Alkaline Phosphatase Relieves Colitis in Obese Mice Subjected to Forced Exercise via Its Anti-Inflammatory and Intestinal Microbiota-Shaping Properties.

Intestinal alkaline phosphatase (IAP) is an enzyme that plays a protective role in the gut. This study investigated the effect of IAP treatment on experimental colitis in mice subjected to forced exercise on a high-fat diet. C57BL/6 mice with TNBS colitis were fed a high-fat diet and subjected to forced treadmill exercise with or without IAP treatment. Disease activity, oxidative stress, inflammatory cytokines, and gut microbiota were assessed. Forced exercise exacerbated colitis in obese mice, as evidenced by increased disease activity index (DAI), oxidative stress markers, and proinflammatory adipokines and cytokines. IAP treatment significantly reduced these effects and promoted the expression of barrier proteins in the colonic mucosa. Additionally, IAP treatment altered the gut microbiota composition, favoring beneficial Verrucomicrobiota and reducing pathogenic Clostridia and Odoribacter. IAP treatment ameliorates the worsening effect of forced exercise on murine colitis by attenuating oxidative stress, downregulating proinflammatory biomarkers, and modulating the gut microbiota. IAP warrants further investigation as a potential therapeutic strategy for ulcerative colitis.

The mitochondria-targeted sulfide delivery molecule attenuates drugs-induced gastropathy. Involvement of heme oxygenase pathway.

Hydrogen sulfide (H2S) signaling and H2S-prodrugs maintain redox balance in gastrointestinal (GI) tract. Predominant effect of any H2S-donor is mitochondrial. Non-targeted H2S-moieties were shown to decrease the non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrotoxicity but in high doses. However, direct, controlled delivery of H2S to gastric mucosal mitochondria as a molecular target improving NSAIDs-pharmacology remains overlooked. Thus, we treated Wistar rats, i.g. with vehicle, mitochondria-targeted H2S-releasing AP39 (0.004-0.5 mg/kg), AP219 (0.02 mg/kg) as structural control without H2S-releasing ability, or AP39 + SnPP (10 mg/kg) as a heme oxygenase (HMOX) inhibitor. Next, animals were administered i.g. with acetylsalicylic acid (ASA, 125 mg/kg) as NSAIDs representative or comparatively with 75% ethanol to induce translational hemorrhagic or necrotic gastric lesions, that were assessed micro-/macroscopically. Activity of mitochondrial complex IV/V, and DNA oxidation were assessed biochemically. Gastric mucosal/serum content of IL-1ß, IL-10, TNF-α, TGF-ß1/2, ARG1, GST-α, or phosphorylation of mTOR, NF-κB, ERK, Akt, JNK, STAT3/5 were evaluated by microbeads-fluorescent xMAP®-assay; gastric mucosal mRNA level of HMOX-1/2, COX-1/2, SOD-1/2 by real-time PCR. AP39 (but not AP219) dose-dependently (0.02 and 0.1 mg/kg) diminished NSAID- (and ethanol)-induced gastric lesions and DNA oxidation, restoring mitochondrial complexes activity, ARG1, GST-α protein levels and increasing HMOX-1 and SOD-2 expression. AP39 decreased proteins levels or phosphorylation of gastric mucosal inflammation/oxidation-sensitive markers and restored mTOR phosphorylation. Pharmacological inhibition of HMOX-1 attenuated AP39-gastroprotection. We showed that mitochondria-targeted H2S released from very low i.g. doses of AP39 improved gastric mucosal capacity to cope with NSAIDs-induced mitochondrial dysfunction and redox imbalance, mechanistically requiring the activity of HMOX-1.

Hydrogen Sulfide-Releasing Indomethacin-Derivative (ATB-344) Prevents the Development of Oxidative Gastric Mucosal Injuries.

