RESUMO
BACKGROUND AND PURPOSE: Recent clinical studies have shown that serum high-density lipoprotein (HDL) levels are correlated with acute pancreatitis (AP) severity. We aimed to investigate the role of HDL in pancreatic necrosis in AP. EXPERIMENTAL APPROACH: ApoA-I is the main constitution and function component of HDL. The roles of healthy human-derived HDL and apoA-I mimic peptide D4F were demonstrated in AP models in vivo and in vitro. Constitutive Apoa1 genetic inhibition on AP severity, especially pancreatic necrosis was assessed in both caerulein and sodium taurocholate induced mouse AP models. In addition, constitutive (Casp1-/-) and acinar cell conditional (Pdx1CreNlrp3Δ/Δ and Pdx1CreGsdmdΔ/Δ) mice were used to explore the effects of HDL on acinar cell pyroptosis in AP. KEY RESULTS: Apoa1 knockout dramatically aggravated pancreatic necrosis. Human-derived HDL protected against acinar cell death in vivo and in vitro. We found that mimic peptide D4F also protected against AP very well. Constitutive Casp1 or acinar cell-conditional Nlrp3 and Gsdmd genetic inhibition could counteract the protective effects of HDL, implying HDL may exert beneficial effects on AP through inhibiting acinar cell pyroptosis. CONCLUSION AND IMPLICATIONS: This work demonstrates the protective role of HDL and apoA-I in AP pathology, potentially driven by the inhibition of NLRP3 inflammasome signaling and acinar cell pyroptosis. Mimic peptides have promise as specific therapies for AP.
Assuntos
Células Acinares , Pancreatite Necrosante Aguda , Animais , Humanos , Camundongos , Células Acinares/metabolismo , Doença Aguda , Apolipoproteína A-I/genética , Apolipoproteína A-I/farmacologia , Caspase 1 , Ceruletídeo/farmacologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pancreatite Necrosante Aguda/patologia , PiroptoseRESUMO
Various organosulfur compounds, such as dimethyl trisulfide (DMTS), display anti-inflammatory properties. We aimed to examine the effects of DMTS on acute pancreatitis (AP) and its mechanism of action in both in vivo and in vitro studies. AP was induced in FVB/n mice or Wistar rats by caerulein, ethanol-palmitoleic acid, or L-ornithine-HCl. DMTS treatments were administered subcutaneously. AP severity was assessed by pancreatic histological scoring, pancreatic water content, and myeloperoxidase activity measurements. The behaviour of animals was followed. Pancreatic heat shock protein 72 (HSP72) expression, sulfide, and protein persulfidation were measured. In vitro acinar viability, intracellular Ca2+ concentration, and reactive oxygen species production were determined. DMTS dose-dependently decreased the severity of AP. It declined the pancreatic infiltration of leukocytes and cellular damage in mice. DMTS upregulated the HSP72 expression during AP and elevated serum sulfide and low molecular weight persulfide levels. DMTS exhibited cytoprotection against hydrogen peroxide and AP-inducing agents. It has antioxidant properties and modulates physiological but not pathophysiological Ca2+ signalling. Generally, DMTS ameliorated AP severity and protected pancreatic acinar cells. Our findings indicate that DMTS is a sulfur donor with anti-inflammatory and antioxidant effects, and organosulfur compounds require further investigation into this potentially lethal disease.
