GLP-2 ameliorates DSS-induced ulcerative colitis in mice by inhibiting inflammatory pathways and regulating gut microbiota

Abstract

Background

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by recurrence and remission of colonic and rectal mucosal inflammation, and its incidence is increasing year by year. Glucagon-like peptide-2 (GLP-2) is a newly discovered enteral nutrition factor, but its efficacy and potential mechanism of action on UC have not been fully elucidated.

Aims

The purpose of this study was to investigate the protective effect of GLP-2 on Dextran sulfate sodium (DSS) -induced UC in mice and its potential mechanism.

Methods

40 C57BL/6J female mice were randomly divided into 4 groups: control group (CON) ,DSS group (DSS) ,DSS + Enterotoxigenic Escherichia coli group (ETEC) ,DSS + ETEC + sitagliptin group (GLP-2). The effect of GLP-2 on UC was evaluated by calculating the disease activity index (DAI), colonic mucosal damage index (CMDI), and histopathological score. The expressions of GLP-2, nuclear factor κB (NF-κB), Interleukin-6 (IL-6), and signal transducer and activator of transcription 3 (STAT3) were detected by the Enzyme-Linked Immunosorbent Assay (ELISA ) and immunohistochemistry. 16SrRNA was used to detect the changes in gut microbiota in mouse colonic tissue.

Results

Compared with the control group, the GLP-2 of mice in the DSS group and ETEC group decreased significantly, and NF-κB, IL-6, and STAT3 were significantly increased(P < 0.0001). Compared with the DSS group, the CMDI score and histopathological score of the GLP-2 group were significantly decreased, GLP-2 expression was significantly increased, and NF-κB, IL-6, and STAT3 were significantly decreased. The results of 16SrRNA detection showed that compared with the DSS group, the dominant bacteria such as Lactobacillus and Prevotellaceae were increased and the diversity of gut microbiota was increased in the GLP-2 group.

Conclusions

GLP-2 reduced the degree of inflammation in UC mice, which may be achieved by inhibiting the inflammatory pathways of NF-κB and Janus Kinase (JAK) /STAT3, increasing the dominant bacteria and improving the diversity of gut microbiota.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory disease of the rectum and colon characterized by mucosal inflammation[1]. The exact pathogenesis of UC is not completely understood, and it is generally believed to be related to the interaction between environment, immune system, gut microbiota and genetic susceptibility[2]. The gut microbiota plays an important role in maintaining the integrity of the intestinal mucosa, and disruption of the intestinal mucosal barrier and bacterial invasion leads to intestinal inflammation[3]. UC is characterized by a dysregulated inflammatory response, manifested by invasion of inflammatory cells into the lamina propria and excessive release of pro-inflammatory cytokines. There is growing evidence that pro-inflammatory cytokines and Janus Kinase (JAK) / signal transducer and activator of transcription 3 (STAT3) pathway play an important role in the pathogenesis of human UC and experimental colitis[4]. NF-κB is a major regulator of innate immunity and inflammatory responses and also upregulates pro-inflammatory cytokine expression in UC, triggering an inflammatory cascade response[5]. After STAT3 activation, nuclear factor (NF) -κB enter the nucleus, bind to the promoter of the target gene, and induce pro-inflammatory mediators such as InducibleNitric Oxide Synthase (iNOS) , Cyclooxygenase-2 (COX-2) , Tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) ,to reactivate the JAK/STAT3 pathway, thus forming a malignant pro-inflammatory cycle[6]. These events lead to the persistence of colonic mucosal inflammation, hinder mucosal regeneration, lead to tissue damage, and eventually lead to the development of the disease in the direction of inflammation-cancer transformation[7].

The drug intervention currently used is aimed at countering the characteristic attacks of intestinal inflammation. The most effective drugs are corticosteroids and TNF-α inhibitors. However, due to serious side effects, the former cannot be used for a long time, while the latter has a considerable number of primary and secondary non-responders[8]. Therefore, it is very important to find new and safer treatments.

GLP-2 is an enteral nutrition hormone released by intestinal endocrine L cells, which indirectly plays a protective role in the intestinal tract through different mediators. However, GLP-2 is degraded rapidly by Dipeptidyl peptidase-4 (DPP-4) in vivo, which reduces its potential physiological effects. Therefore, inhibiting DPP-4 prolonging the half-life of GLP-2 may improve UC. Sitagliptin is the first DPP-4 inhibitor approved by FDA for the treatment of type 2 diabetes. A large number of preclinical and clinical data, as well as post-market monitoring data, have proved that it is an effective and safe DPP-4 inhibitor. In this study, DSS was used to establish a mouse UC model, and Enterotoxigenic Escherichia coli (ETEC) was introduced to aggravate intestinal inflammation in mice, and then to study the efficacy and possible mechanism of GLP-2 in the treatment of UC.