Hydrogen sulfide (H2S) emerged recently as an anti-oxidative signaling molecule that contributes to gastrointestinal (GI) mucosal defense and repair. Indomethacin belongs to the class of non-steroidal anti-inflammatory drugs (NSAIDs) and is used as an effective intervention in the treatment of gout- or osteoarthritis-related inflammation. However, its clinical use is strongly limited since indomethacin inhibits gastric mucosal prostaglandin (PG) biosynthesis, predisposing to or even inducing ulcerogenesis. The H2S moiety was shown to decrease the GI toxicity of some NSAIDs. However, the GI safety and anti-oxidative effect of a novel H2S-releasing indomethacin derivative (ATB-344) remain unexplored. Thus, we aimed here to compare the impact of ATB-344 and classic indomethacin on gastric mucosal integrity and their ability to counteract the development of oxidative gastric mucosal injuries. Wistar rats were pretreated intragastrically (i.g.) with vehicle, ATB-344 (7-28 mg/kg i.g.), or indomethacin (5-20 mg/kg i.g.). Next, animals were exposed to microsurgical gastric ischemia-reperfusion (I/R). Gastric damage was assessed micro- and macroscopically. The volatile H2S level was assessed in the gastric mucosa using the modified methylene blue method. Serum and gastric mucosal PGE2 and 8-hydroxyguanozine (8-OHG) concentrations were evaluated by ELISA. Molecular alterations for gastric mucosal barrier-specific targets such as cyclooxygenase-1 (COX)-1, COX-2, heme oxygenase-1 (HMOX)-1, HMOX-2, superoxide dismutase-1 (SOD)-1, SOD-2, hypoxia inducible factor (HIF)-1α, xanthine oxidase (XDH), suppressor of cytokine signaling 3 (SOCS3), CCAAT enhancer binding protein (C/EBP), annexin A1 (ANXA1), interleukin 1 beta (IL-1ß), interleukin 1 receptor type I (IL-1R1), interleukin 1 receptor type II (IL-1R2), inducible nitric oxide synthase (iNOS), tumor necrosis factor receptor 2 (TNFR2), or H2S-producing enzymes, cystathionine γ-lyase (CTH), cystathionine ß-synthase (CBS), or 3-mercaptopyruvate sulfur transferase (MPST), were assessed at the mRNA level by real-time PCR. ATB-344 (7 mg/kg i.g.) reduced the area of gastric I/R injuries in contrast to an equimolar dose of indomethacin. ATB-344 increased gastric H2S production, did not affect gastric mucosal PGE2 content, prevented RNA oxidation, and maintained or enhanced the expression of oxidation-sensitive HMOX-1 and SOD-2 in line with decreased IL-1ß and XDH. We conclude that due to the H2S-releasing ability, i.g., treatment with ATB-344 not only exerts dose-dependent GI safety but even enhances gastric mucosal barrier capacity to counteract acute oxidative injury development when applied at a low dose of 7 mg/kg, in contrast to classic indomethacin. ATB-344 (7 mg/kg) inhibited COX activity on a systemic level but did not affect cytoprotective PGE2 content in the gastric mucosa and, as a result, evoked gastroprotection against oxidative damage.

Microbiome Profile and Molecular Pathways Alterations in Gastrointestinal Tract by Hydrogen Sulfide-Releasing Nonsteroidal Anti-Inflammatory Drug (ATB-352): Insight into Possible Safer Polypharmacy.

Aims: Nonsteroidal anti-inflammatory drugs, including ketoprofen, induce adverse effects within the gastrointestinal (GI)-tract. Hydrogen sulfide (H2S) is an antioxidative gaseous mediator contributing to GI-protection. We aimed to evaluate the GI safety of a novel H2S-releasing derivative of ketoprofen (ATB-352) versus classic ketoprofen and the molecular mechanisms of their activity after chronic treatment in experimental animal models. Results: Ketoprofen (10 mg/kg/day) administered intragastrically for 7 days in contrast with ATB-352 (14 mg/kg/day) reduced mucosal H2S content inducing GI damage with significantly increased injury score, altered intestinal microbiome profile, and modulation of more than 50% of 36 investigated molecular sensors (e.g., mammalian target of rapamycin or suppressor of cytokine signaling 3 [SOCS3]). Polypharmacy with aspirin (10 mg/kg/day) enhanced ketoprofen toxicity not affecting GI safety of ATB-352. Omeprazole (20 mg/kg/day) decreased ketoprofen-induced injury to the level of ATB-352 alone. Both compounds combined or not with aspirin or omeprazole maintained the ability to inhibit cyclooxygenase (COX) activity manifested by decreased prostaglandin production. Innovation and Conclusions: Ketoprofen-induced H2S-production decrease and intestinal microbiome profile alterations lead to GI toxicity observed on macro-/microscopic and molecular levels. Ketoprofen but not ATB-352 requires concomitant treatment with omeprazole to eliminate GI adverse effects. ATB-352 applied alone or in a polypharmacy setting with aspirin effectively inhibited COX and maintained GI safety due to H2S-release. Neither compound affected DNA oxidation in the GI mucosa, but ATB-352 had lower impact on molecular oxidative/inflammatory response pathways and intestinal microbiome. The GI safety of ATB-352 could be due to the involvement of heme oxygenase 1 and SOCS3 pathway activation. Antioxid. Redox Signal. 36, 189-210.

