Inhibition of endogenous nitric oxide activity impairs the colonic sparing effect of rofecoxib, the cyclooxygenase-2 inhibitor and resveratrol, the preferential cyclooxygenase-1 inhibitor in the course of experimental colitis. Role of oxidative stress biomarkers and proinflammatory cytokines.

The gut mucosal barrier plays a key role in the physiology of gastrointestinal (GI) tract, preventing under homeostatic conditions, the epithelial cells of the gastric mucosa from hydrochloric acid and intestinal mucosa from alkaline secretion, food toxins and pathogenic microbiota. Previous studies have documented that blockade of both isoforms of cyclooxygenase (COX): constitutive (COX-1) and inducible (COX-2), as well NO synthase in the stomach exacerbated the gastric damage induced by various ulcerogens, however, such as effects of non-selective and selective inhibition of COX-1, COX-2 and NOS enzymes on colonic damage have been little studied. The supplementation of NO by intragastric (i.g.) treatment with NO-releasing compound NO-aspirin (NO-ASA) or substrate for NO synthase L-arginine ameliorated the damage of upper GI-tract, but whether similar effect can be observed in colonic mucosa associated with the experimental colitis, and if above mentioned compounds can be effective in aggravation or protection of experimental colitis remains less recognized. In this study rats with experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzosulphonic acid (TNBS) were daily treated for 7 days with: 1) vehicle (i.g.), 2) ASA 40 mg/kg (i.g.), 3) rofecoxib 10 mg/kg (i.g.), 4) resveratrol 10 mg/kg (i.g.), 5) NO-ASA 40 mg/kg (i.g.), 6) L-arginine 200 mg/kg (i.g.) with or without of L-NNA 20 mg/kg (i.p.). The macroscopic and microscopic area of colonic damage was determined planimetrically, the colonic blood flow (CBF) was assessed by Laser flowmetry, and the oxidative stress biomarkers malondialdehyde and 4-hydroxynonenal (MDA+4-HNE), the antioxidative factors superoxide dismutase (SOD) and glutathione (GSH), as well as proinflammatory cytokines in the colonic mucosa (tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1ß)) were measured. We have documented that administration of TNBS produced gross and microscopic colonic damage and significantly decreased CBF (p<0.05). Treatment with ASA significantly increased the area of colonic damage (p<0.05), an effect accompanied by a significant decrease in the CBF, the significant increment of MDA+4-HNE, and the attenuation of the antioxidative properties in colonic mucosa, documented by a significant decrease of SOD activity and GSH concentration, and elevation of the colonic tissue levels of TNF-α and IL-1ß comparing to control Veh-treated TNBS rats. Administration of rofecoxib or resveratrol also significantly increased the colonic damage and significantly decreased the CBF, causing an increase in MDA+4-HNE and mucosal content of TNF-α and IL-1α and a significant decrease of the SOD activity and GSH content (p<0.05), however, these changes were significantly less pronounced as compared with ASA. On the contrary, the treatment with NO-ASA, or L-arginine, significantly diminished the area of colonic lesions, the MDA+4-HNE concentration, attenuated the TNF-α and IL-1ß levels, while increasing the CBF, SOD activity and GSH content (p<0.05). The concomitant treatment of L-NNA with rofecoxib or resveratrol reversed an increase in area of colonic damage and accompanying changes in CBF, colonic mucosa TNF-α and IL-1ß levels, the MDA+4-HNE concentration, and SOD activity and GSH content comparing to those observed in TNBS rats treated with these COX-inhibitors alone (p<0.05). In contrast, co-treatment with L-NNA and NO-ASA or L-arginine failed to significantly affect the decrease of colonic lesions accompanied by the rise in CBF, the attenuation of MDA+4-HNE concentration, TNF-α and IL-1ß levels, SOD activity and GSH content exerted by NO-ASA- or L-arginine treatment of the respective control TNBS-rats without L-NNA administration. These observations suggest that 1) the increase of NO availability either from NO-releasing donors such as NO-ASA or NO precursors such as L-arginine, can inhibit the inflammatory and microvasculature alterations, as well as increase in lipid peroxidation due to the enhanced efficacy of these compounds to increase the antioxidative properties of colonic mucosa, 2) unlike ASA which exacerbated the severity of colitis, the treatment with rofecoxib, the specific 'safer' COX-2 inhibitor or resveratrol, the polyphenolic compound known to act as the dual COX-1 and COX-2 inhibitor, can attenuate the colonic damage during course of TNBS colitis possibly via anti-inflammatory and antioxidative properties, and 3) the blockade of endogenous NO activity by L-NNA which also exacerbated the severity of mucosal damage in colitis, can abolish the sparing effect of rofecoxib and resveratrol indicating the NO bioavailability plays an important role in enhanced efficacy of both specific and dual COX inhibitors to ameliorate the experimental colitis.