Assuntos
Pancreatite , Ratos , Camundongos , Animais , Pancreatite/patologia , Ratos Wistar , Doença Aguda , Pâncreas/metabolismo , Sulfetos/farmacologia , Sulfetos/uso terapêutico , Sulfetos/metabolismo , Antioxidantes/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/metabolismo , Ceruletídeo/farmacologiaRESUMO
BACKGROUND: Acute pancreatitis (AP) is an inflammatory condition of the pancreas characterized by oxidative stress and inflammation in its pathophysiology. Acetyl-11-keto-ß-boswellic acid (AKBA) is an active triterpenoid with antioxidant activity. This article seeks to assess the impact of AKBA on AP and investigate its underlying mechanisms. METHODS: AP was induced in wild-type, Lyz2+/cre Nrf2fl/fl mice and Pdx1+/cre Nrf2fl/fl mice by caerulein. Serum amylase and lipase levels, along with histological grading, were utilized to evaluate the severity of AP. Murine bone marrow-derived macrophages (BMDMs) were isolated, cultured, and polarized to the M1 subtype. Flow cytometry and ELISA were utilized to identify the macrophage phenotype. Alterations in oxidative stress damage and intracellular ROS were observed. Nrf2/HO-1 signaling pathways were also evaluated. RESULTS: In a caerulein-induced mouse model of AP, treatment with AKBA reduced blood amylase and lipase activity and ameliorated pancreatic tissue histological and pathological features. Furthermore, AKBA significantly mitigated oxidative stress-induced damage and induced the expression of Nrf2 and HO-1 protein. Additionally, by using conditional knockout mice (Lyz2+/cre Nrf2fl/fl and Pdx1+/cre Nrf2fl/fl mice), we verified that Nrf2 primarily functions in macrophages rather than acinar cells. In vitro, AKBA inhibits pro-inflammatory M1-subtype macrophage polarization and reduces ROS generation through Nrf2/HO-1 oxidative stress pathway. Moreover, the protective effects of AKBA against AP were abolished in myeloid-specific Nrf2-deficient mice and BMDMs. Molecular docking results revealed interactions between AKBA and Nrf2. CONCLUSION: Our results confirm that AKBA exerts protective effects against AP in mice by inhibiting oxidative stress in macrophages through the Nrf2/HO-1 Pathway.
Assuntos
Pancreatite , Animais , Camundongos , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ceruletídeo/farmacologia , Doença Aguda , Simulação de Acoplamento Molecular , Estresse Oxidativo , Macrófagos/metabolismo , Lipase , AmilasesRESUMO
OBJECTIVE: The pathogenesis of acute pancreatitis mainly involves NLRP3 inflammasome-mediated pancreatic cell injury, although regulators of this inflammasome machinery are still not fully identified. Membrane-associated RING-CH 9 (MARCH9) is a member of MARCH-type finger proteins, which regulates innate immunity through catalyzing polyubiquitination of critical immune factors. The aim of present research is to examine the function of MARCH9 in acute pancreatitis. METHODS: Cerulein-induced acute pancreatitis was established on pancreatic cell line AR42J and rat model. Reactive oxygen species (ROS) accumulation and NLRP3 inflammasome-dependent cell pyroptosis in pancreas were examined by flow cytometry. RESULTS: MARCH9 was downregulated by cerulein, but overexpressing MARCH9 could inhibit NLRP3 inflammasome activation and ROS accumulation, thus suppressing pancreatic cell pyroptosis and mitigating pancreatic injury. We further uncovered that the mechanism underlying such an effect of MARCH9 is through mediating the ubiquitination of NADPH oxidase-2, whose deficiency reduces cellular ROS accumulation and inflammasome formation. CONCLUSIONS: Our results suggested that MARCH9 suppresses NLRP3 inflammasome-mediated pancreatic cell injury through mediating the ubiquitination and degradation of NADPH oxidase-2, which compromises ROS generation and NLRP3 inflammasomal activation.
Assuntos
Inflamassomos , Pancreatite , Ratos , Animais , Inflamassomos/metabolismo , Pancreatite/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Ceruletídeo/farmacologia , Doença Aguda , Pâncreas/patologia , Hormônios Pancreáticos/metabolismo , NADPH Oxidases/metabolismo , NADPH Oxidases/farmacologiaRESUMO
Chronic pancreatitis (CP) is an irreversible and progressive inflammatory disease. Knowledge on the development and progression of CP is limited. The goal of the study was to define the serum profile of pro-inflammatory cytokines and the cell antioxidant defense system (superoxidase dismutase-SOD, and reduced glutathione-GSH) over time in a cerulein-induced CP model and explore the impact of these changes on selected cytokines in the intestinal mucosa and pancreatic tissue, as well as on selected serum biochemical parameters. The mRNA expression of CLDN1 and CDH1 genes, and levels of Claudin-1 and E-cadherin, proteins of gut barrier, in the intestinal mucosa were determined via western blot analysis. The study showed moderate pathomorphological changes in the pigs' pancreas 43 days after the last cerulein injection. Blood serum levels of interleukin (IL)-1-beta, IL-6, tumor necrosis factor alpha (TNF-alpha), C-reactive protein (CRP), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGTP), SOD and GSH were increased following cerulein injections. IL-1-beta, IL-6, TNF-alpha and GSH were also increased in jejunal mucosa and pancreatic tissue. In duodenum, decreased mRNA expression of CDH1 and level of E-cadherin and increased D-lactate, an indicator of leaky gut, indicating an inflammatory state, were observed. Based on the current results, we can conclude that repetitive cerulein injections in growing pigs not only led to CP over time, but also induced inflammation in the intestine. As a result of the inflammation, the intestinal barrier was impaired.
