Si-Ni-San alleviates intestinal and liver damage in ulcerative colitis mice by regulating cholesterol metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Si-Ni-San (SNS), a traditional Chinese medicinal formula derived from Treatise on Febrile Diseases, is considered effective in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice. However, the bioactive ingredients and underlying mechanisms are still unclear and need further investigation. AIM OF THE STUDY: This study aimed to evaluate the effect, explore the bioactive ingredients and the underlying mechanisms of SNS in ameliorating ulcerative colitis (UC) and associated liver injury in dextran sodium sulphate (DSS)-induced mouse colitis models. MATERIALS AND METHODS: The effect of SNS (1.5, 3, 6 g/kg) on 3% DSS-induced acute murine colitis was evaluated by disease activity index (DAI), colon length, inflammatory cytokines, hematoxylin-eosin (H&E) staining, tight junction proteins expression, ALT, AST, and oxidative stress indicators. HPLC-ESI-IT/TOF MS was used to analyze the chemical components of SNS and the main xenobiotics in the colon of UC mice after oral administration of SNS. Network pharmacological study was then conducted based on the main xenobiotics. Flow cytometry and immunohistochemistry techniques were used to demonstrate the inhibitory effect of SNS on Th17 cells differentiation and the amelioration of Th17/Treg cell imbalance. LC-MS/MS, Real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting techniques were performed to investigate the oxysterol-Liver X receptor (LXRs) signaling activity in colon. Targeted bile acids metabolomics was conducted to reveal the change of the two major pathways of bile acid synthesis in the liver, and the expression of key metabolic enzymes of bile acids synthesis was characterized by RT-qPCR and western blotting techniques. RESULTS: SNS (1.5, 3, 6 g/kg) decreased the DAI scores, protected intestinal mucosa barrier, suppressed the production of pro-inflammatory cytokines, improved hepatic and splenic enlargement and alleviated liver injury in a dose-dependent manner. A total of 22 components were identified in the colon of SNS (6 g/kg) treated colitis mice, and the top 10 components ranked by relative content were regarded as the potential effective chemical components of SNS, and used to conduct network pharmacology research. The efficacy of SNS was mediated by a reduction of Th17 cell differentiation, restoration of Th17/Treg cell homeostasis in the colon and spleen, and the experimental results were consistent with our hypothesis and the biological mechanism predicted by network pharmacology. Mechanistically, SNS regulated the concentration of 25-OHC and 27-OHC by up-regulated CH25H, CYP27A1 protein expression in colon, thus affected the expression and activity of LXR, ultimately impacted Th17 differentiation and Th17/Treg balance. It was also found that SNS repressed the increase of hepatic cholesterol and reversed the shift of BA synthesis to the acidic pathway in UC mice, which decreased the proportion of non-12-OH BAs in total bile acids (TBAs) and further ameliorated colitis and concomitant liver injury. CONCLUSIONS: This study set the stage for considering SNS as a multi-organ benefited anti-colitis prescription based on the significant effect of ameliorating intestinal and liver damage, and revealed that derivatives of cholesterol, namely oxysterols and bile acids, were closely involved in the mechanism of SNS anti-colitis effect.

Oral administration of Sophora Flavescens-derived exosomes-like nanovesicles carrying CX5461 ameliorates DSS-induced colitis in mice.

Ulcerative colitis (UC) belongs to chronic inflammatory disease with a relapsing characterization. Conventional oral drugs of UC are restricted in clinical by premature degradation in the gastrointestinal tract, modest efficacy, and adverse effects. CX5461 can treat autoimmune disease, immunological rejection, and vascular inflammation. However, low solubility, intravenous administration, and non-inflammatory targeting limited its clinical application. Herein, this work aims to develop Sophora Flavescens-derived exosomes-like nanovesicles carrying CX5461 (SFELNVs@CX5461) for efficient CX5461 oral delivery for UC therapy. We identified SFELNVs as nano-diameter (80 nm) with negative zeta potential (-32mV). Cellular uptake has shown that SFELNVs were targeted uptake by macrophages, thus increasing drug concentration. Additionally, oral SFELNVs@CX5461 exhibited good safety and stability, as well as inflammation-targeting ability in the gastrointestinal tract of dextran sodium sulfate (DSS)-induced colitis mice. In vivo, oral administration of SFELNVs and CX5461 could relieve mice colitis. More importantly, combined SFELNVs and CX5461 alleviated mice colitis by inhibiting pro-inflammatory factors (TNF-α, IL-1ß, and IL-6) expression and promoting M2 macrophage polarization. Furthermore, SFELNVs promoted M2 polarization by miR4371c using miRNA sequencing. Our results suggest that SFELNVs@CX5461 represents a novel orally therapeutic drug that can ameliorate colitis, and a promising targeting strategy for safe UC therapy.

