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174 Effects of arginine and nitric oxide synthase (NOS) inhibitors in thioacetamide-induced liver fibrosis reversal
Posters
70
on PMN cell counts in ascitic fluid. An alternative method for the early detection of infection using reactive urinalysis tapes has proved to be useful and to be well correlated with standard cytology, with the advantage of being faster, of lower cost and of easy execution. Obl eetives: To assess the usefulness of a reactive urinalysis tape (Mul fist.ix 10SG) for the diagnosis and therapeutic control of SBP in cirrhotic patients. Material and Methods: Ninety-six paracentesis procedures were performed in 58 cirrhotic patients with ascites over a period of 7 months. The results obtained with the reactive tape (Negative, traces, 1+, 2+, 3+) were compared with those obtained by standard cytolog3e. When SBP was diagnosed, comparison was also performed for therapeutic control. Results: Mean patient age was 54 years, the male sex predominated (72%), the most frequent etiologies of cirrhosis were alcohol ingestion (43%) and viral hepatitis (34%), andmost patients had severe liver disease (Child C 55%). Of the 96 paracenteses, 84 showed a neutrophil count <250/rnm 3, and the reactive tape was negative for all of them. Twelve episodes of SBP were diagnosed in 11 patients. The results concerning the SBP episodes are listed in the table. For the diagnosis of SBE the reactive tape showed 67% sensitivity, 100% specificity, 100% positive predictive value, 95% negative predictive value, and 92% diagnostic accuracy. SBP
1 2 3 4 5 6 7 8 9 10 11 12
At diagnosis MULTISTIX
PMN/mm3
After 48 hours MULTISTIX
1+ 3+ Negative 2+ Negative Negative 1+ 2+ Negative 2+ 3+ 2+
4935 170"28 1681 6191 1744 798 868 2893 1220 3977 4432 2990
1+ 1+ Negative DEATH Negative Traces 1+ Negative Negative Negative 1+ 1+
PMNhnm~ 3320 3640 336 116 504 3029 357 390 199 1691 3087
Conclusions: The reactive urinalysis tape Multistix 10SG showed unsatisfactory results for the early diagnosis and therapeutic control of SBP and does not seen) to be a good test for SBP screening. A positive result obtained with the reactive tape indicates the need for antibiotic treatment.
1•
both constitutively expressed NOS (eNOS) and inducible NO-synthase (iNOS) (20 mg/kg, i.g.) and aminoguanidine (AG) as the specific inhibitor of iNOS ( 1 0 0 m g ~ , i.g.). The severity of liver fibrosis was assessed by morphometric evaluation of liver slides stained with Azan-Mallory, hydroxyproline (Hyp) determination and rnRNA steady state levels of collagen I, TGF-bl, MMP-13, -14, TIMP-1 and PAI-1 were quantified by real-time PCR. Serum marker enzyme activities, AsAT, A1AT and alkaline phosphatase, were measured. Results: The TAA treatment during 3 months induced micronodular liver fibrosis with the expressed deposition of collagen fibers. L-Arginine did not affect this deposition and both relative and total liver Hyp contents whereas both NOS inhibitors significandy increased the square of liver connective tissue stained by Azan-Mallory and the above parameters characterizing liver Hyp content. Only both doses of L-arginine decreased AsAT activity, whereas the activity of alkaline phosphatase was diminished by a high dose of L-arginine. Both NOS inhibitors up-regulated collagen I, MMP-13, TIMP-1 and PAI-1 mRNAexpression where the effect of AG was significantly more pronounced. AG increased mRNA expression of TGF-bl and MMP-14. Conclusions: Both NOS inhibitors developed a clear profibrotic effect, where the effect of aminognanidine is more pronounced. Our data propose a significant antifibrotic role for iNOS rather than eNOS. L-Arginine acts more likely as a hepatoprotector, but not as an antifibrotic agent.
