The effect of exenatide (a GLP-1 analogue) and sitagliptin (a DPP-4 inhibitor) on asymmetric dimethylarginine (ADMA) metabolism and selected biomarkers of cardiac fibrosis in rats with fructose-induced metabolic syndrome.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, is a risk factor for endothelial dysfunction, a common pathophysiological denominator for both atherogenesis and cardiac fibrosis. We aimed to investigate whether the cardioprotective and antifibrotic effects of incretin drugs, exenatide and sitagliptin, may be associated with their ability to affect circulating and cardiac ADMA metabolism. Normal and fructose-fed rats were treated with sitagliptin (5.0/10 mg/kg) or exenatide (5/10 µg/kg) for 4 weeks. The following methods were used: LC-MS/MS, ELISA, Real-Time-PCR, colorimetry, IHC and H&E staining, PCA and OPLS-DA projections. Eight-week fructose feeding resulted in an increase in plasma ADMA and a decrease in NO concentration. Exenatide administration into fructose-fed rats reduced the plasma ADMA level and increased NO level. In the heart of these animals exenatide administration increased NO and PRMT1 level, reduced TGF-ß1, α-SMA levels and COL1A1 expression. In the exenatide treated rats renal DDAH activity positively correlated with plasma NO level and negatively with plasma ADMA level and cardiac α-SMA concentration. Sitagliptin treatment of fructose-fed rats increased plasma NO concentration, reduced circulating SDMA level, increased renal DDAH activity and reduced myocardial DDAH activity. Both drugs attenuated the myocardial immunoexpression of Smad2/3/P and perivascular fibrosis. In the metabolic syndrome condition both sitagliptin and exenatide positively modulated cardiac fibrotic remodeling and circulating level of endogenous NOS inhibitors but had no effects on ADMA levels in the myocardium.

The role of PPAR gamma agonists - rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2 in experimental cyclosporine A hepatotoxicity.

Cyclosporine A (CsA) is an immunosuppressive drug used in transplantation and treatment of autoimmune diseases. Experimental studies revealed impairments in liver function and morphology among cyclosporine-treated animals. The aim of the study was to evaluate hepatoprotective activity of peroxisome-proliferator-activated receptors γ (PPARγ) ligands: rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2 (PGDJ2) on CsA-induced hepatotoxicity in experimental animals. CsA was administered subcutaneously at a dose of 15 mg/kg/day for 28 days. Both PPARγ agonists were given for 28 days 0.5 hour before the administration of CsA. Rosiglitazone was administered orally at a dose of 8 mg/kg/day and PGDJ2 was given intraperitoneally at a dose of 30 µg/kg/day. CsA induced liver injury was evidenced by increased serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin. Concentrations of glutathione (GSH) and glutathione disulfide (GSSG), lipid peroxidation products, nicotinamide adenine dinucleotide+/nicotinamide adenine dinucleotide hydrogen (NAD+/NADH), nicotinamide adenine dinucleotide phosphate+/nicotinamide adenine dinucleotide phosphate hydrogen (NADP+/NADPH) and adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratios and caspase 3 activity that were measured in the liver tissue showed, that CsA induced oxidative stress, evoked an imbalanced redox state and apoptosis in the liver. Microscope examination showed sinusoidal dilatation, mononuclear cell infiltration, necrosis of hepatocytes, intracellular vacuolar degeneration and microvesicular steatosis and apoptopic cells. The biochemical and morphological changes induced by CsA were limited by administration of both PPARγ agonist - rosiglitazone and PGDJ2. Our biochemical and liver histopathological examination indicate that both PPARγ agonists may play an important role in protecting against CsA-induced hepatotoxicity.

The effect of the angiotensin II receptor, type 1 receptor antagonists, losartan and telmisartan, on thioacetamide-induced liver fibrosis in rats.

It has been reported previously that the density of angiotensin II receptors is increased in the rat liver in experimentally-induced fibrosis. We hypothesized that pharmacological blockade of angiotensin receptors may produce beneficial effects in models of liver fibrosis. In this study, we used the widely used thioacetamide (TAA)-induced model of liver fibrosis (300 mg/L TAA ad libitum for 12 weeks). Rats received daily injections (i.p), lasting 4 weeks of the angiotensin II type 1 receptor antagonists, losartan 30 mg/kg (TAA + L) or telmisartan 10 mg/kg (TAA + T) and were compared to rat that received TAA alone. Chronic treatment with losartan and telmisartan was associated with a significant reduction in the activity of alkaline phosphatase, and decreased concentrations of tumor necrosis factor-alpha and transforming growth factor beta-1 compared to controls. We also found a significant reduction interleukin-6 in rats receiving telmisartan (P < 0.05) but not losartan. Both treatments increased the concentration of liver glutathione along with a concomitant decrease of GSSG compared to controls. In addition, increased paraoxonase 1 activity was observed in the serum of rats receiving telmisartan group compared to the TAA alone controls. Finally, histological evaluation of liver sections revealed losartan and telmisartan treatment was associated with reduced inflammation and liver fibrosis. Taken together, these results indicate that both telmisartan and losartan have anti-inflammatory and anti-oxidative properties in the TAA model of liver fibrosis. These finding add support to a growing body of literature indicating a potentially important role for the angiotensin system in liver fibrosis and indicate angiotensin antagonists may be useful agents for fibrosis treatment.

