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Quercetin prevents oxidative stress and NF-κB activation in gastric mucosa of portal hypertensive rats

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Quercetin prevents oxidative stress and NF-κB activation in gastric mucosa of portal hypertensive rats
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  Quercetin prevents oxidative stress and NF- k B activation in gastricmucosa of portal hypertensive rats Andrea J. Moreira a , Christina Fraga b , Marı´a Alonso c , Pilar S. Collado c , Claudio Zetller b ,Claudio Marroni b , Norma Marroni a , Javier Gonza´lez-Gallego c, * a Universidade Federal de Rio Grande do Sul and Universidade Luterana do Brasil, Brasil b Fundac¸a˜ o Faculdade Federal de Cieˆ ncias Me´ dicas de Porto Alegre, Brasil c  Department of Physiology, University of Leo´ n, 2401 Leo´ n, Spain Received 29 March 2004; accepted 12 July 2004 Abstract The present study was designed to investigate the effects of quercetin on oxidative stress and activation of nuclear factor kappa B (NF- k B) in an experimental model of portal hypertensive gastropathy induced by partial portal vein ligation (PPVL). Portal pressure wassignificantly elevated in PPVL rats. Transaminase and alkaline phosphatase activities were not significantly modified, indicating absenceof liver injury. Histological analysis of gastric sections showed a lost of normal architecture, with edema and vasodilatation. The cytosolicconcentration of thiobarbituric acid reactive substances and the lipoperoxidation measurement by chemiluminiscence were significantlyincreased. Superoxide dismutase activity in gastric mucosa was significantly reduced. Portal hypertensive gastropathy induced a markedactivation of NF- k B, accompanied by a decrease in I k B protein levels and a significant induction of nitric oxide synthase (iNOS) protein.Administration of quercetin markedly alleviated histological abnormalities and inhibited oxidative stress and NF- k B activation. I k Bdecrease and induction of iNOS protein were partially prevented by quercetin. Quercetin treatment, by abolishing the NF- k B signaltransduction pathway, may block the production of noxious mediators involved in the pathogenesis of portal hypertensive gastropathy. # 2004 Elsevier Inc. All rights reserved. Keywords:  Gastric mucosa; NF- k B; Nitric oxide; Oxidative stress; Portal hypertension; Quercetin 1. Introduction Portal hypertension is one of the most disastrous con-ditions related to chronic hepatic diseases. As the portalpressure elevates, portal-systemic collaterals develop gra-dually to diverse blood flow from the portal system [1].Gastroesophageal varices are most prominent collateralsand hemorrhage from ruptured gastroesophageal varicesleads to a high morbility and mortality [2]. Portal hyper-tensive gastropaty (PHG) is now recognized as a distinctclinical entity characterized by mucosal and submucosalvascular dilatation used to describe the endoscopic appear-ance of gastric mucose, with a characteristic mosaic-likepattern with or without red spots, seen in patients withcirrhotic or noncirrhotic portal hypertension [3]. PHG,usually present in association with either esophageal orgastric varices [4], is one of the leading causes of death inAmericans between the ages of 35–54 [5] and approxi-mately 30% of patients afflicted with PHG develop life –threatening gastric hemorrhage, either spontaneous orcaused by noxious agents [6].There is experimental evidence to suggest that gastricmucosal defense mechanisms are impaired in the presenceof portal hypertension. Factors such as excessive nitricoxide (NO) production and increased generation of oxygenfreeradicalsandlipidperoxidationhavebeenimplicated initsincreasedsusceptibilitytoinjuryinPHG[7–9].Overthe last few years, a number of studies have provided evidenceof an important role of reactive oxygen species (ROS) inmediating the microvascular disturbance that precededgastric mucosal injury [10].ROS may inflict direct damage to vital cell constituentssuch as lipids, proteins and DNA, but also modulate thepatternofgene expressionthrough functionalalterationsof transcription factors such as nuclear factor kappa B (NF- www.elsevier.com/locate/biochempharmBiochemical Pharmacology 68 (2004) 1939–1946* Corresponding author. Tel.: +34 987 291258; fax: +34 987 291267. E-mail address:  dfijgg@unileon.es (J. Gonza´lez-Gallego).0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.bcp.2004.07.016  k B) [11]. NF- k B, in turn, is required for the induction of nitric oxide synthase (iNOS) gene in response to cytokines[12]. NO, a potent endogenous vasodilator synthesized byvascular endothelial cells, contributes to the hyperdynamiccirculation, mesenteric hyperemia