The TRIM28/miR133a/CD47 axis acts as a potential therapeutic target in pancreatic necrosis by impairing efferocytosis.

Efferocytosis, the clearance of apoptotic cells by macrophages, plays a crucial role in inflammatory responses and effectively prevents secondary necrosis. However, the mechanisms underlying efferocytosis in acute pancreatitis (AP) remain unclear. In this study, we demonstrated the presence of efferocytosis in injured human and mouse pancreatic tissues. We also observed significant upregulation of CD47, an efferocytosis-related the "do not eat me" molecule in injured acinar cells. Subsequently, we used CRISPR-Cas9 gene editing, anti-adeno-associated virus (AAV) gene modification, and anti-CD47 antibody to investigate the potential therapeutic role of AP. CD47 expression was negatively regulated by upstream miR133a, which is controlled by the transcription factor TRIM28. To further investigate the regulation of efferocytosis and reduction of pancreatic necrosis in AP, we used miR-133a-agomir and pancreas-specific AAV-shTRIM28 to modulate CD47 expression. Our findings confirmed that CD47-mediated efferocytosis is critical for preventing pancreatic necrosis and suggest that targeting the TRIM28-miR133a-CD47 axis is clinically relevant for the treatment of AP.

Construction and validation of a rat model of acute necrotizing pancreatitis-associated intestinal injury.

Acute pancreatitis (AP) is an acute inflammatory reaction of the pancreatic tissue, which involves auto-digestion, edema, hemorrhage, and necrosis. AP can be categorized into mild, moderately severe, and severe AP, with severe pancreatitis also referred to as acute necrotizing pancreatitis (ANP). ANP is characterized by the accumulation of necrotic material in the peritoneal cavity. This can result in intestinal injury. However, the mechanism of ANP-associated intestinal injury remains unclear. We established an ANP-associated intestinal injury rat model (ANP-IR model) by injecting pancreatitis-associated ascites fluid (PAAF) and necrotic pancreatic tissue at various proportions into the triangular area formed by the left renal artery and ureter. The feasibility of the ANP-IR model was verified by comparing the similar changes in indicators of intestinal inflammation and barrier function between the two rat models. In addition, we detected changes in apoptosis levels and YAP protein expression in the ileal tissues of rats in each group and validated them in vitro in rat epithelial crypt cells (IEC-6) to further explore the potential injury mechanisms of ANP-associated intestinal injury. We also collected clinical data from patients with ANP to validate the effects of PAAF and pancreatic necrosis on intestinal injury. Our findings offer a theoretical basis for restricting the buildup of peritoneal necrosis in individuals with ANP, thus promoting the restoration of intestinal function and enhancing treatment efficacy. The use of the ANP-IR model in further studies can help us better understand the mechanism and treatment of ANP-associated intestinal injury.NEW & NOTEWORTHY We constructed a rat model of acute necrotizing pancreatitis-associated intestinal injury and verified its feasibility. In addition, we identified the mechanism by which necrotic pancreatic tissue and pancreatitis-associated ascites fluid (PAAF) cause intestinal injury through the HIPPO signaling pathway.

Colonic mucin-2 attenuates acute necrotizing pancreatitis in rats by modulating intestinal homeostasis.

Mucin-2 (MUC2) secreted by goblet cells participates in the intestinal barrier, but its mechanism in acute necrotizing pancreatitis (ANP) remains unclear. In acute pancreatitis (AP) patients, the functions of goblet cells (MUC2, FCGBP, CLCA1, and TFF3) decreased, and MUC2 was negatively correlated with AP severity. ANP rats treated with pilocarpine (PILO) (PILO+ANP rats) to deplete MUC2 showed more serious pancreatic and colonic injuries, goblet cell dysfunction, gut dysbiosis, and bacterial translocation than those of ANP rats. GC-MS analysis of feces showed that PILO+ANP rats had lower levels of butyric acid, isobutyric acid, isovaleric acid, and hexanoic acid than those of ANP rats. The expression of MUC2 was associated with colonic injury and gut dysbiosis. All these phenomena could be relieved, and goblet cell functions were also partially reversed by MUC2 supplementation in ANP rats. TNF-α-treated colonoids had exacerbated goblet cell dysfunction. MUC2 expression was negatively correlated with the levels of pro-inflammatory cytokines (IL-1ß and IL-6) (p < .05) and positively related to the expression of tight junction proteins (Claudin 1, Occludin, and ZO1) (p < .05). Downregulating MUC2 by siRNA increased the levels of the pro-inflammatory cytokines in colonoids. MUC2 might maintain intestinal homeostasis to alleviate ANP.

