Hydrogen sulfide is an endogenous mediator that relaxes vascular smooth muscle, exhibits several antiinflammatory activities, and contributes to gastric mucosal defense. This study was performed to ...examine the role of hydrogen sulfide in the resolution of injury; specifically, the healing of gastric ulcers. Ulcers were induced in rats by serosal application of acetic acid. This elicited a marked increase in gastric expression of the two key enzymes in hydrogen sulfide synthesis (cystathionine-β-synthase and cystathionine-γ-lyase) and in hydrogen sulfide synthesis. Twice-daily treatment for a week with hydrogen sulfide donors significantly increased the extent of healing of gastric ulcers as compared to vehicle-treatment. Similar treatment with L-cysteine, a precursor for hydrogen sulfide, also accelerated healing of the ulcers, and the effect was abolished by cotreatment with an inhibitor of cystathionine-γ-lyase. The beneficial effects of hydrogen sulfide on ulcer healing were not dependent on nitric oxide synthesis, nor did they appear to occur through activation of ATP-sensitive K⁺ channels. These results suggest that hydrogen sulfide is produced in the gastric mucosa in response to injury and acts to promote healing. The results further suggest that drugs releasing hydrogen sulfide could be employed to accelerate healing of gastric ulcers, and possibly of other wounds.--Wallace, J. L., Dicay, M., McKnight, W., Martin, G. R. Hydrogen sulfide enhances ulcer healing in rats.
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Aims
Protease‐activated receptor (PAR)2 activation increases the expression of cyclooxygenase (COX)‐2 in intestinal epithelial cells. We tested the hypothesis that PAR2‐induced COX‐2 ...regulates intestinal inflammation and modulates epithelial wound healing.
Methods
PAR2 was activated using the selective activating peptide 2f‐LIGRLO. Circular wounds in Caco2 cell monolayers were monitored with live‐cell imaging.
In vivo,
epithelial damage was induced by giving WT and PAR2 KO (C57Bl/6) mice 2.5% DSS for 5‐7 days.
Results
Activation of PAR2 in Caco2 cells significantly increased COX‐2 protein (4.2‐fold) and PGE
2
metabolites (9.6‐fold).
In vivo
, preliminary results showed less COX‐2 protein in PAR2 KO mice given DSS compared to WT mice. The PAR2 KO mice also lost significantly more weight and had higher histological damage scores compared to WT mice. We next determined the effect of PAR2‐induced COX‐2 on epithelial wound healing
in vitro
. Surprisingly, PAR2 activation significantly inhibited the rate of wound closure over 48 hr (79.3±2.5% wound closure) compared to control (94.3±0.5%), independently of COX‐2 activity. PAR2 activation had no effect on proliferation, but significantly inhibited cell migration.
Conclusions
PAR2 activation was shown to be protective
in vivo
, which correlated with the expression of COX‐2. We also uncovered a novel COX‐2‐independent effect of PAR2, reducing the rate of wound healing by inhibiting cell migration. Our results show that PAR2 plays differential roles in regulating epithelial response to injury and inflammation.
Support:
Crohn's and Colitis Canada, Alberta IBD Consortium, Alberta Cancer Foundation.
Background & Aims Disturbances in the control of ion transport lead to epithelial barrier dysfunction in patients with colitis. Enteric glia regulate intestinal barrier function and colonic ion ...transport. However, it is not clear whether enteric glia are involved in epithelial hyporesponsiveness. We investigated enteric glial regulation of ion transport in mice with trinitrobenzene sulfonic acid− or dextran sodium sulfate−induced colitis and in Il10 −/− mice. Methods Electrically evoked ion transport was measured in full-thickness segments of colon from CD1 and Il10 −/− mice with or without colitis in Ussing chambers. Nitric oxide (NO) production was assessed using amperometry. Bacterial translocation was investigated in the liver, spleen, and blood of mice. Results Electrical stimulation of the colon evoked a tetrodotoxin-sensitive chloride secretion. In mice with colitis, ion transport almost completely disappeared. Inhibiting inducible NO synthase (NOS2), but not neuronal NOS (NOS1), partially restored the evoked secretory response. Blocking glial function with fluoroacetate, which is not a NOS2 inhibitor, also partially restored ion transport. Combined NOS2 inhibition and fluoroacetate administration fully restored secretion. Epithelial responsiveness to vasoactive intestinal peptide was increased after enteric glial function was blocked in mice with colitis. In colons of mice without colitis, NO was produced in the myenteric plexus almost completely via NOS1. NO production was increased in mice with colitis, compared with mice without colitis; a substantial proportion of NOS2 was blocked by fluoroacetate administration. Inhibition of enteric glial function in vivo reduced the severity of trinitrobenzene sulfonic acid−induced colitis and associated bacterial translocation. Conclusions Increased production of NOS2 in enteric glia contributes to the dysregulation of intestinal ion transport in mice with colitis. Blocking enteric glial function in these mice restores epithelial barrier function and reduces bacterial translocation.
