Biological evaluation of Phellinus linteus-fermented broths as anti-inflammatory agents

Running title:P. linteus inhibits inflammation in macrophages

Chun-Jung Lin,1,†Hsiu-Man Lien,2,†Hsiao-Yun Chang,3,†Chao-Lu Huang,4Jau-Jin Liu,5Yun-Chieh Chang,6 Chia-Chang Chen,7and Chih-Ho Lai1,5,*

1Graduate Institute of Clinical Medical Science,ChinaMedicalUniversity,91 Hsuehshih Road, Taichung, 40402,Taiwan

2 Department of Chemistry, TunghaiUniversity, 1727, Section 4, Taiwan Boulevard,Taichung, 40704, Taiwan

3Department of Biotechnology, AsiaUniversity,500, Lioufeng Road, Taichung, 41354,Taiwan

4Department of Life Sciences, NationalChungHsingUniversity, 250 Kuokuang Road, Taichung,40227,Taiwan

5Department of Microbiology and Graduate Institute of Basic Medical Science, China Medical University, 91 Hsuehshih Road,Taichung, 40402, Taiwan

6Department of Optometry, YuanpeiUniversity, 306, Yuanpei Street, Hsinchu, 30015,Taiwan

7School of Management, FengChiaUniversity, 100, Wenhwa Road,Taichung, 40724,Taiwan

†Equal contribution to this work

*Corresponding author

Chih Ho Lai, Ph.D.

Department of Microbiology and Graduate Institute of Basic Medical Science, ChinaMedicalUniversity, Taichung, Taiwan

Tel: 886-4-22052121 ext. 7729; Fax: 886-4-22333641

E-mail:

91, Hsuehshih Road, Taichung, 40402 Taiwan
Abstract

Phellinus linteus and its constituent hispoloninduce potent anti-inflammatory activity in macrophages. Efficient production of the effective constituent and the biological function of P. linteus in the regulation of innate sensing have rarely been investigated. The aim of this study was toefficiently manufacture P. linteus-fermented brothcontaining the effective constituent, hispolon, and evaluate its immunoregulatory functionsin macrophages.Four distinctfermented broths(PL1–4) and the medium dialysate (MD) were prepared to screen suitable culture conditions for the mycelial growth of P. linteus.The P. linteus-fermented brothexhibited a dose-responsive inhibition of lipopolysaccharide(LPS)-induced nitric oxide (NO) production by murine macrophages. In addition, the P. linteus-fermentedbroths suppressed macrophage LPS-mediated nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α. Among the tested samples fromP. linteus, PL4contained vast amounts of hispolon and showedthe greatest anti-inflammatory activity in both the RAW264.7 cells and murine primary peritoneal exudate macrophages (PEMs). This study demonstrates that the purification of the effective constituent from P. linteus-fermentedbrothmay enable the production of apotent therapeutic agent for anti-inflammation in macrophages.

Keywords:Phellinus linteus; hispolon; macrophages;NF-κB;anti-inflammation

INTRODUCTION

Mushrooms have been demonstrated to be physiologically beneficial to humans because of their biological activity, whichincludes anticancer and antimicrobial and immunomodulationeffects(1).Phellinus linteus is a mushroom speciesthat belongs to the mushroom class Basidiomycetes of the Hymenochaetaceae family, which is grown mainly in Asia,particularly in China, Japan, Korea, and Taiwan(2).P. linteusis generally used for the treatment of allergy, cardiovascular disease, diabetes,and gastrointestinal diseases(2,3).Moreover, increasing researchregarding the biological functionsofP. linteushas led to its use in anticancer therapy(4-6).

The most important health benefits of P. linteusrely on its immunomodulation efficacy(3,7-9). A previous study reported that treatment with P. linteus extracts influenced the Th1 and Th2 balance in mice(10).In addition, P. linteushas been found to induce the maturation of dendritic cells (DCs) from endocytotic antigen-capturing cells to antigen-presenting cells(11). In the regulation of innate immune responses, P. linteushas been found to inhibit inflammatory mediators by suppressing redox-based NF-κB activation in lipopolysaccharide (LPS)-induced macrophages(12). These results indicate that P. linteus not only regulates innate immune activation but also modulates cell-mediated immune responses and is therefore able to enhance the immune status of the host.

Despite the previous research investigatingthe biological functions of P. linteus, the sources and quality of mushrooms for herbal medicine require further analysis. In this study, we evaluated whether the samples prepared from P. linteus-fermentedbrothswereable to regulate the innate immunity of murine macrophages. We also investigated the biological effects of P. linteus on inflammatory mediators, including nitric oxide (NO), and cytokine secretions from activated macrophages.Our study showed that P. linteus-fermentedbroth containing large amounts of hispolon may be a potent agent for suppression of inflammatory responses in macrophages.

