Inhibitory Effects and Molecular Mechanism of an Anti-inflammatory Peptide Isolated from Intestine of Abalone, Haliotis Discus Hannai on LPS-Induced Cytokine Production via the p-p38/p-JNK Pathways in RAW264.7 Macrophages

Zhong-Ji Qian¹, BoMi Ryu², Won Sun Park³, IL-Whan Choi^4, and Won-Kyo Jung^5,

¹Key Laboratory of Advanced Processing of Aquatic Products of Guangdong Higher Education Institution, College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China

²School of Pharmacy, the University of Queensland, Brisbane, Qld 4072, Australia

³Department of Physiology, School of Medicine, Kangwon National University, Chuncheon 24341, Republic of Korea

⁴Department of Microbiology, College of Medicine, Inje University, Busan 633-165, Republic of Korea

⁵Department of Biomedical Engineering, and Center for Marine-Intergrated Biomedical Technology (BK21 Plus) Pukyong National University, Busan 608-737, Republic of Korea

Cite this paper:
Zhong-Ji Qian, BoMi Ryu, Won Sun Park, IL-Whan Choi and Won-Kyo Jung. Inhibitory Effects and Molecular Mechanism of an Anti-inflammatory Peptide Isolated from Intestine of Abalone, Haliotis Discus Hannai on LPS-Induced Cytokine Production via the p-p38/p-JNK Pathways in RAW264.7 Macrophages. Journal of Food and Nutrition Research. 2016; 4(10):690-698. doi: 10.12691/jfnr-4-10-9

Abstract

A marine mollusk, abalone (Haliotis discus hannai) is one of the important species in aquaculture industry, but nutraceutical and pharmaceutical benefits of H. discus hannai have been rarely identified and studied. To evaluate beneficial effects of H. discus hannai, an anti-inflammatory peptide (AAIP, abalone anti-inflammatory peptide) was purified from abalone intestines using consecutive HPLC purification system. In tandem MS analysis, the fragmentation results illustrate that the AAIP responsible for the nitric oxide (NO) inhibitory activity (IC₅₀=55.8μM) has amino acid sequence as Pro-Phe-Asn-Glu-Gly-Thr-Phe-Ala-Ser (1175.2 Da). To investigate anti-inflammatory effect of AAIP on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and elucidated the molecular mechanism. The results show that the AAIP peptide suppresses LPS-induced production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. It also significantly reduced the gene transcription of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, AAIP significantly suppresses phosphorylation of mitogen-activated protein kinases (MAPKs) such as p-p38 and p-JNK. These results indicated that AAIP inhibits LPS-induced inflammatory response via blocking of MAPK pathway in murine macrophages. Therefore, potent AAIP might suggest possibility for high valuable utilization and application as nutraceutical and therapeutic substances.

Keywords:
marine mollusk inflammation lipopolysaccharide (LPS) nitric oxide nitric oxide synthase mitogen-activated protein kinases (MAPK)

