Intestinal Epithelial Wound Healing Research Articles

Optimizing postbiotic production through solid-state fermentation with Bacillus amyloliquefaciens J and Lactiplantibacillus plantarum SN4 enhances antibacterial, antioxidant, and anti-inflammatory activities.

Postbiotics are an emerging research interest in recent years and are fairly advanced compared to prebiotics and probiotics. The composition and function of postbiotics are closely related to fermentation conditions. In this study, we developed a solid-state fermentation preparation method for postbiotics with antimicrobial, antioxidant, and anti-inflammatory activities. The antibacterial activity was improved 3.62 times compared to initial fermentation conditions by using optimization techniques such as single factor experiments, Plackett-Burman design (PBD), steepest ascent method (SAM), and central composite design (CCD) methods. The optimized conditions were carried out with an initial water content of 50% for 8 days at 37°C and fermentation strains of Bacillus amyloliquefaciens J and Lactiplantibacillus plantarum SN4 at a ratio of 1:1 with a total inoculum size of 8%. The optimized SSF medium content ratios of peptide powder, wheat bran, corn flour, and soybean meal were 4, 37.4, 30, and 28.6%, respectively. Under these optimized conditions, postbiotics with a concentration of 25 mg/mL showed significant broad-spectrum antibacterial capabilities against Escherichia coli, Salmonella, and Staphylococcus aureus and strong antioxidant activity against ABTS, DPPH, and OH radicals. Moreover, the optimized postbiotics exhibited good anti-inflammatory ability for reducing nitric oxide (NO) secretion in RAW 264.7 macrophage cells in response to LPS-induced inflammation. Furthermore, the postbiotics significantly improved intestinal epithelial wound healing capabilities after mechanical injury, such as cell scratches in IPEC-J2 cells (p < 0.05). In brief, we developed postbiotics through optimized solid-state fermentation with potential benefits for gut health. Therefore, our findings suggested that the novel postbiotics could be used as potential functional food products for improving body health.

A179 MECHANISMS OF ACTION INVOLVED IN THE WOUND HEALING EFFECT OF THE DIETARY FIBRE RHAMNOGALACTURONAN

Abstract Background Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions associated with impaired intestinal epithelial barrier. Mucosal healing is the primary goal for IBD treatment since it is a good predictor of clinical remission. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal), a polysaccharide isolated from the plant Acmella oleracea, on intestinal epithelial barrier function with participation of TLR4 and PKC activation. RNAseq data and pathway analysis have indicated the involvement of the canonical nuclear factor kB (NF-kB) signaling pathway. Purpose We hypothesize that RGal increases intestinal epithelial wound healing through FAK-Src/PI3K/NF-kB signaling pathways. Method Caco-2 and T84 cells, and human primary cell monolayers grown from ulcerative colitis patient-derived organoids, were wounded and treated with vehicle (media or 0.5% DMSO in media) or RGal (1000 μg/ml) for 48 h. Wound healing was assessed using either the IncuCyte or ImageXpress Pico live cell imaging systems. Proliferation and apoptosis of cells were evaluated using EdU and TUNEL assays, respectively. Inhibitors were added at the same time (transcription inhibitor Actinomycin D, 5 μg/ml) or 1 h (FAK inhibitor FAK-14, 10 μM, Src inhibitor PP2, 5 μM, PI3K inhibitor LY294002, 20 μM, NF-kB inhibitors Bay 11-7082 and JSH-23, 10 and 20 μM, respectively, or COX-2 inhibitor NS-398, 20 μM) before RGal treatment. Unwounded Caco-2 monolayers treated with RGal (1000 μg/ml) were collected for Western blotting for COX-2 protein. Result(s) In the wound healing assay, RGal (1000 μg/ml) enhanced wound healing by 12.5% at 48 h in Caco-2 cells and by 14.7% at 24 h in T84 cells, compared to control group. RGal (1000 μg/ml) also accelerated the wound closure in colonoid monolayers obtained from ulcerative colitis patient biopsies by 81.3% at 48 h. Neither proliferation nor apoptosis were involved in the RGal effect on wound healing. Treatment of cells with FAK14 (10 μM), PP2 (5 μM), and PI3K (20 μM) significantly prevented the RGal-induced wound healing. Actinomycin D (5 μg/ml), Bay 11-7082 (10 μM) or JSH-23 (20 μM) treatment significantly reversed the effect of RGal on wound healing, showing that the response was transcriptionally dependent and involved NF-kB signaling. Treatment of cells with NS-398 (20 μM) also reversed the effect of RGal on wound healing. COX-2 protein expression was significantly increased at 6 and 12 h after RGal addition to Caco-2 monolayers. Conclusion(s) These data suggest that the plant-based polysaccharide RGal increases intestinal epithelial cell wound healing by increasing cell migration. The RGal effect is dependent on the activation of the FAK-Src/PI3K signaling pathways and subsequently the transcription factor NF-kB and downstream COX-2 protein expression and activity. Our findings show a novel mechanism of action of RGal in wound healing that could help in the resolution of intestinal inflammation and mucosal healing. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; NSERC Disclosure of Interest None Declared