1. Materials And Methods

1.1 Reagents

DSS with a molecular weight of 36–50 kDa was purchased from MP Biomedicals. Sitagliptin was obtained from Sigma. ETEC was purchased from Beina Chuanglian Biotechnology Institute (Beijing,China) . Mouse GLP-2, NF-κB p65, IL-6, STAT3 ELISA kits were purchased from Jiangsu Meimian industrial Co., Ltd. Rabbit  Anti-GLP-2, Anti-NF-κB p65, Anti-STAT3 Polyclonal Antibodies were purchased from Jiangsu Meimian industrial Co., Ltd. Fecal occult blood reagent was purchased from Shanghai Wei dysprosium Biotechnology Co., Ltd.

1.2 Animals

40 SPF female C57BL/6J mice aged 6-8 weeks and weighing 18-20g were purchased from Liaoning Changsheng Biotechnology Co., Ltd., animal quality license number: SCXK (Liao) 2020-0001. In strict accordance with the standards of the professional committee of animal protection and utilization in the first affiliated Hospital of Harbin Medical University. This experiment was approved by the Animal Ethics Committee of the first affiliated Hospital of Harbin Medical University.

C57BL/6J mice fed adaptively for one week were randomly divided into 4 groups after ear tag clips were marked: control group (CON) ,DSS group,DSS+Enterotoxigenic Escherichia coli group (ETEC) ,DSS+ETEC+sitagliptin（30mg/kg）group (GLP-2) . Except for the control group, the mice in other groups were given 2%DSS for free drinking from 0 to 7 days. On the 3rd day, mice in each group were given 0.2mlETEC (2.27 × 108CFU/ml) or aseptic saline. From 0 to 13, mice in each group were given 0.1 ml 0.6% sioglitine or sterile saline intragastric administration. During the experiment, the body weight, fecal occult blood and drinking water of mice were recorded every day. On the 13th day, mice in each group were given intraperitoneal injection of 4% chloral hydrate according to body weight (0.1ml/10g). The eyeballs were taken for blood, and the colonic tissue was dissected.

1.3 Evaluation of the Disease activity index (DAI)

According to the daily weight change, fecal characteristics and bleeding of mice, the total score of the three items was divided by 3 to get the DAI score. The specific scoring criteria are shown in Table-1.

Table-1 Disease activity index scoring.

1.4 Colonic mucosal damage index(CMDI) and Histological analysis

The colon was cut along the longitudinal axis of the mesentery, and the degree of colonic edema and ulcer was observed. CMDI was scored according to the literature[9]. Remove the contents with PBS, drain the filter paper, cut the anal tissue about 1cm, fix it with 10% formalin for 24 hours or more, routine paraffin embedding, section, HE staining, observe under microscope, and score the histopathology according to Table-2.

Table-2 Colonic histologic scoring.  

1.5 Enzyme-Linked Immunosorbnent Assay (ELISA )

Mice took 0.6-0.8 ml of blood from the eyeball, 4000 r/min, centrifuged for 20 min, and isolated mouse serum. The contents of GLP-2, NF-κB, IL-6 and STAT3 in the serum of mice in each group were determined. According to the kit instructions, the dilution, sample addition, incubation, solution preparation, washing and enzyme addition of the standard sample were carried out. Finally, the OD value was determined by terminating the reaction, and the actual concentration of the sample was calculated. Each sample to be tested was repeated twice and the average value was finally taken.

1.6 Immunohistochemistry

The colonic tissues of mice were embedded in paraffin and sliced, then dewaxed, hydrated, antigen repaired, blocked, first antibody and second antibody incubated, DAB staining, hematoxylin re-staining, dehydration and sealing were performed to observe the expression of GLP-2, NF-κB and STAT3.

1.7 16SrRNA

The purity and concentration of DNA in colonic tissue of mice in each group were detected by NanoDrop2000, and the integrity of DNA was detected by agarose gel electrophoresis. Based on the V3-V4 variable region of 16SrRNA gene, primers were synthesized and amplified by PCR. The purified product was sequenced by IlluminaPE300. After the original data are filtered by quality control, OUT clustering analysis is carried out according to 97% similarity. Based on the results of OTU cluster analysis, diversity index analysis and sequencing depth detection were carried out, and the community structure was statistically analyzed at each classification level based on taxonomic information.

1.8 Statistical Analysis

The data were analyzed by SPSS26.0 and GraphpadPrism8 statistical software packages. The multiple comparisons among these groups were made employing one-way ANOVA test, the results were expressed by`x ±S, and the grade data were plotted by chi-square test. The data of gut microbiota were analyzed on the online platform of Majorbio Cloud Platform (www.majorbio.com). P<0.05 was considered to indicate statistical significance. 

