RESUMO
Abnormal cell proliferation and accumulation of fluid-filled cysts along the nephrons in polycystic kidney disease (PKD) could lead to a decline in renal function and eventual end-stage renal disease (ESRD). Gambogic acid (GA), a xanthone compound extracted from the brownish resin of the Garcinia hanburyi tree, exhibits various pharmacological properties, including anti-inflammation, antioxidant, anti-proliferation, and anti-cancer activity. However, its effect on inhibiting cell proliferation in PKD is still unknown. This study aimed to determine the pharmacological effects and detailed mechanisms of GA in slowing an in vitro cyst growth model of PKD. The results showed that GA (0.25-2.5 µM) significantly retarded MDCK cyst growth and cyst formation in a dose-dependent manner, without cytotoxicity. Using the BrdU cell proliferation assay, it was found that GA (0.5-2.5 µM) suppressed MDCK and Pkd1 mutant cell proliferation. In addition, GA (0.5-2.5 µM) strongly inhibited phosphorylation of ERK1/2 and S6K expression and upregulated the activation of phosphorylation of AMPK, both in MDCK cells and Pkd1 mutant cells. Taken together, these findings suggested that GA could retard MDCK cyst enlargement, at least in part by inhibiting the cell proliferation pathway. GA could be a natural plant-based drug candidate for ADPKD intervention.
Assuntos
Cistos , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Xantonas , Proliferação de Células , Humanos , Rim , Doenças Renais Policísticas/tratamento farmacológico , Rim Policístico Autossômico Dominante/tratamento farmacológico , Xantonas/farmacologia , Xantonas/uso terapêuticoRESUMO
BACKGROUND: Renal bilateral fluid filled-cyst in polycystic kidney disease (PKD) is associated with abnormal epithelial cell proliferation and transepithelial fluid secretion which leads to end-stage renal disease (ESRD). A chalcone derivative, isoliquiritigenin (ISLQ), has been shown to have various pharmacological properties. Since several studies have shown that ISLQ could inhibit CFTR channel activity, it is interesting to see whether it can inhibit renal cyst enlargement. The present study was aimed to determine an inhibitory effect and the mechanism of chalcone derivatives on MDCK cyst progression and Pkd1 mutant cells. METHODS: MDCK cyst growth and cyst formation experiments, MTT assay, Ussing chamber experiment, BrdU cell proliferation assay and western blot analysis were performed in this study. RESULTS: Among four compounds of chalcone derivatives tested, CHAL-005 (100 µM) was found to inhibit MDCK cyst growth in a dose-dependent manner without cytotoxicity. It inhibited short-circuit current of chloride secretion as well as CFTR protein expression in MDCK cells. CHAL-005 significantly suppressed cell proliferation. In addition, CHAL-005 strongly reduced phosphorylation ERK1/2 and phosphorylation S6 kinase in MDCK and Pkd1 mutant cells. Interestingly, CHAL-005 activated phosphorylation of AMP kinase protein expression in MDCK and Pkd1 mutant cells. CONCLUSION: CHAL-005 slowed MDCK cyst progression by inhibiting CFTR expression and reducing ERK1/2 and mTOR/S6K signaling pathways as well as activating AMPK expression. Therefore, a chalcone derivative could represent as a promising drug candidate for polycystic kidney disease intervention.