Overexpression of microRNA-211 in Functional Dyspepsia via Downregulation of the Glial Cell Line-Derived Neurotrophic Factor (GDNF) by Increasing Phosphorylation of p38 MAPK Pathway.

Overexpression of miRNA-211 suppresses the differentiation of bone marrow stem cells into intestinal ganglion cells via downregulation of GDNF, a regulator of intestine barrier function. The study aimed to investigate the interaction between miR-211 and GDNF on intestinal epithelial cells. The expression levels of miR-211 and GDNF in duodenal biopsy specimens from FD patients and healthy controls were compared. Enteric glia cell (EGCs) cell line transfected with miR-211 mimics and inhibitors were used to clarify the expression levels of GDNF were analyzed by qRT-PCR and ELISA. Intestine epithelial cell (IECs) cell line cultured in medium from ECGs in different transfection conditions were used in wound healing assay, cell proliferation assay, and western blotting for evaluation of p38 MAPK phosphorylation level. MiR-211 expression was significantly upregulated in the duodenal tissue of patients with FD compared to healthy subjects, whereas GDNF expression was significantly downregulated (both p < 0.05). Transfection with miR-211 mimics significantly decreased GDNF mRNA expression and protein secretion (p < 0.001). An inhibited intestinal epithelial cell wound healing (p < 0.05) and increased expression levels of phosphorylated p38 MAPK (p < 0.05) were found in IECs cultured with medium from EGCs transfected with miR-211 mimics. MiR-211 may downregulates GDNF mRNA and protein expression via activation of the pp38 MAPK signaling pathway. Targeting miR-211 or the MAPK pathway may be a potential intervention for FD.

Effects of UV/H2O2 degradation and step gradient ethanol precipitation on Sargassum fusiforme polysaccharides: Physicochemical characterization and protective effects against intestinal epithelial injury

In this study, the degraded purified fraction from Sargassum fusiforme polysaccharides (SFP), named DSFP, was produced by the treatment of ultraviolet/hydrogen peroxide (UV/H2O2) degradation and step gradient ethanol precipitation. Results showed that the treatment significantly reduced the molecular weight of polysaccharides, from 282.83 kDa to 18.54 kDa, and influenced their surface morphology and roughness. SFP and DSFP were typical sulfated polysaccharides, mainly composed of fucose, galacturonic acid, glucuronic acid, galactose, and mannose. Both SFP and DSFP increased cell migration during intestinal epithelial wound healing and stimulated the cell cycle progression by promoting the transition from G0/G1 to S phase in the rat intestine epithelium cells (IEC-6). But DSFP had a stronger positive effect on wound healing and cell migration than SFP. It reinforced the intestinal barrier function and attenuated lipopolysaccharides-induced intestinal inflammation. DSFP significantly downregulated the expression of Toll-like receptor 4, tumor necrosis factor-α, interleukin-6, interleukin-1β, and inducible nitric oxide synthase by 53.14%, 92.41%, 66.01%, 68.24%, and 78.09%, respectively, and upregulated that of interleukin-10 by 2.48 folds when compared to the model. Therefore, the treatment (UV/H2O2 degradation and step gradient ethanol precipitation) could effectively improve the protective effects against intestinal epithelial injury.