MATERIALS AND METHODS

Antibodies and reagents

Antibodies specific to inducible nitric oxide synthase (iNOS) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LPS (Escherichia coli O55: B5) was purchased from Sigma-Aldrich (St. Louis, MO).Hispolon was purchased from Enzo biochem (New York, NY, USA). The luciferase assay kit was purchased from Promega (Madison, WI). All other reagents or chemicals were purchased from Sigma-Aldrich or Merck.

Mycelial materials, preparation, and purification

Four different fermented broths(PL1–4) were prepared to screen suitable culture condition to mycelial growth of P. linteus (Table 1). All tested samples were obtained from Yushen Biotechnology Co., Ltd. Briefly, mycelia of P. linteuswere cultured in 300 ml flasks containing 100 ml broth in distilled water at pH5.2 andwere cultured at 28°C for 10 days in a 125 rpm shaking incubator. The culture broths were separated from the mycelia by filtration andthe culture filtrates were freeze-dried and ground to powder samples for experimental use. To prepare themediumdialysate(MD), the purified PL1 culture broth was dialyzed against de-ionized water at 4°C for 72 h by using dialysis tubing with a 12000-14000molecular weight cutoff. The inner dialysate was then collected and lyophilized until experimental use.

To compare the content of hispolon of PL1–4, a previously reported approach was employed with slight modifications(13).All samples were analyzed on aWaterse2695 HPLC system (WatersSeparations Module, USA) using a column (Inertsil-ODS-2 C18, .6 x 250mm, 5μm). The mobile phase consisted of acetonitrile and 0.1% triflouroacetic acid (TFA) in water, 40:60(UV detection at 276 nm). Representative HPLC chromatograms are shown in Fig. 1.

Cell culture

Murine macrophages RAW 264.7 cells (ATCC TIB-71) wasobtained from American Type Culture Collection (ATCC, Rockville, MD), and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA). De-complement fetal bovine serum (10%; HyClone, Logan, UT) was added to the culture medium.

Preparation of murine peritoneal exudate macrophages (PEMs)

Balb/c mice of the 6-8 weeks were used to assess therole of iNOS in LPS-induced NO production by macrophages as described previously(14). Mice were maintained in the animal center of ChinaMedicalUniversity (Taichung, Taiwan). All procedures were performed according to the “Guide for the Care and Use of Laboratory Animals” (NRC, USA) and be approved by the animal experiment committee of ChinaMedicalUniversity. Murine PEMs were obtained after euthanasia by lavaging each mouse with 10ml of cold PBS 3 days after intraperitoneal injection of 2ml of 3% thioglycolate in PBS. Two hours after seeding the cells in culture plates, the non-adherent cells were removed, and the adherent cells were used for further experiments.

Cell viability assay

In order to examine the cytotoxicity of P. linteus extracts on murine macrophages or PEMs, mitochondrial respiration-dependent MTT formazan [1-(4,5-dimethylthiazol-2-yl)-3,5- diphenylformazan]was employed to determine their cytotoxicity as described previously(15). The mean OD value of the content of four wells was used for assessing the cell viability expressed as percentage of control.

Determination of nitric oxide production

Nitric oxide (NO) production was determined from the accumulation of nitrite (NO2−), a stable end product of NO metabolism, in the culture medium, by using the Griess reagent (Sigma-Aldrich) as described previously(16).

Determination of cytokine secretion

To detect TNF-α released by murine macrophages, the levels of these cytokines was measured as described previously(16). Macrophages were added with various concentrations of tested samples in cell culture medium. The supernatants were collected and stored at −80°C before analysis. The level of TNF-α in supernatants from macrophages was determined by using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN), according to the manufacturer's instruction.

Western blot analysis

The treated cells were prepared and boiled in sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) sample bufferfor 10 min. The samples were then resolvedby 10% SDS-PAGE and transferred onto polyvinylidene difluoridemembranes (Millipore, Billerica, MA, USA). The membranes were incubated with anti-iNOS antibody (Santa Cruz Biotechnology)followed byhorseradish peroxidase–conjugated secondary antibodies (Invitrogen). The proteins of interestwere visualized with ECLTM western blotting reagents (GE Healthcare, Buckinghamshire, UK) and analyzed by using Fujifilm ImageQuant LAS 4000 image scanner (GE Healthcare, Buckinghamshire, UK).

Transfection and reporter gene assay

RAW 264.7 cells were grown to 90% confluency in a 12-well plate(Nunc, Roskilde, Denmark) and transfected with NF-κB-Luc reporter plasmid using Lipofectamine 2000 (Invitrogen)(17). After 24 h, cells were incubated with or without LPSand then incubated with various P. linteus extracts. To prepare cell lysates, 100 μl of reporter lysis buffer (Promega, Madison, MA)was added to each well, and cells were scraped from dishes. An equal volume of luciferase substrate (Promega) was added to all samples, and luminescence was measured using a microplate luminometer (Biotek, Winooski, VT). Luciferase activity was normalized to the transfection efficiency as determined by co-transfection of the β-galactosidase expression vector (Promega).

