The influence of erythropoietin on apoptosis and fibrosis in the early phase of chronic pancreatitis in rats.

INTRODUCTION: Chronic pancreatitis (CP) is a continuing, inflammatory process of the pancreas, characterised by irreversible morphological changes. The identification of pancreatic stellate cells resulted in the development of research on the pathogenesis of CP. Erythropoietin (Epo) regulates the interaction between apoptosis and inflammation of the brain, kidney, and heart muscle. Erythropoietin receptors were also found in the pancreas, in particular on the islet cells. Our objective was to evaluate the influence of Epo on fibrosis and apoptosis in experimental CP. MATERIAL AND METHODS: The experiments were performed on 48 male Wistar rats (250-350 g). The animals were divided into six equal groups (I - control, II - chronic cerulein - induced pancreatitis, III - 1 ml of Epo sc, IV - 0.5 ml of Epo sc, V - CP treated with 1 ml Epo, VI - CP treated with 0.5 ml Epo). The blood for gelatinases and pancreata for the morphological examinations and immunohistochemistry were collected. RESULTS: A slight reduction of interstitial oedema and less severe fibrosis were noticed in the groups treated with Epo. Reduced expression of caspase-3 and α-actin, and a lack of Bcl-2 expression were observed in areas with inflammation. There was no expression of caspase-9 observed in all groups. There were no statistically significant differences between the groups in the activity of gelatinases. CONCLUSIONS: Erythropoietin seems to have the effect of reducing fibrosis and apoptosis in an experimental model of CP.

The effect of exenatide (a GLP-1 analog) and sitagliptin (a DPP-4 inhibitor) on plasma platelet-activating factor acetylhydrolase (PAF-AH) activity and concentration in normal and fructose-fed rats.

Inflammation and oxidative stress are the two processes crucial in atherogenesis. Platelet-activating factor acetylhydrolase (PAF-AH), a plasma lipoprotein-associated enzyme, degrades pro-inflammatory lipids generated within oxidatively modified lipoproteins. Extensive evidence shows that incretin-based drugs, a new class of anti-diabetic agents, can provide cardiovascular protection that cannot be attributed to their glucose-lowering effects. The present study was undertaken to determine whether the antiatherogenic effects of the GLP-1(glucagon-like peptide-1) receptor agonist (exenatide) and DPP-4(dipeptidyl peptidase-4) inhibitors (sitagliptin) may occur via the regulation of platelet-activating factor acetylhydrolase (PAF-AH) activity/mass and inhibition of low-density lipoprotein (LDL) oxidation in the fructose-fed rats. Normal and fructose-fed rats (8 wk) were treated (4 wk) with sitagliptin (5 and 10 mg/kg p.o.) or with exenatide (5 and 10 µg/kg, s.c.). Plasma PAF-AH activity and phosphatidylcholine (PC) concentration were measured colorimetrically. Plasma PAF-AH concentration, oxidized LDL (oxLDL), hexanoyl-Lys adduct (HEL), lyso-PC, apolipoprotein A-I (apoA-I), apoB, platelet-activating factor (PAF), monocyte chemoattractant protein-1 (MCP-1) and endothelin-1 (ET-1) were measured by ELISA. The four-week exenatide (5 µg/kg, sc.) treatment of fructose fed-rats significantly increased plasma PAF-AH activity (+33%, P < 0.001) and decreased the level of circulating oxLDL (-42%, P < 0.05) and MCP-1 (-23%, P < 0.01). These changes were accompanied by the decrease in plasma PC/lyso-PC (-47%, P < 0.001) and apoB/apoA-I ratio (-75%, P < 0.001). The effect of exenatide on enzyme activity was associated with only a minor effect on metabolic parameters and was independent of weight reduction. Exenatide but not sitagliptin inhibits oxidative modification of LDL probably due to favorable effect on plasma PAF-AH activity.

The paraoxonase 1 (PON1), platelet-activating factor acetylohydrolase (PAF-AH) and dimethylarginine dimethylaminohydrolase (DDAH) activity in the metformin treated normal and diabetic rats.