A64 THE DIETARY FIBRE RHAMNOGALACTURONAN PROMOTES INTESTINAL EPITHELIAL CELL MIGRATION THROUGH THE NF-κB SIGNALING PATHWAY

BackgroundDamaged intestinal epithelial barrier is characteristic of inflammatory bowel diseases (IBD) and mucosal healing is the primary goal for IBD treatment. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal), a polysaccharide isolated from the plant Acmella oleracea, on intestinal epithelial barrier function with participation of TLR4 and PKC activation. We also observed that RGal accelerates wound healing in human colonic epithelial Caco-2 cells. RNAseq data and pathway analysis have indicated the involvement of the canonical nuclear factor kB (NF-kB) signaling pathway.AimsWe hypothesize that RGal increases intestinal epithelial wound healing through NF-kB signaling pathway.MethodsCaco-2 cells monolayers were scratched and treated with vehicle (media or 0.5% DMSO in media) or RGal (1000 mg/ml) for 48 h. Wound healing was assessed using the IncuCyte live cell imaging system. Proliferation and apoptosis of cells were evaluated using EdU and TUNEL assays, respectively. Inhibitors were added at the same time (transcription inhibitor Actinomycin D, 5 mg/ml) or 1 h (NF-kB inhibitors Bay 11–7082 and JSH-23, 10 and 20 mM, respectively, or COX-2 inhibitor NS-398, 20 mM) before RGal treatment. Unwounded Caco-2 monolayers treated with RGal (1000 mg/ml) were collected for Western blotting for COX-2 protein.ResultsIn the wound healing assay, RGal at a concentration of 1000 µg/ml enhanced wound healing by 12.5% at 48 h compared to control group, under 10% serum conditions. Neither proliferation nor apoptosis were involved in the RGal effect on wound healing, suggesting the response was due solely to cell migration. Actinomycin D (5 mg/ml), Bay 11–7082 (10 mM) or JSH-23 (20 mM) treatment significantly reversed the effect of RGal on wound healing, showing that the response was transcriptionally dependent and involved NF-kB signaling. Treatment of cells with NS-398 (20 mM) also reversed the effect of RGal on wound healing. COX-2 protein expression was significantly increased at 6 and 12 h after RGal addition to Caco-2 monolayers.ConclusionsThese data suggest that the plant-based polysaccharide RGal increases intestinal epithelial cell wound healing by increasing cell migration. The RGal effect is dependent on the activation of the transcription factor NF-kB and downstream COX-2 protein expression and activity. Our findings show a novel mechanism of action of RGal in wound healing that could help in the resolution of intestinal inflammation and mucosal healing.Funding AgenciesNSERC

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing.

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 μg·mL-1) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg-1) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Physicochemical properties and potential beneficial effects of porphyran from Porphyra haitanensis on intestinal epithelial cells

This study examined the beneficial effects of porphyran from Porphyra haitanensis (PHP) on intestinal epithelial cells, in terms of cell proliferation and migration and elucidated the potential molecular mechanism of action of PHP. Purified PHP is a homogenous polysaccharide with a molecular weight of 2.01 × 105 Da, intrinsic viscosity [η] of 463.76 mL/g, and radius of gyration of 61.2 nm. When the intestinal epithelial wound healing activity of PHP was investigated in vitro using the IEC-6 cell line (intestinal epithelial cells-6), it was found that PHP could promote cell migration and proliferation. PHP enhanced the protein expression of cell division control protein 42, paxillin, and focal adhesion kinase, which suggest that PHP might modulate the expression of these proteins to improve intestinal epithelial healing. Thus, this study indicated that PHP could serve as a potential source of functional food constituents for intestinal epithelial protection and restoration.