Effects of renin-angiotensin system inhibitors on fibrosis in patients with alcoholic chronic pancreatitis.

Chronic pancreatitis (CP) results in impairment of exocrine as well as endocrine functions and progressive fibrosis. Previous studies, have demonstrated the presence of renin-angiotensin system receptors within different pancreatic cells. The aim of the present study was to assess the effects of renin-angiotensin system (RAS) inhibitors on serum levels of fibrosis biomarkers (matrix metalloproteinase 2 and 9 (MMP-2, MMP-9), tissue inhibitor of MMP (TIMP- 1, TIMP-2), hyaluronic acid (HA)) and fasting glucose levels in patients with alcoholic CP. Seventy seven outpatients (mean age 43 years, 62 males) with diagnosed alcoholic CP were randomly enrolled into 5 study groups depending on the RAS inhibitors administered and their doses (2.5 or 5 mg and 12.5 or 25 mg for ramipril or losartan, respectively). Venous blood was sampled monthly for a period of one year to monitor serum drug levels. MMP-2, -9, TIMP-1, TIMP-2 and HA were measured with ELISA method on the onset and at the end of the study. Only forty five patients regularly participated in follow-up visits and completed the study. The fluctuations in serum HA levels observed among patients from the remaining groups also did not reach statistical significance. Serum MMP-2 levels (P = 0.06) and MMP-2/TIMP-1 ratio (P = 0.06) showed increasing tendency in the losartan 25 mg group. High doses of ramipril and losartan statistically significantly reduced fasting glucose levels. High doses of losartan can increase the MMP-2 activity in serum of alcoholic CP patients, which potentially is likely to affect turnover of extracellular matrix proteins within the pancreas. Moreover, high doses of both RAS inhibitors decrease the fasting glucose level.

Protective effects of melatonin against thioacetamide-induced liver fibrosis in rats.

The aim of this study was to determine the effect of melatonin on thioacetamide (TAA) induced liver fibrosis in rats. The antifibrotic effects of melatonin were assessed by determining activity indirect markers of fibrosis: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), and proinflammatory cytokines: interleukin 6 (IL-6), interleukin-1beta (IL-1ß), tumour necrosis factor alpha (TNF-α), transforming growth factor-beta (TGF-ß) and platelet-derived growth factor (PDGF). Parameters of oxidative stress: oxidised glutathione (GSSG), reduced glutathione (GSH) and presaged activity of paraoxonase 1 (PON-1), an antioxidative enzyme were determined. Inflammatory changes and fibrosis extent were evaluated histologically. Experiments were carried out in Wistar rats. Animals were divided into 4 groups: I - controls, water ad libitum for 12 weeks, group II - TAA, 300 mg/L ad libitum for 12 weeks, III - melatonin, 10 mg/kg b.w. intraperitoneally (i.p.) daily for 4 weeks, IV - TAA, 300 mg/L ad libitum for 12 weeks followed by melatonin, 10 mg/kg/b.w. i.p. daily for 4 weeks. Results of serum determinations demonstrated significantly lower activity of AST, ALT and AP in the group receiving TAA followed by melatonin compared to the group receiving only TAA. Immunoenzymatic findings on effect of melatonin on concentration of proinflammatory cytokines confirmed these data. Biochemical examinations in liver homogenates revealed statistically significant improvement (concentration of GSH increases and concentration of GSSG decreases) in animals with TAA-induced liver damage receiving melatonin. Moreover, the activity of PON-1 toward phenyl acetate and paraoxon was increased in liver homogenates and serum in the group receiving TAA followed by melatonin compared to the TAA group without melatonin treatment. Microscopic evaluation disclosed inhibitory effects of melatonin on inflammatory changes and extent of liver fibrosis.

The role of peroxisome-proliferator-activating receptor gamma agonists: rosiglitazone and 15-deoxy-delta12,14-prostaglandin J2 in chronic experimental cyclosporine A-induced nephrotoxicity.

