Impact of the Plastein Reaction of Casein Hydrolysates in the Presence of Exogenous Amino Acids on Their Anti-Inflammatory Effect in the Lipopolysaccharide-Stimulated Macrophages

Abstract

In this study, papain-generated casein hydrolysates (CH) with a degree of hydrolysis of 13.7% were subjected to a papain-mediated plastein reaction in the absence or presence of one of the exogenous amino acids—Gly, Pro, and Hyp—to prepare four plastein modifiers, or mixed with one of three amino acids to prepare three mixtures. The assay results confirmed that the reaction reduced free NH2 for the modifiers and caused amino acid incorporation and peptide condensation. When RAW264.7 macrophages were exposed to the CH, modifiers, and mixtures, these samples promoted macrophage growth and phagocytosis in a dose-dependent manner. In addition, the CH shared similar activity in the cells as the mixtures, while the modifiers (especially the PCH-Hyp prepared with Hyp addition) exerted higher potential than CH, the mixtures, and PCH (the modifier prepared without amino acid addition). The plastein reaction thus enhanced CH bioactivity in the cells. When RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS), the inflammatory cells produced more lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) formation, and caused more four inflammatory mediators (NO, PGE2, TNF-α, and IL-6) and two anti-inflammatory mediators (TGF-β1 and IL-10). However, the PCH-Hyp, PCH, and CH at dose levels of 100 μg/mL could combat against the LPS-induced inflammation. Overall, the PCH-Hyp was more active than the CH and PCH in reducing LDH release, ROS formation, and the secretion of these inflammatory mediators, or in increasing the secretion of the anti-inflammatory mediators. The qPCR and Western blot analysis results further confirmed that these samples had anti-inflammatory effects on the stimulated cells by suppressing the LPS-induced activation of the NF-κB signaling pathway, via regulating the mRNA/miRNA expression of iNOS, IL-6, TNF-α, IL-1β, COX-2, TLR4, IL-10, TGF-β1, miR-181a, miR-30d, miR-155, and miR-148, as well as the protein expression of MyD88, p-IKKα, p-IκBα, p-NF-κB p65, and iNOS, involved in this signaling pathway. In addition, the immunofluorescence assay results revealed that these samples could block the LPS-mediated nuclear translocation of the p65 protein and displayed the same function as the NF-κB inhibitor BAY 11-7082. It was concluded that CH could be endowed with higher anti-inflammatory activity to the macrophages by performing a plastein reaction, particularly that in the presence of exogenous Hyp.

Antidiabetic, angiotensin‐converting enzyme inhibitory and anti‐inflammatory activities of fermented camel milk and characterisation of novel bioactive peptides from lactic‐fermented camel milk with molecular interaction study

“…(2012), who assessed the anti‐inflammatory effects of Oyaksung which was fermented with Lactobacillus on RAW 264.7 cells. Similarly, Shi and Zhao (2022) had reported the anti‐inflammatory property of casein hydrolysates using RAW 264.7 cells against LPS‐induced inflammation. Hence, this outcome indicated its profound role of anti‐inflammatory response in the development and progression of inflammatory conditions.…”

Casein Lactose-Glycation of the Maillard-Type Attenuates the Anti-Inflammatory Potential of Casein Hydrolysate to IEC-6 Cells with Lipopolysaccharide Stimulation

“…In LPS-treated IEC-6, bovine lactoferrin also had an anti-inflammatory effect by reducing the secretion of IL-6, TNF-α, and IL-1β, as well as suppressing MAPK activation and the nuclear translocation of NF-κB [ 57 ]. When casein hydrolysates were modified by plastein reactions in the presence of exogenous amino acids, the obtained modifiers demonstrated greater anti-inflammatory activity against LPS-induced macrophage inflammation, mostly by controlling IL-6 and TNF-α production and preventing the activation of the NF-κB signaling pathway [ 58 ]. However, certain protein by-products generated during the Maillard process seem to have an ability to encourage the undesirable cellular inflammatory response.…”

Preventative and therapeutic potential of animal milk components against COVID‐19: A comprehensive review

“…Oxidative stress and inflammation occur concomitantly and act as key players in many disease conditions including COVID‐19 (Delgado‐Roche & Mesta, 2020 ). So far a number of scientific studies have reported the antioxidant (free radical scavenging and metal chelating activities) and anti‐inflammatory activities of CN‐derived peptides (Altmann et al, 2016 ; Bamdad et al, 2017 ; Chen et al, 2022 ; Li, Cheng, et al, 2017 ; Mukhopadhya et al, 2015 ; Oh et al, 2017 ; Shi & Zhao, 2022 ; Yoo et al, 2021 ). Mao et al ( 2011 ) prepared yak milk CN hydrolyzates with enzyme alcalase and observed their significant free radical scavenging activities in terms of 2,2‐diphenylpicrylhydrazyl, superoxide, and hydrogen peroxide (H 2 O 2 ).…”