2',4',6'-Tris(methoxymethoxy) chalcone (TMMC) attenuates the severity of cerulein-induced acute pancreatitis and associated lung injury

Acute pancreatitis (AP) is an inflammatory disease involving acinar cell injury and rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. 2',4',6'-Tris (methoxymethoxy) chalcone (TMMC), a synthetic chalcone derivative, displays potent anti-inflammatory effects. Therefore, we aimed to investigate whether TMMC might affect the severity of AP and pancreatitis-associated lung injury in mice. We used the cerulein hyperstimulation model of AP. Severity of pancreatitis was determined in cerulein-injected mice by histological analysis and neutrophil sequestration. The pretreatment of mice with TMMC reduced the severity of AP and pancreatitis-associated lung injury and inhibited several biochemical parameters (activity of amylase, lipase, trypsin, trypsinogen, and myeloperoxidase and production of proinflammatory cytokines). In addition, TMMC inhibited pancreatic acinar cell death and production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 by inhibiting NF-κB and extracellular signal-regulated protein kinase 1/2 (ERK1/2) activation. Neutralizing antibodies for TNF-α, IL-1β, and IL-6 inhibited cerulein-induced cell death in isolated pancreatic acinar cells. Moreover, pharmacological blockade of NF-κB/ERK1/2 reduced acinar cell death and production of TNF-α, IL-1β, and IL-6 in isolated pancreatic acinar cells. In addition, posttreatment of mice with TMMC showed reduced severity of AP and lung injury. Our results suggest that TMMC may reduce the complications associated with pancreatitis.     

Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis

Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.     

Involvement of thrombopoietin in acinar cell necrosis in l-arginine-induced acute pancreatitis in mice

Thrombopoietin (TPO) plays an important role in injuries of different tissues. However, the role of TPO in acute pancreatitis (AP) is not yet known. The aim of the study was to determine the involvement of TPO in AP. Serum TPO was assayed in necrotizing pancreatitis induced by l-arginine in mice. Recombinant TPO and anti-TPO antibody were given to mice with necrotizing pancreatitis. Amylase, lipase, lactate dehydrogenase, myeloperoxidase activity and pancreatic water content were assayed in serum and tissue samples. Pancreas and lung tissue samples were also collected for histological evaluation. Immunohistochemistry of amylase α and PCNA were applied for the study of acinar regeneration and TUNEL assay for the detection of apoptosis in the pancreas. Increased levels of serum TPO were found in necrotizing pancreatitis. After TPO administration, more severe acinar necrosis was found and blockade of TPO reduced the acinar necrosis in this AP model. Acinar regeneration and apoptosis in the pancreas were affected by TPO and antibody treatment in necrotizing pancreatitis. The severity of pancreatitis-associated lung injury was worsened after TPO treatment, but attenuated after Anti-TPO antibody treatment. In conclusion, serum TPO is up-regulated in the necrotizing pancreatitis induced by l-arginine in mice and may be a risk factor for the pancreatic acinar necrosis in AP. As a pro-necrotic factor, blockade of TPO can attenuate the acinar necrosis in AP and may be a possible therapeutic intervention for AP.     

Probiotics enhance pancreatic glutathione biosynthesis and reduce oxidative stress in experimental acute pancreatitis