2. Results

2.1 Effect of GLP-2 intervention on severity in mice

2.1.1 DAI score for each group of mice

Compared with the control group, the DAI score of DSS group,ETEC group and GLP-2 group increased; compared with DSS group, the DAI score of ETEC group increased and the DAI score of GLP-2 group decreased; compared with ETEC group, the DAI score of GLP-2 group decreased, but the difference was not statistically significant ( Fig. 1-A)

2.1.2 CMDI score for each group of mice

Compared with the control group, mice in the DSS group had congested and edematous colonic tissue, rough mucosa, erosion or ulceration, and the damage index was significantly higher, and the difference was statistically significant (P < 0.001); mice in the ETEC group had necrosis and inflammation on the mucosal surface of colonic tissue and thickened intestinal wall, and the damage index was significantly higher, and the difference was statistically significant (P < 0.0001). Compared with the DSS group, the mice in the ETEC group had heavier colonic tissue damage and higher CMDI score, but the difference was not statistically significant (P > 0.05); the mice in the GLP-2 group had mild colonic tissue congestion and edema, and the damage index was significantly lower, and the difference was statistically significant (P < 0.05); compared with the mice in the ETEC group, the damage index of the mice in the GLP-2 group was significantly lower, and the difference was statistically significant (P < 0.01) (Fig. 1-B).

2.1.3 HE staining and pathological histological score for each group of mice.

Compared with the control group, the colonic histopathology scores of mice in the DSS group were significantly higher (P < 0.001) and also significantly higher in the ETEC group (P < 0.0001); compared with the DSS group, the colonic histopathology scores of mice in the ETEC group were higher, but the difference was not statistically significant (P > 0.05), and the colonic histopathology scores of mice in the GLP-2 group were significantly lower (P < 0.05); the colonic histopathology scores of mice in the GLP-2 group were significantly lower compared with those in the ETEC group (P < 0.001) (Fig. 1-C).

HE staining showed that the colon wall of control mice was intact, with normal morphology and number of cup cells and crypt cells, and few inflammatory cells. the DSS and ETEC groups caused a series of histopathological changes, including mucosal damage and necrosis, submucosal inflammatory cell infiltration, edema and vascular congestion. the GLP-2 group of mice showed reduced histological changes and inflammatory cell infiltration in the colon, and reduced local edema and vascular congestion ( Fig. 1-D).

2.2 ELISA assay for each group of mice serum (Table-3)

Compared with the control group, the serum GLP-2 values were significantly lower (P < 0.0001) and NF-κB, IL-6 and STAT3 values were significantly higher (P < 0.0001) in the DSS and ETEC groups of mice; compared with the DSS group, the GLP-2 values were significantly higher (P < 0.0001) and NF-κB, IL-6 and STAT3 values were significantly lower in the GLP-2 group of mice (P < 0.0001); compared with the ETEC group, the serum NF-κB, IL-6 and STAT3 values of mice in the GLP-2 group were significantly lower ( Figure-2).

Table-3 ELISA for GLP-2, NF-κB, IL-6 and STAT3(`x±S)

^*P<0.0001vsCON;^#P<0.0001vsDSS;^&P<0.01vsETEC;^{^}P<0.0001vsETEC

2.3 Immunohistochemistry of colonic tissues of various groups of mice

Compared with the control group, the GLP-2 positive expression rate was significantly lower (P < 0.05) and the NF-κB and STAT3 positive expression rates were significantly higher (P < 0.05) in the colon tissue of mice in the DSS and ETEC groups; compared with the DSS group, the GLP-2 positive expression rate was significantly higher (P < 0.05) and the STAT3 positive expression rate was significantly lower in the GLP-2 group (P < 0.05) and NF-κB positive expression rate was reduced, but the differences were not statistically significant (P > 0.05); compared with the ETEC group, the GLP-2 positive expression rate was significantly higher (P < 0.05) and STAT3 positive expression rate was significantly lower (P < 0.05) in the GLP-2 group (Figure-3).

2.4 16SrRNA sequencing

2.4.1 Changes in the diversity of the gut microbiota of colonic tissues

A total of 801195 optimized sequences, 336935790 bp, with an average sequence length of 420 bp, were obtained after 16SrRNA sequencing for each group of mouse colon tissues. The α-diversity analysis based on sobs, shannon and chao indices all showed that the gut microbiota diversity was lower in the DSS group and higher in the GLP-2 group compared to the control group (Fig. 4A-C), and the gut microbiota diversity was significantly higher in the GLP-2 group compared to the DSS group (P < 0.05). Analysis of the dilution curve corresponding to the diversity index showed that the curve flattened out as the number of sequences increased, indicating that the number of sequences was sufficient (Fig. 4D-F). Principal Component Analysis (PCA), Principal Co-ordinates Analysis (PCoA) and non-metric multidimensional scaling (NMDS) were performed to measure the similarity of communities between groups by β-diversity index. The results showed that the overlap between the three sample groups was small, suggesting that the results of β-diversity analysis were better, and the differences in colony structure between the groups were obvious (Fig. 4G-H). Venn diagrams showed that there were 564 OTUs in the control group, 286 OTUs in the DSS group, and 706 OTUs in the GLP-2 group; the number of OTUs common to the three groups was 225, and there were 79, 10, and 182 unique OTUs in the CON, DSS, and GLP-2 groups (Fig. 4-I). All the above results indicated that the gut microbiota diversity was decreased in the DSS group and increased in the GLP-2 group.