and vascular hypore-sponsiveness to vasoconstrictors in portal-hypertensivestates [13,14]. In addition, the vascular hyporeactivity phenomenon observed in hemorrhagic shock may bemediated by NO [15].Oxidative stress-induced extracellular signal-regulatedkinase 2 (mitogen-activated protein kinase) has beenshown to be defective in PHG, and this is mediated byan overexpression of mitogen-activated protein kinasephosphatase 1 that normalizes after supplementation withVitamin E. Use of antioxidants may therefore have ther-apeutic implications for the management of PHG. Flavo-noids are phenolic phytochemicals that representsubstantial constituents of the non-energetic part of thehuman diet and are thought to promote optimal health,partly via their antioxidant effects in protecting cellularcomponents against reactive oxygen species (ROS) [16].Quercetin (3,5,7,3 0 4 0 -pentahydroxy flavon) is one of themost widely distributed flavonoids, present in fruit, vege-tables and manyother dietary sources[17].This compoundhas been reported to scavenge superoxide in ischemia-reperfusion injury [18], to protect against oxidative stressinduced by ultraviolet light [19],spontaneous hypertension[20], secondary biliary cirrhosis [21] and bacterial lipo- polysaccharide [22] or to inhibit angiogenesis [23] and carcinogenesis [24].The present study was designed to investigate the effectsof quercetin on oxidative stress, activation of NF- k B andup-regulation of NO in an experimental model of portalhypertensive gastropathy. 2. Materials and methods 2.1. Animal and procedures Male Wistar rats weighing 300–350 g were used for thisstudy. The rats were caged at 24  8 C, with a 12 h light–dark cycle and free access to food and water until the time of experiments.Survivalsurgeryandhemodynamicstudywereperformed with the rats under anesthesia with sodiumpentobarbital (50 mg/kg body weight i.p.). Portal hyperten-sion was induced by partial portal vein ligation (PPVL) asdescribedbyVorbioffetal.[25].Inbrief,theportalveinwasisolated and a 3-0 silk ligature was tied around both theportal vein and an adjacent 20 gauge blunt-tipped needle.The needle was then removed and the vein allowed to re-expand. A second loose ligature was left around the portalvein with two endings of the ligature placed on each side intheabdominalcavity.Theabdomenwasthenclosedandtheanimal allowed to recover. All experiments were performedin accordance with the  Guiding Principles for Research Involving Animals  (NAS). Control rats underwent a similaroperation but without partial occlusion of portal vein.After 8 days, quercetin (Sigma, St. Louis, MO) wassuspended, immediately before administration, in a 0.2%Tween aqueous solution. Groups of control and portalhypertensive animals received daily a 500  m l i.p. injectionof quercetin (50 mg/kg body wt  1 in 0.5 ml) or vehicle forthe last 7 days of the study. Portal venous pressure wasmeasured on a Wilson biscriptual polygraph (WilsonMedical Electronics Inc. Middletown, Wisconsin, USA). 2.2. Biochemical analysis The stomach were excised, weighed, and immediatelyfrozen at  70  8 C. Frozen tissue from each rat was homo-genized in ice-cold phosphate buffer (KCl 140 mM, phos-phate 20 mM, pH 7.4) and centrifuged at 3000 rpm for10 min. Cytosolic superoxide dismutase (SOD) (EC1.15.1.1) was assayed according to Misra and Fridovich[26] at 30  8 C. The rate of autooxidation of epinephrine,which is progressively inhibited by increasing amounts of SOD in the homogenate, is monitored spectrophotometri-cally at 560 nm. The amount of enzyme that inhibitsepinephrine autooxidation at 50% of the maximum inhibi-tion is defined as 1 U of SOD activity.Oxidative stress was determined by measuring the con-centration of thiobarbituric acid reactive substances(TBARS) [27] and the hydroperoxide-initiated chemilu-minescence (QL) [28]. The amount of aldehydic productsgenerated by lipid peroxidation was quantified by thethiobarbituric acid reaction using 3 mg of protein persample. Results were referred as TBARS. The sampleswere incubated at 90  8 C for 30 min after adding 500  m L of 0.37% thiobarbituric acid in 15% trichloroacetic acid, thencentrifuged at 4  8 C at 2000  g  for 15 min. Spectrophoto-metric absorbance was determined in the supernatant a535 nm. For the QL determination, 0.5 ml of homogenatewere added to 120 mM KCl, 30 mM phosphate buffer (pH7.4), and 3 mM  tert  -butyl hydroperoxide at 30  8 C andassayed for chemiluminiscence in a liquid scintillationcounter in the out-of-coincidence mode.Serum levels of alanine aminotransferase (ALT), aspartate aminotransfer-ase (AST) and alkaline phosphatase (AP) were determinedby commercial kits (Boehringer Mannheim, Germany). 