- Email: [email protected]
739 CIRCULATING AND HEPATIC ENDOCANNABINOIDS AND ENDOCANNABINOID-RELATED MOLECULES IN PATIENTS WITH LIVER CIRRHOSIS
02a: CIRRHOSIS AND COMPLICATIONS − a) PATHOPHYSIOLOGY factor-2 (Nrf2) is a key regulator of the cellular response to oxidative stress in multiple tissues and cell types. Nrf2 regulates the expression of many detoxifying genes such as catalase superoxide dismutase (SOD), UDPglucuronosyltransferase, and g-glutamylcysteine synthetase. Nrf2 protects the liver from acute and chronic damage by carbon tetrachloride (CCl4). However, in bile duct ligation model there is still scarce information regarding Nrf2. Aim: To analyze the dynamic of Nrf2 expression and the antioxidant enzyme activity in experimental cholestasis. Methods: Liver fibrosis was induced by bile duct ligation (BDL). Fibrotic animals were sacrificed after 1, 2 and 4 weeks. Nuclear extracts were isolated from whole liver to detect Nrf2 by Western-blotting. Catalase and superoxide dismutase were analyzed by zymography in liver homogenates. Liver sections were stained with hematoxylin/eosin and Masson trichrome to demonstrate liver chronic injury and collagen deposition. Results: Nrf2 expression showed important changes in the progression of liver injury by BDL. We detected a significant increase at the same time that the fibrosis intensified (5 U at 1 week, 44 U at 2 weeks and 129 U at 4 weeks); however, these values were maintained at lower than the control group “sham rats” at 1 and 2 weeks. The catalase activity showed a significant increase at 1 and 2 weeks of ligature (60% and 80%, respectively), as compared with sham rats. SOD activity rose significantly (60%); although, it was observed only at 1 week. In contrast, at 4 weeks both enzymes decreased importantly (55%). Unexpectedly, the highest level of Nrf2 expression was correlated with the lowest enzyme activity during the kinetic assay. Histological analyses showed characteristic alterations of the cholestatic process: inflammation, cell necrosis, bile duct proliferation and collagen deposition. Conclusion: The different changes in Nrf2 expression do not importantly influence the activity of some enzyme detoxifying in the cholestasis model.
737 HISTOLOGICAL AND NON-INVASIVE QUANTITATIVE EVALUATION OF FIBROSIS PROGRESSION IN CHRONIC LIVER DISEASES P. Cales, S. Michalak, I. Valo, M.C. Rousselet, M. Ettalbi, J. Boursier, Y. Gallois, F. Oberti. CHU, Angers, France E-mail: [email protected] Background and Aims: Until now, progression of liver fibrosis has only been measured with pathological semi-quantitative staging. Recently, two meta-analyses have well described the transition between different Metavir fibrosis stages (Thein, Hepatology and AIDS 2008). Our aim was to evaluate the progression rate of fibrosis in a more sensitive way, especially thanks to the quantitative measurement of liver fibrosis via the area of fibrosis, in chronic liver diseases. Methods: Data were recorded in 201 patients with alcoholic or viral chronic liver diseases. Fibrosis was evaluated by Metavir F staging by two independent observers and the area of fibrosis by image analysis. Results: The mean (median) yearly rates of progression were 0.22±0.29 (0.13) MU for Metavir F staging and 1.8±2.6 (1.0) % for the area of fibrosis in patients with one liver biopsy (estimated progression with the starting date of cause) versus 0.17±0.27 (0.09) MU (p = 0.66) and 1.3±3.4 (1.2) % (p: 0.72), respectively, in patients having two biopsies. The progression of F stage and area of fibrosis was not linear, increased as a function of fibrosis F stage, and was faster in alcoholic chronic liver disease than in viral chronic liver disease only with the area of fibrosis. The independent predictors of Metavir F progression were: AST/ALT, cause duration, Metavir F stage and prothrombin index (a R2 : 0.605). Metavir F stage progression could be non-invasively and accurately predicted without any pathological variables (a R2 : 0.488). The independent predictors of area of fibrosis progression were: AST/ALT, cause duration, baseline area of fibrosis, and bglobulins (a R2 : 0.72); steatosis grade had a borderline significance (p: 0.057) but not activity grade (p: 0.53) or chronic liver disease cause (p: 0.39). The area of fibrosis progression could be noninvasively and accurately predicted without any pathological variables (a R2 : 0.649). The area of fibrosis was much more sensitive than Metavir F stage to detect changes due to anti-fibrotic treatment in another population.
