Dietary Bioactive Peptide Alanyl-Glutamine Attenuates Dextran Sodium Sulfate-Induced Colitis by Modulating Gut Microbiota

Inflammatory bowel disease (IBD) is a chronic intestinal disorder threatening human health. Di-peptide alanyl-glutamine (Ala-Gln) has various beneficial effects on gut health. However, its role and functional mechanism in treating IBD are still not clear. Therefore, the protective effects of Ala-Gln and glutamine (Gln) on dextran sulfate sodium- (DSS-) induced colitic mice were investigated in this study. The results showed that oral supplementation of Ala-Gln or Gln significantly attenuated the colitis symptoms in mice, including body weight loss, colon length, disease activity index, histological scores, and tissue apoptosis. The concentrations of interleukin- (IL-) 1β, IL-6, tumor necrosis factor-α, and myeloperoxidase were significantly decreased, while the concentrations of immunoglobulins (IgA, IgG, and IgM) and superoxide dismutase were significantly increased by Ala-Gln or Gln supplementation. The expression of occludin and peptide transporter 1 (PepT1) was significantly increased by Ala-Gln or Gln. Interestingly, Ala-Gln had better beneficial effects than Gln in alleviating colitis. In addition, 16S rDNA sequencing showed that the DSS-induced shifts of the microbiome (community diversity, evenness, richness, and composition) in the mouse colon were restored by Gln and Ala-Gln, including Lactobacillus, Bacteroides_acidifaciens, Bacteroidales, Firmicutes, Clostridia, Helicobacter, and Bacteroides. Correspondingly, the functions of the microflora metabolism pathways were also rescued by Ala-Gln, including fatty acid metabolism, membrane transporters, infectious diseases, and immune system. In conclusion, the results revealed that Ala-Gln can prevent colitis through PepT1, enhancing the intestinal barrier and modulating gut microbiota and microflora metabolites.


Introduction
Inflammatory bowel disease (IBD, including ulcerative colitis) is a chronic gastrointestinal disorder caused by inflammation or oxidative stress in the colon, which has threatened the human health and has various colitis symptoms, such as gut bleeding, bloody diarrhea, body weight (BW) loss, epithelial cell loss, neutrophil infiltration, and the release of proinflammatory mediators (e.g., interleukin-(IL-) 1β, IL-4, IL-5, IL-6, IL-8, and tumor necrosis factor-(TNF-) α) [1]. However, the use of drugs to treat IBDs often has side effects. The application of bioactive peptides with the potential to manage chronic intestinal inflammation comes into people's view [1].
Glutamine (Gln) is a well-studied amino acid with immune-modulating effects [2,3], and it can alleviate intestinal inflammation [4]. The Gln can be utilized to maintain the intestinal structure and function [5], and it also can mediate protein synthesis by intestinal microorganisms [6]. Gln used in human and animal studies is often in the form of alanylglutamine (Ala-Gln) [3]. However, the difference of the effects between Ala-Gln and Gln is still not clear. Intriguingly, Ala-Gln has a superior preventive effect on intestinal damage with more mucosal weight, protein content, and villus height than those in mice with alanine plus Gln mixture [7]. Ala-Gln had a better effect on improving enterocyte proliferation than Gly-Gln or Gln [8]. In other words, Ala-Gln may serve as a bioactive peptide and has better effects than Gln or alanine alone.
It was reported that Ala-Gln can alleviate inflammation via inhibiting cytokine expression and regulating T cells in colitic mice [3,9,10] and alleviates the allergic inflammation of the airways and lungs through modulating gut microbiota and their metabolites in mice [11]. The Ala-Gln can attenuate metabolic stress, enhance immunity [12], and inhibit intestinal mucositis in mice [13]. In addition, Ala-Gln can also improve the gastrointestinal epithelial structure and immune status of early-weaned calves [14]. In a number of studies, dextran sulfate sodium-(DSS-) induced mouse colitis is a widely used model to study IBD with the features of ulcerative colitis [1,15]. The peptide Ala-Gln has attracted an increasing interest of scientists. However, the role and functional mechanism of Ala-Gln on colitis are still unclear, particularly in the gut microbiota. Therefore, the objective of this study is to explore the effects of Ala-Gln on DSSinduced acute colitis in mice.