Epithelial-specific loss of Smad4 alleviates the fibrotic response in an acute colitis mouse model.

Mucosal healing is associated with better clinical outcomes in patients with inflammatory bowel disease. But the epithelial-specific contribution to mucosal healing in vivo is poorly understood. We evaluated mucosal healing in an acute dextran sulfate sodium mouse model that shows an alleviated colitis response after epithelial-specific loss of Smad4. We find that enhanced epithelial wound healing alleviates the fibrotic response. Dextran sulfate sodium caused increased mesenchymal collagen deposition-indicative of fibrosis-within a week in the WT but not in the Smad4 KO colon. The fibrotic response correlated with decreased epithelial proliferation in the WT, whereas uninterrupted proliferation and an expanded zone of proliferation were observed in the Smad4 KO colon epithelium. Furthermore, the Smad4 KO colon showed epithelial extracellular matrix alterations that promote epithelial regeneration. Our data suggest that epithelium is a key determinant of the mucosal healing response in vivo, implicating mucosal healing as a strategy against fibrosis in inflammatory bowel disease patients.

Sex-specific post-inflammatory dysbiosis mediates chronic visceral pain in colitis.

BACKGROUND: Despite achieving endoscopic remission, over 20% of inflammatory bowel disease (IBD) patients experience chronic abdominal pain. Visceral pain and the microbiome exhibit sex-dependent interactions, while visceral pain in IBD shows a sex bias. Our aim was to evaluate whether post-inflammatory microbial perturbations contribute to visceral hypersensitivity in a sex-dependent manner. METHODS: Males, cycling females, ovariectomized, and sham-operated females were given dextran sodium sulfate to induce colitis and allowed to recover. Germ-free recipients received sex-appropriate and cross-sex fecal microbial transplants (FMT) from post-inflammatory donor mice. Visceral sensitivity was assessed by recording visceromotor responses to colorectal distention. The composition of the microbiota was evaluated via 16S rRNA gene V4 amplicon sequencing, while the metabolome was assessed using targeted (short chain fatty acids - SCFA) and semi-targeted mass spectrometry. RESULTS: Post-inflammatory cycling females developed visceral hyperalgesia when compared to males. This effect was reversed by ovariectomy. Both post-inflammatory males and females exhibited increased SCFA-producing species, but only males had elevated fecal SCFA content. FMT from post-inflammatory females transferred visceral hyperalgesia to both males and females, while FMT from post-inflammatory males could only transfer visceral hyperalgesia to males. CONCLUSIONS: Female sex, hormonal status as well as the gut microbiota play a role in pain modulation. Our data highlight the importance of considering biological sex in the evaluation of visceral pain.

Pravastatin prevents colitis-associated carcinogenesis by reducing CX3CR1high M2-like fibrocyte counts in the inflamed colon.

Colorectal cancer (CRC) resulting from chronic inflammation is a crucial issue in patients with inflammatory bowel disease (IBD). Although many reports established that intestinal resident CX3CR1high macrophages play an essential role in suppressing intestinal inflammation, their function in colitis-related CRC remains unclear. In this study, we found that colonic CX3CR1high macrophages, which were positive for MHC-II, F4/80 and CD319, promoted colitis-associated CRC. They highly expressed Col1a1, Tgfb, II10, and II4, and were considered to be fibrocytes with an immunosuppressive M2-like phenotype. CX3CR1 deficiency led to reductions in the absolute numbers of CX3CR1high fibrocytes through increased apoptosis, thereby preventing the development of colitis-associated CRC. We next focused statins as drugs targeting CX3CR1high fibrocytes. Statins have been actively discussed for patients with IBD and reported to suppress the CX3CL1/CX3CR1 axis. Statin treatment after azoxymethane/dextran sulfate sodium-induced inflammation reduced CX3CR1high fibrocyte counts and suppressed colitis-associated CRC. Therefore, CX3CR1high fibrocytes represent a potential target for carcinogenesis-preventing therapy, and statins could be safe therapeutic candidates for IBD.

