Expression of HSP72 in the gastric mucosa is regulated by gastric acid in rats-correlation of HSP72 expression with mucosal protection

BACKGROUND AND AIM: The real mechanism of adaptive cytoprotection in the gastric mucosa is not well established. In the present study, we investigated the effect of acid suppressing agents on a 72-kDa heat shock protein (HSP72) expression, which is known as endogenous cytoprotective factor, in the gastric mucosa. Also, the association of gastric mucosal protective function against HCl-challenge was compared between HSP72-induced and -reduced group. MATERIALS AND METHODS: Expression of HSP72 was measured by Western blotting in the gastric mucosa before and after administration of famotidine or omeprazole. The gastric mucosal protective function against 0.6 N HCl was compared between control group and HSP72-reduced group. Also, the effect of increased expression of gastric HSP72 by additional administration of zinc sulfate or zinc L-carnosine, which is known as HSP72-inducer, on mucosal protective function was studied. RESULTS: HSP72 expression in the gastric mucosa was reduced by acid suppressing agents. The lowest expression level of HSP72 was observed 12 h (famotidine, H2-receptor antagonist) or 48 h (omeprazole, proton pump inhibitor) after administration. The gastric mucosal protective ability against 0.6 N HCl was also reduced when HSP72 expression was decreased by famotidine or omeprazole. This phenomenon was reversed by HSP72 induction by additional administration of zinc derivatives. CONCLUSION: Our results might indicate that the expression of HSP72 in the gastric mucosa is physiologically regulated by gastric acid, and that HSP72 induction could be important in view of mucosal protection especially when HSP72 expression is reduced by administration of acid suppressing agents such as proton pump inhibitor or H2 receptor antagonist.     

Specific induction of a 72-kDa heat shock protein protects esophageal mucosa from reflux esophagitis

AimsThe aim of this study is to investigate the expression and cytoprotective function of a 72-kDa heat shock protein (HSP72) using a reflux esophagitis model in rats.Main methodsExpression of HSP60, HSP72, and HSP90 in rat esophageal mucosa was evaluated by Western blot analysis before and after hyperthermia (42.5 °C, 20 min). Rats received the operation to produce reflux esophagitis with or without pretreatment with hyperthermia to induce HSPs. The esophageal mucosal damage was evaluated 12 h after the operation.Key findingsExpression of HSP72 was significantly increased by hyperthermia in rat esophageal mucosa. Reflux esophagitis was dramatically prevented when HSP72 was preinduced by hyperthermia. Furthermore, activation of TNF-α and IL-1β in esophageal mucosa was also suppressed.SignificanceThese results suggested that hyperthermia protects the esophageal mucosa in reflux esophagitis model by inducing HSP72 and suppressing proinflammatory cytokine activation. These findings might suggest that HSP-inducing therapy could be a novel and unique therapy for reflux esophagitis.     

Is Mongolian gerbil really adequate host animal for study of Helicobacter pylori infection-induced gastritis and cancer?

BACKGROUND: Many researches have been published to understand the pathogenesis and mechanism of Helicobacter pylori (Hp)-associated diseases, including gastritis followed by gastric cancer, using Mongolian gerbil (MG) model because Hp could be hardly inoculated in other animal species. The aim of this study was to evaluate the induction ability of heat shock protein (HSP70) and protective ability in the gastric mucosa of MG comparing with those of Sprague-Dawley (SD) rats, since HSP70 is a key molecule known to be involved in important biological activities such as apoptosis, carcinogenesis, and cytoprotection from cytotoxic damage. MATERIALS AND METHODS: Basal expression level and induction ability of gastric mucosal HSP70 were evaluated by immunoblotting and densitometric analysis in MG and SD rats before and after HSP-induction by zinc l-carnosine, gastric HSP70 inducer, administration. Mucosal protective ability against water-immersion stress-induced mucosal lesion was also compared. RESULTS: Basal expression level of HSP70 was not significantly different between MG and SD rats. However, HSP70-induction by zinc derivatives was not observed in MG. Mucosal lesion induced by water-immersion stress was significantly severe in MG compared with SD rats. CONCLUSIONS: MG might be special (not ordinary) animal, in which HSP70-induction was absent and has extremely poor mucosal protective ability in view of HSP-dependent cytoprotection in the gastric mucosa. Our results may suggest that MG is not an adequate animal to evaluate the effect of Hp-infection-associated gastric inflammation followed by development of gastric cancer.     