Antidiabetic agents per se, apart from their glucose-lowering effect, can have an important impact on modifying the cardiovascular risk. The present study was undertaken to determine whether the known cardio-protective effects of metformin are linked to its potential ability to affect activities of HDL's paraoxonase (PON1) and platelet activating factor acetylohydrolase (PAF-AH) or via its interaction with the asymmetric dimethylarginine (ADMA)- dimethylarginine dimethylaminohydrolase (DDAH) axis. Normal and streptozotocin (STZ)-induced diabetic rats were treated with metformin (300mg/kg; 4 weeks). The activity of PON1, PAF-AH and DDAH were measured spectrophotometrically. The plasma ADMA level was determined by ELISA method. In STZ-induced diabetic rats the long-term administration of metformin normalized reduced PON1 activity assayed toward paraoxon (+42.5%, P<0.05), phenyl acetate (+22.35%, P<0.05) and γ-decanolactone (+108.0%, P<0.01), without affecting elevated PAF-AH activity in the plasma. Moreover, metformin increased DDAH activity in the renal cortex (+38.24%, P<0.01). Additionally metformin administration caused the increase in PON1 activity in the liver (+29.2%, P<0.01) accompanied by the reduction in the lipid peroxidation (-59.8%, P<0.001). Similarly, in non-diabetic treated rats the increase in liver PON1 activity was observed toward both paraoxon (+80.19%, P<0.001) and phenyl acetate (+29.3%, P<0.05), respectively. The present study has demonstrated that insulin-sensitizer metformin is important for preserving antioxidant HDL function in diabetes. Metformin might also exert its effect against diabetic complications by improving DDAH activity in the kidney and increasing PON1 activity in the liver.

Oxidative Stress and Liver Morphology in Experimental Cyclosporine A-Induced Hepatotoxicity.

Cyclosporine A is an immunosuppressive drug used after organ's transplantation. The adverse effects on such organs as kidney or liver may limit its use. Oxidative stress is proposed as one of the mechanisms of organs injury. The study was designed to elucidate CsA-induced changes in liver function, morphology, oxidative stress parameters, and mitochondria in rat's hepatocytes. Male Wistar rats were used: group A (control) receiving physiological saline, group B cyclosporine A in a dose of 15 mg/kg/day subcutaneously, and group C the CsA-vehicle (olive oil). On the 28th day rats were anesthetized. The following biochemical changes were observed in CsA-treated animals: increased levels of ALT, AST, and bilirubin in the serum, statistically significant changes in oxidative stress parameters, and lipid peroxidation products in the liver supernatants: MDA+4HAE, GSH, GSSG, caspase 3 activity, and ADP/ATP, NAD(+)/NADH, and NADP(+)/NADPH ratios. Microscopy of the liver revealed congestion, sinusoidal dilatation, and focal hepatocytes necrosis with mononuclear cell infiltration. Electron microscope revealed marked mitochondrial damage. Biochemical studies indicated that CsA treatment impairs liver function and triggers oxidative stress and redox imbalance in rats hepatocytes. Changes of oxidative stress markers parallel with mitochondrial damage suggest that these mechanisms play a crucial role in the course of CsA hepatotoxicity.

The genetic predisposition and its impact on the diabetes mellitus development in patients with alcoholic chronic pancreatitis.

The most common cause of chronic pancreatitis (CP) is alcohol abuse. The aim of the present study was to identify patients with genetic predisposition to CP abusing alcohol. The question posed was whether CP manifests at a younger age and diabetes mellitus develops earlier in individuals with genetic predisposition. The study encompassed 79 patients with alcoholic chronic pancreatitis (ACP) and control group (100 persons). The following mutations were determined: R122H and N29I of PRSS1 and N34S of SPINK1 as well as E366K and E288V of SERPINA 1. No R122H and N291 mutations were observed in the group of ACP patients and in controls. Moreover, there was no E288V mutation. In 79 ACP patients, six SPINK 1 (N34S/wt) mutations were observed. In the control group, one heterozygous SPINK 1N34S gene mutation was found (P = 0.0238). Two PiZ mutations were identified in patients with ACP and one analogical mutation in controls. Amongst patients with ACP as well as SPINK1 and PiZ mutations, the onset of disease was observed earlier and developed earlier. The prevalence of SPINK1 mutation is higher in patients with ACP than in healthy populations. This mutation together with the effects of alcohol accelerates the development of ACP and of diabetes mellitus.

Role of MMP-2 and MMP-9 and their natural inhibitors in liver fibrosis, chronic pancreatitis and non-specific inflammatory bowel diseases.

