I. De Dios

Time-course of oxygen free radical production in acinar cells during acute pancreatitis induced by pancreatic duct obstruction.

The time-course of oxygen free radicals (OFR) generation within acinar cells was studied at different stages of acute pancreatitis (AP) induced in rats by duct obstruction (PDO) for 48 h by flow cytometry, using dihydrorhodamine-123 (DHR) as fluorescent dye. Parallel measurements of the most common markers of oxidative stress such as glutathione (GSH) depletion and malondialdehyde (MDA) levels in pancreas were also performed. OFR production significantly increased within acinar cells at early stages of AP, concomitant with a marked depletion in pancreatic GSH. Lipid peroxidation was significantly enhanced 6 h after PDO, suggesting that the antioxidant defence system of the cell is overwhelmed by OFR production. Both MDA and OFR production in acinar cells decreased to normal values at late AP stages, thus allowing the recovery of pancreatic GSH levels 48 h after PDO. Among the two types of acinar cells differentiated by flow cytometry, R1 and R2, it was the R2 population that showed higher values of DHR dye. However, no differences between the two cell types were found regarding the amount of OFR generation. Our results demonstrate that individual acinar cells significantly contribute to produce large amounts of OFR at early stages of AP. The two existing populations of acinar cells displayed similar behaviour regarding oxidative stress over the course of the disease.

Mechanisms of dexamethasone-mediated chemokine down-regulation in mild and severe acute pancreatitis

This study aimed to investigate the role of therapeutic dexamethasone (Dex) treatment on the mechanisms underlying chemokine expression during mild and severe acute pancreatitis (AP) experimentally induced in rats. Regardless of the AP severity, Dex (1 mg/kg), administered 1 h after AP, reduced the acinar cell activation of extracellular signal-regulated kinase (ERK) and c-Jun-NH(2)-terminal kinase (JNK) but failed to reduce p38-mitogen-activated protein kinase (MAPK) in severe AP. In both AP models, Dex inhibited the activation of nuclear factor-kappaB (NF-kappaB) and signal transducers and activators of transcription (STAT) factors. All of this resulted in pancreatic down-regulation of the chemokines monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC). Lower plasma chemokine levels as well as decreased amylasemia, hematocrit and plasma interleukin-1beta (Il-1beta) levels were found either in mild or severe AP treated with Dex. Pancreatic neutrophil infiltration was attenuated by Dex in mild but not in severe AP. In conclusion, by targeting MAPKs, NF-kappaB and STAT3 pathways, Dex treatment down-regulated the chemokine expression in different cell sources during mild and severe AP, resulting in decreased severity of the disease.

Effect of chronic administration of hydrocortisone on the induction and evolution of acute pancreatitis induced by cerulein

The effects of chronic administration of hydrocortisone (10 mg/kg/day) on the development and evolution of acute pancreatitis induced by supramaximal stimulation with cerulein were examined in the rat. In these circumstances the potentially therapeutic effect of L-364,718, a CCK-receptor antagonist, was assayed. Administration of hydrocortisone over 7 days did not increase the severity of edematous acute pancreatitis induced by cerulein, since the reduction in pancreatic secretion, the hyperamylasemia and the increase in the levels of hematocrit and fluid in the pancreatic tissue were similar in rats with acute pancreatitis treated and untreated with hydrocortisone previously. When hydrocortisone was administered chronically, before administration of supramaximal doses of cerulein, a spontaneous regression of acute pancreatitis occurred. However, when hydrocortisone administration was continued after inducing pancreatitis, pancreatic recovery was prevented, observing a significantly depressed acinar secretion and elevated values of hematocrit and tissue fluid (edema). L-364,718 administration proved to be detrimental in the evolution of edematous acute pancreatitis when the rats had been treated chronically with hydrocortisone because the blockade exerted on secretion prevented the draining of enzymes stored in excess by hydrocortisone administration.

Therapeutic and protective effect of subcutaneous injections of L-364,718 on caerulein-induced acute pancreatitis.

The prophylactic and therapeutic effects of a potent cholecystokinin (CCK) receptor antagonist, L-364,718, on acute pancreatitis induced by caerulein were evaluated, analyzing morphologic and functional pancreatic parameters jointly. Edematous pancreatitis was induced by four subcutaneous injections of caerulein (20 μkg) in rats at 1-h intervals. Prophylactic administration of L-364,718 (0.1 mg/kg) prevented rise in serum amylase levels, interstitial edema, vacuolization, and impairment of pancreatic enzyme secretion that accompany caerulein-induced acute pancreatitis. After 7 days, a spontaneous regression of the morphologic alterations caused by caerulein-induced acute pancreatitis occurs; however, recovery of the secretory function of the pancreas was only reached after this period of time when L-364,718 was administered therapeutically (0.1 mg/kg/ day). Prophylactically or therapeutically administered, L-364,718 exerts a beneficial effect on caerulein-induced acute pancreatitis, indicating that CCK (exogenous or endogenous) plays an important role in the development of this pathology.

