Impact of a Papain-Catalyzed Plastein Reaction of Salmon Skin Gelatin Hydrolysate and Exogenous Amino Acids on Anti-Inflammatory Activity in LPS-Stimulated Macrophages

The hydrolysate of salmon skin gelatin (HSSG) was generated by hydrolyzing salmon skin gelatin with papain and then modified using a papain-catalyzed plastein reaction with or without one of the three exogenous Gly, Pro, and Hyp. The prepared samples including HSSG, the HSSG modifier alone (namely, PHSSG), three HSSG modifiers with one of the exogenous amino acids (namely, PHSSG-Gly, PHSSG-Pro, and PHSSG-Hyp), and three mixtures of HSSG plus one of the exogenous amino acids (namely, HSSG-Gly, HSSG-Pro, and HSSG-Hyp) were thus assessed for their activities toward the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. These samples were all nontoxic to the macrophages but showed different abilities to promote cell growth. LPS promoted macrophage phagocytosis and lactate dehydrogenase release and enhanced the production levels of intracellular reactive oxygen species, NO, prostaglandin E2, and four inflammatory cytokines including TNF-α, IL-6, TGF-β1, and IL-10. Meanwhile, HSSG, PHSSG, and PHSSG-Hyp alleviated the LPS-induced macrophage inflammation by regulating the expression of 12 inflammation-related genes including iNOS, IL-6, TNF-α, IL-1β, COX-2, TLR4, IL-10, TGF-β1, miR-181a, miR-30d, miR-155, and miR-148a, downregulating the expression of five proteins including p-NF-κB-p65, MyD88, p-IKKα, p-IκBα, and iNOS that are associated with cell inflammation and reducing the nuclear translocation of p65 in the nucleus. Among the three evaluated samples, PHSSG-Hyp and HSSG had the respective highest and lowest anti-inflammatory activities in the LPS-induced macrophages via inactivating the NF-κB signaling pathway. It was thus concluded that the plastein reaction used in the presence of Hyp endowed HSSG with the highest anti-inflammatory activity in the macrophages, highlighting the potential use of this reaction in protein hydrolysates to reach activity enhancement and to produce functional peptide products.