2.4.2 Differences in the species composition of the gut microbiota of colonic tissues

Compared with the control group, the gut microbiota of mouse colonic tissues in the DSS group was significantly (P < 0.05) reduced at the genus level for Lactobacillus, norank_f__Muribaculaceae,Dubosiella,Alloprevotella;Escherichia-Shigella, Mucispirillum,Clostridium_sensu_stricto_1,Romboutsia,Enterococcus,Faecalibaculum significantly increased (P < 0.05) (Fig. 5-D). Norank_f__Muribaculaceae, Lactobacillus, Prevotellaceae_Ga6A1_group, Dubosiella, Alloprevotella were significantly increased in the GLP-2 group compared to the DSS group (P < 0.05); while Escherichia-Shigella, Mucispirillum, Enterococcus significantly decreased (P < 0.05) ( Fig. 5-E).

2.4.3 Association analysis of GLP-2, NF-κB, STAT3 and gut microbiota

To further explore the potential relationship between GLP-2, NF-κB, STAT3 and gut microbiota, this study correlated the three previously detected index values with gut microbiota diversity and generated heat maps to visualize the relationship between flora species and the above markers (Fig. 16-A), and the results showed that gut microbiota diversity was positively correlated with GLP-2 (Fig. 6-B); negatively correlated with NF-κ B (Fig. 6-C); and negative correlation with STAT3 (Fig. 6-D).

The X coordinate indicates the environmental factors (GLP-2, NF-κB, STAT3), the Y coordinate indicates the rank of α-diversity index, and R2 is the determinant indicating the proportion of variation explained by the linear regression line, the larger the R2 index, the higher the degree of explanation of the differences in community composition or α-diversity index by this environmental factor. test1:GLP-2;test2:DSS

3. Discussion

UC is a chronic inflammatory disease characterized by recurrent and remitting inflammation of the mucosa of the colon and rectum. Its exact pathogenesis is not fully understood, but it is generally believed that a complex interplay of gut microbiota, genetic susceptibility and environmental factors may disrupt the immune system, leading to an immune-mediated chronic intestinal inflammatory response[10]. Prolonged inflammation leads to irreversible intestinal damage, which severely affects the quality of life of patients[11]. The main drugs currently used to treat UC include aminosalicylic acid, glucocorticoids, immunosuppressants and biologics, with potential side effects limiting the use of current therapeutic agents[12]. Therefore, there is a need to find safer and more effective drugs for the treatment of UC. In the present study, we demonstrated that GLP-2 attenuates colonic inflammation by mechanisms including inhibition of NF-κB, JAK/STAT3 pathway exerting anti-inflammatory effects and regulation of gut microbiota.

In this experiment, 2% DSS solution was used, and the mice in the modeling group gradually showed depression, laziness, weight loss and positive occult blood from the third day of modeling. Since clinical UC patients are often combined with infections of pathogenic bacteria, this experiment added ETEC to aggravate intestinal inflammation in mice on the basis of modeling to better simulate the changes of clinical UC patients and further investigate the therapeutic effect of GLP-2 on UC. ETEC, the most common causative agent of infectious diarrhea, produces a variety of non- and heat-resistant virulence factors, 26 adhesion factors that promote intestinal epithelial binding and colonization, and three enterotoxins responsible for humoral secretion, molecules that may directly lead to impairment of the tight junctions of selective permeability of intestinal tissues, thereby disrupting the permeability barrier of intestinal epithelial cells[13]. It has also been shown that ETEC has a significant stimulatory effect on the expression of pro-inflammatory cytokines (IL-1α, IL-6, IL-18 and TNF-α), while it has no significant effect on the expression of anti-inflammatory cytokines (IL-4, IL-10)[14]. ETEC aggravated intestinal inflammation in mice in this experiment, as expected from the experiment. GLP-2, as an enterotrophic hormone secreted by enteroendocrine L cells, is rapidly degraded by DPP-IV in vivo and has a very short half-life. In clinical practice, GLP-2 analogs are mainly administered by subcutaneous injection and patient compliance is poor, so in this experiment, the DPP-IV inhibitor sitagliptin was chosen to indirectly increase the level of GLP-2 by gavage in mice, but sitagliptin can also prolong the half-life of GLP-1. In addition to the known role of GLP-1 in regulating blood glucose, it is also worthwhile to conduct research on whether it plays a role in the intestine.

