Infusion Of Taurocholate Research Articles

Systemic injury caused by taurocholate-induced severe acute pancreatitis in rats.

Systemic injury plays a central role in severe acute pancreatitis (SAP). Retrograde biliopancreatic duct infusion of sodium taurocholate (NaT) is commonly used to establish SAP animal models. To better characterize the systemic injury in this model, SAP was induced in Sprague-Dawley rats by NaT administration (3.5 or 5%), followed by sacrifice at 3, 6, 9, 12, 24, 48 and 72 h. Normal saline was used as a control in Sham-operated rats. The mortality rate, ascites volume, and serum and ascitic fluid amylase and lipase activities were assessed. Multiple organ dysfunction, including dysfunction of the pancreas, lung, ileum, liver, and kidney, was investigated using hematoxylin and eosin staining. The interleukin (IL)-1β, IL-6, and tumor necrosis factor-α levels in the ascitic fluid, serum, and ileum tissues were evaluated using an enzyme-linked immunosorbent assay (ELISA). Tight junction proteins, zonula occludens-1 (ZO-1) and occludin, in ileum tissues were studied using immunofluorescence. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE) and urea levels were measured using an automatic biochemical analyzer. The results of the present study indicated that both 3.5 and 5% NaT could induce a stable elevation of pancreatitis indices, with histopathological injury of the pancreas, lungs and ileum (5% NaT). The ascitic fluid levels of IL-6 and IL-1β were increased in the 5% NaT group. ALT and AST levels increased temporarily and recovered in 72 h, without a significant increase in CRE and urea levels or apparent hepatic and renal pathological injury. In conclusion, rats with NaT-induced SAP have characteristics of necrotizing hemorrhagic pancreatitis with multiple organ injuries, including inflammatory lung injury, ischemic intestinal injury and slight liver and kidney injuries.

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice.

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the human clinical condition and reproduction of inflammatory events corresponding to the onset of clinical biliary pancreatitis. The severity of pancreatic damage can be assessed by measuring the concentration, speed, and volume of the infused bile acid. This study provides an updated checklist of the materials and methods used in the protocol reproduction and shows the main results from this acute pancreatitis (AP) model. Most of the previous publications have limited themselves to reproducing this model in rats. We have applied this method in mice, which provides additional advantages (i.e., the availability of an arsenal of reagents and antibodies for these animals along with the possibility of working with genetically modified strains of mice) that may be relevant to the study. For acute pancreatitis induction in mice, we present a systematic protocol, with a defined dose of 2.5% sodium taurocholate at an infusion speed 10 µL/min for 3 min in C57BL/6 mice that reaches its maximal level of severity within 12 h of induction and highlight results with outcomes that validate the method. With practice and technique, the total estimated time, from the induction of anesthesia to the completion of the infusion, is 25 min per animal.

Inhibition of nicotinamide phosphoribosyltransferase protects against acute pancreatitis via modulating macrophage polarization and its related metabolites

Background & objectivesAcute pancreatitis is a common inflammatory disorder of the exocrine pancreas with no specific therapy. Intracellular nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) salvage pathway, is involved in many inflammatory disorders. In this study, we investigated the role of NAMPT in experimental acute pancreatitis. MethodsAcute pancreatitis was induced in mice using three disparate models: (1) caerulein hyperstimulation, (2) ethanol plus palmitoleic acid, and (3) retrograde biliopancreatic ductal infusion of sodium taurocholate. The NAMPT inhibitor FK866 and NAMPT downstream product nicotinamide mononucleotide (NMN) was administered. Serum and pancreas were collected and analyzed biochemically and histologically. Bone marrow derived macrophages were isolated, cultured with cytokines or pancreatic acini, then analyzed by quantitative PCR and non-targeted metabolomics. ResultsThe levels of pancreatic NAMPT and NAD were down-regulated upon acute pancreatitis. NAMPT inhibitor FK866 suppressed M1 macrophage polarization while NMN boosted it. In co-culture of macrophages with acinar cells, inhibition of NAMPT prevented M1-like macrophage differentiation induced by injured pancreatic acini. The injured pancreatic acinar milieu induced a unique metabolic signature linked to macrophage polarization, and inhibition of NAMPT reversed these metabolites changes. Furthermore, NMN supplementation aggravated caerulein hyperstimulation pancreatitis and alcoholic pancreatitis, and inhibition of NAMPT protected against caerulein hyperstimulation, alcoholic and biliary acute pancreatitis and reducing pancreatic macrophage infiltration in vivo. ConclusionsNAMPT inhibition protects against acute pancreatitis via preventing M1 macrophage polarization and restoring the metabolites related to macrophage polarization and that NAMPT could be a promising therapeutic target for acute pancreatitis.