Intestinal Alkaline Phosphatase Combined with Voluntary Physical Activity Alleviates Experimental Colitis in Obese Mice. Involvement of Oxidative Stress, Myokines, Adipokines and Proinflammatory Biomarkers.

Intestinal alkaline phosphatase (IAP) is an essential mucosal defense factor involved in the process of maintenance of gut homeostasis. We determined the effect of moderate exercise (voluntary wheel running) with or without treatment with IAP on the course of experimental murine 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis by assessing disease activity index (DAI), colonic blood flow (CBF), plasma myokine irisin levels and the colonic and adipose tissue expression of proinflammatory cytokines, markers of oxidative stress (SOD2, GPx) and adipokines in mice fed a standard diet (SD) or high-fat diet (HFD). Macroscopic and microscopic colitis in sedentary SD mice was accompanied by a significant decrease in CBF, and a significant increase in the colonic expression of tumor necrosis factor-alpha (TNF-α), IL-6, IL-1ß and leptin mRNAs and decrease in the mRNA expression of adiponectin. These effects were aggravated in sedentary HFD mice but reduced in exercising animals, potentiated by concomitant treatment with IAP, especially in obese mice. Exercising HFD mice demonstrated a substantial increase in the mRNA for adiponectin and a decrease in mRNA leptin expression in intestinal mucosa and mesenteric fat as compared to sedentary animals. The expression of SOD2 and GPx mRNAs was significantly decreased in adipose tissue in HFD mice, but these effects were reversed in exercising mice with IAP administration. Our study shows for the first time that the combination of voluntary exercise and oral IAP treatment synergistically favored healing of intestinal inflammation, strengthened the antioxidant defense and ameliorated the course of experimental colitis; thus, IAP may represent a novel adjuvant therapy to alleviate inflammatory bowel disease (IBD) in humans.

Evidence for Cytoprotective Effect of Carbon Monoxide Donor in the Development of Acute Esophagitis Leading to Acute Esophageal Epithelium Lesions.

Exposure to acidic gastric content due to malfunction of lower esophageal sphincter leads to acute reflux esophagitis (RE) leading to disruption of esophageal epithelial cells. Carbon monoxide (CO) produced by heme oxygenase (HMOX) activity or released from its donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) was reported to protect gastric mucosa against acid-dependent non-steroidal anti-inflammatory drug-induced damage. Thus, we aimed to investigate if CO affects RE-induced esophageal epithelium lesions development. RE induced in Wistar rats by the ligation of a junction between pylorus and forestomach were pretreated i.g. with vehicle CORM-2; RuCl3; zinc protoporphyrin IX, or hemin. CORM-2 was combined with NG-nitro-L-arginine (L-NNA), indomethacin, capsazepine, or capsaicin-induced sensory nerve ablation. Esophageal lesion score (ELS), esophageal blood flow (EBF), and mucus production were determined by planimetry, laser flowmetry, histology. Esophageal Nrf-2, HMOXs, COXs, NOSs, TNF-α and its receptor, IL-1 family and IL-1 receptor antagonist (RA), NF-κB, HIF-1α, annexin-A1, suppressor of cytokine signaling (SOCS3), TRPV1, c-Jun, c-Fos mRNA/protein expressions, PGE2, 8-hydroxy-deoxyguanozine (8-OHdG) and serum COHb, TGF-ß1, TGF-ß2, IL-1ß, and IL-6 content were assessed by PCR, immunoblotting, immunohistochemistry, gas chromatography, ELISA or Luminex platform. Hemin or CORM-2 alone or combined with L-NNA or indomethacin decreased ELS. Capsazepine or capsaicin-induced denervation reversed CORM-2 effects. COHb blood content, esophageal HMOX-1, Nrf-2, TRPV1 protein, annexin-A1, HIF-1α, IL-1 family, NF-κB, c-Jun, c-Fos, SOCS3 mRNA expressions, and 8-OHdG levels were elevated while PGE2 concentration was decreased after RE. CO donor-maintained elevated mucosal TRPV1 protein, HIF-1 α, annexin-A1, IL-1RA, SOCS3 mRNA expression, or TGF-ß serum content, decreasing 8-OHdG level, and particular inflammatory markers expression/concentration. CORM-2 and Nrf-2/HMOX-1/CO pathway prevent esophageal mucosa against RE-induced lesions, DNA oxidation, and inflammatory response involving HIF-1α, annexin-A1, SOCS3, IL-1RA, TGF-ß-modulated pathways. Esophagoprotective and hyperemic CO effects are in part mediated by afferent sensory neurons and TRPV1 receptors activity with questionable COX/PGE2 or NO/NOS systems involvement.

