlation spectroscopy, total correlation spectroscopy (TOCSY), nuclear Overhauser effect and exchange spectroscopy, 13C-edited heteronuclear single quantum coherence (HSQC), HSQC-TOCSY, heteronuclear multiple bond correlation, and heteronuclear 2-bond correlation pulse sequences were used to unambiguously assign all 1H and 13C atoms in OL-2. Based on our investigation, OL-2 consists of a 1,3-ß-glucan backbone chain decorated with a single 6-branched ß-glucosyl side unit on every fourth residue.

Potentiation of Antitumor Activity by Antibody Drugs and Mushroom-Derived ß-Glucans in Natural Killer Cell-Mediated Tumoricidal Activities against Non-Hodgkin's B-Cell Lymphoma.

ß-glucans are polysaccharides that activate innate immunity. We herein investigated whether P-glucans promote the immunological effects of antibody drugs against malignant tumor cells using human peripheral blood mononuclear cells (PBMCs). Rituximab bound to CD20-specific lymphoma and exhibited cytotoxic activity in the presence of human mononuclear cells, but not neutrophils. The addition of Sparassis crispa (cauliflower mushroom)-derived ß-glucan (SCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) to co-cultures of PBMCs and Raji lymphoma cells further promoted antibody-dependent cell-mediated cytotoxicity (ADCC). The GM-CSF treatment increased ß-glucan receptor expression on adherent cells in PBMCs. A co-stimulation with GM-CSF and SCG of PBMCs induced an increase in the number of spreading cells and the activation of natural killer (NK) cells. The enhancement in ADCC was abolished by the removal of NK cells, indicating that SCG and GM-CSF increased ADCC against lymphoma by activating ß-glucan receptor-expressing cells in PBMCs and enhancing NK cell activity. The synergistic mechanisms of action of mushroom-derived ß-glucans and biopharmaceuticals, including recombinant cytokines and antibodies, in the treatment of malignant tumor cells provide important insights into the clinical efficacy of ß-glucans from mushrooms.

Blocking Dectin-1 prevents colorectal tumorigenesis by suppressing prostaglandin E2 production in myeloid-derived suppressor cells and enhancing IL-22 binding protein expression.

Dectin-1 (gene Clec7a), a receptor for ß-glucans, plays important roles in the host defense against fungi and immune homeostasis of the intestine. Although this molecule is also suggested to be involved in the regulation of tumorigenesis, the role in intestinal tumor development remains to be elucidated. In this study, we find that azoxymethane-dextran-sodium-sulfate-induced and ApcMin-induced intestinal tumorigenesis are suppressed in Clec7a-/- mice independently from commensal microbiota. Dectin-1 is preferentially expressed on myeloid-derived suppressor cells (MDSCs). In the Clec7a-/- mouse colon, the proportion of MDSCs and MDSC-derived prostaglandin E2 (PGE2) levels are reduced, while the expression of IL-22 binding protein (IL-22BP; gene Il22ra2) is upregulated. Dectin-1 signaling induces PGE2-synthesizing enzymes and PGE2 suppresses Il22ra2 expression in vitro and in vivo. Administration of short chain ß-glucan laminarin, an antagonist of Dectin-1, suppresses the development of mouse colorectal tumors. Furthermore, in patients with colorectal cancer (CRC), the expression of CLEC7A is also observed in MDSCs and correlated with the death rate and tumor severity. Dectin-1 signaling upregulates PGE2-synthesizing enzyme expression and PGE2 suppresses IL22RA2 expression in human CRC-infiltrating cells. These observations indicate a role of the Dectin-1-PGE2-IL-22BP axis in regulating intestinal tumorigenesis, suggesting Dectin-1 as a potential target for CRC therapy.

Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation.

In this study, we applied a luciferase-fragment complementation assay for chitin detection. When luciferase-fragment fused chitin-binding proteins were mixed with chitin, the reconstituted luciferase became active. The recombinant chitin-binding domain (CBD) and a functionally modified catalytic domain (CatD) of human chitotriosidase were employed for this method. We designed the CatD mutant as a chitin-binding protein with diminished chitinolytic activity. The non-wash assay using the CatD mutant had higher sensitivity than CBD for chitin detection and proved to be a structure-specific biosensor for chitin, including crude biomolecules (from fungi, mites, and cockroaches). The CatD mutant recognized a chitin-tetramer as the minimal binding unit and bound chitin at KD 99 nM. Furthermore, a sandwich ELISA using modified CatD showed a low limit of quantification for soluble chitin (13.6 pg/mL). Altogether, our work shows a reliable method for chitin detection using the potential capabilities of CatD.

