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Research ArticlePleurotus eryngiiAmeliorates Lipopolysaccharide-InducedLung Inflammation in Mice

Junya Kawai,1 Tsugunobu Andoh,2 Kenji Ouchi,1 and Satoshi Inatomi1

Mushroom Research Laboratory, Hokuto Corporation, - Shimokomazawa, Nagano -, Japan Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of oyama,

oyama -, Japan

Correspondence should be addressed to sugunobu Andoh; andoht@pha.u-toyama.ac.jp

Received December ; Revised February ; Accepted March ; Published April

Academic Editor: Evelin iralongo

Copyright Junya Kawai et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pleurotus eryngii(P. eryngii) is consumed as a resh cultivated mushroom worldwide and demonstrated to have multiple benecialeffects. We investigated the anti-inammatory effect oP. eryngii in mice with acute lung injury (ALI). Intranasal instillation olipopolysaccharide (LPS) ( g/site/mouse) induced marked lung inammation (increase in the number o inammatory cells,protein leakage, and production o nitric oxide in bronchoalveolar lavage uid) as well as histopathological damage in the lung, h afer treatment. Mice administered heat-treatedP. eryngii(. g/kg, p.o. (HPE)) h beore LPS challenge showed decreasedpulmonary inammation and ameliorated histopathological damage. Tese results suggest thatHPE has anti-inammatory effectsagainst ALI. Tus,P. eryngiiitsel may also have anti-inammatory effects and could be a benecial ood or the prevention o ALIinduced by bacterial inection.

1. Introduction

Pleurotus eryngii is an edible mushroom native to Europe.Its natural habitat is the dead roots o the weed Eryngiumcampestre.P. eryngiiis cultivated widely, and its productionhas been increasing in Asia, including Japan []. P. eryngiiis considered to be a health ood because it is low in atand calories but rich in amino acids, vitamins, and dietary

ber. P. eryngii is also bioactive, with hypolipidemic [],antitumor [], antioxidant [], and antiallergic activities[]. In particular, in vitro studies have demonstrated thatthe antiallergic activity oP. eryngii is caused by the down-regulation o allergy-related signaling proteins (includinginammation-related proteins) by inhibition o the nuclearactor o activated cells, nuclear actor-kappa B (NF-B),and high-affinity immunoglobulin E receptor (FcRI) medi-ated signaling in antigen-stimulated mast cells. However,whether P. eryngii has clinical effectiveness remains to bedetermined.

Acute respiratory distress syndrome is a result o acuteinammation o the lung and noncardiogenic pulmonary

edema that ofen leads to multiorgan system ailure anddeath [,]. Lipopolysaccharide (LPS) is present in the outermembrane o Gram-negative bacteria. LPS can cause acuteinammation o the lung because o neutrophil recruitmentandpulmonary edema [, ]. Intranasal instillation o LPS inmice (as ananimal model o acute lunginjury(ALI)) has beenshown to result in the release o proinammatory cytokines,which cause aggregation o inammatory cells and, con-

sequently, injury to lung tissue [, ]. Tere have beenreports with regard to these mechanisms and the eatureso this model. Such reports have shown that LPS activatesalveolar macrophages directly and stimulates neutrophilsto migrate into the lung and that the proinammatorymediators released rom these inammatory cells recruitlymphocytes to the lung [,]. Te critical eature o LPS-induced ALI is the destruction o vascular integrity andthe subsequent upregulated permeability results in proteinleakage and pulmonary edema [, , ]. It has also beenreported that nitric oxide (NO) has important roles in thepathogenesis o ALI because inhibitors o NO synthaseinhibit LPS-induced damage[,].

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014, Article ID 532389, 7 pageshttp://dx.doi.org/10.1155/2014/532389

http://dx.doi.org/10.1155/2014/532389http://dx.doi.org/10.1155/2014/532389

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Evidence-Based Complementary and Alternative Medicine

F : Te resh ruiting body oPleurotus eryngii and reeze-dried powder o heat-treatedP. eryngii(HPE).

Te aim o this study was to examine the anti-inamma-tory effect oP. eryngii in vivo using an LPS-induced ALImodel in mice. We used the whole P. eryngiito illustrate the

unctional utility o this mushroom as ood.

2. Materials and Methods

.. Animals. Male BALB/c mice ( weeks o age: Japan SLC,Ltd., Shizuoka, Japan) were used. Tey were kept undercontrolled temperature (C) and humidity (%).Te room was lit rom : am to : pm and during thebehavioral test. Food and water were available ad libitum.Te study was approved by the Committee or AnimalExperiments at the University o oyama (oyama, Japan).

