Proximal Colonic Mucosa Research Articles

Vil-Cre specific Slfn3KO mice exhibit sex-specific differences in lung, stomach, cecum, kidney, and proximal colon differentiation markers and Slfn family members expression levels

The Schlafen (Slfn) family proteins are critical regulators of cell proliferation, induction of immune responses, differentiation, self-restoration, and cell cycle progression. Rodent Slfn3 and human ortholog SLFN12 are critical in the regulation of intestinal epithelial differentiation. Following previous work utilizing Vil-Cre epithelial-specific Slfn3 knockout (VC-Slfn3KO) mice to evaluate Slfn3's role in small intestinal epithelial differentiation, we sought to characterize and distinguish the effects of Slfn3 loss on Slfn family member mRNA expression and differentiation markers for other epithelial cells in the lung, stomach, cecum, and proximal colon. Quantitative PCR analysis of Slfn1, 2, 4, 5, 8, and 9 and multiple differentiation markers were evaluated. We observed gender-specific effects with the loss of Slfn3 on the other Slfn family members and epithelial differentiation markers expression. Lung Slfn4 and 5 were increased only in male VC-Slfn3KO while lung Slfn2 and 8 were decreased only in female VC-Slfn3KO compared to controls. Slfn1, 2, 4, and 9 were increased in the gastric mucosa of male VC-Slfn3KO mice compared to controls. Slfn5 was reduced in female VC-Slfn3KO proximal colonic mucosa compared to controls. Lung AT1 cell differentiation marker Hopx mRNA expression was decreased and Ager was increased in VC-Slfn3KO male mice compared to controls. Lung AT2 differentiation markers and surfactant genes Sftpc and Sftpd were decreased in male VC-Slfn3KO mice. Stomach transcription factors, Lgr5 and Notch1 were increased in male VC-Slfn3KO. Tff1 secretory protein gene was decreased in female VC-Slfn3KO mice. Sucrase isomaltase was greatly increased in male VC-Slfn3KO mice in both cecal and proximal colonic mucosa, but glucose transporter Glut2 was decreased only in the cecum of female VC-Slfn3KO. The changes induced by VC-Slfn3KO in the expression of epithelial differentiation markers and other Schlafen proteins in various target tissues, indicate a complex regulation of gene expression that is sex-dependent.

Dietary Fat and Fiber Alter Rat Colonic Protein Kinase C Isozyme Expression

To better understand the biochemical mechanisms by which select fats and fibers modulate colonic cell proliferation, we determined the profile of protein kinase C (PKC) isozymes and cell proliferation in rat proximal and distal colonic mucosa following diet manipulation, because enhanced cell proliferation has been correlated with colon cancer incidence. Rats were assigned to one of four diets (each with 15 g fat + 6 g fiber/100 g diet) for 3 wk: fiber-free fish oil (FF), fiber-free corn oil (FC), cellulose + corn oil (CC), or pectin + corn oil (PC). Steady-state levels of colonic mucosal cytosolic and membrane PKC isozymes were determined. In vivo cell proliferation was determined by bromodeoxyuridine incorporation into DNA. In addition, viable exfoliated colonic epithelial cells were isolated from feces using Percoll-bovine serum albumin gradients. We found that 1) proximal and distal colonic mucosa possessed different steady-state levels and relative proportions of PKC isozymes; 2) PKC α and δ expression were significantly greater in distal membrane of the PC-fed group compared with the other dietary groups; 3) the number of exfoliated cells per 4-h fecal collection generally was proportional to the diet-induced changes in cell proliferation (PC>FC>CC>FF). These data demonstrate that dietary treatment altered colonic PKC isozyme expression, with animals fed the fiber-containing diets generally expressing higher steady-state levels of PKC α and δ.

Deep learning-based image analysis reveals significant differences in the number and distribution of mucosal CD3 and γδ T cells between Crohn's disease and ulcerative colitis.