Interaction of epidermal growth factor with COX-2 products and peroxisome proliferator-activated receptor-γ system in experimental rat Barrett's esophagus.

Mixed acidic-alkaline refluxate is a major pathogenic factor in chronic esophagitis progressing to Barrett's esophagus (BE). We hypothesized that epidermal growth factor (EGF) can interact with COX-2 and peroxisome proliferator-activated receptor-γ (PPARγ) in rats surgically prepared with esophagogastroduodenal anastomosis (EGDA) with healthy or removed salivary glands to deplete salivary EGF. EGDA rats were treated with 1) vehicle, 2) EGF or PPARγ agonist pioglitazone with or without EGFR kinase inhibitor tyrphostin A46, EGF or PPARγ antagonist GW9662 respectively, 3) ranitidine or pantoprazole, and 4) the selective COX-2 inhibitor celecoxib combined with pioglitazone. At 3 mo, the esophageal damage and the esophageal blood flow (EBF) were determined, the mucosal expression of EGF, EGFR, COX-2, TNFα, and PPARγ mRNA and phospho-EGFR/EGFR protein was analyzed. All EGDA rats developed chronic esophagitis, esophageal ulcerations, and intestinal metaplasia followed by a fall in the EBF, an increase in the plasma of IL-1ß, TNFα, and mucosal PGE2 content, the overexpression of COX-2-, and EGF-EGFR mRNAs, and proteins, and these effects were aggravated by EGF and attenuated by pioglitazone. The rise in EGF and COX-2 mRNA was inhibited by pioglitazone but reversed by pioglitazone cotreated with GW9662. We conclude that 1) EGF can interact with PG/COX-2 and the PPARγ system in the mechanism of chronic esophagitis; 2) the deleterious effect of EGF involves an impairment of EBF and the overexpression of COX-2 and EGFR, and 3) agonists of PPARγ and inhibitors of EGFR may be useful in the treatment of chronic esophagitis progressing to BE.NEW & NOTEWORTHY Rats with EGDA exhibited chronic esophagitis accompanied by a fall in EBF and an increase in mucosal expression of mRNAs for EGF, COX-2, and TNFα, and these effects were exacerbated by exogenous EGF and reduced by removal of a major source of endogenous EGF with salivectomy or concurrent treatment with tyrphostin A46 or pioglitazone combined with EGF. Beneficial effects of salivectomy in an experimental model of BE were counteracted by PPARγ antagonist, whereas selective COX-2 inhibitor celecoxib synergistically with pioglitazone reduced severity of esophageal damage and protected esophageal mucosa from reflux. We propose the cross talk among EGF/EGFR, PG/COX-2, and proinflammatory cytokines with PPARγ pathway in the mechanism of pathogenesis of chronic esophagitis progressing to BE and EAC.

Oxidative gastric mucosal damage induced by ischemia/reperfusion and the mechanisms of its prevention by carbon monoxide-releasing tricarbonyldichlororuthenium (II) dimer.

