The Potential of Food Protein-Derived Bioactive Peptides against Chronic Intestinal Inflammation

Inflammation can cause various chronic diseases like inflammatory bowel diseases. Various food protein-derived bioactive peptides (BAPs) with anti-inflammatory activity have the potential to manage these diseases. The aim of this paper is to overview the mechanisms and the molecular targets of BAPs to exert anti-inflammatory activity. In this review, the in vitro and in vivo effects of BAPs on intestinal inflammation are highlighted. The mechanism, pathways, and future perspectives of BAPs as the potential sources of therapeutic treatments to alleviate intestinal inflammation are provided, including nuclear factor-κB, mitogen-activated protein kinase, Janus kinase-signal transducer and activator of transcription, and peptide transporter 1 (PepT1), finding that PepT1 and gut microbiota are the promising targets for BAPs to alleviate the intestinal inflammation. This review provides a comprehensive understanding of the role of dietary BAPs in attenuating inflammation and gives a novel direction in nutraceuticals for people or animals with intestinal inflammation.


Introduction
Inflammation is a normal immune defense that is generated from the immune system responding to pathogen and infection. Inflammation can cause various chronic diseases, such as inflammatory bowel diseases (IBD), asthma, cancer, cardiovascular diseases, obesity, and diabetes [1]. The intestinal mucosa can be damaged by IBD with chronic inflammatory disorders, including ulcerative colitis (UC) and Crohn's disease (CD). UC is an inflammation of the colon mucosa and submucosa continuity affecting the rectal area, while CD is a full-thickness inflammation discontinuity affecting the terminal ileum and colon or anus [2]. Until now, the aetiology of intestinal inflammation and IBD remains unclear.
In the intestines of human and animals, dietary proteins are digested into free amino acids and peptides by enzymatic hydrolysis. Some peptides consisting of 2-20 AAs with bio-logical function are named bioactive peptides (BAPs), such as anti-inflammation, antihypertension, antioxidation, antidiabetics, anticancer, antimicrobics, antiadhesion, dipeptidyl peptidase IV inhibition, opioid, and immunomodulation [3]. Conventional drug treatments have adverse side effects, such as potential toxicity and immunogenicity [4]. In recent years, BAPs have attracted more and more attention to treat chronic inflammation diseases as a result of their safety [1,5].
However, limited information of the anti-inflammatory mechanisms of the action of these BAPs is available. In this review, food protein-derived BAPs against intestinal inflammation in vitro and in vivo are discussed. Their molecular targets and the action pathways are overviewed and highlighted. Understanding of the anti-inflammatory actions of BAPs can facilitate further research on managing chronic intestinal inflammation and diseases. Therefore, the purpose of this paper is to highlight the roles of BAPs in anti-inflammatory activity and provide future perspectives for the application of BAPs as potential sources of therapeutic management of chronic intestinal diseases.
Progression of inflammation has four steps: inducers, pathways, mediators, and inflammatory response [7]. The inducers (LPS, dextran sodium sulfate (DSS), 2,4,6-trinitrobenzene sulfonic acid (TNBS), or toxicant) stimulate the sensors that can activate pathways, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). Then, inflammatory mediators (IL-8, TNF-α, monocyte chemoattractant protein-1 (MCP-1), or reactive oxygen species (ROS)) are released, leading to the inflammatory response [7]. Proinflammatory cytokines produced mainly by macrophages and mast cells lead to inflammation, while anti-inflammatory cytokines, such as IL-4, IL-10, and transforming growth factor β (TGF-β), reduce the production of procytokines in macrophage cells as agonists of toll-like receptor [7]. In in vivo studies, TNBS and DSS are commonly used to induce intestinal inflammation in animal models, causing immune alterations, gut physiology and morphology changes, and colitis symptoms [8]. Moreover, administration of DSS can lead to higher intraluminal IgG [9]. In UC patients, IgG production is dramatically high in the gut; therefore, IgG is an index to grade IBD. Thus, these cytokines with pathology may be the targets for BAPs to prevent chronic inflammation. In addition, it is also known that oxidative stress is associated with chronic intestinal inflammation, and it can decrease antioxidant defenses in the colonic mucosa. Additionally, ROS are released from immune cells and can be overwhelmed by oxidative stress. Therefore, antioxidative BAPs are the candidates for antioxidant defense in inflammatory gut [10], such as IRW [11], IQW [12], EAMAPK, and AVPYPQ [13]. Soybean-derived lunasin can also enhance antioxidant defenses and inhibit inflammation [14,15].
Along with the above two pathways, the JAK-STAT pathway is also important for inflammatory response and can regulate hematopoietic cell development and inflammatory cytokines. Phosphorylation of JAK and STATs can form the dimer translocated to the nucleus [38]. BAPs can attenuate inflammation by inhibiting phosphorylation of JAK and STATs. However, the role of this pathway needs further verification for the anti-inflammation of BAPs. The translocations and activation of the substrate proteins from the above three pathways, including transcription factors in the nucleus (AP-1, ATF-2, EIK1, and c-Jun), cause the change of target genes, reducing the productions of proinflammatory cytokines, including IL-1β, IL-2, IL-5, IL-8, IL-12, IL-13, IL-17, TNF-α, MCP-1, and IFN-γ. The overexpression of these proinflammatory mediators and the downexpression of anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) can lead to intestinal inflammation. Through regulating these pathways and cytokines, BAPs can attenuate chronic intestinal inflammation and diseases.

