RESUMO
Introduction: Salivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models. Methods: Functional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone. Results: Dexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1ß and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models. Conclusions: Our findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.
Assuntos
Dexametasona , Fator 4 Semelhante a Kruppel , Sialadenite , Animais , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Dexametasona/administração & dosagem , Camundongos , Sialadenite/tratamento farmacológico , Sialadenite/patologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Modelos Animais de Doenças , Organoides/efeitos dos fármacos , Citocinas/metabolismo , Camundongos Endogâmicos C57BL , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/patologia , Glândulas Salivares/metabolismo , Glândulas Salivares/imunologia , Aquaporina 5/metabolismo , Aquaporina 5/genética , Masculino , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , HumanosRESUMO
BACKGROUND: Atypical acinar cell foci (AACF) seen in pancreatic cancer are fatal and have been studied with some causative agents. However, for the first time, the effect of acetylsalicylic acid with nitric oxide (NO-ASA) on AACF was examined in this study. Although NO-ASA has very successful inhibitory effects against some types of cancer, it has not been investigated whether they can exert their inhibition effects on AACFs. METHODS: For experimental purposes, 21 14-day-old male Wistar albino rats were used. Azaserine (30 mg/kg) was dissolved in 0.9% NaCl solution and injected intraperitoneally (i.p.) into 14 rats, except for the Control group (Cont) rats, for three weeks. Rats that were injected with azaserine once a week for three weeks and those that did not receive treatment were divided into experimental groups. 15 days after the end of the azaserine injection protocol, NO-ASA was applied to azaserine with NO-ASA (Az+NO-ASA) group rats three consecutive times with an interval of 15 days by gavage. At the end of the 5-month period, pancreatic tissue was dissected and weighed. Pancreas preparations prepared from histological sections were examined for AACF burden and analyzed via a video image analyzer. One-way analysis of variance (ANOVA) non-parametric statistical analyses were performed to test whether there was a difference between the averages of the experimental and Control groups. RESULTS: AACF burden in both groups injected with azaserine was found to be statistically significant in all categories compared to that of the Control group (p < 0.05). The average Calculated Estimated average AACF volume (mm3) values, the Calculated estimated average AACF diameter (µm), the Estimated average number of AACF per unit volume, AACF rate as a % of Calculated Organ Volume were higher in the AzCont group rats than in the Az+NO-ASA group, when compared, and there was an important level statistical difference between the groups (p < 0.05). It was determined that for all parameters AACFs load in Az+NO-ASA group rats were significantly reduced compared to that of AzCont group rats (p < 0.05). CONCLUSIONS: We observed that, as a result of the NO-ASA application, the experimental AACF focus ratio created by azaserine injection was significantly inhibited. The inhibitory effect of AACFs in Az+NO-ASA group rats may have resulted from the significant and independent chemopreventive and/or chemotherapeutic activity of NO-ASA against exocrine pancreatic AACF foci.
Assuntos
Células Acinares , Aspirina , Óxido Nítrico , Pâncreas Exócrino , Neoplasias Pancreáticas , Ratos Wistar , Animais , Masculino , Aspirina/farmacologia , Aspirina/uso terapêutico , Aspirina/administração & dosagem , Óxido Nítrico/metabolismo , Ratos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Células Acinares/metabolismo , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/patologiaRESUMO
The effect of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a persistent environmental pollutant commonly used as a flame retardant in various consumer products, on pancreatitis has not been clearly elucidated, although it has been reported to be toxic to the liver, nervous system, and reproductive system. Acute pancreatitis (AP) and chronic pancreatitis (CP) models were induced in this study by intraperitoneal injection of caerulein. The aim was to investigate the impact of BDE-47 on pancreatitis by exposing the animals to acute (1 week) or chronic (8 weeks) doses of BDE-47 (30 mg/kg in the low-concentration group and 100 mg/kg in the high-concentration group). Additionally, BDE-47 was utilized to stimulate mouse bone marrow-derived macrophages, pancreatic primary stellate cells, and acinar cells in order to investigate the impact of BDE-47 on pancreatitis. In vivo experiments conducted on mice revealed that chronic exposure to BDE-47, rather than acute exposure, exacerbated the histopathological damage of AP and CP, leading to elevated fibrosis in pancreatic tissue and increased infiltration of inflammatory cells in the pancreas. In vitro experiments showed that BDE-47 can promote the expression of the inflammatory cytokines Tnf-α and Il-6 in M1 macrophages, as well as promote acinar cell apoptosis through the activation of the PERK and JNK pathways via endoplasmic reticulum stress. The findings of this study imply chronic exposure to BDE-47 may exacerbate the progression of both AP and CP by inducing acinar cell apoptosis and dysregulating inflammatory responses.