Assuntos
Pancreatite Crônica , Fator de Necrose Tumoral alfa , Animais , Suínos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ceruletídeo/farmacologia , Projetos Piloto , Interleucina-6/metabolismo , Pancreatite Crônica/patologia , Pâncreas/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Superóxido Dismutase/metabolismo , RNA Mensageiro/metabolismo , Modelos Animais de DoençasRESUMO
Acute pancreatitis (AP) is a major, globally increasing gastrointestinal disease and a biliary origin is the most common cause. However, the effects of bile acids (BAs), given systemically, on the pancreas and on disease severity remains elusive. In this study, we have investigated the roles of different circulating BAs in animal models for AP to elucidate their impact on disease severity and the underlying pathomechanisms. BAs were incubated on isolated acini and AP was induced through repetitive injections of caerulein or L-arginine; pancreatic duct ligation (PDL); or combined biliopancreatic duct ligation (BPDL). Disease severity was assessed using biochemical and histological parameters. Serum cholecystokinin (CCK) concentrations were determined via enzyme immunoassay. The binding of the CCK1 receptor was measured using fluorescence-labeled CCK. In isolated acini, hydrophobic BAs mitigated the damaging effects of CCK. The same BAs further enhanced pancreatitis in L-arginine- and PDL-based pancreatitis, whereas they ameliorated pancreatic damage in the caerulein and BPDL models. Mechanistically, the binding affinity of the CCK1 receptor was significantly reduced by hydrophobic BAs. The hydrophobicity of BAs and the involvement of CCK seem to be relevant in the course of AP. Systemic BAs may affect the severity of AP by interfering with the CCK1 receptor.
Assuntos
Pancreatite , Camundongos , Animais , Pancreatite/patologia , Ceruletídeo/farmacologia , Ácidos e Sais Biliares/metabolismo , Doença Aguda , Colecistocinina/metabolismo , Modelos Animais de Doenças , Pâncreas/metabolismo , Arginina/farmacologia , Arginina/metabolismo , Interações Hidrofóbicas e HidrofílicasRESUMO
Hinokitiol is a natural bio-active tropolone derivative with promising antioxidant and anti-inflammatory properties. This study was conducted to evaluate the ameliorative effects of hinokitiol against acute pancreatitis induced by cerulein. Mice were pre-treated with hinokitiol intraperitoneally for 7 days (50 and 100 mg/kg), and on the final day of study, cerulein (6 × 50 µg/kg) was injected every hour for six times. Six hours after the last dose of cerulein, blood was collected from the mice through retro-orbital plexus for biochemical analysis. After blood collection, mice were euthanized and the pancreas was harvested for studying effects on oxidative stress, pro-inflammatory cytokines, immunohistochemistry and histopathology of tissue sections. Hinokitiol treatment significantly reduced edema of the pancreas and reduced the plasma levels of lipase and amylase in mice with cerulein-induced acute pancreatitis. It also attenuated the oxidative and nitrosative stress related damage as evident from the reduced malondialdehyde (MDA) and nitrite levels, which were significantly increased in the mice with acute pancreatitis. Furthermore, hinokitiol administration significantly reduced the pancreatitis-evoked decrease in the activity of catalase, glutathione (GSH) and superoxide dismutase (SOD) in the pancreatic tissue. Pre-treatment with hinokitiol significantly reduced the elevated levels of pro-inflammatory cytokines like interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α) as well as increased the levels of anti-inflammatory cytokine interleukin-10 (IL-10) in the pancreatic tissue of mice with acute pancreatitis. The immunohistochemical expression of nuclear factor kappa light chain enhancer of activated B cells (NF-κB), cyclooxygenase (COX-2) and TNF-α were significantly decreased by hinokitiol in mice with cerulein-induced acute pancreatitis. In conclusion, the results of the present study demonstrate that hinokitiol has significant potential to prevent cerulein-induced acute pancreatitis.