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Conclusions: The area of fibrosis is more sensitive to evaluate fibrosis progression, and its pharmacological-induced variation, than classical histological semi-quantitative staging. Fibrosis progression is highly predictable by non-invasive scores that can be thus used in clinical practice. 738 BACTERIAL PEPTIDE IDENTIFICATION IN ASCITIC FLUID OF PATIENTS WITH ADVANCED CIRRHOSIS R. Ca˜no1 , P. Zapater2,3 , S. Pascual1,2 , P. Bellot1 , M. P´erez-Mateo1 , J. Such1,2 , R. Franc´es1,2 . 1 Unidad Hep´atica, Hospital General Universitario de Alicante, Alicante, 2 CIBERehd, Instituto de Salud Carlos III., Madrid, 3 Servicio de Farmacolog´ıa Cl´ınica, Hospital General Universitario de Alicante, Alicante, Spain E-mail: [email protected] Background: Bacterial-DNA is associated with the induction of a potent cellular immune response that may influence prognosis in patients with cirrhosis and ascites. However, intensity of the immune response against viable bacteria or bacterial products is frequently different. Aims: To evaluate the presence of bacterial peptides in ascitic fluid (AF) from patients with cirrhosis, as possible complementary markers of bacterial translocation. Methods: Analysis by two-dimensional poliacrylamide gel electrophoresis of AF total protein from patients with cirrhosis and of the most frequently isolated bacterial species in these patients. 2D-maps were digitally compared. The spots identification was performed by MALDI-TOF mass spectrometry. TNF-a, IFN-g, IL-12 and NOx levels were measured in AF samples by ELISA. All samples were inoculated in blood culture bottles. Results: A total of 37 patients with cirrhosis and ascites were included in the study and distributed into groups according to the absence (Group I) or presence (Group II) of bacterial DNA. All samples were culturenegative. No clinical or analytical differences were observed between groups. Bacterial peptides were found in 7 (Group II-b) out of 16 2D-maps from patients with presence of bactDNA (41%), whereas these peptides were not detected in any of the maps from patients without bactDNA. The identified bacterial peptides were GAPDH-A, Porin OmpC and HSP60. Differences in levels of cytokines and NOx are resume in Table 1. Conclusions: For the first time, bacterial peptides are identified in AF of patients with advanced cirrhosis, which implies the transient viability of bacteria, despite negativity of cultures. Their presence is associated with a significant increase in the soluble immune response. Validation of these peptides as markers of bacterial translocation and viability, as well as the clinical consequences of this finding will require further experimental work.
TNF-a (pg/ml) IFN-g (pg/ml) IL-12 (pg/ml) NOx (mmol/ml)
Group I (n = 21) BactDNA−
Group II-a (n = 9) Group II-b (n = 7) BactDNA+/BactProt− BactDNA+/BactProt+
169.3±42.1 227.3±63.5 342.7±73.3 15.1±3.8
427.2±132.8* 361.4±86.5* 720.9±114* 25.9±7.2*
587.9±74.0*# 419.3±76.1*# 935.6±149.8*# 28.4±4.7*#
*p < 0.05 compared with Group I; # p < 0.05 compared with Group II.