Assuntos
Células Acinares , Apoptose , Éteres Difenil Halogenados , Pancreatite Crônica , Pancreatite , Animais , Éteres Difenil Halogenados/toxicidade , Apoptose/efeitos dos fármacos , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/patologia , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Células Acinares/metabolismo , Masculino , Pancreatite/induzido quimicamente , Pancreatite/patologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos , Ceruletídeo/toxicidade , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Inflamação/induzido quimicamente , Inflamação/patologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/patologia , Células Estreladas do Pâncreas/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retardadores de Chama/toxicidade , Células CultivadasRESUMO
This research was carried out to investigate the effect and mechanism of Angelic Shaoyaosan mediated AMPK/SIRT1 positive feedback loop to promote autophagy and regulate systemic inflammatory response in acute pancreatitis. In this study, the rat pancreatic acini AR42J cells were chosen as the research object, the application of hyla induced pancreatic acinar cells made model for acute pancreatitis, application of different concentrations of angelica peony spread effect on building cells, thus divided into control group, built in the module, the low concentration group, concentration and high concentration groups, determined by MTT method was applied to explore the above categories in cell proliferation, cell apoptosis was measured by flow cytometry, the expression of inflammatory factors in cell supernatant was determined by enzyme-linked immunoassay, and the expression of autophagy marker proteins LC3- ? and P62 was determined by Western-Bolt method. In order to explore the relationship between AMPK and SIRT1, immunoco-precipitation method was used to determine the interaction between AMPK and SIRT1, and dual luciferase experiment was used to explore the effect of AMPK on SIRT1. The AICAR group, BLM-275 group and negative control group were established. To explore the effect of SIRT1 on AMPK, we established SRT 1720 group, EX-527 group and control group. Direct binding between AMPK and SIRT1 should be determined by chromatin co-precipitation assay. In order to further explore the effect of AMPK/SIRT1 positive feedback loop on the systemic inflammatory response of acute pancreatitis, this study selected the medium-concentration Danggui Shaoyajiao SAN group as the control group (group C), and applied AMPK inhibitor BLM-275 and SIRT1 inhibitor EX 527 to the effect of medium-concentration Danggui Shaoyajiao SAN cells, respectively. The expression of autophagy marker proteins LC3- ? and P62 in groups A and B were determined by the Western-Bolt method. Results showed that compared with the control group, the cell survival rate, the expression of AMPK, SIRT1 and LC3-II in the model group were decreased, and the apoptosis rate of iNOS, IL-2, TNF-?, P62 and apoptosis were increased in the model group (P<0.05). the levels of iNOS, IL-2, TNF-?, P62 and cell survival rate in low, medium and high concentration groups decreased gradually, while the expressions of AMPK, SIRT1, LC3-II and cell apoptosis rate increased (P<0.05). The levels of iNOS, IL-2 and TNF-? in the three groups were gradually decreased with the increase of the concentration (P<0.05). Immunoprecipitation showed that AMPK and SIRT1 could bind to each other in cells. The double luciferase experiment indicated that the reporter gene containing the SIRT1 binding site was constructed. The luciferase activity was increased in THE AICAR group and decreased in the BLM-275 group (P<0.05). The reporter gene containing the AMPK promoter binding site was constructed. The luciferase activity in SRT1720 group was increased, while that in EX-527 group was decreased. SIRT1 could directly bind to the AMPK promoter. SIRT1 and LC3- ? protein expressions in group A were down-regulated, and P62 protein was increased (P<0.05). The protein expressions of AMPK and LC3- ? in group B were down-regulated, and the protein expression of P62 was increased (P<0.05). It concluded that AMPK can directly bind to activate SIRT1 expression, and SIRT1 expression can also activate AMPK, forming a positive feedback loop between the two. Therefore, Angelic Shaoyaodong decoction can mediate AMPK/SIRT1 positive feedback pathway to promote autophagy and regulate systemic inflammatory response in acute pancreatitis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Angelica sinensis/química , Autofagia/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Pancreatite/metabolismo , Sirtuína 1/metabolismo , Células Acinares/citologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Mediadores da Inflamação/sangue , Pancreatite/patologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and â¼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.
