Introduction

It has emerged that the energy metabolism of colonocytes shapes the gut microbiota, and loss of epithelial hypoxia accompanied by respiration-dependent dysbiosis is observed in experimental colitis^{1, 2}. The inhibitory effect of tacrolimus on inducible nitric oxide synthase (iNOS), a vital protein in nitrate respiration, was reported in human colonic epithelial cell lines³. Our previous study reported that tacrolimus significantly alleviated colitis in mice and induced beneficial alterations of the gut microbiome⁴. However, the mechanism of how tacrolimus improves the gut microbiota in colitis remains to be elucidated.

Aims & Methods

In this study, we aim to explore the effects of tacrolimus on colonocyte metabolism in colitis. Male C57BL/6J mice aged 8 to 10 weeks were employed. Solutions of 2.5% dextran sulfate sodium (DSS) were prepared in the drinking water to induce colitis for 7 consecutive days. Tacrolimus (Tacro) at a dose of 10 mg/kg was administered via gavage, while aminoguanidine hydrochloride (AG), the selective iNOS inhibitor, was given intraperitoneally at a dose of 100 mg/kg per day. The body weight was observed daily. For hypoxia detection, mice were treated with 60 mg/kg of pimonidazole HCl intraperitoneally 1 hour before euthanasia. Colon lengths were measured, and the colon samples were fixed in 4% paraformaldehyde for subsequent hematoxylin and eosin staining and hypoxia staining. Total RNA and protein were extracted from the isolated colonocytes for the measurement of iNOS expression. For the in vitro study, the human colorectal adenocarcinoma cells (Caco-2) were incubated with 10 ug/mL tacrolimus or 0.1% DMSO for 4 hours, then the oxygen consumption rates were measured on the Seahorse XFe24 Analyzers according to the manufacturer’s protocols.

Results

Hypoxia in the colonic epithelium was significantly disrupted in DSS-induced colitis mice, which was partially restored by tacrolimus treatment. The iNOS expression on both mRNA and protein levels was significantly upregulated by DSS and downregulated after tacrolimus administration, while the addition of AG enhanced the effects of tacrolimus. Compared with the individual tacrolimus intervention, the combined treatment of tacrolimus and AG achieved a better remission in weight loss, colonic shortening, and colonic pathological changes including the disruption of crypt-villus structure, goblet cell loss and immune cell infiltration. In vitro measurement of mitochondrial oxidative phosphorylation showed that tacrolimus significantly enhanced oxygen consumption, and promoted ATP production and spare respiratory capacity in Caco-2 cells.

Conclusion

Our results indicated that tacrolimus improved the oxidative metabolism of colonocytes in experimental colitis, which could be an important contributor to the alterations in the gut microbes induced by tacrolimus. The observed effect of tacrolimus in colonocytes may be associated with the inhibition of iNOS.

References

1. Litvak Y, Byndloss MX, Bäumler AJ. Colonocyte metabolism shapes the gut microbiota. Science 2018;362.
2. Cevallos SA, Lee JY, Velazquez EM, et al. 5-Aminosalicylic Acid Ameliorates Colitis and Checks Dysbiotic Escherichia coli Expansion by Activating PPAR-γ Signaling in the Intestinal Epithelium. mBio 2021;12.
3. Hämäläinen M, Lahti A, Moilanen E. Calcineurin inhibitors, cyclosporin A and tacrolimus inhibit expression of inducible nitric oxide synthase in colon epithelial and macrophage cell lines. Eur J Pharmacol 2002;448:239-44.
4. Lv W, Zhang D, He T, et al. Combination of Lactobacillus plantarum improves the effects of tacrolimus on colitis in a mouse model. 2023;13.

Introduction

Inflammatory bowel disease (IBD) is a complex chronic and relapsing inflammatory disorder of the gastrointestinal tract with unclear pathogenesis. Accumulating evidence suggests food emulsifiers as a risk factor for the development of IBD. It is indicated that food emulsifiers carboxymethylcellulose (CMC) can alter gut microbiota and promote intestinal inflammation in IBD under normal diet. Since high-fat diet is the main life scenario for CMC use, little is known about the effect of CMC on colitis model in different high-fat diet.