Endogenous gaseous mediators, such as nitric oxide, hydrogen sulfide or carbon monoxide (CO) are known to exert anti-inflammatory and anti-oxidative activity due to modulation of various molecular pahtways. Therefore, we aimed to investigate if CO released from tricarbonyldichlororuthenium (II) dimer (CORM-2) prevents gastric mucosa against ischemia/reperfusion (I/R)-induced injury in male Wistar rats. Animals were pretreated i.g. With vehicle (DMSO and saline, 1:10), CORM-2 (1, 5 or 10 mg/kg) or zinc protoporphyrin IX (ZnPP, 10 mg/kg i.p.), the HMOXs inhibitor. In separate series, rats were pretreated with CORM-2 (5 mg/kg) applied in combination with glibenclamide (10 mg/kg i.g.), NG-nitro-l-arginine (L-NNA, 20 mg/kg i.p.), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 mg/kg i.p.) or indomethacin (5 mg/kg i.p.). I/R-injuries were induced by clamping celiac artery for 30 min (I) followed by removal of the clamp to obtain R for 3 h. The macroscopic and microscopic area of gastric damage, mucus production and protein expression for HMOX-1/Nrf-2 was determined by planimetry, histology and immunohistochemistry, respectively. Gastric mucosal HMOX-1, HMOX-2, COX-1, COX-2, Kir6.1, Sur2, sGC-α1, sGC-α2, iNOS and eNOS mRNA expression was assessed by real-time PCR. COHb in blood and gastric mucosal CO concentration was analyzed by gas chromatography. Serum content of TGF-ß1, TGF-ß2, TGF-ß3, IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α, IFN-γ, GM-CSF was evaluated using Luminex platform. PGE2 concentration and 8-hydroxyguanozine (8-OHG) concentration in gastric mucosa was determined by ELISA. Exposure to I/R induced extensive hemorrhagic erosions in gastric mucosa pretreated with vehicle as compared with intact rats and the area of this gastric damage was reduced by pretreatment with CORM-2 (5 mg/kg i.g.). This effect of CO donor was accompanied by the increased PGE2 content and a significant decrease in 8-OHG and expression of pro- and anti-inflammatory markers mRNA and proteins. Concurrent treatment of CORM-2 with glibenclamide, L-NNA, ODQ but not with indomethacin significantly increased the area of I/R-induced injury and significantly decreased GBF as compared with the group treated with CORM-2 alone. We conclude that CO releasing CORM-2 prevents gastric mucosal oxidative damage induced by I/R improving GBF, decreasing DNA oxidation and inflammatory response on systemic level. This CO-gastroprotection is mediated by the activity of sGC, NOS and K-ATP channels. CO delivered from its donor maintained physiological gastric mucosal PGE2 concentration but the involvement of endogenous COX in beneficial activity of this gaseous mediator at least in this model is questionable.

Time-dependent course of gastric ulcer healing and molecular markers profile modulated by increased gastric mucosal content of carbon monoxide released from its pharmacological donor.

BACKGROUND AND PURPOSE: Besides hydrogen sulfide (H2S) and nitric oxide (NO), carbon monoxide (CO) contributes to the maintenance of gastric mucosal integrity. We investigated increased CO bioavailability effects on time-dependent dynamics of gastric ulcer healing mediated by particular growth factors, anti-inflammatory and molecular pathways. EXPERIMENTAL APPROACH: Wistar rats with gastric ulcers induced by serosal acetic acid application (day 0) were treated i.g. throughout 3, 6 or 14 days with vehicle or CO-releasing tricarbonyldichlororuthenium (II) dimer (CORM-2, 2.5 mg/kg). Gross and microscopic alterations in gastric ulcer size and gastric blood flow (GBF) at ulcer margin were determined by planimetry, histology and laser flowmetry, respectively. Gastric mRNA/protein expressions of platelet derived growth factors (PDGFA-D), insulin-like growth factor (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGFA) and their receptors, heme oxygenases (HMOX), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), cyclooxygenase (COX-2), hypoxia inducible factor (HIF)-1α, anti-inflammatory annexin-1 and transforming growth factor (TGF-ß1) were assessed by real-time PCR or Western blot. TGF-ß1-3 and IL-10 plasma concentration were measured using Luminex platform. Prostaglandin E2 content at ulcer margin was assessed by ELISA. KEY RESULTS: CORM-2 decreased ulcer area and increased GBF after 6 and 14 days of treatment comparing to vehicle. CO donor upregulated HGF, HGFr, VEGFR1, VEGFR2, TGF-ß1, annexin-1 and maintained increased IGF-1, PDGFC and EGF expression at various time-intervals of ulcer healing. TGF-ß3 and IL-10 plasma concentration were significantly increased after COMR-2 vs. vehicle. CONCLUSIONS: CO time-dependently accelerates gastric ulcer healing and raises GBF at ulcer margin by mechanism involving subsequent upregulation of anti-inflammatory, growth promoting and angiogenic factors response, not observed physiologically.