Kainic acid-induced apoptosis in rat striatum is associated with nuclear factor-kappaB activation

The present study evaluated whether nuclear factor-kappaB (NF-kappaB) activation contributes to the apoptotic-like death of striatal neurons induced by kainic acid (KA) receptor stimulation. Intrastriatally infused KA (1.25-5.0 nmol) produced substantial neuronal loss as indicated by an 8-73% decrease in 67-kDa glutamic acid decarboxylase (p<0.05). KA (1.25-5.0 nmol) elicited internucleosomal DNA fragmentation that was inhibited by the AMPA/KA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dibenzo[f]quinoxaline-7-sulfonamide) but not by the NMDA receptor antagonist MK-801. A decrease in IkappaB-alpha protein levels, which was accompanied by an increase in NF-kappaB binding activity, was found from 6 to 72 h after KA (2.5 nmol) infusion. NF-kappaB was composed mainly of p65 and c-Rel as revealed by supershift assay. In addition, c-Myc and p53 increased from five- to sevenfold from 24 to 72 h after KA (2.5 nmol) administration. Immunohistochemistry revealed high levels of c-Myc and p53 immunoreactivity, mainly in medium-sized striatal neurons. Pretreatment with the cell-permeable recombinant peptide NF-kappaB SN50 (5-20 microg) blocked NF-kappaB nuclear translocation, but had no effect on AP-1 binding. NF-kappaB SN50 also inhibited the KA-induced up-regulation of c-Myc and p53, as well as internucleosomal DNA fragmentation. The apoptotic-like destruction of rat striatal neurons induced by KA receptor stimulation thus appears to involve biochemical mechanisms similar to those mediating the excitotoxic response to NMDA receptor stimulation. The present results provide additional support for the view that NF-kappaB activation contributes to c-Myc and p53 induction and subsequent apoptosis in an excitotoxic model of Huntington's disease.     

Effects of HSP70.1/3 gene knockout on acute respiratory distress syndrome and the inflammatory response following sepsis

Heat shock response has been implicated in attenuating NF-kappaB activation and inflammation following sepsis. Studies utilizing sublethal heat stress or chemical enhancers to induce in vivo HSP70 expression have demonstrated survival benefit after experimental sepsis. However, it is likely these methods of manipulating HSP70 expression have effects on other stress proteins. The aim of this study was to evaluate the role of specific deletion of HSP70.1/3 gene expression on ARDS, NF-kappaB activation, inflammatory cytokine expression, and survival following sepsis. To address this question, we induced sepsis in HSP70.1/3 KO and HSP70.1/3 WT mice via cecal ligation and puncture (CLP). We evaluated lung tissue NF-kappaB activation and TNF-alpha protein expression at 1 and 2 h, IL-6 protein expression at 1, 2, and 6, and lung histopathology 24 h after sepsis initiation. Survival was assessed for 5 days post-CLP. NF-kappaB activation in lung tissue was increased in HSP70.1/3((-/-)) mice at all time points after sepsis initiation. Deletion of HSP70.1/3 prolonged NF-kappaB binding/activation in lung tissue. Peak expression of lung TNF-alpha at 1 and 2 h was also significantly increased in HSP70.1/3((-/-)) mice. Expression of IL-6 was significantly increased at 2 and 6 h, and histopathology revealed a significant increase in lung injury in HSP70.1/3((-/-)) mice. Last, deletion of the HSP70 gene led to increased mortality 5 days after sepsis initiation. These data reveal that absence of HSP70 alone can significantly increase ARDS, activation of NF-kappaB, and inflammatory cytokine response. The specific absence of HSP70 gene expression also leads to increased mortality after septic insult.     

TNF-alpha is crucial for the development of mast cell-dependent colitis in mice

Inflammatory bowel disease (IBD) describes chronic inflammatory conditions of the gastrointestinal tract, and TNF-alpha plays a pivotal role in mediating the response. The proinflammatory cytokine TNF-alpha is rapidly released by mast cells after degranulation. In the present study, we hypothesized TNF-alpha to be an important player in our recently described mast cell-dependent murine model for IBD. The effect of neutralizing anti-TNF-alpha MAb was studied on colonic hypersensitivity in mice induced by a skin application of dinitrofluorobenzene (DNFB) followed by an intrarectal challenge with dinitrobenzene sulfonic acid. Features of the colonic hypersensitivity response included diarrhea, mast cell infiltration and activation, infiltration of inflammatory cells in the colon, colonic patch hypertrophy, and increased mast cell-derived TNF-alpha levels in the colon. Anti-TNF-alpha MAb could effectively abrogate diarrhea in DNFB-sensitized mice 72 h after the challenge. The numbers of colonic patches and total tissue damage scores were reduced by anti-TNF-alpha MAb treatment in DNFB-sensitized mice 72 h after the challenge. Mast cell infiltration and activation remained unaffected by neutralizing anti-TNF-alpha MAb. Treatment with the corticosteroid dexamethasone, a frequently used therapeutic treatment in IBD, resulted in a reduction of diarrhea, cellular infiltration, and total tissue damage scores to the same extent as anti-TNF-alpha MAb. Additionally, dexamethasone treatment could also reduce total TNF-alpha levels in the colon, mast cell numbers, and mast cell activation in both vehicle- and DNFB-sensitized mice 72 h after the challenge. These findings suggest that TNF-alpha can play an instrumental role in causing inflammatory responses in the present murine model for IBD downstream from mast cell activation.     