The role of redox status on chemokine expression in acute pancreatitis.

This study focused on the involvement of oxidative stress in the mechanisms mediating chemokine production in different cell sources during mild and severe acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) and 3.5% NaTc, respectively. N-Acetylcysteine (NAC) was used as antioxidant treatment. Pancreatic glutathione depletion, acinar overexpression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC), and activation of p38MAPK, NF-kappaB and STAT3 were found in both AP models. NAC reduced the depletion of glutathione in BPDO- but not in NaTc-induced AP, in which oxidative stress overwhelmed the antioxidant capability of NAC. As a result, inhibition of the acinar chemokine expression and signalling pathways occurs in mild, but not in severe AP. However, MCP-1 and CINC expressions in whole pancreas and plasma chemokine levels were not reduced by NAC, even in BPDO-induced AP, suggesting that in addition to acini, other pancreatic cells produced chemokines by antioxidant resistant mechanisms. The high Il-6 plasma levels found during AP, both in NAC-treated and non-treated rats, pointed out cytokines as activating factors of chemokine expression in non-acinar cells. In conclusion, from early AP oxidant-mediated MAPK, NF-kappaB and STAT3 activation triggers the chemokine expression in acini but not in non-acinar cells.

Major Pathological Mechanisms of Acute Pancreatitis Are Prevented by N-Acetylcysteine

Aim: To analyze the capability of N-acetylcysteine (NAC) to prevent major intra-acinar pathogenic mechanisms involved in the development of acute pancreatitis (AP). Methods: AP was induced by pancreatic duct obstruction (PDO) in rats. Some animals received NAC (50 mg/kg) 1 h before and 1 h after PDO. During a 24-hour period of PDO, plasma amylase activity and pancreatic glutathione and malondialdehyde levels were measured. Cytosolic Ca2+ levels and enzyme (amylase and trypsinogen) load in acinar cells were also analyzed by flow cytometry, and histological analysis of the pancreas was performed by electron microscopy. Results: NAC avoided glutathione depletion at early AP stages, thereby preventing pancreatic oxidative damage, as reflected by normal malondialdehyde levels. By limiting oxidative stress, NAC treatment effectively prevented the impairment of Ca2+ homeostasis found in acinar cells from early AP onwards, thus protecting the pancreas from damage. In addition, lower quantities of digestive enzymes were accumulated within acinar cells. This finding, together with the significantly lower hyperamylasemia observed in these animals, suggests that NAC treatment palliates the exocytosis blockade induced by PDO. Conclusion: By preventing oxidative stress at early AP stages, NAC administration prevents other pathological mechanisms of AP from being developed inside acinar cells, thus palliating the severity of disease.

Redox-sensitive modulation of CD45 expression in pancreatic acinar cells during acute pancreatitis.

CD45, a transmembrane protein tyrosine phosphatase required for signal transduction in leukocytes, has recently been found in pancreatic acinar cells. We have investigated the relationship between kinetic expression of CD45 on acinar cells during acute pancreatitis (AP) and the ability of these cells to produce tumour necrosis factor‐α (TNF‐α) through mechanisms sensitive to the cellular redox state. Flow cytometric analysis showed a significant decrease in the constitutive expression of CD45 in acinar cells from six hours onwards after inducing AP by bile‐pancreatic duct obstruction (BPDO) in parallel with a significant increase in acinar TNF‐α production. Changes in protein expression on the acinar cell surface preceded CD45 mRNA down‐regulation, which was not found until 12 hours after BPDO. N‐Acetylcysteine treatment delayed and reduced the down‐regulation of CD45 expression induced by AP and prevented acinar cells from producing TNF‐α. Our results show that CD45 expression is down‐regulated in acinar cells during acute pancreatitis by redox‐sensitive mechanisms, and they support the notion that CD45 negatively controls the production of cytokines in pancreatic acinar cells. Copyright

Glucocorticoids effects on exocrine pancreatic secretion in caerulein-induced acute pancreatitis in the rat.

