RESUMEN
OBJECTIVES: To investigate the protective effects of Salidroside (Sal) on AP cell model induced by taurolithocholic acid 3-sulfate (TLC-S) as well as its underlying mechanism. METHODS: AR42J cells were divided into normal group (N group), AP cell model group (Mod group), Sal treated alone group (S+N group) and Sal treated AP cell model group (S+Mod group). The cell viability was examined by CCK-8 assay. Secretion of lipase and trypsin by AR42J cells, quantified using commercial assay kits, was used as the markers of TLC-S-induced pancreatitis. The levels of TNF-α, IL-1ß, IL-8, IL-6 and IL-10 in the cell supernatant were measured by ELISA. The effect of Sal on molecules in the NF-κB signaling pathway and autophagy was investigated by qRT-PCR and western blot. RESULTS: The decreased cell viability in Mod group was increased by Sal (P < 0.01). The upheaved activities of lipase and trypsin in AP cell model were declined by Sal (P < 0.01). The levels of TNF-α, IL-1ß, IL-8 and IL-6 in the cell supernatant, Beclin-1 and LC3-â ¡ mRNA and protein, p-p65/p65 protein, which were increased in AP cell model, were decreased by Sal; and IL-10 in the cell supernatant, LAMP2 mRNA and protein, p-IκBα/IκBα protein which was declined in AP cell model, was increased by Sal (P < 0.05 or 0.01). There were no significant differences in all indexes between the N and S+N groups (P > 0.05). CONCLUSIONS: Sal alleviated AR42J cells injury induced by TLC-S, inhibited the inflammatory responses and modulated the autophagy, mainly through inhibiting the NF-κB signaling pathway.
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Autofagia/efectos de los fármacos , Glucósidos/farmacología , Pancreatitis/prevención & control , Fenoles/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Inflamación/prevención & control , FN-kappa B/metabolismo , Páncreas/citología , Páncreas/efectos de los fármacos , Páncreas/patología , Ratas , Ácido Taurolitocólico/análogos & derivadosRESUMEN
Acute pancreatitis (AP) is a common digestive disorder with high morbidity and mortality. The present study aimed to investigate the expression of early growth response protein 1 (Egr1), and the effect of competing endogenous (ce)RNA network on trypsinogen activation. Pancreatic acinar intracellular trypsinogen activation (PAITA) is an important event in the early stage of AP; however, the underlying mechanisms remain unclear. The present study used taurolithocholic acid 3sulfate (TLCS)treated AR42J cells (pancreatic cell line) to establish a PAITA model. A gene microarray and bioinformatics analysis was performed to identify the potential key targets in PAITA. The results demonstrated that Egr1, an important transcription factor, was significantly overexpressed in PAITA. In Egr1 small interfering (si)RNAtransfected cells, Egr1 expression was decreased and trypsinogen activation was significantly decreased compared with negative control siRNAtransfected cells, indicating that in TLCSinduced PAITA, overexpression of Egr1 enhanced trypsinogen activation. A ceRNA network [mRNAmicroRNA (miRNA/miR)long noncoding (lnc)RNA] generated using the PAITA model revealed that the effects of Egr1 on PAITA may be regulated by multiple ceRNA pairs, and the lncRNAs (including NONRATT022624 and NONRATT031002) and miRNAs [including Rattus norvegicus (rno)miR2143p and rnomiR7645p] included in the ceRNA pairs may serve roles in PAITA by regulating the expression of Egr1. The results of the present study may provide novel targets for researching the underlying mechanisms of, and developing treatments for AP.