3.1 GLP-2 reduces the degree of inflammation in UC mice

GLP-2 is an enterogenic hormone that promotes intestinal growth, digestion, absorption, barrier function and blood flow in healthy animals, and prevents damage and promotes repair in preclinical models of colitis and after massive small bowel resection[15]. Reduced GLP-2 expression in UC due to destruction or inhibition of enteroendocrine L cells in the inflammatory state[16]. Exogenous administration of GLP-2 has been shown to be effective in reducing the symptoms of intestinal injury in animal models[17]. In this experiment, mice in the GLP-2 group showed reduced blood stool condition, degree of weight loss, degree of carnal ulceration, and pathological histological score compared with mice in the DSS and ETEC groups, demonstrating the therapeutic effect of GLP-2 on UC. Ning M et al[18] demonstrated that sitagliptin attenuated DSS-induced experimental colitis and that its effects could be attributed to increased GLP-2 expression and subsequent protection of the intestinal barrier by inhibiting epithelial cell apoptosis and promoting its proliferation. Sitagliptin, a DPP-IV inhibitor, prolongs the half-life of GLP-2, attenuates intestinal inflammation in UC mice, and is expected to be a new drug for the treatment of UC.

3.2 GLP-2 inhibits NF-κB pathway

Among the immunomodulatory factors, the inflammatory response is considered to be a central mechanism in the pathophysiology of UC, and pro-inflammatory cytokines play an active role in the inflammatory response by inducing macrophage migration and the release of inflammatory mediators, which further amplify the inflammatory response[19]. NF-κB plays a central role in the regulation of inflammatory processes[20]. Activation of NF-κB has been reported to upregulate the expression of pro-inflammatory cytokines that trigger positive feedback regulation during inflammatory activation, ultimately damaging colonic tissue[21]. To further explore the anti-inflammatory mechanism of GLP-2, the expression of NF-κBp65, an NF-κB-related protein, was detected in colonic tissues in this experiment. The results showed that NF-κB expression was significantly higher in the DSS group compared with the control group, due to the fact that DSS aggravated intestinal mucosal damage and increased intestinal inflammation in mice, activating the NF-κB inflammatory pathway, consistent with previous studies[22]. Compared with the DSS group, NF-κB expression was significantly reduced in both serum and colonic tissues of mice in the GLP-2 group. Xie S et al[23] demonstrated that GLP-2 significantly reduced lipopolysaccharide-induced production of iNOS, COX-2, IL-1β, IL-6, and TNF-α. Signaling pathway analysis showed that GLP-2 reduced LPS-induced phosphorylation of NF-κBp65, consistent with the present experimental study, suggesting that GLP-2 may reduce inflammation by attenuating NF-κB activation. NF-κB controls the production and secretion of multiple cytokines and chemokines during UC pathophysiology, and it is a central pro-inflammatory gene-induced mediator of inflammation in both natural and acquired immune cells that responds to multiple immune receptors[7]. uncontrolled NF-κB activation is a hallmark of chronic inflammatory diseases, and targeting the NF-κB signaling pathway is an attractive anti-inflammatory therapeutic approach[24].

3.3 GLP-2 inhibits the JAK/STAT3 pathway

Cytokines are the key mediators of inflammatory-mediated intestinal homeostasis imbalance and pathological processes in UC. Most cytokines in UC, such as IL-6, IL-10, IL-2 or IL-22, and those considered to be UC pathological mediators (interferon-γ, IL-12, IL-23 or IL-9) all play a role through the JAK/STAT3 pathway[25]. Activation of STAT3 activates NF-κB, which enters the nucleus, binds to the promoters of target genes, and induces pro-inflammatory mediators such as iNOS, COX-2, TNF-α, and IL-6, which reactivates the JAK/STAT3 pathway, resulting in a (STAT3-NF-κB-IL-6-STAT3) crosstalk that creates a malignant pro-inflammatory cycle and progresses the disease toward inflammatory cancer transformation[6]. Blocking the JAK/STAT pathway has the potential to affect the complex inflammation driven by multiple cytokines associated with UC pathology compared to the more traditional approach of blocking a single cytokine using antibodies[26]. In this experiment, both serum and colonic tissue STAT3 were significantly higher in the DSS group mice than in the control group, and both serum and colonic STAT3 were significantly lower in the GLP-2 group compared with the DSS group, and serum IL-6 showed the same trend. Ivory C et al[27] investigated the mechanism of the anti-inflammatory effect of GLP-2 through an IL-10-deficient colitis mouse model and showed that the anti-inflammatory effect of GLP-2 was not dependent on IL-10, but was attributed to GLP-2 antagonizing IL-6-mediated STAT3 signaling, thereby inhibiting intestinal inflammation, which is consistent with the results of this experiment. GLP-2 may also exert an inhibitory effect on inflammation by inhibiting NF-κB expression, reducing the release of the pro-inflammatory mediator IL-6, and blocking the JAK/STAT3 pathway.