Colon mucosae of ulcerative colitis (UC) and Crohn's disease (CD) display differences in the number and distribution of immune cells that are difficult to assess by eye. Deep learning-based analysis on whole slide images (WSIs) allows extraction of complex quantitative data that can be used to uncover different inflammatory patterns. We aimed to explore the distribution of CD3 and γδ T cells in colon mucosal compartments in histologically inactive and active inflammatory bowel disease. By deep learning-based segmentation and cell detection on WSIs from a well-defined cohort of CD (n=37), UC (n=58), and healthy controls (HCs, n=33), we quantified CD3 and γδ T cells within and beneath the epithelium and in lamina propria in proximal and distal colon mucosa, defined by the Nancy histological index. We found that inactive CD had significantly fewer intraepithelial γδ T cells than inactive UC, but higher total number of CD3 cells in all compartments than UC and HCs. Disease activity was associated with a massive loss of intraepithelial γδ T cells in UC, but not in CD. The total intraepithelial number of CD3 cells remained constant regardless of disease activity in both CD and UC. There were more mucosal CD3 and γδ T cells in proximal versus distal colon. Oral corticosteroids had an impact on γδ T cell numbers, while age, gender, and disease duration did not. Relative abundance of γδ T cells in mucosa and blood did not correlate. This study reveals significant differences in the total number of CD3 and γδ T cells in particularly the epithelial area between CD, UC, and HCs, and demonstrates useful application of deep segmentation to quantify cells in mucosal compartments.

Segmental variability in the regulation of NKCC1 by aldosterone in rat proximal and distal colon

The corticosteroid hormone, aldosterone, markedly enhances K+ secretion throughout the colon – a mechanism critical to its role in maintaining overall K+ balance. Previous studies from our lab have demonstrated that basolateral Na+‐K+‐2Cl‐ cotransporter 1 (NKCC1) is transcriptionally up‐regulated by aldosterone in the distal colon specifically to support large conductance K+ (BK) channel‐mediated K+ secretion. This function is distinct from the more well‐established role of NKCC1 in supporting luminal Cl‐ secretion throughout the gastrointestinal tract. However, considerable segmental variability exists between proximal and distal colonic ion transport processes, especially concerning their regulation by corticosteroids. Although active K+ secretion has also been described in the proximal colon, the molecular identity of the K+ channel(s) that mediates aldosterone‐induced K+ secretion in the proximal colon is not known, nor is the involvement of NKCC1 in the process. Experiments were therefore designed to test the hypothesis that aldosterone up‐regulates NKCC1 in the proximal colon to support BK channel‐mediated K+ secretion, congruent with its effects in the distal colon. Using dietary Na+‐depletion as a model of secondary hyperaldosteronism in rats, we found that NKCC1 protein expression in the proximal colonic mucosa was enhanced 2‐fold (p < 0.05) by Na+ depletion (i.e., aldosterone), whereas mRNA abundance was unaffected (p = 0.32). Surprisingly, electrogenic K+ secretion was not detectable by short‐circuit current (ISC) measurements, in response to either basolateral bumetanide (NKCC1 inhibitor) or luminal Ba2+ (non‐selective K+ channel blocker), despite enhanced K+ secretion in Na+‐depleted vs. normal rats, as measured by 86Rb+ fluxes under voltage‐clamped conditions (net K+ secretion = ‐2.1 ± 0.6 vs ‐0.4 ± 0.2 mEq/cm2.hr; p < 0.05). Expression of both BK and intermediate conductance K+ (IK) channels was also found to be unaltered by dietary Na+ depletion, at both the protein and mRNA level. However, basal ISC (146.7 ± 16.8 vs. 93.7 ± 8.7 mA/cm2; p < 0.05), as well as bumetanide‐sensitive, Ca2+‐ and cAMP‐stimulated ISC (i.e., Cl‐ secretion) were significantly enhanced by dietary Na+ depletion (ΔISC = 239.6 ± 5.6 vs. 105.7 ± 33.5 and 154.3 ± 8.7 vs. 115.3 ± 8.7 mA/cm2 for Ca2+ and cAMP, respectively; p < 0.05 for both). In parallel to this was an increase in cystic fibrosis transmembrane conductance regulator (CFTR) Cl‐ channel protein expression (~1.7‐fold; p < 0.05). We therefore reject our original hypothesis and conclude that NKCC1‐dependent secretory pathways are differentially regulated by aldosterone in proximal and distal colon, where they are biased toward Cl‐ and K+ secretion, respectively. Development of therapeutic strategies in treating pathologies related to aberrant or altered colonic K+/Cl‐ transport – such as pseudo‐obstruction, ulcerative colitis, or end‐stage renal disease (ESRD) – may benefit from these findings.