Assuntos
Proliferação de Células/efeitos dos fármacos , Chalconas/farmacologia , Líquido Cístico/efeitos dos fármacos , Líquido Cístico/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Cães , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Madin Darby de Rim Canino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Fosforilação/efeitos dos fármacos , Doenças Renais Policísticas/tratamento farmacológico , Proteínas Quinases S6 Ribossômicas/metabolismo , Canais de Cátion TRPP/genéticaRESUMO
Human organic cation transporter 1 (hOCT1) and human organic cation transporter 3 (hOCT3) are highly expressed in hepatocytes and play important roles in cationic drug absorption, distribution, and elimination. A previous study demonstrated that downregulation of hOCT1 and hOCT3 mRNA was related to hepatocellular carcinoma (HepG2) prognosis and severity. Whether these transporters expressed in HepG2 cells serve for cationic drug delivery has not been investigated. Besides radioactive transport, options for assessing hOCTs in hepatocytes are limited. This study clarified the significant roles of hOCTs in HepG2 by comparing cationic fluorescent 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+ ) with traditional [3 H]-1-methyl-4-phenylpyridinium (MPP+ ). The results showed ASP+ was preferably transported into HepG2 compared to [3 H]-MPP+ with high affinity and a high maximal transport rate. Selective transport of ASP+ mediated by hOCTs was influenced by extracellular pH, temperature, and membrane depolarization, corresponding to hOCT1 and hOCT3 expressions. Furthermore, transport of cationic drugs, metformin, and paclitaxel in HepG2 cells was blunted by OCT inhibitors, suggesting that hOCT1 and hOCT3 expressed in HepG2 cells exhibit notable impacts on cationic drug actions. The fluorescent ASP+ -based in vitro model may also provide a rapid and powerful analytical tool for further screening of cationic drug actions and interactions with hOCTs, particularly hOCT1 and hOCT3 in hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/metabolismo , Cátions/metabolismo , Neoplasias Hepáticas/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Compostos de Piridínio/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Metformina/metabolismo , Paclitaxel/metabolismoRESUMO
Acute kidney injury (AKI) is a high frequent and common complication following acute myocardial infarction (AMI). This study examined and identified the effect of AMI-induced AKI on organic anion transporter 1 (Oat1) and Oat3 transport using clinical setting of pre-renal AKI in vivo. Cardiac ischaemia (CI) and cardiac ischaemia and reperfusion (CIR) were induced in rats by 30-min left anterior descending coronary artery occlusion and 30-min occlusion followed by 120-min reperfusion, respectively. Renal hemodynamic parameters, mitochondrial function and Oat1/Oat3 expression and function were determined along with biochemical markers. Results showed that CI markedly reduced renal blood flow and pressure by approximately 40%, while these parameters were recovered during reperfusion. CI and CIR progressively attenuated renal function and induced oxidative stress by increasing plasma BUN, creatinine and malondialdehyde levels. Correspondingly, SOD, GPx, CAT mRNAs were decreased, while TNFα, IL1ß, COX2, iNOS, NOX2, NOX4, and xanthine oxidase were increased. Mitochondrial dysfunction as indicated by increasing ROS, membrane depolarisation, swelling and caspase3 activation were shown. Early significant detection of AKI; KIM1, IL18, was found. All of which deteriorated para-aminohippurate transport by down-regulating Oat1 during sudden ischaemia. This consequent blunted the trafficking rate of Oat1/Oat3 transport via down-regulating PKCζ/Akt and up-regulating PKCα/NFκB during CI and CIR. Thus, this promising study indicates that CI and CIR abruptly impaired renal Oat1 and regulatory proteins of Oat1/Oat3, which supports dysregulation of remote sensing and signalling and inter-organ/organismal communication. Oat1, therefore, could potentially worsen AKI and might be a potential therapeutic target for early reversal of such injury.
Assuntos
Infarto do Miocárdio/patologia , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Modelos Animais de Doenças , Regulação para Baixo , Rim/metabolismo , Rim/patologia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Infarto do Miocárdio/metabolismo , NF-kappa B/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Estresse Oxidativo , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Constitutive androstane receptor (CAR) belonging to the nuclear receptor superfamily plays an important role in the xenobiotic metabolism and disposition. It has been reported that CAR regulates the expression of the ATP-binding cassette (ABC) transporters in the intestine, such as multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 2/3 (MRP2 and MRP3). In this study, we investigated the role of CAR in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport in T84 human colonic epithelial cells and mouse intestinal tissues. Treatments of T84 cell monolayers with specific CAR agonists (CITCO and phenytoin at concentrations of 1 µM and 5 µM, respectively) for 24 h decreased transepithelial Cl- secretion in response to cAMP-dependent agonist. This inhibition was abolished by coincubation of CITCO with a CAR antagonist, CINPA1. We confirmed that an inhibitory effect of CAR agonists was not due to their cytotoxicity. Basolateral membrane permeabilization experiments also revealed that activation of CAR decreased apical Cl- current stimulated by both CPT-cAMP and genistein (a direct CFTR activator). Such activation also reduced both mRNA and protein expression of CFTR. Furthermore, CITCO decreased cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers. In ICR mice, administration of TCPOBOP (3 mg/kgBW), a murine-specific CAR agonist, for 7 days produced significant decreases in CFTR mRNA and protein expressions in intestinal tissues. Interestingly, TCPOBOP also inhibited CT-induced intestinal fluid accumulation in mice. This is the first evidence showing that CFTR was downregulated by CAR activation in the intestine. Our findings suggest that CAR has potential as a new drug target for treatment of condition with hyperactivity/ hyperfunction of CFTR especially secretory diarrheas.
Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mucosa Intestinal/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Receptor Constitutivo de Androstano , AMP Cíclico/metabolismo , Diarreia/metabolismo , Regulação para Baixo/fisiologia , Células Epiteliais/metabolismo , Humanos , Intestinos , Camundongos , Camundongos Endogâmicos ICR , Permeabilidade , RNA Mensageiro/metabolismoRESUMO
Tight junctions play key roles in the regulation of airway epithelial barrier function and promotion of tight junction integrity is beneficial to lung health. G-protein coupled receptor (GPR) 40 has been identified as a receptor of polyunsaturated fatty acids. This study aimed to investigate the function of GPR40 in regulating tight junction assembly in human airway epithelial cells (Calu-3 cells) using GW9508, a GPR40 agonist. Immunoblotting and immunofluorescence analyses showed that Calu-3 cells expressed both types of polyunsaturated fatty acid receptors including GPR40 and GPR120. Intracellular Ca2+ measurements confirmed that GW9508 stimulated GPR40, but not GPR120. In Ca2+ switch assays, GW9508 promoted the recovery of transepithelial electrical resistance and re-localization of zonula occludens (ZO)-1 to intercellular areas. These effects were suppressed by inhibitors of GPR40 and phospholipase C (PLC). Interestingly, GW9508 enhanced tight junction assembly in an AMP-activated protein kinase (AMPK)-dependent manner. The effect of GW9508 on inducing tight junction assembly was also confirmed in 16HBE14o- cells. Our results indicate that GPR40 stimulation by GW9508 leads to AMPK activation via calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß). Collectively, this study reveals an unprecedented role of GPR40 in facilitating airway epithelial tight junction assembly via PLC-CaMKKß-AMPK pathways. GPR40 represents a novel regulator of airway epithelial integrity and its stimulation may be beneficial in the treatment of airway diseases.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais/fisiologia , Junções Íntimas/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , HumanosRESUMO
Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) is mediated by abnormal cyst-ling cell proliferation and fluid accumulation. Liver X receptor (LXR)-activating ligands suppresses renal cyst enlargement by modulation of cysticfibrosis transmembrane conductance regulator (CFTR)-mediated fluid accumulation. Lansoprazole has been reported as agonist of LXR, and shows an anti-proliferative effect in cancer cells. Here, lansoprazole's pharmacological effect and underlying mechanism on renal cyst development and expansion in in vitro; human ADPKD cyst-lining epithelial cell line and Type I Mardin Darby Canine Kidney (MDCK) cells, and in vivo models was investigated. Lansoprazole inhibited cyst development via inhibition of cell proliferation. In renal cells, lansoprazole's anti-proliferative effect was mediated by inhibition of mTOR/S6K and extracellular signal-regulated kinase (ERK) signaling proteins. In addition, lansoprazole inhibited CFTR-mediated fluid secretion via reduction of CFTR protein expression. In PCK rats, administering lansoprazole (50â¯mg/kgBW) for 4â¯weeks produced significant decreases in the cystic area and improved renal function by reduction of plasma creatinine and blood urea nitrogen. Inhibition of mTOR/S6K, ERK, and CFTR protein expression was observed in PCK rat kidney following lansoprazole treatment. The findings point to potential therapeutic application of lansoprazole in ADPKD.