The effect of hydrogen sulfide-releasing naproxen (ATB-346) versus naproxen on formation of stress-induced gastric lesions, the regulation of systemic inflammation, hypoxia and alterations in gastric microcirculation.

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin or naproxen is limited due to the gastrotoxicity evoked by these compounds. Endogenous hydrogen sulfide (H2s) and delivered via an H2s donor have been shown to play important role in the maintenance of gastric mucosal integrity. This study aimed to compare the effects of naproxen and an H2s-releasing naproxen derivative (ATB-346) on gastric lesion induction by water immersion and restraint stress (WRS), the alterations in gastric blood flow (GBF) and the influence of these drugs on systemic inflammation. Wistar rats were pretreated i.g. with vehicle, naproxen (20 mg/kg) or ATB-346 (equimolar dose) or NaHS (5 mg/kg), the H2s donor, combined with naproxen and exposed to 3.5 hours of WRS. The gastric lesion number and GBF were assessed by planimetry and laser Doppler flowmetry, respectively. Plasma concentrations of interleukins: IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and GM-CSF were determined by Luminex system and gastric mucosal protein expression of cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), hypoxia inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1) and cyclooxygenase (COX-2) were analyzed by Western blot. Pretreatment with naproxen increased the number of WRS stress-induced gastric lesions and significantly decreased GBF as compared with vehicle (p < 0.05). In contrast, pretreatment with ATB-346 or naproxen combined with NaHS significantly reduced WRS-lesions number and elevated GBF as compared with naproxen (p < 0.05). Naproxen significantly increased gastric mucosal protein expression of CSE, Nrf-2 and HIF-1α as compared with vehicle (p < 0.05), but failed to affect CBS, 3-MST and HO-1. ATB-346 significantly increased Nrf-2 and HO-1 protein expression as compared with vehicle (P < 0.05) but did not affect the protein expression of CSE, CBS, 3-MST or HIF-1α. ATB-346 but not naproxen decreased COX-2 protein expression in gastric mucosa compromised by WRS (p < 0.05). Exposure to WRS increased plasma concentration of all investigated cytokines (p < 0.05). ATB-346 but not naproxen decreased plasma content of IL-1α, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α and IFN-γ in rats exposed to WRS (p < 0.05). We conclude that H2s through its vasoactive properties attenuates the gastrotoxic effects of naproxen, which increased stress-induced hypoxia in gastric mucosa. In contrast to naproxen, ATB-346 decreased stress-induced systemic inflammation and pro-inflammatory COX-2 expression in the gastric mucosa. The decreased gastrotoxicity of ATB-346 could be due to upregulation of Nrf-2/HO-1 pathway mediated by the release of H2s.

Role of sensory afferent nerves, lipid peroxidation and antioxidative enzymes in the carbon monoxide-induced gastroprotection against stress ulcerogenesis.