Assuntos
Células Acinares/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Catepsinas/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Fenilefrina/farmacologia , Via Secretória/efeitos dos fármacos , Lágrimas/metabolismo , Células Acinares/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Carbacol/farmacologia , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Modelos Animais de Doenças , Feminino , Inativação Gênica , Aparelho Lacrimal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , beta-N-Acetil-Hexosaminidases/metabolismo , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
Hypoxia-inducible factor-1α (Hif1α) is activated in hypoxia and is closely related to oxidative stress, immunity and cell metabolism. Recently, it is reported that Hif1α is involved in atherosclerosis, ischemia-reperfusion (I/R) injury, alcoholic liver disease and pancreatic tumors. In this study, we found that Hif1 signal pathway is significantly changed in pancreas of acute pancreatitis (AP) mice. Meanwhile, we verified that the high expression of Hif1α injured pancreatic tissues of cerulean-induced AP mice, which prompting that Hif1α participated in the progress of histopathology on AP. We applied a Hif1α inhibitor PX478 and observed that it could alleviate histological injury of pancreas as well as the levels of serum amylase, lipase and proinflammatory cytokine in the murine model of AP induced by caerulein. In addition, PX478 could reduce the formation of necrosome (RIP3 and p-MLKL) and the generation of reactive oxygen species (ROS) in AP mice. Correspondingly, we further confirmed the effectiveness of PX478 in vitro and found that inhibiting Hif1α could mitigated the necrosis of pancreatic acinar cells via reducing the RIP3 and p-MLKL expression and the ROS production. In conclusion, inhibiting Hif1α could protect against acinar cells necrosis in AP, which may provide a new target for the prevention and treatment of AP clinically.
Assuntos
Células Acinares/efeitos dos fármacos , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Compostos de Mostarda/farmacologia , Necrose/tratamento farmacológico , Pancreatite/tratamento farmacológico , Fenilpropionatos/farmacologia , Células Acinares/metabolismo , Animais , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Necrose/metabolismo , Pancreatite/metabolismoRESUMO
Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.
Assuntos
Células Acinares/metabolismo , Ceruletídeo/química , Interleucina-6/metabolismo , Pâncreas/metabolismo , Resistina/química , Células Acinares/efeitos dos fármacos , Adipocinas/metabolismo , Animais , Anti-Inflamatórios/química , Cálcio/química , Cálcio/metabolismo , Linhagem Celular , Quelantes/química , NADPH Oxidases/metabolismo , Obesidade/metabolismo , Estresse Oxidativo , Pâncreas/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio , Xantofilas/farmacologiaRESUMO
Acute pancreatitis (AP) is serious inflammatory disease of the pancreas. Accumulating evidence links diabetes with severity of AP, suggesting that endogenous insulin may be protective. We investigated this putative protective effect of insulin during cellular and in vivo models of AP in diabetic mice (Ins2Akita) and Pancreatic Acinar cell-specific Conditional Insulin Receptor Knock Out mice (PACIRKO). Caerulein and palmitoleic acid (POA)/ethanol-induced pancreatitis was more severe in both Ins2Akita and PACIRKO vs control mice, suggesting that endogenous insulin directly protects acinar cells in vivo. In isolated pancreatic acinar cells, insulin induced Akt-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) which upregulated glycolysis thereby preventing POA-induced ATP depletion, inhibition of the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) and cytotoxic Ca2+ overload. These data provide the first mechanistic link between diabetes and severity of AP and suggest that phosphorylation of PFKFB2 may represent a potential therapeutic strategy for treatment of AP.