Complement Component 3 Is Required for Tissue Damage, Neutrophil Infiltration, and Ensuring NET Formation in Acute Pancreatitis

Background: Neutrophil extracellular traps (NETs) are known to play an important role in the pathophysiology of acute pancreatitis (AP). Activation of the complement cascade has been shown to occur in AP. The aim of this study was to examine whether complement component 3 is involved in the generation of NETs in AP. Methods: AP was induced in wild-type and C3-deficient mice by retrograde infusion of taurocholate into the pancreatic duct. Blood, lung, and pancreas tissue were collected and MPO activity was determined in lung and pancreas tissue. Histological examination of the inflamed pancreas was performed. Plasma levels of CXCL2, MMP-9, IL-6, and DNA-histone complexes as well as pancreatic levels of CXCL1 and CXCL2 were determined by use of enzyme-linked immunosorbent assay. NETs were detected in the pancreas by electron microscopy. The amount of MPO and citrullinated histone 3 in neutrophils isolated from bone marrow was examined using flow cytometry. Results: In C3-deficient mice, challenge with taurocholate yielded much fewer NETs in the pancreatic tissue compared with wild-type controls. Taurocholate-induced blood levels of amylase, tissue injury, and neutrophil recruitment in the pancreas were markedly reduced in the mice lacking C3. Furthermore, MPO levels in the lung, and plasma levels of IL-6, MMP-9, and CXCL2 were significantly lower in the C3-deficient mice compared to wild-type mice after the induction of AP. In vitro studies revealed that neutrophils from C3-deficient mice had normal NET-forming ability and recombinant C3a was not capable of directly inducing NETs formation in the wild-type neutrophils. Conclusion: C3 plays an important role in the pathophysiology of AP as it is necessary for the recruitment of neutrophils into the pancreas and ensuring NETs formation. Targeting C3 could hence be a potential strategy to ameliorate local damage as well as remote organ dysfunction in AP.

Extracellular cold-inducible RNA-binding protein regulates neutrophil extracellular trap formation and tissue damage in acute pancreatitis

AbstractNeutrophil extracellular traps (NETs) play a key role in the development of acute pancreatitis (AP). In the present study, we studied the role of extracellular cold-inducible RNA-binding protein (eCIRP), a novel damage-associated-molecular-pattern molecule, in severe AP. C57BL/6 mice underwent retrograde infusion of taurocholate into the pancreatic duct. C23, an eCIRP inhibitor, was given 1 h prior to induction of AP. Pancreatic, lung, and blood samples were collected and levels of citrullinated histone 3, DNA-histone complexes, eCIRP, myeloperoxidase (MPO), amylase, cytokines, matrix metalloproteinase-9 (MMP-9), and CXC chemokines were quantified after 24 h. NETs were detected by electron microscopy in the pancreas and bone marrow-derived neutrophils. Amylase secretion was analyzed in isolated acinar cells. Plasma was obtained from healthy individuals and patients with mild and moderate severe or severe AP. Taurocholate infusion induced NET formation, inflammation, and tissue injury in the pancreas. Pretreatment with C23 decreased taurocholate-induced pancreatic and plasma levels of eCIRP and tissue damage in the pancreas. Blocking eCIRP reduced levels of citrullinated histone 3 and NET formation in the pancreas as well as DNA-histone complexes in the plasma. In addition, administration of C23 attenuated MPO levels in the pancreas and lung of mice exposed to taurocholate. Inhibition of eCIRP reduced pancreatic levels of CXC chemokines and plasma levels of IL-6, HMGB-1, and MMP-9 in mice with severe AP. Moreover, eCIRP was found to be bound to NETs. Coincubation with C23 reduced NET-induced amylase secretion in isolated acinar cells. Patients with severe AP had elevated plasma levels of eCIRP compared with controls. Our novel findings suggest that eCIRP is a potent regulator of NET formation in the inflamed pancreas. Moreover, these results show that targeting eCIRP with C23 inhibits inflammation and tissue damage in AP. Thus, eCIRP could serve as an effective target to attenuate pancreatic damage in patients with AP.The authors investigated the role of extracellular cold-inducible RNA-binding protein (eCIRP) in acute pancreatitis (AP) and found that eCIRP acts as a potent regulator of neutrophil extracellular traps (NETs) formation in AP. They observed that eCIRP itself is one of the NETs associated proteins. Furthermore, they demonstrate that C23 (a potent eCIRP inhibitor) reduces NETs formation and inflammation in AP.