Aims & Methods

C57/BL6 mice (3-5 weeks old) were randomly divided into three high-fat diet (HFD) groups, which only differed by fat composition (40 %), derived from corn oil (HCO, n-6 polyunsaturated fatty acids), olive oil (HOO, monounsaturated fatty acids) and milk fat (HMF, saturated fatty acids), and exposed to CMC (1.0% w/v) in drinking water for 4 weeks before the establishment of dextran sulfate sodium (DSS) induced acute colitis mice model. Colon inflammation severity was evaluated using survival rate, HE staining, colon length, body weight change and DAI score. Ki67 and TUNEL assays were performed to detect proliferation and apoptosis.

Results

Compared with the CMC+DSS group, the HMF+CMC+DSS group markedly increased survival rate (P=0.013), colon length (P=0.027) and body weight (day4, day5, day6 and day7, P<0.05, respectively), and relieved DAI score (day4 and day6, P<0.05, respectively) and histological damage (P<0.0001). The HOO+CMC+DSS group seemly had no significant difference from the CMC+DSS group. In contrast, the HCO+CMC+DSS group developed more aggressive colitis with increased DAI score (day5, P<0.01) and decreased body weight (day1, day2, day5 and day6, P<0.05, respectively). In terms of maintaining homeostasis of the intestinal barrier, the HMF+CMC+DSS group showed the highest Ki67 (P=0.012, P=0.0002 P=0.042, respectively) and TUNEL positive cells (P=0.324, P<0.0001, P<0.0001, respectively) compared with the CMC+DSS, HOO+CMC+DSS or HCO+CMC+DSS group. Between CMC+DSS and HOO+CMC+DSS or HCO+CMC+DSS group, there was no difference in Ki67 positive cells, while TUNEL positive cells significantly decreased (P=0.002, P=0.008, respectively).

Conclusion

The function of CMC on DSS colitis varies in different high-fat. Unexpectedly, CMC protects against the development of high-milk-fat related colitis by maintaining homeostasis of proliferation and apoptosis.

References

The function of CMC on DSS colitis varies in different high-fat. Unexpectedly, CMC protects against the development of high-milk-fat related colitis by maintaining homeostasis of proliferation and apoptosis.

Disclosure

none

Introduction

Oxidative stress disruption are involved in the occurrence and development of inflammatory bowel disease(IBD). Previous reports have shown that melanin nanoparticles (MNPs) have the function of regulating oxidative stress. The aim of this study is to explore the preventive effect of MNPs on DSS-induced colitis in mice and its mechanism.

Aims & Methods

In this study, C57BL/6 wild-type mice were divided into 7 groups according to whether induced colitis by DSS, whether prophylactic application of MNPs or mesalazine, and different administration method. Mice were randomly assigned to control group, DSS group, MNPs gavage (MNPs-ig+DSS), MNPs enema (MNPs-en+DSS), MNPs enema + gavage (MNPs-bo+DSS), MSL gavage (MSL-ig+DSS), and MSL gavage + enema group (MSL-bo+DSS). The effect of MNPs on DSS-mice was evaluated by clinical manifestation, length of colon, pathological scores of colon tissues, and the level of inflammatory cytokines. The distribution and expression of intestinal mucosal junction proteins Occludin (OCC) and ZO-1 were detected to evaluate the effect of MNPs on colon mucosal barrier. In addition, the abundance and species distribution of intestinal microbiota were determined by 16S rRNA sequencing in each group. In the mechanism exploration part, the level of superoxide dismutase (SOD) and malondialdehyde (MDA) were used to evaluate the activity of reactive oxygen species (ROS) in mouse colon tissue, and the expression of ROS-related NLRP3/NF-κB signaling pathway-related proteins was further detected. In addition, this study verified the effect of MNPs on liver and kidney through the evaluation of organ morphology and function.