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Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Pancreatitis/genética , Ácido Taurolitocólico/análogos & derivados , Tripsinógeno/metabolismo , Animales , Línea Celular , Biología Computacional , Proteína 1 de la Respuesta de Crecimiento Precoz/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , MicroARNs/genética , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pancreatitis/inducido químicamente , Pancreatitis/metabolismo , ARN Largo no Codificante/genética , ARN Interferente Pequeño/farmacología , Ratas , Ácido Taurolitocólico/efectos adversos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Activation of trypsinogen (formation of trypsin) inside the pancreas is an early pathological event in the development of acute pancreatitis. In our previous studies we identified the activation of trypsinogen within endocytic vacuoles (EVs), cellular organelles that appear in pancreatic acinar cells treated with the inducers of acute pancreatitis. EVs are formed as a result of aberrant compound exocytosis and subsequent internalization of post-exocytic structures. These organelles can be up to 12 µm in diameter and can be actinated (i.e. coated with F-actin). Notably, EVs can undergo intracellular rupture and fusion with the plasma membrane, providing trypsin with access to cytoplasmic and extracellular targets. Unraveling the mechanisms involved in cellular processing of EVs is an interesting cell biological challenge with potential benefits for understanding acute pancreatitis. In this study we have investigated autophagy of EVs and discovered that it involves a non-canonical LC3-conjugation mechanism, reminiscent in its properties to LC3-associated phagocytosis (LAP); in both processes LC3 was recruited to single, outer organellar membranes. Trypsinogen activation peptide was observed in approximately 55% of LC3-coated EVs indicating the relevance of the described process to the early cellular events of acute pancreatitis. We also investigated relationships between actination and non-canonical autophagy of EVs and concluded that these processes represent sequential steps in the evolution of EVs. Our study expands the known roles of LAP and indicates that, in addition to its well-established functions in phagocytosis and macropinocytosis, LAP is also involved in the processing of post-exocytic organelles in exocrine secretory cells. ABBREVIATIONS: AP: acute pancreatitis; CCK: cholecystokinin; CLEM: correlative light and electron microscopy; DPI: diphenyleneiodonium; EV: endocytic vacuole; LAP: LC3-associate phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PACs: pancreatic acinar cells; PFA: paraformaldehyde; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; Res: resveratrol; TAP: trypsinogen activation peptide; TEM: transmission electron microscopy; TLC-S: taurolithocholic acid 3-sulfate; TRD: Dextran Texas Red 3000 MW Neutral; ZGs: zymogen granules.
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Células Acinares/metabolismo , Autofagia , Endocitosis , Proteínas Asociadas a Microtúbulos/metabolismo , Páncreas/citología , Fagocitosis , Vacuolas/metabolismo , Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico/farmacología , Células Acinares/efectos de los fármacos , Células Acinares/ultraestructura , Actinas/metabolismo , Animales , Autofagia/efectos de los fármacos , Homólogo de la Proteína 1 Relacionada con la Autofagia/antagonistas & inhibidores , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/metabolismo , Cloroquina/farmacología , Colecistoquinina/farmacología , Ratones Endogámicos C57BL , Compuestos Onio/farmacología , Fagocitosis/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Dominios Proteicos , Inhibidores de Proteínas Quinasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Resveratrol/farmacología , Ácido Taurolitocólico/análogos & derivados , Tripsinógeno/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/efectos de los fármacosRESUMEN
Sodium taurocholate cotransporting polypeptide (NTCP, encoded by Slc10a1/SLC10A1) deficiency can result in hypercholanemia but no obvious symptoms in both mice and humans. However, the consequence of and response to long-term hypercholanemia caused by NTCP deficiency remain largely unexplored. Here, we analyzed lifelong dynamics of serum total bile acid (TBA) levels in Slc10a1-/- mice, and we also assessed changes of TBA levels in 33 young individuals with SLC10A1 loss-of-function variant p.Ser267Phe. We found that overall serum TBA levels tended to decrease gradually with age in both Slc10a1-/- mice and p.Ser267Phe individuals. Liver mRNA profiling revealed notable transcription alterations in hypercholanemic Slc10a1-/- mice, including inhibition of bile acid (BA) synthesis, enhancement of BA detoxification, and altered BA transport. Members of the sulfotransferase (SULT) family showed the most dramatic increases in livers of hypercholanemic Slc10a1-/- mice, and one of their BA sulfates, taurolithocholic acid 3-sulfate, significantly increased. Importantly, consistent with the mouse studies, comprehensive profiling of 58 BA species in sera of p.Ser267Phe individuals revealed a markedly increased level of BA sulfates. Together, our findings indicate that the enhanced BA sulfation is a major mechanism for BA detoxification and elimination in both mice and humans with Slc10a1/SLC10A1 deficiency.