.. Agents. LPS (Escherichia coli :B), dexamethasone,

and pentobarbital sodium were purchased rom Sigma-Aldrich (St. Louis, MO, USA). Other agents used in thisstudy were purchased rom Wako Pure Chemical Indus-tries Ltd. (Osaka, Japan). LPS was dissolved in phosphate-buffered saline (PBS) and instilled intranasally. Dexametha-sone, which was dissolved in saline with % ethanol, wasgiven to mice in the dexamethasone group by intraperi-toneal injection h beore LPS administration. Pentobarbitalsodium was dissolved in saline containing .% propyleneglycol and .% ethanol, and given by intraperitoneal injec-tion.

.. Preparation of P. eryngii Intakes. Te ruiting body oP.

eryngii (Figure ) was obtained rom Hokuto Co. (Nagano,Japan). It was then cut into small pieces and boiled in anequal amount o distilled water or min. Heat-treated P.eryngii (HPE) was reeze-dried and powdered. HPE wasresuspended in tap water and administered orally h beoreintranasal administration o LPS.

.. LPS-Induced ALI Model in Mice. BALB/c mice werechallenged with intranasal instillation (i.n.) o LPS ( g in L PBS per mouse) to induce lung inammation. Controlmice were given PBS (i.n.) without LPS. Afer h, collectiono bronchoalveolar lavage uid (BALF) was carried outollowing the method o Chu et al. under anesthesia (sodium

pentobarbital, mg/kg, i.p.) []. Afer centriugation (25g,C, min), cell pellets were resuspended in PBS or total cellcounts using a hemacytometer. Te supernatant was used orNO analyses and protein analyses.

.. Measurement for Protein Concentration. Te protein

concentration in BALF was determined using a protein assaykit (Bio-Rad Laboratories, Hercules, CA, USA).

.. Measurement for NO Production. A metabolite o NO,

nitrite (NO2), in BALF was measured using Griess reagent(% sulanilamide, .% N-(-naphthyl)ethylenediaminedihydrochloride, .% phosphoric acid). Briey, Lo BALF and L o Griess reagent were mixed in a-well plate. Te azo dye ormed was determined with aspectrophotometer (Multiskan FC: Termo Fisher ScienticK.K., Yokohama, Japan) at nm using sodium nitrite asthe standard.

.. Hematoxylin and Eosin (H&E) Staining. Anesthesia(sodium pentobarbital, mg/kg, i.p.) was induced in mice h afer LPS treatment. Mice were then decapitated. Lungswere removed and, afer washing with PBS, placed in %ormalin solution. Preparation o paraffin-embedded sec-tions and staining with H&E were undertaken using standardprocedures. Staining was observed under a light microscope(AX: Olympus, Osaka,Japan) with a charge-coupleddevicecamera (Axio Cam; Carl Zeiss, Jena, Germany).

.. Immunohistochemical Staining. Anesthesia (sodiumpentobarbital, mg/kg, i.p.) was induced in mice hafer LPS treatment. Mice were then decapitated. Lungs

were removed and, afer washing with PBS, placed in% ormalin solution. Preparation o paraffin-embeddedsections and deparaffinization were undertaken usingstandard procedures. Deparaffinized sections were treatedwith methanol containing .% hydrogen peroxide and thenwith .% riton X- in PBS. Afer treatment with .%etal bovine serum in PBS to block immunoglobulin binding,sections were incubated with rabbit anti-myeloperoxidase(MPO) antibody (DAKO, Glostrup, Denmark) or ratanti-Mac- antibody (Cedarlane, Ontario, Canada) at Covernight, ollowed by horseradish peroxidase-conjugatedanti-rabbit IgG antibody (DAKO) or horseradish peroxidase-conjugated anti-rat IgG antibody (DAKO). Color was

developed using DAKO liquid with a ,-diaminobenzidinetetrahydrochloride (DAB) substrate chromogen system(DAKO) and counterstained with hematoxylin. Tesestained sections were observed under a light microscope(AX-; Olympus) coupled to a CCD camera (Axio Cam;Carl Zeiss).

.. Statistical Analyses. Data are presented as the mean standard error o the mean (SEM). Statistical signicancebetween groups was assessed using one-way analysis o

variance andpost hoc Holm-Sidak multiple comparisons.