Elsevier

Journal of Ethnopharmacology

Volume 114, Issue 3, 3 December 2007, Pages 307-315
Journal of Ethnopharmacology

Phellinus linteus inhibits inflammatory mediators by suppressing redox-based NF-κB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophage

https://doi.org/10.1016/j.jep.2007.08.011Get rights and content

Abstract

The mushroom Phellinus linteus has been known to exhibit potent biological activity. In contrast to the immuno-potentiating properties of Phellinus linteus, the anti-inflammatory properties of Phellinus linteus have rarely been investigated. Recently, ethanol extract and n-BuOH fractions from Phellinus linteus were deemed most effective in anti-inflammatory activity in RAW 264.7 macrophages. The regulatory mechanisms of Phellinus linteus butanol fractions (PLBF) on the pharmacological and biochemical actions of macrophages involved in inflammation have not been clearly defined yet. In the present study, we tested the role of PLBF on anti-inflammation patterns in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. To investigate the mechanism by which PLBF inhibits NO and PGE2 production as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, we examined the activation of IκB and MAPKs in LPS-activated macrophages. PLBF clearly inhibited nuclear translocation of NF-κB p65 subunits, which correlated with PLBF's inhibitory effects on IκBα phosphorylation and degradation. PLBF also suppressed the activation of mitogen-activated protein (MAP) kinases including p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Furthermore, macrophages stimulated with LPS generated ROS via activation of membrane-bound NADPH oxidase, and ROS played an important role in the activation of nuclear factor-κB (NF-κB) and MAPKs. We demonstrated that PLBF directly blocked intracellular accumulation of reactive oxygen species in RAW 264.7 cells stimulated with LPS much as the NADPH oxidase inhibitors, diphenylene iodonium, and antioxidant pyrrolidine dithiocarbamate did. The suppression of NADPH oxidase also inhibited NO production and iNOS protein expression. Cumulatively, these results suggest that PLBF inhibits the production of NO and PGE2 through the down-regulation of iNOS and COX-2 gene expression via ROS-based NF-κB and MAPKs activation. Thus, PLBF may provide a potential therapeutic approach for inflammation-associated disorders.

Introduction

Phellinus linteus is a member of Hymenochaetaceae, which has been used for the treatment of gastric cancer, insulin non-dependent diabetes, diarrhea, and menstrual irregularity. A few pharmacological actions of Phellinus linteus (PL) have been elucidated. The polysaccharide isolated from PL acts as an effective immunomodulator and exhibits a wider range of immuno-stimulation, anti-tumor activity and prevention of metastasis (Han et al., 1999, Kim et al., 2004a). Phellinus linteus stimulation could induce the phenotypic and functional maturation of dendritic cells via Toll-like receptor-2 (TLR2), TLR4 mediated-NF-κB, and ERK and p38 MAPK signal pathways (Kim et al., 2004b). Among the sub-fractions, the n-BuOH fraction is the most effective in anti-inflammation, and also contains anti-angiogenic activity (Kim et al., 2004c). The n-BuOH fraction from Phellinus linteus has showed its in vitro anti-inflammatory activity via induction of heme oxygenase-1 in RAW 264.7 macrophage (Kim et al., 2006a). Although Phellinus linteus has been investigated for anti-inflammatory effect, the regulatory mechanisms are not well established.

Macrophages that are ubiquitously distributed in tissues are derived from precursors in the bone marrow via the monocytes of the peripheral blood marrow via the mononuclear phagocyte system essential for the support of homeostasis and host defense against intracellular parasitic bacteria, pathogenic protozoa and fungi (Fujiwara and Kobayashi, 2005). Activated macrophage play an important role in inflammatory diseases by the production of cytokines, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and other inflammatory mediators such as nitric oxide (NO), and prostaglandins (PGE2) (Paul et al., 1999, Fujiwara and Kobayashi, 2005). Overproduction of the inflammatory mediators involves many diseases, such as rheumatoid arthritis (Manzi and Wasko, 2000), atherosclerosis (Libby et al., 2000), asthma (Tak and Firestein, 2001) and pulmonary fibrosis (Coker and Laurent, 1998). Thus, inhibition of the production of these inflammatory mediators in activated macrophage may prevent or suppress a variety of inflammatory diseases, including atherosclerosis, sepsis, and endotoxemia.

Inflammation of macrophage is activated upon appropriate extracellular stimulation, most often by stress or pro-inflammatory cytokines, including TNF-α and IL-1β (Berenbaum, 2000), and pathogens such as bacterial components including LPS (Zhang and Ghosh, 2000) through the NF-κB. These pro-inflammatory proteins such as cyclooxygenase-2 (COX-2) (Kim et al., 2003, Shin et al., 2004) and inducible nitric oxide synthase (iNOS) (Pan et al., 2000a, Pan et al., 2000b, Islam et al., 2004) act through distinct signaling pathways that converge on the activation of an IκBα kinase (IKK). IKK activation initiates IκBα phosphorylation at specific amino-terminal serine residue (Surh et al., 2001). The phosphorylation of IκBα is then ubiquitinated, which is degraded by the 26S proteasome, thereby releasing NF-κB dimers from the cytoplasmic NF-κB–IκBα complex and allowing them to translocate to the nucleus. Therefore, IκBα is a negative-feedback regulator of NF-κB (Makarov, 2000, Moynagh, 2005).