Assuntos
Células Acinares/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Glicólise/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Pancreatite/metabolismo , Substâncias Protetoras/farmacologia , Células Acinares/efeitos dos fármacos , Doença Aguda , Animais , ATPases Transportadoras de Cálcio/metabolismo , Ceruletídeo , Diabetes Mellitus Experimental/metabolismo , Ácidos Graxos Monoinsaturados , Masculino , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/patologiaRESUMO
Antioxidant agents are promising pharmaceuticals to prevent salivary gland (SG) epithelial injury from radiotherapy and their associated irreversible dry mouth symptoms. Epigallocatechin-3-gallate (EGCG) is a well-known antioxidant that can exert growth or inhibitory biological effects in normal or pathological tissues leading to disease prevention. The effects of EGCG in the various SG epithelial compartments are poorly understood during homeostasis and upon radiation (IR) injury. This study aims to: (1) determine whether EGCG can support epithelial proliferation during homeostasis; and (2) investigate what epithelial cells are protected by EGCG from IR injury. Ex vivo mouse SG were treated with EGCG from 7.5-30 µg/mL for up to 72 h. Next, SG epithelial branching morphogenesis was evaluated by bright-field microscopy, immunofluorescence, and gene expression arrays. To establish IR injury models, linear accelerator (LINAC) technologies were utilized, and radiation doses optimized. EGCG epithelial effects in these injury models were assessed using light, confocal and electron microscopy, the Griess assay, immunohistochemistry, and gene arrays. SG pretreated with EGCG 7.5 µg/mL promoted epithelial proliferation and the development of pro-acinar buds and ducts in regular homeostasis. Furthermore, EGCG increased the populations of epithelial progenitors in buds and ducts and pro-acinar cells, most probably due to its observed antioxidant activity after IR injury, which prevented epithelial apoptosis. Future studies will assess the potential for nanocarriers to increase the oral bioavailability of EGCG.
Assuntos
Células Acinares/efeitos dos fármacos , Células Acinares/efeitos da radiação , Catequina/análogos & derivados , Protetores contra Radiação/farmacologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/efeitos da radiação , Animais , Apoptose/efeitos dos fármacos , Catequina/farmacologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Humanos , Imuno-Histoquímica , Estresse Oxidativo , Lesões por Radiação/prevenção & controleRESUMO
High alcohol intake results in the accumulation of non-oxidative ethanol metabolites such as fatty acid ethyl esters (FAEEs) in the pancreas. High FAEE concentrations mediate pancreatic acinar cell injury and are associated with alcoholic pancreatitis. Treatment with ethanol and the fatty acid palmitoleic acid (EtOH/POA) increased the levels of palmitoleic acid ethyl ester and induced zymogen activation and cytokine expression in pancreatic acinar cells. EtOH/POA induces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production and pancreatic acinar cell injury. Lycopene, a bright-red carotenoid, is a potent antioxidant due to its high number of conjugated double bands. This study aimed to investigate whether lycopene inhibits the EtOH/POA-induced increase in ROS production, zymogen activation, and expression of the inflammatory cytokine IL-6 in EtOH/POA-stimulated pancreatic acinar AR42J cells. EtOH/POA increased the ROS levels, NADPH oxidase and NF-κB activities, zymogen activation, IL-6 expression, and mitochondrial dysfunction, which were inhibited by lycopene. The antioxidant N-acetylcysteine and NADPH oxidase 1 inhibitor ML171 suppressed the EtOH/POA-induced increases in ROS production, NF-κB activation, zymogen activation, and IL-6 expression. Therefore, lycopene inhibits EtOH/POA-induced mitochondrial dysfunction, zymogen activation, and IL-6 expression by suppressing NADPH oxidase-mediated ROS production in pancreatic acinar cells.