Assuntos
Ceruletídeo , Pancreatite , Doença Aguda , Animais , Anti-Inflamatórios/farmacologia , Ceruletídeo/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos , Monoterpenos , NF-kappa B/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/patologia , Tropolona/análogos & derivados , Tropolona/metabolismo , Tropolona/farmacologia , Tropolona/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Necroptosis is a form of regulated necrosis mainly controlled by receptor-interacting protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Necroptosis has important roles in defensing against pathogenic infections, but it is also implicated in various inflammatory diseases including pancreatitis. Baicalin, a flavonoid from Scutellaria baicalensis Georgi, has been shown to possess anti-inflammatory and anti-pyroptosis properties, yet it is unclear whether baicalin can inhibit necroptosis and confer protection against necroptosis-related diseases. Here we reported that baicalin significantly inhibited necroptosis in macrophages induced by lipopolysaccharide plus pan-caspase inhibitor (IDN-6556), or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). Mechanistically, baicalin did not inhibit the phosphorylation of RIPK1, RIPK3 and MLKL, nor membrane translocation of p-MLKL, during necroptotic induction, but instead inhibited p-MLKL oligomerization that is required for executing necroptosis. As intracellular reactive oxygen species (ROS) has been reported to be involved in p-MLKL oligomerization, we assessed the effects of N-acetyl-L-cysteine (NAC), an ROS scavenger, on necroptosis and found that NAC significantly attenuated TSI-induced necroptosis and intracellular ROS production concomitantly with reduced levels of oligomerized p-MLKL, mirroring the effect of baicalin. Indeed, inhibitory effect of baicalin was associated with reduced TSI-induced superoxide (indicating mitochondrial ROS) production and increased mitochondrial membrane potential within cells during necroptosis. Besides, oral administration of baicalin significantly reduced the severity of caerulein-induced acute pancreatitis in mice, an animal model of necroptosis-related disease. Collectively, baicalin can inhibit necroptosis through attenuating p-MLKL oligomerization and confers protection against caerulein-induced pancreatitis in mice.
Assuntos
Necroptose , Pancreatite , Doença Aguda , Animais , Apoptose , Ceruletídeo/farmacologia , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Camundongos , Necrose/tratamento farmacológico , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.
Assuntos
Neoplasias Pancreáticas , Pancreatite , Células Acinares/patologia , Animais , Ceruletídeo/farmacologia , Camundongos , Pâncreas/patologia , Neoplasias Pancreáticas/induzido quimicamente , Neoplasias Pancreáticas/genética , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/patologiaRESUMO
Proper mitochondrial function and adequate cellular ATP are necessary for normal pancreatic protein synthesis and sorting, maintenance of intracellular organelles and enzyme secretion. Inorganic phosphate is required for generating ATP and its limited availability may lead to reduced ATP production causing impaired Ca2+ handling, defective autophagy, zymogen activation, and necrosis, which are all features of acute pancreatitis. We hypothesized that reduced dietary phosphate leads to hypophosphatemia and exacerbates pancreatitis severity of multiple causes. We observed that mice fed a low-phosphate diet before the induction of pancreatitis by either repeated caerulein administration or pancreatic duct injection as a model of pressure-induced pancreatitis developed hypophosphatemia and exhibited more severe pancreatitis than normophosphatemic mice. Pancreatitis severity was significantly reduced in mice treated with phosphate. In vitro modeling of secretagogue- and pressure-induced pancreatic injury was evaluated in isolated pancreatic acini using cholecystokinin and the mechanoreceptor Piezo1 agonist, Yoda1, under low and normal phosphate conditions. Isolated pancreatic acini were more sensitive to cholecystokinin- and Yoda1-induced acinar cell damage and mitochondrial dysfunction under low-phosphate conditions and improved following phosphate supplementation. Importantly, even mice on a normal phosphate diet exhibited less severe pancreatitis when treated with supplemental phosphate. Thus, hypophosphatemia sensitizes animals to pancreatitis and phosphate supplementation reduces pancreatitis severity. These appear to be direct effects of phosphate on acinar cells through restoration of mitochondrial function. We propose that phosphate administration may be useful in the treatment of acute pancreatitis.NEW & NOTEWORTHY Impaired ATP synthesis disrupts acinar cell homeostasis and is an early step in pancreatitis. We report that reduced phosphate availability impairs mitochondrial function and worsens pancreatic injury. Phosphate supplementation improves mitochondrial function and protects against experimental pancreatitis, raising the possibility that phosphate supplementation may be useful in treating pancreatitis.