The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases that are part of the signal transduction pathways, which connect inflammatory and various other extracellular signals to intracellular response, e.g., gene expression. These classical MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK), have been implicated in the transcriptional regulation of inflammatory gene (Herlaar and Brown, 1999). p38 MAPK and JNK are members of the MAPK family, and they are activated by chemical and physical stress. They regulate inflammatory proteins as well as immune responses and expression of various cytokines, e.g., tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) (Hommes et al., 2003). The p38 (Chen and Wang, 1999, Chen et al., 2006, Kim et al., 2006c) and JNK (Uto et al., 2005) signaling pathway are involved in LPS-induced COX-2 and iNOS expression in macrophage. Also, Paul et al. (1999) report showed that the Erk and p38 in the regulation of COX-2 but not iNOS induction following exposure to LPS. Until now, activation of MAPKs in LPS-induced macrophages remains a lack of understanding of inflammatory molecular mechanism.

The generation of reactive oxygen species (ROS) by phagocytic leukocytes (neutrophils, monocytes, macrophages, and eosinophils) is one of the most important hallmarks in the inflammatory process. The ROS are mediators of cellular injury and are involved in the onset of cellular damage during endotoxemia (Ginn-Pease and Whisler, 1998, Forman and Torres, 2001). ROS are involved in a variety of cellular stress mechanisms. Several lines of evidence indicate that the redox status of cells participates in modulating NF-κB activation (D’Acquisto et al., 2002). A number of reports have shown that a broad range of antioxidants abolish NF-κB activation (Lo et al., 2002, Bai et al., 2005, Suh et al., 2006) and intracellular ROS (Han et al., 2001, Kamata et al., 2002). Nevertheless, both the mechanism for ROS induced by LPS and the role of induced ROS in cytokine expression of macrophage is still unclear.

In this study, we investigated whether the butanol fraction from Phellinus linteus (PLBF) would inhibit the production of NO and PGE2 as well as iNOS and COX-2 expression in macrophages following stimulation with LPS, because these effects may play an important role in disease therapy. Our results demonstrated that PLBF suppresses the production of these inflammatory mediators in LPS-activated macrophages, which may play an important role in inflammation-associated disorders.

Section snippets

Chemicals

Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Life Technology (Rockville, USA). LPS (Escherichia coli O11:B4) was obtained from Sigma (St. Louis, USA). Monoclonal iNOS, Phospho-p44/42 MAPK, and Akt were purchased from Signal Transduction Laboratory (Lexington, KY). Also Polyclonal COX-2, p44/42 MAP kinase, Phospho-Akt and actin antibodies were purchased from Signal Transduction Laboratory (Lexington, KY). Polyclonal p-IκB

Effect of PLBF on NO and iNOS protein and mRNA expression in LPS-stimulated macrophage

Several studies have demonstrated that induction of iNOS produces a large amount of NO under inflammatory conditions (Berenbaum, 2000, Makarov, 2000, Pan et al., 2000b). To recently, the n-butanol sub-fraction of Phellinus linteus (PL) suppresses NO production and iNOS protein in LPS-stimulated RAW 264.7 macrophages through the mediated of heme-oxygenase-1 (Kim et al., 2006a). Therefore, the butanol fraction from Phellinus linteus (PLBF) was examined to determine whether it affects NO

Discussion

Phellinus linteus has been used as a traditional medicine in oriental countries. Among the sub-fractions, the n-BuOH fraction is the most effective in anti-inflammation, and also contains anti-angiogenic activity (Kim et al., 2004c, Kim et al., 2006a). However, the mechanism of the anti-inflammatory action of Phellinus linteus in RAW 264.7 macrophage is not yet fully understood. We examined the effects of PLBF on the release of several inflammatory mediators, the expression levels of iNOS and

Acknowledgements

We wish to acknowledge the financial support from Agricultural R&D Promotion Center (ARPC), Korea. Institute for Cordyceps Research of Kangwon National University is also thanked for providing research facilities to carry out this study.

References (40)

  • H.G. Kim et al.

    Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells

    European Journal of Pharmacology

    (2006)
  • J.H. Kim et al.

    Rengyolone inhibits inducible nitric oxide synthase expression and nitric oxide production by down-regulation of NF-κB and p38 MAP kinase activity in LPS-stimulated RAW 264.7 cells

    Biochemical Pharmacology

    (2006)
  • S.H. Kim et al.

    Anti-inflammatory and related pharmacological activities of the n-BuOH subfraction of mushroom Phellinus linteus

    Journal of Ethnopharmacology

    (2004)
  • S.S. Makarov

    NF-κB as a therapeutic target in chronic inflammation: recent advance

    Molecular Medicine

    (2000)
  • M.H. Pan et al.

    Suppression of lipopolysaccharide-induced nuclear factor-κB activity by theaflavin-3,3′-digallate from black tea and other polyphenols through down-regulation of IκB kinase activity in macrophages

    Biochemical Pharmacology

    (2000)
  • M.H. Pan et al.

    Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IκB kinase and NFκB activation in macrophages

    Biochemical Pharmacology

    (2000)
  • A. Paul et al.

    Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages

    Cellular Signalling

    (1999)
  • S.J. Suh et al.

    The naturally occurring biflavonoid, ochnaflavone, inhibits LPS-induced iNOS expression, which is mediated by ERK1/2 via NF-κB regulation, in RAW 264.7 cells

    Archives of Biochemistry and Biophysics

    (2006)
  • T. Uto et al.

    6-(Methylsulfinyl)hexyl isothiocyanate suppresses inducible nitric oxide synthase expression through the inhibition of Janus kinase 2-mediated JNK pathway in lipopolysaccharide-activated murine macrophages

    Biochemical Pharmacology

    (2005)
  • T.L. Wadsworth et al.

    Effects of the wine polyphenolic quercetin and reveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages

    Biochemical Pharmacology

    (1999)
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