Effect of moxibustion on colonic mucosal injury and expression of CGRP positive nerve fibers of distal colonic mucosa in ulcerative colitis mice

To observe the effect of moxibustion at "Zusanli" (ST 36) on distal, middle and proximal colonic mucosal injury and expression of calcitonin gene-related peptide (CGRP) positive nerve fibers of distal colonic mucosa in ulcerative colitis (UC) mice at different time points. A total of 51 C57BL/6N mice were randomized into a 7-day control group (n=8), a 7-day model group (n=7), a 7-day moxibustion group (n=7), a 14-day control group (n=6), a 14-day model group (n=14) and a 14-day moxibustion group (n=9). In the model groups and the moxibustion groups, 2% dextran sulfate sodium (DSS) was given for 7-day free drinking to establish the UC model. Three days into modeling, moxibustion was applied at "Zusanli" (ST 36) in the 7-day moxibustion group and the 14-day moxibustion group, once a day, 10 min a time for 5 days and 12 days respectively. HE staining was used to observe the morphology of colonic tissue, the percentages of distal, middle and proximal colonic mucosal injury were calculated. Immunofluorescence staining was used to detected the expressions of positive nerve fibers of distal, middle and proximal colonic mucosa and CGRP positive nerve fibers of distal colonic mucosa. Mucosal injury can be observed in mice after modeling, displaying epithelial layer disappearance, abnormal crypt structure or crypt disappearance. Compared with the 7-day control group, colon length was shortened (P<0.001), percentages of overall, distal, middle colonic mucosal injury were increased (P<0.001), the expressions of positive nerve fibers of distal, middle and proximal colonic mucosa and CGRP positive nerve fibers of distal colonic mucosa were increased (P<0.001, P<0.05, P<0.01) in the 7-day model group. Compared with the 7-day model group, the expressions of positive nerve fibers of middle and distal colonic mucosa and CGRP positive nerve fibers of distal colonic mucosa were decreased in the 7-day moxibustion group (P<0.05). Compared with the 14-day control group, the colon length was shortened (P<0.01), percentage of overall colonic mucosal injury was increased (P<0.001) in the 14-day model group. Compared with the 14-day model group, colon length was lengthened (P<0.05), percentage of overall colonic mucosal injury was decreased (P<0.05) in the 14-day moxibustion group. Moxibustion at "Zusanli" (ST 36) can reduce the expressions of positive nerve fibers of colonic mucosa and CGRP positive nerve fibers of distal colonic mucosa, thus, improve the colonic mucosal injury.

Differential genetic influences over colorectal cancer risk and gene expression in large bowel mucosa.

Site-specific variation in colorectal cancer (CRC) incidence, biology and prognosis are poorly understood. We sought to determine whether common genetic variants influencing CRC risk might exhibit topographical differences on CRC risk through regional differences in effects on gene expression in the large bowel mucosa. We conducted a site-specific genetic association study (10 630 cases, 31 331 controls) to identify whether established risk variants exert differential effects on risk of proximal, compared to distal CRC. We collected normal colorectal mucosa and blood from 481 subjects and assessed mucosal gene expression using Illumina HumanHT-12v4 arrays in relation to germline genotype. Expression quantitative trait loci (eQTLs) were explored by anatomical location of sampling. The rs3087967 genotype (chr11q23.1 risk variant) exhibited significant site-specific effects-risk of distal CRC (odds ratio [OR]=1.20, P=8.20 × 10-20 ) with negligible effects on proximal CRC risk (OR=1.05, P=.10). Expression of 1261 genes differed between proximal and distal colonic mucosa (top hit PRAC gene, fold-difference=10, P=3.48 × 10-57 ). In eQTL studies, rs3087967 genotype was associated with expression of 8 cis- and 21 trans-genes. Four of these (AKAP14, ADH5P4, ASGR2, RP11-342M1.7) showed differential effects by site, with strongest trans-eQTL signals in proximal colonic mucosa (eg, AKAP14, beta=0.61, P=5.02 × 10-5 ) and opposite signals in distal mucosa (AKAP14, beta=-0.17, P=.04). In summary, genetic variation at the chr11q23.1 risk locus imparts greater risk of distal rather than proximal CRC and exhibits site-specific differences in eQTL effects in normal mucosa. Topographical differences in genomic control over gene expression relevant to CRC risk may underlie site-specific variation in CRC. Results may inform individualised CRC screening programmes.

