Leonurine Alleviates DSS‐Induced Colitis in Mice by Regulating Pancreatic Secretion Pathway and Gut Microbiota

Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease

Role of the Protein C Pathway in the Extraintestinal Thrombosis Associated With Murine Colitis

Background: Disulfiram (DSF), a Food and Drug Administration (FDA)-approved drug for chronic alcohol addiction, has anti-inflammatory effects that help prevent various cancers, and Cu2+ can enhance the effects of DSF. Inflammatory bowel diseases (IBD) are characterized by chronic or recurrent relapsing gastrointestinal inflammation. Many drugs targeting the immune responses of IBD have been developed, but their application has many problems, including side effects and high costs. Therefore, there is an urgent need for new drugs. In this study, we investigated the preventive effects of DSF+Cu2+ on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Methods: The anti-inflammatory effects were investigated using the DSS-induced colitis mouse model and lipopolysaccharide (LPS)-induced macrophages. DSS-induced TCRβ-/- mice were used to demonstrate the effect of DSF in conjunction with Cu2+ on CD4+ T cell-secreted interleukin 17 (IL-17). In addition, the effect of DSF+Cu2+ on intestinal flora was studied by 16S rRNA microflora sequencing. Results: DSF and Cu2+ could significantly reverse the symptom of DSS-induced UC in mice, such as weight loss, disease activity index score, colon length shortening, and reversal of colon pathological changes. DSF and Cu2+ could inhibit colonic macrophage activation by blocking the nuclear factor kappa B (NF-κB) pathway, reducing nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3)-inflammasome-derived interleukin 1 beta (IL-1β) secretion and caspase-1 (CASP1) activation, and decreasing IL-17 secretion by CD4+ T cells. Moreover, the treatment of DSF and Cu2+ could protect the intestinal barrier by reversing the expression of tight junction proteins, zonula occluden-1 (ZO-1), occludin, and mucoprotein-2 (MUC2). Additionally, DSF+Cu2+ could reduce the abundance of harmful bacteria and increase beneficial bacteria in the intestinal tract of mice, effectively improving intestinal microecology. Conclusion: Our study evaluated the effect of DSF+Cu2+ on the immune system and gut microbiota in colonic inflammation and highlighted its potential to treat UC in the clinic.

Latent Cytomegalovirus Infection Exacerbates Experimental Colitis

Akkermansia muciniphila in inflammatory bowel disease and colorectal cancer.

Quality Is as Important as the Quantity: Role of Mucin Glycosylation on Intestinal Barrier Function