Assuntos
Células Acinares/patologia , Inflamação/patologia , Licopeno/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Pâncreas Exócrino/patologia , Acetilcisteína/farmacologia , Células Acinares/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Etanol , Ácidos Graxos Monoinsaturados , Interleucina-6/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Radiation therapy for head and neck cancers causes salivary gland dysfunction leading to permanent xerostomia. Limited progress in the discovery of new therapeutic strategies is attributed to the lack of in vitro models that mimic salivary gland function and allow high-throughput drug screening. We address this limitation by combining engineered extracellular matrices with microbubble (MB) array technology to develop functional tissue mimetics for mouse and human salivary glands. We demonstrate that mouse and human salivary tissues encapsulated within matrix metalloproteinase-degradable poly(ethylene glycol) hydrogels formed in MB arrays are viable, express key salivary gland markers, and exhibit polarized localization of functional proteins. The salivary gland mimetics (SGm) respond to calcium signaling agonists and secrete salivary proteins. SGm were then used to evaluate radiosensitivity and mitigation of radiation damage using a radioprotective compound. Altogether, SGm exhibit phenotypic and functional parameters of salivary glands, and provide an enabling technology for high-content/throughput drug testing.
Assuntos
Células Acinares/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Lesões por Radiação/prevenção & controle , Glândulas Salivares/efeitos dos fármacos , Análise Serial de Tecidos , Xerostomia/prevenção & controle , Células Acinares/metabolismo , Células Acinares/efeitos da radiação , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Hidrogéis , Masculino , Camundongos Endogâmicos C57BL , Microbolhas , Pessoa de Meia-Idade , Glândula Parótida/efeitos dos fármacos , Glândula Parótida/metabolismo , Glândula Parótida/efeitos da radiação , Fenótipo , Polietilenoglicóis/química , Lesões por Radiação/etiologia , Lesões por Radiação/metabolismo , Glândulas Salivares/metabolismo , Glândulas Salivares/efeitos da radiação , Xerostomia/etiologia , Xerostomia/metabolismoRESUMO
Thiamin (vitamin B1) plays critical roles in normal metabolism and function of all mammalian cells. Pancreatic acinar cells (PACs) import thiamin from circulation via specific carrier-mediated uptake that involves thiamin transporter-1 and -2 (THTR-1 and -2; products of SLC19A2 and SLC19A3, respectively). Our aim in this study was to investigate the effect(s) of proinflammatory cytokines on thiamin uptake by PACs. We used human primary (h)PACs, PAC 266-6 cells, and mice in vivo as models in the investigations. First, we examined the level of expression of THTR-1 and -2 mRNA in pancreatic tissues of patients with chronic pancreatitis and observed severe reduction in their expression compared with normal control subjects. Exposing hPACs and PAC 266-6 to proinflammatory cytokines (hyper IL-6, TNF-α, and IL-1ß) was found to lead to a significant inhibition in thiamin uptake. Focusing on hyper-IL-6 (which also inhibited thiamin uptake by primary mouse PACs), the inhibition in thiamin uptake was found to be associated with significant reduction in THTR-1 and -2 proteins and mRNA expression as well as in activity of the SLC19A2 and SLC19A3 promoters; it was also associated with reduction in level of expression of the transcription factor Sp1 (which is required for activity of these promoters). Finally, blocking the intracellular Stat3 signaling pathway was found to lead to a significant reversal in the inhibitory effect of hyper IL-6 on thiamin uptake by PAC 266-6. These results show that exposure of PACs to proinflammatory cytokines negatively impacts thiamin uptake via (at least in part) transcriptional mechanism(s).NEW & NOTEWORTHY Findings of the current study demonstrate, for the first time, that exposure of pancreatic acinar cells to proinflammatory cytokines (including hyper IL-6) cause significant inhibition in vitamin B1 (thiamin; a micronutrient that is essential for normal cellular energy metabolism) and that this effect is mediated at the level of transcription of the thiamin transporter genes SLC19A2 and SLC19A3.