Alterations in Gastric Mucosal Expression of Calcitonin Gene-Related Peptides, Vanilloid Receptors, and Heme Oxygenase-1 Mediate Gastroprotective Action of Carbon Monoxide against Ethanol-Induced Gastric Mucosal Lesions.

Carbon monoxide (CO) has been reported to contribute to the maintenance of gastric mucosal integrity, gastroprotection, and ulcer healing. However, involvement of transient receptor potential vanilloid receptor type 1 (TRPV1) located on afferent sensory fibers endings and sensory neuropeptide calcitonin gene-related peptide (CGRP) in CO-mediated gastroprotection against ethanol-induced gastric damage has not been explored. Male Wistar rats with and without denervation of afferent sensory neurons induced by capsaicin (total dose 125 mg/kg within 3 days) were pretreated with vehicle, CO donor tricarbonyldichlororuthenium (II) dimer (CORM-2, 5 mg/kg i.g.), administered alone or with CGRP-α (10 µg/kg i.p.) or TRPV1 antagonist capsazepine (5 mg/kg i.g.), followed 30 min later by intragastric (i.g.) administration of 75% ethanol. The area of gastric damage and gastric blood flow (GBF) were assessed planimetrically and by laser flowmetry, respectively. Microscopic evaluation of ethanol-induced gastric lesions was performed after haematoxylin/eosin (H&E) or alcian blue/periodic acid-Schiff/alcian blue (AB/PAS) staining. Gastric mucosal mRNA fold change for heme oxygenase (HMOX)-1, HMOX-2, CGRP-α, CGRP-ß, inducible nitric oxide synthase (iNOS), endothelial (e)NOS, neuronal (n)NOS, cyclooxygenase (COX)-1, COX-2, and protein expression for HMOX-1 and TRPV1 was determined by real-time PCR or Western blot, respectively. Pretreatment with CORM-2 combined or not with CGRP reduced ethanol-induced gastric lesions and elevated GBF. Capsaicin-denervation or co-treatment with capsazepine or CGRP and CORM-2 in capsaicin-denervated animals failed to affect these beneficial effects of CO donor. In rats with intact sensory nerves, CORM-2 increased gastric mRNA level for HMOX-1 and CGRP-α. In capsaicin-denervated rats, CORM-2 increased eNOS mRNA fold change and TRPV1 protein expression while capsaicin denervation itself decreased HMOX-1 protein expression and eNOS mRNA level. We conclude that CO prevents gastric mucosa from ethanol-induced lesions due to activation of TRPV1/CGRP-α system and accompanying increase in gastric microcirculation but independently on afferent sensory nerve activity despite the stimulation of TRPV1 protein and CGRP-α mRNA expression.

Treatment with Obestatin-A Ghrelin Gene-Encoded Peptide-Reduces the Severity of Experimental Colitis Evoked by Trinitrobenzene Sulfonic Acid.