3.4 GLP-2 regulates gut microbiota

Disturbances in the interaction between the gut microbiota and the mucosal immune system play a key role in the development of UC and are usually associated with reduced flora diversity and imbalances in strain composition[28]. Dysbiosis usually leads to a decrease in the production of short-chain fatty acids(SCFAs), which reduces an important source of energy for intestinal epithelial cells, leading to increased intestinal permeability and inflammation[29]. Compared to healthy gut microbiota, the diversity of UC gut microbiota is generally reduced, with lower levels of bacterial phylum[30]. It is unclear whether the reported changes in gut microbiota are a cause or a consequence of UC. In this experiment, 16SrRNA gene sequencing of mouse colon tissue showed that the gut microbiota diversity was reduced in the DSS group, and the dominant bacteria such as Lactobacillus, norank_f__Muribaculaceae were significantly reduced; the inferior bacteria such as Escherichia-Shigella, Mucispirillum, Clostridium_sensu_stricto_1, Romboutsia, Enterococcus, Faecalibaculum, etc. increased significantly. Compared with the DSS group, the GLP-2 group had higher gut microbiota diversity, with significant increases in the dominant bacteria norank_f__Muribaculaceae, Lactobacillus, Prevotellaceae_Ga6A1_group, etc.; and significant decreases in the inferior bacteria Escherichia-Shigella, Mucispirillum, Enterococcus, etc. Jang, YJ et al[31]demonstrated that Lactobacillus can improve DSS-induced colitis by modulating the immune response and altering the gut microbiota. Cani et al[32]showed that prebiotics improve gut microbiota and mucosal barrier function by increasing the production of endogenous GLP-2, which improves intestinal permeability and reduces plasma LPS levels by regulating the expression of the tight junction proteins ZO-1 and ocdcludin, thereby blunting inflammation and oxidative stress. Li D et al[33]demonstrated that GLP-2 improves colonizing bacteria and reduces the severity of UC by enhancing the diversity and abundance of intestinal mucosa, all of which corroborate the results of this paper. The association analysis of GLP-2, NF-κB, STAT3 and microbiota in this experiment showed that the gut microbiota diversity increased with the increase of GLP-2 expression, which showed that GLP-2 may play an intestinal protective role by regulating the microbiota diversity and increasing the dominant strains of bacteria such as Lactobacillus.

This experiment also has many shortcomings. The DSS + GLP-2 group was not set up separately to directly study the therapeutic effect of GLP-2, and the GLP-2 prodrug intervention was not given exogenously directly, but the DPP-IV inhibitor sitagliptin was chosen to act indirectly. Since the introduction of ETEC aggravated the destruction of intestinal mucosa in mice, it was possible to reduce the production of endogenous GLP-2, thus decreasing the efficacy of sitagliptin in treating UC. However, in this experiment, sitagliptin still played a role in suppressing UC inflammation by increasing the expression of GLP-2. It is still necessary to investigate whether GLP-1 plays a role in intestinal protection in the future, and it is also crucial to seek new compounds with longer half-life and higher stability. The crosstalk between various inflammatory pathways also deserves to be investigated, and it is expected that a more desirable goal of UC treatment can be achieved by GLP-2 in the future.

Conclusions

In summary, GLP-2 was able to significantly reduce the clinical symptoms of UC mice, and significantly reduce the DAI score, visual CMDI score, HE staining and pathological histological score, which reflect the degree of inflammation, even though the intestinal inflammation was aggravated by ETEC infection in UC mice. This experiment provides preliminary evidence that GLP-2 may block the malignant pro-inflammatory cycle by inhibiting the NF-κB pathway and inhibiting the JAK/STAT3 inflammatory pathway, and exert intestinal protective effects by increasing the dominant bacterial species and regulating microbiota diversity.

Abbreviations

Declarations

Ethics approval and consent to participate:

All animal experiments were performed in accordance with the Animal Experimental Ethics Committee of the First Hospital of Harbin Medical University (IACUC: 2021068).

Consent for publication:

All authors gave their consent for publications.

Availability of data and materials:

Raw sequences generated by 16S rRNA sequencing have been deposited in the NCBI Sequence Read Archive (SRA: SUB8157907; Study PRJNA664267), and the relevant meta-data can be found at https://www.ncbi.nlm.nih.gov/Traces/study/ under the SRA accession number.

Competing interests:

The authors declare that they have no competing interests.

Funding:

This study was supported by a grant from the Provincial Education Department project (2019-KYYMF-0335)

Authors' contributions:

Dongyue Li and Hongyu Xu designed the study; Yanhong Gao wrote the manuscript;  Dongyue Li revised the manuscript. Yanhong Gao, Lanrong Cui, Yang Li, Hao Ling, and Xin Tan carried out the experiments; Dongyue Li analyzed the data. All authors read and approved the final manuscript.