Disease Activity Index (DAI) and Colon Histologic
Analysis. The DAI was assessed with the score sum according to BW loss, stool consistency and bleeding, and mouse condition as previous reports [16,17]. After sacrificing mice on day 15, the colon length was measured, and the colon tissue (~3.5 cm proximal to the anus) was fixed with 10% buffered formalin and stained with hematoxylin and eosin (HE) to obtain the morphology structure. The tissue damage was evaluated using the degree of inflammatory cell infiltration based on previous descriptions [16,18]. In addition, the cell apoptotic level was assessed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay by using a cell death detection kit with the nuclei stained with DAPI (Roche, Basel, Switzerland). The morphology was observed using fluorescent microscopy (DMIL LED, Leica, Germany).
2.3. Enzyme-Linked Immunosorbent Assay (ELISA). The serum was obtained with blood being centrifuged for 15 min at 1500 g. The concentrations of immunoglobulin (Ig) A, IgG, and IgM in the serum were detected by using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The contents of IL-1β, IL-6, TNF-α, superoxide dismutase (SOD), and myeloperoxidase (MPO) in colon tissues were measured by using ELISA kits according to the manufacturer's instructions [19]. The content of tissue protein was measured using the Bicinchoninic Acid Protein Assay Kit (P0011, Beyotime, Shanghai, China) and analyzed using a microplate reader (Bio-Rad, 680, Hercules, CA, USA).
2.5. Gut Microbiota. The DNA of mouse colon luminal content was extracted by using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The primers targeting V3 to V4 regions of the 16S rDNA sequence were amplified using polymerase chain reaction (PCR). The PCR products were purified using Agencourt AMPure XP and sequenced using a HiSeq platform. The raw data was filtered, and the Operational Taxonomic Units (OTUs; >97% similarity) were clustered by using USEARCH and the data was analyzed by using an RDP classifier (>80% confidence). The diversity indices of alpha and beta were analyzed using Quantitative Insights into Microbial Ecology (QIIME; http://qiime.org/). The distances among samples were computed using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). In addition, linear discriminant analysis (LDA) effect size (LEfSe) was used to determine taxa, which can characterize each population (LDA score > 4) to discover biomarkers. A Venn diagram was drawn by using R software and used to present OTU overlap among samples. Additionally, the cladogram plot was drawn using Figure   2 Oxidative Medicine and Cellular Longevity Tree software to identify corresponding group biomarkers (http://tree.bio.ed.ac.uk/software/figtree/). The taxonomical levels were carried out at the phylum, class, order, genus, and species levels. Moreover, the function of the metagenome was predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt; http://picrust.github.com) and Kyoto Encyclopedia of Genes and Genomes (KEGG) levels 1, 2, and 3.
2.6. Statistical Analysis. Data analysis was performed by Student's t-test between two groups and one-way ANOVA followed by Tukey's comparison among multiple groups by using SPSS software (SPSS Inc., Chicago, IL, USA). The results were presented as means ± standard error of mean (SEM). p < 0:05 was considered statistically significant.

Ala-Gln Alleviated DSS-Induced Mouse Colitis.
The experiment was carried out according to a timeline as shown in Figure 1(a). Colitis symptoms had appeared in mice after oral treatment with DSS for a week, including low BW ( Figure 1(b)), short colon length (Figure 1(c)), and high DAI (Figure 1(d)). However, the BW and colon length were significantly upregulated by Ala-Gln (p < 0:05) (Figures 1(b) and 1(c)). The DSS-induced high DAI was significantly decreased by Ala-Gln (p < 0:05) (Figure 1(d)). However, the treatment of Gln did not significantly alleviate DSS-induced mouse colitis. These results suggested that the DSS-induced colitis symptom was dramatically alleviated by Ala-Gln.