Jerveratrum-Type Steroidal Alkaloids Inhibit ß-1,6-Glucan Biosynthesis in Fungal Cell Walls.

The limited number of available effective agents necessitates the development of new antifungals. We report that jervine, a jerveratrum-type steroidal alkaloid isolated from Veratrum californicum, has antifungal activity. Phenotypic comparisons of cell wall mutants, K1 killer toxin susceptibility testing, and quantification of cell wall components revealed that ß-1,6-glucan biosynthesis was significantly inhibited by jervine. Temperature-sensitive mutants defective in essential genes involved in ß-1,6-glucan biosynthesis, including BIG1, KEG1, KRE5, KRE9, and ROT1, were hypersensitive to jervine. In contrast, point mutations in KRE6 or its paralog SKN1 produced jervine resistance, suggesting that jervine targets Kre6 and Skn1. Jervine exhibited broad-spectrum antifungal activity and was effective against human-pathogenic fungi, including Candida parapsilosis and Candida krusei. It was also effective against phytopathogenic fungi, including Botrytis cinerea and Puccinia recondita. Jervine exerted a synergistic effect with fluconazole. Therefore, jervine, a jerveratrum-type steroidal alkaloid used in pharmaceutical products, represents a new class of antifungals active against mycoses and plant-pathogenic fungi. IMPORTANCE Non-Candida albicans Candida species (NCAC) are on the rise as a cause of mycosis. Many antifungal drugs are less effective against NCAC, limiting the available therapeutic agents. Here, we report that jervine, a jerveratrum-type steroidal alkaloid, is effective against NCAC and phytopathogenic fungi. Jervine acts on Kre6 and Skn1, which are involved in ß-1,6-glucan biosynthesis. The skeleton of jerveratrum-type steroidal alkaloids has been well studied, and more recently, their anticancer properties have been investigated. Therefore, jerveratrum-type alkaloids could potentially be applied as treatments for fungal infections and cancer.

Recombinant Human Soluble Thrombomodulin Suppresses Arteritis in a Mouse Model of Kawasaki Disease.

INTRODUCTION AND OBJECTIVE: Kawasaki disease (KD) is associated with diffuse and systemic vasculitis of unknown aetiology and primarily affects infants and children. Intravenous immunoglobulin (IVIG) treatment reduces the risk of developing coronary aneurysms, but some children have IVIG-resistant KD, which increases their risk of developing coronary artery injury. Here, we investigated the effect of recombinant human soluble thrombomodulin (rTM), which has anticoagulant, anti-inflammatory, and cytoprotective properties on the development of coronary arteritis in a mouse model of vasculitis. METHODS: An animal model of KD-like vasculitis was created by injecting mice with Candida albicans water-soluble fraction (CAWS). This model was used to investigate the mRNA expression of interleukin (IL)-10, tumour necrosis factor alpha (TNF-α), and tissue factor (TF), in addition to histopathology of heart tissues. RESULTS: rTM treatment significantly reduces cardiac vascular endothelium hypertrophy by 34 days after CAWS treatment. In addition, mRNA expression analysis revealed that rTM administration increased cardiac IL-10 expression until day 27, whereas expression of TNF-α was unaffected. Moreover, in the spleen, rTM treatment restores IL-10 and TF expression to normal levels. CONCLUSION: These findings suggest that rTM suppresses CAWS-induced vasculitis by upregulating IL-10. Therefore, rTM may be an effective treatment for KD.

Polymeric Caffeic Acid Acts as a Nasal Vaccine Formulation against Streptococcus pneumoniae Infections in Mice.

Infectious diseases are the second leading cause of death worldwide, highlighting the importance of the development of a novel and improved strategy for fighting pathogenic microbes. Streptococcus pneumoniae is a highly pathogenic bacteria that causes pneumonia with high mortality rates, especially in children and elderly individuals. To solve these issues, a mucosal vaccine system would be the best solution for the prevention and treatment of these diseases. We have recently reported that enzymatically polymerized caffeic acid (pCA) acts as a mucosal adjuvant when co-administered with antigenic proteins via the nasal route. Moreover, the sources of caffeic acid and horseradish peroxidase are ingredients found commonly in coffee beans and horseradish, respectively. In this study, we aimed to develop a pneumococcal nasal vaccine comprising pneumococcal surface protein A (PspA) and pCA as the mucosal adjuvant. Intranasal immunization with PspA and pCA induced the production of PspA-specific antibody responses in the mucosal and systemic compartments. Furthermore, the protective effects were tested in a murine model of S. pneumoniae infection. Intranasal vaccination conferred antigen-dependent protective immunity against a lethal infection of S. pneumoniae. In conclusion, pCA is useful as a serotype-independent universal nasal pneumococcal vaccine formulation.