Nonsteroidal anti-inflammatory drugs elevate gastric acid secretion, possibly contributing to their ability to interfere with gastric ulcer healing. Inhibitors of cyclooxygenase-2 have been shown to ...delay experimental gastric ulcer healing. In the present study, we tested the hypothesis that cyclooxygenase-2-derived prostaglandins modulate gastric acid secretion. Studies were performed in normal rats and in rats with iodoacetamide-induced gastritis. Inflammation in the latter group was confirmed histologically and by a threefold increase in tissue levels of the granulocyte marker myeloperoxidase and was also associated with overexpression of cyclooxygenase-2 in the stomach. Basal acid secretion in both groups of rats was not affected by pretreatment with DuP-697, a selective inhibitor of cyclooxygenase-2. A nonselective cyclooxygenase inhibitor, indomethacin, had no effect on acid secretion in normal rats but caused a doubling of acid secretion in the rats with gastritis. DuP-697 had no effect on pentagastrin-induced secretion in either group of rats. Gastritis itself was associated with significantly increased pentagastrin-induced acid secretion, and this was further increased in rats pretreated with indomethacin. These results suggest that in a setting of gastric inflammation, prostaglandins derived from cyclooxygenase-1, not cyclooxygenase-2, exert inhibitory effects on acid secretion.
The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional ...epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease–susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress–ATF6–DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.
Salvinorin A (SA) has a potent inhibitory action on mouse gastrointestinal (GI) motility and ion transport, mediated primarily by kappa-opioid receptors (KOR). The aim of the present study was to ...characterize possible antiinflammatory and antinociceptive effects of SA in the GI tract of mice.
Colonic damage scores and myeloperoxidase activity were determined after intraperitoneal (i.p.), intracolonic (i.c.), and oral (p.o.) administration of SA using the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis in mice. Additionally, KOR, cannabinoid (CB)1, and CB2 western blot analysis of colon samples was performed. The antinociceptive effect of SA was examined based on the number of behavioral responses to i.c. instillation of mustard oil (MO).
The i.p. (3 mg/kg, twice daily) and p.o. (10 mg/kg, twice daily) administration of SA significantly attenuated TNBS and DSS colitis in mice. The effect of SA was blocked by KOR antagonist nor-binaltorphimine (10 mg/kg, i.p.). Western blot analysis showed no influence of SA on KOR, CB1, or CB2 levels. SA (3 mg/kg, i.p. and 10 mg/kg, i.c.) significantly decreased the number of pain responses after i.c. instillation of MO in the vehicle- and TNBS-treated mice. The antinociceptive action of SA was blocked by KOR and CB1 antagonists. The analgesic effect of i.c. SA was more potent in TNBS-treated mice compared to controls.
Our results suggest that the drugs based on the structure of SA have the potential to become valuable antiinflammatory or analgesic therapeutics for the treatment of GI diseases.
Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFNγ) is centrally involved in ...intestinal inflammation, the effect of this cytokine on AQP3 expression remains unknown. HT-29 human colonic epithelial cells were treated with IFNγ to assess AQP3 mRNA expression by real-time RT-PCR and functional protein expression through the uptake of radiolabelled glycerol. Transient knockdown of signal transducer and activator of transcription 1 (STAT1), STAT3, Sp1, and Sp3 were performed to determine the involvement of these transcription factors in the IFNγ-induced signalling cascade. AQP3 promoter regions involved in the response to IFNγ were assessed using a luciferase reporter system. Likewise, enteroids derived from human colonic biopsies were also treated with IFNγ to assess for changes in AQP3 mRNA expression. IFNγ decreased AQP3 mRNA expression in HT-29 cells in a time- and concentration-dependent manner and reduced functional AQP3 protein expression (decreased
3
H-labelled glycerol uptake). IFNγ also reduced AQP3 expression in enteroids derived from human colonic biopsies. Knockdown of STAT1 partially prevented the IFNγ-induced downregulation of AQP3 expression, whereas STAT3 and Sp3 knockdowns resulted in increased baseline expression of AQP3 but did not alter IFNγ-induced downregulation. Constitutive transcription of AQP3 is downregulated by IFNγ as demonstrated using the luciferase reporter system, with Sp3 bound to the AQP3 promoter as shown by chromatin immunoprecipitation. AQP3 constitutive transcription in intestinal epithelial cells is downregulated by IFNγ. This response requires STAT1 that is postulated to drive the downregulation of AQP3 expression through increased acetylation or decreased deacetylation the AQP3 promoter, ultimately resulting in decreased constitutive transcription of AQP3.