BACKGROUND: There is a growing evidence that matrix metalloproteinase (MMP)-2 and MMP-9 (gelatinases) play an important role in the pathogenesis of numerous disorders, especially with inflammatory etiology and extracellular matrix (ECM) remodeling. Despite the fact that gelatinases involve in liver cirrhosis is provided in the literature, their role in the pathogenesis of chronic pancreatitis and non-specific inflammatory bowel diseases is still under investigation. DATA SOURCES: We carried out a PubMed search of English-language articles relevant to the involvement of gelatinases in the pathogenesis of liver fibrosis, pancreatitis, and non-specific inflammatory bowel diseases. RESULTS: The decreased activity of gelatinases, especially MMP-2, is related to the development of liver fibrosis, probably due to the decrease of capability for ECM remodeling. Similar situation can be found in chronic pancreatitis; however, reports on this matter are rare. The presence of non-specific inflammatory bowel diseases results in MMP-9 activity elevation. CONCLUSION: The fluctuation of gelatinases activity during liver fibrosis, chronic pancreatitis and non-specific inflammatory bowel diseases is observed, but the exact role of these enzymes demands further studies.

The effects of the adenosine A3 receptor agonist IB-MECA on sodium taurocholate-induced experimental acute pancreatitis.

The role of adenosine A3 receptors and their distribution in the gastrointestinal tract have been widely investigated. Most of the reports discuss their role in intestinal inflammations. However, the role of adenosine A3 receptor agonist in pancreatitis has not been well established. The aim of this study is [corrected] to evaluate the effects of the adenosine A3 receptor agonist on the course of sodium taurocholate-induced experimental acute pancreatitis (EAP). The experiments were performed on 80 male Wistar rats, 58 of which survived, subdivided into 3 groups: C--control rats, I--EAP group, and II--EAP group treated with the adenosine A3 receptor agonist IB-MECA (1-deoxy-1-6[[(3-iodophenyl) methyl]amino]-9H-purin-9-yl)-N-methyl-B-D-ribofuronamide at a dose of 0.75 mg/kg b.w. i.p. at 48, 24, 12 and 1 h before and 1 h after the injection of 5% sodium taurocholate solution into the biliary-pancreatic duct. Serum for α-amylase and lipase determinations and tissue samples for morphological examinations were collected at 2, 6, and 24 h of the experiment. In the IB-MECA group, α-amylase activity was decreased with statistically high significance compared to group I. The activity of lipase was not significantly different among the experimental groups but higher than in the control group. The administration of IB-MECA attenuated the histological parameters of inflammation as compared to untreated animals. The use of A3 receptor agonist IB-MECA attenuates EAP. Our findings suggest that stimulation of adenosine A3 receptors plays a positive role in the sodium taurocholate-induced EAP in rats.

The decrease of serum MMP-2 activity corresponds to alcoholic cirrhosis stage.

Because of numerous limitations for liver biopsy, a noninvasive marker of liver cirrhosis is sought. Promising indicators seem to be matrix metalloproteinases (MMPs) that are responsible for degradation of extracellular matrix. The aim of the study was to evaluate the gelatinase activities (MMP-2 and MMP-9) in patients with different stages of alcoholic cirrhosis. Sixty-seven outpatients who presented various stages of alcoholic cirrhosis according to Child-Turcotte-Pugh criteria and 26 healthy control subjects were enrolled. Blood samples were collected for MMP-2 and MMP-9 activities. A significant decrease of serum MMP-2 activity was noted in stages B and C of cirrhosis in comparison with control. Serum MMP-9 activity did not depend on the stage of cirrhosis. The MMP-2 levels, but not those of MMP-9, may be of value in understanding the pathogenesis and progression of alcoholic cirrhosis.

Erythropoietin inhibits liver gelatinases during galactosamine-induced hepatic damage in rats.