Obestatin is a 23-amino acid peptide derived from proghrelin, a common prohormone for ghrelin and obestatin. Previous studies showed that obestatin exhibited some protective and therapeutic effects in the gut. The aim of our presented study was to examine the effect of treatment with obestatin on trinitrobenzene sulfonic acid (TNBS)-induced colitis. In rats anesthetized with ketamine, colitis was induced through intrarectal administration of 25 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Obestatin was administered intraperitoneally at doses of 4, 8, or 16 nmol/kg, twice per day for four consecutive days. The first dose of obestatin was given one day before the induction of colitis, and the last one was given two days after administration of TNBS. Fourteen days after the induction of colitis, rats were anesthetized again with ketamine, and the severity of colitis was determined. The administration of obestatin had no effect on the parameters tested in rats without the induction of colitis. In rats with colitis, administration of obestatin at doses of 8 or 16 nmol/kg reduced the area of colonic damage, and improved mucosal blood flow in the colon. These effects were accompanied by a reduction in the colitis-evoked increase in the level of blood leukocytes, and mucosal concentration of pro-inflammatory interleukin-1ß. Moreover, obestatin administered at doses of 8 or 16 nmol/kg reduced histological signs of colonic damage. The administration of obestatin at a dose of 4 nmol/kg failed to significantly affect the parameters tested. Overall, treatment with obestatin reduced the severity of TNBS-induced colitis in rats. This effect was associated with an improvement in mucosal blood flow in the colon, and a decrease in local and systemic inflammatory processes.

Cross-talk between hydrogen sulfide and carbon monoxide in the mechanism of experimental gastric ulcers healing, regulation of gastric blood flow and accompanying inflammation.

Hydrogen sulfide (H2S) and carbon monoxide (CO) exert gastroprotection against acute gastric lesions. We determined the cross-talk between H2S and CO in gastric ulcer healing process and regulation of gastric blood flow (GBF) at ulcer margin. Male Wistar rats with acetic acid-induced gastric ulcers were treated i.g. throughout 9 days with vehicle (control), NaHS (0.1-10 mg/kg) +/- zinc protoporphyrin (ZnPP, 10 mg/kg), d,l-propargylglycine (PAG, 30 mg/kg), CO-releasing CORM-2 (2.5 mg/kg) +/- PAG. GBF was assessed by laser flowmetry, ulcer area was determined by planimetry/histology. Gastric mucosal H2S production was analysed spectrophotometrically. Protein and/or mRNA expression at ulcer margin for vascular endothelial growth factor (VEGF)A, epidermal growth factor receptor (EGFr), cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST), heme oxygenases (HOs), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), IL-1ß, TNF-α and hypoxia inducible factor (HIF)-1α were determined by real-time PCR or western blot. IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, TNF-α, GM-CSF plasma concentration was assessed using Luminex platform. NaHS dose-dependently decreased ulcer area and increased GBF but ZnPP attenuated these effects. PAG decreased H2S production but failed to affect CORM-2-mediated ulcer healing and vasodilation. NaHS increased Nrf-2, EGFr, VEGFA and decreased pro-inflammatory markers expression and IL-1ß, IL-2, IL-13, TNF-α, GM-CSF plasma concentration. CORM-2 decreased IL-1ß and GM-CSF plasma levels. We conclude that NaHS accelerates gastric ulcer healing increasing microcirculation and Nrf-2, EGFr, VEGFA expression. H2S-mediated ulcer healing involves endogenous CO activity while CO does not require H2S. NaHS decreases systemic inflammation more effectively than CORM-2.

Carbon monoxide released from its pharmacological donor, tricarbonyldichlororuthenium (II) dimer, accelerates the healing of pre-existing gastric ulcers.

BACKGROUND AND PURPOSE: Carbon monoxide (CO), a gaseous mediator produced by haem oxygenases (HOs), has been shown to prevent stress-, ethanol-, aspirin- and alendronate-induced gastric damage; however, its role in gastric ulcer healing has not been fully elucidated. We investigated whether CO released from tricarbonyldichlororuthenium (II) dimer (CORM-2) can affect gastric ulcer healing and determined the mechanisms involved in this healing action. EXPERIMENTAL APPROACH: Gastric ulcers were induced in Wistar rats by serosal application of acetic acid. Animals received 9 days of treatment with RuCl3 [2.5 mg·kg-1 intragastrically (i.g.)], haemin (5 mg·kg-1 i.g.), CORM-2 (0.1-10 mg·kg-1 i.g.) administered alone or with zinc protoporphyrin IX (ZnPP, 10 mg·kg-1 i.g.), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 mg·kg-1 i.g.), NG -nitro-l-arginine (l-NNA, 15 mg·kg-1 i.g.), indomethacin (5 mg·kg-1 i.g.) or glibenclamide (10 mg·kg-1 i.g.). Gastric ulcer area and gastric blood flow (GBF) were assessed planimetrically, microscopically and by laser flowmeter respectively. Gastric mRNA/protein expressions of EGF, EGF receptors, VEGFA, HOs, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), COX-2, hypoxia-inducible factor (HIF)-1α and pro-inflammatory iNOS, IL-1ß and TNF-α were determined by real-time PCR or Western blots. KEY RESULTS: CORM-2 and haemin but not RuCl3 or ZnPP decreased ulcer size while increasing GBF. These effects were reduced by ODQ, indomethacin, l-NNA and glibenclamide. CORM-2 significantly decreased the expression of pro-inflammatory markers, Nrf2/HO1 and HIF-1α, and up-regulated EGF. CONCLUSIONS AND IMPLICATIONS: CO released from CORM-2 or endogenously produced by the HO1/Nrf2 pathway accelerates gastric ulcer healing via an increase in GBF, an up-regulation in EGF expression and down-regulation of the inflammatory response.