RanBP2/Nup358 enhances miRNA activity by sumoylating Argonautes.

Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.

Fentanyl but Not Morphine or Buprenorphine Improves the Severity of Necrotizing Acute Pancreatitis in Rats.

Opioids are widely used for the pain management of acute pancreatitis (AP), but their impact on disease progression is unclear. Therefore, our aim was to study the effects of clinically relevant opioids on the severity of experimental AP. Various doses of fentanyl, morphine, or buprenorphine were administered as pre- and/or post-treatments in rats. Necrotizing AP was induced by the intraperitoneal injection of L-ornithine-HCl or intra-ductal injection of Na-taurocholate, while intraperitoneal caerulein administration caused edematous AP. Disease severity was determined by laboratory and histological measurements. Mu opioid receptor (MOR) expression and function was assessed in control and AP animals. MOR was expressed in both the pancreas and brain. The pancreatic expression and function of MOR were reduced in AP. Fentanyl post-treatment reduced necrotizing AP severity, whereas pre-treatment exacerbated it. Fentanyl did not affect the outcome of edematous AP. Morphine decreased vacuolization in edematous AP, while buprenorphine pre-treatment increased pancreatic edema during AP. The overall effects of morphine on disease severity were negligible. In conclusion, the type, dosing, administration route, and timing of opioid treatment can influence the effects of opioids on AP severity. Fentanyl post-treatment proved to be beneficial in AP. Clinical studies are needed to determine which opioids are best in AP.

Elevated CA125 levels are associated with adverse clinical outcomes in acute pancreatitis: A propensity score-matched study.

OBJECTIVES: Glycosylation alterations are indicative of tissue inflammation and neoplasia. However, there are no large-sample, real-world studies assessing the levels of serum carbohydrate antigen 125 (CA125) in patients with acute pancreatitis (AP). We aimed to identify the association between elevated CA125 levels and adverse clinical outcomes in AP. METHODS: This was a retrospective cohort study with an analysis of 3939 patients with AP who were admitted to the First Affiliated Hospital of Nanchang University between January 2015 and September 2019 that used data from a prospectively maintained database. Multivariate logistic regression analysis and a propensity score-matched analysis were conducted to reveal the relationship between elevated CA125 levels and poor prognosis. RESULTS: The overall prevalence of elevated CA125 (>35 U/mL) levels was 38.51% (1517/3939) in AP patients. Elevated CA125 levels were independently associated with higher risks of mortality (adjusted odds ratio (AdjOR), 1.82; 95% confidence interval (CI), 1.30-2.54; P < 0.001), severe acute pancreatitis (SAP) (AdjOR, 2.40; 95% CI, 2.00-2.88; P < 0.001), and infected pancreatic necrosis (IPN) (AdjOR, 3.54; 95% CI, 2.65-4.71; P < 0.001). The propensity score-matched cohort analysis also demonstrated that mortality (OR, 1.57; 95% CI, 1.06-2.23; P < 0.05), SAP (OR, 2.20; 95% CI, 1.77-2.73; P < 0.001), and IPN (OR, 2.79; 95% CI, 1.98-3.92; P < 0.001) were more common in the elevated CA125 group than in the normal CA125 group. CONCLUSIONS: Elevated CA125 levels (>35 U/mL) are independently associated with adverse clinical outcomes in AP patients. These observations justify ongoing efforts to understand the role of CA125 in the pathogenesis and prognosis of AP.