Colon Tissue Damage and Apoptosis.
The challenge with DSS in mice damaged colon tissue as shown in HE straining morphology, including crypt loss and leukocyte infiltration; however, these damage signs were attenuated by Gln and Ala-Gln. DSS challenge increased the colon histological index. Interestingly, this increase was significantly ameliorated by Gln and Ala-Gln ( Figure 2(a)). In addition, DSS treatment enhanced colon apoptosis as shown in TUNELpositive nuclei; however, this increase was significantly counteracted by Gln and Ala-Gln ( Figure 2(b)). These results indicated that Gln and Ala-Gln restored the DSS-induced colon damage.

Effects of Ala-Gln on Inflammatory Mediators and
Immunoglobulins. The contents of inflammatory mediators IL-1β, IL-6, and TNF-α in colon tissue of DSS-challenged mice were significantly reduced by Gln or Ala-Gln supplementation (p < 0:05) ( In mouse colon tissues, SOD activity was significantly increased and MPO concentration was significantly decreased by Ala-Gln administration (p < 0:05) (Figures 3(g) and 3(h)). These results indicated that Gln and Ala-Gln rescued the immunity status of mice. However, IL-1β, TNF-α, IgG, SOD, and MPO were not significantly affected by Gln, indicating that Gln is not so effective as Ala-Gln to suppress DSSinduced colitis.

Western Blot. The expression of the tight junction (TJ) protein occludin was significantly increased by Gln and
Ala-Gln (p < 0:05) (Figures 4(a) and 4(b)), indicating that the intestinal barrier was enhanced by Gln and Ala-Gln to alleviate the colitis. The expression of the peptide transporter PepT1 (Figures 4(a) and 4(c)) was also significantly upregulated by Gln and Ala-Gln (p < 0:05), indicating that PepT1 was involved in colitis alleviation by Ala-Gln. However, no significant difference was detected in the expression of phosphorylation MAPK signaling (p38, JNK, and ERK) (data not shown), suggesting that the MAPK signaling pathway was not involved in alleviating intestinal inflammation of Ala-Gln.
3.5. Gut Microbiota. The colon microbial community of mice was analyzed by the 16S rDNA phylogenetic method with OTU similarity higher than 97%. Alpha diversity of the microbial communities was estimated by using Shannon and Simpson indices. The Shannon index was significantly decreased by DSS treatment (p < 0:01), while the Simpson index was significantly increased by DSS (p < 0:05), indicating that DSS decreased the diversity of gut microbial community of mice (Figures 5(a) and 5(b)). Moreover, this decrease was drastically rescued by both Gln and Ala-Gln. The microbiota structure was analyzed by PCA, which showed that the gut microbiota was separated by DSS, Gln, and Ala-Gln treatments (Figures 5(c) and 5(d)). Compared with the DSS group, gut microbiota was separated by Gln and Ala-Gln treatments using OTU-based partial least squares discriminant analysis (PLS-DA) ( Figure 5(d)). In addition, the Venn diagram illustrated that 261 universal OTUs were detected out of 521 total OTUs in all samples. There were 105, 6, 3, and 11 unique OTUs in control (total 432), DSS (total 346), Gln (total 356), and Ala-Gln (total 381) groups, respectively ( Figure 5(e)). The key bacterial alterations in the taxonomic cladogram showed that Sphingobacteriaceae, Sphingobacteriales, Sphingobacteria, Turicibacteraceae, and Turicibacterales were the unique cluster markers in the Gln group ( Figure 5(f)). The LDA score of taxon abundance illustrated that DSS treatment significantly increased the abundances of Dorea and Defluviitalea, while Gln significantly increased the abundances of Ruminococcus and Coprococcus (p < 0:05), and Ala-Gln significantly increased p_75_a5 abundance (p < 0:05). Notably, compared with the control group, DSS challenge in all other groups significantly decreased the abundance of probiotics, such as Prevotellaceae, Lactobacillales, Lactobacillaceae, and Lactobacillus (p < 0:05, Figure 5(g)).