Schizophyllum commune ß-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria.

ß-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of ß-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment.

Dectin-1-mediated suppression of RANKL-induced osteoclastogenesis by glucan from baker's yeast.

Immunoreceptors expressed on osteoclast precursor cells modify osteoclast differentiation and bone resorption activity. Dectin-1 is a lectin receptor of ß-glucan and is specifically expressed in osteoclast precursor cells. In this study, we evaluated the bioactivity of ß-glucan on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and observed that glucan from baker's yeast inhibited this process in mouse bone marrow cells and dectin-1-overexpressing RAW264.7 (d-RAW) cells. In conjunction, RANKL-induced nuclear factor of activated T cell c1 expression was suppressed, subsequently downregulating TRAP and Oc-stamp. Additionally, nuclear factor-kappa B activation and the expression of c-fos and Blimp1 were reduced in d-RAW cells. Furthermore, glucan from baker's yeast induced the degradation of Syk protein, essential factor for osteoclastogenesis. These results suggest that glucan from baker's yeast suppresses RANKL-induced osteoclastogenesis and can be applied as a new treatment strategy for bone-related diseases.

Quantification of ß-Glucan from Culinary-Medicinal Mushrooms Using Novel Artificial ß-Glucan Recognition Protein.

(1->3)-ß-D-glucans (BGs), found in culinary-medicinal mushrooms, exhibit an immunostimulatory effect; hence, it is important to measure the content of BGs contained in mushrooms. BGs content in a mushroom extract was measured using a recombinant BG-binding protein, supBGRP, and compared with the existing BG assay using BGs antibody. The specificity of supBGRP enzyme immunoassay (EIA) was evaluated using a commercially available polysaccharide reagent. The supBGRP did not react to barley glucan, dextran, mannan, pustulan, and xylan, but reacted to sonifilan, and only slightly to curdlan. Among the BGs tested, supBGRP was most reactive to lentinan. The glucans were extracted using hot water and alkaline solution from the fruit body of the following edible mushrooms: Pleurotus ostreatus, Grifola frondosa, Lentinus edodes, Hypsizygus marmoreus, Flammulina velutipes, and Auricularia polytricha. All BGs extracted from edible mushrooms were detectable; in particular, the reactivity of supBGRP toward the alkaline-extracted fraction from Lentinus edodes was higher than that toward polyclonal antibody for BGs. The results suggest that supBGRP had a specific reaction to BG. The supBGRP seems to be superior to antibodies due to easy availability as a reagent and stability as a protein molecule for measurement of BGs.

Outdoor-Cultivated Royal Sun Medicinal Mushroom Agaricus brasiliensis KA21 (Agaricomycetes) Reduces Anticancer Medicine Side Effects.

We investigated whether outdoor-cultivated Agaricus brasiliensis (KA21) could reduce the side effects caused by the anticancer medicine 5-fluorouracil (5-FU). The adverse effects of 5-FU were analyzed in mice by orally administering the drug every day for 5 days. Leukopenia, diarrhea, body weight loss, anorexia, kidney injury, gastrointestinal tract injury, and hair loss were evaluated as side effects. We determined whether these side effects were reduced by the intake of outdoor-cultivated A. brasiliensis. The side effects were reduced in mice receiving the outdoor-cultivated A. brasiliensis but not in those receiving the indoor-cultivated A. brasiliensis. These results suggest that outdoor-cultivated A. brasiliensis is beneficial in reducing the side effects of the anticancer medicine and might, therefore, be useful in improving the quality of life of patients with cancer. Furthermore, because antioxidants have been reported to contribute to the suppression of the side effects of anticancer medicines, the antioxidant activities of different mushrooms were determined. The antioxidant activity of the outdoor-cultivated A. brasiliensis was the highest among all the tested mushrooms. These findings make it clear that the cultivation conditions of A. brasiliensis are important in suppressing the side effects of anticancer medicines. Strong antioxidant activity might be one of the mechanisms through which this pharmacological activity is mediated.