Factors determining severity of acute pancreatitis (AP) are poorly understood. Oxidative stress causes acinar cell injury and contributes to the severity, whereas prophylactic probiotics ameliorate experimental pancreatitis. Our objective was to study how probiotics affect oxidative stress, inflammation, and acinar cell injury during the early phase of AP. Fifty-three male Sprague-Dawley rats were randomly allocated into groups: 1) control, 2) sham procedure, 3) AP with no treatment, 4) AP with probiotics, and 5) AP with placebo. AP was induced under general anesthesia by intraductal glycodeoxycholate infusion (15 mM) and intravenous cerulein (5 microg.kg(-1).h(-1), for 6 h). Daily probiotics or placebo were administered intragastrically, starting 5 days prior to AP. After cerulein infusion, pancreas samples were collected for analysis including lipid peroxidation, glutathione, glutamate-cysteine-ligase activity, histological grading of pancreatic injury, and NF-kappaB activation. The severity of pancreatic injury correlated to oxidative damage (r = 0.9) and was ameliorated by probiotics (1.5 vs. placebo 5.5; P = 0.014). AP-induced NF-kappaB activation was reduced by probiotics (0.20 vs. placebo 0.53 OD(450nm)/mg nuclear protein; P < 0.001). Probiotics attenuated AP-induced lipid peroxidation (0.25 vs. placebo 0.51 pmol malondialdehyde/mg protein; P < 0.001). Not only was AP-induced glutathione depletion prevented (8.81 vs. placebo 4.1 micromol/mg protein, P < 0.001), probiotic pretreatment even increased glutathione compared with sham rats (8.81 vs. sham 6.18 miccromol/mg protein, P < 0.001). Biosynthesis of glutathione (glutamate-cysteine-ligase activity) was enhanced in probiotic-pretreated animals. Probiotics enhanced the biosynthesis of glutathione, which may have reduced activation of inflammation and acinar cell injury and ameliorated experimental AP, via a reduction in oxidative stress.     

Chaiqin chengqi decoction alleviates severity of acute pancreatitis via inhibition of TLR4 and NLRP3 inflammasome: Identification of bioactive ingredients via pharmacological sub-network analysis and experimental validation

BackgroundChaiqin chengqi decoction (CQCQD) is a Chinese herbal formula derived from dachengqi decoction. CQCQD has been used for the management of acute pancreatitis (AP) in the West China Hospital for more than 30 years. Although CQCQD has a well-established clinical efficacy, little is known about its bioactive ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory effects.PurposeToll-like receptor 4 (TLR4) and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pro-inflammatory signaling pathways, play a central role in AP in determining the extent of pancreatic injury and systemic inflammation. In this study, we screened the bioactive ingredients using a pharmacological sub-network analysis based on the TLR4/NLRP3 signaling pathways followed by experimental validation.MethodsThe main CQCQD bioactive compounds were identified by UPLC-QTOF/MS. The TLR4/NLRP3 targets in AP for CQCQD active ingredients were confirmed through a pharmacological sub-network analysis. Mice received 7 intraperitoneal injections of cerulein (50 μg/kg; hourly) to induce AP (CER-AP), while oral gavage of CQCQD (5, 10, 15 and 20 g/kg; 3 doses, 2 hourly) was commenced at the 3rd injection of cerulein. Histopathology and biochemical indices were used for assessing AP severity, while polymerase chain reaction, Western blot and immunohistochemistry analyses were used to study the mechanisms. Identified active CQCQD compounds were further validated in freshly isolated mouse pancreatic acinar cells and cultured RAW264.7 macrophages.ResultsThe main compounds from CQCQD belonged to flavonoids, iridoids, phenols, lignans, anthraquinones and corresponding glycosides. The sub-network analysis revealed that emodin, rhein, baicalin and chrysin were the compounds most relevant for directly regulating the TLR4/NLRP3-related proteins TLR4, RelA, NF-κB and TNF-α. In vivo, CQCQD attenuated the pancreatic injury and systemic inflammation of CER-AP and was associated with reduced expression of TLR4/NLRP3-related mRNAs and proteins. Emodin, rhein, baicalin and chrysin significantly diminished pancreatic acinar cell necrosis with varied effects on suppressing the expression of TLR4/NLRP3-related mRNAs. Emodin, rhein and chrysin also decreased nitric oxide production in macrophages and their combination had synergistic effects on alleviating cell death as well as expression of TLR4/NLRP3-related proteins.ConclusionsCQCQD attenuated the severity of AP at least in part by inhibiting the TLR4/NLRP3 pro-inflammatory pathways. Its active ingredients, emodin, baicalin, rhein and chrysin contributed to these beneficial effects.     