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Ácidos y Sales Biliares/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Simportadores/genética , Ácido Taurolitocólico/análogos & derivados , Animales , Ácidos y Sales Biliares/sangre , Cromatografía Líquida de Alta Presión , Femenino , Homocigoto , Humanos , Hipercolesterolemia/patología , Hipercolesterolemia/veterinaria , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Dependiente/deficiencia , Simportadores/deficiencia , Espectrometría de Masas en Tándem , Ácido Taurolitocólico/sangre , Ácido Taurolitocólico/metabolismo , Ácido Taurolitocólico/orinaRESUMEN
The present study examined the significance of enterohepatic circulation and the effect of rifampicin [an inhibitor of organic anion-transporting polypeptide 1B (OATP1B)] on the plasma concentrations of bile acid-O-sulfates (glycochenodeoxycholate-O-sulfate, lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate) in monkeys and human liver-transplanted chimeric mice (PXB mouse). Rifampicin significantly increased the area under the curve of bile acid-O-sulfates in monkeys (13-69 times) and PXB mice (13-25 times) without bile flow diversion. Bile flow diversion reduced the concentration of plasma bile acid-O-sulfates under control conditions in monkeys and the concentration of plasma glycochenodeoxycholate-O-sulfate in PXB mice. It also diminished diurnal variation of plasma lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate in PXB mice under control conditions. Bile flow diversion did not affect the plasma concentration of bile acid-O-sulfates in monkeys and PXB mice treated with rifampicin. Plasma coproporphyrin I and III levels were constant in monkeys throughout the study, even with bile flow diversion. This study demonstrated that bile acid-O-sulfates are endogenous OATP1B biomarkers in monkeys and PXB mice. Enterohepatic circulation can affect the baseline levels of plasma bile acid-O-sulfates and modify the effect of OATP1B inhibition.
Asunto(s)
Ácido Glicocólico/análogos & derivados , Ácido Litocólico/análogos & derivados , Transportador 1 de Anión Orgánico Específico del Hígado/antagonistas & inhibidores , Rifampin/farmacología , Ácido Taurolitocólico/análogos & derivados , Animales , Ácido Glicocólico/sangre , Humanos , Ácido Litocólico/sangre , Hígado/metabolismo , Trasplante de Hígado , Macaca fascicularis , Masculino , Ratones , Rifampin/administración & dosificación , Ácido Taurolitocólico/sangreRESUMEN
OBJECTIVES: Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP). METHODS: Changes in mitochondrial membrane potential (Δψm), cytosolic Ca ([Ca]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology. RESULTS: TRO40303 prevented loss of Δψm and necrosis induced by 100 µM palmitoleic acid ethyl ester or 500 µM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP. CONCLUSIONS: TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP.
