Novel mechanisms and clinical trial endpoints in intestinal fibrosis.

The incidence of inflammatory bowel diseases (IBD) worldwide has resulted in a global public health challenge. Intestinal fibrosis leading to stricture formation and bowel obstruction is a frequent complication in Crohn's disease (CD), and the lack of anti-fibrotic therapies makes elucidation of fibrosis mechanisms a priority. Progress has shown that mesenchymal cells, cytokines, microbial products, and mesenteric adipocytes are jointly implicated in the pathogenesis of intestinal fibrosis. This recent information puts prevention or reversal of intestinal strictures within reach through innovative therapies validated by reliable clinical trial endpoints. Here, we review the role of immune and non-immune components of the pathogenesis of intestinal fibrosis, including new cell clusters, cytokine networks, host-microbiome interactions, creeping fat, and their translation for endpoint development in anti-fibrotic clinical trials.

Mechanical Stress-induced Connective Tissue Growth Factor Plays a Critical Role in Intestinal Fibrosis in Crohn's-like Colitis.

Crohn's disease (CD) is an inflammatory bowel disease characterized by transmural inflammation and intestinal fibrosis. Mechanisms of fibrosis in CD are not well understood. Transmural inflammation is associated with inflammatory cell infiltration, stenosis, and distention, which present mechanical stress (MS) to the bowel wall. We hypothesize that MS induces gene expression of pro-fibrotic mediators such as connective tissue growth factor (CTGF), which may contribute to fibrosis in CD. A rodent model of CD was induced by intracolonic instillation of TNBS to the distal colon. TNBS instillation induced a localized transmural inflammation (site I), with a distended colon segment (site P) proximal to site I. We detected significant fibrosis and collagen content not only in site I, but also in site P in CD rats by day 7. CTGF expression increased significantly in sites P and I, but not in the segment distal to the inflammation site. Increased CTGF expression was detected mainly in the smooth muscle cells (SMC). When rats were fed exclusively with clear liquid diet to prevent mechanical distention in colitis, expression of CTGF in sites P and I was blocked. Direct stretch led to robust expression of CTGF in colonic SMC. Treatment of CD rats with anti-CTGF antibody FG-3149 reduced fibrosis and collagen content in both sites P and I and exhibited consistent trends towards normalizing expression of collagen mRNAs. In conclusion, our studies suggest that mechanical stress, by up-regulating pro-fibrotic mediators i.e. CTGF, may play a critical role in fibrosis in CD.

Differential expression of small bowel TGFß1 and TGFß3 characterizes intestinal strictures in patients with fibrostenotic Crohn's disease.

Small bowel strictures remain a debilitating consequence of Crohn's disease and contribute to poor outcomes for patients. Recently, TGFß has been identified as an important driver of intestinal fibrosis. We studied the localization of TGFß isoforms in ileal strictures of patients with Crohn's disease using in situ hybridization to understand TGFß's role in stricture formation. The mucosa of strictures was characterized by higher TGFß1 while the stricture submucosa showed higher TGFß3 compared to normal ileum from patients without Crohn's disease (p = 0.02 and p = 0.044, respectively). We correlated these findings with single-cell transcriptomics which demonstrated that TGFß3 transcripts overall are very rare, which may partially explain why its role in intestinal fibrosis has remained unclear to date. There were no significant differences in fibroblast or B cell TGFß1 and/or TGFß3 expression in inflamed vs. noninflamed ileum. We discuss the implications of these findings for therapeutic development strategies to treat patients with fibrostenotic Crohn's disease.

Faecalibacterium prausnitzii-derived extracellular vesicles alleviate chronic colitis-related intestinal fibrosis by macrophage metabolic reprogramming.