739 CIRCULATING AND HEPATIC ENDOCANNABINOIDS AND ENDOCANNABINOID-RELATED MOLECULES IN PATIENTS WITH LIVER CIRRHOSIS P. Caraceni1 , A. Viola1 , M. Domenicali1 , F. Piscitelli2 , S. Petrosino2 , F. Giannone1 , A. Berzigotti3 , M. Zoli3 , M. Cescon4 , G. Grazi4 , V. Di Marzo2 , M. Bernardi1 . 1 Clinical Medicine, University of Bologna, Bologna, 2 Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Naples, 3 Internal Medicine and Aging, University of Bologna, 4 Surgical Sciences, University of Bologna, Bologna, Italy E-mail: [email protected] Background and Aims: Endogenous cannabinoids (EC) include anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The EC-
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related molecules, oleoyl-ethanolamine (OEA) and palmitoyl-ethanolamine (PEA), have been recently identified. AEA contributes to the pathogenesis of cardiovascular alterations in experimental cirrhosis, but data in humans are scarce. Thus, we assessed whether: 1. the EC system is up-regulated in cirrhosis by comparing plasma and hepatic EC levels in cirrhotic pts and controls; 2. the cirrhotic liver contributes to circulating EC by measuring their concentrations in brachial and hepatic veins; and 3. the EC levels correlate with systemic hemodynamics, portal pressure, liver and renal functions. Methods: Blood samples were collected from the brachial vein of 18 cirrhotic pts (Child–Pugh A/B/C:2/6/10) and 14 age- and sex-comparable controls, and from the hepatic veins during HVPG measurement in 8 cirrhotic pts. Tissue samples were also collected from 10 cirrhotic and 10 non-cirrhotic livers during surgery. EC concentrations were measured by liquid chromatography-mass spectroscopy. Systemic hemodynamics were assessed by trans-thoracic electrical bioimpedance. Results: AEA, but not 2-AG levels of cirrhotics were higher than controls in peripheral plasma (1.78±0.24vs1.13±0.15 pmol/mg lipid, p = 0.034) and, to a lower extent, in liver tissue. Circulating and hepatic EC-related molecules were also increased in cirrhosis (plasma OEA: 9.9±1.6vs2.3±0.2 pmol/mg lipid, p < 0.001; plasma PEA: 50.6±5.3vs23.2±6.5 pmol/mg lipid, p < 0.001). In cirrhotic pts, AEA, OEA and PEA were significantly lower in hepatic than peripheral veins, possibly because the liver more efficiently degrades these molecules than other organs and/or these compounds by-pass the liver through the portosystemic shunts. Finally, circulating AEA, OEA and PEA significantly correlated with liver function tests, while only the hepatic vein AEA levels correlated with HVPG. Conclusions: These data show that the EC system is up-regulated in cirrhosis. The circulating AEA levels are increased and, particularly in hepatic veins, may reflect liver dysfunction and portal hypertension. In contrast, the 2-AG levels, the other major EC, are not affected by cirrhosis. The pattern of OEA and PEA, with no activity at cannabinoid receptor sites, but capable to activate PPAR-a and TRPV1 receptors, mirrors that of AEA. However, the meaning of the latter finding remains elusive, prompting investigations on these novel compounds.
740 REACTIVE OXYGEN SPECIES FROM NAD(P)H OXIDASE CONTRIBUTE CARDIAC DYSFUNCTION IN CIRRHOTIC RATS G. Ceolotto1 , A. Bortoluzzi1 , I. Papparella1 , M. Cavalli2 , A. Sticca1 , S. Bova2 , A. Semplicini1 , A. Gatta1 , P. Angeli1 . 1 Clinical and Experimental Medicine, 2 Pharmacology and Anaesthesiology, University of Padova, Padova, Italy E-mail: [email protected] Background and Aim: Cirrhotic cardiomiopathy is characterized by diastolic dysfunction, electrophysiological abnormalities and by a reduced b-adrenergic contractility. Among the pathogenic mechanisms, an important role is played by oxidative stress due to excessive production of reactive oxygen species (ROS). ROS generation is mainly derived from NAD(P)H oxidase, formed by 2 membrane subunits p22phox and gp91phox and by 3 cytosolic subunits: p47phox , p67phox and Rac-1. Recently, it has shown that ROS may attenuate the b-adrenergic cardiac signal transduction by altering the function of adenylyl cyclase and G protein alpha inhibiting subunit 2 (Gai2 ), with a mechanism not completely clear. Aim of our study was to verify whether the abnormal contractile response to b adrenergic agonists may be related to an overactivation of NAD(P)H in an animal model of cirrhosis. Methods: 12 Wistar Kyoto rats were treated for 15 weeks by inhalation with carbon tetrachloride (CCl4 ) to induce cirrhosis and 12 rats were used as control animals. Left ventricular contractility was determined in isolated hearts with a b-agonist, isoproterenol. NAD(P)H oxidase enzymatic activity was measured by cytochrome c reduction assay. The activation and expression of NAD(P)H oxidase subunits were determined by Western Blot analysis with specific antibodies. b adrenergic receptors (b1 -AR and b2 -AR), Gai2 were evaluated by PCR real time.