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Ésteres/farmacología , Ácidos Grasos/farmacología , Mitocondrias/efectos de los fármacos , Oximas/farmacología , Pancreatitis Alcohólica/prevención & control , Secoesteroides/farmacología , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Enfermedad Aguda , Animales , Ceruletida , Ésteres/metabolismo , Ácidos Grasos/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Necrosis/prevención & control , Oximas/farmacocinética , Pancreatitis/inducido químicamente , Pancreatitis/prevención & control , Pancreatitis Alcohólica/metabolismo , Pancreatitis Alcohólica/patología , Secoesteroides/farmacocinética , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
Acute pancreatitis is a disease associated with inflammation and tissue damage. One protein that protects against acute injury, including ischemic injury to both the kidney and heart, is renalase, which is secreted into the blood by the kidney and other tissues. However, whether renalase reduces acute injury associated with pancreatitis is unknown. Here, we used both in vitro and in vivo murine models of acute pancreatitis to study renalase's effects on this condition. In isolated pancreatic lobules, pretreatment with recombinant human renalase (rRNLS) blocked zymogen activation caused by cerulein, carbachol, and a bile acid. Renalase also blocked cerulein-induced cell injury and histological changes. In the in vivo cerulein model of pancreatitis, genetic deletion of renalase resulted in more severe disease, and administering rRNLS to cerulein-exposed WT mice after pancreatitis onset was protective. Because pathological increases in acinar cell cytosolic calcium levels are central to the initiation of acute pancreatitis, we also investigated whether rRNLS could function through its binding protein, plasma membrane calcium ATPase 4b (PMCA4b), which excretes calcium from cells. We found that PMCA4b is expressed in both murine and human acinar cells and that a PMCA4b-selective inhibitor worsens pancreatitis-induced injury and blocks the protective effects of rRNLS. These findings suggest that renalase is a protective plasma protein that reduces acinar cell injury through a plasma membrane calcium ATPase. Because exogenous rRNLS reduces the severity of acute pancreatitis, it has potential as a therapeutic agent.
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Monoaminooxidasa/metabolismo , Páncreas/metabolismo , Pancreatitis/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Células Acinares/patología , Animales , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/uso terapéutico , Biomarcadores/metabolismo , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Línea Celular , Ceruletida/toxicidad , Activación Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente Indirecta , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Hipertensión/etiología , Hipertensión/prevención & control , Ligandos , Moduladores del Transporte de Membrana/farmacología , Ratones , Ratones Noqueados , Monoaminooxidasa/sangre , Monoaminooxidasa/genética , Monoaminooxidasa/uso terapéutico , Páncreas/efectos de los fármacos , Páncreas/inmunología , Páncreas/patología , Pancreatitis/inducido químicamente , Pancreatitis/tratamiento farmacológico , Pancreatitis/patología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , ATPasas Transportadoras de Calcio de la Membrana Plasmática/química , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
The present study aimed to screen for differentially expressed extracellular microRNAs (miRNAs) during the development of acute pancreatitis (AP) and validate the miRNA expression in the plasma of patients with AP. The culture medium of taurolithocholic acid3 sulfatetreated rat pancreatic acinar AR42J cells was collected to extract total RNA for miRNA microarray analysis. Compared with the miRNA test results of the AP rats in the GEO databases, the differentially expressed extracellular miRNAs were screened. The TargetScan, miRanda, and PicTar programs were used for target gene prediction of the identified miRNAs, and gene ontologybiological processes (GOBP) functional annotation was performed. Finally, the results from the combined microarray analyses (in vitro cell line and in vivo rat samples) were validated using plasma samples from patients with mild and moderately severe AP by reverse transcriptionpolymerase chain reaction. The results demonstrated that extracellular miR24 was differentially expressed by microarray and bioinformatics analysis in both the cell line and the animal model of AP. Bioinformatics prediction analysis revealed that downstream target genes of miR24 included Vav2, Syk, Lhcgr, Slc9a3r1, Cacnb1, Cacna1b, Bcl10, and Fgd3. Functional enrichment analysis revealed that the main GOBP predicted functional presentations were positive regulation of calciummediated signaling, activation of cJun Nterminal kinase activity, calcium ion transport, regulation of Rho protein signal transduction, negative regulation of the protein kinase B signaling cascade, and the T cell receptor signaling pathway. Validation analysis for the plasma miR24 expression in humans revealed a significant upregulation of miR24 in the plasma samples of AP patients compared with the healthy controls, while no significant difference was observed in the miR24 expression between the mild and the moderately severe AP groups. The present study confirmed the high expression of miR24 in peripheral blood during AP, suggesting that miR24 might have an intercellular communication role contributing to the APassociated distant organ injury.
