Anti-inflammatory activity of lupine (Lupinus angustifolius L.) protein hydrolysates in THP-1-derived macrophages
Introduction
The consumption of dietary protein drives many fundamental metabolic processes and is particularly important in nutrient-based biological functions (Gersh, Sliwa, Mayosi, & Yusuf, 2010). In addition to providing essential amino acids to various systemic modulatory pathways, proteins and peptides produced by the hydrolysis of food proteins may also elicit potent anticancer, antimicrobial, hypocholesterolemic, antihypertensive, antithrombotic, and anti-inflammatory effects (Möller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008). Bioactive peptides can be cleaved from polypeptide chains through gastrointestinal digestion, by fermentation or ripening during food processing, and by controlled hydrolytic processes using exogenous proteases (Pedroche et al., 2007). Many of these biologically active peptides are derived from both plant and animal sources, with most potentially stemming from milk-based products and legumes, such as soybean. Many reports have shown the potential health benefits of enzymatic hydrolysates prepared from milk, egg, and soy proteins (Möller et al, 2008, Shahidi, Zhong, 2008). Much research has focused on hydrolysates with angiotensin-converting enzyme inhibitory and antioxidant activities whereas other bioactive properties such as anti-inflammatory effect are less studied. In fact, very few studies report anti-inflammatory properties of protein hydrolysates (Vo et al, 2013, Xu et al, 2012). Regarding the bioactive properties of lupine, it was previously reported that protein extracts from white lupine can lower plasma cholesterol (Weiβe et al., 2010) and triacylglycerol concentrations (Spielmann et al., 2007) in hypercholesterolemic animal models. Furthermore, lupine has shown anti-atherogenic effects in laboratory animals due to its lipid-lowering properties. In this study, Marchesi et al. (2008) demonstrated that a protein isolate from lupine reduces focal plaque development in the common carotid arteries in a rabbit model of atherosclerosis. However, no information has been reported about the anti-inflammatory properties of lupine proteins and peptides.
Inflammation is an important normal immune response during lesions and infections. However, an excessive inflammation can contribute to several acute and chronic diseases characterized by uncontrolled production of pro-inflammatory cytokines, eicosanoids derived from arachidonic acid, reactive oxygen species (ROS) and adhesion molecules (Calder, 2006). Therefore, inhibitors of the pro-inflammatory cytokines have been considered as a candidate of anti-inflammatory drugs. Chronic inflammation is a hallmark of several pathologies, such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and cancer. The macrophage is the key player of the chronic inflammatory response, which the monocytic cell line THP-1 represents an appropriate model system to study immune responses (Weldon, Mullen, Loscher, Hurley, & Roche, 2007). It is well known that macrophages are key players during inflammatory responses and their phenotype determines the cytokine secretion profile. Thus, classically activated M1 macrophages contribute to the development and enhancement of inflammatory and immunity processes and are, therefore, associated with high microbicidal activity, supporting the activity of Th1 cells (Mills, Kincaid, Alt, Heilman, & Hill, 2000). This type of activation is associated with an elevated production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF), IL-6, and IL-1; ROS; and nitrogen intermediates (Cathcart, 2004). Alternative macrophage activation (M2) is triggered in response to IL-4 or IL-13 and is associated with tissue remodeling and immunoregulation (Gordon & Martinez, 2010). M2 macrophages produce anti-inflammatory cytokines, such as IL-10, chemokine (C-C motif) ligand 18 (CCL18), and IL-1 receptor antagonist. Due to their anti-inflammatory profile, M2 macrophages are frequently associated with all types of activation triggered by anti-inflammatory stimuli.
The increase in the incidence of inflammation related disorders has led to the search of proteins and peptides with anti-inflammatory properties (Ndiaye, Vuong, Duarte, Aluko, & Matar, 2012). There is evidence of the ability of distinct food compounds, including proteins, to modulate inflammation in experimental models involving macrophages (Boesch-Saadatmandi et al, 2011, Hämäläinen et al, 2011, Yu et al, 2002). Indeed some peptides with anti-inflammatory activity have been purified from plants (Dia, Wang, Oh, de Lumen, & Gonzalez de Mejia, 2009).
In a previous paper, we have described that blue lupine protein hydrolysates (LPHs) inhibited some enzymes involved in the inflammatory pathway, such as phospholipase A2 and cyclooxygenase-2 (Millán-Linares, Yust, Alcaide-Hidalgo, Millán, & Pedroche, 2014). In this work, we investigated the potential anti-inflammatory activity of two lupine protein hydrolysates (LPHs) in a THP-1-derived macrophage model. LPHs were obtained by hydrolysis of lupine protein isolate (LPI) with Izyme AL and Alcalase 2.4 L, two food-grade proteases produced by Novozymes. Izyme AL has trypsin-like activity whereas Alcalase is a non-specific endoprotease. Both trypsin and Alcalase have previously been used for the generation of bioactive peptides (Korhonen & Pihlanto, 2006).
Section snippets
Materials
LPI was prepared according to Yust, Pedroche, Millán-Linares, Alcaide-Hidalgo, and Millán (2010). Izyme AL and Alcalase 2.4 L were provided by Novozymes (Bagsvaerd, Denmark). The cell type used was THP-1 monocytes, ATCC® Number TIB-202TM. The medium for this line was Gibco® RPMI 1640 (Life Technologies SA, Alcobendas, Spain). PBS, fetal bovine serum (FBS), and penicillin/streptomycin (P/S) solution were obtained from Gibco® as well. Dimethyl sulfoxide (DMSO),
LPHs do not alter the cellular integrity of THP-1-derived macrophages
Before assessing the anti-inflammatory activity, cell viability and the potential cytotoxicity of the LPHs were evaluated (Gülden & Seibert, 2003).
To investigate whether LPHs may cause a cytotoxic effect, an MTT assay was performed on THP-1-derived macrophages after separately adding each LPHs at increasing concentrations to conditioned RPMI culture medium for 24 h. The LPHs I + 15A and 15A had no significant effects (Figs. 1A, B) compared with the untreated control group. Moreover, using flow
Acknowledgments
This work was supported by the grant AGL2012–40247-C02-01 from the Spanish Ministry of Economy and Competitiveness. We thank Prof. F.J.G. Muriana for his critical reading of this manuscript.
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