Resolvin E1 is a pro-repair molecule that promotes intestinal epithelial wound healing

Resolution of intestinal inflammation and wound repair are active processes that mediate epithelial healing at mucosal surfaces. Lipid molecules referred to as specialized proresolving mediators (SPMs) play an important role in the restorative response. Resolvin E1 (RvE1), a SPM derived from omega-3 fatty acids, has been reported to dampen intestinal inflammation by promoting anti-inflammatory responses including increased neutrophil spherocytosis and macrophage production of IL-10. Despite these observations, a role for RvE1 in regulating intestinal epithelial cell migration and proliferation during mucosal wound repair has not been explored. Using an endoscopic biopsy-based wound healing model, we report that RvE1 is locally produced in response to intestinal mucosal injury. Exposure of intestinal epithelial cells to RvE1 promoted wound repair by increasing cellular proliferation and migration through activation of signaling pathways including CREB, mTOR, and Src-FAK. Additionally, RvE1-triggered activation of the small GTPase Rac1 led to increased intracellular reactive oxygen species (ROS) production, cell-matrix adhesion, and cellular protrusions at the leading edge of migrating cells. Furthermore, in situ administration of RvE1-encapsulated synthetic targeted polymeric nanoparticles into intestinal wounds promoted mucosal repair. Together, these findings demonstrate that RvE1 functions as a prorepair lipid mediator by increasing intestinal epithelial cell migration and proliferation, and highlight potential therapeutic applications for this SPM to promote mucosal healing in the intestine.

Targeting Epithelial Expressed Sialylated Lewis glycans improves colonic mucosal wound healing and protects against colitis

Delayed healing of mucosal wounds is a hallmark of many pathological conditions including inflammatory bowel disease (IBD). Mucosal wound healing involves a complex interplay between epithelial cells and infiltrating immune cells that trigger cell signaling events and epithelial cell migration, proliferation and differentiation that are necessary for restoration of mucosal homeostasis. Specific alterations in surface glycosylation have been reported in intestinal epithelial cells following injury and we have shown that targeting the inflammation induced glycan sialyl Lewis A (sLea) on epithelial CD44v6 (with a specific mAb termed GM35) results in inhibition of neutrophil (PMN) trafficking in the inflamed intestine. Despite this observation, the role of glycans in intestinal wound healing has not been examined. Here we report that selective targeting of sialylated Lewis glycans on epithelial glycoprotein CD44v6 increases human intestinal epithelial cell proliferation, migration and wound healing. Furthermore, direct injection of GM35 into biopsy‐induced colonic wounds as well as systemic injection resulted in a significant increase in epithelial proliferation and mucosal wound closure in vivo. These glycan mediated increases in epithelial proliferation were found to be mediated through the activation of Src, Fak and Akt (kinases previously shown to signal downstream of CD44v6 engagement). Finally, systemic administration of GM35 to C57BL/6J mice followed by induction of colitis with dextran sodium sulfate resulted in reduced disease activity, as assessed clinically and histologically, when compared to mice treated with an isotype control mAb. Given our observations of robust upregulation of sialylated Lewis glycans during intestinal inflammation in mice and humans, these data suggest that targeting sLea on epithelial CD44v6 may represent a promising new target for improved recovery of epithelial barrier function and restitution of intestinal homeostasis following inflammation or injury.Support or Funding InformationThis work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – 410855404), a senior Research Award from the Crohn’s and Colitis Foundation (CCF) and R01 awards from the NIH (DK072564 and DK079392).

Resolvin E1 is a pro‐repair mediator that promotes intestinal epithelial wound healing