Mechanism of Food-Derived Anti-Inflammatory Peptides to Exert Bioactivities
The potential anti-inflammatory mechanisms of BAPs derived from food proteins through regulating various cytokines or systems are shown in Figure 3 [7,48]. The secretions and expressions of proinflammatory cytokines IL-1β, IL-2, IL-5, IL-6, IL-8, IL-12, IL-17, TNF-α, and IFN-γ can be inhibited by BAPs, as well as the activations of NF-κB and MAPK pathways, COX-2, ROS, iNOS, and nitric oxide (NO). ROS are associated with inflammatory diseases, and NO is synthesized by NO synthase (NOS) enzyme (iNOS), and the inhibition of iNOS and ROS activities can suppress NO production. BAPs can also inhibit the expression and release of a transcription factor that drives treg phenotypic differentiation (Foxp3) and T-helper-cell-associated cytokines (Th1, Th2, and Th17) and the secretions of IgG, IgE, and IgA. On the other side, secretions and expressions of anti-inflammatory cytokines (IL-4, IL-10, and TGF-β), CD4 + /CD8 + , numbers of macrophages, and superoxide dismutase (SOD) activity can be increased by BAPs. In addition, the gut microbiome, which is an active topic in health, can be normalized by BAPs [7,48]. In conclusion, these cytokines and pathways are the molecular targets and mechanisms for BAPs to regulate the intestinal inflammation of human and animals. Milk-derived VPP and IPP can exhibit beneficial effect in an animal colitis model through anti-inflammatory action for these targets [49]. VPP also reduced TNF-α and IL-1β expression and macrophage accumulation and activation, inhibited adipose inflammation in mice via angiotensinconverting enzyme-dependent cascade [31], and moderated monocyte adhesion to inflamed endothelia via the MAPK-JNK pathway [50]. In addition, tripeptides IRW and IQW downregulated the expression of inflammatory proteins via the NF-κB pathway [12,24]. Generally, these BAPs can inhibit the expression of cytokines and mediate the NF-κB and MAPK pathways [1].