Effect of Clostridium butyricum and Butyrate on Intestinal Barrier Functions: Study of a Rat Model of Severe Acute Pancreatitis With Intra-Abdominal Hypertension.

Background/AimsSevere acute pancreatitis (SAP) is associated with intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS), but treatment of these conditions is difficult. We studied a rat model of SAP + IAH to determine the effect of oral administration of Clostridium butyricum and butyrate (its major metabolite) on intestinal barrier functions.MethodsA total of 48 rats were assigned to four groups, with 12 rats per group: Sham, SAP+IAH, SAP+IAH+C. butyricum, and SAP + IAH + butyrate. SAP was induced by sodium taurocholate infusion into the biliopancreatic duct, intra-abdominal pressure (IAP), mortality was measured 24 h later, and then rats were euthanized. The plasma levels of several markers [amylase, diamine oxidase (DAO), fluorescein isothiocyanate (FITC)-dextran, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, IL-12, lipopolysaccharide (LPS)] and fecal butyric acid level were determined. The pancreas and intestine were examined using histology, and RT-PCR and Western blotting of intestinal tissues were used to measure the expression of six markers {tight junction proteins [zonula occludens protein-1 (ZO-1), claudin-1, claudin-2, occluding], matrix metalloproteinase 9 [MMP9], and TNF-α}. The gut flora of the rats was examined by 16S rRNA sequencing.ResultsInduction of SAP + IAH altered several functions of the intestinal barrier, and enhanced intestinal permeability, decreased the levels of ZO-1, claudin-1, occludin, the richness and diversity of the microflora community, the relative abundance (RA) of Firmicutes, and the number of probiotic organisms. However, induction of SAP+IAH increased the expression of claudin-2, MMP9, and TNF-α, and the RA of Proteobacteria and pathogens in the feces. Rats that received oral C. butyricum or butyrate had reduced intestinal injury and plasma levels of DAO, LPS, and inflammatory cytokines.ConclusionThis study of rats with SAP+IAH indicated that oral dosing of C. butyricum or butyrate reduced intestinal injury, possibly by altering the functions of the intestinal mucosal barrier.

C-Abl kinase regulates neutrophil extracellular trap formation, inflammation, and tissue damage in severe acute pancreatitis

Neutrophil extracellular traps (NETs) are involved in acute pancreatitis (AP) but mechanisms controlling NET expulsion in AP are incompletely understood. Herein, we examined the role of c-Abelson (c-Abl) kinase in NET formation and tissue damage in severe AP. AP was induced by taurocholate infusion into pancreatic duct or intraperitoneal administration of l-arginine in mice. Pancreatic, lung, and blood samples were collected and levels of phosphorylated c-Abl kinase, citrullinated histone 3, DNA-histone complexes, myeloperoxidase, amylase, cytokines, and CXC chemokines were quantified. Citrullinated histone 3, reactive oxygen species (ROS), and NET formation were determined in bone marrow neutrophils. Taurocholate challenge increased phosphorylation of c-Abl kinase and levels of citrullinated histone 3 in the pancreas as well as DNA-histone complexes in the plasma. Administration of the c-Abl kinase inhibitor GZD824 not only abolished activation of c-Abl kinase but also decreased levels of citrullinated histone 3 in the pancreas and DNA-histone complexes in the plasma of animals with AP. Moreover, GZD824 decreased plasma levels of amylase, IL-6, and MMP-9 as well as edema, acinar cell necrosis, hemorrhage, CXC chemokine formation, and neutrophil infiltration in the inflamed pancreas. A beneficial effect of c-Abl kinase inhibition was confirmed in l-arginine-induced pancreatitis. In vitro, inhibition of c-Abl kinase reduced TNF-α-induced formation of ROS, histone 3 citrullination, and NETs in isolated bone marrow neutrophils. Our findings demonstrate that c-Abl kinase regulates NET formation in the inflamed pancreas. In addition, inhibition of c-Abl kinase reduced pancreatic tissue inflammation, and damage in AP. Thus, targeting c-Abl kinase might be a useful way to protect the pancreas in severe AP.