Results

C57BL/6 wild-type mice showed weight loss, colon shortening, increase of DAI and intestinal pathological scores, accompanied by increased levels of intestinal inflammatory cytokines TNF-α, IL-1β and IL-6 and decreased levels of IL-10 in colon tissue, which can be prevented by the application of MNPs to a certain extent. MNPs can help protected the integrity of the intestinal mucosal barrier, which is manifested by the restoration of the distribution and expression of tight junction proteins ZO-1 and Occludin. However, 16S rRNA sequencing failed to show a significant regulatory effect of MNPs on the intestinal microbiota. MNPs can significantly reduce the level of colonic SOD and increase the level of MDA induced by DSS, and this effect was related to the inhibition of the activation of NLRP3 inflammasome and the expression of downstream NF-κB inflammatory signaling pathway proteins. Finally, MNPs had no effect on the morphology and function of liver and kidney.

Conclusion

MNPs can improve colitis and regulate mucosal barrier function in mice, which may be related to reduced oxidative stress and inhibited activation of NLRP3 inflammasome and downstream NF-κB inflammatory signaling pathway. Our findings suggest the potential value of MNPs as a treatment for IBD.

Introduction

Glucocorticoids (GC) are important antiinflammatory and immunosuppresive agents in the management of inflammatory bowel disease and many other conditions. However, GC also display deleterious effects on the intestinal barrier which may limit their clinical benefit. These effects may be ascribed partly to actions on the epithelium [1]. In this regard, mice carrying a deletion of the GC receptor in intestinal epithelial cells are protected against experimental colitis [2]. It may be therefore beneficial to limit GC epithelial actions in the inflamed intestine. On the other hand, recent evidence shows that dexamethasone-treated mice aggravate acute experimental colitis due to the activation of mTOR pathway in intestinal epithelial cells [3].

Aims & Methods

Our aim is to limit harmful GC effects in intestinal epithelium in experimental colitis, with the ultimate goal of improving the management of inflammatory bowel disease with GC.
This study features two experiments. First, experimental colitis was induced by administration of 2.5% dextran sulfate sodium (DSS) for 7 days to C57BL/6J mice and treated with prednisolone 200 µg/day by either the oral or parenteral route (as prednisolone phosphate). Secondly, effects of glucocorticoid-dependent mTOR pathway activation were assessed by administering prednisolone at the same dose in combination with rapamycin 60 µg/day, a specific mTOR signal inhibitor, both by the oral route, to DSS-induced colitic C57BL/6J mice, in the same conditions as the first experiment.

Results

​Prednisolone ameliorated colitis, based on downregulated expression of inflammatory markers (S100a8, S100a9, Cxcl1, Il6) and lower colon thickening. However, oral (but not intraperitoneal) prednisolone augmented 4 kDa FITC-dextran permeability. This correlated with plasma lipopolysaccharide (LPS) levels, which were higher with oral prednisolone. In addition, colonic hemorrhage was enhanced by prednisolone treatment, particularly when given orally, as it both accelerated and enhanced blood loss. Besides, mice treated with oral prednisolone showed an increased disease activity index (DAI, based on body weight loss, feces consistency and rectal bleeding). On the other hand, GC administration suppressed Cyp11a1 expression in the colonic mucosa and also in jejunal organoids, suggesting inhibition of colonic steroidogenesis by exogenous treatment. These findings were largely reproduced using 6 µg/day of budesonide but, in this case, body weight loss was also augmented by glucocorticoid treatment. In the second experiment, rapamycin reduced DAI compared to prednisolone-only treatment, and additionally decreased bacterial adherence to colonic mucosa. This is consistent with previous findings with dexamethasone [3].

Conclusion

Our data are consistent with GC weakening of the mucosal barrier coexisting with an antiinflammatory response, which may be enhanced by oral administration. Addition of rapamycin appears to limit this detrimental effect of prednisolone, thus improving its benefit:risk ratio in this model.

References

1. Ocon, B, et al., Biochem Pharmacol, 2016. 116: p. 73-88.
2. Arredondo-Amador, M, et al., Br J Pharmacol, 2021. 178(12): p. 2482-2495.
3. Zhang, Z, et al., Cancers, 2020. 12(4), 945.