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Células Acinares/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Páncreas/metabolismo , Pancreatitis/genética , Células Acinares/efectos de los fármacos , Células Acinares/patología , Enfermedad Aguda , Adulto , Anciano , Animales , Línea Celular , Biología Computacional , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Masculino , MicroARNs/sangre , Análisis por Micromatrices , Persona de Mediana Edad , Anotación de Secuencia Molecular , Páncreas/efectos de los fármacos , Páncreas/patología , Pancreatitis/metabolismo , Pancreatitis/patología , Ratas , Índice de Severidad de la Enfermedad , Transducción de Señal , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
OBJECTIVES: To evaluate the therapeutic potential of I-BET-762, an inhibitor of the bromodomain and extra-terminal (BET) protein family, in experimental acute pancreatitis (AP). METHODS: AP was induced by retrograde infusion of taurolithocholic acid sulphate into the biliopancreatic duct (TLCS-AP) or 2 intraperitoneal (i.p.) injections of ethanol and palmitoleic acid 1 h apart (FAEE-AP) or 12 hourly i.p. injections of caerulein (CER-AP). In all treatment groups, I-BET-762 (30 mg/kg, i.p.) was administered at the time of disease induction and again 12 h later. AP severity was assessed at 24 h by serum biochemistry, multiple cytokines and histopathology. RESULTS: TLCS-AP, FAEE-AP and CER-AP resulted in characteristic elevations in serum amylase and cytokine levels, increased pancreatic trypsin and myeloperoxidase activity, typical pancreatic histopathological changes and lung injury. Treatment with I-BET-762 significantly reduced biochemical, cytokine and histopathological responses in TLCS-AP and FAEE-AP, but not CER-AP. CONCLUSIONS: These results suggest that in different forms of AP there are significant differences in the epigenetic control of gene transcription contributing to the severity of disease responses. There is therapeutic potential in targeting bromodomains for the treatment of gallstone- and alcohol-related pancreatitis.
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Benzodiazepinas/farmacología , Ácidos y Sales Biliares/toxicidad , Ceruletida/toxicidad , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Pancreatitis/inducido químicamente , Receptores de Superficie Celular/antagonistas & inhibidores , Ácido Taurolitocólico/análogos & derivados , Enfermedad Aguda , Amilasas/sangre , Amilasas/metabolismo , Animales , Citocinas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inflamación/prevención & control , Pulmón/enzimología , Masculino , Ratones , Páncreas/enzimología , Páncreas/patología , Pancreatitis/terapia , Peroxidasa/genética , Peroxidasa/metabolismo , Ácido Taurolitocólico/toxicidad , Tripsina/metabolismoRESUMEN
OBJECTIVE: Caffeine reduces toxic Ca2+ signals in pancreatic acinar cells via inhibition of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated signalling, but effects of other xanthines have not been evaluated, nor effects of xanthines on experimental acute pancreatitis (AP). We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP. DESIGN: Isolated pancreatic acinar cells were exposed to secretagogues, uncaged IP3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca2+]C), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry. RESULTS: Caffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP3-mediated Ca2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP3-induced Ca2+ rises, toxin-induced Ca2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25â mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25â mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at >2â mM with 25â mg/kg caffeine but at <100â µM with 25â mg/kg paraxanthine or theophylline. CONCLUSIONS: Caffeine and its dimethylxanthine metabolites reduced pathological IP3R-mediated pancreatic acinar Ca2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.
