Polyozellin Inhibits Nitric Oxide Production by Down-Regulating LPS-Induced Activity of NF-κB and SAPK/JNK in RAW 264.7 Cells

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Polyozellin, isolated from Polyozellus multiplex (Thelephoraceae), was investigated for its anti-inflammatory activity in the murine macrophage cell line RAW 264.7. Polyozellin inhibited both lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in a dose-dependent manner. The effects of polyozellin on the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein (MAP) kinases in these cells were studied in order to elucidate the underlying mechanism. Polyozellin suppressed the activation of both LPS-induced NF-κB and the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), but had no effect on the extracellular signal-regulated kinase (ERK) or p38. These data suggest that polyozellin suppresses iNOS expression by inhibiting the activation of NF-κB and SAPK/JNK, leading to the inhibition of NO production.

Nitric oxide (NO) is an important cellular second messenger produced from L-arginine via nitric oxide synthase (NOS) [1]. Three types of NOS have been identified: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). An increased output of NO produced by iNOS is associated with diverse disorders, such as septic and hemorrhagic shock, rheumatoid arthritis, and chronic infections [2], [3], [4]. Therefore, the suppression of excessive NO production may be a useful therapeutic strategy for treating inflammatory disorders.

Polyozellin (6,12-diacetoxy-2,3,8,9-tetrahydroxybenzol[1,2-b;4,5-b′] bisbenzofuran) (Fig. [1]), was isolated from Polyozellus multiplex (Thelephoraceae). Previous studies have demonstrated that polyozellin exhibits a variety of biological activities, including the inhibition of prolyl endopeptidase [5] and antioxidant [6] and anticancer properties [7]. Several natural antioxidant compounds reduce inflammation, and suppression of the effects of these compounds on NO production is associated with their antioxidant activities [8]. Therefore, we evaluated the effects of polyozellin on NO production in murine macrophage RAW 264.7 cells stimulated by lipopolysaccharide (LPS). As shown in Fig. [2] A, LPS (1 μg/mL) increased the nitrite concentration in the culture medium 13-fold, compared with the untreated control cells. Polyozellin at concentrations of 5, 10, and 20 μM significantly reduced nitrite production by 32.6 ± 10.8 %, 48.2 ± 8.1 % and 73.7 ± 4.2 %, respectively. It also inhibited LPS-induced nitrite production in RAW264.7 cells with an IC₅₀ value of 11.6 ± 0.9 μM. A positive control, N ^G-monomethyl-L-arginine acetate (L-NMMA), which is a non-specific inhibitor of NOS [9], also suppressed nitrite production by 33.5 ± 8.8 % and 72.5 ± 6.4 % at 50 μM and 100 μM, respectively, showing weaker inhibition than polyozellin. To further evaluate the inhibition of NO production in RAW 264.7 cells affected by the cytotoxic effects of polyozellin, cell viability was determined using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay (data not shown). Polyozellin showed no cytotoxicity to RAW 264.7 cells at concentrations of 20 μM or below.

The inhibitory effect of polyozellin on LPS-induced NO production was then examined in order to determine if this is a direct effect on iNOS expression or if it is mediated by some other mechanism. We examined iNOS protein expression levels by Western blot analysis and iNOS mRNA expression by reverse transcription polymerase chain reaction (RT-PCR) analysis. Polyozellin significantly inhibited the LPS-induced expression of both iNOS protein and mRNA in macrophages (Fig. [2] B). Thus, polyozellin appears to act on LPS-induced NO production in RAW 264.7 cells via suppression of iNOS expression.

Nuclear factor-κB (NF-κB) is a major transcription factor involved in the regulation of iNOS gene expression [10], [11]. Activation of NF-κB results in degradation of the inhibitor-κB (I-κB) protein, followed by translocation of NF-κB to the nucleus and induction of gene transcription through its binding to the cis-acting NF-κB element. As shown in Fig. [2] B, pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor [12], was used as a positive control and inhibited the expression of iNOS protein. Therefore, we examined the effect of polyozellin on the activation of NF-κB. As shown in Fig. [3] A, polyozellin clearly inhibited NF-κB DNA binding and the nuclear translocation of NF-κB/p65 in LPS-stimulated RAW 264.7 cells. To gain further insight into the mechanism of polyozellin-mediated regulation of NF-κB, we examined the effects of polyozellin on the degradation of I-κB using Western blotting. Fig. [3] B shows that polyozellin inhibited LPS-induced I-κBα degradation in a dose-dependent manner. Thus, polyozellin inhibition of LPS-induced iNOS expression is mediated via inhibition of NF-κB activation.