Bone marrow-derived mesenchymal stem cells alleviate severe acute pancreatitis-induced multiple-organ injury in rats via suppression of autophagy.

Patients with severe acute pancreatitis (SAP) represent a substantial challenge to medical practitioners due to the high associated rates of morbidity and mortality and a lack of satisfactory therapeutic outcomes. In a previous study, our group demonstrated that bone marrow-derived mesenchymal stem cells (BMSCs) can ameliorate SAP; however, the mechanisms of action remain to be fully understood. BMSCs were intravenously injected into SAP rats 12 h after experimental induction of SAP using sodium taurocholate (NaT). Histopathological changes and the levels of pro-inflammatory mediators were assessed by hematoxylin and eosin (H&E) staining and ELISA, respectively. Autophagy levels were assessed using qRT-PCR, western blotting, immunohistochemistry, immunofluorescence, and transmission electron microscopy. AR42J cells and human umbilical vein endothelial cells (HUVECs) were administered BMSC-conditioned media (BMSC-CM) after NaT treatment, and cell viability was measured using a Cell Counting Kit-8 (CCK-8) and flow cytometry. In vivo, BMSCs effectively reduced multiple systematic inflammatory responses, suppressed the activation of autophagy, and improved intestinal dysfunction. In vitro, BMSC-CM significantly improved the viability of injured cells, promoted angiogenesis, and decreased autophagy. We therefore propose that the administration of BMSCs alleviates SAP-induced multiple organ injury by inhibiting autophagy.

Genistein protects against acute pancreatitis via activation of an apoptotic pathway mediated through endoplasmic reticulum stress in rats.

Acute pancreatitis (AP) is a severe and frequently lethal disorder, but the precise mechanisms are not well understood and there is lack of effective drugs. Therefore, our study examined the in vivo intervention effects of genistein and elucidated its mechanism in acute experimental pancreatitis models. We used cerulein or taurocholate to induce acute pancreatitis (AP) in Sprague-Dawley rats with prior genistein treatment. Histological examination of the pancreas was performed and the expression of unfolded protein response (UPR) components and apoptotic mediators like caspase 12 and c-Jun N-terminal protein kinase (JNK) were measured. The amount of apoptosis in pancreatic acinar cells was also determined. Our studies found that the severity of cerulein- or taurocholate-induced AP was rescued by prior genistein treatment. Genistein stimulated the activation of multiple endoplasmic reticulum (ER) stress-related regulators like GRP78, PERK, eIF2α, and upregulated the expression of the apoptotic genes, caspase 12 and CHOP. Moreover, TUNEL assays showed that genistein treatment promoted acinar cell apoptosis. Taken together, we speculated that ER stress-associated apoptotic pathways in AP are induced by genistein, which showed cytoprotective capacity in the exocrine pancreas. These data suggest novel therapeutic strategies that employ genistein in the prevention of AP.

High-mobility group box-1 inhibition stabilizes intestinal permeability through tight junctions in experimental acute necrotizing pancreatitis.

