Pretreatment of a matrix metalloproteases inhibitor and aprotinin attenuated the development of acute pancreatitis-induced lung injury in rat model.

OBJECTIVE: Acute lung injury (ALI) is one of the most common extra-pancreatic complications of acute pancreatitis. In this study, we examined the protective effect of protease inhibitor aprotinin and a matrix metalloproteinase inhibitor (MMPi) on pulmonary inflammation in rats with severe pancreatitis-associated ALI. METHOD: A rat model of acute pancreatitis (AP) was established by injecting sodium glycodeoxycholate (GDOC) into the pancreatic duct. Pharmacological interventions included pretreatment with a protease inhibitor aprotinin (10mg/kg) and a matrix metalloproteinase inhibitor (MMPi, 100g/kg). The extent of pancreatic and lung injury and systemic inflammation was assessed by examinations of blood, bronchoalveolar lavage (BAL), and lung tissue. Pancreatic or lung tissue edema was evaluated by tissue water content. Pulmonary arterial pressure and alveolar-capillary membrane permeability were evaluated post-injury via a catheter inserted into the pulmonary artery in an isolated, perfused lung model. RESULTS: Pre-treatment with aprotinin or MMPi significantly decreased amylase and lactate dehydrogenase (LDH), and the wet/dry weight ratio of the lung and pancreas in AP rats. Compared to the GDOC alone group, administration of aprotinin or MMPi prevented pancreatitis-induced IL-6 increases in the lung. Similarly, treatment with aprotinin or MMPi significantly decreased the accumulation of white blood cells, oxygen radicals, nitrite/nitrates in both blood and BAL, and markedly reduced lung permeability. CONCLUSION: Pretreatment with either aprotinin or MMPi attenuated the systemic inflammation and reduced the severity of lung and pancreas injuries. In short, our study demonstrated that inhibition of protease may be therapeutic to pulmonary inflammation in this GDOC-induced AP model.

The Effects of Pancreatic Microcirculatory Disturbances on Histopathologic Tissue Damage and the Outcome in Severe Acute Pancreatitis.

OBJECTIVES: Severe acute pancreatitis is an inflammatory disease of the pancreas with a high morbidity and mortality. To date, no causal treatment is known. The aim of the present study was to analyze the impact of pancreatic microcirculatory disturbances in severe acute pancreatitis and to correlate the effects with histopathologic tissue damage and outcome. METHODS: Severe acute pancreatitis was induced in 129 pigs by injection of glycodeoxycholic acid into the pancreatic duct. Pancreatic microcirculation, pancreatic tissue oxygenation, histopathologic tissue damage, and survival were measured and analyzed. RESULTS: Our study demonstrates a strong correlation between pancreatic microcirculatory disturbances and histopathologic tissue damage (r = 0.728; P < 0.001). Furthermore, we showed a strong correlation between tissue oxygenation and the severity of the pancreatitis according to an established porcine pancreatitis score (r = 0.694; P < 0.001). In addition, disturbances of the pancreatic microcirculation were shown to be associated with an increased mortality rate in severe acute pancreatitis. CONCLUSIONS: We found that pancreatic microcirculatory disturbances have significant effects on histopathologic tissue damage and the outcome of severe acute pancreatitis. For a better survival of severe acute pancreatitis, the treatment should focus on an improvement of pancreatic microcirculation.

Effects of glutamine alone on the acute necrotizing pancreatitis in rats.

BACKGROUND: The effects of the glutamine on the acute pancreatitis are controversial in the clinical and experimental studies. The aim of this study was to investigate the influence of glutamine alone on acute necrotizing pancreatitis (ANP) induced by glycodeoxycholic acid in rats. MATERIAL AND METHODS: Fifty-two male Sprague-Dawley rats weighing 300-350 g were used. Rats were divided into four groups as sham + saline, sham + glutamine, ANP + saline and ANP + glutamine. ANP in rats was induced by glycodeoxycholic acid. The extent of acinar cell injury, mortality, systemic cardiorespiratory variables, functional capillary density, renal/hepatic functions, and changes in some enzyme markers for pancreatic and lung tissue were investigated during ANP in rats. RESULTS: The induction of ANP resulted in a significant increase in the mortality rate, pancreatic necrosis, and serum activity of amylase, alanine aminotransferase, interleukin-6, lactate dehydrogenase in bronchoalveolar lavage fluid, serum concentration of urea, and tissue activity of myeloperoxidase and malondialdehyde in the pancreas and lung, and a significant decrease in concentrations of calcium, blood pressure, urine output, pO2, and functional capillary density. The use of glutamine alone improved these changes. CONCLUSIONS: Glutamine demonstrated beneficial effect on the course of ANP in rats. Therefore, it may be used by itself in the treatment of acute pancreatitis.

Capsaicin reduces tissue damage in experimental acute pancreatitis.

OBJECTIVES: Calcitonin gene-related peptide (CGRP) is released from perivascular pancreatic nerves. It effects vasomotion and cytokine liberation in inflammatory processes, including acute pancreatitis (AP). Calcitonin gene-related peptide liberation is stimulated by capsaicin, a substance of red hot chili peppers. Aim of the study was to investigate the influence of exogenous capsaicin on experimental AP. METHODS: Acute pancreatitis was induced in rats by glycodeoxycholic acid and cerulein. Animals were divided into 4 groups: (1) severe AP, (2) severe AP+capsaicin, (3) control without AP, and (4) control+capsaicin. After 24 hours, survival, histology, and CGRP were evaluated (n=6/group). In additional animals, erythrocyte flow and leukocyte activation were evaluated by intravital microscopy 6 hours after AP induction (n=6/group). RESULTS: In the control groups, all animals survived without histological alterations. Mortality in severe AP was 67%. Capsaicin reduced mortality to 16% (P<0.05). Acute pancreatitis animals developed pancreatic inflammation and necrosis, which was significantly less after capsaicin application. Intravital microscopy in severe AP showed reduced erythrocyte velocity and increased leukocyte adhesion, which was nearly normalized by capsaicin (P<0.01). Calcitonin gene-related peptide increased in both capsaicin groups, indicating endogenous CGRP liberation (P<0.01). CONCLUSION: Capsaicin releases endogenous CGRP with improved pancreatic microcirculation and reduced inflammation in experimental AP. This underlines neuropeptide activity in the pathogenesis of AP.