Interferon regulatory factor 7 alleviates the experimental colitis through enhancing IL-28A-mediated intestinal epithelial integrity.

BACKGROUND: The incidence of inflammatory bowel disease (IBD) is on the rise in developing countries, and investigating the underlying mechanisms of IBD is essential for the development of targeted therapeutic interventions. Interferon regulatory factor 7 (IRF7) is known to exert pro-inflammatory effects in various autoimmune diseases, yet its precise role in the development of colitis remains unclear. METHODS: We analyzed the clinical significance of IRF7 in ulcerative colitis (UC) by searching RNA-Seq databases and collecting tissue samples from clinical UC patients. And, we performed dextran sodium sulfate (DSS)-induced colitis modeling using WT and Irf7-/- mice to explore the mechanism of IRF7 action on colitis. RESULTS: In this study, we found that IRF7 expression is significantly reduced in patients with UC, and also demonstrated that Irf7-/- mice display heightened susceptibility to DSS-induced colitis, accompanied by elevated levels of colonic and serum pro-inflammatory cytokines, suggesting that IRF7 is able to inhibit colitis. This increased susceptibility is linked to compromised intestinal barrier integrity and impaired expression of key molecules, including Muc2, E-cadherin, ß-catenin, Occludin, and Interleukin-28A (IL-28A), a member of type III interferon (IFN-III), but independent of the deficiency of classic type I interferon (IFN-I) and type II interferon (IFN-II). The stimulation of intestinal epithelial cells by recombinant IL-28A augments the expression of Muc2, E-cadherin, ß-catenin, and Occludin. The recombinant IL-28A protein in mice counteracts the heightened susceptibility of Irf7-/- mice to colitis induced by DSS, while also elevating the expression of Muc2, E-cadherin, ß-catenin, and Occludin, thereby promoting the integrity of the intestinal barrier. CONCLUSION: These findings underscore the pivotal role of IRF7 in preserving intestinal homeostasis and forestalling the onset of colitis.

Intestinal Lactobacillus murinus-derived small RNAs target porcine polyamine metabolism.

Gut microbiota plays a vital role in host metabolism; however, the influence of gut microbes on polyamine metabolism is unknown. Here, we found germ-free models possess elevated polyamine levels in the colon. Mechanistically, intestinal Lactobacillus murinus-derived small RNAs in extracellular vesicles down-regulate host polyamine metabolism by targeting the expression of enzymes in polyamine metabolism. In addition, Lactobacillus murinus delays recovery of dextran sodium sulfate-induced colitis by reducing polyamine levels in mice. Notably, a decline in the abundance of small RNAs was observed in the colon of mice with colorectal cancer (CRC) and human CRC specimens, accompanied by elevated polyamine levels. Collectively, our study identifies a specific underlying mechanism used by intestinal microbiota to modulate host polyamine metabolism, which provides potential intervention for the treatment of polyamine-associated diseases.

Human IL-22 receptor-targeted small protein antagonist suppress murine DSS-induced colitis.

BACKGROUND: Human interleukin-22 (IL-22) is known as a "dual function" cytokine that acts as a master regulator to maintain homeostasis, structural integrity of the intestinal epithelial barrier, and shielding against bacterial pathogens. On the other hand, the overexpression of IL-22 is associated with hyper-proliferation and recruitment of pathologic effector cells, leading to tissue damage and chronic inflammation in specific diseases including inflammatory bowel disease (IBD). To study a role of IL-22-mediated signaling axis during intestinal inflammation, we generated a set of small protein blockers of IL-22R1 and verified their inhibitory potential on murine model of colitis. METHODS: We used directed evolution of proteins to identify binders of human IL-22 receptor alpha (IL-22R1), designated as ABR ligands. This approach combines the assembly of a highly complex combinatorial protein library derived from small albumin-binding domain scaffold and selection of promising protein variants using ribosome display followed by large-scale ELISA screening. The binding affinity and specificity of ABR variants were analyzed on transfected HEK293T cells by flow cytometry and LigandTracer. Inhibitory function was further verified by competition ELISA, HEK-Blue IL-22 reporter cells, and murine dextran sulfate sodium (DSS)-induced colitis. RESULTS: We demonstrate that ABR specifically recognizes transgenic IL-22R1 expressed on HEK293T cells and IL-22R1 on TNFα/IFNγ-activated HaCaT cells. Moreover, some ABR binders compete with the IL-22 cytokine and function as IL-22R1 antagonists in HEK-Blue IL22 reporter cells. In a murine model of DSS-induced acute intestinal inflammation, daily intraperitoneal administration of the best IL-22R1 antagonist, ABR167, suppressed the development of clinical and histological markers of colitis including prevention of mucosal inflammation and architecture deterioration. In addition, ABR167 reduces the DSS-induced increase in mRNA transcript levels of inflammatory cytokines such as IL-1ß, IL-6, IL-10, and IL-17A. CONCLUSIONS: We developed small anti-human IL-22R1 blockers with antagonistic properties that ascertain a substantial role of IL-22-mediated signaling in the development of intestinal inflammation. The developed ABR blockers can be useful as a molecular clue for further IBD drug development.