Carbon monoxide (CO) is a physiological gaseous mediator recently implicated in the mechanism of gastric mucosal defense due to its vasodilatory and antioxidative properties. Small quantities of endogenous CO are produced during heme degradation by heme oxygenase (HO-1), however, the involvement of the capsaicin-sensitive afferent neurons releasing calcitonin gene related peptide (CGRP) and anti-oxidative factors and mechanisms in the CO-induced gastroprotection against stress ulcerogenesis has been little studied. We investigated the possible role of CO released from the CO donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) in the protection against water immersion and restraint stress (WRS)-induced lesions in rats with intact sensory nerves and those with capsaicin denervation and the accompanying changes in malondialdehyde (MDA) content considered as an index of lipid peroxidation, the activity of GSH and SOD-2 and gastric mucosal expression of antioxidative enzymes glutathione peroxidase (GPx) and SOD-2. Wistar rats with intact sensory nerves or those with capsaicin administered in total dose of 125 mg/kg s.c. within 3 days (capsaicin denervation) were pretreated either with 1) vehicle (saline) or 2) CORM-2 (0.1 - 0 mg/kg i.g.) with or without exogenous CGRP (10 µg/kg i.p.) and 30 min later exposed to 3.5 h of WRS. At the termination of WRS, the number of gastric lesions was counted and gastric blood flow (GBF) was assessed by H2-gas clearance technique. The mucosal content of MDA and reduced glutathione (GSH) and the activity of SOD-2 were determined and the expression of GPx-1 and SOD-2 mRNA in the gastric mucosa was analyzed by real-time PCR. The exposure of rats to 3.5 h of WRS resulted in numerous hemorrhagic gastric lesions and significantly decreased the GBF, raised MDA content and significantly decreased the mucosal SOD and GSH contents compared with intact gastric mucosa and these changes were exacerbated in rats with capsaicin denervation. Pretreatment with CORM-2 (1 mg/kg i.g.) which in our previous studies significantly reduced the ethanol and aspirin-induced gastric damage, significantly decreased the number of WRS-induced gastric lesions while raising the GBF and significantly increasing the activity of SOD and GSH (P < 0.05). The pretreatment with CORM-2 significantly decreased MDA content as compared with vehicle-pretreated rats exposed to WRS (P < 0.05). The reduction of WRS damage and the accompanying increase in the GBF as well as the significant decrease in MDA content and the increase in GSH content and SOD activity induced by CORM-2 (1 µg/kg i.g.) were all significantly altered in rats with capsaicin denervation (P < 0.05). The concurrent treatment of CORM-2 with exogenous CGRP in rats with or without sensory nerves tended to decrease the number of WRS lesions as compared with CORM-2 alone pretreated animals and significantly increased the GBF over the values measured in gastric mucosa of CORM-2 alone pretreated rats with or without capsaicin denervation. Such combined administration of CORM-2 and CGRP in rats with capsaicin denervation significantly inhibited an increase in MDA and 4-HNE content and evoked a significant increase in the GSH concentration (P < 0.05) remaining without significant effect on the increase in SOD activity observed with CORM-2 alone. The gastric mucosal expression of SOD-2- and GPx-1 mRNA was significantly increased as compared with those in intact gastric mucosa (P < 0.05). The pretreatment with CORM-2 applied with or without CGRP failed to significantly alter the mRNA expression for SOD-2 and GPx in the gastric mucosa of rats exposed to WRS. Both, the expression of SOD-2- and GPx-1 mRNA was significantly increased in capsaicin denervated rats exposed to WRS rats (P < 0.05) and this effect was abolished by the pretreatment with CORM-2. The expression of SOD-2 tended to decrease, though insignificantly, in rats pretreated with the combination of CORM-2 and CGRP as compared with that detected in CORM-2 alone in rats with capsaicin denervation. In contrast, the mRNA expression of GPx-1 was significantly decreased in gastric mucosa of capsaicin-denervated rats treated with the combination of CORM-2 and CGRP as compared with CORM-2 alone pretreated animals. We conclude that 1) CORM-2 releasing CO exerts gastroprotective activity against stress ulcerogenesis and this effect depends upon an increase in the gastric microcirculation and the vasodilatory activity of this gaseous mediator, and 2) the sensory nerve endings releasing CGRP can contribute, at least in part, to the CO-induced gastric hyperemia, the attenuation of gastric mucosal lipid peroxidation and prevention of oxidative stress as indicated by the CORM-2-induced normalization of the antioxidative enzyme expression enhanced in gastric mucosa of capsaicin-denervated rats.