The Firmicutes and Bacteroidetes were the predominant bacteria at the phylum level, and Clostridia and Bacteroidia were the predominant bacteria at the class level, while Clostridia and Bacteroidales were the predominant bacteria at the order level (Figures 6(a)-6(c)). At the phylum level, Bacteroidetes abundance and the ratio of Bacteroidetes/Firmicutes were significantly decreased by Gln and Ala-Gln (p < 0:05, Figure 6(a)). At the class level, Clostridia abundance was significantly increased by Gln, while the abundances of Bacteroidia and Epsilonproteobacteria were significantly decreased by Gln or Ala-Gln (p < 0:05, Figure 6(b)). At the order level, the abundance of Clostridiales was significantly increased by Gln, while the abundances of Bacterioidales and 3 Oxidative Medicine and Cellular Longevity Campylobacterales (gastrointestinal pathogens) were significantly decreased by Gln or Ala-Gln (p < 0:05, Figure 6(c)). At the genus level, the abundances of pathogens Helicobacter spp. and Bacteroides spp. were increased by DSS challenge, while they were significantly decreased by Gln or Ala-Gln to counteract DSS-induced increases (p < 0:05, Figure 6(d)). At the species level, the abundance of Bacteroides_acidifaciens was significantly increased by DSS challenge, while it was significantly decreased by Gln (p < 0:05, Figure 6(e)). These results indicated that the DSS-induced change of gut microbiota was restored by Gln and Ala-Gln.  Oxidative Medicine and Cellular Longevity communities was predicted by PICRUSt. At KEGG level 1, the function of environmental information processing was significantly enhanced by Ala-Gln compared with DSS treatment (p < 0:05, Figure 7(a)). At KEGG level 2, the functions of membrane transport and enzyme families were significantly enhanced by Ala-Gln (p < 0:05, Figure 7(b)). However, the functions of replication and repair, genetic information processing, infectious diseases, immune system, and glycan biosynthesis and metabolism were significantly inhibited by Ala-Gln (p < 0:05, Figure 7(b)). At KEGG level 3, compared with the DSS group, the metabolism functions of transporters, ABC transporters, and biosynthesis of ansamycins were significantly enhanced by Ala-Gln treatment. However, the functions of DNA replication proteins, fatty acid biosynthesis, propanoate metabolism, folate biosynthesis, epithelial cell signaling in Helicobacter pylori infection, polycyclic aro-matic hydrocarbon degradation, restriction enzyme, and bacterial toxins were significantly inhibited by Ala-Gln compared with the DSS treatment (p < 0:05, Figure 7(c)). From the above results, the underlying mechanism of Ala-Gln in alleviating colitis of mice can be concluded as follows: (1) the transport of Ala-Gln via PepT1 into intestinal epithelial cells, (2) the decrease in IL-1β, IL-6, TNF-α, and MPO and the increase in IgA, IgG, IgM, and SOD, and (3) the normalization of microbiota and microbial metabolism (Figure 8).

11
Oxidative Medicine and Cellular Longevity having bioactive function; therefore, the efficiency to transport amino acids can be enhanced by peptides [20,34]. In addition, Gln is a therapeutic candidate for improving intestinal health based on its influence on repairing the intestinal epithelial wall and enhancing immunity [35]. Several studies have been performed to investigate the beneficial effects of Gln and Ala-Gln on colitis and intestinal health [3,9,15,36]. However, little is known about the difference between Gln and Ala-Gln in colitis and gut microbiota in the intestinal inflammation. In addition, a dose of 750 mg Ala-Gln/kg BW/d (equal 500 mg Gln/kg BW/d) was used to manage mice based on previous reports of the safe and effective treated dose [9,10,15].