Resolution of inflammation and epithelial repair are active processes mediated by bioactive lipids known as specialized pro‐resolving mediators (SPMs). Resolvin E1 (RvE1), a SPM derived from omega‐3 fatty acid, has been reported to mediate anti‐inflammatory response in different tissues. Nevertheless, the role of RvE1 in mediating intestinal epithelial wound healing is not well understood.We identified increased RvE1 in healing colonic wounds. RvE1 treatments enhanced intestinal epithelial wound repair in‐vitro. Analysis of the signaling pathways revealed activation of pathways such as CREB, mTOR and Src‐FAK in response to RvE1 exposure. Additionally, RvE1 increased activation of the small GTPase Rac1, leading to increased intracellular reactive oxygen species (ROS) production, cell‐matrix adhesion and cellular protrusions at the leading edge of migrating cells. To harness this mechanism of repair, we performed intramucosal injection of synthetic nanoparticles containing RvE1 in injured murine colon. RvE1 nanoparticles increased wound closure compared to naked RvE1 and empty nanoparticles. In summary, the above findings provide important insight on mechanisms of intestinal mucosal wound repair driven by RvE1 and determine its potential as a therapeutic agent aimed at facilitating mucosal wound closure and epithelial barrier recovery in the intestineSupport or Funding InformationThis work was supported by a Crohn’s and Colitis Foundation Career Development Award (544599, to MQ) and NIH grants (HL127236 to AJG; DK055679, DK089763, DK059888, to AN; and DK61739, DK72564, DK79392, to CAP).

Targeting Epithelial Expressed Sialyl Lewis A Improves Intestinal Mucosal Wound Healing and Protects Against Colitis

Dysregulated healing of mucosal wounds is a hallmark of many pathological conditions including inflammatory bowel disease (IBD). Mucosal wound healing involves a complex interplay between epithelial cells and infiltrating immune cells that trigger cell signaling events and epithelial cell migration, proliferation and differentiation that are necessary for mucosal homeostasis. Specific alterations in surface glycosylation have been reported in intestinal epithelial cells following injury, and we have shown that targeting the inflammation induced epithelial glycan sialyl Lewis A (sLea) with a specific mAb termed GM35 results in inhibition of neutrophil (PMN) trafficking in the inflamed intestine. Despite this observation, the role of glycans in intestinal wound healing has not been examined. Here we report that, in addition to inhibiting PMN transepithelial migration, targeting epithelial expressed sLea increases intestinal epithelial wound healing and recovery from barrier disruption in vitro. We report that these protective effects are mediated through deactivation of Akt (a kinase previously shown to destabilize epithelial adherens junctions) downstream of sLea engagement. Furthermore, wound bed injection of GM35 directly into biopsy‐induced colonic wounds resulted in a significant increase in mucosal wound healing in vivo. Finally, administration of GM35 to C57BL/6J mice followed by induction of colitis with dextran sodium sulfate resulted in reduced disease activity as assessed clinically and histologically, compared to mice treated with an isotype control mAb. Given our observations of robust upregulation of epithelial sLea during intestinal inflammation in mice and humans, these data suggest that targeting sLea may represent a promising new target for regulating intestinal trafficking of PMNs. In addition, targeting of this glycan may also provide an additional therapeutic strategy for improved recovery of epithelial barrier function and restitution of intestinal homeostasis following inflammation or injury.Support or Funding InformationThis work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, 410855404), a senior Research Award from the Crohn's and Colitis Foundation (CCF) and R01 awards from the NIH (DK072564 and DK079392).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ginsenoside Rb1 promotes intestinal epithelial wound healing through extracellular signal-regulated kinase and Rho signaling.

Daikenchuto, a traditional Japanese herbal medicine, has been reported to exhibit anti-inflammatory effects against intestinal inflammation. However, whether daikenchuto has a therapeutic effect against intestinal mucosal injuries remains unclear. Thus, the aim of this study was to determine the effect of daikenchuto on intestinal mucosal healing. Colitis was induced in male Wistar rats by using trinitrobenzenesulfonic acid. Daikenchuto (900mg/kg/day) was administered for 7days after the induction of colitis. Thereafter, intestinal mucosal injuries were evaluated by determining the colonic epithelial regeneration ratio ([area of epithelial regeneration/area of ulcer]×100). Restoration of rat intestinal epithelial cells treated with daikenchuto and its constituent herbs (Zanthoxylum fruit, processed ginger, and ginseng) and ginsenoside Rb1, which is a ginseng ingredient, was evaluated using a wound-healing assay. The colon epithelial regeneration ratio in the daikenchuto-treated rats was significantly higher than that in the control rats. Daikenchuto, ginseng, and ginsenoside Rb1 enhanced wound healing, and the ginsenoside Rb1-induced enhancement was inhibited by extracellular signal-regulated kinase and Rho inhibitors. Daikenchuto and its constituent, ginsenoside Rb1, promoted wound healing. Because mucosal healing is one of the most important therapeutic targets in patients with inflammatory bowel disease, ginsenoside Rb1 may be a novel therapeutic agent against intestinal mucosal damage such as that occurring in intestinal bowel disease.