BACKGROUND: In acute necrotizing pancreatitis (ANP), bacterial translocation (BT) from the gastrointestinal tract is the essential pathogenesis in the development of septic complications. Although high-mobility group box-1 (HMGB1) is associated with BT and organ dysfunction in ANP, the mechanism of HMGB1 in the intestinal barrier dysfunction and BT has not been well addressed. In this study, we intend to address the role of HMGB1 in ANP involving BT and intestinal barrier dysfunction. METHODS: Experimental ANP was achieved in male Sprague-Dawley rats through a retrograde injection of taurocholate into the common biliopancreatic duct following a laparotomy operation. HMGB1 blockade intervention was conducted with a subcutaneous injection of anti-HMGB1 antibody immediately before the laparotomy procedure. Twenty-four hours after ANP induction, pancreatic and intestinal tissues and blood samples were collected for a histopathological assessment and lipid peroxidation or glutathione (GSH) evaluation. AP-induced barrier dysfunction was determined by an intestinal permeability assessment. Tight junction proteins and autophagy regulators were investigated by western blotting, immunohistological analysis and confocal immunofluorescence imaging. RESULTS: ANP developed as indicated by microscopic parenchymal necrosis and fat necrosis, which were associated with intestinal mucosal barrier dysfunction. HMGB1 inhibition played a protective role in intestinal mucosal barrier dysfunction, protected against microbiome changes in ANP, and relieved intestinal oxidative stress. Additionally, HMGB1 inhibition attenuated intestinal permeability; preserved the expression of TJs, such as claudin-2 and occludin; and decreased autophagy. Furthermore, the autophagy regulator LC3 and TJ protein claudin-2 were both upregulated in ANP according to dual immunofluorescence analysis. CONCLUSION: HMGB1 inhibition ameliorated the severity of experimental ANP though beneficial effects on BT, mainly involving in TJ function.

Paneth Cell Ablation Aggravates Pancreatic and Intestinal Injuries in a Rat Model of Acute Necrotizing Pancreatitis after Normal and High-Fat Diet.

We previously reported that acute necrotizing pancreatitis (ANP) after normal or high-fat diet is associated with a decreased number of Paneth cells in ileal crypts. Here, we ablated Paneth cells in a rat model of ANP after normal and high-fat diet to investigate the effects on disease symptoms. Adult male Sprague-Dawley rats received standard rat chow or a high-fat diet for 2 weeks, after which they were treated with dithizone to deplete Paneth cells. Six hours later, ANP was established by retrograde injection of sodium taurocholate into the biliopancreatic duct. Rats were sacrificed at 6, 12, and 24 h for assessment. We found dithizone aggravated ANP-associated pathological injuries to the pancreas and ileum in rats on high-fat or standard diets. Lysozyme expression in ileal crypts was decreased, while serum inflammatory cytokines (TNFα, IL-1ß, and IL-17A) and intestinal permeability (serum DAO activity and D-lactate) were increased. Expression of tight junction proteins (claudin-1, zo-1, and occludin) was decreased. Using high-throughput 16S rRNA sequencing, we found dithizone reduced microbiota diversity and altered microbiota composition in rats on high-fat or standard diets. Dithizone decreased fecal short-chain fatty acids (SCFAs) in rats on high-fat or standard diets. Changes in intestinal microbiota correlated significantly with SCFAs, lysozyme, DAO activity, D-lactate, inflammatory cytokines, and pathological injury to the pancreas and ileum in rats on high-fat or standard diets. In conclusion, ablation of Paneth cells exacerbates pancreatic and intestinal injuries in ANP after normal and high-fat diet. These symptoms may be related to changes in the intestinal microbiota.

Murine Models of Acute Pancreatitis: A Critical Appraisal of Clinical Relevance.

Acute pancreatitis (AP) is a severe disease associated with high morbidity and mortality. Clinical studies can provide some data concerning the etiology, pathophysiology, and outcomes of this disease. However, the study of early events and new targeted therapies cannot be performed on humans due to ethical reasons. Experimental murine models can be used in the understanding of the pancreatic inflammation, because they are able to closely mimic the main features of human AP, namely their histologic glandular changes and distant organ failure. These models continue to be important research tools for the reproduction of the etiological, environmental, and genetic factors associated with the pathogenesis of this inflammatory pathology and the exploration of novel therapeutic options. This review provides an overview of several murine models of AP. Furthermore, special focus is made on the most frequently carried out models, the protocols used, and their advantages and limitations. Finally, examples are provided of the use of these models to improve knowledge of the mechanisms involved in the pathogenesis, identify new biomarkers of severity, and develop new targeted therapies.