Role of curcumin in protection of gastric mucosa against stress-induced gastric mucosal damage. Involvement of hypoacidity, vasoactive mediators and sensory neuropeptides.

The antioxidizing properties of curcumin, a highly pleiotropic substance used for centuries in traditional medicine has been confirmed by numerous experimental and clinical studies. Curcumin exhibits anti-inflammatory, antiproliferative and anti-angiogenic actions inhibiting the development and progression of tumors but the efficacy of this compound to influence gastric acid secretion n in the stomach and to affect the gastric mucosal damage induced by non-topical ulcerogenes such as stress has been little studied. We determined the effect of curcumin on basal and pentagastrin- or histamine-stimulated gastric secretion, in rats with surgically implemented gastric fistulas and we assessed the contribution of gastric secretion, endogenous prostaglandin (PG), endogenous nitric oxide (NO), as well as sensory afferent nerves in the mechanisms underlying the potential gastroprotective effects of curcumin against stress-induced gastric mucosal lesions. Rats exposed to water immersion and restraint stress (WRS) for 3.5 h were pretreated either with: 1) vehicle (saline); 2) curcumin (2.5 - 100 mg/kg i.g.) or 3) curcumin (50 mg/kg i.g.) combined with or without indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg i.g.) or rofecoxib (10 mg/kg i.g.); 4) curcumin (50 mg/kg i.g.) co-administered with (L-NNA (20 mg/kg i.p.) with or without L-arginine (200 mg/kg i.g.), a substrate for NO-synthase; 5) curcumin (50 mg/kg i.g.) administered in rats with intact or capsaicin-induced functional ablation of sensory nerve fibers, and 6) curcumin (50 mg/kg i.g.) administered with capsazepine (5 mg/kg i.g.), the antagonist of vanilloid TRPV1 receptor. The number of gastric lesions was determined by planimetry, the gastric blood flow (GBF) was assessed by H2-gas clearance technique, the plasma gastrin concentrations were measured using the radioimmunoassay (RIA) and the expression of mRNA for tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in gastric mucosa was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Curcumin dose-dependently reduced the WRS-induced gastric lesions, the dose inhibiting these lesions by 50% being about 50 mg/kg. These effects of curcumin were accompanied by an increase in GBF and the reduction in basal and histamine- or pentagastrin-stimulated gastric acid secretion. The protective and hyperemic activities of curcumin (50 mg/kg i.g.) against WRS lesions were significantly attenuated (P < 0.05) in rats pretreated with rofecoxib and SC-560 and completely reversed (P < 0.01) by indomethacin. L-NNA significantly reduced (P < 0.05) the decrease in WRS-induced lesions and the accompanying rise in GBF caused by curcumin and these effects were restored by concurrent treatment with L-arginine (200 mg/kg i.g.). The curcumin-induced decrease in the number of WRS-induced gastric lesions and accompanying increase in the GBF were significantly attenuated (P < 0.05) in capsaicin-denervated rats and in those pretreated with capsazepine. These effects of curcumin in rats with capsaicin denervation were restored by concomitant treatment with exogenous calcitonin gene related pepetide (CGRP) combined with curcumin and subsequently exposed to WRS. The expression of mRNA for TNF-α, COX-2 and iNOS was significantly increased (P < 0.05) in vehicle-pretreated control rats exposed to WRS and significantly attenuated (P < 0.05) by curcumin administered in graded dosages. We conclude that curcumin exerts gastroprotective and hyperemic activities against experimental stress-induced gastric lesions by mechanism involving endogenous prostaglandins, NO, the neuropeptides such as CGRP released from capsaicin-sensitive afferent nerves and the activation of vanilloid TRPV1 receptors located on these sensory nerve terminals.