Assuntos
Proliferação de Células/efeitos dos fármacos , Líquido Cístico/efeitos dos fármacos , Líquido Cístico/metabolismo , Lansoprazol/uso terapêutico , Doenças Renais Policísticas/tratamento farmacológico , Doenças Renais Policísticas/metabolismo , Animais , Proliferação de Células/fisiologia , Cães , Relação Dose-Resposta a Droga , Humanos , Lansoprazol/farmacologia , Células Madin Darby de Rim Canino , Masculino , Doenças Renais Policísticas/patologia , Inibidores da Bomba de Prótons/farmacologia , Inibidores da Bomba de Prótons/uso terapêutico , Ratos , Ratos TransgênicosRESUMO
Overexpression of aquaporin 2 (AQP2) was observed and suggested to be involved in fluid secretion leading to cyst enlargement in polycystic kidney disease (PKD). The cyst expansion deteriorates the renal function and, therefore, therapies targeting cyst enlargement are of clinical interest. Of note, inhibition of vasopressin function using vasopressin 2 receptor (V2R) antagonist which decreased cAMP production along with AQP2 production and function can slow cyst growth in ADPKD. This finding supports the role of AQP2 in cyst enlargement. Steviol, a major metabolite of the sweetening compound stevioside, was reported to retard MDCK cyst growth and enlargement by inhibiting CFTR activity. Interestingly, its efficacy was found to be higher than that of CFTRinh-172. Since steviol was also found to produce diuresis in rodent, it is likely that steviol might have an additional effect in retarding cyst progression, such as inhibition of AQP2 expression and function. Here, we investigated the effect of steviol on AQP2 function and on cyst growth using an in vitro cyst model (MDCK and Pkd1-/- cells). We found that steviol could markedly inhibit cyst growth by reducing AQP2 expression in both Pkd1-/- and MDCK cells. Real-time PCR also revealed that steviol decreased AQP2 mRNA expression level as well. Moreover, a proteasome inhibitor, MG-132, and the lysosomotropic agent, hydroxychloroquine (HCQ) were found to abolish the inhibitory effect of steviol in Pkd1-/- cells. Increased lysosomal enzyme marker (LAMP2) expression following steviol treatment clearly confirmed the involvement of lysosomes in steviol action. In conclusion, our finding showed for the first time that steviol slowed cyst growth, in part, by reducing AQP2 transcription, promoted proteasome, and lysosome-mediated AQP2 degradation. Due to its multiple actions, steviol is a promising compound for further development in the treatment of PKD.
Assuntos
Aquaporina 2/antagonistas & inibidores , Aquaporina 2/biossíntese , Cistos/metabolismo , Diterpenos do Tipo Caurano/farmacologia , Doenças Renais Policísticas/metabolismo , Animais , Aquaporina 2/genética , Cistos/tratamento farmacológico , Cistos/patologia , Diterpenos do Tipo Caurano/uso terapêutico , Cães , Relação Dose-Resposta a Droga , Expressão Gênica , Células Madin Darby de Rim Canino , Doenças Renais Policísticas/tratamento farmacológico , Doenças Renais Policísticas/patologia , Canais de Cátion TRPP/deficiênciaRESUMO
Overstimulation of CFTR-mediated Cl- secretion plays an important role in the pathogenesis of secretory diarrheas, which remain an important global health problem. This study aimed to identify inhibitors of CFTR-mediated Cl- secretion from a library of fungus-derived compounds and to evaluate their pharmacological properties and anti-diarrheal utility. We identified zearalenone, 7'-dehydrozearalenone and 8'-hydroxyzearalenone isolated from the seagrass-derived fungus Fusarium sp. PSU-ES123 as inhibitors of CFTR-mediated Cl- secretion in human intestinal epithelial (T84) cells. Being the most potent fungal metabolite capable of inhibiting CFTR-mediated Cl- secretion, zearalenone reversibly inhibited CFTR Cl- channel activity in T84 cells with an IC50 of â¼0.5⯵M. Functional and biochemical analyses and molecular docking studies indicate that zearalenone binds to the ß-estradiol binding sites in the ATP-binding pockets on NBD1 and NBD2 of CFTR. Mechanisms of CFTR inhibition by zearalenone do not involve activation of phosphodiesterases, protein phosphatases, multidrug-resistance protein 4 and AMP-activated protein kinases. Importantly, zearalenone significantly inhibited cholera toxin (CT)-induced Cl- secretion in T84 cells and blocked CT-induced intestinal fluid secretion in mice. Collectively, our study indicates that zearalenone represents the first class of fungus-derived CFTR inhibitors. Further development of this class of compounds may provide an effective treatment of secretory diarrheas.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Diarreia/tratamento farmacológico , Diarreia/metabolismo , Fusarium , Zearalenona/metabolismo , Zearalenona/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular/métodos , Estrutura Secundária de Proteína , Zearalenona/farmacologiaRESUMO
Hyperglycemia-induced oxidative stress is usually found in diabetic condition. 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors, statins, are widely used as cholesterol-lowering medication with several "pleiotropic" effects in diabetic patients. This study aims to evaluate whether the protective effects of atorvastatin and insulin on renal function and renal organic anion transporter 3 (Oat3) function involve the modulation of oxidative stress and pancreatic function in type 1 diabetic rats. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin (50 mg/kg BW). Atorvastatin and insulin as single or combined treatment were given for 4 weeks after diabetic condition had been confirmed. Diabetic rats demonstrated renal function and renal Oat3 function impairment with an increased MDA level and decreased SOD protein expression concomitant with stimulation of renal Nrf2 and HO-1 protein expression. Insulin plus atorvastatin (combined) treatment effectively restored renal function as well as renal Oat3 function which correlated with the decrease in hyperglycemia and oxidative stress. Moreover, pancreatic inflammation and apoptosis in diabetic rats were ameliorated by the combined drugs treatment. Therefore, atorvastatin plus insulin seems to exert the additive effect in improving renal functionby alleviating hyperglycemiaand the modulation of oxidative stress, inflammation and apoptosis.