Vitamin B12 ameliorates gut epithelial injury via modulating the HIF-1 pathway and gut microbiota.

Inflammatory bowel diseases (IBDs) are immune chronic diseases characterized by recurrent episodes, resulting in continuous intestinal barrier damage and intestinal microbiota dysbiosis. Safe strategies aimed at stabilizing and reducing IBDs recurrence have been vigorously pursued. Here, we constructed a recurrent intestinal injury Drosophila model and found that vitamin B12 (VB12), an essential co-factor for organism physiological functions, could effectively protect the intestine and reduce dextran sulfate sodium-induced intestinal barrier disruption. VB12 also alleviated microbial dysbiosis in the Drosophila model and inhibited the growth of gram-negative bacteria. We demonstrated that VB12 could mitigate intestinal damage by activating the hypoxia-inducible factor-1 signaling pathway in injured conditions, which was achieved by regulating the intestinal oxidation. In addition, we also validated the protective effect of VB12 in a murine acute colitis model. In summary, we offer new insights and implications for the potential supportive role of VB12 in the management of recurrent IBDs flare-ups.

5-Methoxytryptophan Alleviates Dextran Sulfate Sodium-Induced Colitis by Inhibiting the Intestinal Epithelial Damage and Inflammatory Response.

Background: Colitis is a refractory intestinal inflammatory disease significantly affecting the quality of a patient's life and increasing the risk of exacerbation. The primary factors leading to colitis encompass infections, insufficient blood flow, and the buildup of collagen as well as white blood cells. Among various available therapeutics, 5-methoxytryptophan (5-MTP) has emerged as one of the protectants by inhibiting inflammatory damage. Nonetheless, there is no report on the role of 5-MTP in the treatment of colitis. Materials and Methods: To verify the anti-inflammatory effect of 5-MTP in vivo, we first constructed mouse model with dextran sulfate sodium-induced colitis. Furthermore, the macrophage infiltration and release of inflammatory factors through western blot (WB) and hematoxylin-eosin staining analyses were examined. Intestinal epithelial cell tight junction damage and apoptosis were investigated by WB analysis, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Finally, we examined the generation of cellular inflammation and analyzed the influence of 5-MTP on M1 polarization at the cellular level. Results: This study initially confirmed that 5-MTP possessed an excellent therapeutic effect on colitis. 5-MTP inhibits macrophage infiltration and the generation of inflammatory factors. In addition to its effects on immune cells, 5-MTP significantly inhibits intestinal epithelial cell tight junction damage and apoptosis in vivo. Moreover, it inhibits inflammation and M1 polarization response in vitro. Conclusion: 5-MTP counteracts excessive inflammation, thereby preventing intestinal epithelial tight junction damage. In addition, inhibition of apoptosis suggests that 5-MTP may be a potential therapeutic agent for colitis.

Sirtuin 6 inhibits group 3 innate lymphoid cell function and gut immunity by suppressing IL-22 production.