Lipid peroxidation, reactive oxygen species and antioxidative factors in the pathogenesis of gastric mucosal lesions and mechanism of protection against oxidative stress - induced gastric injury.

The gastric mucosa plays an important role in the physiological function of the stomach. This mucosa acts as gastric barrier, which protects deeper located cells against the detrimental action of the gastric secretory components, such as acid and pepsin. Integrity of the gastric mucosa depends upon a variety of factors, such as maintenance of microcirculation, mucus-alkaline secretion and activity of the antioxidizing factors. The pathogenesis of gastric mucosal damage includes reactive oxygen species (ROS), because of their high chemical reactivity, due to the presence of uncoupled electron within their molecules. Therefore they cause tissue damage, mainly due to enhanced lipid peroxidation. Lipid peroxides are metabolized to malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). The local increase of MDA and 4-HNE concentration indicates ROS-dependent tissue damage. Superoxide dismutase (SOD) is the main enzyme, which neutralizes ROS into less noxious hydrogen peroxide. A decrease of SOD activity is an indicator of impairment of the protective mechanisms and significantly contributes to cell damage. Hydrogen peroxide is further metabolized to water in the presence of reduced glutathione (GSH). GSH can also work synergetically with SOD to neutralize ROS. The reactions between GSH and ROS yields glutathione free radical (GS(•)), which further reacts with GSH leading to free radical of glutathione disulphide (GSSG(•)). This free radical of GSSG can then donate an electron to the oxygen molecule, producing O2 (•-) Subsequently, O2 (•-) is eliminated by SOD. Adecrease of the GSH level has detrimental consequences for antioxidative defense cellular properties. Gastric mucosa, exposed to stress conditions, exhibits an enhancement of lipid peroxidation (increase of MDA and 4-HNE), as well as a decrease of SOD activity and GSH concentration. This chain reaction of ROS formation triggered by stress, appears to be an essential mechanism for understanding the pathogenesis of stress - induced functional disturbances in the gastric mucosa leading to ulcerogenesis.

Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, interacts with gastric oxidative metabolism and enhances stress-induced gastric lesions.

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase known to exert vasoconstriction of vascular bed. The elevation of ADMA has been considered as the cardiovascular risk factor associated with hyperlipidemia, hypercholesterolemia and metabolic syndrome. ADMA is produced by the action of dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to L-citrulline and dimethylamine. Previous studies have shown that endogenous NO plays an important role in the mechanism of gastric mucosal defense, but the role of ADMA in the pathogenesis of serious clinical entity, such as the acute gastric mucosal injury induced by stress has been little studied. In present study, we determined the effect of intragastric (i.g.) pretreatment with ADMA applied in graded doses ranging from 0.1 up to 20 mg/kg on gastric mucosal lesions induced by 3.5 h of water immersion and restraint stress (WRS). The number of gastric lesions was determined by planimetry and the gastric blood flow (GBF) was assessed by laser Doppler technique. The malondialdehyde and 4-hydroxynonenal (MDA+4-HNE) concentration, as an index of oxygen radical-lipid peroxidation was assessed in the gastric mucosa in rats exposed to WRS with or without ADMA administration. Proinflammatory cytokines IL-1ß, TNF-α, superoxide dismutase (SOD) and glutathione peroxidase (GPx) mRNAs in the gastric mucosa and plasma levels of ADMA, IL-1ß and TNF-α were analyzed by RT-PCR and ELISA, respectively. The exposure of rats to WRS for 3.5 h produced acute gastric lesions accompanied by a significant rise in the plasma ADMA levels and a significant fall in the GBF, an increase in MDA+4-HNE concentrations and the significant increase in the expression and release of IL-1ß and TNF-α. The pretreatment with ADMA, applied i.g. 30 min before WRS dose-dependently, aggravated WRS damage and this effect was accompanied by a further significant fall in the GBF. The ADMA induced exacerbation of WRS lesions and the accompanying rise in the plasma ADMA levels and the fall in GBF were significantly attenuated by concurrent treatment with glyceryl trinitrate (GTN) (10 mg/kg i.g.) in the presence of ADMA. Administration of ADMA resulted in a significant decrease in the expression of SOD and GPx mRNAs and the up-regulation of mRNA for IL-1ß and TNF-α followed by an increase in these plasma cytokine levels as compared to respective values observed in vehicle-pretreated animals. We conclude that 1) ADMA could be implicated in the mechanism of WRS-induced ulcerogenesis, 2) ADMA exacerbates WRS-induced gastric lesions due to enhancement in neutrophil dependent lipid peroxidation and overexpression and release of proinflammatory cytokines IL-1ß and TNF-α and a potent depletion of antioxidative enzymes SOD and GPx expression and activity.