FFA3 and TRPM5 Are Involved in the Sodium Propionate‐Induced Anion Secretion in Mouse Proximal Colon

Luminal short-chain fatty acids (SCFA) triggers cholinergic signals for anion secretion in the rodent colonic mucosa, while exact mechanisms are still unclear. Tuft cells, one of luminal chemosensing components in the intestine, expresses choline acetyltransferase (ChAT), transient receptor potential menthol 5 (TRPM5) and free fatty acid receptor 3 (FFA3). We thus examined the roles of TRPM5 and FFA3 in SCFA-induced anion secretion in mouse proximal colon. Short-circuit current (Isc) was measured in the muscle-stripped mouse proximal colonic mucosa mounted in an Ussing chamber. Sodium propionate (NaP, 10 mM, m), atropine (10 µM, s), the selective free fatty acid receptor 3 (FFA3) antagonist AR399519 (1 µM, m or s), or the TRPM5 antagonist triphenylphosphine oxide (TPPO, 1 - 300 µM, m or s) were applied to the mucosal (m) or serosal (s) baths of an Ussing chamber. Luminal NaP transiently increased Isc with atropine-sensitive manner. NaP-induced Isc increase was dose-dependently inhibited by luminal application of TPPO (IC50 0.2 µM) by 76%, while serosal TPPO had higher IC50 (53.5 µM). Luminal AR399519 inhibited NaP-induced Isc increase by 77%, while serosal AR399519 inhibited by 48%. Doublecortin-like kinase 1 (DCLK1)-positive tuft cells were scattered in the epithelium, whereas FFA3 was localized in the surface epithelial cells and in the myenteric nerves. These results suggest that tuft cell TRPM5 and epithelial cell FFA3 are both involved in the NaP-induced anion secretion in mouse proximal colon.

The role of nitric oxide in the inhibitory effects of exogenous hydrogen sulfide on rat colonic smooth muscle contraction

Objective: To investigate the role of nitric oxide (NO) in hydrogen sulfide (H(2)S)-induced inhibition upon colonic smooth muscle contraction. Methods: Immunohistochemistry was applied to observe the distribution of H(2)S-producing enzymes CBS and CSE in adult male Wistar rats. Organ bath system was used to observe the spontaneous contraction of colonic smooth muscle. Patch clamp technique was applied to record currents of L-type calcium channel (I(Ca,L)) in smooth muscle cells. Results: Specific immunoreactivity for CSE and CBS was observed in mucosa, smooth muscle and enteric plexus of rat proximal colon. NaHS elicited relaxation in a concentration-dependent manner upon muscle contraction in the presence of tetrodotoxin. The NaHS IC(50) of LM was 917.6 μmol/L (95% CI: 776.3-1 085 μmol/L, n=6) and the NaHS IC(50) of CM was 730.4 μmol/L (95% CI: 592.2-900.8 μmol/L, n=6). The SNP-induced relaxation in muscle strips was partially reversed by NaHS (P<0.05). Instead, the relaxation caused by NaHS was decreased by the sGC inhibitor ODQ but affected neither by NO precursor L-arginine, the NO inhibitor L-NNA nor the competitive cGMP antagonist PET-cGMP. NaHS (100 μmol/L) increased I(Ca,L) while NaHS (300 μmol/L) decreased the peak I(Ca,L) with modifying the ion channel characteristics (P<0.05). Conclusions: Exogenous hydrogen sulfide might have a dual effect on colonic motility and its inhibitory effect might be independent of NO signaling system. L-type calcium channel may play an important role during the process of H(2)S modulating colonic contraction.

The ASAMET trial: a randomized, phase II, double-blind, placebo-controlled, multicenter, 2\u2009\xd7\u20092 factorial biomarker study of tertiary prevention with low-dose aspirin and metformin in stage I-III colorectal cancer patients

BackgroundEpidemiological studies and cardiovascular prevention trials have shown that low-dose aspirin can reduce colorectal cancer (CRC) incidence and mortality, including inhibition of distant metastases. Metformin has also been associated with decreased colon adenoma recurrence in clinical trials and lower CRC incidence and mortality in epidemiological studies in diabetics. While both drugs have been tested as single agents, their combination has not been tested in cancer prevention trials.Methods/designThis is a randomized, placebo-controlled, double-blind, 2 × 2 biomarker trial of aspirin and metformin to test the activity of either agent alone and the potential synergism of their combination on a set of surrogate biomarkers of colorectal carcinogenesis. After surgery, 160 patients with stage I-III CRC are randomly assigned in a four-arm trial to either aspirin (100 mg day), metformin (850 mg bis in die), their combination, or placebo for one year. The primary endpoint biomarker is the change of IHC expression of nuclear factor kappa-B (NFκB) in the unaffected mucosa of proximal and distal colon obtained by multiple biopsies in two paired colonoscopies one year apart. Additional biomarkers will include: 1) the measurement of circulating IL-6, CRP and VEGF; 2) the IHC expression of tissue pS6K, p53, beta-catenin, PI3K; 3) the associations of genetic markers with treatment response as assessed by next generation sequencing of primary tumors; 4) the genomic profile of candidate genes, pathways, and overall genomic patterns in tissue biopsies by genome wide gene expression arrays; and 5) the evaluation of adenoma occurrence at 1 year.DiscussionA favorable biomarker modulation by aspirin and metformin may provide important clues for a subsequent phase III adjuvant trial aimed at preventing second primary cancer, delaying recurrence and improving prognosis in patients with CRC.Trial registrationEudraCT Number: 2015–004824-77; ClinicalTrial.gov Identifier: NCT03047837. Registered on February 1, 2017.