Assuntos
Células Acinares/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Citocinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Acinares/metabolismo , Animais , Citocinas/metabolismo , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Camundongos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/metabolismoRESUMO
Imbalance of sexual steroids milieu and oxidative stress are often observed during aging and correlated to prostate disorders. Likewise, high-fat intake has been related to prostate damage and tumor development. Melatonin (MLT) is an antioxidant whose secretion decreases in elderly and is also suggested to protect the gland. This study evaluated the impact of a long-term high-fat diet during aging on prostate morphology and antioxidant system of rats and tested the effects of MLT supplementation under these conditions. Male rats were assigned into four groups: control, treated with MLT, high-fat diet and high-fat diet treated with MLT. The high-fat diet was provided from the 24th week of age, MLT from the 48th (100 µg/kg/day) and rats were euthanized at the 62nd week. The high-fat diet increased body weight, retroperitoneal fatness, glycaemia, and circulating estrogen levels. It aggravated the aging effects, leading to epithelial atrophy (â¼32% reduction of epithelial height) and collagen fibers increase (83%). MLT alone did not alter biometric and physiological parameters, except for the prostate weight decrease, whereas it alleviated biometric as well as ameliorated acinar atrophy induced by high-lipid intake. Systemic oxidative stress increased, and prostatic glutathione peroxidase activity decreased fivefold with the high-fat diet despite the indole. Regardless of the diet, MLT triggered epithelial desquamation, reduced androgen receptor-positive cells, increased smooth muscle layer thickness (12%), decreased at least 50% corpora amylacea formation, and stimulated prostatic gluthatione-S-transferase activity. In conclusion, MLT partially recovered prostate damage induced by aging and the long-term high-fat diet and ameliorated degenerative prostate alterations.
Assuntos
Melatonina/farmacologia , Próstata/patologia , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Adiposidade/efeitos dos fármacos , Animais , Colágeno/metabolismo , Dieta Hiperlipídica , Epitélio/efeitos dos fármacos , Epitélio/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Próstata/efeitos dos fármacos , Ratos , Receptores Androgênicos/metabolismo , Espaço Retroperitoneal/patologiaRESUMO
A human microfluidic four-cell liver acinus microphysiology system (LAMPS), was evaluated for reproducibility and robustness as a model for drug pharmacokinetics and toxicology. The model was constructed using primary human hepatocytes or human induced pluripotent stem cell (iPSC)-derived hepatocytes and 3 human cell lines for the endothelial, Kupffer and stellate cells. The model was tested in two laboratories and demonstrated to be reproducible in terms of basal function of hepatocytes, Terfenadine metabolism, and effects of Tolcapone (88 µM), Troglitazone (150 µM), and caffeine (600 µM) over 9 days in culture. Additional experiments compared basal outputs of albumin, urea, lactate dehydrogenase (LDH) and tumor necrosis factor (TNF)α, as well as drug metabolism and toxicity in the LAMPS model, and in 2D cultures seeded with either primary hepatocytes or iPSC-hepatocytes. Further experiments to study the effects of Terfenadine (10 µM), Tolcapone (88 µM), Trovafloxacin (150 µM with or without 1 µg/mL lipopolysaccharide), Troglitazone (28 µM), Rosiglitazone (0.8 µM), Pioglitazone (3 µM), and caffeine (600 µM) were carried out over 10 days. We found that both primary human hepatocytes and iPSC-derived hepatocytes in 3D culture maintained excellent basal liver function and Terfenadine metabolism over 10 days compared the same cells in 2D cultures. In 2D, non-overlay monolayer cultures, both cell types lost hepatocyte phenotypes after 48 h. With respect to drug effects, both cell types demonstrated comparable and more human-relevant effects in LAMPS, as compared to 2D cultures. Overall, these studies show that LAMPS is a robust and reproducible in vitro liver model, comparable in performance when seeded with either primary human hepatocytes or iPSC-derived hepatocytes, and more physiologically and clinically relevant than 2D monolayer cultures.