The In Vivo Studies of BAPs on Inflammation
For the in vivo studies of BAPs, various inflammatory models have been used, typically colitis in mice induced by DSS and TNBS. As observed in human CD, the administration of TNBS to mice can release proinflammatory cytokines, followed by infiltration of T cell CD4 + phenotype. In these studies, the mice with colitis were orally administered with BAPs mostly with an amount of 50-500 mg/kg body weight/day for several days to weeks (Table 2). Then, the tissues are collected for common evaluation of anti-inflammation of BAPs using morphological, immunological, and biochemical assays [51], such as body weight, colonic length, disease activity index (DAI), lymphocyte proliferation, CD4 + /CD8 + determination, secretory-immunoglobulin-A      Table 2).
Oral administration of dipeptide AQ reduced inflammatory cytokine expression, enhancing the mucosa recovery in DSS-induced mice [53]. Likewise, intravenous infusion with AQ to calves with early weaned stress can increase concentrations of IgA, IgG, s-IgA, CD2 + and CD4 + lymphocytes, and CD4 + /CD8 + ratio; therefore, the diarrhea occurrence was decreased [52]. Bean protein is also a rich resource for BAPs. For example, bean-and yeast extract-derived flavor peptide γ-EC and γ-EV can inhibit the inflammation in IBD mice [47]. Soybean-derived dipeptides and tripeptides decreased the colonic expressions of proinflammatory IFNG, IL-1B, IL-12B, TNF, and IL-17A and MPO activity and increased Foxp3 expression and CD4 + CD25 + T cells; therefore, the colon and ileum inflammation of piglets with DSS-induced colitis was attenuated [74]. In addition, with the infusion of 150 mg/kg of egg white protein-derived EWP, weight loss, crypt distortion, IL-6 and TNF-α concentrations, and expressions of IL-1β, IL-8, IL-17, and IFN-γ in the colon of piglets with DSS-induced colitis can be reduced, and gut barrier function was restored [55], as well as the barrier protection effects of milk-derived β-Casofensin [77] and dipeptide AQ [53]. Therefore, food-derived BAPs can contribute to disease treatment through modifying intestinal barrier function [79].
Milk protein is a rich source for BAPs, which has potential beneficial effects to the gut of humans and animals [80,81]. Milk casein-derived VPP and IPP are two famous BAPs with antihypertensive and anti-inflammatory activities. Proinflammatory IL-6 and IL-1β were reduced, and atherosclerosis was attenuated by oral administration of VPP and IPP [71]. Arterial dysfunction was attenuated by drinking water with VPP and IPP through increasing vasorelaxation and nitrite and nitrate and reducing pulse wave velocity and cardiac and renal damage [72]. It was reported that VPP attenuated inflammation via the MAPK-JNK pathway by reducing monocytes, macrophages, CD18, IL-6, and MCP-1 in adipose inflammatory mice [46]. Milk casein-derived QEPVL and QEPV reduced nitric oxide (NO) release, increased anti-inflammatory IL-4 and IL-10 production, and decreased productions of IFN-γ and TNF-α in LPS-induced mice [6]. Milk κ-casein-derived glycomacropeptide inhibited inflammation and attenuated colitis via normalizing the inflammatory cytokine and the NF-κB and MAPK pathways in previous studies [57][58][59][60].
From these in vivo studies, the evidences that the intestinal inflammation can be attenuated by oral administration of food protein-derived BAPs have been presented. As many studies have been performed recently, large-scale human and animal trials are still lacking [2]. It has been reviewed that numbers of BAPs can be transported into the bloodstream of humans or animals to exert bioactivities [3,81]. However, there is still limitation for such in vivo studies due to the possible degradation of BAPs by peptidases in the gut and plasma or insufficient absorption [82]. In the future, more studies of humans and animals are needed to evaluate the anti-inflammatory effects of BAPs, as well as the doses, times, and kinetics in the body.
It was reported that anti-inflammatory tripeptide KPV can attenuate intestinal inflammation associated with PepT1 expression, and KPV lost the anti-inflammatory function without PepT1 expression, suggesting that PepT1 mediates the anti-inflammation of KPV [66]. It was reported that soy protein-derived tripeptide VPY exerted anti-inflammatory activity in cells also through PepT1, which can transport VPY into cells [33]. In addition, pharmacological inhibition of PepT1 can counteract the inhibition of IL-8 expression mediated by peptide IPAV [23]. Moreover, the antiinflammatory effect of meat-derived carnosine (β-Ala-His) was inhibited by dipeptide Gly-Sar, a PepT1 substrate [35]. These findings indicate that PepT1 is a promising target to treat intestinal inflammation by transporting sufficient short-chain BAPs into colonic cells [10]. In conclusion, PepT1 is a possible mechanism for the inhibition of intestinal inflammation by BAPs. However, this PepT1 pathway involved in anti-inflammation of BAPs still needs to be verified by further researches in the future (Figure 2).