Introduction

Inflammatory bowel disease (IBD), encompassing ulcerative colitis and Crohn’s disease, is characterized by chronic intestinal inflammation linked to dysbiosis and impaired bile acid (BA) metabolism. Recent studies have underscored the pivotal role of the gut microbiota–BA axis in modulating intestinal immunity [1]. Disruptions in this axis, particularly reduced microbial conversion of primary to secondary BAs, have been implicated in the pathogenesis of IBD [2–4]. Probiotic interventions have emerged as promising strategies to restore microbial balance and modulate BA profiles. Some probiotic bacteria express enzymes that can modify bile acids thereby influencing BA receptor signaling pathways such as GPBAR1, FXR and receptors that have recently been identified as BA-binding receptors, such as RORγt [4–6]. Building upon this framework, our previous research demonstrated that specific microbial-derived BAs, notably 3-oxo-DCA, act as dual agonists for GPBAR1 and inverse agonists for RORγt, leading to reduced Th17-mediated inflammation in murine models of colitis [7]. These findings suggest that targeted modulation of the gut microbiota–BA axis via probiotic formulations could offer therapeutic benefits in IBD management.

Aims & Methods

To investigate the therapeutic potential of a novel multi-strain probiotic formulation in reducing colitis. For this purpose, we employed murine models of colitis induced in male C57BL/6 mice. In the acute colitis model, disease was induced by administering a 2% dextran sulfate sodium (DSS) solution in drinking water for 9 days. Chronic colitis was induced using three cycles of 1.5% DSS administration (1 week) followed by 15 days of water to mimic the exacerbation-remission phases of human IBD. The new probiotic formulation, named Vivomixx neo9®, was administered daily by gavage at 50 × 10^9 CFU/kg. Vivomixx neo9® included nine bacterial strains: Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus (two distinct bacterial strains), Lactobacillus lactis, and Streptococcus thermophilus. Vivomixx®, a commercial probiotic, served as the control.

Results

Our data demonstrated that treatment with both probiotic strains effectively mitigated disease severity in both acute and chronic murine models of colitis. Furthermore, the novel probiotic formulation notably ameliorated colonic mucosal injury at both macroscopic and microscopic levels and decreased fibrosis indices. DSS administration induced severe gut dysbiosis, reflected by substantial alterations in intestinal microbiota composition and disrupted BA profiles. Specifically, fecal BA analysis revealed a pronounced dysregulation characterized by diminished secondary BA levels and an elevated primary-to-secondary BA ratio. Probiotic treatment effectively reversed these dysbiotic changes, restoring fecal BA profiles comparable to those observed in naive mice. Of particular interest, the novel multi-strain probiotic enhanced the generation of oxo- and allo-BA derivatives, recognized as dual agonists for GPBAR1 and inverse agonists for RORγt, regulators of innate and adaptive immune responses.

Conclusion

Our findings indicate that Vivomixx neo9® significantly reduces colitis severity through modulation of intestinal microbiota composition and normalization of fecal bile acid profiles. These results underscore the therapeutic promise of targeting microbiota–bile acid interplay as a supplementary approach in the clinical management of IBD.

References

[1] Pan Y, Zhang H, Li M, He T, Guo S, Zhu L, et al. Novel approaches in IBD therapy: targeting the gut microbiota-bile acid axis. Gut Microbes 2024;16:2356284. https://doi.org/10.1080/19490976.2024.2356284.
[2] Bai SH, Chandnani A, Cao S. Bile Acids in Inflammatory Bowel Disease: From Pathophysiology to Treatment. Biomedicines 2024;12. https://doi.org/10.3390/biomedicines12122910.
[3] Lavelle A, Sokol H. Gut microbiota-derived metabolites as key actors in inflammatory bowel disease. Nat Rev Gastroenterol Hepatol 2020;17:223–37. https://doi.org/10.1038/s41575-019-0258-z.
[4] Biagioli M, Marchianò S, Carino A, Di Giorgio C, Santucci L, Distrutti E, et al. Bile Acids Activated Receptors in Inflammatory Bowel Disease. Cells 2021;10:1281. https://doi.org/10.3390/cells10061281.
[5] Fiorucci S, Carino A, Baldoni M, Santucci L, Costanzi E, Graziosi L, et al. Bile Acid Signaling in Inflammatory Bowel Diseases. Dig Dis Sci 2021;66:674–93. https://doi.org/10.1007/s10620-020-06715-3.
[6] Paik D, Yao L, Zhang Y, Bae S, D’Agostino GD, Zhang M, et al. Human gut bacteria produce Τ(Η)17-modulating bile acid metabolites. Nature 2022;603:907–12. https://doi.org/10.1038/s41586-022-04480-z.
[7] Biagioli M, Di Giorgio C, Massa C, Marchianò S, Bellini R, Bordoni M, et al. Microbial-derived bile acid reverses inflammation in IBD via GPBAR1 agonism and RORγt inverse agonism. Biomed Pharmacother 2024;181:117731. https://doi.org/10.1016/j.biopha.2024.117731.