Obesity Aggravates Acute Pancreatitis via Damaging Intestinal Mucosal Barrier and Changing Microbiota Composition in Rats

Obesity may aggravate acute pancreatitis (AP) through damaging the intestinal mucosal barrier (IMB). The underlying mechanism remains unclear. This study was aimed to provide further data to clarify the mechanism. 48 rats were divided into 4 groups: 1) normal control (NC), chow-fed rats with sham operation, 2) no-obese rats with AP (NAP), chow-fed rats with taurocholate infusion, 3) obese control (OC), high-fat diet (HFD)-fed rats with sham operation, and 4) obese rats with AP (OAP), HFD-fed rats with taurocholate infusion. Pancreatic pathologic score (11.39 ± 1.76 vs. 14.11 ± 1.05, p = 0.005), intestinal permeability to FD4 (0.91 ± 0.25 μg/ml vs. 7.06 ± 3.67 μg/ml, p < 0.001), serum leptin (10.25 ± 5.59 ng/ml vs. 79.73 ± 38.44 ng/ml, p < 0.001) and ileal apoptosis (2.05 ± 0.73% vs. 4.53 ± 2.28%, p = 0.006) were significantly higher in OAP than in NAP group. The intestinal bacterial richness (Chao 1 and OTUs) was significantly lower in OAP than in NAP rats. The higher abundance of Proteobacteria and reduced proportions of intestinal Actinobacteria, Allobaculum and Barnesiella were detected in OAP group. Obesity may result in decreased intestinal leptin/ObR-b binding, distinct phylogenetic clusters of ileal bacterial communities, increased intestinal inflammatory injury and the insufficient intestinal epithelial cells proliferation during AP attack. Pancreatic injury was aggravated due to obesity associated dysfunction of IMB.

Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis.

Recent evidence suggests that neutrophil extracellular traps (NETs) play an important role in the development of acute pancreatitis (AP). Herein, we examined the role of peptidylarginine deiminase (PAD), which has been shown to regulate NET formation, in severe AP. AP was induced by retrograde of taurocholate infusion into pancreatic duct in C57BL/6 mice. PAD was pharmacologically inhibited using Cl-amidine, a pan-PAD inhibitor. Pancreata were collected, and histones, citrullinated histone 3, chemokines, myeloperoxidase, and NETs were quantified. Chemokines, matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), and DNA-histone complexes were determined in plasma samples. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice. Pretreatment with Cl-amidine markedly reduced the NET formation in the inflamed pancreas. Moreover, inhibition of PAD decreased the levels of blood amylase as well as edema, acinar cell necrosis, hemorrhage, and neutrophil infiltration in the pancreas of animals with AP. Administration of Cl-amidine attenuated the myeloperoxidase levels in the pancreas and lung of mice exposed to taurocholate. In addition, Cl-amidine decreased pancreatic levels of CXC chemokines, plasma levels of IL-6, and MMP-9 in mice with severe AP. This study shows that Cl-amidine is a potent inhibitor of NET formation in severe AP. Also, our results suggest that PAD regulates pathological inflammation and tissue damage in the inflamed pancreas. Thus, targeting PAD might be a useful strategy to treat patients with severe AP.

Development of an acute pancreatitis porcine model based on endoscopic retrograde infusion of contrast medium or sodium taurocholate.