Assuntos
Hipofosfatemia , Pancreatite , Doença Aguda , Trifosfato de Adenosina/metabolismo , Animais , Ceruletídeo/farmacologia , Colecistocinina/metabolismo , Hipofosfatemia/metabolismo , Canais Iônicos/metabolismo , Camundongos , Pâncreas/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Fosfatos/metabolismoRESUMO
Triptolide (TP), the main active ingredient of Tripterygium wilfordii Hook.f., displays potent anti-inflammatory, antioxidant, and antiproliferative activities. In the present study, the effect of TP on acute pancreatitis and the underlying mechanisms of the disease were investigated using a caerulein-induced animal model of acute pancreatitis (AP) and an in vitro cell model. In vivo, pretreatment with TP notably ameliorated pancreatic damage, shown as the improvement in serum amylase and lipase levels and pancreatic morphology. Meanwhile, TP modulated the infiltration of neutrophils and macrophages (Ly6G staining and CD68 staining) and decreased the levels of proinflammatory factors (TNF-α and IL-6) through inhibiting the transactivation of nuclear factor-κB (NF-κB) in caerulein-treated mice. Furthermore, TP reverted changes in oxidative stress markers, including pancreatic glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA), in acute pancreatitis mice. Additionally, TP pretreatment inhibited intracellular reactive oxygen species (ROS) levels via upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes expression (HO-1, SOD1, GPx1 and NQO1) in vitro. Taken together, our data suggest that TP exert protection against pancreatic inflammation and tissue damage by inhibiting NF-κB transactivation, modulating immune cell responses and activating the Nrf2-mediated antioxidative system, thereby alleviating acute pancreatitis.
Assuntos
Diterpenos/farmacologia , Pancreatite/tratamento farmacológico , Fenantrenos/farmacologia , Doença Aguda , Animais , Antioxidantes/farmacologia , Ceruletídeo/efeitos adversos , Ceruletídeo/farmacologia , China , Modelos Animais de Doenças , Diterpenos/metabolismo , Compostos de Epóxi/metabolismo , Compostos de Epóxi/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/metabolismo , Pancreatite/imunologia , Pancreatite/fisiopatologia , Fenantrenos/metabolismo , Espécies Reativas de OxigênioRESUMO
The excessive inflammatory response mediated by macrophage is one of the key factors for the progress of acute pancreatitis (AP). Paeonol (Pae) was demonstrated to exert multiple anti-inflammatory effects. However, the role of Pae on AP is not clear. In the present study, we aimed to investigate the protective effect and mechanism of Pae on AP in vivo and vitro. In the caerulein-induced mild acute pancreatitis (MAP) model, we found that Pae administration reduced serum levels of amylase, lipase, IL-1ß and IL-6 and alleviated the histopathological manifestations of pancreatic tissue in a dose-dependent manner. And Pae decrease the ROS generated, restore mitochondrial membrane potential (ΔΨm), inhibit M1 macrophage polarization and NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) in vitro. In addition, specific NLRP3 inhibitor MCC950 eliminated the protective effect of Pae on AP induced by caerulein in mice. Correspondingly, the inhibitory effect of Pae on ROS generated and M1 polarization was not observed in BMDMs with MCC950 in vitro. Taken together, our datas for the first time confirmed the protective effects of Pae on AP via the NLRP3 inflammasomes Pathway.
Assuntos
Inflamassomos , Pancreatite , Acetofenonas , Doença Aguda , Animais , Ceruletídeo/farmacologia , Inflamassomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/tratamento farmacológico , Espécies Reativas de Oxigênio/efeitos adversosRESUMO
Chronic pancreatitis (CP) is a pathological fibroinflammatory syndrome of the pancreas. Currently, there are no therapeutic agents available for treating CP-associated pancreatic fibrosis. Fraxinus rhynchophylla (FR) reportedly exhibits anti-inflammatory, antioxidative and antitumor activities. Although FR possesses numerous properties associated with the regulation of diverse diseases, the effects of FR on CP remain unknown. Herein, we examined the effects of FR on CP. For CP induction, mice were intraperitoneally administered cerulein (50 µg/kg) 6 times a day, 4 days per week for 3 weeks. FR extract (100 or 400 mg/kg) or saline (control group) was intraperitoneally injected 1 hour before the first cerulein injection. After 3 weeks, the pancreas was harvested for histological analysis. In addition, pancreatic stellate cells (PSCs) were isolated to examine the antifibrogenic effects and regulatory mechanisms of FR. Administration of FR significantly inhibited histological damage in the pancreas, increased pancreatic acinar cell survival, decreased PSC activation and collagen deposition, and decreased pro-inflammatory cytokines. Moreover, FR treatment inhibited the expression of fibrotic mediators, such as α-smooth muscle actin (α-SMA), collagen, fibronectin 1, and decreased pro-inflammatory cytokines in isolated PSCs stimulated with transforming growth factor (TGF)-ß. Furthermore, FR treatment suppressed the phosphorylation of Smad 2/3 but not of Smad 1/5 in TGF-ß-stimulated PSCs. Collectively, these results suggest that FR ameliorates pancreatic fibrosis by inhibiting PSC activation during CP.