Inflammatory bowel disease (IBD) is a chronic disease involving multiple genes, and the current available targeted drugs for IBD only deliver moderate efficacy. Whether there is a single gene that systematically regulates IBD is not yet known. MiR-146a plays a pivotal role in repression of innate immunity, but its function in the intestinal inflammation is sort of controversy, and the genetic regulatory networks regulated by miR-146a in IBD has not been revealed. RT-qPCR was employed to detect the expression of miR-146a in IBD patients and in a mouse IBD model induced by dextran sulfate sodium (DSS), and then we generated a miR-146a knock-out mouse line with C57/Bl6N background. The disease activity index was scored in DSS-treated miR-146a deficiency mice and their wild type (WT) littermates. Bulk RNA-sequencing, RT-qPCR and immunostaining were done to illustrate the downstream genetic regulatory networks of miR-146a in flamed colon. Finally, the modified miR-146a mimics were used to treat DSS-induced IBD in miR-146a knock-out and WT IBD mice. We showed that the expression of miR-146a in the colon was elevated in dextran sulfate sodium (DSS)-induced IBD mice and patients with IBD. DSS induced dramatic body weight loss and more significant rectal bleeding, shorter colon length, and colitis in miR-146a knock-out mice than WT mice. The miR-146a mimics alleviated DSS-induced symptoms in both miR-146a-/- and WT mice. Further RNA sequencing illustrated that the deficiency of miR-146a de-repressed majority of DSS-induced IBD-related genes that cover multiple genetic regulatory networks in IBD, and supplementation with miR-146a mimics inhibited the expression of many IBD-related genes. Quantitative RT-PCR or immunostaining confirmed that Ccl3, Saa3, Csf3, Lcn2, Serpine1, Serpine2, MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, P65, p-P65, and IRAK1 were regulated by miR-146a in DSS induced IBD. Among them, MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, CSF3, and IL1A were involved in the active stage of IBD in humans. Our date demonstrated that miR-146a acts as a top regulator in C57/BL6N mice to systematically repress multiple genetic regulatory networks involved in immune response of intestine to environment factors, and combinatory treatment with miR-146a-5p and miR-146a-3p mimics attenuates DSS-induced IBD in mice through down-regulating multiple genetic regulatory networks which were increased in colon tissue from IBD patients. Our findings suggests that miR-146a is a top inhibitor of IBD, and that miR-146a-5p and miR-146a-3p mimics might be potential drug for IBD.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD), and its pathogenesis is related to intestinal mucosal barrier damage and gut microbiota imbalance. Protopine (PRO), an isoquinoline alkaloid, is one of the main anti-inflammatory ingredients of traditional Chinese medicine Macleaya cordata (Willd.) R. Br. This study investigated the effects of PRO on the intestinal mucosal barrier and gut microbiota in dextran sodium sulfate (DSS)-induced colitis mice. C57BL/6J mice were treated with 3% DSS in drinking water to induce acute colitis, while PRO was administered orally once daily for 7 days. The results showed that PRO administration significantly alleviated the symptoms of DSS-induced colitis in mice and inhibited the expression of inflammation-related genes. In addition, PRO restored the integrity of the intestinal barrier in colitis mice by restoring colonic mucin secretion and promoting the expression of tight junction proteins. Furthermore, PRO alleviated the DSS-induced gut microbiota dysbiosis by decreasing the abundance of Proteobacteria, Escherichia-Shigella and Enterococcus, as well as enhancing the abundance of beneficial bacteria, such as Firmicutes and Akkermansia. These findings suggested that PRO effectively alleviated DSS-induced ulcerative colitis by suppressing the expression of inflammation-related genes, maintaining the intestinal mucosal barrier and regulating the intestinal microbiota.

BackgroundIntestinal hyper-permeability plays a critical role in the etiopathogenesis of inflammatory bowel disease (IBD) by affecting the penetration of pathogens, toxic compounds and macromolecules. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D, has been shown to be an important regulator of IBD and recent epidemiology suggests that patients with IBD have an impaired vitamin D status. The purpose of this study is to investigate the possible protective effects of 1,25(OH)2D3 on mucosal injury and epithelial barrier disruption on dextran sulfate sodium (DSS)-induced acute colitis model.MethodsWe used DSS-induced acute colitis model to investigate the protective effects of 1,25(OH)2D3 on mucosal injury and epithelial barrier integrity. Severity of colitis was evaluated by disease activity index (DAI), body weight (BW) change, colon length, histology, myeloperoxidase (MPO) activity, and proinflammatory cytokine production including tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). In vitro the protective role of 1,25(OH)2D3 was assessed by incubating Caco-2 cells with or without DSS and measuring transepithelial electrical resistance (TEER) and fluorescein isothiocyanate dextran (FITC-D). The intestinal permeability was analyzed by FITC-D, bacterial translocation and measurement of lipopolysaccharide (LPS). Ultrastructural features of the colon tissue and Caco-2 cell monolayer were observed by electron microscopy. Expressions of tight junction (TJ) proteins in the colon mucosa and Caco-2 cells were detected by immunohistochemistry, immunofluorescence, Western blot and real-time fluorescent quantitative PCR, respectively.ResultsDSS-induced acute colitis model was characterized by a reduced BW, AUC of BW, serum calcium, higher DAI, AUC of DAI, shortened colon length, elevated MPO activity, worsened histologic inflammation, increased mononuclear cell numbers in mesenteric lymph nodes (MLNs) and colonic lamina propria (LP), and enhanced proteins and mRNA levels of TNF-α and IFN-γ. 1,25(OH)2D3 markedly increased expressions of TJ proteins and mRNA and decreased the FITC-D permeability and the level of LPS. Furthermore, 1,25(OH)2D3 abrogated bacterial translocation to MLNs and ameliorated ultrastructural features of the colon epithelium by scanning electron microscopy (SEM). In vitro, 1,25(OH)2D3 increased TEER, TJ proteins and mRNA expressions, decreased the FITC-D permeability, and preserved structural integrity of the TJ in Caco-2 cells.Conclusions1,25(OH)2D3 may play a protective role in mucosal barrier homeostasis by maintaining the integrity of junction complexes and in healing capacity of the colon epithelium. 1,25(OH)2D3 may represent an attractive and novel therapeutic agent for the adjuvant therapy of IBD.