Quantification of Mushroom-Derived Soluble ß-1,6-Glucan Using the Function-Modified Recombinant ß-1,6-Glucanase.

Mushroom is one of the major sources of ß-glucan used in medical applications and traditional therapies. Thus, structure analysis and quantification of ß-glucan content is crucial to evaluate medicinal mushrooms. Most studies concerning mushroom-derived ß-glucan have been focused on ß-1,3-glucans. However, recent investigations suggest that ß-1,6 glucans have important roles for immunomodulating activity. Therefore, to elucidate the fine structure of various mushroom-derived ß-glucans, we recently developed a novel ß-1,6 glucan detection system using the function-modified recombinant ß-1,6-glucanase. In this study, we performed an ELISA-like assay using modified ß-1,6-glucanase and soluble dectin-1-Fc as the probes for ß-1,6-glucan and ß-1,3-glucan, respectively. Reactivity of ELISA to crude hot water extracts of edible mushrooms (Grifola frondosa, Agaricus bisporus, Pleurotus tuoliensis, P. eryngii, P. ostreatus, Hypsizygus marmoreus, and Lentinus edodes) was compared and L. edodes showed the strongest reactivity among them. An additional 19 different products of fresh L. edodes (shiitake mushroom) commercially available in Japan were also analyzed. This revealed limited differences in amounts of ß-1,6-glucan and ß-1,3-glucan in each shiitake mushroom. Furthermore, structural analysis of some purified ß-glucans derived from medicinal mushrooms was performed, and their action for inducing tumor necrosis factor-α production from the murine bone marrow-derived dendritic cells was investigated. We found relation between reactivity to modified ß-1,6-glucanase and its cytokine inducing activity. This assay could be useful for evaluating the strains of edible or medicinal mushrooms, which may be used as alternative medicines.

The Core Fucose on an IgG Antibody is an Endogenous Ligand of Dectin-1.

The core fucose, a major modification of N-glycans, is implicated in immune regulation, such as the attenuation of the antibody-dependent cell-mediated cytotoxicity of antibody drugs and the inhibition of anti-tumor responses via the promotion of PD-1 expression on T cells. Although the core fucose regulates many biological processes, no core fucose recognition molecule has been identified in mammals. Herein, we report that Dectin-1, a known anti-ß-glucan lectin, recognizes the core fucose on IgG antibodies. A combination of biophysical experiments further suggested that Dectin-1 recognizes aromatic amino acids adjacent to the N-terminal asparagine at the glycosylation site as well as the core fucose. Thus, Dectin-1 appears to be the first lectin-like molecule involved in the heterovalent and specific recognition of characteristic N-glycans on antibodies.

Dectin-2-mediated signaling triggered by the cell wall polysaccharides of Cryptococcus neoformans.

Cryptococcus neoformans is rich in polysaccharides of the cell wall and capsule. Dectin-2 recognizes high-mannose polysaccharides and plays a central role in the immune response to fungal pathogens. Previously, we demonstrated Dectin-2 was involved in the activation of dendritic cells upon stimulation with C. neoformans, suggesting the existence of a ligand recognized by Dectin-2. In the present study, we examined the cell wall structures of C. neoformans contributing to the Dectin-2-mediated activation of immune cells. In a NFAT-GFP reporter assay of the reported cells expressing Dectin-2, the lysates, but not the whole yeast cells, of an acapsular strain of C. neoformans (Cap67) delivered Dectin-2-mediated signaling. This activity was detected in the supernatant of ß-glucanase-treated Cap67 and more strongly in the semi-purified polysaccharides of this supernatant using ConA-affinity chromatography (ConA-bound fraction), in which a large amount of saccharides, but not protein, were detected. Treatment of this supernatant with periodic acid and the addition of excessive mannose, but not glucose or galactose, strongly inhibited this activity. The ConA-bound fraction of the ß-glucanase-treated Cap67 supernatant was bound to Dectin-2-Fc fusion protein in a dose-dependent manner and strongly induced the production of interleukin-12p40 and tumour necrosis factor-α by dendritic cells; this was abrogated under the Dectin-2-deficient condition. Finally, 98 kDa mannoprotein (MP98) derived from C. neoformans showed activation of the reporter cells expressing Dectin-2. These results suggested that a ligand with mannose moieties may exist in the cell walls and play a critical role in the activation of dendritic cells during infection with C. neoformans.

Solubilized melanin suppresses macrophage function.