Epigenetic Regulation of Early- and Late-Response Genes in Acute Pancreatitis.

Chromatin remodeling seems to regulate the patterns of proinflammatory genes. Our aim was to provide new insights into the epigenetic mechanisms that control transcriptional activation of early- and late-response genes in initiation and development of severe acute pancreatitis as a model of acute inflammation. Chromatin changes were studied by chromatin immunoprecipitation analysis, nucleosome positioning, and determination of histone modifications in promoters of proinflammatory genes in vivo in the course of taurocholate-induced necrotizing pancreatitis in rats and in vitro in rat pancreatic AR42J acinar cells stimulated with taurocholate or TNF-α. Here we show that the upregulation of early and late inflammatory genes rely on histone acetylation associated with recruitment of histone acetyltransferase CBP. Chromatin remodeling of early genes during the inflammatory response in vivo is characterized by a rapid and transient increase in H3K14ac, H3K27ac, and H4K5ac as well as by recruitment of chromatin-remodeling complex containing BRG-1. Chromatin remodeling in late genes is characterized by a late and marked increase in histone methylation, particularly in H3K4. JNK and p38 MAPK drive the recruitment of transcription factors and the subsequent upregulation of early and late inflammatory genes, which is associated with nuclear translocation of the early gene Egr-1 In conclusion, specific and strictly ordered epigenetic markers such as histone acetylation and methylation, as well as recruitment of BRG-1-containing remodeling complex are associated with the upregulation of both early and late proinflammatory genes in acute pancreatitis. Our findings highlight the importance of epigenetic regulatory mechanisms in the control of the inflammatory cascade.

Resolvin D1 Resolve Inflammation in Experimental Acute Pancreatitis by Restoring Autophagic Flux.

BACKGROUND: Acute pancreatitis (AP) is a common acute gastrointestinal disorders. Increasing evidence indicated that autophagy is involved in the development of AP. Resolvin D1 is an endogenous pro-resolving lipid mediator, which can protect mice from cerulein-induced acute pancreatitis and facilitate autophagy in macrophage, but its mechanism remians unclear. AIMS: To investigate the effect of resolvin D1 on autophagy in mouse models of cerulein-induced AP. METHODS: C57BL/6 mice were randomly divided into control group, AP group and resolvin D1 group. The models of cerulein-induced AP were constructed by intraperitoneally cerulein. Resolvin D1 group was established by intraperitoneally resolvin D1 based on AP models, simultaneously, control group received normal saline. The severity of AP, the level of inflammatory cytokines, the number of autophagic vacuoles, and the expression of autophagy-related markers were evaluated among three groups. RESULTS: The AP models were established successfully. Compared to control group, the number of autophagic vacuoles and expressions of autophagy-related markers including Beclin-1, p62 and LC3-II were increased in AP models, In contrast, the degree of inflammation and levels of inflammatory cytokines in AP models were reduced after resolvin D1 treatment. Moreover, resolvin D1 attenuated the number of autophagic vacuoles and expressions of autophagy-related markers. CONCLUSIONS: Autophagic flux is impaired in cerulein-induced AP. Resolvin D1 ameliorate the severity of mice with cerulein-induced acute pancreatitis, possible attributing to its reducing impaired autophagy and restoring autophagic flux.

Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice.

Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis.

Association of Systemic Inflammatory and Anti-inflammatory Responses with Adverse Outcomes in Acute Pancreatitis: Preliminary Results of an Ongoing Study.