Faecalibacterium prausnitzii (F. prausnitzii) has been recognized for its various intestinal and extraintestinal benefits to human. And reduction of F. prausnitzii has been linked to an increased risk of intestinal fibrosis in patients of Crohn's disease (CD). In this study, oral administration of either live F. prausnitzii or its extracellular vesicles (FEVs) can markedly mitigate the severity of fibrosis in mice induced by repetitive administration of DSS. In vitro experiment revealed that FEVs were capable of directing the polarization of peripheral blood mononuclear cells (PBMCs) towards an M2b macrophage phenotype, which has been associated with anti-fibrotic activities. This effect of FEV was found to be stable under various conditions that promote the development of pro-fibrotic M1/M2a/M2c macrophages. Proteomics and RNA sequencing were performed to uncover the molecular modulation of macrophages by FEVs. Notably, we found that FEVs reprogramed every metabolism of macrophages by damaging the mitochondria, and inhibited oxidative phosphorylation and glycolysis. Moreover, FEV-treated macrophages showed a decreased expression of PPARγ and an altered lipid processing phenotype characterized by decreased cholesterol efflux, which may promote energy reprogramming. Taken together, these findings identify FEV as a driver of macrophage reprogramming, suggesting that triggering M2b macrophage polarization by oral admiration of FEV may serve as strategy to alleviate hyperfibrotic intestine conditions in CD.

27-Hydroxycholesterol activates the GSK-3ß/ß-catenin signaling pathway resulting in intestinal fibrosis by inducing oxidative stress: effect of dietary interventions.

OBJECTIVE: Intestinal fibrosis, a common and serious complication of inflammatory bowel disease (IBD), results from chronic inflammation. A high-cholesterol diet may be a risk factor for IBD and 27-hydroxylcholesterol (27HC) is the main human cholesterol metabolite. This study investigated whether 27HC can induce intestinal fibrosis. METHODS: The effects of cholesterol and 27HC on intestinal fibrosis were assessed in zebrafish and human intestinal epithelial Caco-2 cells. RESULTS: Cholesterol and 27HC induced intestinal inflammation and collagen deposition, inhibited E-cadherin (E-ca) expression in the intestinal epithelium, and promoted nuclear translocation of ß-catenin in zebrafish. Cholesterol and 27HC up-regulated expression of COL-1, α-SMA, CTGF, TIMP1, N-cadherin, vimentin, glycogen synthesis kinase-3ß (GSK-3ß) and ß-catenin, but inhibited E-ca, in Caco-2 cells. The expression of these proteins was inhibited by CYP27A1 knockdown and ß-catenin knockdown. 27HC-induced nuclear translocation of ß-catenin occurs in Caco-2 cells. p38, ERK, and AKT activate ß-catenin and thereby participate in 27HC-induced epithelia-mesenchymal transition (EMT) and fibrosis. 27HC-increased oxidative stress and the fibrosis and EMT markers, the nuclear translocation of ß-catenin, and the up-regulation of p-cell kinase proteins promoted by 27HC were inhibited by N-acetyl-L-cysteine (NAC). Folic acid (FA), resveratrol (RES), and NAC all ameliorated the 27HC-induced effects in Caco-2 cells and zebrafish. CONCLUSION: A high-cholesterol diet caused intestinal fibrosis in zebrafish, mediated by a major cholesterol metabolite, 27HC. 27HC increased oxidative stress and activated p38, ERK, AKT, and ß-catenin, leading to EMT of epithelial cells and intestinal fibrosis. FA and RES both ameliorated intestinal fibrosis by restraining 27HC-induced ß-catenin activation.

Matrix gla protein, a potential marker of tissue remodelling and physiological ageing of the gut in crohn's disease.