Melanin-producing Cryptococcus and Aspergillus are highly invasive and can suppress or escape the immune system of the host. Since non-melanin-producing strains do not affect the immune system, melanin may play a role in immune system suppression. Artificial melanin synthesized using conventional methods is insoluble, making structural and functional analysis of this chemical difficult. In this study, we describe a melanin solubilization method based on polymerization of homogentisic acid (solubilizing component) and an equivalent amount of L-DOPA in the presence of laccase. In addition, we investigated the effect of melanin on the immune system. Homogentisic acid and L-DOPA mixed melanin (HALD), the synthetic solubilized melanin, did not exert a cytotoxic effect on mouse macrophages. HALD suppressed cytokine and reactive oxygen species production by macrophages when they were stimulated by fungal components. HALD also suppressed the phagocytosis of fungal components by macrophages. These results suggest that HALD can suppress the function of macrophages without causing cytotoxicity.

1O, 20O-diacetyl kamebakaurin protects against acetaminophen-induced hepatotoxicity in mice.

The present study aimed to investigate the protective effects of kamebakaurin (KA) and 1O, 20O-diacetyl kamebakaurin (Ac2KA) on acetaminophen (APAP)-induced hepatotoxicity and compare the hepatoprotective mechanisms of the two chemicals. Seven-week-old male C57BL/6J mice were orally administered KA, Ac2KA, or an ethanol/olive oil emulsion once per day for 7-days. Twenty-four hours after the final administration, the mice were fasted and then intraperitoneally injected with 450 mg/kg APAP or saline. At 16 h after injection, the mice were euthanized and blood samples were collected for plasma analysis. Pretreatment with KA and Ac2KA significantly attenuated APAP-induced hepatic injury. The protective effect of Ac2KA was stronger than that of KA. These two chemicals attenuated oxidative stress, inflammatory cytokine production, c-jun N-terminal kinase activation, and receptor-interacting protein (RIP)-3 activation. Ac2KA also decreased APAP-induced RIP-1 activation and nuclear factor kappa B (NF-κB) p65 translocation. Moreover, Ac2KA repressed mRNA expression of Cyp1a2/2e1 in the liver. Our results showed that KA and Ac2KA exerted protective effects against APAP-induced hepatotoxicity. The responsible mechanisms may be related to the chemicals' antioxidant activity and the inhibition of c-jun N-terminal kinase activation and RIP-3 activation. The effects of Ac2KA included those of KA, as well as RIP-1 inactivation, NF-κB inhibition, and Cyp inhibition.

Protection against Gut Inflammation and Sepsis in Mice by the Autodigested Product of the Lingzhi Medicinal Mushroom, Ganoderma lingzhi (Agaricomycetes).

Ganoderma lingzhi (reishi) (GL) is a widely used medicinal mushroom in the treatment of several diseases, including metabolic syndrome and cancer. We recently performed autodigestion of GL and found enhanced release of hypotensive peptides and immunomodulating beta-1,3-glucan. In the present study, we examined the protective effects of G. lingzhi and its autodigested product (AD-GL) against gut inflammation and endogenous sepsis induced in mice by the oral administration of indomethacin (IND). Gut inflammation was assessed by measuring the lengths of the intestines and colon, and sepsis was evaluated by the survival period. G. lingzhi and AD-GL were mixed with animal feed (2.5%) that was available ad libitum during the experimental period. The murine model was established by the repeated oral administration of IND (once a day, 5 mg/kg from day 0). On day 3, the lengths of the small intestine and colon were measured, and the average lengths of the intestines were significantly shorter in the control and G. lingzhi-administered groups than in the AD-GL-administered group. This finding suggests that AD-GL protected against gut inflammation due to IND-induced ulceration and subsequent microbial translocation. Furthermore, the median numbers of survival days in the control group, the G. lingzhi group, and the AD-GL group were 5, 6, and 11, respectively. The concentrations of the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin-6, in the blood were significantly reduced in the mice administered AD-GL. In the in vitro cell culture, G. lingzhi and AD-GL fractions released a significantly higher concentration of TNF-α from the spleen, and the splenocytes of mice administered AD-GL hot water extract showed a greater potential to produce cytokines in response to pathogen-associated molecular patterns. These results strongly suggest the protection of the gut mucosa from inflammation, and therefore the prevention of sepsis, by the administration of AD-GL. Autodigestion appears to be a promising protocol that enhances the usefulness of G. lingzhi as a functional food.