Interaction between selective cyclooxygenase inhibitors and capsaicin-sensitive afferent sensory nerves in pathogenesis of stress-induced gastric lesions. Role of oxidative stress.

Gastric microcirculation plays an important role in the maintenance of the mucosal gastric integrity and the mechanism of injury as well as providing protection to the gastric mucosa. Disturbances in the blood perfusion, through the microcapillaries within the gastric mucosa may result in the formation of mucosal damage. Acute gastric mucosal lesions constitute an important clinical problem. Originally, one of the essential component of maintaining the gastric mucosal integrity was the biosynthesis of prostaglandins (PGs), an issue that has captured the attention of numerous investigations. PGs form due to the activity of cyclooxygenase (COX), an enzyme which is divided into 2 isoforms: constitutive (COX-1) and inducible (COX-2) ones. The inhibition of COX-1 by SC-560, or COX-2 by rofecoxib, reduces gastric blood flow (GBF) and impairs gastric mucosal integrity. Another detrimental effect on the gastric mucosal barrier results from the ablation of sensory afferent nerves by neurotoxic doses of capsaicin. Functional ablation of the sensory afferent nerves by capsaicin attenuates GBF and also renders the gastric mucosa more susceptible to gastric mucosal damage induced by ethanol, aspirin and stress. However, the role of reactive oxygen species (ROS) in the interaction between COX specific inhibitors and afferent sensory nerves has not been extensively studied. The aim of our present study was to determine the participation of ROS in pathogenesis of stress-induced gastric lesions in rats administered with SC-560 or rofecoxib, with or without ablation of the sensory afferent nerves. ROS were estimated by measuring the gastric mucosal tissue level of MDA and 4-HNE, the products of lipid peroxidation by ROS as well as the SOD activity and reduced glutathione (GSH) levels, both considered to be scavengers of ROS. It was demonstrated that exposure to 3.5 h of WRS resulted in gastric lesions, causing a significant increase of MDA and 4-HNE in the gastric mucosa, accompanied by a decrease of SOD activity and mucosal GSH level. Pretreatment with COX-1 and COX-2 inhibitors (SC-560 and rofecoxib, respectively) aggravated the number of gastric lesions, decreased GBF, attenuated GSH level without further significant changes in MDA and 4-HNE tissue levels and SOD activity. Furthermore, the capsaicin--nactivation of sensory nerves resulted in exaggeration of gastric mucosal damage induced by WRS and this was further augmented by rofecoxib. We conclude that oxidative stress, as reflected by an increase of MDA and 4-HNE tissue concentrations (an index of lipid peroxidation), as well as decrease of SOD activity and the fall in GSH tissue level, may play an important role in the mechanism of interaction between the inhibition of COX activity and afferent sensory nerves releasing vasoactive neuropeptides. This is supported by the fact that the addition of specific COX-1 or COX-2 inhibitors to animals with capsaicin denervation led to exacerbation of gastric lesions, and further fall in the antioxidizing status of gastric mucosa exposed to stress.