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease (IBD) that has become a major gastroenterologic problem during recent decades. Numerous complicating factors are involved in UC development such as oxidative stress, inflammation, and microbiota disorder. These factors exacerbate damage to the intestinal mucosal barrier. Spirulina platensis is a commercial alga with various biological activity that is widely used as a functional ingredient in food and beverage products. However, there have been few studies on the treatment of UC using S. platensis aqueous extracts (SP), and the underlying mechanism of action of SP against UC has not yet been elucidated. Herein, we aimed to investigate the modulatory effect of SP on microbiota disorders in UC mice and clarify the underlying mechanisms by which SP alleviates damage to the intestinal mucosal barrier. Dextran sulfate sodium (DSS) was used to establish a normal human colonic epithelial cell (NCM460) injury model and UC animal model. The mitochondrial membrane potential assay 3-‍‍(4,5-dimethylthiazol-2-yl)-2,‍5-diphenyltetrazolium bromide (MTT) and staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and Hoechst 33258 were carried out to determine the effects of SP on the NCM460 cell injury model. Moreover, hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), western blot, and 16S ribosomal DNA (rDNA) sequencing were used to explore the effects and underlying mechanisms of action of SP on UC in C57BL/6 mice. In vitro studies showed that SP alleviated DSS-induced NCM460 cell injury. SP also significantly reduced the excessive generation of intracellular reactive oxygen species (ROS) and prevented mitochondrial membrane potential reduction after DSS challenge. In vivo studies indicated that SP administration could alleviate the severity of DSS-induced colonic mucosal damage compared with the control group. Inhibition of inflammation and oxidative stress was associated with increases in the activity of antioxidant enzymes and the expression of tight junction proteins (TJs) post-SP treatment. SP improved gut microbiota disorder mainly by increasing antioxidant enzyme activity and the expression of TJs in the colon. Our findings demonstrate that the protective effect of SP against UC is based on its inhibition of pro-inflammatory cytokine overproduction, inhibition of DSS-induced ROS production, and enhanced expression of antioxidant enzymes and TJs in the colonic mucosal barrier.