BACKGROUND: The inactive dephosphorylated and uncarboxylated form of the matrix Gla protein (dp-ucMGP) has been shown to be increased in plasma of inflammatory bowel disease (IBD) patients. Our aim was to assess if the plasmatic level of dp-ucMGP could reflect disease endoscopic activity, presence of strictures and cumulative structural bowel damage in Crohn's disease (CD) patients. METHODS: The plasmatic level of dp-ucMGP was measured in a monocentric cohort of prospectively recruited patients. The analysis was done by chemiluminescent immunoassay on blood samples collected the day of a planned ileocolonoscopy. In addition to classical clinical data (gender, age, body mass index (BMI), disease duration, current treatment), endoscopic data (disease location, Crohn's Disease Endoscopic Index of Severity (CDEIS), mucosal healing (MH), presence of 9 CD lesion types) and biological markers (faecal calprotectin and C-reactive protein (CRP)) were collected. The association between dp-ucMGP level and Lémann index was also investigated. Univariate linear regression was used to investigate the relationship between dp-ucMGP level and different parameters collected. RESULTS: A total of 82 ileocolonoscopies and dp-ucMGP assays were performed in 75 CD patients (45 females; 37 ileocolonic, 19 ileal and 19 colonic diseases) between October 2012 and November 2019. A total of 24 patients (29.3%) showed MH. The dp-ucMGP levels were not associated with MH, CDEIS, faecal calprotectin or CRP levels. Plasmatic dp-ucMGP levels increased significantly with age (p = 0.0032), disease duration (p = 0.0033), corticosteroids use (p = 0.019) and tended to increase in patients with intestinal strictures (p = 0.086) but not with the Lémann index. CONCLUSION: The significant increase of plasmatic dp-ucMGP levels with age, disease duration and the trend observed in patients with non-ulcerated strictures may suggest that this extracellular matrix protein could be a marker of tissue remodelling and physiological ageing of the gut.

Total flavone of Abelmoschus manihot regulates autophagy through the AMPK/mTOR signaling pathway to treat intestinal fibrosis in Crohn's disease.

BACKGROUND AND AIM: Drug therapy is the treatment of choice for Crohn's disease because it effectively controls or prevents intestinal inflammation. The purpose was to research the molecular mechanism of the total flavones of Abelmoschus manihot (TFA) on intestinal fibrosis in Crohn's disease. METHODS: A 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis model and IGF-1-treated intestinal fibroblasts were established. Then, TFA, 3-MA, and compound C were used treatments. Hematoxylin and eosin, Masson, and Picrosirius red staining were performed to observe the colon tissue. Immunohistochemical staining was used to detect α-SMA expression. Flow cytometry, CCK8, wound healing, and Transwell assays were conducted to determine apoptosis, proliferation, invasion, and migration. Col1a1 and Col3a1 levels were detected using quantitative polymerase chain reaction. Proteins related to autophagy and apoptosis were detected using western blotting. RESULTS: TFA treated intestinal fibrosis in chronic Crohn's disease. Colon length was the shortest in the ethanol + TNBS group, and TFA treatment significantly improved the situation. Intestinal fibrosis and the percentage of collagen area decreased after TFA treatment. TFA reduced fibrosis by enhancing autophagy stimulation, whereas an autophagy inhibitor reversed the TFA effect. TFA also inhibited migration, proliferation, and collagen synthesis in intestinal fibroblasts. Moreover, it enhanced autophagy and apoptosis of intestinal fibroblasts. TFA upregulated p-AMPK expression and decreases p-mTOR levels. Compound C partially rescued the effect of TFA, indicating that TFA affected intestinal fibroblasts via the AMPK/mTOR pathway in vitro and in vivo. TFA also downregulated Col1a1 and Col3a1 expression. CONCLUSION: TFA regulates autophagy through AMPK/mTOR signaling pathway to treat intestinal fibrosis, which may provide a new therapy for Crohn's disease treatment.

Fibrostenosing Crohn's Disease: Pathogenetic Mechanisms and New Therapeutic Horizons.

Bowel strictures are well recognized as one of the most severe complications in Crohn's disease, with variable impacts on the prognosis and often needing surgical or endoscopic treatment. Distinguishing inflammatory strictures from fibrotic ones is of primary importance due to the different therapeutic approaches required. Indeed, to better understand the pathogenesis of fibrosis, it is crucial to investigate molecular processes involving genetic factors, cytokines, alteration of the intestinal barrier, and epithelial and endothelial damage, leading to an increase in extracellular matrix synthesis, which ultimately ends in fibrosis. In such a complex mechanism, the gut microbiota also seems to play a role. A better comprehension of molecular processes underlying bowel fibrosis, in addition to radiological and histopathological findings, has led to the identification of high-risk patients for personalized follow-up and testing of new therapies, primarily in preclinical models, targeting specific pathways involving Transforming Growth Factor-ß, interleukins, extracellular matrix balance, and gut microbiota. Our review aims to summarize current evidence about molecular factors involved in intestinal fibrosis' pathogenesis, paving the way for potential diagnostic biomarkers or anti-fibrotic treatments for stricturing Crohn's disease.