Assuntos
Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Técnicas de Cultura de Células/métodos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Microfluídica/métodos , Células Acinares/patologia , Hepatócitos/patologia , Antagonistas não Sedativos dos Receptores H1 da Histamina/toxicidade , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Terfenadina/toxicidadeRESUMO
PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , Adenocarcinoma/metabolismo , Animais , Cálcio/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Ceruletídeo/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Progressão da Doença , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas RGS/metabolismo , Transdução de Sinais/efeitos dos fármacos , Gencitabina , Neoplasias PancreáticasRESUMO
Fatty acid ethyl esters (FAEEs), non-oxidative metabolites of ethanol, are the main causative agents of severe acute pancreatitis resulting from alcohol abuse. Pancreatic acinar cells exposed to ethanol in combination with the fatty acid palmitoleic acid (EtOH/POA) display increased levels of palmitoleic acid ethyl ester and cell death. Oxidative stress and acinar cell necroptosis are implicated in the pathology of severe acute pancreatitis. Docosahexaenoic acid (DHA) serves as a powerful anti-oxidant that reduces pancreatic inflammation and improves the outcomes of patients with acute pancreatitis. We investigated whether treatment of EtOH/POA, as an in vitro model of alcoholic pancreatitis, increases reactive oxygen species (ROS), necroptosis-regulating proteins, and cell death by increasing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and intracellular calcium. Also, we investigated whether DHA inhibits EtOH/POA-induced alterations in pancreatic acinar AR42J cells. As a result, EtOH/POA increased intracellular and mitochondrial ROS levels, NADPH oxidase activity, necroptosis-regulating proteins, and cell death, which was inhibited by NADPH oxidase inhibitor apocynin, the Ca2+ chelator BAPTA, and DHA. However, DHA did not reduce EtOH/POA-induced increases in Ca2+ oscillation or levels in AR42J cells. Furthermore, EtOH/POA induced mitochondrial dysfunction by reducing mitochondrial membrane polarization and hence, adenosine triphosphate (ATP) production. DHA treatment attenuated EtOH/POA-induced mitochondrial dysfunction. In conclusion, DHA inhibits EtOH/POA-induced necroptosis by suppressing NADPH oxidase activity, reducing ROS levels, preventing mitochondrial dysfunction, and inhibiting activation of necroptosis-regulating proteins in AR42J cells.
Assuntos
Células Acinares/efeitos dos fármacos , Antioxidantes/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Etanol/toxicidade , Ácidos Graxos Monoinsaturados/toxicidade , Necroptose/efeitos dos fármacos , Pâncreas Exócrino/efeitos dos fármacos , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Linhagem Celular Tumoral , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/patologia , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Perfluoroalkyl substances, such as perfluorooctanoic acid (PFOA), are widely used in consumer and industrial applications. Human epidemiologic and animal studies suggest that PFOA exposure elicits adverse effects on the pancreas; however, little is known about the biological effects of PFOA in this organ. In this study, we show that PFOA treatment of mouse pancreatic acinar cells results in endoplasmic reticulum (ER) stress and activation of the protein kinase-like endoplasmic reticulum kinase (PERK), inositol-requiring kinase/endonuclease 1α (IRE1α), and activating transcription factor 6 arms of the unfolded protein response (UPR) pathway. PFOA-stimulated activation of the UPR was blocked by pretreatment with specific PERK and IRE1α inhibitors and the chemical chaperone 4-phenyl butyrate, but not the antioxidants N-acetyl- l-cysteine and Tiron. PFOA treatment led to increased cytosolic Ca+2 levels and induction of the UPR was blocked by an inhibitor of the inositol 1,4,5-trisphosphate receptor. These findings indicate that PFOA-induced ER stress may be the mechanistic trigger leading to oxidative stress in the pancreas.