Inhibition of Intestinal Epithelial Wound Healing through Protease-Activated Receptor-2 Activation in Caco2 Cells.

The mechanisms of epithelial wound healing are not completely understood, especially in the context of proteases and their receptors. It was recently shown that activation of protease-activated receptor-2 (PAR2) on intestinal epithelial cells induced the expression of cyclooxygenase-2 (COX-2), which has protective functions in the gastrointestinal tract. It was hypothesized that PAR2-induced COX-2 could enhance wound healing in intestinal epithelial cells. Caco2 cells were used to model epithelial wound healing of circular wounds. Cellular proliferation was studied with a 5-ethynyl-2'-deoxyuridine assay, and migration was studied during wound healing in the absence of proliferation. Immunofluorescence was used to visualize E-cadherin and F-actin, and the cellular transcription profile during wound healing and PAR2 activation was explored with RNA sequencing. PAR2 activation inhibited Caco2 wound healing by reducing cell migration, independently of COX-2 activity. Interestingly, even though migration was reduced, proliferation was increased. When the actin dynamics and cell-cell junctions were investigated, PAR2 activation was found to induce actin cabling and prevent the internalization of E-cadherin. To further investigate the effect of PAR2 on transcriptionally dependent wound healing, RNA sequencing was performed. This analysis revealed that PAR2 activation, in the absence of wounding, induced a similar transcriptional profile compared with wounding alone. These findings represent a novel effect of PAR2 activation on the mechanisms of epithelial cell wound healing that could influence the resolution of intestinal inflammation.

High MUC2 Mucin Biosynthesis in Goblet Cells Impedes Restitution and Wound Healing by Elevating Endoplasmic Reticulum Stress and Altered Production of Growth Factors

Intestinal epithelial cell wound healing involves cell migration, proliferation, and differentiation. Although numerous studies have analyzed the migration of absorptive epithelial cells during wound healing, it remains unclear how goblet cells restitute and how MUC2 mucin production affects this process. In this study, we examined the role of high MUC2 production in goblet cell migration during wound healing and demonstrated that during high MUC2 output, goblet cells migrated slower because of impaired production of wound healing factors and endoplasmic reticulum (ER) stress. Two goblet cell lines, HT29-H and HT29-L, that produced high and low MUC2 mucin, respectively, were used. HT29-L healed wounds faster than HT29-H cells by producing significantly higher amounts of fibroblast growth factor (FGF) 1, FGF2, vascular endothelial growth factor-C, and matrix metallopeptidase 1. Predictably, treatment of HT29-H cells with recombinant FGF2 significantly enhanced migration and wound healing. High MUC2 biosynthesis in HT29-H cells induced ER stress and delayed migration that was abrogated by inhibiting ER stress with tauroursodeoxycholic acid and IL-22. FGF2- and IL-22-induced wound repair was dependent on STAT1 and STAT3 signaling. During wound healing after dextran sulfate sodium-induced colitis, restitution of Math1M1GFP+ goblet cells occurred earlier in the proximal colon, followed by the middle and then distal colon, where ulceration was severe. We conclude that high MUC2 output during colitis impairs goblet cell migration and wound healing by reducing production of growth factorscritical in wound repair.

