Prior peritoneal lavage with hot 0.9 % saline induces HSP70 expression and protects against cerulein-induced acute pancreatitis in rats

Recent studies have indicated that pre-induction of heat shock protein 70 (HSP70) expression in the pancreas protects against secretagogue-induced pancreatitis. In those studies, the HSP70 was mostly induced by unfeasible conditions. The aim of this current study was to investigate the effect of peritoneal lavage with hot 0.9 % saline (42 °C) on the pancreatic expression of HSP70 and its protective effect on cerulein-induced acute pancreatitis in rats. Male Wistar rats were peritoneally lavaged with 0.9 % saline at 42 °C for 30 min. HSP70 expression was evaluated by western blotting analysis. Prior peritoneal lavages with hot and warm saline were performed. Acute pancreatitis was induced by administration of intraperitoneal injection of cerulein (20 μg/kg) four times, and its severity was assessed by measuring serum amylase, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and trypsinogen activation peptide (TAP) levels. Pancreatic sections were stained with hematoxylin and eosin for histological evaluation. Peritoneal lavage with hot 0.9 % saline increased intrapancreatic HSP70 expression and ameliorated the cerulein-induced pancreatitis in rats, judged by the significantly reduced serum amylase, TNF-α, and IL-6 concentrations; histopathological scores, and serum TAP levels. Peritoneal lavage with hot 0.9 % saline can induce HSP70 expression and prevent cerulein-induced acute pancreatitis in rats. The results suggest that HSP70 protects against cerulein-induced pancreatitis by preventing proinflammatory cytokine synthesis and trypsinogen activation during acute pancreatitis.     

Specific type IV phosphodiesterase inhibitor ameliorates cerulein-induced pancreatitis in rats

BACKGROUND AND AIMS: Type IV phosphodiesterase is a key enzyme to metabolize intracellular adenosine 3',5'-cyclic monophosphate (cAMP) expressed in inflammatory cells. The specific type IV phosphodiesterase inhibitor that increases intracellular cAMP is known to be potent suppressor of proinflammatory cytokines. However, the effect of phosphodiesterase inhibitors on the development of pancreatitis has not been well understood. In the present study, we examined the effect of a specific type IV phosphodiesterase inhibitor on experimentally induced pancreatitis. METHODS: Severity of cerulein-induced pancreatitis and pancreatic proinflammatory cytokine levels were studied with or without pretreatment with a specific type IV phosphodiesterase inhibitor (rolipram) in Sprague-Dawley rats. RESULTS: Administration of rolipram clearly ameliorated severity of pancreatitis evaluated by edema, serum amylase (P<0.05), and lipase levels (P<0.05) in rats. Also, the level of pancreatic proinflammatory cytokine (interleukin-1beta (IL-1beta)) was significantly reduced when rats were treated with rolipram prior cerulein injection (P<0.05). CONCLUSIONS: Our results demonstrated that intracellular cAMP and pancreatic proinflammatory cytokine level, which are regulated by type IV phosphodiesterase, might play an important role in the pathogenesis of acute pancreatitis.     

1,8-cineole (eucalyptol) ameliorates cerulein-induced acute pancreatitis via modulation of cytokines,oxidative stress and NF-κB activity in mice

AimsAcute pancreatitis (AP) is an inflammatory condition wherein pro-inflammatory mediators, oxidative stress, and NF-κB signaling play a key role. Currently, no specific therapy exists and treatment is mainly supportive and targeted to prevent local pancreatic injury and systemic inflammatory complications. This study was aimed to examine whether 1,8-cineole, a plant monoterpene with antioxidant and anti-inflammatory properties could ameliorate cerulein-induced acute pancreatitis.Main methodsAP was induced in Swiss mice by six one hourly injections of cerulein (50 μg/kg, i.p.). 1,8-cineole (100, 200 and 400 mg/kg, p.o.) was administered 1 h prior to first cerulein injection, keeping vehicle and thalidomide treated groups as controls. Blood samples were taken 6-h later to determine serum levels of amylase and lipase, and cytokines. The pancreas was removed for morphological examination, myeloperoxidase (MPO) and malondialdehyde (MDA) assays, reduced glutathione (GSH) levels, and for nuclear factor (NF)-κB immunostaining.Key findings1,8-cineole effectively reduced the cerulein-induced histological damage, pancreatic edema and NF-κB expression, levels of MPO activity and MDA, and replenished the GSH depletion. Cerulein increased serum levels of amylase and lipase, and pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were also decreased by 1,8-cineole pretreatment, similar to thalidomide, a TNF-α inhibitor. The anti-inflammatory IL-10 cytokine level was, however, enhanced by 1,8-cineole.SignificanceThese findings indicate that 1,8-cineole can attenuate cerulein-induced AP via an anti-inflammatory mechanism and by combating oxidative stress. Further studies are needed to clearly elucidate its benefits in patients on acute pancreatitis.     