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Sebban, H., Courtois, G. NF-kB and inflammation in genetic disease. Biochemical Pharmacology, 7. 1153-1160. 2006.
[2]	Lee, H.J., Hyun, E.A., Yoon, W.J., Kim, B.H., Rhee, M.H., Kang, H.K., Cho, J.Y., Yoo, E.S. In vitro anti-inflammatory and anti-oxidative effects of Cinnamomum camphora extracts. Journal of Ethnopharmacology, 103. 208-216. 2006.
[3]	Walsh, N.C., Crotti, T.N., Goldring, S.R., Gravallese, E.M. Rheumatic diseases: the effects of inflammation on bone. Immunological Reviews, 208. 228-251. 2005.
[4]	Wong, J.M., Billiar, T.R. Regulation and function of inducible nitric oxide synthase during sepsis and acute inflammation. Advances in Pharmacology, 34. 155-170. 1995.
[5]	Inoue, H., Kubo, M. SOCS proteins in T helper cell differentiation: implications for allergic disorders?. Expet Reviews in Molecular Medicine, 6. 1-11. 2004.
[6]	Jung, W.K., Rajapakse, N., Kim SK. Antioxidative activity of low molecular peptide derived from the sauce of fermented blue mussel, Mytilus edulis. European Food Research Technology, 220. 535-539. 2005.
[7]	Rajapakse, N., Mendis, E., Jung, W.K., Je, J.Y., Kim, S.K. Purification of radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Research International, 38. 175-182. 2005.
[8]	Ryu, B.M., Qian, Z.J., Kim, S.K. SHP-1, a novel peptide isolated from seahorse inhibits collagen release through the suppression of collagenases 1 and 3, nitric oxide products regulated by NF-kB/p38 kinase. Peptides, 31. 79-87. 2010.
[9]	Kapsokefalou, M., Miller, D.D. Effects of meat and selected food components on the valence of nonheme iron during in vitro digestion. Journal of Food Science, 56. 352-355. 1991.
[10]	Qian, Z.J., Kim, S.A., Lee, J.S., Kim, H.J., Choi, I.W., Jung, W.K. The Antioxidant and Anti-inflammatory Effects of Abalone Intestine Digest, Haliotis discus hannai in RAW 264.7 Macrophages. Biotechnology and Bioprocess Engineering, 17. 475-484. 2012.
[11]	Hansen, M.B., Nielson, S.E., Berg, K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Journal of Immunological Methods, 119. 203-210. 1989.
[12]	Lee, M.H., Lee, J.M., Jun, S.H., Lee, S.H., Kim, N.W., Lee, J.H., Ko, N.Y., Kun, S.H., Kim, B.K., Lim, B.O., Choi, D.K., Choi, W.S. The anti-inflammatory effects of Pyrolae herba extract through the inhibition of the expression of inducible nitric oxide synthase (iNOS) and NO production. Journal of Ethnopharmacology, 112. 49-54. 2007.
[13]	Ren, G.L., Bai, X.F., Zhang, Y., Chen, H.M., Huang, C.X., Wang, P.Z, et al. Stable inhibition of hepatitis B virus expression and replication by expressed siRNA. Biochemical Biophysical Research Communications, 335. 1051-1059. 2005.
[14]	Mayer, A.M.D., Rodríguez, A.D., Berlinck, R., Fusetani, N. Marine pharmacology in 2007-2008: Marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous system, and other miscellaneous mechanisms of action. Comparative Biochemistry and Physiology C, 153. 191-222. 2011.
[15]	Sun, L., Zhu, B., Li, D., Wang, L., Dong, X., Murata, Y., Xing, R., Dong, Y. Purification and bioactivity of a sulphated polysaccharide conjugate from viscera of abalone Haliotis discus hannai Ino. Food and Agriculture Immunology, 21. 15-26. 2010.
[16]	Ekanayake, P.M., De, Zoysa M., Kang, H.S., Wan, Q., Jee, Y., Lee, Y.H., Kim, S.J., Lee, J. Cloning, characterization and tissue expression of disk abalone (Haliotis discus discus) catalase. Fish and Shellfish Immunology, 24. 267-278. 2008.
[17]	Zoysa, M.D., Whang, I., Lee, Y.D., Lee, S.K., Lee, J.S., Lee, J. Transcriptional analysis of antioxidant and immune defense genes in disk abalone (Haliotis discus discus) during thermal, low-salinity and hypoxic stress/comparative biochemistry and physiology. Comparative Biochemistry and Physiology B, 154. 387-395. 2009.
[18]	Wan, Q., Whang, I., Lee, J. Molecular characterization of mu class glutathione-Stransferase from disk abalone (Haliotis discus discus), a potential biomarker of endocrine-disrupting chemicals. Comparative Biochemistry and Physiology B, 150. 187-199. 2005.
[19]	Vo, T.S., Ryu, B.M., Kim, S.K. Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina maxima. Journal of Functional Foods, 5. 1336-1246. 2013.
[20]	Harnedy, P.A., FitzGerald, R.J. Bioactive peptides from marine processing waste and shellfish: A review. Journal of Functional Foods, 4. 6-24. 2012.