The Diagnosis of Intestinal Fibrosis in Crohn's Disease-Present and Future.

Crohn's disease (CD) progresses with periods of remission and exacerbations. During exacerbations, chronic inflammation leads to tissue destruction. As a result, intestinal fibrosis may develop in response to the ongoing inflammatory process. Fibrosis in CD should be considered the result of the response of the intestinal wall (over) to the presence of inflammation in the deep structures of the intestinal wall. In the absence of ideal noninvasive methods, endoscopic evaluation in combination with biopsy, histopathological analysis, stool analysis, and blood analysis remains the gold standard for assessing both inflammation and fibrosis in CD. On the contrary, the ability to identify markers of intestinal fibrosis would help to develop new diagnostic and therapeutic methods to detect early stages of fibrosis. It is speculated that miRNAs may, in the future, become biomarkers for early noninvasive diagnosis in the treatment of intestinal fibrosis. The purpose of this review is to summarise existing diagnostic methods for Crohn's disease and present recent scientific reports on molecular testing.

Dynamic alterations in metabolomics and transcriptomics associated with intestinal fibrosis in a 2,4,6-trinitrobenzene sulfonic acid-induced murine model.

BACKGROUND & AIMS: Intestinal fibrosis is a common and severe complication of inflammatory bowel disease without clear pathogenesis. Abnormal expression of host genes and metabolic perturbations might associate with the onset of intestinal fibrosis. In this study, we aimed to investigate the relationship between the development of intestinal fibrosis and the dynamic alterations in both fecal metabolites and host gene expression. METHODS: We induced intestinal fibrosis in a murine model using 2,4,6-trinitrobenzene sulfonic acid (TNBS). TNBS-treated or control mice were sacrificed after 4 and 6 weeks of intervention; alterations in colonic genes and fecal metabolites were determined by transcriptomics and metabolomics, respectively. Differential, tendency, enrichment, and correlation analyses were performed to assess the relationship between host genes and fecal metabolites. RESULTS: RNA-sequencing analysis revealed that 679 differential genes with enduring changes were mainly enriched in immune response-related signaling pathways and metabolism-related biological processes. Among them, 15 lipid metabolism-related genes were closely related to the development of intestinal fibrosis. Moreover, the fecal metabolic profile was significantly altered during intestinal fibrosis development, especially the lipid metabolites. Particularly, dynamic perturbations in lipids were strongly associated with alterations in lipid metabolism-related genes expression. Additionally, six dynamically altered metabolites might serve as biomarkers to identify colitis-related intestinal fibrosis in the murine model. CONCLUSIONS: Intestinal fibrosis in colitis mice might be related to dynamic changes in gene expression and metabolites. These findings could provide new insights into the pathogenesis of intestinal fibrosis.

Transcriptomic analyses of treatment-naïve pediatric ulcerative colitis patients and exploration of underlying disease pathogenesis.

BACKGROUND: Ulcerative colitis (UC) is a form of chronic inflammatory bowel disease of nonspecific origin. This study used an RNA-Sequencing (RNA-Seq) approach to evaluate the transcriptomic landscape of a well-stratified treatment-naïve pediatric UC patient population by comparing them with healthy control children. The data were analyzed to evaluate the mechanisms driving UC-related intestinal inflammation and fibrosis. METHODS: Intestinal mucosal samples from five pediatric UC patients and five healthy controls were analyzed by RNA-Seq, and results were verified by qPCR. A CRISPR/Cas9 approach was used to knock out the expression of HLA-DRB5, and molecular biology techniques were used for additional mechanistic studies. RESULTS: In these analyses, 2290 genes were found to be differentially expressed between the UC and control samples, of which 1258 and 1032 were upregulated and downregulated, respectively. Gene Ontology analysis showed that these genes were enriched in extracellular matrix (ECM)-related processes and that 7 of 8 differentially expressed genes of interest (PIK3CD, IL1ß, IL1α, TIMP1, MMP1, MMP12, COL6A3, and HLADRB5) were upregulated and involved in ECM-receptor interaction and inflammatory bowel disease-related pathways. Increased HLA-DRB5 expression driven by intestinal bacteria was found to promote IL-1α secretion, leading to intestinal inflammation and fibrosis, suggesting a possible target for the treatment of UC. CONCLUSION: These data suggest that intestinal inflammation is present in pediatric UC patients for extended periods before the onset of symptoms, and intestinal fibrosis begins even during the early stages of UC. Intestinal bacteria were also found to trigger intestinal inflammation and fibrosis, with HLA-DRB5 playing a central role in this process.