Statistical analysis

The data are presented as mean ± standard deviation of triplicate experiments. The Student's t test was used to calculate statistical significance; a P value <0.05was considered significant.

RESULTS

Preparation and characterization of P.linteus-fermented broths

Hispolon, an active phenolic compound of P. linteus, has recently been demonstrated to have anti-inflammatory and analgesic effects(18).To manufacture large amounts of hispolon-containing extracts, several compositionsfor the preparation ofP. linteus-fermentedbrothswere assessed and the hispoloncontent was analyzed by HPLC (Fig. 1). The concentrations ofhispolon were 0.07, 0.001, 0.03, and 0.08 mg/g in the PL1, PL2, PL3, and PL4 samples, respectively (Table 2). However, hispolon was not present in the MD.

Inhibitory effects of P. linteusagainst LPS-induced NO in murine macrophages

We then analyzedwhether P. linteus-fermented broths inhibit LPS-induced inflammatory mediators. To this end, the levels of NOproduction and expression of iNOS in the LPS-stimulated macrophage RAW264.7 cellswere evaluated. As shown in Fig. 2A, PL1–PL4 and MD dose-dependently inhibited NO production at concentrations of 50–400 μg/ml. The positive control,hispolon (10 μg/ml), inhibited NO production by 72.1%. The effect of each P. linteus-fermented brothon the proliferation of murine macrophage (RAW264.7) cells was than assessed by MTT assay. Our data did not show any considerable cytotoxicity in theRAW264.7 cells upon incubation with the P. linteussamples for 24 h at 50–400 g/ml(Fig. 2B). Therefore, the P. linteus-fermented broths with a maximal concentration of 400 g/mlwere chosen to examine the effect on anti-inflammation in macrophages.

Ex vivomurine primary PEMs were then employed to further investigate the suppressive effects of P. linteus-fermentedbrothson LPS-induced NO production. Our data showed that LPS-induced NO production was reduced in a dose-dependent manner when the primary PEMs were treated with PL1–PL4, MD, as well as hispolon (Fig. 3A). Moreover, PL4 exhibited the greatest inhibitory effect among the tested P. linteus-fermented broths. Consistently, the cell viability of the PEMswas not influenced by treatment with the P. linteus-fermentedbroths and hispolon (Fig. 3B).These results again indicate that P. linteusinhibits LPS-induced NO production in a dose-dependent manner in both RAW 264.7 cells and murine primary PEMs.

To further explore the inhibitory effects of P. linteus-fermentedbrothon LPS-stimulatedinflammatory responses in macrophages, the inflammatory protein iNOS was examined by western blot analysis. Our data showed that treatment of cells with P. linteus-fermentedbroths did not alter iNOS expression in the absence of LPS (Fig. 4A).Treatment of cells with LPS followed by PL1, PL2, and MD, only slightly decreased the iNOS expression upon compared to LPS-stimulated along (Fig. 4B). However, the expression levels of iNOS were significantly reduced in the LPS-induced RAW264.7 cells that were treated with PL3and PL4, as well as hispolon (Fig. 4C).In particular, the PL4 samplewas themost effective inhibitor of LPS-mediated inflammation. These results indicate that P. linteus-fermentedbrothscontaininghispolon may play an important role in the suppression of iNOS expression and NO production in macrophages.

Suppressionof LPS-induced inflammatory responses in murine macrophages byP. linteus

We furtheranalyzed whether the P. linteus-fermentedbroths affectednuclear factor (NF)-κB activation.Our data showed that the PL1–PL4 and MD samples significantly suppressed the luciferase activity, compared to theresults for the LPS treatment alone (Fig. 5A). Consistently, the PL4 sample most effectively inhibited NF-κB activation. In addition, the positive control, hispolon, dramatically inhibited the luciferase activity by 61.3% at 10μg/mlcompared to LPS treatment alone. We then examined TNF-α secretion in the LPS-stimulated RAW264.7 cells in the presence of P. linteus-fermented broths or hispolon. As shown in Fig. 5B, PL2–PL4 and MDsuppressed theTNF-α production by LPS-induced RAW264.7 cells in a dose-dependent manner. These results indicate thatP. linteuscontaining vast amounts of hispolon inhibited the LPS-induced inflammatory responses through attenuation of NF-κB activity followed by suppression of NO production and TNF-α secretion in murine macrophages.