Ulcerative colitis (UC) is a type of inflammatory bowel disease associated with immune system dysfunction caused by gut dysbiosis. This study aimed to investigate the alleviating effect of Lactobacillus acidophilus LA85 on colitis and its underlying mechanism using mouse models of dextran sulfate sodium (DSS)-induced UC. The UC mouse models were established by treating C57BL/6J male mice with 2.5% (w/v) DSS in drinking water for 7 days. These mice received supplementation with either L. acidophilus LA85 (1 × 109 colony-forming units/day) or 200 µL of sterile water once daily (LA85-treated and UC model mice, respectively). The disease activity index (DAI), colon length, and histological changes in the colons of mice were then analyzed at Day 21, and the effects of L. acidophilus LA85 on the gut microbiota and serum inflammatory cytokines were also investigated. Compared with the UC model mice, L. acidophilus LA85-treated UC mice showed significant reductions in a variety of colitis symptoms, including weight loss, the DAI score, colon shortening, and colon tissue damage. Lactobacillus acidophilus LA85 supplementation also significantly decreased the serum concentrations of tumor necrosis factor α and interleukin-6 while increasing the serum concentration of IL-10. Furthermore, LA85 supplementation improved the diversity and composition of the gut microbiota, both of which had been decreased by DSS. In particular, L. acidophilus LA85-treated UC mice showed higher relative abundances of Akkermansia and Romboutsia than the UC model mice. These results demonstrate that L. acidophilus LA85 can alleviate inflammatory diseases of the intestine, such as inflammatory bowel disease, by regulating immune responses and restoring the gut microbiota. PRACTICAL APPLICATION: Ulcerative colitis is a type of inflammatory bowel disease caused by imbalance of gut microbiota. This study showed that L. acidophilus LA85 can alleviate DSS-induced colitis in mice through regulation of inflammatory cytokines, protection of intestinal barrier, and regulation of specific gut microbiota. L. acidophilus LA85 is a promising probiotic candidate for the treatment of UC.

ABSTRACTMicrobiological treatments are expected to have a role in the future management of inflammatory bowel disease (IBD). Clostridium butyricum (C. butyricum) is a probiotic microorganism that exhibits beneficial effects on various disease conditions. Although many studies have revealed that C. butyricum provides protective effects in mice with colitis, the way C. butyricum establishes beneficial results in the host remains unclear. In this study, we investigated the mechanisms by which C. butyricum modifies the gut microbiota, produces bacterial metabolites that may be involved, and, specifically, how microbial extracellular vesicles (EVs) positively influence IBD, using a dextran sulfate sodium (DSS)-induced colitis murine model in mice. First, we showed that C. butyricum provides a protective effect against colitis, as evidenced by the prevention of body weight loss, a reduction in the disease activity index (DAI) score, a shortened colon length, decreased histology score, and an improved gut barrier function, accompanied by reduced levels of pathogenic bacteria, including Escherichia/Shigella, and an increased relative abundance of butyrate-producing Clostridium sensu stricto-1 and Butyricicoccus. Second, we also confirmed that the gut microbiota and metabolites produced by C. butyricum played key roles in the attenuation of DSS-induced experimental colitis, as supported by the profound alleviation of colitis effects following fecal transplantation or fecal filtrate insertion supplied from C. butyricum-treated mice. Finally, C. butyricum-derived EVs protected the gut barrier function, improved gut microbiota homeostasis in ulcerative colitis, and contributed to overall colitis alleviation.IMPORTANCE This study indicated that C. butyricum provided a prevention effect against colitis mice, which involved protection of the intestinal barrier and positively regulating gut microbiota. Furthermore, we confirmed that the gut microbiota and metabolites that were induced by C. butyricum also contributed to the attenuation of DSS-induced colitis. Importantly, C. butyricum-derived EVs showed an effective impact in alleviating colitis.

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease, and the intestinal barrier is an important line of defense against intestinal disease. Herein, we investigated the effect of Lactobacillus gasseri JM1 at different doses (1 × 106, 1 × 107, 1 × 108 CFU/day) on colitis mice and explored the possible mechanism. The results showed that L. gasseri JM1 alleviated DSS-induced colitis in mice, with reductions in disease activity index (DAI), histological scores and myeloperoxidase activity as well as alleviation of colonic shortening. Furthermore, L. gasseri JM1 regulated the levels of inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-10; restored the expression of Claudin-3, Occludin, ZO-1, and MUC2; and increased the number of goblet cells and acidic mucin. The 16S rDNA sequencing results indicated that intervention with L. gasseri JM1 balanced the gut microbiota structure by elevating the abundance of beneficial bacteria (Oscillospira, Clostridium and Ruminococcus) and decreasing that of harmful bacteria (Shigella and Turicibacter). Meanwhile, the contents of short-chain fatty acids (SCFAs) increased. In conclusion, L. gasseri JM1 could alleviate intestinal barrier damage in colitis mice by modulating the tight junction structures, intestinal mucus layer, inflammatory cytokines, gut microbiota, and SCFAs. It can be considered a potential preventive strategy to alleviate colitis injury.