Integrin αvß6 contributes to the development of intestinal fibrosis via the FAK/AKT signaling pathway.

Intestinal fibrosis is one of the most severe complications of inflammatory bowel disease (IBD) and frequently requires surgery due to intestinal obstruction. Integrin αvß6, which is mainly regulated by the integrin ß6 subunit gene (ITGB6), is a special integrin subtype expressed only in epithelial cells. In our previous study, we found integrin αvß6 can promote the development of IBD, but the role of integrin αvß6 in intestinal fibrosis remains unclear. In this study, we observed a gradual increase of ITGB6 mRNA expression from normal region to stenotic region of IBD patients' intestinal specimens. Next, we established a dextran sulfate sodium (DSS)-induced intestinal fibrosis model and a heterotopic intestinal transplant model, and found intestinal fibrosis was decreased in ITGB6-deficient mice compared to wild-type (WT) mice. Furthermore, we performed RNA-sequencing and KEGG pathway analysis on intestinal tissues from ITGB6-overexpressing transgenic mice and WT mice, and found multiple pathways containing ITGB6, are related to the activation of focal adhesion kinase (FAK); finding was confirmed by Western blot. At last, we generated a heterotopic intestinal transplant model found the FAK/AKT pathway was inhibited in ITGB6-deficient mice. In conclusion, our data demonstrate that integrin αvß6 promotes the pathogenesis of intestinal fibrosis by FAK/AKT pathway, making integrin αvß6 a potential therapeutic target to prevent this condition.

Natural compound fraxinellone ameliorates intestinal fibrosis in mice via direct intervention of HSP47-collagen interaction in the epithelium.

Intestinal fibrosis is a common complication of inflammatory bowel disease. There is still a lack of effective drugs for the prevention or treatment of intestinal fibrosis. Heat shock protein 47 (HSP47) plays a key role in the development of intestinal fibrosis. In this study we investigated the therapeutic potential and underlying mechanisms of fraxinellone, a degraded limonoid isolated from the root bark of Dictamnus dasycarpus, in the treatment of intestinal fibrosis. Intestinal fibrosis was induced in mice by dextran sodium sulfate (DSS) treatment. DDS-treated mice were administered fraxinellone (7.5, 15, 30 mg·kg-1·d-1, i.g.) for 45 days. We showed that fraxinellone administration dose-dependently alleviated DSS-induced intestinal impairments, and reduced the production of intestinal fibrosis biomarkers such as α-smooth muscle actin (SMA), collagen I, hydroxyproline, fibronectin and laminin, and cytokines such as TGF-ß, TNF-α and IL-ß. We then established in vitro intestinal fibrosis cell models in SW480 and HT-29 cells, and demonstrated that treatment with fraxinellone (3, 10, 30 µM) significantly relieved TGF-ß-induced fibrosis responses by inhibiting the TGF-ß/Smad2/3 signaling pathway. Molecular docking suggested that the fraxinellone might disrupt the interaction between HSP47 and collagen, which was confirmed by coimmunoprecipitation experiments. SPR analysis showed that fraxinellone had a high affinity for HSP47 with a Kd value of 3.542 × 10-5 M. This study provides a new example of HSP47-collagen intervention by a natural compound and has important implications for the clinical treatment of inflammation-induced issue fibrosis.