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Células Acinares/efectos de los fármacos , Cafeína/farmacología , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Páncreas/patología , Pancreatitis/prevención & control , Inhibidores de Fosfodiesterasa/farmacología , Células Acinares/metabolismo , Animales , Cafeína/uso terapéutico , Muerte Celular/efectos de los fármacos , Células Cultivadas , Ceruletida , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Citosol/metabolismo , Etanol , Ácidos Grasos Monoinsaturados , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Ratones , Microscopía Confocal , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Necrosis/diagnóstico por imagen , Pancreatitis/sangre , Pancreatitis/inducido químicamente , Inhibidores de Fosfodiesterasa/uso terapéutico , Transducción de Señal/efectos de los fármacos , Ácido Taurolitocólico/análogos & derivados , Xantinas/sangre , Xantinas/farmacologíaRESUMEN
In the current study, we have characterized the global miRNA expression profile in mouse pancreatic acinar cells and during acute pancreatitis using next-generation RNA sequencing. We identified 324 known and six novel miRNAs that are expressed in mouse pancreatic acinar cells. In the basal state, miR-148a-3p, miR-375-3p, miR-217-5p, and miR-200a-3p were among the most abundantly expressed, whereas miR-24-5p and miR-421-3p were the least abundant. Treatment of acinar cells with caerulein (100 nM) and taurolithocholic acid 3-sulfate [TLC-S (250 µM)] induced numerous changes in miRNA expression profile. In particular, we found significant overexpression of miR-21-3p in acini treated with caerulein and TLC-S. We further looked at the expression of miR-21-3p in caerulein, l-arginine, and caerulein + LPS-induced acute pancreatitis mouse models and found 12-, 21-, and 50-fold increased expression in the pancreas, respectively. In summary, this is the first comprehensive analysis of global miRNA expression profile of mouse pancreatic acinar cells in normal and disease conditions. Our analysis shows that miR-21-3p expression level correlates with the severity of the disease.
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Células Acinares/metabolismo , MicroARNs/metabolismo , Pancreatitis/metabolismo , Células Acinares/efectos de los fármacos , Animales , Ceruletida/farmacología , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , MicroARNs/genética , Pancreatitis/genética , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
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.
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Bilis/metabolismo , Señalización del Calcio , Células Estrelladas Pancreáticas/metabolismo , Pancreatitis Aguda Necrotizante/metabolismo , Sodio/metabolismo , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Células Acinares/patología , Animales , Bradiquinina/farmacología , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Estrelladas Pancreáticas/efectos de los fármacos , Células Estrelladas Pancreáticas/patología , Pancreatitis Aguda Necrotizante/etiología , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/toxicidadRESUMEN
The expression of microRNA-19b (miR-19b) in acute necrotizing pancreatitis (ANP) and its functional role in acinar cell necrosis of SD rats were investigated. Twelve SD rats were divided into two groups randomly, including control group and ANP group. The rat ANP models were established by intraperitoneal injection of L-arginine (2400 mg/kg body weight), and equal volume of 0.9% NaCl was injected in the control group. MiRNA chip assay was performed to examine the expression of miRNAs in the pancreas in two different groups. Besides, to further explore the role of miR-19b in ANP in vitro, taurolithocholic acid 3-sulfate disodium salt (TLC-S) (200 µmol/L) was administrated to treat the rat pancreatic acinar cell line, AR42J, for establishing the ANP cells model. The quantitative real-time PCR (qRT-PCR) was adopted to measure the miR-19b expression. Moreover, the mimic miRNA, miRNA antisense oligonucleotide (AMO) and control vector were used to transfect AR42J cells, the expression of miR-19b was confirmed by qRT-PCR and the necrotizing rate of AR42J cells was detected with AO/EB method. The expression of miR-19b was significantly higher in ANP group than in control group as displayed by the miRNA chip assay. Furthermore, after inducing necrosis of AR42J cells in vitro, the expression of miR-19b was significantly increased by 2.51±0.14 times in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-19b was 5.94±0.95 times higher in the mimic miRNA group than in the control vector group, companied with an obviously increased acinar cell necrotizing rate (50.3%±1.5% vs. 39.6%±2.3%, P<0.05). Moreover, the expression of miR-19b in the miRNA AMO group was 0.38±0.15 times lower than in the control vector group, and the cell necrosis rate was much lower accordingly (23.1%±3.3% vs. 39.6%±2.3%, P<0.05). Besides, there was no significant difference between the control vector cells and the cells without treatment (P>0.05). The expression of miR-19b was significantly induced in ANP. In addition, up-regulation of miR-19b could promote the necrosis of pancreatic acinar cells and miR-19b deficiency could decrease the rate of pancreatic acinar cell necrosis.