Gut dysbiosis in the development of cerebral small vessel disease

The importance of a healthy gut microbiota on host physiology is becoming increasingly evident. Recent studies suggest that alterations to the microbiota (i.e., dysbiosis) can have adverse effects beyond the GI tract, and has been linked to hypertension and stroke. We have demonstrated that the microbiota of the spontaneously hypertensive stroke prone rat (SHRSP), a model of cerebral small vessel disease (CSVD), is significantly different than that of WKY controls. Thus we hypothesized that gut dysbiosis contributes to the development of CSVD. Phenotypically, CSVD includes hypertension, vessel remodeling, BBB breakdown and neuroinflammation. If our hypothesis is valid, then creating a gut microbiota in a WKY rat that resembles one from an SHRSP rat should produce CSVD phenotypes. Conversely, creating a gut microbiota in a SHRSP rat that resembles one from a healthy WKY strain should abolish or attenuate the onset of CSVD phenotypes, including hypertension. Given that rat pups acquire their microbiota from their mother and the immediate environment, we used foster mothers to shape the microbiota of rat pups. WKY and SHRSP pups were removed from their birthing mother at 2–3 days of age and fostered on mothers of the same or opposite strain. Upon weening, fostered animals were co‐housed with animals of the same strain as their fostered mother. Systolic blood pressure (SBP) was measured using tail cuff plethysmography from 6–20 weeks of age. At 20 weeks of age, rats were sacrificed and brain and gut tissue were collected to assess for CSVD phenotypes and gene expression for inflammatory markers and toll‐like receptor signaling. Feces were collected at each BP measurement to evaluate the composition of the gut microbiota using 16s rRNA sequencing. Repeated measures two‐way ANOVA of SBP from week 6–20 showed a 15 mmHg increase in WKY rats fostered by an SHRSP mother (sWKY)compared to WKY fostered on WKY mothers (wWKY; p&lt;.001), and a 7 mmHg decrease in SHRSPs fostered by a WKY mother (wSHRSP) compared to SHRSPs fostered by a SHRSP mother (sSHRSP) (p=.027). Independent of genotype, rats nursed by SHRSP mothers had evidence of brain and gut inflammation including increases in TLR2, TLR4, NF‐kB1, and Il‐1α expression in the brain and proximal colon mucosa, compared to animals fostered by WKYs (p&lt;.01). Furthermore, by using IgG extravasation into the brain parenchyma as a marker for blood brain barrier integrity, we discovered that wSHRSP animals had improved blood brain barrier integrity as compared to sSHRSP (p&lt;.05). Through sequencing of fecal 16s rRNA it was determined that cross‐fostering animals on opposite strains lead to significant alterations in the gut microbiota composition. Most striking was that animals nursing on SHRSP mothers led to significant decreases in Akkermansia, a genus associated with improving gut barrier integrity (p&lt;.05). This study supports the hypothesis that gut dysbiosis contributes to the onset and development of CSVD, possibly by creating an inflammatory environment in the colon. Furthermore, maintaining a healthy gut microbiota in a CSVD strain has the potential to reduce CSVD phenotypes, gut inflammation and attenuate hypertension. These findings support the idea that the gut is a possible therapeutic target to treat CSVD and other cardiovascular diseases.Support or Funding InformationAHA Predoctoral Fellowship 16PRE29640005This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural and functional rearrangement of the large intestine after first stage of the reconstructive and restore operations

The morphological changes of the large bowel mucous membrane of the patients after reconstructive and restore operations (RRO) were analyzed. The progressive reduction of the colon mucosa columnar epithelial cells with their replacement by monolayer of the goblet cells that secrete mainly sialomucins during the six-month follow-up was established. Recovery of the proximal colon mucosa with decrease of hyperplastic and reactive inflammatory changes, stabilization of intracellular mucin secretion by goblet cells in the proximal part, the low degree of atrophy and fibrosis in the distal part of colon mucosa were observed after the third month, indicating the most favorable time for RRO.

ASAMET: A randomized, 2x2 biomarker prevention trial of low-dose aspirin and metformin in colorectal cancer.