Assuntos
Fraxinus , Pancreatite Crônica , Animais , Ceruletídeo/metabolismo , Ceruletídeo/farmacologia , Ceruletídeo/uso terapêutico , Colágeno/metabolismo , Colágeno/farmacologia , Colágeno/uso terapêutico , Fibrose , Humanos , Camundongos , Pâncreas/patologia , Pancreatite Crônica/tratamento farmacológico , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologia , Casca de Planta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND AND AIMS: Acute pancreatitis (AP) is an acute inflammatory disease that can lead to death. Mir-325-3p is strongly and abnormally expressed in many diseases, necessitating exploration of its function and mechanism in AP. METHODS: Blood samples from AP patients and mice were analyzed. The expression levels of miR-325-3p in AP patients and mouse were detected. Whether miR-325-3p targets RIPK3 gene was predicted by TargetScan online database and dual luciferase reporter assay. In vitro experiments verified the effect of miR-325-3p overexpression on caerulein-induced MPC83 pancreatic acinar cancer cell line. In vivo experiments verified the effect of overexpression of miR-325-3p on the disease degree of pancreatic tissues in AP mice. RESULTS: Analysis of blood samples from AP patients and experiments in mice demonstrated that expression of miR-325-3p was significantly reduced during the process of AP in humans and mice. Predicted using the TargetScan online database and through dual luciferase reporter assay detection, miR-325-3p directly targets the RIPK3 gene. In vitro experiments revealed that overexpression of miR-325-3p reversed caerulein-induced apoptosis and necroptosis in MPC83 pancreatic acinar cancer cell line. We used Z-VAD-FMK to assess necroptosis and demonstrated that miR-325-3p targets necroptosis to reduce cell damage. In subsequent experiments in mice, we verified that overexpression of miR-325-3p reduces inflammation, edema, hemorrhage, and necrosis in acute pancreatitis. Characteristic western blot, immunohistochemistry, and transmission electron microscopy results revealed that overexpression of miR-325-3p reduces the severity of acute pancreatitis by inhibiting pancreatic necroptosis in AP mice. CONCLUSIONS: The current research results indicate that miR-325-3p directly targets RIPK3 and exerts a protective role in mouse AP. Necroptosis is still the primary mechanism of RIPK3 regulation. MiR-325-3p inhibits acute pancreatitis by targeting RIPK3-dependent necroptosis, which may represent a novel treatment method for acute pancreatitis.
Assuntos
MicroRNAs , Pancreatite , Proteína Serina-Treonina Quinases de Interação com Receptores , Células Acinares/metabolismo , Doença Aguda , Animais , Ceruletídeo/farmacologia , Humanos , Camundongos , MicroRNAs/genética , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease's progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson's trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFß, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.
Assuntos
Pancreatite/fisiopatologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Células Acinares/metabolismo , Doença Aguda , Animais , Ceruletídeo/farmacologia , Modelos Animais de Doenças , Fibrose , Inflamação/patologia , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/metabolismo , Pancreatite/imunologia , Pancreatite Crônica/patologia , Poli(ADP-Ribose) Polimerase-1/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS: Acute pancreatitis (AP) is a common inflammatory disorder with high incidence and mortality. AMPK-SIRT1 pathway is involved in a variety of diseases, but its role in AP remains elusive. This study was aimed to explore the role of AMPK-SIRT1 pathway in AP. MAIN METHODS: AP models in vivo and vitro were constructed by intraperitoneal administration of L-arginine and caerulein-stimulated respectively. Rat serum amylase, IL-6 and TNF-α were determined by ELISA. The expression levels of AMPK, SIRT1, Beclin-1, LC3 and p62 were determined by qRT-PCR and western blot. The number of autophagosome was checked by transmission electron microscope. KEY FINDINGS: Compared with NC rats, serum amylase, IL-6 and TNF-α were increased in AP rats. The expressions of AMPK and SIRT1 were decreased, while Beclin-1, LC3II/Iratio and p62 were markedly increased in AP rats. After activation of AMPK by metformin, expressions of p-AMPKα, SIRT1 were significantly raised, while expressions of Beclin-1, LC3 II/I, p62, TNF-α, IL-6 were reduced, and the number of autophagosome was decreased significantly in caerulein-stimulated AR42J cells. The inhibition of AMPK by compound C obtained opposite results. SIGNIFICANCE: During AP occurrence, p-AMPK and SIRT1 were down-regulated, leading to the accumulation of p62, increase of autophagic vacuoles, damage of autophagy, and the occurrence of inflammation. It hinted that activation of AMPK restored impaired autophagy and inhibited inflammation reaction by up-regulating SIRT1. Our findings might provide important theoretical basis for explaining the pathogenesis of AP and investigating therapeutic target to treat and prevent AP.