Local Pentraxin-2 Deficit Is a Feature of Intestinal Fibrosis in Crohn's Disease.

BACKGROUND: Pentraxin-2 (PTX-2) is a homo-pentameric plasma protein showing evidence of antifibrotic activity in Phase 2 clinical trials in idiopathic pulmonary fibrosis (IPF). Whether PTX-2 plays a role in other fibrotic diseases, including intestinal fibrosis which commonly occurs in inflammatory bowel disease (IBD), remains unknown. AIMS: This study aimed to qualitatively and quantitatively assess PTX-2 expression in fibrostenotic Crohn's disease (FCD) and determine whether expression is correlated with postsurgical restenosis. METHODS: Immunohistochemistry was performed in histologic sections of small bowel resected from patients with fibrostenotic Crohn's disease (FCD), comparing strictured segments with adjacent surgical margins from the same patient. Ileal resections from patients without inflammatory bowel disease were examined as controls. RESULTS: PTX-2 signal was analyzed in 18 patients with FCD and 15 patients without IBD and localized predominantly to submucosal vasculature, including arterial subendothelium and internal elastic lamina, and perivascular connective tissue. PTX-2 signal in the surgical margins from patients with FCD strictures (where tissue architecture was normal) was consistently lower than non-IBD samples. Fibrostenotic regions showed increased PTX-2 signal relative to surgical margins from the same patient in 14/15 paired samples. Submucosal/mural PTX-2 signal in fibrostenotic tissue was lower in patients who subsequently experienced re-stenosis (P = 0.015). CONCLUSIONS: This exploratory study is the first analysis of PTX-2 within the intestine, and demonstrates that PTX-2 signal is reduced in the architecturally normal bowel of patients with FCD. Lower submucosal PTX-2 levels in patients with re-stenosis raises the possibility of a protective role of PTX-2 in intestinal fibrosis.

Orally administration of cerium oxide nanozyme for computed tomography imaging and anti-inflammatory/anti-fibrotic therapy of inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic nonspecific disease with unknown etiology. Currently, the anti-inflammatory therapeutic approaches have achieved a certain extent of effects in terms of inflammation alleviation. Still, the final pathological outcome of intestinal fibrosis has not been effectively improved yet. RESULTS: In this study, dextran-coated cerium oxide (D-CeO2) nanozyme with superoxide dismutase (SOD) and catalase (CAT) activities was synthesized by chemical precipitation. Our results showed that D-CeO2 could efficiently scavenge reactive oxide species (ROS) as well as downregulate the pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and iNOS) to protect cells from H2O2-induced oxidative damage. Moreover, D-CeO2 could suppress the expression of fibrosis-related gene levels, such as α-SMA, and Collagen 1/3, demonstrating the anti-fibrotic effect. In both TBNS- and DSS-induced colitis models, oral administration of D-CeO2 in chitosan/alginate hydrogel alleviated intestinal inflammation, reduced colonic damage by scavenging ROS, and decreased inflammatory factor levels. Notably, our findings also suggested that D-CeO2 reduced fibrosis-related cytokine levels, predicting a contribution to alleviating colonic fibrosis. Meanwhile, D-CeO2 could also be employed as a CT contrast agent for noninvasive gastrointestinal tract (GIT) imaging. CONCLUSION: We introduced cerium oxide nanozyme as a novel therapeutic approach with computed tomography (CT)-guided anti-inflammatory and anti-fibrotic therapy for the management of IBD. Collectively, without appreciable systemic toxicity, D-CeO2 held the promise of integrated applications for diagnosis and therapy, pioneering the exploration of nanozymes with ROS scavenging capacity in the anti-fibrotic treatment of IBD.

P2X7 Receptor Regulates Collagen Expression in Human Intestinal Fibroblasts: Relevance in Intestinal Fibrosis.