Acknowledgements:

Not applicable.

References

1. Abdulrazeg, O, Li, B,Epstein, J. Management of ulcerative colitis: summary of updated NICE guidance. Bmj 2019; doi:10.1136/bmj.l5897.
2. Du, L,Ha, C. Epidemiology and Pathogenesis of Ulcerative Colitis. Gastroenterology clinics of North America 2020; doi:10.1016/j.gtc.2020.07.005.
3. Guo, XY, Liu, XJ,Hao, JY. Gut microbiota in ulcerative colitis: insights on pathogenesis and treatment. Journal of digestive diseases 2020; doi:10.1111/1751-2980.12849.
4. Cordes, F, Lenker, E, Weinhage, T, Spille, LJ, Bettenworth, D, Varga, G et al. Impaired IFN-gamma-dependent STAT3 Activation Is Associated With Dysregulation of Regulatory and Inflammatory Signaling in Monocytes of Ulcerative Colitis Patients. Inflamm Bowel Dis 2021; doi:10.1093/ibd/izaa280.
5. Lin, JC, Wu, JQ, Wang, F, Tang, FY, Sun, J, Xu, B et al. QingBai decoction regulates intestinal permeability of dextran sulphate sodium-induced colitis through the modulation of notch and NF-κB signalling. Cell proliferation 2019; doi:10.1111/cpr.12547.
6. Fan, Y, Mao, R,Yang, J. NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein & cell 2013; doi:10.1007/s13238-013-2084-3.
7. Yao, D, Dong, M, Dai, C,Wu, S. Inflammation and Inflammatory Cytokine Contribute to the Initiation and Development of Ulcerative Colitis and Its Associated Cancer. Inflamm Bowel Dis 2019; doi:10.1093/ibd/izz149.
8. Ungaro, R, Mehandru, S, Allen, PB, Peyrin-Biroulet, L,Colombel, JF. Ulcerative colitis. Lancet (London, England) 2017; doi:10.1016/S0140-6736(16)32126-2.
9. Xie, Q, Li, H, Ma, R, Ren, M, Li, Y, Li, J et al. Effect of Coptis chinensis franch and Magnolia officinalis on intestinal flora and intestinal barrier in a TNBS-induced ulcerative colitis rats model. Phytomedicine: international journal of phytotherapy and phytopharmacology 2022; doi:10.1016/j.phymed.2022.153927.
10. Deng, L, Guo, H, Wang, S, Liu, X, Lin, Y, Zhang, R et al. The Attenuation of Chronic Ulcerative Colitis by (R)-salbutamol in Repeated DSS-Induced Mice. Oxidative medicine and cellular longevity 2022; doi:10.1155/2022/9318721.
11. Clooney, AG, Eckenberger, J, Laserna-Mendieta, E, Sexton, KA, Bernstein, MT, Vagianos, K et al. Ranking microbiome variance in inflammatory bowel disease: a large longitudinal intercontinental study. Gut 2021; doi:10.1136/gutjnl-2020-321106.
12. Zhou, A, Zhang, S, Yang, C, Liao, N,Zhang, Y. Dandelion root extracts abolish MAPK pathways to ameliorate experimental mouse ulcerative colitis. Advances in clinical and experimental medicine: official organ Wroclaw Medical University 2022; doi:10.17219/acem/146234.
13. Dubreuil, JD. Enterotoxigenic Escherichia coli targeting intestinal epithelial tight junctions: An effective way to alter the barrier integrity. Microbial pathogenesis 2017; doi:10.1016/j.micpath.2017.10.037.
14. Bahrami, B, Macfarlane, S,Macfarlane, GT. Induction of cytokine formation by human intestinal bacteria in gut epithelial cell lines. Journal of applied microbiology 2011; doi:10.1111/j.1365-2672.2010.04889.x.
15. Li, M,Weigmann, B. A Novel Pathway of Flavonoids Protecting against Inflammatory Bowel Disease: Modulating Enteroendocrine System. Metabolites 2022; doi:10.3390/metabo12010031.
16. Zatorski, H, Sałaga, M,Fichna, J. Role of glucagon-like peptides in inflammatory bowel diseases-current knowledge and future perspectives. Naunyn-Schmiedeberg's archives of pharmacology 2019; doi:10.1007/s00210-019-01698-z.
17. Wu, J, Qi, K, Xu, Z,Wan, J. Glucagon-like peptide-2-loaded microspheres as treatment for ulcerative colitis in the murine model. Journal of microencapsulation 2015; doi:10.3109/02652048.2015.1065923.