Impact of Anti-Inflammatory Peptides on Gut Microbiota
When intestinal inflammation or IBD occurs, the gut microbial community would also change, such as the decrease of Firmicutes (particularly Clostridium groups) and the increase of Bacteroides, Lactobacillus, Eubacterium, and Proteobacteria [91]. In DSS-induced colitis mice, compositions and varieties of the gut microorganism  Figure 3: The potential mechanisms of anti-inflammatory action of food-derived bioactive peptides. CD4 + /CD8 + : splenic T lymphocyte subpopulations; COX-2: cyclooxygenase-2; Foxp3: a transcription factor that drives treg phenotypic differentiation; iNOS: inducible oxide nitric synthase; IFN-γ: interferon-γ; IL-1β: interleukin-1β; MAPK: mitogen-activated protein kinase; MPO: myeloperoxidase; NF-κB: nuclear factor-κB; NO: nitric oxide; ROS: reactive oxygen species; SOD: superoxide dismutase; TNF-α: tumor necrosis factor α; TGF-β: transforming growth factor β; Th1/2/17: T-helper-cellassociated cytokine 1/2/17. This figure was adapted from previous reports [7,48].   Treat IBD via gut microbiota [76] 10 Mediators of Inflammation (Anaerotruncus, Bacteroides, Enterobacteriaceae, Lactobacilli, and Parabacteroides) have changed [92]. In general, when defensins decline, the abundance of bacteria from Bacteroides and Firmicutes would be increased [93]. It was reported that BAPs can exert anti-inflammation via changing the gut microbiota in several studies [62,63,76]. For example, oral administration of anti-inflammatory peptide pyroGlu-Leu derived from wheat gluten can normalize the population of Bacteroidetes and Firmicutes in the colon of colitis mice [76]. Shannon and Simpson indices represent species richness and species evenness, respectively. The Simpson index and the abundance of Coprococcus-1, Desulfovibrio, and Ruminococcaceae-UCG-014 were increased by tripeptides IRW and IQW. Additionally, IQW decreased the abundance of Bacteroides and increased Parabacteroides, while the levels of Anaerotruncus, Ruminiclostridium-9, and Oscillibacter were increased by IRW [63]. Firmicutes and Actinobacteria species were increased, and the proportions of Bacteroidetes and Proteobacteria species were decreased by oral administration of IRW and IQW; therefore, the colonic inflammation was inhibited via regulation of intestinal microorganisms [62]. In addition, dietary dipeptide GQ changed the gut microbiota beneficially through increasing alpha diversity, bacterial loading, abundance of anaerobes and fiber-degrading bacteria (Phylum Fibrobacteres), and short-chain fatty acids in the gut [94].

Mediators of Inflammation
In conclusion, the gut microbiota is a promising mechanism for BAPs to inhibit intestinal inflammation. However, the information of the mechanism underlying the effects of BAPs on gut microbiota is still lacking, and it needs more studies to explore the interaction between antiinflammation of BAPs and gut microbiota in the future.

Conclusions and Future Perspectives
In this review, the mechanism and pathways of food proteinderived BAPs to exert anti-inflammatory bioactivities were highlighted, including pathways (NF-κB, MAPK, and JAK-STAT), PepT1, inflammatory mediators, and gut microbiota. Moreover, various in vitro and in vivo studies of BAPs on inflammation were reviewed, finding that PepT1 and gut microbiota are promising targets for the inhibition of BAPs on intestinal inflammation; however, their roles still need more further studies to be verified in the future.
The discovery of novel BAP sequences and their corresponding action mechanisms as well as gut microbiota and PepT1 involved in the mediation can provide new opportunities for better targeting of intestinal inflammation. More in vivo data, including pharmacokinetics and proper dosage and time of administration of BAPs, are needed before their application to humans and animals. The role of dietary BAPs in inhibiting intestinal inflammation represents a novel direction in nutraceuticals for people or animals with intestinal inflammation.

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Mediators of Inflammation