Background/AimsA reproducible, endoscope-based, large animal model, of acute pancreatitis was developed to meet the need for a suitable means of preclinically testing treatments. The aim of this study was to develop an endoscope-based animal model of acute pancreatitis.MethodsThis experimental study was conducted on six mini-pigs. The pancreatitis model was induced by infusing contrast medium (CM) or sodium taurocholate (TCA) under high pressure (100 mmHg) into the main pancreatic duct by endoscopic retrograde pancreatography. Animals were randomly allocated to three groups: a CM group, a 10% TCA group, and a 20% TCA group. Pancreatic injuries were evaluated histologically, and serum amylase and lipase levels were measured.ResultsAcute pancreatitis was observed in all animals during hematologic and histologic examinations. Serum amylase and lipase levels were significantly higher (> 10 times baseline), and pancreatic edema, vacuolization of acinar cells, and hemorrhagic necrosis were observed. Severity of pancreatitis tended to be greater in the TCA groups than in the CM group as assessed using histologic scores, and degrees of pancreatitis were found to be dose-dependently related to TCA concentration.ConclusionsThe two endoscopic procedures described are effective and safe for creating a swine model of acute pancreatitis. The authors hope the described endoscopic methods will assist in the development of a suitable treatment strategy.

Involvement of Nucleotide-Binding and Oligomerization Domain-Like Receptors in the Intestinal Injury of Severe Acute Pancreatitis in Rats.

The aim of the study was to observe the role of nucleotide-binding and oligomerization domain (NOD)-like receptors (NLR) in intestinal injury of severe acute pancreatitis (SAP) in rats. Severe acute pancreatitis was induced by retrograde infusion of sodium taurocholate into the biliopancreatic duct. Rats were divided into the following 6 groups: sham operation, SAP treated with saline, and SAP treated with interleukin 1β (IL-1β)-converting enzyme inhibitor, killed at 6 or 12 hours after operation. Serum IL-18 and IL-1β concentrations were measured. mRNA expression and protein levels of NOD1, NOD2, and NLRP3 in the intestine were measured. Severe acute pancreatitis resulted in significantly higher serum IL-18 and IL-1β concentration, higher mRNA expression, and protein levels of NOD1, NOD2, and NLRP3 in intestine in SAP treated with saline groups compared with sham operation groups. This effect was attenuated by administration of IL-1β-converting enzyme inhibitor. The NLRs, including NOD1, NOD2, and NLRP3, were involved in the intestinal injury in SAP through a caspase-1 pathway.

Effects of ω-3 Fatty Acids on Toll-like Receptor 4 and Nuclear Factor κB p56 in the Pancreas of Rats With Severe Acute Pancreatitis.

The aims of this study were to determine the effects of ω-3 fatty acids (ω-3FAs) on the Toll-like receptor 4 (TLR4)/nuclear factor κB p56 (NF-κBp56) signaling pathway in the pancreas of rats with severe acute pancreatitis (SAP). Sixty-four Sprague-Dawley rats were randomly divided into 4 groups: the control, SAP-saline, SAP-soybean oil, and SAP-ω-FA groups. Severe acute pancreatitis was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the pancreas was evaluated by immunohistochemistry and Western blot analysis. The levels of the proinflammatory cytokines interleukin 6 and tumor necrosis factor α in the pancreas were measured by enzyme-linked immunosorbent assays. Toll-like receptor 4, NF-κBp56, and inflammatory cytokine expression in the pancreas was increased significantly in the SAP group compared with that in the control group (P < 0.05), but was significantly decreased in the ω-3FA group compared with that in the soybean oil group at 24 and 48 hours (P < 0.05). Our results suggest that during the initial stage of SAP ω-3FAs could efficiently lower the inflammatory response by activating the TLR4/NF-κBp56 signaling pathway.

Poly(ADP-ribose) polymerase inhibition suppresses inflammation and promotes recovery from adrenal injury in a rat model of acute necrotizing pancreatitis.