LPS initiates intracellular signaling pathways that lead to the activation of three distinct mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 [13]. Moreover, MAP kinases are implicated in LPS-induced iNOS expression and NF-κB activation [14], [15]. Therefore, we investigated the effects of polyozellin on LPS-induced phosphorylation of MAP kinases in RAW 264.7 cells. As shown in Fig. [4], treatment with polyozellin caused a significant inhibition of the phosphorylation levels of SAPK/JNK, but not of ERK1/2 or p38, suggesting that SAPK/JNK is involved in the inhibitory effect of polyozellin on LPS-stimulated iNOS gene expression and NF-κB activation.

In summary, polyozellin inhibits LPS-induced NO production and iNOS expression via suppressing the activation of both NF-κB and SAPK/JNK in macrophages. This is the first report that polyozellin has anti-inflammatory action. Thus, polyozellin may be effective therapeutically for alleviating the symptoms of inflammatory diseases.

Materials and Methods

The fruiting bodies of Polyozellus multiplex were collected from Mt. Odae, Kangwondo, Korea, and identified by Prof. K. S. Song as reported [16]. The voucher specimen (No. KNU-NPC-PM-005) was deposited at the Natural Products Chemistry Laboratory Kyungpook National University, Daegu, Korea. Polyozellin was prepared and identified according to a published method [7]. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. RAW 264.7 cells were maintained at 5 × 10⁵ cells/mL culture in DMEM containing 10 % FBS (Gibco-BRL; Grand Island, NY, USA), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine. Cells were grown at 37 °C under 5 % CO₂. Nitrite production in the culture supernatant was determined by the Griess reaction [17]. Whole-cell lysates, cytosolic fractions (for I-κBα), and nuclear fractions (for NF-κB/p65) were prepared as described [18]. Immunoblot analysis was performed using specific antibodies to iNOS, I-κBα, NF-κB/p65 (RelA) (Santa Cruz Biotechnology; Santa Cruz, CA, USA), to actin and to phosphorylated or non-phosphorylated forms of the MAPK family (ERK1/2, SAPK/JNK, and p38; Cell Signaling Technology; Danvers, MA, USA) [18]. Total RNA isolated from RAW 264.7 cells with Trizol reagent (Invitrogen; Carlsbad, CA, USA) was reverse-transcribed into cDNA, and PCR was performed as described [18]. NF-κB DNA binding was analyzed using Gel Shift Assay Systems (Promega; Madison, WI, USA) according to the manufacturer’s instructions. Values are expressed as mean ± SD. Student’s t test (P < 0.05) was used to assess the statistical significance of any differences.

Acknowledgements

This work was supported by the Korea Research Foundation Grant (KRF-2004-005-E00093). We thank Ms. H. M. Song for excellent technical assistance.

References

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  CrossrefPubMedSearch in Google Scholar