Key messages
• IFNγ suppresses the expression of AQP3 in intestinal epithelial cells.
• Proximal AQP3 promoter elements are sufficient to drive constitutive expression and mediate the IFNγ-induced downregulation of AQP3 mRNA expression.
• IFNγ-induced suppression of AQP3 is dependent upon STAT1 expression, but not STAT3, Sp1, or Sp3.
Abstract only
Background
Patients with IBD commonly present deregulated barrier function of the intestinal epithelium. We have shown that treatment with dinitrophenol (DNP: uncouples oxidative ...phosphorylation) dramatically impairs the barrier function of cultured epithelial monolayers, particularly internalization of non‐invasive commensal
E. coli
. Mitochondria do not function in isolation and are linked to the endoplasmic reticulum (ER). We hypothesized that interaction of mitochondrial dysfunction and ER stress is a key determinant of epithelial‐bacterial interaction, specifically the fate of commensal bacteria that gain access to the intracellular compartment as a consequence of metabolic stress.
Methods
Human colonic biopsies were mounted in Ussing chambers and human colon‐derived epithelial cell lines were cultured on transwell filters or on plasticware. Bacteria were added to the luminal buffer and the cells treated with DNP ± the ER stressor, tunicamycin (TM), and bacterial internalization and translocation assessed. Mechanistic studies involved measuring ATP production, assessment of autophagy (i.e. LC3 activation) and gene knock‐down (KD) by siRNA and CRISPR/cas9.
Results
DNP promoted the translocation and internalization of
E. coli
in colon tissue and epithelial monolayers: TM reduced this barrier defect, but did not prevent the DNP‐evoked drop in ATP or the rate of epithelial update of inert beads.
E. coli
, DNP + TM‐treated cells had increased autophagy. The TM antagonist of the DNP effect was lost in cells lacking the autophagy protein, ATG16L1, suggesting that the ER‐stress promoted killing of the internalized bacteria. Of the 3 major arms of the ER stress response, only KD of ATF6 ablated the TM antagonism of the DNP‐evoked barrier effect; the increase in autophagy evoked by TM was absent in ATF6 KD epithelia.
Conclusion
An effective ER stress response, via mobilization of xenophagic response, can ameliorate epithelial barrier function in terms of enhanced killing of bacteria that gain access to the cell as a consequence of mitochondrial dysfunction. We suggest that target induction of ATF6 activity could be beneficial in treating disease characterized by bacterial ‘invasion’ of epithelial cells.
Support or Funding Information
CIHR AIHS HPI NSERC
Abstract only
Chronic diseases like IBD are characterized by high levels of IFNγ and abnormal intestinal transepithelial fluid flux. The water channel protein Aquaporin 1 (AQP1) is widely expressed ...on intestinal epithelial cells with the primary purpose of regulating transmembrane water flux. To investigate the deficiencies in water transport associated with IBD, we sought to determine the cellular signaling mechanisms by which IFNγ modulates AQP1 expression in the mouse intestinal epithelium.
CMT93 cells treated with IFNγ significantly decreased constitutive AQP1 levels detected by immunofluorescence. Pre‐treatment with either the JAK1 inhibitor or STAT3 siRNA, prior to IFNγ administration resulted in the amelioration of the IFNγ‐induced suppression of AQP1 protein expression. Conversely, IRF‐2 siRNA pre‐treatment further decreased AQP1 protein levels following IFNγ treatment.
Inflammatory cytokines present during IBD are able to influence AQP1 levels and may contribute to the water transport deficiencies associated with these disorders. In this study, the results suggest that decreased epithelial AQP1 expression exerted by IFNγ in CMT93 cells is regulated by JAK1 and STAT3. Of note, IRF‐2 which is normally considered to be a negative regulator of IFNγ signaling, acted in a positive regulatory fashion by further decreasing AQP1 protein signal.
Source of funding: Crohn's & Colitis Foundation of Canada
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