BackgroundPoly(ADP-ribose) polymerase (PARP) participates in multi-organ failure in various inflammatory diseases including acute necrotizing pancreatitis (ANP). Since pancreatitis-associated adrenal insufficiency is partly caused by inflammatory damage to the adrenal cortex, we examined whether PARP antagonism could alleviate adrenal insufficiency in a rat model of ANP.MethodsANP was induced by retrograde infusion of sodium taurocholate into the bile-pancreatic duct. At 30 min prior to taurocholate infusion, rats were pretreated with the PARP inhibitor 3-Aminobenzamide (3-AB, 20 mg/kg) or vehicle. Pancreatic pathological injury, adrenal histology, neutrophil infiltration, cell apoptosis, and serum corticosterone level were assessed at various times points. Activities of poly(ADP-ribosyl)ated protein (PAR), nuclear factor-kappaB (NF-kB), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) in the adrenal were also examined.ResultsPARP overactivation in ANP rats is associated with reduced serum corticosterone level and marked cellular alterations in adrenocortical tissue. Inflammatory stress caused by ANP reduced adrenal corticosterone release. 3-AB reduced the activation of PARP and inflammatory markers, decreased myeloperoxidase activity, attenuated adrenal morphologic lesions and cells apoptosis, simultaneously improved the impaired adrenal function.ConclusionsOur data demonstrate the involvement of PARP overactivation in the pathogenesis of adrenal dysfunction after ANP. PARP inhibition may suppress inflammation and promote functional recovery from adrenal injury.

Platelet-derived CXCL4 regulates neutrophil infiltration and tissue damage in severe acute pancreatitis

Platelets are known to play an important role in acute pancreatitis (AP) via promotion of neutrophil accumulation, although mechanisms behind platelet-dependent accumulation of neutrophils in the pancreas remain elusive. Platelets contain a wide spectrum of different pro-inflammatory compounds, such as chemokines. CXCL4 (platelet factor 4) is one of the most abundant chemokine in platelets, and we hypothesized that CXCL4 might be involved in platelet-dependent accumulation of neutrophils in the inflamed pancreas. The aim of this study was to examine the role of CXCL4 in severe AP. Pancreatitis was provoked by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine in C57BL/6 mice. Animals were treated with an antibody against platelets or CXCL4 before induction of pancreatitis. Plasma and lung levels of CXCL2, CXCL4, and interleukin (IL)-6 were determined by use of enzyme-linked immunosorbent assay. Flow cytometry was used to examine surface expression of macrophage-1 (Mac-1) on neutrophils. Plasma was obtained from healthy individuals (controls) and patients with AP. Challenge with taurocholate increased plasma levels of CXCL4, and depletion of platelets markedly reduced plasma levels of CXCL4 indicating that circulating levels of CXCL4 are mainly derived from platelets in AP. Inhibition of CXCL4 reduced taurocholate-induced neutrophil recruitment, IL-6 secretion, edema formation, amylase release, and tissue damage in the pancreas. However, immunoneutralization of CXCL4 had no effect on CXCL2-evoked neutrophil expression of Mac-1 or chemotaxis invitro, suggesting an indirect effect of CXCL4 on neutrophil recruitment in AP. Targeting CXCL4 significantly attenuated plasma and lung levels of CXCL2, which is a potent neutrophil chemoattractant, and inhibition of the CXCL2 receptor attenuated neutrophil infiltration and tissue damage in the inflamed pancreas. A significant role of CXCL4 was confirmed in an alternate model of AP induced by L-arginine challenge. Moreover, patients with AP had significantly increased plasma levels of CXCL4 compared with healthy controls. These findings' results suggest that platelet-derived CXCL4 is a potent stimulator of neutrophil accumulation in AP and that this is mediated via generation of CXCL2 in the inflamedpancreas. We conclude that CXCL4 plays an important role in pancreatic inflammation and that targeting CXCL4 might be a useful way to ameliorate tissue damage in AP.

Effect of N-acetylcysteine on neutrophil functions during experimental acute pancreatitis.

Systemic inflammatory responses and extrapancreatic vital organ impairment are mediated by activated neutrophil functions and products, such as oxygen-derived free radicals, in patients with acute pancreatitis (AP). The present study is an examination of effects of an antioxidant, N-acetylcysteine (NAC), on local and systemic histopathological changes and neutrophil functions during AP. This experimental study was performed on 24 Wistar albino rats equally divided into 3 groups: Group 1 comprised sham laparotomy, Group 2 had AP induced with taurocholate infusion, and Group 3 consisted of AP with NAC treatment. Histopathological features in pancreas, kidney, and lung tissues were examined for local and systemic changes during AP. Neutrophil functions were evaluated using flow cytometry. Serum levels of pancreatic enzymes were elevated, and histopathological parameters showed acinar cell damage and pancreatic tissue necrosis in the 2 groups with AP. Severe histopathological changes were found in pulmonary and renal tissues, and flow cytometry results indicated defective neutrophil functions in the group with AP alone. NAC treatment significantly ameliorated phagocytosis, chemotaxis, and opsonization of neutrophils (p<0.05). NAC treatment also ameliorated systemic changes in pulmonary and renal tissue damage in all microscopic parameters (p<0.05). Uncontrolled and defective neutrophil functions could provoke severe systemic inflammatory responses. In addition to local inflammation and necrosis, severe systemic responses and histopathological changes in extrapancreatic vital organs occur during AP. Treatment with antioxidant NAC significantly reverses detrimental systemic responses in extrapancreatic vital organs by significantly ameliorating neutrophil functions despite ongoing AP.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis.