The present work reports on exocrine pancreatic secretion in control rats, adrenalectomized rats and hydrocortisone-treated (10 mg/Kg/d) rats during 7 days, under normal conditions and after induction of acute pancreatitis with caerulein (20 micrograms/Kg) by 4 subcutaneous injections at hourly intervals. Pancreatic secretion was seen to be affected by the procedure of adrenalectomy, which led to a marked reduction in the secretion of proteins and amylase with respect to control values. This was probably due to the decrease occurring in the zymogen granules in the acinar cells of the exocrine pancreas, a phenomenon which also led to a decrease in pancreatic weight observed in these animals. Treatment with hydrocortisone induced a decrease in the secretion of proteins and amylase, as well as an increase in pancreatic weight. This agrees with the accepted hypothesis that large amounts glucocorticoids stimulate the synthesis and storage of proteins in the exocrine pancreas, reducing the secretory phase. The administration of high doses of caerulein under these conditions led to acute pancreatitis in the three groups of animals. This was paralleled by a dramatic decrease in protein and amylase secretion and by severe interstitial edema of the pancreas and by increases in serum amylase values. In the case of the animals treated previously with hydrocortisone, the latter were tripled with respect to the control animals. The conclusion is offered that since the storage of enzyme proteins is governed by glucocorticoids, which furthermore increase the sensitivity of the acinar cells to stimulation by secretagogues, the administration of these substances during the development of pancreatic lesions such as acute pancreatitis is highly compromising to the organism.

Cross-talk between TLR4 and PPARγ pathways in the arachidonic acid-induced inflammatory response in pancreatic acini

Arachidonic acid (AA) is generally associated with inflammation in different settings. We assess the molecular mechanisms involved in the inflammatory response exerted by AA on pancreatic acini as an approach to acute pancreatitis (AP). Celecoxib (COX-2 inhibitor), TAK-242 (TLR4 inhibitor) and 15d-PGJ2 (PPARγ agonist) were used to ascertain the signaling pathways. In addition, we examine the effects of TAK-242 and 15d-PGJ2 on AP induced in rats by bile-pancreatic duct obstruction (BPDO). To carry out in vitro studies, acini were isolated from pancreas of control rats. Generation of PGE2 and TXB2, activation of pro-inflammatory pathways (MAPKs, NF-κB, and JAK/STAT3) and overexpression of CCL2 and P-selectin was found in AA-treated acini. In addition, AA up-regulated TLR4 and down-regulated PPARγ expression. Celecoxib prevented the up-regulation of CCL2 and P-selectin but did not show any effect on the AA-mediated changes in TLR4 and PPARγ expression. TAK-242, reduced the generation of AA metabolites and repressed both the cascade of pro-inflammatory events which led to CCL2 and P-selectin overexpression as well as the AA-induced PPARγ down-regulation. Thus, TLR4 acts as upstream activating pro-inflammatory and inhibiting anti-inflammatory pathways. 15d-PGJ2 down-regulated TLR4 expression and hence prevented the synthesis of AA metabolites and the inflammatory response mediated by them. Reciprocal negative cross-talk between TLR4 and PPARγ pathways is evidenced. In vivo experiments showed that TAK-242 and 15d-PGJ2 treatments reduced the inflammatory response in BPDO-induced AP. We conclude that through TLR4-dependent mechanisms, AA up-regulated CCL2 and P-selectin in pancreatic acini, partly mediated by the generation of PGE2 and TXB2, which activated pro-inflammatory pathways, but also directly by down-regulating PPARγ expression with anti-inflammatory activity. In vitro and in vivo studies support the role of TLR4 in AP and the use of TLR4 inhibitors and PPARγ agonists in AP treatment.

N‐acetylcysteine induces beneficial changes in the acinar cell cycle progression in the course of acute pancreatitis

Abstract.  Oxygen free radicals (OFR) are produced in the course of acute pancreatitis (AP). In addition to injurious oxidative effects, they are also involved in the regulation of cell growth. The aim of the present study was to examine the relationship between the effectiveness of N‐acetyl‐l‐cysteine (NAC) to prevent the generation of OFR and the changes in the cell‐cycle pattern of acinar cells in the course of AP induced in rats by pancreatic duct obstruction (PDO). NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Flow‐cytometric measurement of OFR generation in acinar cells was carried out using dihydrorhodamine as fluorescent dye. Plasma amylase activity, pancreatic glutathione (GSH) content and TNF‐α plasma levels were also measured. The distribution of acinar cells throughout the different cell‐cycle phases was analysed at different AP stages by flow cytometry using propidium iodide staining. NAC administration reduced the depletion of pancreatic GSH content and prevented OFR generation in acinar cells of rats with PDO‐induced acute pancreatitis. As a result, AP became less severe as reflected by the significant improvement of hyper‐amylasaemia and maintenance of plasma TNF‐α levels at values not significantly different from controls were found. NAC administration inhibited progression of cell‐cycle phases, maintaining acinar cells in quiescent state at early PDO times. The protection from oxidative damage by NAC treatment during early AP, allows the pancreatic cell to enter S‐phase actively at later stages, thereby allowing acinar cells to proliferate and preventing the pancreatic atrophy provoked by PDO‐induced AP. The results provide evidence that OFR play a critical role in the progression of acinar cell‐cycle phases. Prevention of OFR generation of acinar cells in rats with PDO‐induced AP through NAC treatment, not only protects pancreas from oxidative damage but also promotes beneficial changes in the cell cycle progression which reduce the risk of pancreatic atrophy.