Results: Cardiac contractility induced by isoproterenol (10−6 M) was significantly reduced in the left ventricular tissue of cirrhotic rats in comparison to controls (p < 0.01). In cardiac tissue, NAD(P)H oxidase activity was significantly increased in cirrhotic in comparison to control rats (3.4±0.3 mU/mg vs 1.2±0.2 mU/mg protein, p < 0.01). Furthermore, Rac-1 traslocation from cytosol to membranes was increased by 3 fold in cirrhotic in comparison to control rats (p < 0.01). No differences were seen for the other NAD(P)H subunits. Finally, we found an overexpression of Gai2 in cardiac tissue from cirrhotic rats, while b1 -AR and b2 -AR gene expression were unchanged. Conclusion: These results indicate that the overactivation of Rac-1 subunit may be important in the pathophysiology of cardiac dysfunction by contributing to alter the b-adrenergic system at a post-receptor level in a model of cirrhosis. 741 PHARMACOLOGICAL EFFECTS OF ACETYL SALICYLIC ACID, SULFASALAZINE, IBUPROFEN AND CELECOXIB ON LIVER FIBROSIS INDUCED BY CHRONIC ADMINISTRATION OF CCL4 E. Ch´avez, M.G. Moreno, P. Muriel. Pharmacology, CINVESTAV-IPN, Distrito Federal, Mexico E-mail: [email protected] Liver fibrosis is the accumulation of extracellular matrix proteins including collagen. Is now known that nucler factor-úB (NF-úB) activity has an important role in the mechanism of liver injury regulating transcription of several genes like profibrogenic cytokine transforming growth factor-b (TGF-b) and the enzyme cyclooxygenase (COX)-2. There is evidence of an increment of both, NF-úB binding activity and COX-2 expression in liver fibrosis induced by chronic administration of CCl4 in rats. The aim of this work was to evaluate the pharmacological effect of sulfasalazine (SF), acetyl salicylic acid (ASA), ibuprofen (IBP) and celecoxib (CLC), which inhibit NF-úB and/or COX, in CCl4 liver fibrosis. Ten groups were formed: control group, which received the vehicles only; CCl4 group that received the toxin (0.4g/kg, i.p., three times per week, for eight weeks); CCl4 plus SF (100 mg/kg, daily); CCl4 plus ASA (100 mg/kg, daily); CCl4 plus IBP (30 mg/kg, daily); CCl4 plus CLC (6 mg/kg, daily) and the last four corresponding to control drugs groups. Enzyme activity of alanine aminotransferase (ALT), g-glutamyl transpeptidase (g-GTP), content of malondialdehyde (MDA) and collagen were determined as indicators of necrosis, cholestasis, oxidative stress and fibrosis, respectively. Masson’s trichromic stains of liver sections were performed to corroborate results of collagen content. ALT and g-GTP increased by CCl4 , all of drugs avoid the cholestatic damage but none of the drugs prevented the increse in ALT activity produced by CCl4 . We observed an important antioxidant effect of all drugs since they preserved normal values of MDA. CCl4 intoxication increased the collagen content about four-times over control. We found that ASA had partial but significant antifibrotic effect. SF, IBP and CLC preserved normal collagen content despite treatment with CCl4 . These results were corroborated by the histopathological analysis under light microscopy. Our findings indicate that drugs used in this study, possess a strong antifibrogenic effect in the CCl4 model of cirrhosis, probably due to inhibition of NF-úB and/or COX. Because these drugs are currently used in humans, clinical trials appear atractive. 