[21]	Lee, M., Kovacs-Nolan, J., Archbold, T., Fan, M.Z., Juneja, L.R., Okubo, T., Mine, Y. Therapeutic potential of hen egg white peptides for the treatment of intestinal inflammation. Journal of Functional Foods, 1(2). 161-169. 2009.
[22]	Hwang, J.W., Lee, S.J., Kim, Y.S., Kim, E.K., Ahn, C.B., Jeon, Y.J., Moon, S.H., Jeon, B.T., Park, P.J. Purification and characterization of a novel peptide with inhibitory effects on colitis induced mice by dextran sulfate sodium from enzymatic hydrolysates of Crassostrea gigas. Fish and Shellfish Immunology, 33(4). 993-999. 2012.
[23]	Lee, S.J., Kim, E.K., Kim, Y.S., Hwang, J.W., Lee, K.H., Choi, D.K., Kang, H., Moon, S.H., Jeon, B.T., Park, P.J. Purification and characterization of a nitric oxide inhibitory peptide from Ruditapes philippinarum. Food Chemistry and Toxicology, 50(5). 1660-1666. 2012.
[24]	Fujiwara, N., Kobayashi, K. Macrophages in inflammation. Current Drug Targets, 4. 281-286. 2004.
[25]	Mosser, D.M., Edwards, J.P. Exploring the full spectrum of macrophage activation. Nature Reviews Immunology, 8. 958-969. 2008.
[26]	Kim, E.Y., Moudgil, K.D. Regulation of autoimmune inflammation by proinflammatory cytokines. Immunology Letters, 120. 1-5. 2008.
[27]	Yoon, W.J., Ham, Y.M., Kim, S.S., Yoo, B.S., Moon, J.Y., Baik, J.S., Lee, N.H., Hyun, C.G. Suppression of pro-inflammatory cytokines, iNOS, and COX-2 expression by brown algae Sargassum micracanthum in RAW 264.7 macrophages. European and Asian Journal of BioScience, 3. 130-143. 2009.
[28]	Dinarello, C.A. Proinflammatory cytokines. Chest, 118. 503-508. 2000.
[29]	Feghali, C.A., Wright, T.M. Cytokines in acute and chronic inflammation. Frontieres in Bioscience, 2. 12-26. 1997.
[30]	Dinarello, C.A. Anti-inflammatory agents: present and future. Cell, 140. 935-950. 2010.
[31]	Petersen, A.M., Pedersen, B.K. The role of IL-6 in mediating the anti-inflammatory effects of exercise. Journal of physiology and pharmacology, 57. 43-51. 2006.
[32]	Opal, S.M., DePalo, V.A. Anti-inflammatory cytokines. Chest, 117. 1162-1172. 2000.
[33]	Nishimoto, N. Interleukin-6 as a therapeutic target in candidate inflammatory diseases. Clinical Pharmacology and Therapeutics, 87. 483-487. 2010.
[34]	Park, J.S., Lee, E.J., Lee, J.C., Kim, W.K., Kim, H.S. Anti-inflammatory effects of short chain fatty acids in IFN-[gamma]-stimulated RAW 264.7 murine macrophage cells: involvement of NF-[kappa] B and ERK signaling pathways. International immunopharmacology, 7, 70-77. 2007.
[35]	Tedelind, S., Westberg, F., Kjerrulf, M., Vidal, A. Anti-inflammatory properties of the short-chain fatty acids acetate and propionate: a study with relevance to inflammatory bowel disease. World Journal of Gastroenterology, 13. 2826-2832. 2007.
[36]	Lee, J.H., Jung, H.S., Giang, P.M., Jin, X., Lee, S., Son, P.T et al. Blockade of nuclear factorkappaBsignaling pathway and anti-inflammatory activity of cardamomin, a chalcone analog from Alpinia conchigera. Journal of Pharmacology and Experimental Therapeutics, 316. 271-278. 2006.
[37]	Jung, H.W., Yoon, C.H., Park, K.M., Han, H.S., Park, Y.K. Hexane fraction of Zingiberis Rhizoma crudus extract inhibits the production of nitric oxide and pro-inflammatory cytokines in LPS stimulated BV2 microgilial cells via the NF-kB pathway. Food Chemistry and Toxicology, 47. 1190-1197. 2009b.
[38]	Himaya, S.W.A., Ryu, B.M., Qian, Z.J., Kim, S.K. Sea cucumber, Stichopus japonicus ethyl acetate fraction modulates the lipopolysaccharide induced iNOS and COX-2 via MAPK signaling pathway in murine macrophages. Environmental Toxicolocy and Pharmacology, 30. 68-75. 2010.
[39]	Cobb, M.H., Goldsmith, E.J. How MAP kinases are regulated. Journal of Biological Chemistry, 270. 14843-14846. 1995.
[40]	Hommes, D.W., Peppelenbosch, M.P., van Deventer, S.J. Mitogen activated protein (MAP) kinase signal transduction pathways and novel anti-inflammatory targets. Gut, 52, 144-151. 2004.
[41]	Cario, E., Rosenberg, I.M., Brandwein, S.L., Beck, P.L., Reinecker, H.C., Podolsky, D.K. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. Journal of Immunology, 164, 966-972. 2000.
[42]	Jongeneel, C.V. Transcriptional regulation of the tumor necrosis factor gene. Immunobiology, 193. 210-216. 1995.