Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)？[extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4th and the 5th h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5th h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5th h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.
References
[1]
Huang GJ, Huang SS, Lin SS, Shao YY, Chen CC, et al. (2010) Analgesic effects and the mechanisms of anti-inflammation of ergostatrien-3β-ol from Antrodia camphorata submerged whole broth in mice. J Agric Food Chem 58: 7445–7452.
[2]
Chang CT, Huang SS, Lin SS, Amagaya S, Ho HY, et al. (2011) Anti-inflammatory activities of tormentic acid from suspension cells of Eriobotrya Japonica ex vivo and in vivo. Food Chem 127: 1131–1137.
[3]
Chang HY, Sheu MJ, Yang CH, Leu ZC, Chang YS, et al. (2009) Analgesic effects and the mechanisms of anti-inflammation of hispolon in mice. Evidence-Based Compl Altern Med. doi:10.1093/ecam/nep027.
[4]
Huang MH, Wang BS, Chiu CS, Amagaya S, Hsieh WT, et al. (2011) Antioxidant, antinociceptive, and anti-inflammatory activities of Xanthii fructus extract. J Ethnopharmacol 135: 545–552.
[5]
Huang GJ, Yang CM, Chang YS, Amagaya S, Wang HC, et al. (2010) Hispolon suppresses SK-Hep1 human hepatoma cell metastasis by inhibiting matrix metalloproteinase-2/9 and urokinase-plasminogen activator through the PI3K/Akt and ERK signaling pathways. J Agric Food Chem 58: 9468–9475.
[6]
Huang GJ, Deng JS, Huang SS, Hu ML (2011) Hispolon Induces Apoptosis and Cell Cycle Arrest of Human Hepatocellular Carcinoma Hep3B Cells by Modulating ERK Phosphorylation. J Agric Food Chem 59: 7104–13.
[7]
Lai CS, Lee JH, Ho CT, Liu CB, Wang JM, et al. (2009) Rosmanol potently inhibits lipopolysaccharide-Induced iNOS and COX-2 expression through downregulating MAPK, NF-κB, STAT3 and C/EBP signaling pathways. J Agric Food Chem 57: 10990–10998.
[8]
Park EJ, Kim SA, Choi YM, Kwon HK, Shim W, et al. (2011) Capric acid inhibits NO production and STAT3 activation during LPS-induced osteoclastogenesis. PLoS One 6: e27739.
[9]
Huang GJ, Hsieh WT, Chang HY, Huang SS, Lin YC, et al. (2011) Inhibitory constituents of α-glucosidase and aldose reductase in the fruiting body of Phellinus merrillii. J Agric Food Chem 59: 5702–5706.
[10]
Lee IK, Yun BS (2011) Styrylpyrone-class compounds from medicinal fungi Phellinus and Inonotus spp., and their medicinal importance. J Antibiot (Tokyo) 64: 349–359.
[11]
Jung JY, Lee IK, Seok SJ, Lee HJ, Kim YH, et al. (2008) Antioxidant polyphenols from the mycelial culture of the medicinal fungi Inonotus xeranticus and Phellinus linteus. J Appl Microbiol 104: 1824–32.
[12]
Kuo YC, Lai CS, Wang JM, Badmaev V, Nagabhushanam K, et al. (2009) Differential inhibitory effects of inotilone on inflammatory mediators, inducible nitric oxide synthase and cyclooxygenase-2, in LPS-stimulated murine macrophage. Mol Nutr Food Res 53: 1386–1395.
[13]
Committee for Research and Ethical Issues of the IASP (1983) Ethical standards for investigators of experimental pain in animals. Pain 16: 161–164.
[14]
Chang TN, Huang SS, Chang YS, Chang CI, Yang HL, et al. (2011) Analgesic effects and the mechanisms of anti-inflammation of taraxeren-3-one from Diospyros maritima in mice. J Agric Food Chem 59: 9112–9119.
[15]
Liao YC, Shih YW, Chao CH, Lee XY, Chiang TA (2009) Involvement of the ERK signaling pathway in fisetin reduces invasion and migration in the human lung cancer cell line A549. J Agric Food Chem 57: 8933–8941.
[16]
Wen CL, Chang CC, Huang SS, Kuo CL, Hsu SL, et al. (2011) Anti-inflammatory effects of methanol extract of Antrodia cinnamomea mycelia both in vitro and in vivo. J Ethnopharmacol 137: 575–584.