Alpha-melanocyte stimulating hormone has beneficial effects on cerulein-induced acute pancreatitis

We investigated the effect of alpha-melanocyte stimulating hormone (alpha-MSH) on cerulein induced acute pancreatitis in rats. alpha-MSH treatment (50 microg per rat, intraperitoneally) prior to cerulein reduced the plasma amylase level, pancreatic weight, pancreatic myeloperoxidase activity and the severity of the lesions microscopically. These data suggest that alpha-MSH has a protective effect on cerulein-induced acute pancreatitis and this effect could be attributed, at least in part, to decreased tissue leukocyte infiltration and thus, to decreased pro-inflammatory cytokine production and/or oxygen- and nitrogen-derived reactive metabolite release.     

Adrenomedullin reduces the severity of cerulein-induced acute pancreatitis

We investigated the effect of Adrenomedullin (AM) on cerulein-induced acute pancreatitis in rats. AM treatment (100 ng/kg per rat, subcutaneous) after one hour of cerulein injection reduced the plasma amylase levels, pancreatic weight, pancreatic malondialdehyde (MDA) levels, and the severity of the lesions microscopically. These data suggest that AM has a protective effect on cerulein-induced acute pancreatitis. These could be due to anti-inflammatory properties of AM, inhibition of proinflammatory cytokine secretion, reducing the endothelial permeability increased by reactive oxygen species, endotoxins or cytokines.     

Altered systemic and tissue prostacyclin in cerulein induced acute pancreatitis in rats.

Prostacyclin metabolism in rat acute pancreatitis was evaluated by measuring the tissue levels of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and the urinary excretion of 2, 3-dinor 6-keto-PGF1 alpha. Acute pancreatitis was induced by i.v. cerulein perfusion and was confirmed by the pancreas enzyme changes and the histological findings. Significantly enhanced tissue and urinary prostacyclin levels were found in acute pancreatitis rats, when compared to the controls. Concomitantly, an enhanced tissue phospholipase A2 (PLA2) activity was also found. These data show the importance of 2, 3-dinor PGF1 alpha as an inflammatory marker in cerulein-induced pancreatitis.     

Protective effect of central thyrotropin-releasing hormone analog on cerulein-induced acute pancreatitis in rats

Central neuropeptides play a role in many physiological functions through the autonomic nervous system. We have recently demonstrated that central injection of a thyrotropin-releasing hormone (TRH) analog increases pancreatic blood flow through vagal and nitric oxide-dependent pathways. In this study, the central effect of a TRH analog on experimental acute pancreatitis was investigated in rats. Acute pancreatitis was induced by two intraperitoneal injections of cerulein (40 microg/kg) at 1-h interval. Either stable TRH analog, RX 77368 (5-100 ng), or saline was injected intracisternally 15 min before the first cerulein injection under ether anesthesia. Serum amylase level was measured before and 5 h after the first cerulein injection. Pancreatic wet/dry weight ratio and histological changes were also evaluated. Intracisternal TRH analog inhibited cerulean-induced elevation of serum amylase level, increase in pancreatic wet/dry weight ratio and pancreatic histological changes, such as interstitial edema, inflammation and vacuolization. The pancreatic cytoprotection induced by central TRH analog was abolished by subdiaphragmatic vagotomy and N(G)-nitro-L-arginine-methyl ester (L-NAME), but not by 6-hydroxydopamine (6-OHDA). Intravenous administration of the TRH analog did not influence cerulein-induced acute pancreatitis. These results indicate that the TRH analog acts in the central nervous system to protect against acute pancreatitis through vagal and nitric oxide-dependent pathways.     

Effects of the S2-serotonergic receptor antagonist, ketanserin, on cerulein-induced pancreatitis in the rat.