EFFECTS OF ARGININE AND NITRIC OXIDE S Y N T H A S E (NOS) INHIBITORS IN THIOACETAMIDEINDUCED LIVER FIBROSIS REVERSAL
O. LuldvskavaI , Y.U. Popov 1'2, E. Patsenker 1'2, R. Lis3, D. Schuppan 2, V Buko I . 1Department of Experimental Hepatology, Institute of
Biochemistry, Grodno, Belarus," 2Department of Medicine I, University of Erlangen, Erlangen, Germany," 3Grodno Medical University, Grodno, Belarus Nitric oxide (NO) is likely to be involved in the pathogenesis of liver fibrosis. Many authors believe that the pharmacological modulation o f NO synthesis is promising in future treatment o f liver fibrosis. However, there is considerable controversy in understanding the role of NO in fibrogenesis and fibrolysis. The aim of our study was to evaluate the effects of L-arginine, as NO metabolic precursor, and inhibitors of NOS on the thioacetamide (TAA)-induced liver fibrosis reversal. Methods: Male Wistar rats, 230-240g, received TAA (200mg/kg, i.p.) during 3 months, 2 times per week. After TAA withdrawal, the TAA-treated groups were daily administered with: L-arginine (100 and 300mg/kg); L-nitroarginine methyl ester (L-NAME) as the inhibitor of
1 1 ~ OPIOID-DEPENDENT MODULATION OF AUTONOMIC FUNCTION AND S-NITROSYLATION OF PROTEINS IN RATS WITH C HO LESTASIS M.R. Ebrahirnkhani I , S. Kiani I , S. Payabvash 1, A.R. Dehpour 1, K.E Moore 2, A.R. Mani 2. 1Department of Pharmacology, Tehran
University of Medical Sciences, Tehran, Iran; 2Department of Medicine, Royal Free & University College Medical School, UCL, London, UK Background: Administration of the opioid antagonist naltrexone increases cellular concentrations of glutathione (GSH) in acute cholestasis. Since cellular GSH is important in redox regulation of vascular function in liver disease, we tested the hypothesis that opiods may modulate cardiovascular responses in cholestatic liver disease. Aim: The aim of tile present study was to determine tile role of the endogenous opioid system on the abnormal cardiovascular responses observed in rats with acute cholestasis. Methods: Cholestasis was induced by bile duct ligation in rats. Naltrexone (20 rng/k4~ per day) was administered to cholestatic and sham-operated control animals for 1 week, at which point cardiovascular autonomic function was assessed using spectral analysis of heart rate variability (HRV). Liver and plasma samples were obtained for measurement of GSH, S-nitrosothiol and nitrotyrosine concentrations to assess nitrosative stress. Results: Cholestasis was associated with a significant increase in sympathetic activity, and a decrease ofvagal activity as assessed by HRV analysis (P < 0.05). Opioid receptor blockade improved the sympatho-vagal balance towards normal values. Acute bile duct ligation (1 week) was associated with a decrease of liver GSH/GSSH ratio (9.335-0.22 and 3.085-0.45, P < 0.01), and this was significantly attenuated following opioid-receptor blockade with naltrexone (P < 0.05). Hepatic nitrotyrosine levels increased in acute cholestasis, but did not increase in rats treated with naltrexone. The levels of hepatic S-nitrosothiols increased significantly in cholestatic animals (554-10 and 1475_28 pmol/g, P < 0.01), and these decreased significantly following opioid-receptor blockade. Conclusions: This is the first study to demonstrate that administration of an opioid antagonist is protective against nitrosative stress and improves sympatho-vagal balance in a rat model of cholestasis.
70
on PMN cell counts in ascitic fluid. An alternative method for the early detection of infection using reactive urinalysis tapes has proved to be useful and to be well correlated with standard cytology, with the advantage of being faster, of lower cost and of easy execution. Obl eetives: To assess the usefulness of a reactive urinalysis tape (Mul fist.ix 10SG) for the diagnosis and therapeutic control of SBP in cirrhotic patients. Material and Methods: Ninety-six paracentesis procedures were performed in 58 cirrhotic patients with ascites over a period of 7 months. The results obtained with the reactive tape (Negative, traces, 1+, 2+, 3+) were compared with those obtained by standard cytolog3e. When SBP was diagnosed, comparison was also performed for therapeutic control. Results: Mean patient age was 54 years, the male sex predominated (72%), the most frequent etiologies of cirrhosis were alcohol ingestion (43%) and viral hepatitis (34%), andmost patients had severe liver disease (Child C 55%). Of the 96 paracenteses, 84 showed a neutrophil count <250/rnm 3, and the reactive tape was negative for all of them. Twelve episodes of SBP were diagnosed in 11 patients. The results concerning the SBP episodes are listed in the table. For the diagnosis of SBE the reactive tape showed 67% sensitivity, 100% specificity, 100% positive predictive value, 95% negative predictive value, and 92% diagnostic accuracy. SBP
1 2 3 4 5 6 7 8 9 10 11 12
At diagnosis MULTISTIX
PMN/mm3
After 48 hours MULTISTIX
1+ 3+ Negative 2+ Negative Negative 1+ 2+ Negative 2+ 3+ 2+
4935 170"28 1681 6191 1744 798 868 2893 1220 3977 4432 2990
1+ 1+ Negative DEATH Negative Traces 1+ Negative Negative Negative 1+ 1+
PMNhnm~ 3320 3640 336 116 504 3029 357 390 199 1691 3087
Conclusions: The reactive urinalysis tape Multistix 10SG showed unsatisfactory results for the early diagnosis and therapeutic control of SBP and does not seen) to be a good test for SBP screening. A positive result obtained with the reactive tape indicates the need for antibiotic treatment.