AIMTo determine the effects of ω-3 fatty acids (ω-3FA) on the toll-like receptor 4 (TLR4)/nuclear factor κB p56 (NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis (SAP).METHODSA total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay.RESULTSThe expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group (P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h (P < 0.05).CONCLUSIONDuring the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

BCL3 Reduces the Sterile Inflammatory Response in Pancreatic and Biliary Tissues

Under conditions of inflammation in the absence of micro-organisms (sterile inflammation), necrotic cells release damage-associated molecular patterns that bind to Toll-like receptors on immune cells to activate a signaling pathway that involves activation of IκB kinase and nuclear factor κB (NF-κB). Little is known about the mechanisms that control NF-κB activity during sterile inflammation. We analyzed the contribution of B-cell CLL/lymphoma 3 (BCL3), a transcription factor that associates with NF-κB, in control of sterile inflammation in the pancreas and biliary system of mice. Acute pancreatitis (AP) was induced in C57BL/6 (control) and Bcl3(-/-) mice by intraperitoneal injection of cerulein or pancreatic infusion of sodium taurocholate. We also studied Mdr2(-/-) mice, which develop spontaneous biliary inflammation, as well as Bcl3(-/-)Mdr2(-/-) mice. We performed immunohistochemical analyses of inflamed and noninflamed regions of pancreatic tissue from patients with AP or primary sclerosing cholangitis (PSC), as well as from mice. Immune cells were characterized by fluorescence-activated cell sorting analysis. Control or Bcl3(-/-) mice were irradiated, injected with bone marrow from Bcl3(-/-) or control mice, and AP was induced. Pancreatic or biliary tissues from patients with AP or PSC had higher levels of BCL3 and phosphorylated RelA and IκBα in inflamed vs noninflamed regions. Levels of BCL3 were higher in pancreata from control mice given cerulein than from mice without AP, and were higher in biliary tissues from Mdr2(-/-) mice than from control mice. Bcl3(-/-) mice developed more severe AP after administration of cerulein or sodium taurocholate than control mice; pancreata from the Bcl3(-/-) mice with AP had greater numbers of macrophages, myeloid-derived suppressor cells, dendritic cells, and granulocytes than control mice with AP. Activation of NF-κB was significantly prolonged in Bcl3(-/-) mice with AP, compared with control mice with AP. Bcl3(-/-)Mdr2(-/-) mice developed more severe cholestasis and had increased markers of liver injury and increased proliferation of biliary epithelial cells and hepatocytes than Mdr2(-/-) mice. In experiments with bone marrow chimeras, expression of BCL3 by acinar cells, but not myeloid cells, was required for reduction of inflammationduring development of AP. BCL3 inhibited ubiquitination and proteasome-mediated degradation of p50 homodimers, which prolonged binding of NF-κB heterodimers to DNA. BCL3 is up-regulated in inflamed pancreatic orbiliary tissues from mice and patients with AP or cholangitis. Its production appears to reduce the inflammatory response in these tissues via blocking ubiquitination and proteasome-mediated degradation of p50 homodimers.

Circulating Histone Levels Reflect Disease Severity in Animal Models of Acute Pancreatitis.

