Anti-inflammatory action of high molecular weight Mytilus edulis hydrolysates fraction in LPS-induced RAW264.7 macrophage via NF-κB and MAPK pathways
Introduction
Inflammation is considered to be a major risk factor for the pathogenesis of chronic diseases and macrophages are important immune cells which regulate inflammation and host defense by secretion of pro-inflammatory cytokines, chemokines, NO, and PGE2 as well as the expression of inflammatory proteins (Abarikwu, 2014). However, excessive activation of macrophages has damaging effects, such as septic shock, which can lead to multiple organ dysfunction syndrome and death (Valledor, Comalada, Santamaria-Babi, Lloberas, & Celada, 2010). In addition, persistence of proinflammatory activity results in the development of chronic inflammation, such as rheumatoid arthritis, psoriasis, and inflammatory bowel diseases (Valledor et al., 2010). Thus, modulation of macrophage activation is a good strategy to prevent undesirable effects.
To date, the health benefits of fish and shellfish has been extensively studied. The most well known health benefits are antihypertensive and antioxidant activities through production of bioactive peptides by enzymatic hydrolysis (Gu et al., 2011, Je et al., 2015). These bioactive peptides are generally inactive forms and can be generated from the parent proteins by enzymatic hydrolysis. In addition, bioactive peptides have demonstrated notable modulatory effects against inflammatory reactions by inhibiting the production of proinflammatory markers, such as NO, IL-1β, IL-6, TNF-α, iNOS and COX-2 (Mao et al., 2011, Ndiaye et al., 2012, Udenigwe et al., 2013, Young et al., 2012). However, only scanty information, with regard to the anti-inflammatory activity of marine protein-derived bioactive peptides, has been available until now.
Mytilus edulis (blue mussel) is widely consumed as foodstuff in Korea and it contains approximately 55% protein (in dry basis), which is a good source to generate bioactive peptides by enzymatic hydrolysis. Our previous study demonstrated that membrane fractions from M. edulis hydrolysates (MEH) exhibited antioxidant activity and inhibition activity for NO production in LPS-stimulated macrophage cells (Park, Ahn, & Je, 2014). In particular, high molecular weight MEH membrane fraction exhibited the highest NO inhibition activity. In the present study, therefore, molecular mechanisms underlying anti-inflammatory activity of high molecular weight MEH membrane fraction were investigated in LPS-induced RAW264.7 macrophages.
Section snippets
Materials
The Griess reagents, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lipopolysaccharides (LPSs, from Escherichia coli serotype 026: B6) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies for iNOS, COX-2, NF-κB, p-IκBα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Preparation and fractionation of M. edulis hydrolysates (MEH) by peptic hydrolysis
MEH were prepared according to our previous report (Park et al., 2014). A 10% substrate and enzyme (enzyme/substrate ratio of 1:500) were mixed and then hydrolyzed for 30
Inhibitory effect of M. edulis hydrolysates (MEH) and its membrane fractions on NO production in LPS-induced RAW264.7 macrophages
According to our previous method, MEH was prepared and fractionated into three fractions based on molecular weight, namely a <1 kDa, 1–5 kDa, and >5 kDa peptide fraction (Park et al., 2014). As shown in Fig. 1A, no cytotoxic effects of MEH fractions in the tested concentrations were observed. The result of NO inhibitory activities of MEH fractions exhibited that all fractions and unfractionated material inhibited NO production in LPS-induced RAW264.7 macrophages (Fig. 1B). NO inhibitory activities
Conclusion
In this study, our findings demonstrated that high molecular weight peptide fraction (>5 kDa) from M. edulis hydrolysates obtained by peptic hydrolysis exhibited anti-inflammatory action in LPS-stimulated RAW264.7 macrophages through modulation of pro-inflammatory mediators. Modulation effects are attributed to its inhibition of inflammatory gene expressions through blocking of NF-κB and MAPK signaling pathways (Fig. 5). These findings may indicate the potential utility of the peptide fraction
Acknowledgments
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2057530).
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These authors contributed equally to this work.