Introduction: Group 3 innate lymphoid cells (ILC3s) are enriched in the intestinal mucosa and play important roles in host defense against infection and inflammatory diseases. Sirtuin 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD+)- dependent deacetylase and has been shown to control intestinal epithelial cell differentiation and survival. However, the role of SIRT6 in ILC3s remains unknown. Methods: To investigate the role of SIRT6 in gut ILC3s, we generated SIRT6 conditional knockout mice by crossing Rorccre and Sirt6flox/flox mice. Cell number and cytokine production was examined using flow cytometry. Citrobacter rodentium infection and dextran sodium sulfate-induced colitis models were used to determine the role of SIRT6 in gut defense. RT-qPCR, flow cytometry and immunohistochemistry were used to assess the intestinal inflammatory responses. Results: Here we show that SIRT6 inhibits IL-22 expression in intestinal ILC3s in a cell-intrinsic manner. Deletion of SIRT6 in ILC3s does not affect the cell numbers of total ILC3s and subsets, but results in increased IL-22 production. Furthermore, ablation of SIRT6 in ILC3s protects mice against Citrobacter rodentium infection and dextran sodium sulfate-induced colitis. Our results suggest that SIRT6 may play a role in ILC3 function by regulating gut immune responses against bacterial infection and inflammation. Discussion: Our finding provided insight into the relation of epigenetic regulators with IL-22 production and supplied a new perspective for a potential strategy against inflammatory bowel disease.

Protection against DSS-induced colitis in mice through FcεRIα deficiency: the role of altered Lactobacillus.

The role of mast cells (MCs) in ulcerative colitis (UC) development is controversial. FcεRI, the IgE high-affinity receptor, is known to activate MCs. However, its role in UC remains unclear. In our study, Anti-FcεRI showed highly diagnostic value for UC. FcεRIα knockout in mice ameliorated DSS-induced colitis in a gut microbiota-dependent manner. Increased Lactobacillus abundance in FcεRIα deficient mice showed strongly correlation with the remission of colitis. RNA sequencing indicated activation of the NLRP6 inflammasome pathway in FcεRIα knockout mice. Additionally, Lactobacillus plantarum supplementation protected against inflammatory injury and goblet cell loss, with activation of the NLRP6 inflammasome during colitis. Notably, this effect was absent when the strain is unable to produce lactic acid. In summary, colitis was mitigated in FcεRIα deficient mice, which may be attributed to the increased abundance of Lactobacillus. These findings contribute to a better understanding of the relationship between allergic reactions, microbiota, and colitis.

Bacteroides salyersiae Is a Candidate Probiotic Species with Potential Anti-Colitis Properties in the Human Colon: First Evidence from an In Vivo Mouse Model.

Previous studies have indicated a critical role of intestinal bacteria in the pathogenesis of ulcerative colitis (UC). B. salyersiae is a commensal species from the human gut microbiota. However, what effect it has on UC development has not been investigated. In the present study, we explored this issue and demonstrated for the first time that oral administration of B. salyersiae CSP6, a bacterium previously isolated from the fecal sample of a healthy individual, protected against dextran sulfate sodium (DSS)-induced colitis in C57BL/6J mice. In particular, B. salyersiae CSP6 improved mucosal damage and attenuated gut dysbiosis in the colon of DSS-fed mice. Specifically, B. salyersiae CSP6 decreased the population of pathogenic Escherichia-Shigella spp. and increased the abundance of probiotic Dubosiella spp. and Bifidobacterium pseudolongum. Additionally, by reshaping the colonic microbiota, B. salyersiae CSP6 remarkably increased the fecal concentrations of equol, 8-deoxylactucin, and tiglic acid, three beneficial metabolites that have been well documented to exert strong anti-inflammatory effects. Altogether, our study provides novel evidence that B. salyersiae is a candidate probiotic species with potential anti-colitis properties in the human colon, which has applications for the development of next-generation probiotics.

[Sanguinarine alleviates ulcerative colitis in mice by regulating the Nrf2/NF-κB pathway].