2.3. Western blot  For Western blot analysis of iNOS and I k B proteinformation, stomach tissue was homogenized with140 mM NaCl, 15 mM EDTA, 10% glycerol, 20 mM anda protease inhibitor cocktail. Themixturewasincubated for30 min at 4  8 C and centrifuged 30 min at 17,000    g and4  8 C. The supernatant was kept as stomach tissue lysate.Samples containing 100  m g of protein were separated bysodium dodecyl sulfate-polyacrylamide gel electropho-resis (10% acrylamide) and transferred to nitrocellulose.  A.J. Moreira et al./Biochemical Pharmacology 68 (2004) 1939–1946  1940  Non-specific binding was blocked by preincubation of thenitrocellulose in phosphate-buffered saline containing 5%bovine serum albumin for 1 h. The nitrocellulose was thenincubated overnight at 4  8 C with polyclonal anti-iNOS oranti-I k B antibodies (Santa Cruz Biotechnology). Boundprimary antibody was detected with HRP-conjugated anti-rabbit antibody (DAKO) by chemiluminiscence. The den-sityofthespecificiNOSandI k Bbandswasquantitatedwithan imaging densitometer. 2.4. Electrophoretic mobility shift assay Nuclear extracts were prepared from stomach lysates asdescribed previously [29]. Activation of transcription fac-torNF- k BwasexaminedusingconsensusoligonucleotidesofNF- k B(5 0 -AGTTGAGGGGACTTT CCC AGG C-3 0 ).Probes were labeled by T4 polynucleotide kinase. Bindingreactions included 10  m g of nuclear extracts in incubationbuffer (50 mM Tris–HCl pH 7.5, 200 mM NaCl, 5 mMEDTA, 5 mM mercaptoethanol, 20% glycerol and 1  m gpoly (dI-dC)). After 15 min on ice, the labeled oligonu-cleotide (30,000 cpm) was added and the mixture incu-bated 20 min at room temperature. For competitionstudies, 3.5 pmol of unlabeled (cold) NF- k B oligonucleo-tide (competitor) or 3.5 pmol of labelled NF- k B oligonu-cleotidemutate(noncompetitor)weremixed15 minbeforethe incubation with the labelled oligonucleotide. Themixturewas electrophoresed through a 6% polyacrylamidegel for 90 min at 150 V. The gel was then dried andautoradiographed at  70  8 C overnight. Signals were den-sitometrically analyzed. 2.5. Histology For histological examination a piece of the liver andstomach was trimmed and fixed by immersion in 10%buffered formalin for 24 h. The blocks were dehydrated ina graded series of ethanol and embedded in paraffin wax.Serial 3  m m sections were stained with hematoxilin andeosin. 2.6. Statistical analysis Means and S.E.M.’s were calculated for all data.Significant differences between means were evaluatedby analysis of variance and in the case of significance, aNewman–Keul’s test was also applied. Significance wasaccepted at  P  <  0.05. 3. Results 3.1. Portal pressure and transaminase activities No significant differences were found for any of testedparameters between untreated controls and those treatedwith quercetin, and hence data were pooled together. Therewas a statistically significant difference in portal pressurebetween control animals and thosewithportal vein ligation(+46%). No significant difference from the control groupwasobservedinPPVLratstreatedwithquercetin(Table1).Transaminase and alkaline phosphatase activities did notsignificantly differ between the different experimentalgroups. 3.2. Histology Histological analysis of gastric sections showed mod-ifications of normal architecture, with congestion andedema in the submucosa and a proliferation of blood vesselin PPVL animals. Administration of quercetin markedlyalleviated histological abnormalities (Fig. 1). No histolo-gical alterations were detected in liver of different groups(data not shown). 3.3. Markers of oxidative stress and SOD activity The cytosolic concentration of TBARS increased in theanimals with portal hypertension (PPVL group) (+93%),whilevalues did not significantly differ from the controls inPPVL rats treated with quercetin (Fig. 2). Lipoperoxidationmeasurement by chemiluminiscence also demonstrated asignificant increase in PVL rats (+101%) that was absent inthose animals receiving quercetin (Fig. 2). SOD activitytended to decrease in PPVL animals and this effect wasprevented by quercetin administration (Fig. 3). 3.4. NF- k  B activation and I  k  B expression NF- k B binding activity was evaluated by EMSA per-formed with a NF- k B consensus nucleotide sequence. Toconfirm that the result shifted band is the specific bindingof NF- k B to its sequence specific oligonucleotide, a com-petition binding assay was performed. As shown in Fig. 4,portal hypertensive gastropathy induced a marked activa-tion of NF- k B (+98%) that was absent in stomach of animals treated with quercetin. Since it has been welldocumented that activation of NF- k B correlates with rapidproteolytic degradation of I k B, we assessed protein levels  A.J. Moreira et al./Biochemical Pharmacology 68 (2004) 1939–1946   1941Table 1Effects of partial portal vein ligation (PPVL) and quercetin (Q) adminis-tration on portal pressure and serum AST, ALT and AP activitiesControl PPVL PPVL + QPortal pressure (mmHg) 11.9    1.6 19.7    1.9 * 13.0    0.9 a AST (U/L) 166.0    14.6 159.2    21.3 174.0    15.7ALT (U/L) 95.1    8.0 91.0    9.2 72.3    6.2AP (U/L) 191    16 170    20 164    29Rats were partially ligated or sham operated as described in Section 2.Values are means    S.E.M. for 6–8 rats. AS: aspartate aminotransferase;ALT: alanine aminotransferase; AP: alkaline phosphatase. * P  <  0.05 against control. a P  <  0.05 against PPVL.   