INTRODUCTION: This paper reports preliminary data of an ongoing study that evaluates the association of systemic inflammatory response (SIRS) with early severe acute pancreatitis (ESAP) and compensatory anti-inflammatory response syndrome (characterized by HLA-DR down-regulation) with infected pancreatic necrosis (IPN). METHODS: Consecutive patients presenting within 72 h of symptom onset with organ dysfunction and/or local complications were included. Following parameters were recorded: demographics, etiology, SIRS, APACHE II, creatinine, BUN. Circulating IL-8, IL-6, IL-10, TNF-alpha concentrations and expression of HLA-DR and IL-10 by qRT-PCR in PBMCs were measured. Strength of associations of cytokine concentration and HLA-DR/IL-10 expression with outcomes was expressed as Hedges' G and relative risk (95% CI). RESULTS: Twenty-eight patients (10 MSAP; 18 SAP) fulfilled inclusion criteria. Twelve patients had ESAP and eight presented with organ failure. Admission SIRS worsened in eight (28.6%) patients over 48 h. Sixteen (57.1%) patients developed primary IPN. Twenty-one (75%) patients had HLA-DR down-regulation during the first week, which persisted to the second week in 12 (42.9%) patients. IL-8, IL-6, and TNF-α progressively increased from healthy controls to MAP to MSAP to SAP. IL-6 and TNF-α was higher in the patients who developed ESAP (p = 0.01 and 0.05, respectively). Patients who died within the first week also had a significantly elevated concentration of IL-6 and TNF-α (p = 0.02 and 0.01, respectively). The relative risk (95% CI) of developing primary IPN with persistent HLA-DR down-regulation till the second week of illness was 11.3 (1.6-82.4; p = 0.01). CONCLUSIONS: Our study objectively demonstrates significant association of ESAP and early mortality with primary cytokine response, and development of IPN with persistent HLA-DR down-regulation.

Peripancreatic fat necrosis worsens acute pancreatitis independent of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis collections.

BACKGROUND AND AIMS: Peripancreatic fat necrosis occurs frequently in necrotising pancreatitis. Distinguishing markers from mediators of severe acute pancreatitis (SAP) is important since targeting mediators may improve outcomes. We evaluated potential agents in human pancreatic necrotic collections (NCs), pseudocysts (PCs) and pancreatic cystic neoplasms and used pancreatic acini, peripheral blood mononuclear cells (PBMC) and an acute pancreatitis (AP) model to determine SAP mediators. METHODS: We measured acinar and PBMC injury induced by agents increased in NCs and PCs. Outcomes of caerulein pancreatitis were studied in lean rats coadministered interleukin (IL)-1ß and keratinocyte chemoattractant/growth-regulated oncogene, triolein alone or with the lipase inhibitor orlistat. RESULTS: NCs had higher fatty acids, IL-8 and IL-1ß versus other fluids. Lipolysis of unsaturated triglyceride and resulting unsaturated fatty acids (UFA) oleic and linoleic acids induced necro-apoptosis at less than half the concentration in NCs but other agents did not do so at more than two times these concentrations. Cytokine coadministration resulted in higher pancreatic and lung inflammation than caerulein alone, but only triolein coadministration caused peripancreatic fat stranding, higher cytokines, UFAs, multisystem organ failure (MSOF) and mortality in 97% animals, which were prevented by orlistat. CONCLUSIONS: UFAs, IL-1ß and IL-8 are elevated in NCs. However, UFAs generated via peripancreatic fat lipolysis causes worse inflammation and MSOF, converting mild AP to SAP.

Mechanism of mitochondrial permeability transition pore induction and damage in the pancreas: inhibition prevents acute pancreatitis by protecting production of ATP.

OBJECTIVE: Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. DESIGN: We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. RESULTS: MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. CONCLUSIONS: This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.

Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium-driven bile uptake.

KEY POINTS: Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas. Bile acids are known to induce Ca2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored. Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3-sulfate primarily affects acinar cells. Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+ ; and Na+ -dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells. Bile acid-mediated pancreatic damage can be further escalated by bradykinin-induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. ABSTRACT: Acute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca2+ signals and necrosis in acinar cells. However, bile acid-elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3-sulfate (TLC-S), known to induce Ca2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca2+ signals on extracellular Na+ and the presence of sodium-taurocholate cotransporting polypeptide (NTCP) indicate a Na+ -dependent bile acid uptake mechanism in stellate cells. Bile acid treatment caused necrosis predominantly in stellate cells, which was abolished by removal of extracellular Ca2+ and significantly reduced in the absence of Na+ , showing that bile-dependent cell death was a downstream event of Ca2+ signals. Finally, combined application of TLC-S and the inflammatory mediator bradykinin caused more extensive necrosis in both stellate and acinar cells than TLC-S alone. Our findings shed new light on the mechanism by which bile acids promote pancreatic pathology. This involves not only signalling in acinar cells but also in stellate cells.