Effects of pleuran (beta-glucan isolated from Pleurotus ostreatus) on experimental colitis in rats

The effects of pleuran, beta-1,3 glucan isolated from Pleurotus ostreatus, were studied in a model of acute colitis induced by intracolonic administration of acetic acid. There was a reduction of the colonic damage score, colonic wet weight and wet/dry weight ratio 48 h after single luminal 2% pleuran suspension pretreatment. Similar results were obtained after repeated intraperitoneal administration of pleuran in doses of 30 and 100 mg/kg. Pleuran given orally as a 10% food component over 4 weeks was effective in reducing the extent of mucosal damage, but did not prevent the increase of myeloperoxidase in the injured colonic segment. In the segment without macroscopic evidence of inflammation, myeloperoxidase activity was significantly lower as documented by histological examination. The results indicate a possible role of this immunomodulator in the treatment of ulcerative colitis.     

Effects of histamine H1 antagonist dithiaden on acetic acid-induced colitis in rats.

To assess the possible involvement of mast cells and/or their mediators in inflammatory bowel diseases, the effect of the histamine H1 antagonist Dithiaden was studied on a model of acetic acid-induced colitis in rats. Dithiaden pretreatment by intracolonic administration was found to reduce the extent of acute inflammatory colonic injury. This was manifested by a decrease in the score of gross mucosal injury, by lowered colonic wet weight and by diminished myeloperoxidase activity reflecting reduced leukocyte infiltration. Vascular permeability and gamma-glutamyl transpeptidase activity, elevated by acetic acid exposure, were decreased after Dithiaden pretreatment. The results indicate that locally administered Dithiaden may protect the colonic mucosa against an acute inflammatory attack by interfering with the action of the major mast cell mediator histamine.     

Effect of nor-trimebutine on neuronal activation induced by a noxious stimulus or an acute colonic inflammation in the rat

Nor-trimebutine is the main metabolite of trimebutine that is used in the treatment of patients with irritable bowel syndrome. Nor-trimebutine has a blocking activity on sodium channels and a potent local anesthetic effect. These properties were used to investigate the effect of nor-trimebutine on spinal neuronal activation induced by models of noxious somato-visceral stimulus and acute colonic inflammation. Nor-trimebutine was administered in rats either subcutaneously 30 min before intraperitoneal administration of acetic acid or intracolonically 30 min before intracolonic infusion of trinitrobenzenesulfonic acid. Abdominal contractions were counted for 1 h as a marker of abdominal pain. c-fos expression was used as a marker of neuronal activation and revealed by immunohistochemistry 1h after intraperitoneal acetic acid injection and 2 h after colonic inflammation. Nor-trimebutine decreased Fos expression in the thoraco-lumbar (peritoneal irritation) and lumbo-sacral (colonic inflammation) spinal cord in laminae I, IIo V, VII and X. This effect was also observed in the sacral parasympathetic nucleus after colonic inflammation. Nor-trimebutine induced a significant decrease of abdominal contractions following intraperitoneal acetic acid injection. These data may explain the effectiveness of trimebutine in the therapy of abdominal pain in the irritable bowel syndrome.     