Rat hepatocellular apoptosis induced by glycodeoxycholate.

OBJECTIVE: To explore the relationship between glycodeoxycholate (GDC) and rat hepatocellular apoptosis. METHODS: GDC was used to treat rat hepatocytes cultured in vitro and the apoptotic cells were observed and analyzed with light microscope, transmission electron microscope, TUNEL in situ hybridization, DNA agarose gel electrophoresis and flow cytometry. RESULTS: When rat hepatocytes were incubated with GDC (final concentration of 100 micromol/L) for 2h, apoptotic hepatocytes were observed with light and transmission electron microscope, and TUNEL in situ hybridization. When the hepatocytes were incubated with GDC (final concentration of 50, 100, 150 micromol/L, respectively) for 2h, or with GDC (final concentration of 100 micromol/L) for 2, 4, 6, 8h, respectively, agarose gel electrophoresis of the hepatocytes DNA demonstrated the typical ladder patterns. CONCLUSION: GDC with final concentration of 50, 100, 150 micromol/L could induce rat hepatocellular apoptosis.

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.

Co-mutagenicity of glyco- and tauro-deoxycholic acids in the Ames test.

Mutagenicity and co-mutagenicity of glyco- and tauro-deoxycholic acids (GDCA and TDCA), which are abundant in human bile, were examined by the Ames test. The two chemicals were not mutagenic for themselves to Salmonella typhimurium TA98 and TA100, with and without S9 mix. They enhanced, however, the mutagenic activities of the pro-mutagens, 2-aminoanthracene (2AA) and benzo[a]pyrene (BaP), for both TA98 and TA100 with S9 mix. They were more co-mutagenic for the pro-mutagens on TA98 than on TA100. On TA98, the mutagenic activities of 2AA with GDCA (5 mumol/plate) and with TDCA (5 mumol/plate) were 9.7-fold and 11.8-fold as high as that of the corresponding control (2AA only), respectively. BaP with GDCA (2.5 mumol/plate) and with TDCA (2.5 mumol/plate) showed 2.8-fold and 3.0-fold increases over the corresponding control level (BaP only), respectively. It is hence concluded that GDCA and TDCA may enhance the activity of some mutagens existing in bile.

Toxicity order of cholanic acids using an immobilised cell biosensor.

There is considerable published evidence of the use of cells of various species to evaluate the toxicity of numerous compounds, many of pharmaceutical interest. The coupling of cell colonies with a suitable transduction device has led to the development in recent years of toxicity biosensors based on the alteration of a process or a cell metabolic function by the toxic substance under examination. A biosensor based on immobilised yeast cells (Saccharomyces cerevisiae) has been developed recently in this department for the purpose of performing a rapid toxicity test in aqueous environmental matrices. This biosensor has now been used in the toxicity screening of a number of sodium salts of conjugated and free cholanic acids. The "toxicity degree" scale, which was found by placing in decreasing order the values of the slopes of the straight lines obtained by quantifying changes in the behaviour of the respirometric curve, plotted before and after incubation, using known concentrations of cholanic acid sodium salts, was: deoxycholic acid > chenodeoxycholic acid > ursodeoxycholic acid > cholic acid, for free cholanic acids; and glycodeoxycholic acid > glycochenodeoxycholic acid > glycocholic acid, for glycocholanic acids. These values are in good agreement with published toxicity data obtained in vitro. This sensor can thus be considered to provide a valid instrument for the preliminary evaluation of the toxicity of organic compounds or drugs.

A model of hemorrhagic pancreatitis in cats--role of 16,16-dimethyl prostaglandin E2.

Acute edematous pancreatitis was induced in cats by perfusing activated pancreatic enzymes through their pancreatic ducts. The ducts had been made permeable to large molecules by one of two techniques. The cats either received ethanol (2 ml/kg every 8 h) and aspirin (25 mg/kg every 8 h) orally for 48 h or had their pancreatic ducts perfused for 1 h with 7.5 mM glycodeoxycholate. When the same procedure was followed, but using 16,16-dimethyl prostaglandin E2 (dmPGE2) (2 micrograms/kg X h infused intravenously for 1 h before and during ductal perfusion with activated enzymes), hemorrhagic pancreatitis developed instead. To investigate whether an increase in pancreatic blood flow or microvascular permeability (both caused by dmPGE2) was important in this phenomenon, we tested the effects of isoproterenol (which increased blood flow) and histamine (which increased microvascular permeability) in the model. Thus in similar experiments, either isoproterenol (0.3 micrograms/kg . min) or histamine phosphate (2 micrograms/kg . min) was infused instead of dmPGE2. The animals that received histamine also developed hemorrhagic pancreatitis. Those that received isoproterenol did not. These observations suggested that an increase in microvascular permeability in the pancreas converted edematous pancreatitis to hemorrhagic pancreatitis. These findings suggest also that clinical studies using prostaglandins to treat patients with pancreatitis should be approached with caution.