TPS1591 Background: Epidemiological studies and cardiovascular prevention trials have shown that low-dose aspirin (ASA) can inhibit colorectal cancer (CRC) incidence and mortality, including inhibition of distant metastases. Metformin (MET) has also been associated with decreased CRC incidence and mortality in meta-analyses of epidemiological studies in diabetics and has been shown to decrease by 40% colorectal adenoma recurrence in a randomized trial. Recent studies have shown that ASA is an inhibitor of mTOR/S6K1 and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism, and that the combination of ASA and MET, another AMPK activator and S6K1 inhibitor, has a striking additive effect on AMPK activation and mTOR inhibition, with increased autophagy and decreased cell growth in CRC cell lines. While both drugs are being tested as single agents, their combination has not been tested in clinical trials. Methods: This is a randomized, placebo-controlled, double-blind, 2x2 biomarker trial of ASA and MET to test the activity of either agent alone and the potential synergism of their combination on a set of surrogate biomarkers of colorectal carcinogenesis. After surgery, 160 patients with stage I-III CRC will randomly be assigned in a 4-arm trial to either ASA, 100mg qd, MET 850mg bid, their combination, or placebo for 1 year. The primary endpoint is the change, defined as the difference between pre- and post-treatment expression, of NFκBin the unaffected mucosa of proximal and distal colon obtained by multiple biopsies in paired colonoscopies one year apart. Additional biomarkers will include: 1) the genomic profile of candidate genes, pathways, and overall genomic patterns in tissue biopsies by genome-wide gene expression arrays; 2) the IHC expression of tissue pS6K, p53, beta-catenin, PI3K; 3) the associations of mutations and SNPs with treatment response by NGS of primary tumors; 4) the measurement of circulating IL-6, CRP and VEGF, and 5) plasma and colonic MET concentrations and their correlation with biomarker profiles. A favorable biomarker modulation may provide important clues for a subsequent phase III adjuvant trial. Clinical trial information: EudraCT Number: 2015-004824-77.

Expression of transient receptor potential ankyrin 1 correlating to the recovery of colonic transit after pelvic nerve denervation in rats

BackgroundIt has been reported that colorectal motility dysfunction due to pelvic nerve (PN) damage is restored overtime. However, the adaptive mechanism is unknown. Previous studies implied that transient receptor potential ankyrin 1 (TRPA1) mediated sensory nerve signal input plays a crucial role in gut motility regulation. The present study aimed to observe the colorectal motility restoration in rats after PN transection and to explore the change of TRPA1 protein expression in this adaptive process. MethodsSeventy-eight adult rats were divided into two groups randomly: sham and PN cut. Colonic transit function was determined with radioisotope method by calculating the geometric center (GC) of the distribution of 51Cr at postoperative days (POD) 1, 3, and 7. Expression of TRPA1 in the proximal and distal colon mucosa was detected with Western blotting at POD 1, 3, and 7. ResultsAt POD 1, the colonic transit in PN cut group was significantly delayed (GC = 4.91 ± 0.41, P < 0.05), when compared with the sham group (GC = 5.76 ± 0.85). A significant trend toward recovery was noted in the PN cut group at POD 3 (GC = 5.58 ± 0.36) and POD 7 (GC = 6.44 ± 0.78). Western blot demonstrated attenuated expression of TRPA1 in the distal colon mucosa after PN denervation at POD 1 (0.39 ± 0.12) compared with that of the shams. A significant trend of increasing expression of TRPA1 was demonstrated in the PN cut group at POD 3 (0.78 ± 0.10) and at POD 7 (1.06 ± 0.13). ConclusionsDelayed colonic motility due to PN denervation gradually restored overtime, which may relate to the corresponding expression of TRPA1 in the distal colonic mucosa of rats.

Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon

Luminal short-chain fatty acids (SCFAs) influence gut physiological function via SCFA receptors and transporters. The contribution of an SCFA receptor, free fatty acid receptor (FFA)3, to the enteric nervous system is unknown. FFA3 is expressed in enteric cholinergic neurons. Activation of neural FFA3 suppresses Cl(-) secretion induced by nicotinic ACh receptor activation via a Gi/o pathway. Neural FFA3 may have an anti-secretory function by modulating cholinergic neural reflexes in the enteric nervous system. The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl(-) secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13μm. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the Gi/o pathway. Luminal propionate induced Cl(-) secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression throughFxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells