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Células Acinares/metabolismo , MicroARNs/genética , Pancreatitis Aguda Necrotizante/metabolismo , Células Acinares/patología , Animales , Arginina/toxicidad , Línea Celular , MicroARNs/metabolismo , Necrosis , Pancreatitis Aguda Necrotizante/etiología , Ratas , Ratas Sprague-Dawley , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/toxicidad , Regulación hacia ArribaRESUMEN
Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.
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Antioxidantes/química , Mitocondrias/metabolismo , Compuestos Organofosforados/química , Pancreatitis/metabolismo , Ubiquinona/análogos & derivados , Células Acinares/metabolismo , Enfermedad Aguda , Animales , Apoptosis , Ceruletida/química , Colecistoquinina/química , Modelos Animales de Enfermedad , Inflamación/metabolismo , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Necrosis/metabolismo , Estrés Oxidativo , Páncreas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/química , Ubiquinona/químicaRESUMEN
Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 µM) or carbachol (1 mM). Ryanodine (100 µM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 µM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 µM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.
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Células Acinares/metabolismo , Páncreas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Células Acinares/efectos de los fármacos , Animales , Calcio/metabolismo , Carbacol/farmacología , Muerte Celular , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ratones , Páncreas/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/genética , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
Biliary pancreatitis is the most common etiology of acute pancreatitis, accounting for 30-60% of cases. A dominant theory for the development of biliary pancreatitis is the reflux of bile into the pancreatic duct and subsequent exposure to pancreatic acinar cells. Bile acids are known to induce aberrant Ca(2+) signals in acinar cells as well as nuclear translocation of NF-κB. In this study, we examined the role of the downstream Ca(2+) target calcineurin on NF-κB translocation. Freshly isolated mouse acinar cells were infected for 24 h with an adenovirus expressing an NF-κB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate caused NF-κB activation at concentrations (500 µm) that were associated with cell injury. We show that the NF-κB inhibitor Bay 11-7082 (1 µm) blocked translocation and injury. Pretreatment with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the calcineurin inhibitors FK506 and cyclosporine A, or use of acinar cells from calcineurin Aß-deficient mice each led to reduced NF-κB activation with taurolithocholic acid-3-sulfate. Importantly, these manipulations did not affect LPS-induced NF-κB activation. A critical upstream regulator of NF-κB activation is protein kinase C, which translocates to the membranes of various organelles in the active state. We demonstrate that pharmacologic and genetic inhibition of calcineurin blocks translocation of the PKC-δ isoform. In summary, bile-induced NF-κB activation and acinar cell injury are mediated by calcineurin, and a mechanism for this important early inflammatory response appears to be upstream at the level of PKC translocation.
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Células Acinares/metabolismo , Ácidos y Sales Biliares/farmacología , Calcineurina/metabolismo , FN-kappa B/metabolismo , Páncreas/patología , Células Acinares/efectos de los fármacos , Células Acinares/patología , Animales , Humanos , Lipopolisacáridos/farmacología , Masculino , Ratones , Modelos Biológicos , Proteína Quinasa C-delta/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/farmacologíaRESUMEN
Trypsinogen activation is the initial factor involved in the development of all types of acute pancreatitis (AP) and has been suggested to be regulated by protein kinases. In the present study, AR42J rat pancreatic acinar cells were treated with taurolithocholic acid 3-sulfate (TLC-S), and trypsinogen activation was detected with bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) dihydrochloride (BZiPAR) staining and flow cytometry. Differentially expressed protein kinase genes were screened by Gene Chip analysis, and the functions of these kinases were analyzed. A significantly increased activation of trypsinogen in AR42J cells following treatment with TLC-S was observed. A total of 22 differentially expressed protein kinase genes were found in the TLC-S group, among which 19 genes were upregulated and 3 were downregulated. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, kinase genes of the same KEGG pathways were connected to create a network through signaling pathways, and 10 nodes of kinases were identified, which were mitogen-activated protein kinase (Mapk)8, Mapk14, Map2k4, interleukin-1 receptor-associated kinase 3 (Irak3), ribosomal protein S6 kinase, 90 kDa, polypeptide 2 (Rps6ka2), protein kinase C, alpha (Prkca), v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (Lyn), protein tyrosine kinase 2 beta (Ptk2b), p21 protein (Cdc42/Rac)-activated kinase 4 (Pak4) and FYN oncogene related to SRC, FGR, YES (Fyn). The interactions between signaling pathways were further analyzed and a network was created. MAPK and calcium signaling pathways were found to be located at the center of the network. Thus, protein kinases constitute potential drug targets for AP treatment.