OBJECTIVE: To investigate the mechanism of sanguinarine (SA) for alleviating ulcerative colitis (UC) induced by dextran sodium sulfate (DSS) in mice. METHODS: Male C57BL/6 mouse models of 3.5% DSS-induced UC were randomized for treatment with 1, 5 and 10 mg/kg SA by gavage, 400 mg/kg sulfasalazine by gavage, or 10 mg/kg SA combined with intraperitoneal injection of 30 mg/kg ML385 (a Nrf2 inhibitor). The changes in intestinal inflammation was assessed by monitoring weight changes, disease activity index (DAI) score, colon length measurement, and HE staining. After the treatments, the colon tissues were collected for detection of malondialdehyde (MDA) content using colorimetry, mRNA expressions of inflammatory factors using RT-qPCR, and the expressions of Nrf2, HO-1, Keap-1, p-p65, p65, occludin, and ZO-1 proteins were detected using Western blotting. RESULTS: SA treatment obviously alleviated weight loss, colon length shortening and DAI score increase and ameliorated structural destruction of the colon glands and colonic crypts in mice with DSSinduced UC. SA intervention significantly decreased the levels of TNF-α, IL-1ß and IL-6 mRNA and lowered ROS and MDA levels in the colon tissue of UC mice. The mouse models receiving SA treatment showed significantly increased expressions of Nrf2, HO-1, occludin and ZO-1 and lowered expressions of Keap-1 and P-P65 in the colon tissue without significant changes of p65 expression, and these changes were SA dose-dependent. Treatment with ML385 obviously attenuated the effect of highdose SA for improving UC in the mouse models. CONCLUSION: SA can improve UC-like enteritis in mice possibly by activating the Nrf2 pathway and inhibiting the NF-κB pathway in the colon tissue.

Cultured fecal microbial community and its impact as fecal microbiota transplantation treatment in mice gut inflammation.

The fecal microbiome is identical to the gut microbial communities and provides an easy access to the gut microbiome. Therefore, fecal microbial transplantation (FMT) strategies have been used to alter dysbiotic gut microbiomes with healthy fecal microbiota, successfully alleviating various metabolic disorders, such as obesity, type 2 diabetes, and inflammatory bowel disease (IBD). However, the success of FMT treatment is donor-dependent and variations in gut microbes cannot be avoided. This problem may be overcome by using a cultured fecal microbiome. In this study, a human fecal microbiome was cultured using five different media; growth in brain heart infusion (BHI) media resulted in the highest microbial community cell count. The microbiome (16S rRNA) data demonstrated that the cultured microbial communities were similar to that of the original fecal sample. Therefore, the BHI-cultured fecal microbiome was selected for cultured FMT (cFMT). Furthermore, a dextran sodium sulfate (DSS)-induced mice-IBD model was used to confirm the impact of cFMT. Results showed that cFMT effectively alleviated IBD-associated symptoms, including improved gut permeability, restoration of the inflamed gut epithelium, decreased expression of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1, IL-6, IL-12, and IL-17), and increased expression of anti-inflammatory cytokines (IL-4 and IL-10). Thus, study's findings suggest that cFMT can be a potential alternative to nFMT. KEY POINTS: • In vitro fecal microbial communities were grown in a batch culture using five different media. • Fecal microbial transplantation was performed on DSS-treated mice using cultured and normal fecal microbes. • Cultured fecal microbes effectively alleviated IBD-associated symptoms.

Lemairamin (Wgx-50) Attenuates DSS-Induced Intestinal Inflammation in Zebrafish.

Inflammatory bowel disease (IBD) is a chronic non-specific intestinal inflammatory disease that affects millions of people worldwide, and current treatment methods have certain limitations. This study aimed to explore the therapeutic potential and mechanism of action of lemairamin (Wgx-50) in inflammatory bowel disease (IBD). We used dextran sulfate sodium (DSS)-treated zebrafish as an inflammatory bowel disease model, and observed the effect of Wgx-50 on DSS-induced colitis inflammation. The results of the study showed that Wgx-50 could reduce the expression of pro-inflammatory cytokines induced by DSS and inhibit the recruitment of neutrophils to the site of intestinal injury. Further experiments revealed that Wgx-50 exerted its anti-inflammatory effect by regulating the activation of the Akt pathway. These research findings indicate that Wgx-50 possesses anti-inflammatory activity.

SIX4 Activation in Inflammatory Response Drives the Transformation of Colorectal Epithelium into Inflammation and Tumor via Feedback-Enhancing Inflammatory Signaling to Induce Tumor Stemness Signaling.