Extracellular histones are rapidly cleared by the liver and rarely detectable in the circulation unless there is extensive cell death, as in severe trauma and sepsis. This study investigated whether circulating histones are elevated in experimental acute pancreatitis models and correlate to disease severity. Acute pancreatitis was induced in mice by: (1) 4 or (2) 12 intraperitoneal injections of cerulein (50 μg/kg) at 1 hour apart; (3) retrograde infusion of 3.5% sodium taurocholate into the biliopancreatic duct. Mice were sacrificed at various time points to collect blood and tissues. Severity of pancreatitis was assessed by biochemical markers and histopathology. Circulating histones were determined by Western blotting. Four cerulein injections induced edematous pancreatitis, whereas 12 cerulein injections and ductal taurocholate infusion caused necrotizing pancreatitis. Circulating histones were barely detectable in the blood of animals with edematous pancreatitis but significantly increased in necrotizing pancreatitis. The levels of circulating histones were strongly correlated to histopathological scores of necrosis of the pancreas. Circulating histones increased significantly in necrotizing pancreatitis due to extensive pancreatic acinar cell death. Levels of circulating histones may have translational potential as a biomarker of disease severity.

Neutrophil Extracellular Traps Induce Trypsin Activation, Inflammation, and Tissue Damage in Mice With Severe Acute Pancreatitis

Neutrophils are involved in the development of acute pancreatitis (AP), but it is not clear how neutrophil-induced tissue damage is regulated. In addition to secreting antimicrobial compounds, activated neutrophils eliminate invading microorganisms by expelling nuclear DNA and histones to form extracellular web-like structures called neutrophil extracellular traps (NETs). However, NETs have been reported to contribute to organ dysfunction in patients with infectious diseases. We investigated whether NETs contribute to the development of AP in mice. AP was induced in C57BL/6 mice by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine. Pancreata were collected and extracellular DNA was detected by Sytox green staining, levels of CXC chemokines, histones, and cytokines also were measured. Cell-free DNA was quantified in plasma samples. Signal transducer and activator of transcription 3 phosphorylation and trypsin activation were analyzed in isolated acinar cells. NETs were depleted by administration of DNase I to mice. Plasma was obtained from healthy individuals (controls) and patients with severe AP. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice and increased levels of cell-free DNA in plasma. Neutrophil depletion prevented taurocholate-induced deposition of NETs in the pancreas. Administration of DNase I to mice reduced neutrophil infiltration and tissue damage in the inflamed pancreas and lung, and decreased levels of blood amylase, macrophage inflammatory protein-2, interleukin 6, and high-mobility groups protein 1. In mice given taurocholate, DNase I administration also reduced expression of integrin α M (macrophage-1 antigen) on circulating neutrophils. Similar results occurred in mice with L-arginine-induced AP. Addition of NETs and histones to acinar cells induced formation of trypsin and activation of signal transducer and activator of transcription 3; these processes were blocked by polysialic acid. Patients with severe AP had increased plasma levels of NET components compared with controls. NETs form in the pancreata of mice during the development of AP, and NET levels are increased in plasma from patients with AP, compared with controls. NETs regulate organ inflammation and injury in mice with AP, and might be targeted to reduce pancreatic tissue damage and inflammation in patients.

Intraductal infusion of taurocholate followed by distal common bile duct ligation leads to a severe necrotic model of pancreatitis in mice.

The most common etiology of acute pancreatitis results from the impaction of gallstones or sludge in the distal common bile duct (CBD). The result is pancreatic duct obstruction, diversion of bile into the pancreas, or cholestasis. In the current study, we examined whether combining both aspects, that is, infusion of the bile acid taurocholate (TC) followed by bile duct ligation (BDL), could yield a more severe form of pancreatitis that mimics biliary pancreatitis. In mice, after laparotomy, the CBD was infused with either normal saline (NS) or TC. Subsequently, the CBD was ligated at the ampulla. Mice receiving TC infusion followed by BDL (TC + BDL) had higher mortality compared with animals receiving intraductal NS with BDL (NS + BDL). The TC + BDL arm developed more severe and diffuse pancreatic necrosis. In addition, serum amylase, IL-6, and bilirubin were significantly higher. However, pancreatic edema as well as lung and liver injury were unchanged between TC + BDL and NS + BDL. In summary, the combination of bile infusion into the pancreas followed by BDL causes a more severe, necrotizing pancreatitis. We believe that this novel model of pancreatitis is useful because it can be used in transgenic mice and recapitulates several aspects of biliary pancreatitis.