Microbiota metabolites SCFA promote intestinal epithelial repair and wound healing through promoting epithelial cell production of milk fat globule-EGF factor 8

Abstract The mechanisms by which gut microbiota regulate intestinal homeostasis are still largely unknown. The short chain fatty acids (SCFA), mainly acetate, propionate, and butyrate, are bacterial metabolites produced from the fermentation of dietary fibers, which have been shown to regulate intestinal homeostasis. In this study, we investigated whether SCFAs promote intestinal epithelial repair and wound healing through promoting epithelial cell production of milk fat globulin E8 (MFG-E8), a known molecule promoting wound healing and cell migration via actin reorganization. Treatment with SCFAs enhanced intestinal epithelial cell (IEC) migration significantly, and promoted wound healing in both mouse and rat IEC in vitro. We then investigated how SCFAs regulated IEC gene expression by microarray. Microarray analysis showed 401 genes differentially expressed upon stimulation with butyrate, of which 196 were upregulated and 205 were downregulated. Among upregulated genes, MFG-E8 has been shown in several systems to promote IEC proliferation and tissue repair. We confirmed MFG-E8 upregulation by qRT-PCR for mRNA and Western blot for protein. Interestingly, SCFAs also upregulated several genes pertinent to actin reorganization including RhoA, Rho GTPase, and PAK1 as well as genes known to effect proliferation such as cyclin D2 and PKCδ, which are down stream of MFG-E8 signaling. Our study thus demonstrated that SCFAs promote intestinal epithelial cell tissue repair, possibly through upregulation of MFG-E8.

Transcription factor CUX1 is required for intestinal epithelial wound healing and targets the VAV2-RAC1 Signalling complex

Intestinal epithelial cells form a protective barrier in limiting gut luminal content potentially harmful to the host. Upon gut epithelium injury, several signals instruct epithelial cells to undergo a rapid healing process. Defects in this process induce inflammatory responses and can further evolve into chronic gut inflammatory diseases. We previously identified the transcription factor CUX1 as crucial for protecting against experimental colitis in mice. However, the precise molecular mechanisms by which CUX1 intervenes during this biological process are unknown. Our aim was to evaluate CUX1 biological and functional roles during intestinal epithelial cell wound healing. RNAi knockdown of CUX1 in intestinal epithelial cells revealed a crucial role for this regulator in migratory response following wounding assays. Gene expression profiling identified several gene transcripts modulated in absence of CUX1 during wound healing for which a significant number was associated with cell motility and cytoskeleton function. Chromatin immunoprecipitation assays identified the guanine nucleotide exchange factor Vav2 gene as a direct target for CUX1. Coincidently, reduction of VAV2 in absence of CUX1 was associated with a significant decrease of RAC1 activity in response to epithelial wounding. Our results identify a novel pathway by which CUX1 regulates normal intestinal epithelial cell restitution.

Constitutive androstane receptor regulates the intestinal mucosal response to injury.

The pathogenesis of the inflammatory bowel diseases (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), involves aberrant interactions between a genetically susceptible individual, their microbiota and environmental factors. Alterations in xenobiotic receptor expression and function are associated with increased risk for IBD. Here, we have assessed the role of the constitutive androstane receptor (CAR), a xenobiotic receptor closely related to the pregnane X receptor, in the regulation of intestinal mucosal homeostasis. CAR expression was assessed in intestinal mucosal biopsies obtained from CD and UC patients, and in C57/Bl6 mice exposed to dextran sulphate sodium (DSS; 3.5% w/v in drinking water) to evoke intestinal inflammation and tissue damage. CAR-deficient mice were exposed to DSS and mucosal healing assessed. Modulation of wound healing by CAR was assessed in vitro. The therapeutic potential of CAR activation was evaluated, using 3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP), a selective rodent CAR agonist. CAR expression was reduced in CD and UC samples, compared with expression in healthy controls. This was reproduced in our DSS studies, where CAR expression was reduced in colitic mice. CAR-deficient mice exhibited reduced healing following DSS exposure. In vitro, CAR activation accelerated intestinal epithelial wound healing by enhancing cell migration. Lastly, treating mice with TCPOBOP, following induction of colitis, enhanced mucosal healing. Our results support the notion that xenobiotic sensing is altered during intestinal inflammation, and suggest that CAR activation may prove effective in enhancing mucosal healing in patients with IBD.

P-290 Role of the Pro-resolving Lipid Mediator Resolvin E1 in Intestinal Epithelial Wound Healing

P-290 Role of the Pro-resolving Lipid Mediator Resolvin E1 in Intestinal Epithelial Wound Healing

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing.

The incidence of inflammatory bowel disease, i.e., Crohn's disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn's disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.