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. Prolonged inflammation leads to irreversible intestinal damage, which severely affects the quality of life of patients. 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. 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. 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). 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. Reduced GLP-2 expression in UC due to destruction or inhibition of enteroendocrine L cells in the inflammatory state. Exogenous administration of GLP-2 has been shown to be effective in reducing the symptoms of intestinal injury in animal models. 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 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. NF-κB plays a central role in the regulation of inflammatory processes. 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. 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. 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 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. 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.
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. 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. 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. 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 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. 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. Compared to healthy gut microbiota, the diversity of UC gut microbiota is generally reduced, with lower levels of bacterial phylum. 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 aldemonstrated that Lactobacillus can improve DSS-induced colitis by modulating the immune response and altering the gut microbiota. Cani et alshowed 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 aldemonstrated 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.