742 SYMPATHETIC NERVE REGRESSION AS A POSSIBLE FACTOR CONTRIBUTING TO SUPERIOR MESENTERIC ARTERY VASODILATION IN EXPERIMENTAL PORTAL HYPERTENSION M. Coll1 , M. Martell1 , I. Raurell1 , J. Bosch2 , J. Genesc`a1 . 1 Hospital Unversitari Vall d’Hebron, Institut de Recerca, Universitat Aut`onoma de Barcelona, 2 Hospital Clinic de Barcelona. IMDiM. IDIBAPS. Ciberehd. University of Barcelona., Barcelona, Spain E-mail: [email protected] Background: Portal hypertension induces down-regulation of mRNA and proteins involved in adrenergic transmission in the superior mesenteric
737 HISTOLOGICAL AND NON-INVASIVE QUANTITATIVE EVALUATION OF FIBROSIS PROGRESSION IN CHRONIC LIVER DISEASES P. Cales, S. Michalak, I. Valo, M.C. Rousselet, M. Ettalbi, J. Boursier, Y. Gallois, F. Oberti. CHU, Angers, France E-mail: [email protected] Background and Aims: Until now, progression of liver fibrosis has only been measured with pathological semi-quantitative staging. Recently, two meta-analyses have well described the transition between different Metavir fibrosis stages (Thein, Hepatology and AIDS 2008). Our aim was to evaluate the progression rate of fibrosis in a more sensitive way, especially thanks to the quantitative measurement of liver fibrosis via the area of fibrosis, in chronic liver diseases. Methods: Data were recorded in 201 patients with alcoholic or viral chronic liver diseases. Fibrosis was evaluated by Metavir F staging by two independent observers and the area of fibrosis by image analysis. Results: The mean (median) yearly rates of progression were 0.22±0.29 (0.13) MU for Metavir F staging and 1.8±2.6 (1.0) % for the area of fibrosis in patients with one liver biopsy (estimated progression with the starting date of cause) versus 0.17±0.27 (0.09) MU (p = 0.66) and 1.3±3.4 (1.2) % (p: 0.72), respectively, in patients having two biopsies. The progression of F stage and area of fibrosis was not linear, increased as a function of fibrosis F stage, and was faster in alcoholic chronic liver disease than in viral chronic liver disease only with the area of fibrosis. The independent predictors of Metavir F progression were: AST/ALT, cause duration, Metavir F stage and prothrombin index (a R2 : 0.605). Metavir F stage progression could be non-invasively and accurately predicted without any pathological variables (a R2 : 0.488). The independent predictors of area of fibrosis progression were: AST/ALT, cause duration, baseline area of fibrosis, and bglobulins (a R2 : 0.72); steatosis grade had a borderline significance (p: 0.057) but not activity grade (p: 0.53) or chronic liver disease cause (p: 0.39). The area of fibrosis progression could be noninvasively and accurately predicted without any pathological variables (a R2 : 0.649). The area of fibrosis was much more sensitive than Metavir F stage to detect changes due to anti-fibrotic treatment in another population.