Prof. Dong Hwan Sohn

College of Pharmacy

Medicinal Resources Research Institute

Wonkwang University

Iksan, Jeonbuk 570-749

Republic of Korea

Phone: +82-63-850-6822

Fax: +82-63-854-6038

Email: dhsohn@wonkwang.ac.kr

References

• 1 Marletta M A. Nitric oxide synthase structure and mechanism.  J Biol Chem. 1993;  268 12 231-4
  PubMedSearch in Google Scholar
• 2 Zingarelli B, Squadrito F, Altavilla D, Calapei G, Campo G M, Calo M. et al . Evidence for a role of nitric oxide in hypovolemic hemorrhagic shock.  J Cardiovasc Pharmacol. 1992;  19 982-6
  PubMedSearch in Google Scholar
• 3 Kaur H, Halliwell B. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients.  FEBS Lett. 1994;  350 9-12
  CrossrefPubMedSearch in Google Scholar
• 4 Ohshima H, Bartsch H. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis.  Mutat Res. 1994;  305 253-64
  PubMedSearch in Google Scholar
• 5 Hwang J S, Song K S, Kim W G, Lee T H, Koshino H, Yoo I D. Polyozellin, a new inhibitor of prolyl endopeptidase from Polyozellus multiplex .  J Antibiot. 1997;  50 773-7
  CrossrefPubMedSearch in Google Scholar
• 6 Chung S K, Jeon S Y, Lee H J, Kim S K, Kim S I, Kim G S. et al . Antioxidative effects of polyozellin and thelephoric acid isolated from Polyozellus multiplex .  J Korean Soc Appl Biol Chem. 2004;  47 283-6
  PubMedSearch in Google Scholar
• 7 Kim J H, Lee J S, Song K S, Kwon C S, Kim Y K, Kim J S. Polyozellin isolated from Polyozellus multiplex induces phase 2 enzymes in mouse hepatoma cells and differentiation in human myeloid leukaemic cell lines.  J Agric Food Chem. 2004;  52 451-5
  PubMedSearch in Google Scholar
• 8 Kim K M, Chun S B, Koo M S, Choi W J, Kim T W, Kwon Y G. et al . Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine.  Free Radic Biol Med. 2001;  30 747-56
  CrossrefPubMedSearch in Google Scholar
• 9 Banskota A H, Tezuka Y, Nguyen N T, Awale S, Nobukawa T, Kadota S. DPPH radical scavenging and nitric oxide inhibitory activities of the constituents from the wood of Taxus yunnanensis .  Planta Med. 2003;  69 500-5
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Xie Q W, Kashiwabara Y, Nathan C. Role of transcription factor NF-κB/Rel in induction of nitric oxide synthase.  J Biol Chem. 1994;  269 4705-8
  PubMedSearch in Google Scholar
• 11 Karin M. The beginning of the end: IκB kinase (IKK) and NF-κB activation.  J Biol Chem. 1999;  274 27 339-42
  PubMedSearch in Google Scholar
• 12 Jang S I, Jeong S I, Kim K J, Kim H J, Yu H H, Park R. et al . Tanshinone IIA from Salvia miltiorrhiza inhibits inducible nitric oxide synthase expression and production of TNF-α, IL-1β and IL-6 in activated RAW 264.7 cells.  Planta Med. 2003;  69 1057-9
  Thieme ConnectPubMedSearch in Google Scholar
• 13 Sanghera J S, Weinstein S L, Aluwalia M, Girn J, Pelech S L. Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages.  J Immunol. 1996;  156 4457-65
  PubMedSearch in Google Scholar
• 14 Caivano M. Role of MAP kinase cascades in inducing arginine transporters and nitric oxide synthetase in RAW264 macrophages.  FEBS Lett. 1998;  429 249-53
  CrossrefPubMedSearch in Google Scholar
• 15 Kim Y H, Lee S H, Lee J Y, Choi S W, Park J W, Kwon T K. Triptolide inhibits murine-inducible nitric oxide synthase expression by down-regulating lipopolysaccharide-induced activity of nuclear factor-κB and c-Jun NH2-terminal kinase.  Eur J Pharmacol. 2004;  494 1-9
  CrossrefPubMedSearch in Google Scholar
• 16 Hwang J S, Song K S, Kim Y S, Seok S J, Lee T H, Yoo I D. Lipid peroxidation inhibitors from Polyozellus multiplex .  Kor J Appl Microbiol Biotechnol. 1996;  24 591-6
  PubMedSearch in Google Scholar
• 17 Green L C, Wagner D A, Glogowski J, Skipper P L, Wishnok J S, Tannenbaum S R. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.  Anal Biochem. 1982;  126 131-8
  CrossrefPubMedSearch in Google Scholar
• 18 Lee S H, Seo G S, Sohn D H. Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by butein in RAW 264.7 cells.  Biochem Biophys Res Commun. 2004;  323 125-32
  CrossrefPubMedSearch in Google Scholar

Prof. Dong Hwan Sohn

College of Pharmacy

Medicinal Resources Research Institute

Wonkwang University

Iksan, Jeonbuk 570-749

Republic of Korea

Phone: +82-63-850-6822

Fax: +82-63-854-6038

Email: dhsohn@wonkwang.ac.kr