We investigated the effects of ketanserin, a S 2 (5-hydroxytryptamine 2; 5-HT 2)-serotonergic receptor antagonist, on cerulein-induced pancreatitis in the rat. Large pharmacological doses of cerulein induced acute pancreatitis in the rat. Ketanserin reduced the cerulein-induced increase in serum amylase concentration in a dose-dependent manner. Treatment with 10 mg/kg of ketanserin per os markedly improved cerulein-induced pancreatitis and was associated with a significant reduction of the increase in serum amylase concentration. In addition, a very specific serotonin S 2 antagonist, ritanserin which has no antihypertensive effect, also reduced the cerulein-induced increase in the serum amylase concentration. These results suggest that S 2 (5-HT 2) may play a role in pathophysiology of cerulein-induced pancreatitis in the rat.     

Organ microcirculatory disturbances in experimental acute pancreatitis. A role of nitric oxide

Microcirculatory disturbances are important early pathophysiological events in various organs during acute pancreatitis (AP). The aim of the study was to investigate an influence of L-arginine (nitric oxide substrate) and N(G)-nitro-L-arginine (L-NNA, nitric oxide synthase inhibitor) on organ microcirculation in experimental acute pancreatitis induced by four consecutive intraperitoneal cerulein injections (15 microg/kg/h). The microcirculation of pancreas, liver, kidney, stomach, colon and skeletal muscle was measured by laser Doppler flowmeter. Serum interleukin 6 and hematocrit levels were analyzed. AP resulted in a significant drop of microperfusion in all examined organ. L-arginine administration (2 x 100 mg/kg) improved the microcirculation in the pancreas, liver, kidney, colon and skeletal muscle, and lowered hematocrit levels. L-NNA treatment (2 x 25 mg/kg) caused aggravation of edematous AP to the necrotizing situation, and increased IL-6 and hematocrit levels. A further reduction of blood perfusion was noted in the stomach only. It is concluded that L-arginine administration has a positive influence on organ microcirculatory disturbances accompanying experimental cerulein-induced AP. NO inhibition aggravates the course of pancreatitis.     

Sodium arsenite induces heat shock protein 70 expression and protects against secretagogue-induced trypsinogen and NF-kappaB activation

Heat shock proteins (HSPs), induced by a variety of stresses, are known to protect against cellular injury. Recent studies have demonstrated that prior beta-adrenergic stimulation as well as thermal or culture stress induces HSP70 expression and protects against cerulein-induced pancreatitis. The goal of our current studies was to determine whether or not a non-thermal, chemical stressor like sodium arsenite also upregulates HSP70 expression in the pancreas and prevents secretagogue-induced trypsinogen and NF-kappaB activation. We examined the effects of sodium arsenite preadministration on the parameters of cerulein-induced pancreatitis in rats and then monitored the effects of preincubating pancreatic acini with sodium arsenite in vitro. Our results showed that sodium arsenite pretreatment induced HSP70 expression both in vitro and in vivo and significantly ameliorated the severity of cerulein-induced pancreatitis, as evidenced by the markedly reduced degree of hyperamylasemia, pancreatic edema, and acinar cell necrosis. Sodium arsenite pretreatment not only inhibited trypsinogen activation and the subcellular redistribution of cathepsin B, but also prevented NF-kappaB translocation to the nucleus by inhibiting the IkappaBalpha degradation both in vivo and in vitro. We also examined the effect of sodium arsenite pretreatment in a more severe model of pancreatitis induced by L-arginine and found a similarly protective effect. Based on our observations we conclude that, like thermal stress, chemical stressors such as sodium arsenite also induce HSP70 expression in the pancreas and protect against acute pancreatitis. Thus, non-thermal pharmacologically induced stress can help prevent or treat pancreatitis.     

Lipid peroxidation inhibition reduces NF-kappaB activation and attenuates cerulein-induced pancreatitis

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF-kappaB) activation and augmented tumor necrosis factor-alpha (TNF-alpha) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF-kappaB activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250g body weight received administration of cerulein (80 microg/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER + DMSO = 3.075 +/- 0.54 micromol/g; CER + raxofelast = 0.693 +/- 0.18 micromol/g; p < 0.001), decreased myeloperoxidase (MPO) activity (CER + DMSO = 22.2 +/- 3.54 mU/g; CER + raxofelast = 9.07 +/- 2.05 mU/g, p < 0.01), increased glutathione levels (GSH) (CER + DMSO = 5.21 +/- 1.79 micromol/g; CER + raxofelast = 15.71 +/- 2.14 micronol/g; p < 0.001), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase (CER + DMSO = 4063 +/- 707.9 U/l; CER + raxofelast = 1198 +/- 214.4 U/l; p < 0.001), and lipase (CER + DMSO = 1654 +/- 330 U/l; CER + raxofelast = 386 +/- 118.2 U/l; p < 0.001), Furthermore, raxofelast reduced pancreatic NF-kappaB activation and the TNF-alpha mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.     