The DSS-induced colitic mice are an intensively used model to study IBD with the evaluation of DAI, BW, colon length, signaling pathways, inflammatory cytokines, and gut microbiome in mice [1,17,37]. In this study, high DAI, short colon length, and colon tissue damage were observed in DSSchallenged mice, indicating that the colitis model was established successfully [38]. However, these IBD markers were restored by the oral supplement of Ala-Gln, indicating that the mouse colitis was attenuated by Ala-Gln. As shown in this study, it has been confirmed that Ala-Gln attenuated colon inflammation and increased colon length [9]. The cytokines IL-1β, IL-6, IL-8, and TNF-α can cause intestinal inflammation [1], and their release can induce intestinal dysfunction [37]. The intestinal inflammatory cytokine disorders always are associated with oxidative stress imbalance [39,40]. In this study, the decreases in the contents of IL-1β, IL-6, and TNF-α caused by Ala-Gln were confirmed by a previous study which showed that Gln reduced the expression of these cytokines in mice with Pasteurella multocida vaccine [41]. These results can be explained partly by Gln bioactivity, which can inhibit DSS-induced colitis via the decrease in proinflammatory cytokines and SOD [36]. The Gln has also been found to inhibit inflammatory response by regulating NF-κB activation, which can cause proinflammatory cytokine release [42], while Gln can inhibit inflammatory cytokines and is beneficial for intestinal mucosa in endotoxemic rats [43]. In the current study, the decrease in inflammatory cytokines by Ala-Gln (not by Gln with IL-1β and TNF-α) indicated that the extent of colitis was ameliorated by Ala-Gln, which has better beneficial effects than Gln. Additionally, the contents of IgA, IgG, and IgM were increased by Ala-Gln to enhance the immune system. It was reported that Gln can increase IL-6 and SIgA concentrations and protect against Escherichia coli infection via intestinal innate immunity in mice [2,44]. These results indicated that dietary Ala-Gln can ameliorate colitis via enhancing immunoglobulin levels.
The TJ barrier is important to intestinal health by modulating gut permeability and IBD pathogenesis [45], and the impairment of its integrity is a crucial reason to cause colitis. The Gln had defensive effects on the maintenance of intestinal barrier integrity in Caco-2 cells [46]. In the present study, the expression of the TJ protein occludin in the colon was upregulated by Ala-Gln. Another reason for the better suppression effect of Ala-Gln than Gln is that Ala-Gln is the substrate of intestinal PepT1, which can transport Ala-Gln efficiently into the blood circulation [20]. Although PepT1 expression can be upregulated in the IBD colon due to its transport of bacterial peptides into cells, PepT1 can also transport Ala-Gln into intestinal cells and bloodstream to inhibit inflammation [47], such as anti-inflammatory peptides VPY [29], KPV [26], IPAV [48], and β-Ala-His [49]. Therefore, PepT1 is a promising potential target for managing colitis; however, its mechanism underlying the inhibition of colitis by bioactive peptides still needs further investigation. However, in this study, no significant effect of Ala-Gln

12
Oxidative Medicine and Cellular Longevity was observed on MAPK phosphorylation, indicating that MAPK signaling is not the mechanism for Ala-Gln to ameliorate colitis. In addition, oxidative stress is another mechanism that causes IBD [50], and the SOD activity can be decreased by DSS challenge [16,22]. As an indicator for colitis extent, MPO can induce tissue damage due to the release of oxyradicals from leukocytes [3]. In this study, Ala-Gln showed defensive effects by increasing SOD, demonstrating that Ala-Gln could inhibit colitis through mediating oxidation resistance. The MPO concentration was increased by DSS and reversed by Ala-Gln, indicating that Ala-Gln has alleviated colitis inflammation. The microbiota plays a critical role in maintaining gut oxidative environment and altering chronic inflammation of human and animals [51]. In the current study, intestinal