18. Ning, MM, Yang, WJ, Guan, WB, Gu, YP, Feng, Y,Leng, Y. Dipeptidyl peptidase 4 inhibitor sitagliptin protected against dextran sulfate sodium-induced experimental colitis by potentiating the action of GLP-2. Acta Pharmacol Sin 2020; doi:10.1038/s41401-020-0413-7.
19. Zhao, XX, Ma, SB, Wen, JP, Hu, DT, Gao, JS, Peng, Q et al. Reactive Oxygen Species-Responsive Polyether Micelle Nanomaterials for Targeted Treatment of Ulcerative Colitis. Journal of biomedical nanotechnology 2022; doi:10.1166/jbn.2022.3233.
20. Lee, SY, Lee, BH, Park, JH, Park, MS, Ji, GE,Sung, MK. Bifidobacterium bifidum BGN4 Paraprobiotic Supplementation Alleviates Experimental Colitis by Maintaining Gut Barrier and Suppressing Nuclear Factor Kappa B Activation Signaling Molecules. Journal of medicinal food 2022; doi:10.1089/jmf.2021.K.0150.
21. Pandurangan, AK, Mohebali, N, Hasanpourghadi, M,Esa, NM. Caffeic Acid Phenethyl Ester Attenuates Dextran Sulfate Sodium-Induced Ulcerative Colitis Through Modulation of NF-kappaB and Cell Adhesion Molecules. Appl Biochem Biotechnol 2022; doi:10.1007/s12010-021-03788-2.
22. Gu, LM, Li, H, Xia, JQ, Pan, CY, Gu, C,Tian, YZ. Huangqin Decoction Attenuates DSS-Induced Mucosal Damage and Promotes Epithelial Repair via Inhibiting TNF-alpha-Induced NF-kappaB Activation. Chinese journal of integrative medicine 2022; doi:10.1007/s11655-021-3343-4.
23. Xie, S, Liu, B, Fu, S, Wang, W, Yin, Y, Li, N et al. GLP-2 suppresses LPS-induced inflammation in macrophages by inhibiting ERK phosphorylation and NF-κB activation. 2014; doi:10.1159/000363025.
24. Liu, T, Zhang, L, Joo, D,Sun, SC. NF-κB signaling in inflammation. Signal Transduct Target Ther 2017; doi:10.1038/sigtrans.2017.23.
25. Moon, SY, Kim, KD, Yoo, J, Lee, JH,Hwangbo, C. Phytochemicals Targeting JAK-STAT Pathways in Inflammatory Bowel Disease: Insights from Animal Models. Molecules (Basel, Switzerland) 2021; doi:10.3390/molecules26092824.
26. Salas, A, Hernandez-Rocha, C, Duijvestein, M, Faubion, W, McGovern, D, Vermeire, S et al. JAK-STAT pathway targeting for the treatment of inflammatory bowel disease. Nature reviews. Gastroenterology & hepatology 2020; doi:10.1038/s41575-020-0273-0.
27. Ivory, CP, Wallace, LE, McCafferty, DM,Sigalet, DL. Interleukin-10-independent anti-inflammatory actions of glucagon-like peptide 2. American journal of physiology. Gastrointestinal and liver physiology 2008; doi:10.1152/ajpgi.90494.2008.
28. Bunt, DV, Minnaard, AJ,El Aidy, S. Potential Modulatory Microbiome Therapies for Prevention or Treatment of Inflammatory Bowel Diseases. Pharmaceuticals (Basel) 2021; doi:10.3390/ph14060506.
29. Wu, J, Wei, Z, Cheng, P, Qian, C, Xu, F, Yang, Y et al. Rhein modulates host purine metabolism in intestine through gut microbiota and ameliorates experimental colitis. Theranostics 2020; doi:10.7150/thno.43528.
30. Shen, ZH, Zhu, CX, Quan, YS, Yang, ZY, Wu, S, Luo, WW et al. Relationship between intestinal microbiota and ulcerative colitis: Mechanisms and clinical application of probiotics and fecal microbiota transplantation. World journal of gastroenterology 2018; doi:10.3748/wjg.v24.i1.5.
31. Jang, YJ, Kim, WK, Han, DH, Lee, K,Ko, G. Lactobacillus fermentum species ameliorate dextran sulfate sodium-induced colitis by regulating the immune response and altering gut microbiota. Gut Microbes 2019; doi:10.1080/19490976.2019.1589281.
32. Cani, PD, Possemiers, S, Van de Wiele, T, Guiot, Y, Everard, A, Rottier, O et al. Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability. Gut 2009; doi:10.1136/gut.2008.165886.
33. Li, D, Yang, Y, Yin, X, Liu, Y, Xu, H, Ni, Y et al. Glucagon-like peptide (GLP) -2 improved colonizing bacteria and reduced severity of ulcerative colitis by enhancing the diversity and abundance of intestinal mucosa. Bioengineered 2021; doi:10.1080/21655979.2021.1958600.