Assuntos
Células Acinares/efeitos dos fármacos , Caprilatos/toxicidade , Fluorocarbonos/toxicidade , Pâncreas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Células Acinares/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/citologia , Pâncreas/metabolismoRESUMO
The inflammatory response in acute pancreatitis (AP) is associated with acinar-to-dendritic cell transition. The CD4+ T-cell-mediated adaptive immune response is necessary for pancreatic inflammatory damage. However, the effect of acinar-to-dendritic cell transition on the CD4+ T-cell response and the regulatory mechanism remain undefined. A mouse animal model of AP was established by repeated intraperitoneal injection of CAE. The mTOR inhibitor rapamycin was administered before AP induction. Primary acinar cells were isolated and co-incubated with subsets of differentiated CD4+ T cells. The expression of DC-SIGN was also assessed in pancreatic tissues from human AP patients. We found acinar cells expressed DC-SIGN and displayed the phenotype of dendritic cells (DCs), which promoted the differentiation of naive CD4+ T cells into CD4+/IFN-γ+ Th1 and CD4+/IL-17A+ Th17 cells in pancreatic tissues during AP. DC-SIGN was the target gene of Myc. The mTOR inhibitor rapamycin inhibited AP-induced DC-SIGN expression, CD4+ Th1/Th17 cell differentiation and the pro-inflammatory response via Myc. Acinar cells expressed DC-SIGN in pancreatic tissues of human patients with AP. In conclusion, acinar-to-dendritic cell transition is implicated in the CD4+ T-cell immune response via mTOR-Myc-DC-SIGN axis, which might be an effective target for the prevention of local pancreatic inflammation in AP.
Assuntos
Células Acinares/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/metabolismo , Pancreatite/imunologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Humanos , Inflamação/patologia , Lectinas Tipo C/metabolismo , Camundongos Endogâmicos C57BL , Pâncreas/imunologia , Pâncreas/patologia , Pancreatite/patologia , Receptores de Superfície Celular/metabolismo , Sirolimo/farmacologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: Cholecystokinin (CCK) may potentially be used to treat obesity. However, it is well-known to induce acute pancreatitis and pancreas neoplasia in rodents, but not in primates. Here we report the nonclinical safety profile of a long-acting CCK-1 receptor (CCK-1R) agonist, NN9056, in rats and monkeys to support a First-in-Man clinical trial with NN9056. METHODS: Thirteen-week toxicological studies were conducted in rats and non-human primates followed by histopathological evaluation of affected tissues. NN9056 was characterised in vitro, and CCK-1R expression was assessed by in situ hybridization in cynomolgus monkey and human pancreas tissues. RESULTS: Affinity and potency of NN9056 was comparable to native sulphated CCK-8 (CCK-8) across species on the CCK-1R while it had no effect on the CCK-2 receptor (CCK-2R). In situ hybridization demonstrated abundant expression of CCK-1Rs in the exocrine pancreas of the rat. In contrast, it was only discreetly expressed on pancreatic acinar cells in the periphery of scattered lobules in monkeys. A similar expression pattern was observed in human pancreas. 13-weeks daily dosing with NN9056 produced the expected pancreatic pathological findings in rats. In monkeys, NN9056 increased pancreas weight and induced histopathological changes despite the low expression level of CCK-1Rs. CONCLUSION: Surprisingly, chronic CCK-1R activation constitutes a risk for pancreatitis and trophic actions on the exocrine pancreas in monkeys. Since similar CCK-1R expression patterns were found in pancreas of monkeys and humans this risk is likely translatable to humans and clinical development of NN9056 was therefore halted.
Assuntos
Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/patologia , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Receptores da Colecistocinina/agonistas , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Animais , Células COS , Chlorocebus aethiops , Colecistocinina/metabolismo , Humanos , Macaca fascicularis , Primatas , RatosRESUMO
The present study aimed to elucidate the mechanisms by which leucine impacts the secretion of pancreatic enzymes, especially amylase, by studying the proteomics profiles of pancreatic acinar (PA) cells from dairy cows. PA cells, the experimental model, were treated with four concentrations of leucine (0, 0.23, 0.45, and 0.90 mM). The abundance of different proteins in the four leucine treatment groups was detected. Label-free proteomic analysis enabled the identification of 1,906 proteins in all four treatment groups, and 1,350 of these proteins showed common expression across the groups. The primary effects of leucine supplementation were increased (P < 0.05) citrate synthase and ATPase activity, which enlarged the cytosolic ATP pool, and the upregulation of secretory protein 61 (Sec61) expression, which promoted protein secretion. In summary, these results suggest that leucine increases citrate synthase in the TCA cycle and ATPase activity and promotes the Sec signaling pathway to increase the exocrine function of PA cells.