Src kinases play a novel dual role in acute pancreatitis affecting severity but no role in stimulated enzyme secretion.

In pancreatic acinar cells, the Src family of kinases (SFK) is involved in the activation of several signaling cascades that are implicated in mediating cellular processes (growth, cytoskeletal changes, apoptosis). However, the role of SFKs in various physiological responses such as enzyme secretion or in pathophysiological processes such as acute pancreatitis is either controversial, unknown, or incompletely understood. To address this, in this study, we investigated the role/mechanisms of SFKs in acute pancreatitis and enzyme release. Enzyme secretion was studied in rat dispersed pancreatic acini, in vitro acute-pancreatitis-like changes induced by supramaximal COOH-terminal octapeptide of cholecystokinin (CCK). SFK involvement assessed using the chemical SFK inhibitor (PP2) with its inactive control, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3), under experimental conditions, markedly inhibiting SFK activation. In CCK-stimulated pancreatic acinar cells, activation occurred of trypsinogen, various MAP kinases (p42/44, JNK), transcription factors (signal transducer and activator of transcription-3, nuclear factor-κB, activator protein-1), caspases (3, 8, and 9) inducing apoptosis, LDH release reflective of necrosis, and various chemokines secreted (monocyte chemotactic protein-1, macrophage inflammatory protein-1α, regulated on activation, normal T cell expressed and secreted). All were inhibited by PP2, not by PP3, except caspase activation leading to apoptosis, which was increased, and trypsin activation, which was unaffected, as was CCK-induced amylase release. These results demonstrate SFK activation is playing a dual role in acute pancreatitis, inhibiting apoptosis and promoting necrosis as well as chemokine/cytokine release inducing inflammation, leading to more severe disease, as well as not affecting secretion. Thus, our studies indicate that SFK is a key mediator of inflammation and pancreatic acinar cell death in acute pancreatitis, suggesting it could be a potential therapeutic target in acute pancreatitis.

Resolvin D1 protects against inflammation in experimental acute pancreatitis and associated lung injury.

Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure with considerable mortality. Recently, resolvin D1 (RvD1) as an endogenous anti-inflammatory lipid mediator has been confirmed to protect against many inflammatory diseases. This study was designed to investigate the effects of RvD1 in acute pancreatitis and associated lung injury. Acute pancreatitis varying from mild to severe was induced by cerulein or cerulein combined with LPS, respectively. Mice were pretreated with RvD1 at a dose of 300 ng/mouse 30 min before the first injection of cerulein. Severity of AP was assessed by biochemical markers and histology. Serum cytokines and myeloperoxidase (MPO) levels in pancreas and lung were determined for assessing the extent of inflammatory response. NF-κB activation was determined by Western blotting. The injection of cerulein or cerulein combined with LPS resulted in local injury in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the cerulein and LPS group. Pretreated RvD1 significantly reduced the degree of amylase, lipase, TNF-α, and IL-6 serum levels; the MPO activities in the pancreas and the lungs; the pancreatic NF-κB activation; and the severity of pancreatic injury and associated lung injury, especially in the severe acute pancreatitis model. These results suggest that RvD1 is capable of improving injury of pancreas and lung and exerting anti-inflammatory effects through the inhibition of NF-κB activation in experimental acute pancreatitis, with more notable protective effect in severe acute pancreatitis. These findings indicate that RvD1 may constitute a novel therapeutic strategy in the management of severe acute pancreatitis.