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Ceruletídeo/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/prevenção & controle , Pancreatite/patologia , Sirtuína 1/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Apoptose , Inflamação/metabolismo , Inflamação/patologia , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sirtuína 1/genéticaRESUMO
Acute pancreatitis (AP) is a potential gastrointestinal problem most commonly associated with pancreatic inflammation and acinar cells injury. Nimbolide (NB), isolated from the tree Azadirachta indica, possesses antioxidant and anti-inflammatory effects. Here, we aimed to investigate the pancreatic protective effects of NB in ameliorating cerulein-induced pancreatic inflammation and apoptosis in AP model and evaluate the potential mechanism of action. AP was induced in Swiss albino mice by six-hourly intraperitoneal exposures of cerulein (50⯵g/kg/hr) and pre-treatment of NB (0.3 and 1â¯mg/kg) 7â¯days prior to the cerulein exposure. Various parameters associated with AP in plasma and pancreatic tissues were evaluated. Severity of AP was effectively ameliorated by NB as shown by reducing pancreatic edema, plasma amylase and lipase levels, MPO levels and in cerulein-induced histological damage. Further, the antioxidant effect of NB was associated with a significant inhibition of oxidative-nitrosative stress in Raw 264.7 cells and cerulein-induced AP mice. Moreover, NB suppressed proinflammatory cytokines, iNOS and nitrotyrosine expression. In addition, NB inhibited NF-κB activation and increased SIRT1 expression in cerulein challenged mice. Furthermore, NB also inhibited pancreatic apoptosis by downregulating cleaved caspase 3 and Bax while upregulating Bcl2 expression in cerulein-treated mice. Inhibition of pancreatic inflammation and apoptosis resulted in attenuation of cerulein-induced AP. These results suggest that NB exerts strong anti-pancreatitis effects against cerulein-induced AP by combating inflammatory and apoptosis signaling via SIRT1 activation.
Assuntos
Apoptose/efeitos dos fármacos , Limoninas/uso terapêutico , NF-kappa B , Pancreatite/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1 , Animais , Ceruletídeo/farmacologia , Edema/patologia , Edema/prevenção & controle , Injeções Intraperitoneais , Limoninas/farmacologia , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/patologia , Pancreatite/patologia , Células RAW 264.7 , alfa-Amilases/metabolismoRESUMO
Chronic pancreatitis (CP) is associated with elevated plasma levels of bacterial lipopolysaccharide (LPS) and we have demonstrated reduced acinar cell autophagy in human CP tissue. Therefore, we investigated the role of autophagy in experimental endotoxin-induced pancreatic injury and aimed to identify LPS in human CP tissue. Pancreatic Atg7-deficient mice were injected with a single sub-lethal dose of LPS. Expression of autophagy, apoptosis, necroptosis, and inflammatory markers was determined 3 and 24 h later utilizing immunoblotting and immunofluorescence. The presence of LPS in pancreatic tissue from mice and from patients and healthy controls was determined using immunohistochemistry, immunoblots, and chromogenic assay. Mice lacking pancreatic autophagy exhibited local signs of inflammation and were particularly sensitive to the toxic effect of LPS injection as compared to control mice. In response to LPS, Atg7Δpan mice exhibited enhanced vacuolization of pancreatic acinar cells, increase in TLR4 expression coupled to enhanced expression of NF-κΒ, JNK, and pro-inflammatory cytokines by acinar cells and enhanced infiltration by myeloid cells (but not Atg7F/F controls). Cell death was enhanced in Atg7Δpan pancreata, but only necroptosis and trypsin activation was further amplified following LPS injection along with elevated pancreatic LPS. The presence of LPS was identified in the pancreata from all 14 CP patients examined but was absent in the pancreata from all 10 normal controls. Altogether, these results support a potential role for metabolic endotoxemia in the pathogenesis of CP. Moreover, the evidence also supports the notion that autophagy plays a major cytoprotective and anti-inflammatory role in the pancreas, and blunting metabolic endotoxemia-induced CP.