The effect of malotilate, a derivative of malotilate and a flavenoid on eicosanoid production in inflammatory bowel disease in rats

Acetic acid induced colitis in rats was used to investigate the effects of malotilate, a drug which has been shown to inhibit 5-1ipoxygenase in human macrophages, the malotilate derivate ZY16268 and the flavenoid ZY16369 on the eicosanoid production and the colonic morphology in inflammatory bowel disease. Acetic acid produced an acute inflammatory response in the colon, associated with a markedly raised inflammation score (15.8 vs. < 0.5), based on a seven-scaled scoring system which includes observation of haemorrhage, submucosal oedema, cellular infiltration, goblet cell depletion, loss of architecture, crypt abscesses and serosal involvement, of which every item was subdivided as mild, moderate and severe. Incubation of colonic mucosa from rats treated with arachidonic acid and stimulated with A23187 showed an increase of the cyclooxygenase product 12-hydroxy-heptadecatrienoic acid (HHT) and the 12-1ipoxygenase product (12-HETE) and a decrease in the formation of 6-keto-prostaglandin F(1alpha)(6kPGF(1alpha)) in comparison with normal rat mucosa. Malotilate, ZY16268 and ZY16369 all resulted in a decrease in HHT, leukotriene B(4) (LTB(4))-like compounds and 12-hydroxyeicosaenoic acid (12-HETE) production. None of the tested compounds significantly reduced the colonic damage by acetic acid although the formation of 12-HETE was proportional to the histologically obtained inflammation score. There were marked differences in eicosanoid formation patterns between rat and human mucosa, both normal and inflamed. In view of the hyperacute nature of the mucosal damage and the marked differences in eicosanoid production, acetic acid induced colitis in rats is probably not a suitable model of ulcerative colitis in humans.     

The chemokine RANTES is a crucial mediator of the progression from acute to chronic colitis in the rat

Chemokines have well characterized proinflammatory actions, including the ability to induce extravasation of leukocytes that participate in chronic inflammation. In this study, we evaluated the role of a C-C chemokine, RANTES, in the chronic phase of a rat model of colitis. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various timepoints thereafter (2 h to 14 days), colonic tissue levels of several chemokines were measured. Unlike the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant, the expression of RANTES was significantly elevated during the chronic phase of colitis (> or =7 days after induction). Colonic RANTES mRNA expression was also significantly elevated during the chronic phase of colitis. The numbers of macrophages and monocytes in the colonic mucosa increased substantially during the chronic phase, as did expression of two of the receptors (CCR1 and CCR5) to which RANTES is known to bind. Administration on days 7 through 14 after trinitrobenzene sulfonic acid administration of a CCR1/CCR5 receptor antagonist, Met-RANTES, resulted in a significant reduction of both macroscopic and microscopic colonic damage, as well as reducing the recruitment into the colon of monocytes, mast cells, and neutrophils. In some rats, treatment with Met-RANTES resulted in a near-complete resolution of colonic damage and inflammation. These results suggest a crucial role of RANTES in the progression from acute to chronic inflammation in a rat model of colitis.     

Reduction of Smad3 accelerates re-epithelialization in a murine model of colitis

To determine the role of Smad3 in re-epithelialization and inflammation, experimental colitis was induced in Smad3 heterozygous mice and their wild-type littermates by single intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol. The area of epithelial deficiency was significantly reduced in the heterozygotes on the 4th-6th day after TNBS administration as compared to the controls although the number of inflammatory cells in the colonic mucosa in the heterozygotes and their wild-type littermates varied similarly throughout the course of colitis. Proliferation of the intestinal epithelium in the heterozygotes was significantly accelerated as compared to that in the wild-type controls on the 1st and 2nd days after TNBS administration. These results suggest that reduction of Smad3 significantly accelerates re-epithelialization of the intestinal mucosa without enhancing inflammation. Suppression of TGF-beta1 induction in the colonic mucosa of the heterozygotes may lead to a higher level of proliferation of intestinal epithelial cells.     

Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium

Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.     

Resistance to apoptosis is a mechanism of adaptation of rat stomach to aspirin

Adaptation of the gastric mucosa to nonsteroidal anti-inflammatory drug-induced injury is a well-documented phenomenon, but the mechanisms are not known. We investigated whether changes in stress protein expression and apoptosis play roles in adaptation of rat stomach to aspirin. RT-PCR and Western blotting techniques were used to analyze mRNA and protein expression of HSP72 and HSP90 and cleavage of caspase 3 protein. Apoptosis was detected by the TUNEL method and quantified. HSP72 mRNA and protein expression was unchanged in adapted mucosa, whereas HSP90 mRNA and protein levels decreased. Caspase 3 protein was activated, and the number of apoptotic cells increased in mucosa after one aspirin dose. However, in adapted mucosa after aspirin, activated caspase 3 and the number of apoptotic cells had returned to basal levels. Induction of the stress response was found not to be a mechanism of mucosal adaptation against multiple doses of aspirin. Our results lead us to propose instead that resistance to aspirin-induced apoptosis plays a role in the protective phenomenon of adaptation.     