Cyclosporine A(CsA) is an immunosuppressor frequently used in the transplant surgery and in the treatment of autoimmune diseases. The therapeutic benefits of CsA are often limited by it's main side effect-nephrotoxicity. Mechanisms of chronic CsA- induced renal damage include: activation of renin-angiotensin-aldosterone system, upregulation of transforming growth factor beta (TGF-ß), oxidative stress. This study was undertaken to investigate the protective effect of the peroxisome-proliferator-activated receptors gamma (PPARs-γ) agonists: rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2 (PGDJ2), against CsA-induced kidney injury in male Wistar rats. CsA was administered subcutaneously at a dose of 15 mg/kg/day for 28 days. Both PPAR-γ agonists were given for 28 days 0.5 hour before the administration of CsA. Rosiglitazone was administered orally at a dose of 8 mg/kg/day and PGDJ2 was given intraperitoneally at a dose of 30 µg/kg/day. CsA induced renal failure was evidenced by increased serum levels of urea, uric acid and creatinine. Serum concentrations of GSH and GSSG, lipid peroxidation products as well as NAD+/NADH, NADP+/NADPH and ADP/ATP ratios showed, that CsA induced oxidative stress and evoked an imbalanced red-ox state in the kidney. Light and electron microscope studies showed degenerative changes within renal tubules with damage to their mitochondria, interstitial fibrosis and arteriolopathy. Immunohistochemical expression of profibrotic TGF-ß was assessed. The biochemical and morphological changes induced by CsA were limited by administration of both rosiglitazone and PGDJ2. Ultrastructural examination of renal tubular epithelial cells showed marked improvement within mitochondria. Our results indicate that both PPAR-γ agonists used in the experiment may play an important role in protecting against CsA-induced damage in the kidney.

Influence of the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, rosiglitazone and antagonist, biphenol-A-diglicydyl ether (BADGE) on the course of inflammation in the experimental model of colitis in rats.

PPAR-γ plays a role in the development of immune response, particularly in inflammation. The inflammatory reaction may be stimulated or suppressed by the presence of PPAR ligands. Some researchers suggest positive influence of the PPAR-γ agonist on suppression of the intestinal inflammatory process, yet there has not been much evidence showing that the antagonist of PPAR-γ can affect the inflammatory process. The aim of the present study was to define the mechanism by which PPAR-γ ligands affect the course of experimentally induced colitis in rats. Colitis was induced in rats by rectal administration of TNBS (trinitrobenzene sulfonate). Rosiglitazone was administrated to animals at the dose of 8 mg/kg four times via an intra-gastric probe. Biphenol-A-diglicydyl ether (BADGE) was administrated intraperitoneally at the dose of 120 mg/kg, three times every second day. One group of animals received rosiglitazone together with BADGE before the induction of inflammation. Histological and ELISA examinations of large intestine samples were performed. Levels of IL-1ß, IL-6, TNF-α cytokines were determined in serum and homogenates. Rats exposed to rosiglitazone had higher body weight yet lower large intestine weight. Histological findings showed less ulceration, lower expression of crypts' loss and smaller oedema. Animals, which did not receive rosiglitazone, and those receiving it together with BADGE, developed more severe inflammatory changes. Rosiglitazone decreased the expression of inflammatory cytokines, such as IL-6 and TNF-α, both in serum and in intestinal homogenates. BADGE used with TNBS did not increase the expression of inflammatory cytokines; however, applied together with rosiglitazone, it caused inflammation similar to that observed among rats with experimentally induced colitis. Rosiglitazone reduces inflammation by decreasing the expression of IL-6 and TNF-α. BADGE administered with rosiglitazone blocks the activity of PPAR-γ and abolishes the protective effects of PPAR-γ agonist.

RAS inhibitors decrease apoptosis of acinar cells and increase elimination of pancreatic stellate cells after in the course of experimental chronic pancreatitis induced by dibutyltin dichloride.