Beneficial Effect of Voluntary Exercise on Experimental Colitis in Mice Fed a High-Fat Diet: The Role of Irisin, Adiponectin and Proinflammatory Biomarkers.

Inflammatory bowel diseases (IBDs) are a heterogeneous group of disorders exhibited by two major phenotypic forms: Crohn's disease and ulcerative colitis. Although the aetiology of IBD is unknown, several factors coming from the adipose tissue and skeletal muscles, such as cytokines, adipokines and myokines, were suggested in the pathogenesis of ulcerative colitis; however, it has not been extensively studied whether voluntary exercise can ameliorate that disorder. We explored the effect of moderate exercise (i.e., voluntary wheel running) on the disease activity index (DAI), colonic blood flow (CBF), plasma irisin and adiponectin levels and real-time PCR expression of proinflammatory markers in mesenteric fat in mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis fed a high-fat diet (HFD) compared to those on a standard chow diet (SD). Macroscopic and microscopic colitis in sedentary SD mice was accompanied by a significant fall in CBF, some increase in colonic tissue weight and a significant increase in the plasma levels of tumour necrosis factor-alpha (TNF-α), IL-6, monocyte chemotactic protein 1 (MCP-1) and IL-13 (p < 0.05). In sedentary HFD mice, colonic lesions were aggravated, colonic tissue weight increased and the plasma TNF-α, IL-6, MCP-1, IL-1ß and leptin levels significantly increased. Simultaneously, a significant decrease in the plasma irisin and adiponectin levels was observed in comparison with SD mice (p < 0.05). Exercise significantly decreased macroscopic and microscopic colitis, substantially increased CBF and attenuated the plasma TNF-α, IL-6, MCP-1, IL-1ß and leptin levels while raising the plasma irisin and the plasma and WAT concentrations of adiponectin in HFD mice (p < 0.05). We conclude that: (1) experimental colitis is exacerbated in HFD mice, possibly due to a fall in colonic microcirculation and an increase in the plasma and mesenteric fat content of proinflammatory biomarkers; and (2) voluntary physical activity can attenuate the severity of colonic damage in mice fed a HFD through the release of protective irisin and restoration of plasma adiponectin.

The kinetics of haemolysis of spherocytic erythrocytes.

Spherocytosis is a hereditary disease. It results from mutations in genes that encode proteins participating in the attachment of the membrane skeleton to the plasma membrane bilayer of the erythrocyte. In affected cells, interaction between the spectrin-actin meshwork and integral membrane proteins is altered. This results in the weakening of plasma membrane mechanical resistance and diminishing its elasticity. Since defective cells are prone to mechanical destruction and phagocytosis in the spleen, the fraction of morphologically-altered erythrocytes is rather small; this in turn means such an examination is prone to errors. In this paper, we describe a simple method which could be useful in the identification of red blood cells with altered osmotic properties. The method is based on the measurement of the amount of light scattered by a suspension of the red blood cells, during which cells are exposed to osmotic stress in the stopped-flow regime. The obtained plots are fitted to a mathematical formula, the parameters of which can be used as quantitative indicators of the changes in red blood cells' osmotic features. Two types of spherocytotic samples were examined: those with a proven deficiency in ankyrin and those with a decrease in the band 3 anion transporting protein. The presented data show that this method gives a reliable indication of altered osmotic properties of the spherocytic cells.