DISCUSSION

Treatment of macrophages with LPS, the NF-κB rapidly translocatedinto the nucleus and activated the transcription of target genes, such as iNOS and several pro-inflammatory cytokines(19). Additionally, the MAPK pathways (i.e. p38, ERK, and JNK) can be activated in response to LPS(20,21). Activation of NF-κB and MAPKs pathways in macrophages may play a critical role in the regulation of inflammatory mediators and stimulation of host inflammation. Excessive production of inflammatory mediators including cytokines and NO were observed in hemorrhagic shock, septic shock, and several chronic inflammatory diseases(22,23). Therefore, inhibition of LPS-induced NF-κB activation and MAPK pathways might be a useful therapeutic strategy in the treatment of circulation shock as well as infectious diseases.

Our current study demonstrated that P. linteus-fermentedbroths are potent immunosuppressive agents that inhibit LPS-stimulated macrophage activation. Here, we show that P. linteus-fermentedbroths can suppress LPS-induced inflammation in macrophages, including the production of NO, expression of iNOS, and secretion of TNF-α.In addition, LPS-activated NF-κB was decreased by treatment withthe P. linteus-fermentedbroths. NO and pro-inflammatory cytokines, which are mediators involved in inflammatory signaling pathways, are triggered by NF-κB(24),thereby suggesting that P. linteus-fermentedbroths may perform their anti-inflammatory function by attenuatingNF-κB triggeredinflammatory mediators in macrophages.

The most active P. linteus-fermentedbroth, PL4, exhibited immunomodulatory activity viasuppression of LPS-induced inflammatory responses in macrophages.Moreover, PL4 consistently demonstrated the greatest inhibition of NF-κB activation, iNOS expression, NO production,as well asTNF-α secretion. Our data show that the P. linteus-fermentedbrothPL4 contained a large amount of hispolon (Table 2), which has been reported to possessanti-inflammatory activity(18).In addition,hispolonis able to block bacteria-induced NO, cyclooxygenase-2, and prostaglandin E2 productions in macrophages(25).These lines of evidence indicate that the effects are mechanistically related. Therefore,P. linteusthat hashigh levels of hispolon might be beneficial for the development of anti-inflammatory therapeutics.

The most extensive studies of P. linteus are focused on its immunoregulation activity(10,26).Several components isolated from P. linteus have been demonstrated to contain the immunomodulatory properties. For example, proteoglycan from P. linteus fruit-bodies exerted the anti-inflammatory effect through altering the ratio between Th1 and Th2 cells, resulting in the prevention of autoimmune joint inflammation in mice(27).A similar study was reported to attenuate the LPS-induced septic shock in mice with acidic polysaccharide isolated from P. linteus fruit-bodies(28).Additionally, polysaccharides isolated from P. linteuswere found to contain the immunomodulating activity in macrophages(8).These findings indicated that not only hispolon but other components isolated from P. linteus may control the immunoregulation in macrophages and might be explained that the content of hispolon in PL1 was similar to the PL4; however, the anti-inflammation activity by PL4 was much better than PL1. The investigation of potent components that involved in PL4 against LPS-induced inflammation in macrophages is required in future study.

In this study, hispolon was the major active component thatelicitedanti-inflammation activity, but it was absent from the MD. We foundthat MD inhibited NO production in a dose-dependent manner (Fig. 2A), and suppressed NF-κB luciferase activity and TNF-α production, when compared to the treatment with LPS alone (Fig. 5). In a preliminary investigation, we determined that the MD contained abundant polysaccharides (98 mg/L; unpublished data), which have previously been reported to regulate multiple biological effects,including the acceleration of humoral and cell-mediated immunity(29,30), and anti-oxidant and anti-cancer activities (31). In addition, Phellinus linteus polysaccharides have been shown its anti-inflammatory activity via mediation of heme oxygenase-1 (HO-1)in RAW 264.7 cells(32), and to inhibit the secretion of the pro-inflammatory cytokines TNF-α and IL-12 in a mouse model (28). These findings explain that polysaccharides from MD may suppress NO production and NF-κB luciferase activity.

In summary, this study demonstrates that P. linteus-fermentedbroth exhibitsinhibitory effects against LPS-induced inflammatory mediators in macrophages. Although the precise molecular mechanisms for this effect requires furtherinvestigation, our study indicates that the inhibitory effects involve attenuated expression of iNOS, NO, and TNF-α, all of which are triggered by NF-κB. The P. linteus-fermentedbroth PL4,which contained large amounts of hispolon,possessed the greatest inhibitory activity across all the tests used in this study, thereby indicating the anti-inflammatory activity of this component. The results from this study suggest that P. linteus-fermentedbroth containing large amounts of hispolonmay be a suitable candidate for the development of a new therapeutic agent for suppression of inflammatory responses in macrophages.

ACKNOWLEDGEMENTS

This study was supported by the grants from CentralTaiwanSciencePark, National Science Council of Taiwan (101-RB01 and NSC101-2313-B-039-004-MY3).

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