Intestinal fibrosis is a common complication that affects more than 50% of Crohn´s Disease (CD) patients. There is no pharmacological treatment against this complication, with surgery being the only option. Due to the unknown role of P2X7 in intestinal fibrosis, we aim to analyze the relevance of this receptor in CD complications. Surgical resections from CD and non-Inflammatory Bowel Disease (IBD) patients were obtained. Intestinal fibrosis was induced with two different murine models: heterotopic transplant model and chronic-DSS colitis in wild-type and P2X7-/- mice. Human small intestine fibroblasts (HSIFs) were transfected with an siRNA against P2X7 and treated with TGF-ß. A gene and protein expression of P2X7 receptor was significantly increased in CD compared to non-IBD patients. The lack of P2X7 in mice provoked an enhanced collagen deposition and increased expression of several profibrotic markers in both murine models of intestinal fibrosis. Furthermore, P2X7-/- mice exhibited a higher expression of proinflammatory cytokines and a lower expression of M2 macrophage markers. Moreover, the transient silencing of the P2X7 receptor in HSIFs significantly induced the expression of Col1a1 and potentiated the expression of Col4 and Col5a1 after TGF-ß treatment. P2X7 regulates collagen expression in human intestinal fibroblasts, while the lack of this receptor aggravates intestinal fibrosis.

Vitamin D receptor inhibits EMT via regulation of the epithelial mitochondrial function in intestinal fibrosis.

We previously showed that the vitamin D receptor (VDR) plays a crucial role in acute inflammatory bowel disease and that intestinal fibrosis is a common complication of Crohn's disease (CD). Epithelial-mesenchymal transition (EMT) is an important hallmark of fibrogenesis through which epithelial cells lose their epithelial phenotype and transform into mesenchymal cells. It is known that the VDR plays an essential role in epithelial integrity and mitochondrial function, but its role in intestinal fibrosis remains unknown. Here, we investigated whether the VDR is involved in epithelial mitochondrial dysfunction that results in EMT in intestinal fibrosis. Using human CD samples, intestine-specific VDR-KO mice, and fibroblast cellular models, we showed that the expression of the VDR was significantly lower in intestinal stenotic areas than in nonstenotic areas in patients with chronic CD. Genetic deletion of the VDR in the intestinal epithelium exacerbated intestinal fibrosis in mice administered with dextran sulfate sodium or 2,4,6-trinitrobenzene sulfonic acid, two experimental colitis inducers. In addition, we found that vitamin D dietary intervention regulated intestinal fibrosis by modulating the intestinal expression of the VDR. Mechanistically, knocking down the VDR in both CCD-18Co cells and human primary colonic fibroblasts promoted fibroblast activation, whereas VDR overexpression or VDR agonist administration inhibited fibroblast activation. Further analysis illustrated that the VDR inhibited EMT in the HT29 cell model and that mitochondrial dysfunction mediated epithelial integrity and barrier function in VDR-deficient epithelial cells. Together, our data for the first time demonstrate that VDR activation alleviates intestinal fibrosis by inhibiting fibroblast activation and epithelial mitochondria-mediated EMT.

Resolvin D1 Prevents Epithelial-to-Mesenchymal Transition and Reduces Collagen Deposition by Stimulating Autophagy in Intestinal Fibrosis.

BACKGROUND: Intestinal fibrosis is the most common complication of inflammatory bowel disease; nevertheless, specific therapies are still unavailable. Resolvin D1 (RvD1), a typical endogenous ω-3 fatty acid-derived lipid mediator, has attracted wide attention due to its remarkable anti-fibrosis effects. However, the efficacy and mechanisms of RvD1 in intestinal fibrosis remain unclear. AIM: To investigate the protective effect of RvD1 in a dextran sulfate sodium (DSS)-induced intestinal fibrosis model and explore the molecular mechanisms underlying its anti-fibrotic effect. METHODS: A DSS-induced intestinal fibrosis model and intestinal epithelial-to-mesenchymal transition (EMT) model were used to observe the efficacy of RvD1, and fibroblasts were stimulated with conditioned medium with or without TGF-ß1 to investigate the probable mechanisms of RvD1 in intestinal fibrosis disease. RESULTS: Intestinal fibrosis was effectively alleviated by RvD1 in a DSS-induced model, both preventively and therapeutically, and autophagy inhibition-induced EMT in intestinal epithelial cells was significantly suppressed in vivo and in vitro. Furthermore, RvD1 reduced epithelial cell EMT paracrine signaling, which promoted the differentiation of local fibroblasts into myofibroblasts. CONCLUSIONS: Our results suggested that RvD1 reduces autophagy-induced EMT in intestinal epithelial cells and ameliorates intestinal fibrosis by disrupting epithelial-fibroblast crosstalk.