Matrix metalloproteinase (MMP)-2 and -9 (gelatinases) participate in extracellular protein remodeling. Moreover, they are involved in the development of hepatic fibrosis. The goal of this study was to evaluate liver gelatinase activities after erythropoietin (Epo) treatment (1U/dose, sc) in experimentally damaged livers of rats treated with D-galactosamine (Gal, 800 mg/kg/dose, ip). Sixty rats were divided into six equal groups: I - received 5 doses of Epo and a single dose of Gal [the experiment duration (ED): 10 days]; II - received 5 doses of Epo and 3 doses of Gal (ED: 14 days); III - received only 5 doses of Epo (ED: 9 days); IV - received 3 doses of Gal (ED: 5 days);V - received a single dose of Gal (ED: 1 day); VI - control group (ED: 9 days). The animals were sacrificed and the livers were collected 48 h after the last drug administration. The activity of gelatinases was measured using gelatin zymography. No fluctuations in gelatinase activities were observed after the administration of a single dose of Gal in comparison to the control group. However, a significant increase in gelatinase activities was observed after treatment with three doses of Gal. Five doses of Epo administrated before Gal treatment prevented elevated gelatinase activities: MMP-9 activity was comparable to control, and MMP-2 activity was decreased (group II). The gelatinase activities was lower in group I and II in comparison to the control group. These results revealed that Epo decreases MMP-2 and MMP-9 activity, suggesting that it is a hepatoprotective agent against hepatic damage induced by galactosamine injection.

Catalase, superoxide dismutase, and glutathione peroxidase activities in various rat tissues after carbon tetrachloride intoxication.

BACKGROUND: The aim of this study was to determine the possible relationship between the activity of three different antioxidant enzymes--peroxidase superoxide dismutase, catalase, and glutathione peroxidase--and carbon tetrachloride-induced injury. METHODS: Male Wistar rats weighing 200-250 g were used in the experiments. Rats of the experimental groups were given carbon tetrachloride 0.5 ml/kg i.p. in olive oil (5 mmol/kg body mass) for 1 or 3 days. Control group rats were injected with olive oil only for the same period. Brain, liver, kidney, and heart supernatants were used for measurement of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) activities. RESULTS: No statistically significant changes in SOD and GPX activities were observed in the liver after CCl4 administration, but catalase activity was significantly increased after 24 h and remained at that level during the course of the study. In the brain, SOD and catalase activities decreased after 24 h of experiment, but GPX activity statistically significantly increased at all time points studied. Increased activities of SOD, catalase, and GPX were found in heart after CCl4 intoxication. The CCl4 injection in our experiment caused a reduction of SOD and catalase activities and increased GPX activity in the kidney. CONCLUSIONS: The results suggest that change in antioxidant enzyme activities may be relevant to the ability of the liver and other investigated organs to cope with oxidative stress during CCl4 poisoning.

The influence of interferon alpha on the rat liver injured by chronic administration of carbon tetrachloride.

Due to their complex and not fully known etiopathogenesis as well as difficulties in treatment, chronic hepatitis and cirrhosis still remain one of the main problems of hepatologists. Nowadays, the use of IFN alpha is considered the most effective method of treatment in chronic hepatitis. Recently, a new property of IFN, i.e. its effects on the reduction of fibrosis, has been discovered. The aim of the paper was to examine the effects of IFN alpha on biochemical parameters (AlAt and AspAt activities), on the metabolic function of the liver and its morphologic picture observed under the light and electron microscope after the 3- and 6-week CCl4-induced damage. The experiments were carried out in Wistar male rats. To evaluate the liver function, the test of aminophenazone elimination in the isolated perfused rat livers was used according to Miller modified by Hafte. Additionally, AspAt and AlAt activities were determined. The liver specimens were analysed under the light and electron microscope and using immunohistochemical methods. The findings show that after the 3-week CCl4-induced liver damage, IFN alpha does not significantly affect AlAt and AspAt activities, irrespective of the dose used. IFN alpha administered after the 6-week damage significantly changes those activities when the doses used are high. It was found that carbon tetrachloride does not result in evident cirrhotic changes, however it activates Ito cells, causes focal retraction of the stroma and fibrosis. The increased number of Ito cells in Disse's space observed in immunohistochemical and ultrastructural examinations is indicative of the activation of liver fibrotic processes following CCl4 administration in both variants used. IFN alpha substantially weakens fibrogenesis of the CCl4-damaged liver which is visible in the decreased number of Ito cells and weaker expression of the stroma retraction. Moreover, IFN alpha administered to the experimental animals after the CCl4-induced injury of the liver increases aminophenazone clearance, especially when used in higher doses. Positive effects of IFN confirmed in the studies suggest that the drug may be used in patients with chronic hepatitis and early cirrhosis since it is likely not only to eliminate the virus but also to improve the liver function and reduce fibrosis.