[17]
Huang SS, Chiu CS, Chen HJ, Lin SS, Hsieh IC, et al. (2011) Antinociceptive activities and the mechanisms of anti-inflammation of asiatic acid in mice. Evidence-Based Compl Altern Med. doi:10.1155/2011/895857.
Aebi H (1984) Catalase in vitro. Methods Enzymol 105: 121–126.
[20]
Paglia ED, Valentine WN (1967) Studies on the quantitative and qualitative characterization of erythrocytes glutathione peroxidase. J Lab Clin Med 70: 158–169.
[21]
Hsing CH, Lin MC, Choi PC, Huang WC, Kai JI, et al. (2011) Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKβ/NF-κB signaling. PLoS One 6: e17598.
[22]
Achoui M, Appleton D, Abdulla MA, Awang K, Mohd MA, et al. (2010) In vitro and in vivo anti-inflammatory activity of 17-O-acetylacuminolide through the inhibition of cytokines, NF-κB translocation and IKKβ activity. PLoS One 5: e15105.
[23]
Chapelsky S, Batty S, Frost M, Mogridge J (2008) Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol. PLoS One 3: e1421.
[24]
Huang GJ, Huang SS, Lin SS, Hsieh IC, Hou WC, et al. (2011) Anti-inflammatory activities of 6beta-acetoxy-7alpha-hydroxyroyleanone from Taiwania cryptomerioides Hayata ex vivo and in vivo. J Agric Food Chem 59: 11211–11218.
[25]
Ku KT, Huang YL, Huang YJ, Chiou WF (2008) Miyabenol A inhibits LPS-induced NO production via IKK/IkappaB inactivation in RAW 264.7 macrophages: possible involvement of the p38 and PI3K pathways. J Agric Food Chem 56: 8911–8918.
[26]
Huang MH, Huang SS, Wang BS, Wu CH, Sheu MJ, et al. (2011) Antioxidant and anti-inflammatory properties of Cardiospermum halicacabum and its reference compounds ex vivo and in vivo. J Ethnopharmacology 133: 743–750.
[27]
Rajapakse N, Kim MM, Mendis E, Kim SK (2008) Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-kappaB signaling. Immunol 123: 348–357.
[28]
Ajizian SJ, English BK, Meals EA (1999) Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma. J Infect Dis 179: 939–944.
[29]
Bhat NR, Zhang P, Lee JC, Hogan EL (1998) Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures. J Neurosci 18: 1633–1641.
[30]
Yan C, Boyd DD (2007) Regulation of matrix metalloproteinase gene expression. J Cell Physiol 211: 19–26.
[31]
Benbow U, Brinckerhoff CE (1997) The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol 15: 519–526.
[32]
Pan MH, Chiou YS, Chen WJ, Wang JM, Badmaev V, et al. (2009) Pterostilbene inhibited tumor invasion via suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells. Carcinogenesis 30: 1234–1242.
[33]
Woo CH, Lim JH, Kim JH (2004) Lipopolysaccharide induces matrix metalloproteinase-9 expression via a mitochondrial reactive oxygen species-p38 kinase-activator protein-1 pathway in Raw 264.7 cells. J Immunol 173: 6973–6780.
[34]
Handy RL, Moore PK (1998) A comparison of the effects of L-NAME, 7-NI and L-NIL on carrageenan-induced hindpaw oedema and NOS activity. Brit. J Pharmacol 123: 1119–1126.
[35]
Chaturvedi P (2008) Inhibitory response of Raphanus sativus on lipid peroxidation in albino rats. Evidence-Based Compl. Altern Med 5: 55–59.
[36]
Bhattacharyya S, Dudeja PK, Tobacman JK (2008) Carrageenan-induced NFkappaB activation depends on distinct pathways mediated by reactive oxygen species and Hsp27 or by Bcl10. Biochim Biophys Acta 1780: 973–982.
[37]
Haddad JJ, Land SC (2002) Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-alpha biosynthesis. Brit. J Pharmacol 135: 520–536.
[38]
Jeong JB, Jeong HJ (2010) Rheosmin, a naturally occurring phenolic compound inhibits LPS-induced iNOS and COX-2 expression in RAW264.7 cells by blocking NF-kappaB activation pathway. Food Chem Toxicol 48: 2148–2153.
[39]
Lu Y, Hong TG, Jin M, Yang JH, Suh SJ, et al. (2010) Saucerneol G, a new lignan, from Saururus chinensis inhibits matrix metalloproteinase-9 induction via a nuclear factor κB and mitogen activated protein kinases in lipopolysaccharide-stimulated RAW264.7 cells. Biol Pharm Bull 33: 1944–1948.