S271
Conclusions: The area of fibrosis is more sensitive to evaluate fibrosis progression, and its pharmacological-induced variation, than classical histological semi-quantitative staging. Fibrosis progression is highly predictable by non-invasive scores that can be thus used in clinical practice. 738 BACTERIAL PEPTIDE IDENTIFICATION IN ASCITIC FLUID OF PATIENTS WITH ADVANCED CIRRHOSIS R. Ca˜no1 , P. Zapater2,3 , S. Pascual1,2 , P. Bellot1 , M. P´erez-Mateo1 , J. Such1,2 , R. Franc´es1,2 . 1 Unidad Hep´atica, Hospital General Universitario de Alicante, Alicante, 2 CIBERehd, Instituto de Salud Carlos III., Madrid, 3 Servicio de Farmacolog´ıa Cl´ınica, Hospital General Universitario de Alicante, Alicante, Spain E-mail: [email protected] Background: Bacterial-DNA is associated with the induction of a potent cellular immune response that may influence prognosis in patients with cirrhosis and ascites. However, intensity of the immune response against viable bacteria or bacterial products is frequently different. Aims: To evaluate the presence of bacterial peptides in ascitic fluid (AF) from patients with cirrhosis, as possible complementary markers of bacterial translocation. Methods: Analysis by two-dimensional poliacrylamide gel electrophoresis of AF total protein from patients with cirrhosis and of the most frequently isolated bacterial species in these patients. 2D-maps were digitally compared. The spots identification was performed by MALDI-TOF mass spectrometry. TNF-a, IFN-g, IL-12 and NOx levels were measured in AF samples by ELISA. All samples were inoculated in blood culture bottles. Results: A total of 37 patients with cirrhosis and ascites were included in the study and distributed into groups according to the absence (Group I) or presence (Group II) of bacterial DNA. All samples were culturenegative. No clinical or analytical differences were observed between groups. Bacterial peptides were found in 7 (Group II-b) out of 16 2D-maps from patients with presence of bactDNA (41%), whereas these peptides were not detected in any of the maps from patients without bactDNA. The identified bacterial peptides were GAPDH-A, Porin OmpC and HSP60. Differences in levels of cytokines and NOx are resume in Table 1. Conclusions: For the first time, bacterial peptides are identified in AF of patients with advanced cirrhosis, which implies the transient viability of bacteria, despite negativity of cultures. Their presence is associated with a significant increase in the soluble immune response. Validation of these peptides as markers of bacterial translocation and viability, as well as the clinical consequences of this finding will require further experimental work.
TNF-a (pg/ml) IFN-g (pg/ml) IL-12 (pg/ml) NOx (mmol/ml)
Group I (n = 21) BactDNA−
Group II-a (n = 9) Group II-b (n = 7) BactDNA+/BactProt− BactDNA+/BactProt+
169.3±42.1 227.3±63.5 342.7±73.3 15.1±3.8
427.2±132.8* 361.4±86.5* 720.9±114* 25.9±7.2*
587.9±74.0*# 419.3±76.1*# 935.6±149.8*# 28.4±4.7*#
*p < 0.05 compared with Group I; # p < 0.05 compared with Group II.
739 CIRCULATING AND HEPATIC ENDOCANNABINOIDS AND ENDOCANNABINOID-RELATED MOLECULES IN PATIENTS WITH LIVER CIRRHOSIS P. Caraceni1 , A. Viola1 , M. Domenicali1 , F. Piscitelli2 , S. Petrosino2 , F. Giannone1 , A. Berzigotti3 , M. Zoli3 , M. Cescon4 , G. Grazi4 , V. Di Marzo2 , M. Bernardi1 . 1 Clinical Medicine, University of Bologna, Bologna, 2 Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Naples, 3 Internal Medicine and Aging, University of Bologna, 4 Surgical Sciences, University of Bologna, Bologna, Italy E-mail: [email protected] Background and Aims: Endogenous cannabinoids (EC) include anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The EC-
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Poster Session − Saturday, April 25