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Células Acinares/efectos de los fármacos , Células Acinares/enzimología , Perfilación de la Expresión Génica , Páncreas/citología , Proteínas Quinasas/genética , Ácido Taurolitocólico/análogos & derivados , Tripsinógeno/metabolismo , Animales , Línea Celular , Activación Enzimática/efectos de los fármacos , Redes Reguladoras de Genes/genética , Proteínas Quinasas/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Ácido Taurolitocólico/farmacologíaRESUMEN
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 Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) 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 Ca(2+). 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 Aß (CnAß) 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 Ca(2+) 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.
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Células Acinares/citología , Ácidos y Sales Biliares/química , Calcineurina/metabolismo , Calcio/química , Citosol/metabolismo , Páncreas/metabolismo , Pancreatitis/metabolismo , Células Acinares/metabolismo , Animales , Calcio/metabolismo , Quimotripsina/química , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , L-Lactato Deshidrogenasa/metabolismo , Ratones , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Isoformas de Proteínas , Tacrolimus/farmacología , Ácido Taurolitocólico/análogos & derivados , Ácido Taurolitocólico/química , Factores de TiempoRESUMEN
BACKGROUND: Bile acids are the initiating factors of biliary acute pancreatitis. Bile acids can induce the activation of intracellular zymogen, thus leading injury in pancreatic acinar cells. Pathological zymogen activation in pancreatic acinar cells is a common feature of all types of acute pancreatitis. The proteins expressed in pancreatic acinar cells during the activation of zymogen may determine the severity of acute pancreatitis. The present study aims to determine the differentially expressed proteins in taurolithocholic acid 3-sulfate-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. METHODS: Rat pancreatic acinar AR42J cells were treated with taurolithocholic acid 3-sulfate for 20 min. Laser confocal scanning microscopy and flow cytometry were used to detect activated trypsinogen in pancreatic acinar AR42J cells. After the determination of trypsinogen activation, proteome analysis was performed to identify the proteins differentially expressed in taurolithocholic acid 3-sulfate-treated cells and non-treated cells. RESULTS: After treatment with taurolithocholic acid 3-sulfate for 20 min, the activation of trypsinogen in AR42J cells was concurrent with changes in the protein expression profile. Thirty-nine differentially expressed proteins were detected; among these, 23 proteins were up-regulated and 16 proteins were down-regulated. KEGG analysis indicated that these proteins are involved in cellular metabolic pathways, cellular defensive mechanisms, intracellular calcium regulation and cytoskeletal changes. CONCLUSION: The expression of proteins in the pancreatic acinar cell changes at the early stage of biliary acute pancreatitis. These differentially expressed proteins will provide valuable information to understand the pathophysiologic mechanism biliary acute pancreatitis and may be useful for prognostic indices of acute pancreatitis.
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Células Acinares/efectos de los fármacos , Pancreatitis/fisiopatología , Ácido Taurolitocólico/análogos & derivados , Células Acinares/enzimología , Enfermedad Aguda , Animales , Células Cultivadas , Regulación hacia Abajo , Activación Enzimática , Perfilación de la Expresión Génica , Páncreas/patología , Proteoma , Ratas , Ácido Taurolitocólico/farmacología , Tripsinógeno/metabolismo , Regulación hacia ArribaRESUMEN
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.