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Inflammation is a complex and highly regulated adaptive response that is triggered by a variety of stimuli including pathogens, noxious mechanical and chemical agents. Macrophages play an important role in the initiation, maintenance and resolution of inflammation. Various egg white proteins, including ovalbumin, ovotransferrin, ovomucin, lysozyme, and avidin, have been reported to have impact on inflammation. Several egg yolk components, including the carotenoids lutein and zeaxanthin, and bioactive phospholipids have been shown to possess anti-inflammatory property.The objective of this study was to demonstrate the anti-inflammatory activity of egg yolk livetins and its enzymatic hydrolysates in LPS-stimulated murine RAW 264.7 macrophage, as in vitro model.
Freshly laid white shell eggs were obtained. Egg yolks were manually separated from the white and carefully rolled on Whatman paper (grade No. 4) to remove all albumen from the vitellin membrane. Egg yolk livetins were extracted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of livetins and its hydrolysates was performed under reducing conditions using 4–20% Mini-PROTEAN TGX precast protein gels. Then the effects of livetins and their peptides on the proliferation of RAW 264.7 cells were tested in cell culture. Inflammatory (TNF-α, IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines levels were determined in cell culture supernatants using solid phase sandwich ELISA kits from Invitrogen Life Technologies Corp. according to the manufacturer’s instructions.
A significant increase (p < 0.001, p < 0.01, p < 0.05) of phagocytosis activity of RAW 264.7 cells was noticed in livetins and its enzymatic hydrolysates treatment groups in comparison with the untreated control group. The results demonstrated that lower concentrations of livetins (25 μg/mL), pepsin peptides (25, 50 and 100 μg/mL), and alcalase peptides (25 μg/mL) significantly stimulated phagocytosis whereas higher concentrations (200 and 400 μg/mL) did not show effect on the phagocytosis in RAW 264.7 cells. Livetins most significantly inhibited NO production by 39.2%, followed by pepsin (32.9%) and Alcalase (22.7%) hydrolysates. This suggested that livetins and its hydrolysates inhibit the NO production in LPS induced RAW 264.7 cells. Livetins and its enzymatic hydrolysates dose-dependently inhibited LPS-induced TNF-α, IL-1β, and IL-6 production compared with the LPS control. Livetins, pepsin and Alcalase hydrolysates exhibited maximum inhibition of TNF-α (37.1%, 43.2% & 36.9%), IL-1β (26.1%, 28.2% & 50.9%), and IL-6 (63.3%, 60.4% & 69.0%) at concentration of 400 μg/mL. Livetins and pepsin hydrolysate significantly (p < 0.001) decreased the IL-10 secretion in a dose dependent manner, whereas there was no significant difference in Alcalase hydrolysate treated cells, in comparison with LPS control group. In the present study, livetins and its enzymatic hydrolysates decreased the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in a similar way to how they reduced NO production.
The present study demonstrated, for the first time, that livetins and its enzymatic hydrolysates possess anti-inflammatory and phagocytosis enhancing capacity. The current study results suggest that egg yolk livetins fraction (including IgY) and its enzymatic hydrolysates exhibit anti-inflammatory effects in RAW 264.7 macrophages through inhibition of NO/iNOS and PGE2/COX-2 pathways as well as inhibition of the production of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Livetins fraction and its enzymatic hydrolysates enhanced phagocytic capacity of the macrophages. The present study results showed that livetins and its enzymatic hydrolysates with anti-inflammatory and immune enhancing properties could be useful in health foods/nutraceuticals to control the inflammation and inflammatory markers. However, further in vivo animal experiments are needed to confirm their benefits before being exploited for human nutrition.