related molecules, oleoyl-ethanolamine (OEA) and palmitoyl-ethanolamine (PEA), have been recently identified. AEA contributes to the pathogenesis of cardiovascular alterations in experimental cirrhosis, but data in humans are scarce. Thus, we assessed whether: 1. the EC system is up-regulated in cirrhosis by comparing plasma and hepatic EC levels in cirrhotic pts and controls; 2. the cirrhotic liver contributes to circulating EC by measuring their concentrations in brachial and hepatic veins; and 3. the EC levels correlate with systemic hemodynamics, portal pressure, liver and renal functions. Methods: Blood samples were collected from the brachial vein of 18 cirrhotic pts (Child–Pugh A/B/C:2/6/10) and 14 age- and sex-comparable controls, and from the hepatic veins during HVPG measurement in 8 cirrhotic pts. Tissue samples were also collected from 10 cirrhotic and 10 non-cirrhotic livers during surgery. EC concentrations were measured by liquid chromatography-mass spectroscopy. Systemic hemodynamics were assessed by trans-thoracic electrical bioimpedance. Results: AEA, but not 2-AG levels of cirrhotics were higher than controls in peripheral plasma (1.78±0.24vs1.13±0.15 pmol/mg lipid, p = 0.034) and, to a lower extent, in liver tissue. Circulating and hepatic EC-related molecules were also increased in cirrhosis (plasma OEA: 9.9±1.6vs2.3±0.2 pmol/mg lipid, p < 0.001; plasma PEA: 50.6±5.3vs23.2±6.5 pmol/mg lipid, p < 0.001). In cirrhotic pts, AEA, OEA and PEA were significantly lower in hepatic than peripheral veins, possibly because the liver more efficiently degrades these molecules than other organs and/or these compounds by-pass the liver through the portosystemic shunts. Finally, circulating AEA, OEA and PEA significantly correlated with liver function tests, while only the hepatic vein AEA levels correlated with HVPG. Conclusions: These data show that the EC system is up-regulated in cirrhosis. The circulating AEA levels are increased and, particularly in hepatic veins, may reflect liver dysfunction and portal hypertension. In contrast, the 2-AG levels, the other major EC, are not affected by cirrhosis. The pattern of OEA and PEA, with no activity at cannabinoid receptor sites, but capable to activate PPAR-a and TRPV1 receptors, mirrors that of AEA. However, the meaning of the latter finding remains elusive, prompting investigations on these novel compounds.
740 REACTIVE OXYGEN SPECIES FROM NAD(P)H OXIDASE CONTRIBUTE CARDIAC DYSFUNCTION IN CIRRHOTIC RATS G. Ceolotto1 , A. Bortoluzzi1 , I. Papparella1 , M. Cavalli2 , A. Sticca1 , S. Bova2 , A. Semplicini1 , A. Gatta1 , P. Angeli1 . 1 Clinical and Experimental Medicine, 2 Pharmacology and Anaesthesiology, University of Padova, Padova, Italy E-mail: [email protected] Background and Aim: Cirrhotic cardiomiopathy is characterized by diastolic dysfunction, electrophysiological abnormalities and by a reduced b-adrenergic contractility. Among the pathogenic mechanisms, an important role is played by oxidative stress due to excessive production of reactive oxygen species (ROS). ROS generation is mainly derived from NAD(P)H oxidase, formed by 2 membrane subunits p22phox and gp91phox and by 3 cytosolic subunits: p47phox , p67phox and Rac-1. Recently, it has shown that ROS may attenuate the b-adrenergic cardiac signal transduction by altering the function of adenylyl cyclase and G protein alpha inhibiting subunit 2 (Gai2 ), with a mechanism not completely clear. Aim of our study was to verify whether the abnormal contractile response to b adrenergic agonists may be related to an overactivation of NAD(P)H in an animal model of cirrhosis. Methods: 12 Wistar Kyoto rats were treated for 15 weeks by inhalation with carbon tetrachloride (CCl4 ) to induce cirrhosis and 12 rats were used as control animals. Left ventricular contractility was determined in isolated hearts with a b-agonist, isoproterenol. NAD(P)H oxidase enzymatic activity was measured by cytochrome c reduction assay. The activation and expression of NAD(P)H oxidase subunits were determined by Western Blot analysis with specific antibodies. b adrenergic receptors (b1 -AR and b2 -AR), Gai2 were evaluated by PCR real time.