Some colorectal cancer patients have experienced normal epithelial transformation into inflammatory and tumor states, but the molecular basis still needs to be further determined. The expression levels of SIX4 are gradually increased in dextran sodium sulfate (DSS) and azoxymethane (AOM)/DSS-induced colonic epithelial inflammation and tumors, respectively, in mice. Targeting SIX4 alleviates intestinal inflammation occurrence and reduces adenoma formation in mice. Clinical sample assays indicated that SIX4 is upregulated in inflammatory bowel disease (IBD) and colorectal cancer (CRC) tissues compared to normal colorectal tissues. In a subsequent study, we found that SIX4, transcriptionally activated by the proinflammatory IL-6/STAT3 signal, binds to c-Jun to transcribe IL-6, thus forming a positive IL-6/STAT3/SIX4/c-Jun feedback loop, which further induces intestinal inflammation occurrence. In addition, elevated SIX4 also induces the expression of DeltaNp63, rather than wild-type p63, by binding to its promoter and thus facilitates the activation of tumor stemness signals, which ultimately leads to the formation of colorectal cancer. Our study first observes that activated SIX4 in inflammation induction drives the transformation of colorectal epithelium into inflammation and tumor, which demonstrates SIX4 as a significant therapeutic target in IBD and colitis-associated colorectal cancer (CAC) and CRC pathogenesis.

Pericentriolar material 1 promotes intestinal inflammation in ulcerative colitis by activating NLRP3/gasdermin D-mediated macrophage pyroptosis.

Excessive proinflammatory cytokine release induced by pyroptosis plays a vital role in intestinal mucosal inflammation in ulcerative colitis (UC). Several pyroptosis-related factors are regulated by the centrosome. Pericentriolar material 1 (PCM1) is a primary component of centriolar satellites that is present as cytoplasmic granules around the centrosome. Our previous study revealed that PCM1 was highly expressed in UC patients, but the role of PCM1 in UC remains unknown. This study aimed to elucidate the role of PCM1 in the development of UC, especially the mechanism in pyroptosis process of UC. Clinical mucosal sample and dextran sulfate sodium (DSS)-induced colitis mouse were used to reveal the association between PCM1 and intestinal inflammation. Intestinal epithelial cell-specific PCM1-knockout mice were constructed to determine the role of PCM1 in colitis. Finally, PCM1 RNA interference and overexpression assays in THP1 cells were employed to study the molecular mechanisms of PCM1 in inflammatory responses and pyroptosis. We found that PCM1 expression was upregulated in the colonic mucosa of UC patients and positively correlated with inflammatory indicators. PCM1 expression was elevated in DSS-induced colitis mice and was reduced after methylprednisolone treatment. In the DSS colitis model, intestinal-specific PCM1-knockout mice exhibited milder intestinal inflammation and lower pyroptosis levels than wild-type mice. In cell level, PCM1 exerted a proinflammatory effect by activating the NLRP3 inflammasome and triggering subsequent gasdermin D-mediated pyroptosis to release IL-1ß and IL-18. In conclusion, PCM1 mediates activation of the NLRP3 inflammasome and gasdermin D-dependent pyroptosis, ultimately accelerating intestinal inflammation in UC. These findings revealed a previously unknown role of PCM1 in initiating intestinal mucosal inflammation and pyroptosis in UC, and this factor is expected to be a regulator in the complex inflammatory network of UC.

[Changes in the expression of genes related to intestinal fatty acid oxidation and carnitine metabolism in patients with ulcerative colitis].