Apoptosis versus necrosis in acute pancreatitis

Acute pancreatitis is a disease of variable severity in which some patients experience mild, self-limited attacks, whereas others manifest a severe, highly morbid, and frequently lethal attack. The events that regulate the severity of acute pancreatitis are, for the most part, unknown. It is generally believed that the earliest events in acute pancreatitis occur within acinar cells and result in acinar cell injury. Other processes, such as recruitment of inflammatory cells and generation of inflammatory mediators, are believed to occur subsequent to acinar cell injury, and these "downstream" events are believed to influence the severity of the disease. Several recently reported studies, however, have suggested that the acinar cell response to injury may, itself, be an important determinant of disease severity. In these studies, mild acute pancreatitis was found to be associated with extensive apoptotic acinar cell death, whereas severe acute pancreatitis was found to involve extensive acinar cell necrosis but very little acinar cell apoptosis. These observations led to the hypothesis that apoptosis could be a favorable response to acinar cells and that interventions that favor induction of apoptotic, as opposed to necrotic, acinar cell death might reduce the severity of an attack of acute pancreatitis. Indeed, in an experimental setting, the induction of pancreatic acinar cell apoptosis protects mice against acute pancreatitis. Little is known about the mechanism of apoptosis in the pancreatic acinar cell, although some early attempts have been made in that direction. Also, clinical relevance of these experimental studies remains to be investigated.     

Necro-inflammatory response of pancreatic acinar cells in the pathogenesis of acute alcoholic pancreatitis

The role of pancreatic acinar cells in initiating necro-inflammatory responses during the early onset of alcoholic acute pancreatitis (AP) has not been fully evaluated. We investigated the ability of acinar cells to generate pro- and anti-inflammatory mediators, including inflammasome-associated IL-18/caspase-1, and evaluated acinar cell necrosis in an animal model of AP and human samples. Rats were fed either an ethanol-containing or control diet for 14 weeks and killed 3 or 24 h after a single lipopolysaccharide (LPS) injection. Inflammasome components and necro-inflammation were evaluated in acinar cells by immunofluorescence (IF), histology, and biochemical approaches. Alcohol exposure enhanced acinar cell-specific production of TNFα, IL-6, MCP-1 and IL-10, as early as 3 h after LPS, whereas IL-18 and caspase-1 were evident 24 h later. Alcohol enhanced LPS-induced TNFα expression, whereas blockade of LPS signaling diminished TNFα production in vitro, indicating that the response of pancreatic acinar cells to LPS is similar to that of immune cells. Similar results were observed from acinar cells in samples from patients with acute/recurrent pancreatitis. Although morphologic examination of sub-clinical AP showed no visible signs of necrosis, early loss of pancreatic HMGB1 and increased systemic levels of HMGB1 and LDH were observed, indicating that this strong systemic inflammatory response is associated with little pancreatic necrosis. These results suggest that TLR-4-positive acinar cells respond to LPS by activating the inflammasome and producing pro- and anti-inflammatory mediators during the development of mild, sub-clinical AP, and that these effects are exacerbated by alcohol injury.     

N-acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruction-induced acute pancreatitis

Although oxygen free radicals (OFR) are considered to be one of the pathophysiological mechanisms involved in acute pancreatitis (AP), the contribution of acinar cells to their production is not well established. The aim of the present study was to determine the effect of N-acetylcysteine (NAC) in the course of AP induced by pancreatic duct obstruction (PDO) in rats, directly analysing by flow cytometry the quantity of OFR generated in acinar cells. NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Measurements by flow cytometry of OFR generated in acinar cells were taken at different PDO times over 24 h, using dihydrorhodamine-123 as fluorescent dye. Histological studies of pancreas and measurements of neutrophil infiltration in the pancreas, pancreatic glutathione (GSH), malondialdehyde (MDA) levels, plasma amylase activity and hemoconcentration were carried out in order to assess the severity of AP at different stages. NAC effectively blunted GSH depletion at early AP stages and prevented OFR generation found in acinar cells as a consequence of AP induced by PDO. This attenuation of the redox state impairment reduced cellular oxidative damage, as reflected by less severe pancreatic lesions, normal pancreatic MDA levels, as well as diminished neutrophil infiltration in pancreas. Hyperamylasemia and hemoconcentration following AP induction were ameliorated by NAC administration at early stages, when oxidative stress seems to be critical in the development of pancreatitis. In conclusion, NAC reinforces the antioxidant defences in acinar cells, preventing OFR generation therefore attenuating oxidative damage and subsequently reducing the severity of PDO-induced AP at early stages of the disease.     