Results: Cardiac contractility induced by isoproterenol (10−6 M) was significantly reduced in the left ventricular tissue of cirrhotic rats in comparison to controls (p < 0.01). In cardiac tissue, NAD(P)H oxidase activity was significantly increased in cirrhotic in comparison to control rats (3.4±0.3 mU/mg vs 1.2±0.2 mU/mg protein, p < 0.01). Furthermore, Rac-1 traslocation from cytosol to membranes was increased by 3 fold in cirrhotic in comparison to control rats (p < 0.01). No differences were seen for the other NAD(P)H subunits. Finally, we found an overexpression of Gai2 in cardiac tissue from cirrhotic rats, while b1 -AR and b2 -AR gene expression were unchanged. Conclusion: These results indicate that the overactivation of Rac-1 subunit may be important in the pathophysiology of cardiac dysfunction by contributing to alter the b-adrenergic system at a post-receptor level in a model of cirrhosis. 741 PHARMACOLOGICAL EFFECTS OF ACETYL SALICYLIC ACID, SULFASALAZINE, IBUPROFEN AND CELECOXIB ON LIVER FIBROSIS INDUCED BY CHRONIC ADMINISTRATION OF CCL4 E. Ch´avez, M.G. Moreno, P. Muriel. Pharmacology, CINVESTAV-IPN, Distrito Federal, Mexico E-mail: [email protected] Liver fibrosis is the accumulation of extracellular matrix proteins including collagen. Is now known that nucler factor-úB (NF-úB) activity has an important role in the mechanism of liver injury regulating transcription of several genes like profibrogenic cytokine transforming growth factor-b (TGF-b) and the enzyme cyclooxygenase (COX)-2. There is evidence of an increment of both, NF-úB binding activity and COX-2 expression in liver fibrosis induced by chronic administration of CCl4 in rats. The aim of this work was to evaluate the pharmacological effect of sulfasalazine (SF), acetyl salicylic acid (ASA), ibuprofen (IBP) and celecoxib (CLC), which inhibit NF-úB and/or COX, in CCl4 liver fibrosis. Ten groups were formed: control group, which received the vehicles only; CCl4 group that received the toxin (0.4g/kg, i.p., three times per week, for eight weeks); CCl4 plus SF (100 mg/kg, daily); CCl4 plus ASA (100 mg/kg, daily); CCl4 plus IBP (30 mg/kg, daily); CCl4 plus CLC (6 mg/kg, daily) and the last four corresponding to control drugs groups. Enzyme activity of alanine aminotransferase (ALT), g-glutamyl transpeptidase (g-GTP), content of malondialdehyde (MDA) and collagen were determined as indicators of necrosis, cholestasis, oxidative stress and fibrosis, respectively. Masson’s trichromic stains of liver sections were performed to corroborate results of collagen content. ALT and g-GTP increased by CCl4 , all of drugs avoid the cholestatic damage but none of the drugs prevented the increse in ALT activity produced by CCl4 . We observed an important antioxidant effect of all drugs since they preserved normal values of MDA. CCl4 intoxication increased the collagen content about four-times over control. We found that ASA had partial but significant antifibrotic effect. SF, IBP and CLC preserved normal collagen content despite treatment with CCl4 . These results were corroborated by the histopathological analysis under light microscopy. Our findings indicate that drugs used in this study, possess a strong antifibrogenic effect in the CCl4 model of cirrhosis, probably due to inhibition of NF-úB and/or COX. Because these drugs are currently used in humans, clinical trials appear atractive. 742 SYMPATHETIC NERVE REGRESSION AS A POSSIBLE FACTOR CONTRIBUTING TO SUPERIOR MESENTERIC ARTERY VASODILATION IN EXPERIMENTAL PORTAL HYPERTENSION M. Coll1 , M. Martell1 , I. Raurell1 , J. Bosch2 , J. Genesc`a1 . 1 Hospital Unversitari Vall d’Hebron, Institut de Recerca, Universitat Aut`onoma de Barcelona, 2 Hospital Clinic de Barcelona. IMDiM. IDIBAPS. Ciberehd. University of Barcelona., Barcelona, Spain E-mail: [email protected] Background: Portal hypertension induces down-regulation of mRNA and proteins involved in adrenergic transmission in the superior mesenteric