Chronic pancreatitis (CP) is a progressive disease, in which the exocrine function of the gland is gradually lost and fibrosis develops due to repeated episodes of acute pancreatitis. The aim of the study was to investigate the effects of RAS inhibitors on the apoptosis of acinar cells and pancreatic stellate cells (PSCs) elimination in experimental CP induced by dibutyltin dichloride (DBTC). CP was induced by administration of DBTC to the femoral vein. Simultaneously captopril, losartan, enalapril and lisinopril were administered intraperitoneally. The rats were decapitated after 60 days and tissue of pancreas was collected. In rats treated by DBTC the features of inflammatory infiltration, ductal lumen dilatation, fibrosis were found. Strong reactivity with caspase2(L) and clusterin-beta antibodies was observed in areas of fibrosis. In animals treated with RAS inhibitors inflammatory changes and fibrosis were less severe. In groups of rats treated with DBTC and RAS inhibitors immunoreactivity of caspase(2L) and clusterin-beta was weak. Positive immunostaining against smooth muscle actine and desmin was observed in the elongated cells (PSC-s). This reaction was weak in groups of rat treated with DBTC and RAS inhibitors. Treatment of CP rats with RAS inhibitors alleviate apoptosis of pancreatic acinar cells and induces PSCs elimination.

The role of adenosine A2a receptors in experimental acute pancreatitis.

PURPOSE: The role of adenosine and its receptors in acute pancreatitis remains unelucidated. The aim was to evaluate the effects of the adenosine A2a receptor agonist and antagonist in the severe, taurocholate-induced experimental acute pancreatitis (EAP). MATERIAL AND METHODS: The experiments were performed on 80 male Wistar rats, subdivided into 4 groups: C--the control rats, I--the EAP group, IIA--EAP group treated with the A2a adenosine receptor agonist CGS 21680, IIB--EAP group treated with the A2a adenosine receptor antagonist ZM 241385. The blood for alpha-amylase and lipase and tissues samples for the morphological examinations and immunohistochemistry for A2a receptors were collected in 2, 6, 24 hours of the experiment. RESULTS: The serum alpha-amylase tended to decrease in the group IIA as compared to EAP untreated after 6 and 24 h. No significant effect of both treatments on serum lipase was noted. The administration of CGS 21680 resulted in favorable decrease of the inflammatory cell infiltration, hemorrhagic changes, necrosis and vacuolization of acinar cells, without an evident effect on the edema of the interstitial tissue. The administration of ZM 241385 did not affect the scores of necro-hemorrhagic changes and inflammatory infiltration, whereas it decreased the scores of vacuolization and edema. In all groups the expression of A2a receptors was similar. CONCLUSIONS: Our findings suggest, that A2a adenosine receptors are involved in the course of sodium taurocholate EAP. It is probable that the modulation of some subgroups of adenosine receptors could alleviate the course of severe experimental AP.

Effects of antiepileptic drugs on rat platelet aggregation: ex vivo and in vitro study.

The influence of conventional antiepileptic drugs (valproate, phenobarbital, diazepam, clonazepam, carbamazepine and diphenylhydantoin) on rat platelet activation induced by arachidonic acid (AA) or adenosine-5'-diphosphate (ADP) was investigated both ex vivo and in vitro on platelet-rich plasma (PRP). It was found that only diazepam, and to a smaller extent clonazepam, impaired rat platelet function. These benzodiazepines did not affect ex vivo platelet aggregation induced by ADP but dose-dependent inhibition of platelet aggregation and malondialdehyde (MDA) synthesis were observed, when the platelets were stimulated with AA (ED(50) of diazepam for aggregation was 2.7 mg/kg and that for MDA synthesis - 3.9 mg/kg). In in vitro study, diazepam was found to be a potent inhibitor of AA-induced platelet aggregation (IC(50) 1.2 microg/ml) and MDA synthesis (IC(50) 4.0 microg/ml). Higher concentrations of diazepam were required to inhibit ADP-induced aggregation (IC(50) 29.0 microg/ml). Clonazepam also exhibited a concentration-dependent inhibitory effect on AA-induced platelet aggregation and MDA synthesis but this effect was weaker when compared to diazepam. The present data demonstrate that diazepam possesed a strong inhibitory effect on rat platelet activation. The correlation between the reduction of platelet aggregation and the synthesis of MDA may suggest that the observed effect of diazepam is due to the inhibition of the cyclooxygenase pathway of the AA metabolism in platelet.

The effect of caerulein-induced acute pancreatitis on the levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in various rat tissues.

The effects of caerulein-induced acute pancreatitis (AP) on the levels of 5-HT and 5-HIAA in pancreas, liver, brain, kidney and different parts of the digestive tract in rats have been studied. The acute pancreatitis was induced by the continuous intravenous infusion of caerulein in the doses of 5 x 10(-6) g/kg/h and 7.5 x 10(-6) g/kg/h for 12 hours, and the 5-HT and 5-HIAA concentrations were determined by the method of Curzon and Green. The changes evoked by caerulein varied qualitatively and quantitatively between organs. The significant decrease of both 5-HT and 5-HIAA was observed in pancreas, liver, brain and kidney while in the stomach there was a significant fall of 5-HT level accompanied by the relevant increase of 5-HIAA level. In the duodenum, an opposite effect was noticed, the level of 5-HT was higher than normal whereas the 5-HIAA level was reduced. The significant decrease of the 5-HT and 5-HIAA levels in pancreas observed in the present study suggests the importance of 5-HT in the development of acute pancreatitis.