Adiponectin Alleviates Intestinal Fibrosis by Enhancing AMP-Activated Protein Kinase Phosphorylation.

BACKGROUND: Intestinal fibrosis is a common complication of Crohn's disease (CD). Adiponectin reportedly exerts anti-inflammatory effects in various disease models, including colitis models. AIMS: In this study, we aimed to determine the effects of adiponectin on intestinal fibrosis and the underlying mechanisms. METHODS: A murine model of intestinal fibrosis was established by administering increasing doses of 2,4,6-trinitrobenzene sulfonic acid to Balb/c mice via enema for 7 weeks. Primary human fibroblasts were isolated from the colon tissues of patients with CD. The fibroblasts were incubated with transforming growth factor (TGF)-ß1 to establish a fibrosis model in vitro. Pathway inhibitors were used to verify the potential signaling pathways involved in the anti-fibrogenic effect of adiponectin. RESULTS: Compared with the normal mesentery, adiponectin expression was significantly increased in the hypertrophic mesentery of patients with CD. Intraperitoneal injection of adiponectin significantly decreased the activity of myeloperoxidase and the expression of pro-inflammatory cytokines (tumor necrosis factor α and interleukin 6) in the colon of fibrosis model mice, whereas the expression of the anti-inflammatory cytokine interleukin 10 was substantially increased. Moreover, adiponectin treatment inhibited colon shortening, decreased colon weight, and reduced fibrotic protein deposition in the model mice. Adiponectin reduced the phosphorylation of Smad2 and collagen deposition induced by TGF-ß1 in primary human intestinal fibroblasts, with an increase in AMP-activated protein kinase (AMPK) phosphorylation. Furthermore, this phenomenon was reversed by the AMPK inhibitor. CONCLUSIONS: Adiponectin can protect against intestinal fibrosis by enhancing the phosphorylation of AMPK and inhibiting the activity of the TGF-ß1/Smad signaling pathway.

ZNF281 Promotes Colon Fibroblast Activation in TGFß1-Induced Gut Fibrosis.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract. Chronic inflammation is the main factor leading to intestinal fibrosis, resulting in recurrent stenosis, especially in CD patients. Currently, the underlying molecular mechanisms of fibrosis are still unclear. ZNF281 is a zinc-finger transcriptional regulator that has been characterized as an epithelial-to-mesenchymal transition (EMT)-inducing transcription factor, suggesting its involvement in the regulation of pluripotency, stemness, and cancer. The aim of this study is to investigate in vivo and in vitro the role of ZNF281 in intestinal fibrogenesis. Intestinal fibrosis was studied in vivo in C57BL/6J mice with chronic colitis induced by two or three cycles of administration of dextran sulfate sodium (DSS). The contribution of ZNF281 to gut fibrosis was studied in vitro in the human colon fibroblast cell line CCD-18Co, activated by the pro-fibrotic cytokine TGFß1. ZNF281 was downregulated by siRNA transfection, and RNA-sequencing was performed to identify genes regulated by TGFß1 in activated colon fibroblasts via ZNF281. Results showed a marked increase of ZNF281 in in vivo murine fibrotic colon as well as in in vitro human colon fibroblasts activated by TGFß1. Moreover, abrogation of ZNF281 in TGFß1-treated fibroblasts affected the expression of genes belonging to specific pathways linked to fibroblast activation and differentiation into myofibroblasts. We demonstrated that ZNF281 is a key regulator of colon fibroblast activation and myofibroblast differentiation upon fibrotic stimuli by transcriptionally controlling extracellular matrix (ECM) composition, remodeling, and cell contraction, highlighting a new role in the onset and progression of gut fibrosis.