microbial composition was changed and the microbial   13 Oxidative Medicine and Cellular Longevity diversity was reduced in colitic mice. Gut microbial diversity was decreased by DSS challenge, while it was significantly increased by Gln and Ala-Gln, indicating that Gln and Ala-Gln reversed DSS-induced decrease in microbial diversity. In this study, the DSS challenge decreased the OTU numbers, while Ala-Gln treatment increased OTUs to counteract their shifts, indicating that the richness of the gut microbiome was normalized by Ala-Gln to a certain extent. It was reported that the increase in Bacteroidetes and the ratio of Bacteroidetes/Firmicutes were the indicators of IBD [17,37]. In the current study, the microbiota composition was reversed and the ratio of Bacteroidetes/Firmicutes was decreased by Ala-Gln treatment. It was also observed that Gln increased the Firmicutes/Bacteroidetes ratio, decreased the abundance of Firmicutes, and beneficially changed the bacterial community and activated the innate immunity in the intestine [52]. It was reported that the decrease in Bacteroides and the increase in Firmicutes have a defensive effect on IBD [53]. The abundance of Firmicutes in DSS-induced colitic mice was also increased by serine [37]. The decrease in Bacteroides abundance linked to intestinal inflammation can explain the colitis amelioration by Ala-Gln [17]. In our study, Gln increased Clostridiales abundance, which can promote the development of intestinal IgA-producing cells and benefits the immunological responses [54]. The Ala-Gln or Gln restored DSS-induced change of Lactobacillus, Bacteroidales, Firmicutes, Bacteroides_acidifaciens, Clostridia, Helicobacter, and Bacteroides. These results indicated that Ala-Gln alleviated the DSS-induced decrease in microbial diversity, evenness, and composition and rescued gut microbiota dysbiosis, which was confirmed by a previous study that Gly-Gln reduced the inflammatory response in piglets by modulating gut microbial community and metabolism [5]. The reason may be that Gln can regulate amino acid utilization via intestinal microorganisms [6].
The metabolism functions of gut microbiota were also inhibited by Ala-Gln, such as epithelial cell signaling in Helicobacter pylori infection, infectious diseases, polycyclic aromatic hydrocarbon degradation, and bacterial toxins. Among them, Helicobacter pylori infection is a key factor in intestinal disease etiology [55]. However, the metabolism of membrane transporters was enhanced by Ala-Gln, indicating that nutritional transporters (e.g., PepT1) may be enhanced to transport more nutrients into epithelial cells and bloodstream to resist inflammation. Furthermore, the role of gut microbiota mediated by Ala-Gln is still lacking; therefore, the modulating mechanism of Ala-Gln on gut microbiota needs to be investigated in the future.

Conclusion
This study reveals that the oral administration of Gln and Ala-Gln can protect against DSS-induced colitis through alleviating mucosa damage and inflammatory responses, upregulating the expression of TJ and PepT1 proteins and modulating gut microbiota in mice. The DSS-induced shifts of the microbiome (community diversity, evenness, richness, and composition) in the mouse colon were restored by Gln and Ala-Gln supplementation, including Lactobacillus, Bac-teroides_acidifaciens, Bacteroidales, Firmicutes, Clostridia, Helicobacter, and Bacteroides. The metabolism functions of gut microflora were also rescued by Ala-Gln, including membrane transporters (PepT1), fatty acid and propanoate metabolism, infectious diseases, immune system, and bacterial toxins. Interestingly, Ala-Gln has a better beneficial effect than Gln in alleviating colitis. In conclusion, Ala-Gln can prevent IBD through PepT1, enhancing the intestinal barrier and modulating gut microbiota and their metabolites. This study provides a new insight into the biological function and protective effect of Ala-Gln in managing IBD. Furthermore, the role and the underlying mechanism of PepT1 and gut microbiota in alleviating colitis need to be investigated in the future.