Amelioration of dextran sulfate colitis by butyrate: role of heat shock protein 70 and NF-kappaB

Butyrate enemas have been demonstrated to ameliorate inflammation in ulcerative colitis. The mechanism of this protective effect of butyrate is not known, and this study examines the effect of butyrate on epithelial function, inducible heat shock protein 70 (HSP70) expression, and NF-kappaB activation in experimental colitis. Colitis was induced in rats by oral dextran sulfate sodium (DSS) and by butyrate or saline enemas. Mucosal barrier function was assessed by electrical resistance and [14C]mannitol permeability. HSP70 production was determined by [35S]methionine labeling, Western blot analysis, and immunohistochemistry. Activation of heat shock factors (HSFs) and NF-kappaB was evaluated by EMSA. Butyrate showed a significant protection against the decrease in cell viability, increase in mucosal permeability, and polymorphonuclear neutrophil infiltration seen in DSS colitis. Butyrate inhibited HSP70 expression in DSS colitis and also inhibited the activation of HSF and NF-kappaB. Thus butyrate enema was found to be cytoprotective in DSS colitis, an effect partly mediated by suppressing activation of HSP70 and NF-kappaB.     

Effect of supplementing fava bean (Vicia faba L.) on ulcerative colitis and colonic mucosal DNA content in rats fed a high-sucrose diet

Ulcerative colitis (UC) is an inflammatory bowel disease with high morbidity. Acetic acid-induced damage of colonic mucosa in rats is a commonly used experimental animal model of UC. This research aimed to explore for the first time the ameliorative effect of dietary supplementation with fava bean on the incidence of UC in rats fed with sucrose containing diet. Rats were divided into five groups as follows: G1, control healthy rats; G2, colitic rats; G3, colitic rats fed diets containing 30% sucrose, G4, healthy rats fed diets containing 30% sucrose and G5, colitic rats fed diets containing 30% sucrose supplemented with dried ground fava bean. Colonic injury and inflammation were evaluated through a disturbance of oxidative biomarkers, a significant increase in inflammatory biomarkers and inflammatory cytokines, and histological abnormalities in colonic tissues accompanied by colonic mucosal DNA damage. Colitic rats fed on sucrose containing diet demonstrated additional histological, biochemical, and DNA alterations in colonic mucosa of rats. Dietary supplementation with dried ground fava bean significantly corrected the impaired oxidative and inflammatory biomarker levels and modulated histological features and DNA alterations. Finally, fava bean attenuated the oxidative damage and colonic injury induced by acetic acid, which confirmed its high anti-oxidant and anti-incendiary properties.     

The antioxidant properties of a novel zinc-carnosine chelate compound, N-(3-aminopropionyl)-L-histidinato zinc.

A zinc-carnosine chelate compound, Z-103, attenuates gastric mucosal injuries and inhibits the increase of lipid peroxide in the gastric mucosa induced by burn shock or ischemia-reperfusion. However, the exact mechanism of the antioxidative effect of Z-103 is not clear. The antioxidant properties of a novel anti-peptic ulcer agent Z-103 in vitro were compared with those of zinc ion and L-carnosine. Z-103 scavenged superoxide anion radicals. Z-103 and ZnSO4, but not L-carnosine, inhibited the superoxide generation from polymorphonuclear leukocytes stimulated by opsonized zymosan, and also inhibited the generation of hydroxyl radicals by the Fenton reaction. The increase of lipid peroxides produced by rat brain homogenates and liver microsomes was also inhibited by Z-103 and ZnSO4. These findings indicate that the strong anti-ulcer and antioxidative actions of Z-103 in vivo are due to a combination of these antioxidant actions in vitro.     

Rapid onset of (R)-α-methylhistamine protection in response to ethanol-induced histologic damage in rat gastric mucosa

The influence of pretreatment with (R)-α-methylhistamine, selective agonist of histamine H₃ receptors, has been investigated on gastric mucosal lesions at different time intervals, from 5 to 60 minutes, after administration of absolute ethanol in the rat. The amount and depth of lesions were quantitatively evaluated by light microscopy. In rats pretreated with (R)-α-methylhistamine, the integrity of the mucosa was preserved in approximately 80% of the total mucosal length measured despite ethanol challenge. Prevention of lesion formation was as great at 5 min after ethanol administration as at 60 min. When present, damage involved mainly superficial mucosa and lesions extending deeply into the gland region were evident in 1–2.5% of the total mucosa. Present findings indicate that mechanisms by which (R)-α-methylhistamine operates enable the mucosa to counteract damage just from the moment of exposure to ethanol and that protection is exerted both on surface and pit cells and on gastric glands.