Morphometric measurements to quantify the cerulein induced hyperstimulatory pancreatitis of rats under the protective effect of lectins.

In preceding papers we demonstrated an inhibitory effect of wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) on the cholecystokinin (CCK) binding to the CCK receptor of rat pancreatic cells and also on the CCK induced Ca2+ release and alpha-amylase secretion in vitro as well as on pancreatic secretion of intact rats in vivo. In the present study we show the same inhibitory effect of both lectins on the cerulein pancreatitis of rats. This acute pancreatitis was induced by supramaximal injections (5 microg/kg/h i.v. or 10 microg/kg/h i.p.) of the CCK analogue cerulein in rats every hour. To monitor the degree of pancreatitis, we measured the number and diameter of injury vacuoles in the pancreatic acinar cells as one of the most important signs of this type of pancreatitis by light microscopic morphometry with two different systems on paraffin sections. Furthermore, the serum alpha-amylase activity was measured biochemically. We found a correlation between the diameter of vacuoles inside the acinar cells and the serum enzyme activity up to 24 h. The simultaneous i.p. administration of cerulein and WGA or UEA in a dosage of 125 microg/kg/h for 8 h led to a reduction of vacuolar diameter from 13.1+/-2.0 microm (cerulein) to 7.5+/-1.1 microm (cerulein + WGA) or 7.2+/-1.3 microm (cerulein + UEA). The serum amylase activity was reduced from 63.7+/-15.8 mmol/l x min (cerulein) to 37.7+/-11.8 (cerulein + WGA) or 39.4; +52.9; -31.1 (cerulein + UEA-I). Both parameters allow the grading this special type of pancreatitis to demonstrate the protective effect of the lectins.     

Role of parathyroid hormone-related protein in the pro-inflammatory and pro-fibrogenic response associated with acute pancreatitis

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1-36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1-36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7-34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.     

Prophylactic and therapeutic treatments with AT 1 and AT 2 receptor antagonists and their effects on changes in the severity of pancreatitis

Previous studies showed that a local pancreatic renin-angiotensin system (RAS) was upregulated in experimental acute pancreatitis. RAS inhibition could attenuate pancreatic inflammation and fibrosis, which casts a new light on the role of the pancreatic RAS in pancreatitis. The present study explores the prophylactic and therapeutic potentials, and possible molecular mechanism for the antagonism of angiotensin II receptors on the changes in the severity of pancreatic injury induced by acute pancreatitis. Experimental pancreatitis was induced by an intraperitoneal injection of supra-maximal dose of cerulein. The differential effects of angiotensin II receptors inhibitors losartan and PD123319 on the pancreatic injury were assessed by virtue of using the pancreatic water content, biochemical and histological analyses. Blockade of the AT(1) receptor by losartan at a dose of 200microg/kg could markedly ameliorate the pancreatic injury induced by cerulein, as evidenced by biochemical and histopathological studies. However, blockade of the AT(2) receptor by PD123319 appeared not to provide any beneficial role in cerulein-induced pancreatic injury. Both prophylactic and therapeutic treatments with losartan were effective against cerulein-induced pancreatic injury. The protective action of losartan was linked to an inhibition of NAD(P)H oxidase activity, thus consequential oxidative modification of pancreatic proteins in the pancreas. Inhibition of the AT(1) receptor, but not AT(2) receptor, may play a beneficial role in ameliorating the severity of acute pancreatitis. The differential effects of AT(1) and AT(2) inhibitors on cerulein-induced pancreatic injury might be due to the distinctive mechanism of the AT(1) and AT(2) receptors on the activation of NAD(P)H oxidase. Thus the protective role of AT(1) receptor antagonist, losartan, could be mediated by the inhibition of NAD(P)H oxidase-dependent generation of reactive oxygen species (ROS).