Ardisiacrispin B (AB) has demonstrated anti-inflammatory and anti-tumor activities, yet its therapeutic potential in inflammatory bowel disease (IBD) remains unexplored. This study evaluated the efficacy of AB in dextran sulfate sodium (DSS)-induced IBD in mice by monitoring body weight, disease activity index, stool consistency, rectal bleeding, and colon length. Intestinal barrier integrity and inflammatory responses were assessed via ELISA, FITC-dextran permeability, Western blot, and immunohistochemistry. Gut microbiota composition was profiled using 16S rRNA sequencing, along with bioinformatics to predict potential mechanisms, which were subsequently validated through immunofluorescence and flow cytometry. The results showed that AB significantly mitigated body weight loss, DAI scores, colon length shortening, and splenomegaly in mice, and alleviated the pathological damage to the colon. AB strengthened intestinal barrier integrity by increasing ZO-1, Occludin, claudin-1, MUC2, and EPO levels, and reducing ET-1 and DAO levels. AB suppressed inflammation by reducing IL-1β, IL-6, and TNF-α levels, and inhibiting JAK2/STAT3 and TLR4/MyD88/NF-κB pathways. Additionally, AB rebalanced the gut microbiota by increasing beneficial bacteria (Akkermansia) and decreasing pathogenic bacteria (Bacteroides and Helicobacter). Interestingly, AB rebalanced Th17/Treg by decreasing the colonic IL-17 level, increasing IL-10, IL-22, and TGF-β levels, and reversing the proportion of Th17/Treg cells in the blood, mesenteric lymph nodes, and spleen of IBD mice. These findings demonstrate that AB mitigates DSS-induced IBD through coordinated regulation of intestinal barrier integrity, gut microbiota composition, and Th17/Treg immune balance, identifying AB as a promising candidate for IBD therapy.

BackgroundHymenolepis nana (H. nana) is a zoonotic parasitic worm that parasitizes the small intestines of humans and rodents. Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease. Current symptom-based clinical treatments do not alter the natural course of UC, and mucosal healing has become a primary therapeutic goal for UC. However, the regulatory role of excretory/secretory products (ESPs) from H. nana adult worms in repairing the damaged intestinal mucosal barrier remains unclear.MethodsThis study investigated the protective effects of ESPs on intestinal mucosal integrity by using a dextran sulfate sodium (DSS)-induced colitis mouse model and a mouse small intestine organoid inflammation model. Histopathological alterations of mouse intestinal tissues were determined by pathological staining; the alterations in mucins, tight junction proteins, cytokines, and the number of various intestinal cells were detected by Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF) and real-time quantitative polymerase chain reaction (RT-qPCR), etc.ResultsESPs significantly improved DSS-induced intestinal damage in mice. Meanwhile, ESPs increased mucins and tight junction proteins expression and promoted intestinal stem cell proliferation and differentiation, thereby maintaining intestinal mucosal barrier integrity and alleviating UC in mice. In the DSS-induced inflamed small intestinal organoid model, ESPs reduced organoid damage and promoted the proliferation and differentiation of intestinal stem cells. The protective mechanism of ESPs might be related to the activation of the tuft/IL-13 signaling pathway, regulating intestinal barrier function and promoting the regeneration of intestinal stem cells.ConclusionsIn conclusion, H. nana-derived ESPs intervention facilitates healing of intestinal mucosa to alleviate UC in mice, enriching the feasibility and selectivity of “helminthic therapy.”Graphical abstract