Background: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention.Objective: We investigated howAPC inactivation influences the regulation ofFXR expression in colonic mucosal cells. We hypothesized thatAPC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation.Methods: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J andApc-deficient (ApcMin/+) male mice, respectively. The expression ofFxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation ofFxr in bisulfonated genomic DNA. In vitro, we measured the impact ofAPC inactivation and deoxycholic acid (DCA) treatment onFXR expression in human colon cancer HCT-116 cells transfected with silencing RNA forAPC and HT-29 cells carrying inactivatedAPC.Results: InApcMin/+ mice, constitutive CpG methylation of theFxrα3/4 promoter was linked to reduced (60–90%) baselineFxr,Ibabp, andShp and increasedCox-2 expression in apparently normal adjacent mucosa and colon tumors.Apc knockdown in HCT-116 cells increased cellular myelocytomatosis(c-MYC) and lowered (∼50%)FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA inducedFXR expression and lowered CpG methylation ofFXR.Conclusions: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.

Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis-associated cancer

BackgroundKeratins are intermediate filament (IF) proteins, which form part of the epithelial cytoskeleton and which have been implicated pathology of inflammatory bowel diseases (IBD).MethodsIn this study biopsies were obtained from...

Docosahexaenoic Acid Attenuated Experimental Chronic Colitis in Interleukin 10-Deficient Mice by Enhancing Autophagy Through Inhibition of the mTOR Pathway.

In the battle against Crohn's disease, autophagy stimulation is a promising therapeutic option-one both new and newly rediscovered. In experimental models, docosahexaenoic acid (DHA)-a long-chain polyunsaturated fatty acid-has been demonstrated to be useful in the treatment of inflammatory bowel disease through inhibition of the nuclear factor-κB pathway. However, the impact of DHA on autophagy in the colon remains unclear. Mice were divided into 3 groups: wild type (placebo), the interleukin 10 knockout group (IL-10-/-, placebo), and the DHA group (IL-10-/-, DHA). DHA was administered to IL-10-/- mice by gavage at a dosage of 35.5 mg/kg/d for 2 weeks. The severity of colitis, expression of proinflammatory cytokines, expression/distribution of LC3B, and mTOR signaling pathway were evaluated in the proximal colon tissues collected from all mice at the end of the experiment. DHA administration ameliorated experimental colitis in the IL-10-/- mice, as demonstrated by decreased proinflammatory cytokines (TNF-α and IFN-γ), reduced infiltration of inflammatory cells, and lowered histologic scores of the proximal colon mucosa. Moreover, in the DHA-treated mice, enhanced autophagy was observed to be associated with (1) increased expression and restoration of the distribution integrity of LC3B in the colon and (2) inhibition of the mTOR signaling pathway. This study showed that DHA therapy could attenuate experimental chronic colitis in IL-10-/- mice by triggering autophagy via inhibition of the mTOR pathway.

Luminal 5-HT stimulates colonic bicarbonate secretion in rats.

The bioactive monoamine 5-HT, implicated in the pathogenesis of functional gastrointestinal disorders, is abundantly synthesized and stored in rat proximal colonic mucosa and released to the gut lumen and subepithelial space. Despite much data regarding its expression and function, the effects of luminal 5-HT on colonic anion secretion have not been fully investigated. We measured short-circuit current (Isc ) as an indicator of ion transport in mucosa-submucosa or mucosa-only preparations of rat proximal colon. Total CO2 output was measured in vitro and in vivo. Immunohistochemistry was performed to investigate the localization of 5-HT4 , NOS1 and NOS2. Luminal 5-HT gradually increased the amplitude and sustained the elevation of Isc . Luminal 5-HT-evoked ΔIsc was acetazolamide sensitive and HCO3 (-) dependent, consistent with cytosolic carbonic anhydrase-dependent electrogenic HCO3 (-) secretion, while not affected by tetrodotoxin (TTX), atropine or indomethacin. Pretreatment with the selective 5-HT4 antagonist GR113808, but not antagonists for 5-HT3 , 5-HT6 or 5-HT7 , inhibited luminal 5-HT-evoked ΔIsc . Furthermore, luminal cisapride and tegaserod increased Isc to the same extent as did 5-HT in the presence of indomethacin and TTX. Removal of the submucosa or pretreatment with NOS inhibitors enhanced luminal 5-HT-evoked ΔIsc , suggesting that NO synthesized in the submucosa suppresses mucosal anion secretion. NOS1 and NOS2 were immunostained in the submucosal neurons and glial cells respectively. Luminal 5-HT-evoked HCO3 (-) secretion was confirmed in vivo, inhibited by co-perfusion of GR113808, but not by ondansetron. A novel apical 5-HT4 -mediated HCO3 (-) secretory pathway and an NO-dependent inhibitory mechanism are present in the proximal colon. Luminal 5-HT-evoked HCO3 (-) secretion may be important for the maintenance of mucosal integrity by regulating luminal pH.