Introduction

Inflammatory bowel disease (IBD), a chronic inflammatory disease of the gastrointestinal tract, is caused by various factors, including genetic factors and environmental factors, as well as dysregulated immune responses. LMT503, a novel organic small-molecule compound, has the potential to suppress pro-inflammatory cells and induce anti-inflammatory cells by modulating their cell metabolisms. We evaluated the anti-inflammatory effects of LMT503 in a murine adoptive T cell transfer-induced colitis model.

Aims & Methods

This study was conducted to evaluate the anti-inflammatory effect of LMT503 in the murine chronic colitis model. Naïve CD4⁺CD25^-CD45RB^high CD62L^high T cells that were isolated from wild-type mice by flow cytometry based-method and transferred to Rag1 knock-out mice by intraperitoneal injection to induce murine adoptive T cell transfer-induced colitis. LMT503 was orally administrated (50mg/kg, 100mg/kg) for 2 weeks starting at 5 weeks after naïve T cell transfer. The disease activity index (DAI) was checked daily, and histopathological score, macroscopic injury score, and colon length were evaluated upon sacrifice. Macrophages and T cells from the spleen were analyzed by flow cytometry. Gene expression in colon tissue was measured by qRT-PCR and cytokine profiles in plasma was measured by cytometric bead array. Myeloperoxidase (MPO) activity was measured using the MPO activity kit.

Results

Oral administration of LMT503 ameliorated adoptive T cell transfer-induced colitis in mice. The DAI, histopathological score, and macroscopic injury score were significantly decreased after the administration of LMT503 in a dose-dependent manner. The LMT503-treated group showed significantly increased mucin production in the colon. Th1, Th17 cells, and M1 macrophages in the spleen were suppressed, and M2 macrophages were induced by the administration of LMT503. MPO activity and gene expressions of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, in colon tissue were suppressed by the administration of LMT503. And the level of inflammatory cytokines, such as IL-2, TNF-α, and IFN-γ, in plasma were significantly suppressed by the administration of LMT503. Especially, LMT503 treatment significantly increased the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a), a master regulator of mitochondrial biogenesis, suggesting that LMT503 modulates cellular metabolism towards an anti-inflammatory metabolism.

Conclusion

Our data suggest that the anti-inflammatory effect of LMT503 ameliorates murine adoptive T cell transfer-induced colitis by modulating cellular metabolism and immune cell modulation. Therefore, LMT503 may be a novel therapeutic drug that opens new avenues for the treatment of IBD.

References

1. Erben U, Loddenkemper C, Doerfel K, et al. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7(8):4557-4576.
2. Ostanin DV, Bao J, Koboziev I, et al. T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am J Physiol Gastrointest Liver Physiol. 2009;296(2):G135-G146.
3. Ghoreschi K, Jesson MI, Li X, et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol. 2011;186(7):4234-4243.
4. Burke JR, Cheng L, Gillooly KM, et al. Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain. Sci Transl Med. 2019;11(502):eaaw1736.
5. Kiesler P, Fuss IJ, Strober W. Experimental Models of Inflammatory Bowel Diseases. Cell Mol Gastroenterol Hepatol. 2015;1(2):154-170.