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

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-alpha 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-alpha 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.     

JAK and STAT proteins are expressed and activated by IFN-gamma in rat pancreatic acinar cells

The development of acute pancreatitis (AP) is triggered by acinar events, but the subsequent extra-acinar events, particularly a distinct immune response, appear to determine its severity. Cytokines modulate this immune response and are derived not only from immunocytes but also from pancreatic acinar cells. We studied whether pancreatic acinar cells were also capable of responding to cytokines. The JAK/STAT-pathway represents the main effector for many cytokines. Therefore, expression and regulation of JAK and STAT proteins were investigated in rat pancreatic acinar cells. Western blotting showed expression of JAK1, JAK2, Tyk2, and STAT1, STAT2, STAT3, STAT5, STAT6. In addition, STAT1 was reversibly tyrosine-phosphorylated upon the procedure of acinar cell isolation. In contrast, STAT3-phosphorylation occurred spontaneously after pancreas removal and was not reversible within 8 h. STAT1 phosphorylation was also observed upon treatment with IFN-gamma but not upon EGF, TNF-alpha or IL-6, and inhibited by the JAK2-inhibitor AG-490. Immunohistochemistry revealed cytoplasmic expression of unphosphorylated STAT1 in untreated acinar cells and nuclear translocation of phosphorylated STAT1 following IFN-gamma-treatment. Interestingly, although CCK leads to the activation of multiple stress pathways in pancreatic acinar cells, we found no influence of CCK on phosphorylation of STAT1, STAT3, or STAT5 in the pancreas. In conclusion, our data provide further evidence that pancreatic acinar cells are able to interact with immune cells. Besides stimulating immune cells via cytokine secretion, acinar cells are in turn capable of responding to IFN-gamma via JAK2 and STAT1 which may have an impact on the development of AP.     

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Effect of long-term CCK blockade on the pancreatic acinar cell renewal in rats with acute pancreatitis

This study determines the effect of 7-day pretreatment with L364,718 (a potent cholecystokinin (CCK) receptor antagonist) on pancreatic cell turnover during the course of acute pancreatitis (AP) induced in the rat by bile-pancreatic duct obstruction (BPDO). Cell cycle distribution and apoptosis were analyzed by flow cytometry using propidium iodide (PI) and Annexin V staining. Besides altering the pancreatic redox status, long-term CCK blockade inhibited the normal proliferation of acinar cells as indicated by the significant increase in G(0)/G(1)-phase cells and the decrease in G(2)/M-cells found in control rats treated with L364,718 for 7 days. A progressive depletion in pancreatic GSH was found from 3 to 24h after BPDO with similar values in L364,718-pretreated and non-treated rats, which led to a maximum peak in malondialdehyde (MDA) levels 6h after BPDO. However, plasma amylase activity and ascites volume indicated higher severity of AP in L364,718-pretreated rats. CCK blockade enhanced the alterations that appear in cell cycle distribution of acinar cells during AP demonstrated by the significantly higher increase in G(0)/G(1)-cells and decrease in S-cells found in L364,718-treated rats 48h after BPDO. Our results indicate that the renewal of acinar cells deleted by apoptosis 48h after BPDO worsens if CCK is blocked before inducing AP.     