Objective: To investigate the changes in gene expression related to intestinal fatty acid oxidation and carnitine metabolism in patients with ulcerative colitis (UC). Methods: A retrospective study was conducted involving patients with UC (UC group) and non-UC controls (control group) who underwent routine colonoscopy to exclude polyps at Peking Union Medical College Hospital between January 1, 2018, to December 31, 2023. Colon tissue samples were collected from both groups and RNA was extracted. Real-time fluorescence quantitative polymerase chain reaction technology was used to detect the mRNA expression levels of genes related to fatty acid oxidation and carnitine metabolism and to analyze their correlation with inflammatory gene expression. The expression of genes linked to fatty acid oxidation and carnitine metabolism was analyzed by analyzing the colonic mucosal transcriptome data of UC patients and controls in high-throughput gene expression database (GEO). Immunohistochemistry was used to examine the expression of the carnitine transporter SLC6A14 in the intestinal tissues of both groups at the protein level. Eight-week-old male C57BL/6 mice were selected and divided into a drinking water group (drinkind daily water) and a dextran sodium sulfate (DSS) group (drinking 2.5% DSS solution) with 4 mice in each group. DSS was used to induce an acute colitis model in mice and detect the difference in mRNA expression levels of SLC6A14 and interleukin-6 (IL-6) in the intestinal tissues of the both groups of mice. Results: A total of 22 patients were included in the UC group, with 12 males and 10 females, aged 16-64 (40±12) years. The control group consisted of 10 patients, with 3 males and 7 females, aged 43-72 (64±8) years. The UC group had lower mRNA expression levels of genes related to fatty acid oxidation and transport in the intestine compared to those in the control group, such as CD36 [0.40 (0.27, 0.55) vs 0.93 (0.39, 2.93)], CPT1A [0.39 (0.07, 0.54) vs 0.93 (0.41, 1.71)], CPT1B (0.37±0.36 vs 1.37±0.89), CPT2 [0.36 (0.30, 0.43) vs 1.14 (0.68, 1.34)], CRAT [0.31 (0.25, 0.41) vs 1.06 (0.64, 1.73)], CROT [0.14 (0.10, 0.21) vs 0.95 (0.77, 1.27)] (all P<0.05). The mRNA expression levels of genes related to carnitine transport in the UC group were lower than those in the control group, such as OCTN1 [0.18 (0.10, 0.41) vs 0.83 (0.41, 1.47)], OCTN2 [0.01 (0.00, 0.01) vs 0.47 (0.35, 2.15)] (both P<0.05). The mRNA expression levels of the carnitine transporter gene SLC6A14 in the intestine of UC patients was higher than that of the control group [11.31 (5.34, 23.50) vs 0.78 (0.07, 3.70), P<0.001], and showed a positive correlation with the inflammatory gene IL-6 (r=0.425, 95%CI: 0.076-0.681, P=0.019). Analysis of the GEO database revealed lower expression levels of CD36, CPT1A, CPT2, CRAT and CROT in UC group compared to controls (all P<0.05), while the expression levels of SLC6A14 were higher than those in control group (P<0.05). The protein expression level of SLC6A14 in colon tissue of UC group was higher than that of control group (0.45±0.07 vs 0.30±0.01, P=0.019). The mRNA expression of SLC6A14 in the intestine of DSS group was higher compared to that in the drinking water group (1.83±0.90 vs 0.60±0.10, P=0.035). Conclusion: The expression levels of genes associated with intestinal fatty acid oxidation and carnitine metabolism (CD36, CPT1A, CPT1B, CPT2, CRAT, CROT, OCTN1, and OCTN2) are decreased in UC patients, while the expression level of SLC6A14, a gene capable of transporting both amino acids and carnitine, is increased.

Prophylactic Effects of Betaine on Depression and Anxiety Behaviors in Mice with Dextran Sulfate Sodium-Induced Colitis.

Ulcerative colitis (UC) is a typical type of inflammatory bowl disease, which is accompanied by an increased risk of depression and anxiety-related psychological symptoms. Betaine is a naturally derived compound that can function as an anti-inflammatory drug and a neuromodulator. In-depth exploration of the potential role of betaine in treating UC-related depression and anxiety is crucial. This study aimed to elucidate the effects of betaine on UC-related depression and anxiety and clarify the underlying mechanisms. A dextran sulfate sodium (DSS)-induced mice model was established by 4% DSS drinking ad libitum for 7 days. The colonic injury was measured using hematoxylin-eosin (HE) staining and Alcian blue-periodic acid Schiff (AB-PAS) staining. Depression and anxiety-like behaviors were separately evaluated using a forced swimming test (FST), a tail suspension test (TST), a light-dark box test (LDBT), and an open field test (OFT). Immunohistochemistry was used to detect DNA damage and neurogenesis in the hippocampus. Western blotting was applied to detect the protein levels of macrophage polarization in mice colons and the alteration of mitochondrial dysfunction and the cGAS-STING pathway in the hippocampus. Betaine strongly alleviated mucosal structural disorder and mucin secretion reduction and promoted M2-macrophage polarization in the colon of DSS-treated mice. In addition, betaine could mitigate depression- and anxiety-like behaviors in DSS-treated mice, reduce the DNA damage and mitochondrial dysfunction, and inhibit the cGAS-STING signaling pathway. Our study reveals the antidepression/anxiety effects of betaine and further demonstrates the potential mechanism by which betaine inhibits DNA damage and mitochondrial dysfunction to block the cGAS-STING pathway, thereby repairing neurogenesis in the hippocampus.