A.J. Moreira et al./Biochemical Pharmacology 68 (2004) 1939–1946  1942Fig. 1. Micrographs of gastric tissue in a control rats (A), rats with partial portal vein ligation (PPVL) (B) and rats with portal vein ligation and quercetintreatment (PPVL + Q) (C). G: glandular tissue, MM: muscular mucosa, SM: submucosa. Hematoxilin and eosin staining, srcinal magnification, 10  .Congestion, edema and a proliferation of blood vessel were evident in B.  of I k B using Western blot analysis. Protein levels weredecreased in animals from the PPVL group (  25%) andthis effect was partially blocked by quercetin (Fig. 5). 3.5. iNOS expression To evaluate the effects of experimental gastropathy andquercetin treatment on nitric oxide production, the expres-sion of iNOS was quantified by measurement of protein(Western blot) levels. PPVL coursed with a significantinduction of iNOS protein (+346%). This effect waspartially abrogated by quercetin (Fig. 6). 4. Discussion The partial portal vein ligation used in this study is ananimal model characterized by prehepatic portal hyperten-sion, with a maintained hepatic structure and a hyperdy-namic circulation that develops in a short and predictedperiod of time [1]. This model has been extensivelystudiedand found to be a useful tool for understanding thepathophysiology of portal hypertension [1,2,30]. More- over, it can provide measurements in the portal circulationwhich cannot be performed accurately in humans becauseof ethical and technical limitations [31].Inourstudy,portalhypertensionwasaccompaniedbythepresenceofedemaanddilatedvesselsinthegastricmucosa,but no alteration was detected in liver histology or transa-minase activities. This confirms previous findings that theprocedure of PPVL, although causing a transient reductionin the metabolic activity of the liver [32], do not producehepatocellular damage [33] and support the suggestion thatportal hypertension seems to be the key factor for thedevelopment of PHG, being equally common in portalhypertensive patients with or without liver disease [34].The fact that partial portal vein ligation results in oxidantinjury was first demonstrated by Fernando et al. [7], whichconcludedthattheformationofROSmaybeimportantinthepathogenesisofhemodynamicchangesandthedevelopmentofthehyperdynamiccirculation.Inmodelsofinjuryinducedbynonsteroidalanti-inflammatorydrugsanumberofstudieshave also provided evidence of an important role of ROS inmediating the microvascular disturbance that preceedsgastricmucosalinjury[10].Kawanakaetal.[5]haveshown that mitogen-activated protein kinase ERK2 activation isimpairedinthegastricmucosaofportalhypertensiveratsasaresultoftheunderlyingandcontinualoxidativestress.Thepotential role ofROSformationinthepathogenesis ofPHGis further supported by the prevention of the hyperdymamiccirculation induced by  N  -acetylcysteine [7] and the reverseby Vitamin E of the increased susceptibility of the gastricmucosatoalcohol-inducedinjuryinportalhypertensiverats[5].Oxidative stress has been defined as an imbalancebetween pro- and antioxidants, and ROS-induced lipidperoxidationcanoccureitherinsituationsinwhichscaven-ging systems are overwhelmed (excessive production of ROS) or by impairing the antioxidant systems [35]. Super-oxide dismutase is an intracellular metalloenzyme whichowes its antioxidant properties to its elevated capacity of scavenging O 2  radicals. In the present investigation SODactivity was significantly decreased in the gastric mucosa,in line with previous studies which have shown a similareffect in NSAID-induced gastropathy [10,36]. The  A.J. Moreira et al./Biochemical Pharmacology 68 (2004) 1939–1946   1943Fig. 2. Effects of partial portal vein ligation (PPVL) and quercetin (Q)administration on gastric TBARS concentration and chemiluminiscence(QL). Values are means  S.E.M. for 6–8 rats.  * P < 0.05 against control.  # P <  0.05 against PPVL.Fig. 3. Effects of partial portal vein ligation (PPVL) and quercetin (Q)administration on gastric superoxide dismutase activities. Values are means  S.E.M. for 6–8 rats.  * P < 0.05 against control.  # P < 0.05 against PPVL.
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