PTU-069 The fate of epithelial keratins in active ulcerative colitis

<h3>Introduction</h3> Intermediate filaments (IF), which mainly consist of keratins (K), are one of the main components of the human cell cytoskeleton. K8, K18 and K19 constitute the main keratins in the intestinal epithelial cells. Keratin alterations may play a role in the pathophysiology of ulcerative colitis (UC). We have previously shown reduced expression of Keratins in IF fraction from distal active UC relative to proximal inactive mucosa as well as controls using proteomic iTRAQ-based analysis.¹Whether this reduced expression is due to change in solubilisation, reduced total expression and/or degradation, is unknown. We aimed to clarify the fate of epithelial keratins during UC disease activity. <h3>Method</h3> Soluble proteome fractions were extracted from rectal biopsies in patients (n = 10) with active distal colitis (ACT) as well as endoscopically and histologically uninflamed proximal colonic mucosa (INACT) in the same patients. Median histological activity index in ACT and INACT were 2 (range 1–3) and 0 (range 0). Control colonic biopsies (n = 7) from normal individuals were fractionated similarly. An iTRAQ-compatible extraction protocol for soluble proteins was developed and applied. Labelled peptides from pooled patients in each group were analysed by SCX-LC-MS/MS and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction. Validation of iTRAQ results was carried out with immunoblotting on pooled and individual samples using monoclonal antibody against K8. These outcomes were then compared with our previous insoluble proteome analyses.¹ <h3>Results</h3> Comparative iTRAQ analysis of the soluble fraction showed significantly lower log fold changes in K8, K18 and K19 levels in ACT relative to INACT (0.42, 0.53, 0.42) and normal controls (0.61, 0.76, 0.41), respectively. The results were similar to our recent iTRAQ analysis of the IF fraction.¹Immunoblotting further confirmed these findings: median relative K8 concentration in IF/soluble proteome fractions in individual ACT and INACT samples was 0.18/1 (p = 0.02) and 1.21/1.02 (p = 0.02), respectively. <h3>Conclusion</h3> This study suggests relative total reduction in expression of epithelial keratins in actively inflamed colonic mucosa of UC patients compared to un-inflamed proximal mucosa and normal controls. Keratins may have utility as a tissue biomarker of UC disease activity. <h3>Disclosure of interest</h3> None Declared. <h3>Reference</h3> Corfe BM, Majumdar D, Assadsangabi A, Marsh AMR, Cross SS, Connolly JB, Evans CA, Lobo AJ. Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis-associated-cancer. <i>BMJ Open Gastroenterol.</i>, In press

Immune and inflammatory responses in pigs infected with Trichuris suis and Oesophagostomum dentatum

The aim of the present study was to investigate parasite induced immune responses in pigs co-infected with Trichuris suis and Oesophagostomum dentatum as compared to mono-species infected pigs. T. suis is known to elicit a strong immune response leading to rapid expulsion, and a strong antagonistic effect on O. dentatum populations has been observed in co-infected pigs. Forty-eight helminth naïve pigs were allocated into 4 groups in a 2-factorial design. Two groups were trickle inoculated with either 10 T. suis eggs/kg/day (Group T) or 20 O. dentatum L3/kg/day (Group O). Group OT was infected with same levels of both T. suis and O. dentatum (Group OT) and Group C remained uninfected. In each group, six pigs were necropsied after 35 days and the remaining pigs after 71 days. Parasite E/S-antigen specific serum antibodies were quantified by an in-direct ELISA. qPCR was used to measure the expression of immune function related genes in the mucosa of proximal colon and the draining lymph node. Highly significant interactions were identified for O. dentatum specific IgG1 (p<0.0001) and IgG2 (p<0.0006) antibodies with a remarkable 2-fold higher antibody response in group OT pigs as compared to group O. These findings indicated that T. suis enhanced the antibody response against O. dentatum in Group OT. The gene expression data confirmed a strong Type 2 response to T. suis (e.g. marked increase in IL-13, ARG1 and CCL11) and clearly weaker in amplitude and/or delayed onset response to O. dentatum in the single infected group. Interactions were found between the two nematodes with regard to several cytokines, e.g. the increase in IL-13 observed in Group T was absent in Group OT (p=0.06, proximal colon mucosa, 35 and 71p.i.). Some of these immune response-related interactions may support, or even partially explain, the observed interactions between the two worm populations in co-infected pigs.