Substance P treatment stimulates chemokine synthesis in pancreatic acinar cells via the activation of NF-kappaB

Acinar cell injury early in acute pancreatitis leads to a local inflammatory reaction and to the subsequent systemic inflammatory response, which may result in multiple organ dysfunction and death. Inflammatory mediators, including chemokines and substance P (SP), are known to play a crucial role in the pathogenesis of acute pancreatitis. It has been shown that pancreatic acinar cells produce the chemokine monocyte chemoattractant protein-1 (MCP-1) in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Similarly, SP levels in the pancreas and pancreatic acinar cell expression of neurokinin-1 receptor, the primary receptor for SP, are both increased during secretagogue-induced experimental pancreatitis. This study aims to examine the functional consequences of exposing mouse pancreatic acinar cells to SP and to determine whether it leads to proinflammatory signaling, such as production of chemokines. Exposure of mouse pancreatic acini to SP significantly increased synthesis of MCP-1, macrophage inflammatory protein-1alpha (MIP-1alpha), as well as MIP-2. Furthermore, SP also increased NF-kappaB activation. The stimulatory effect of SP was specific to chemokine synthesis through the NF-kappaB pathway, since the increase in chemokine production was completely attenuated when pancreatic acini were pretreated with the selective NF-kappaB inhibitor NF-kappaB essential modulator-binding domain peptide. This study shows that SP-induced chemokine synthesis in mouse pancreatic acinar cells is NF-kappaB dependent.     

Pro- and anti-inflammatory response of acinar cells during acute pancreatitis. Effect of N-acetyl cysteine

We investigate the ability of acinar cells to produce tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) at different stages of acute pancreatitis (AP). Since oxidative stress is involved in the inflammatory response, the effect of N-acetyl cysteine (NAC) has also been evaluated. AP was induced in rats by bile-pancreatic duct obstruction (BPDO). NAC (50 mg/kg) was administered 1h before and 1h after BPDO. Acinar cells were incubated for 4 h at 37 degrees C in 5% CO2 atmosphere in absence and presence of 24-h BPDO-PAAF (20%, v/v) as stimulant agent. Acinar production of TNF-alpha and IL-10 was analysed by flow cytometry. Plasma amylase activity and histological studies of the pancreas indicated the severity of AP. PAAF significantly stimulated the acinar production of TNF-alpha and IL-10 in control rats. TNF-alpha production was also significantly stimulated in acinar cells of rats with AP, although a decrease in the pro-inflammatory response was found from 6 h after BPDO onwards. However, acinar cells failed to produce IL-10 from 3 h after BPDO. The protective effect of NAC treatment against oxidative cell damage reduced the pancreatic injury and maintained and enhanced the ability of acinar cells to produce IL-10 at early AP stages. As long as acinar cells were not severely damaged in the course of AP, greater ability to produce cytokines in response to PAAF was found in those with higher forward scatter (R2 cells). We suggest that the capability of acinar cells to maintain an appropriate balance between the production of pro- and anti-inflammatory mediators could contribute to determine the degree of severity of AP.     

Inhibition of cyclooxygenase-2 ameliorates the severity of pancreatitis and associated lung injury

Cyclooxygenase-2 (COX-2), a widely distributed enzyme, plays an important role in inflammation. We have studied the role of COX-2 in acute pancreatitis and pancreatitis-associated lung injury using both the pharmacological inhibition of COX-2 and genetic deletion of COX-2. Pancreatitis was induced in mice by 12 hourly injections of cerulein. The severity of pancreatitis was assessed by measuring serum amylase, pancreatic trypsin activity, intrapancreatic sequestration of neutrophils, and acinar cell necrosis. The severity of lung injury was evaluated by measuring lactate dehydrogenase levels in the bronchoalveolar lavage fluid and by quantitating neutrophil sequestration in the lung. In both the pharmacologically inhibited and genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was reduced compared with the noninhibited strains of COX-2-sufficient mice. This reduction in injury indicates that COX-2 plays an important proinflammatory role in pancreatitis and its associated lung injury. Our findings support the concept that COX-2 inhibitors may play a beneficial role in the prevention of acute pancreatitis or in the reduction of its severity.     

Bile Acids Induce Pancreatic Acinar Cell Injury and Pancreatitis by Activating Calcineurin

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca²⁺. Thus, it would be clinically relevant to know the targets of this aberrant Ca²⁺ signal. We hypothesized that the Ca²⁺-activated phosphatase calcineurin is such a Ca²⁺ target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca²⁺. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca²⁺ generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.     

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-κB 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-κB and STAT3 activation triggers the chemokine expression in acini but not in non-acinar cells.