Competitive NMDA receptor antagonists enhance the antielectroshock activity of various antiepileptics.

CGP 37849 (1 mg/kg i.p.) enhanced the protective action of carbamazepine, diphenylhydantoin and phenobarbital against maximal electroshock-induced convulsions in mice. At 0.25 mg/kg CGP 37849 was inactive and at 0.5 mg/kg it potentiated the anticonvulsive activity of phenobarbital. CGP 39551 (5 mg/kg i.p.) reduced the ED50 values of diphenylhydantoin and phenobarbital, being without influence on carbamazepine. In the dose of 1.25 mg/kg, CGP 39551 potentiated the antielectroshock action of diphenylhydantoin and at 2.5 mg/kg that of phenobarbital. Neither NMDA receptor antagonist elevated the total plasma levels of antiepileptic drugs. Consequently, a pharmacokinetic interaction (in terms of total plasma levels at least) seems unlikely to be responsible for the observed potentiation of the antiepileptic drugs' activity. Combinations of CGP 37849 with either carbamazepine or phenobarbital resulted in a motor and memory impairment quantified by the chimney test and passive avoidance task, respectively. Moreover, combined treatment with phenobarbital and CGP 39551 caused a memory deficit. In contrast, diphenylhydantoin combined with either CGP 37849 or 39551 was devoid of adverse effects. It may be concluded that NMDA receptor blockade results in enhanced anticonvulsive action of common antiepileptics against maximal electroshock-induced seizures.

Competitive antagonists of NMDA receptors, CGP 37849 and CGP 39551, enhance the anticonvulsant activity of valproate against electroconvulsions in mice.

Two novel N-methyl-D-aspartic acid (NMDA) competitive antagonists, CGP 37849 (1.25 and 2.5 mg/kg) and CGP 39551 (10 mg/kg), significantly raised the threshold for electroconvulsions in mice. CGP 37849 in doses of 0.125-1.0 mg/kg and CGP 39551 in doses of 0.625-5 mg/kg i.p. considerably potentiated the protective activity of magnesium valproate against maximal electroshock-induced convulsions. The anticonvulsant activity of sodium valproate was potentiated by CGP 37849 (1 mg/kg) to a similar degree, which suggests that magnesium is not involved in the observed interaction. Neither CGP agent influenced the plasma level of valproate, so a pharmacokinetic interaction, in terms of total plasma levels, is not probable. Furthermore, the performance of mice injected with magnesium valproate (91 mg/kg) and CGP 37849 (0.25 mg/kg), which provided 50% protection against maximal electroshock-induced convulsions, in the long-term memory test and chimney test did not differ significantly from that of the control animals. The combination of magnesium valproate and CGP 39551 had a neurotoxic potential comparable to that of valproate alone. The results suggest that a combined treatment of valproate and some competitive NMDA antagonists may be important from a clinical point of view.

Synthesis and pharmacological properties of new derivatives of 1-diphenylacetamide-2-butanol.

Newly synthesized derivatives of 1-diphenylacetamide-2-butanol were investigated pharmacologically for their central properties in mice and rats. The most active were 2 compounds: racemic (RS) and enantiomer S (+) form of N-diphenylacetamide-2-butanol which produced hypothermia in normothermic mice, showed anxiolytic action in the four-plate test and reversed reserpine-induced hypothermia.

Synthesis and pharmacological properties of diphenylimidazolidine acetic and propionic acids derivatives.

Several derivatives of diphenylimidazolidine-2,4-dione and diphenylimidazolidin-4-one acetic and propionic acids have been synthesized. Some of them were screened for their effect on the CNS in mice and rats. All the investigated compounds showed an analgesic activity. The most active one was 1-benzyl-5,5-diphenyl-3-imidazolidine-2,4-dione acetic acid. That compound exerted an inhibitory activity against the CNS, anxiety-relieving, anticonvulsant and antidepressive effects.

Pharmacological studies on the central action of novel benzylidene-imidazothiazolone derivatives.

Pharmacological studies on the central action of novel benzylidene-imidazothiazolone derivatives were carried out on mice and rats. The highest activity showed two compounds: a chloro- and a methoxy- derivative. They produced analgesic, anticonvulsant, anti-anxiety and "antidepressant" effects in mice.