1•
both constitutively expressed NOS (eNOS) and inducible NO-synthase (iNOS) (20 mg/kg, i.g.) and aminoguanidine (AG) as the specific inhibitor of iNOS ( 1 0 0 m g ~ , i.g.). The severity of liver fibrosis was assessed by morphometric evaluation of liver slides stained with Azan-Mallory, hydroxyproline (Hyp) determination and rnRNA steady state levels of collagen I, TGF-bl, MMP-13, -14, TIMP-1 and PAI-1 were quantified by real-time PCR. Serum marker enzyme activities, AsAT, A1AT and alkaline phosphatase, were measured. Results: The TAA treatment during 3 months induced micronodular liver fibrosis with the expressed deposition of collagen fibers. L-Arginine did not affect this deposition and both relative and total liver Hyp contents whereas both NOS inhibitors significandy increased the square of liver connective tissue stained by Azan-Mallory and the above parameters characterizing liver Hyp content. Only both doses of L-arginine decreased AsAT activity, whereas the activity of alkaline phosphatase was diminished by a high dose of L-arginine. Both NOS inhibitors up-regulated collagen I, MMP-13, TIMP-1 and PAI-1 mRNAexpression where the effect of AG was significantly more pronounced. AG increased mRNA expression of TGF-bl and MMP-14. Conclusions: Both NOS inhibitors developed a clear profibrotic effect, where the effect of aminognanidine is more pronounced. Our data propose a significant antifibrotic role for iNOS rather than eNOS. L-Arginine acts more likely as a hepatoprotector, but not as an antifibrotic agent.
EFFECTS OF ARGININE AND NITRIC OXIDE S Y N T H A S E (NOS) INHIBITORS IN THIOACETAMIDEINDUCED LIVER FIBROSIS REVERSAL
O. LuldvskavaI , Y.U. Popov 1'2, E. Patsenker 1'2, R. Lis3, D. Schuppan 2, V Buko I . 1Department of Experimental Hepatology, Institute of
Biochemistry, Grodno, Belarus," 2Department of Medicine I, University of Erlangen, Erlangen, Germany," 3Grodno Medical University, Grodno, Belarus Nitric oxide (NO) is likely to be involved in the pathogenesis of liver fibrosis. Many authors believe that the pharmacological modulation o f NO synthesis is promising in future treatment o f liver fibrosis. However, there is considerable controversy in understanding the role of NO in fibrogenesis and fibrolysis. The aim of our study was to evaluate the effects of L-arginine, as NO metabolic precursor, and inhibitors of NOS on the thioacetamide (TAA)-induced liver fibrosis reversal. Methods: Male Wistar rats, 230-240g, received TAA (200mg/kg, i.p.) during 3 months, 2 times per week. After TAA withdrawal, the TAA-treated groups were daily administered with: L-arginine (100 and 300mg/kg); L-nitroarginine methyl ester (L-NAME) as the inhibitor of
1 1 ~ OPIOID-DEPENDENT MODULATION OF AUTONOMIC FUNCTION AND S-NITROSYLATION OF PROTEINS IN RATS WITH C HO LESTASIS M.R. Ebrahirnkhani I , S. Kiani I , S. Payabvash 1, A.R. Dehpour 1, K.E Moore 2, A.R. Mani 2. 1Department of Pharmacology, Tehran
University of Medical Sciences, Tehran, Iran; 2Department of Medicine, Royal Free & University College Medical School, UCL, London, UK Background: Administration of the opioid antagonist naltrexone increases cellular concentrations of glutathione (GSH) in acute cholestasis. Since cellular GSH is important in redox regulation of vascular function in liver disease, we tested the hypothesis that opiods may modulate cardiovascular responses in cholestatic liver disease. Aim: The aim of tile present study was to determine tile role of the endogenous opioid system on the abnormal cardiovascular responses observed in rats with acute cholestasis. Methods: Cholestasis was induced by bile duct ligation in rats. Naltrexone (20 rng/k4~ per day) was administered to cholestatic and sham-operated control animals for 1 week, at which point cardiovascular autonomic function was assessed using spectral analysis of heart rate variability (HRV). Liver and plasma samples were obtained for measurement of GSH, S-nitrosothiol and nitrotyrosine concentrations to assess nitrosative stress. Results: Cholestasis was associated with a significant increase in sympathetic activity, and a decrease ofvagal activity as assessed by HRV analysis (P < 0.05). Opioid receptor blockade improved the sympatho-vagal balance towards normal values. Acute bile duct ligation (1 week) was associated with a decrease of liver GSH/GSSH ratio (9.335-0.22 and 3.085-0.45, P < 0.01), and this was significantly attenuated following opioid-receptor blockade with naltrexone (P < 0.05). Hepatic nitrotyrosine levels increased in acute cholestasis, but did not increase in rats treated with naltrexone. The levels of hepatic S-nitrosothiols increased significantly in cholestatic animals (554-10 and 1475_28 pmol/g, P < 0.01), and these decreased significantly following opioid-receptor blockade. Conclusions: This is the first study to demonstrate that administration of an opioid antagonist is protective against nitrosative stress and improves sympatho-vagal balance in a rat model of cholestasis.