Assuntos
Autofagia/efeitos dos fármacos , Endotoxinas/farmacologia , Inflamação/induzido quimicamente , Pancreatite Crônica/metabolismo , Células Acinares/metabolismo , Animais , Ceruletídeo/farmacologia , Endotoxinas/metabolismo , Humanos , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Camundongos Transgênicos , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/tratamento farmacológicoRESUMO
Acute pancreatitis (AP), a sudden inflammatory process of pancreas, is painful and may contribute to death. The aberrant expression of miR-27a-5p has been reported in many types of cancers and diseases including AP. Thus, it is urgent to manifest the functions and mechanism of miR-27a-5p in AP. The levels of miR-27a-5p, tumor necrosis factor (TNF) receptor-associated factor 3 (Traf3) in serum of AP patient, or cerulein-treated AR42J cells were detected by qRT-PCR. Functionally, the apoptotic rate, the protein levels of Bcl-2 and Bax, the caspase-3 activity, and the levels of IL-1ß, IL-6, and TNF-α in cerulein-treated AR42J cells were measured by flow cytometry, Western blot, caspase-3 activity assay, and qRT-PCR and ELISA assay, respectively. In addition, the putative target of miR-27a-5p was predicted by TargetScan online database, and the dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify this interaction. Cerulein-treated mouse AP model was established to explore the role of miR-27a-5p in AP in vivo. The level of miR-27a-5p was notably downregulated in AP patients and cerulein-treated AR42J cells. The functional experiments indicated that miR-27a-5p mimics attenuated the promotion effects on cell apoptosis and the inflammatory response in AR42J cells caused by cerulein. The interaction between miR-27a-5p and Traf3 was predicted by TargetScan online database and validated by dual luciferase reporter assay and RIP assay. Following qRT-PCR results exhibited that Traf3 was apparently enhanced in cerulein-treated AR42J cells. The further functional experiments disclosed that Traf3 overexpression relieved the inhibitory effects on cell apoptosis and the inflammatory response induced by miR-27a-5p mimics in cerulein-treated AR42J cells. Moreover, miR-27a-5p alleviated cerulein-induced injury in vivo. In this study, we established the cerulein-treated AR42J cells as AP model in vitro. We validated that miR-27a-5p was significantly downregulated, and Traf3 was strikingly upregulated in AP patient and/or cerulein-treated AR42J cells. The further mechanistical and functional experiments unraveled that miR-27a-5p regulated Traf3 to relieve the cerulein-induced cell apoptosis and inflammatory injury of AR42J cells. Therefore, this novel regulatory network may provide therapeutic target for AP patients.
Assuntos
Apoptose/efeitos dos fármacos , Ceruletídeo/farmacologia , Mediadores da Inflamação/metabolismo , MicroRNAs/biossíntese , Pancreatite/metabolismo , Fator 3 Associado a Receptor de TNF/biossíntese , Animais , Apoptose/fisiologia , Linhagem Celular , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/antagonistas & inibidores , Pancreatite/patologia , RatosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumour with an extremely poor prognosis due to its insidious initiation and a lack of therapeutic strategies. Resveratrol suppresses pancreatic cancer progression and attenuates pancreatitis by modulating multiple targets, including nuclear factor kappa B (NFκB) signalling pathways. However, the effect of resveratrol on pancreatic cancer initiation and its mechanisms remain unclear. In this study, we utilised the LSL-KrasG12D/+; Pdx1-Cre (KC) spontaneous pancreatic precancerous lesion mouse model to explore the anti-tumourigenesis mechanisms of resveratrol in vivo. In vitro acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasias (PanINs) formation assays were performed by pancreatic acinar cell 3-dimensional (3D) culture. Histopathological analysis was used to examine the pathological morphology of pancreatic tissues. Resveratrol prevented the progression of pancreatic precancerous lesions and inhibited the activation of NFκB signalling pathway-related molecules in KC mouse pancreatic tissues. In addition, resveratrol reduced the severity of cerulein-induced pancreatitis and the formation of ADM/PanINs in vivo and in vitro, which may be related to its effect on NFκB inactivation. Furthermore, pancreatic acinar 3D culture demonstrated that activation of the NFκB signalling pathway promoted the formation of ADM/PanINs in vitro, and this initiating effect of NFκB was blocked by resveratrol. Resveratrol slowed the tumourigenesis of pancreatic cancer by inhibiting NFκB activation.