Assuntos
Atorvastatina/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Insulina/uso terapêutico , Rim/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Animais , Atorvastatina/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Insulina/farmacologia , Interleucina-6/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
Malfunction of polycystin 1 (PC1) is linked to abnormally high epithelial cell proliferation and fluid secretion, eventually leading to renal cyst development and declined renal function as found in autosomal dominant polycystic kidney disease (ADPKD). Currently, there is no effective therapy for ADPKD. Recent studies report PC1 regulates CFTR chloride channels and ß-catenin levels in normal renal epithelial cells. Concurrently, our previous study found steviol retarded renal cyst enlargement in an in vitro and in an in vivo models by reducing CFTR expression and activity. Therefore, a potential relationship between steviol and PC1 is worthy of exploration. The present study was aimed to determine the effect of steviol on PC1, CFTR, and ß-catenin levels in renal epithelial cells with defective PC1 biogenesis and expression (Prkcsh-/- cell) and postnatal Pkd1 homozygous cell (Pkd1-/- cells). Using western blot analysis, it was found that steviol treatment at 100µM for 24-48h substantially enhanced and stabilized PC1 C-terminal expression, while decreasing CFTR and ß-catenin protein expression in both Prkcsh-/- and Pkd1-/- cells. In addition, steviol promoted LAMP2 expression, a lysosomal enzyme marker. Interestingly, hydroxychloroquine (a lysosome inhibitor) treatment abolished steviol's effect in reducing CFTR and ß-catenin protein expression. Taken together, these findings suggest steviol slows cyst progression in cells and animal models of PKD, in part, by enhancing and stabilizing PC1 protein expression as well as by promoting lysosomal degradation of CFTR and ß-catenin. Therefore, steviol may represent a promising compound for treatment of polycystic kidney disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Diterpenos do Tipo Caurano/farmacologia , Canais de Cátion TRPP/genética , beta Catenina/efeitos dos fármacos , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Rim/citologia , Rim/efeitos dos fármacos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Lisossomos/metabolismo , Camundongos , Doenças Renais Policísticas/tratamento farmacológico , Doenças Renais Policísticas/fisiopatologia , beta Catenina/metabolismoRESUMO
Intestinal Cl- secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl- secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl- secretion in T84 cell monolayers with IC50 of â¼8 µM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl- channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K+ channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca2+-dependent Cl- secretion with IC50 of â¼10 µM. FFA inhibited activities of Ca2+-activated Cl- channels and KCa3.1, a Ca2+-activated basolateral K+ channels, but had no effect on activities of Na+-K+-Cl- cotransporters and Na+-K+ ATPases. These results indicate that FFA inhibits both cAMP and Ca2+-dependent Cl- secretion by suppressing activities of both apical Cl- channels and basolateral K+ channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Canais de Cloreto/antagonistas & inibidores , Cloretos/metabolismo , Células Epiteliais/efeitos dos fármacos , Ácido Flufenâmico/farmacologia , Intestinos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Canais de Cloreto/metabolismo , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Secreções Intestinais/efeitos dos fármacos , NF-kappa B/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/metabolismo , Sódio/metabolismoRESUMO
Chitosan oligosaccharide (COS) is an oligomer of ß-(1â4)-linked d-glucosamine. COS can be prepared from the deacetylation and hydrolysis of chitin, which is commonly found in the exoskeletons of arthropods and insects and the cell walls of fungi. COS is water soluble, non-cytotoxic, readily absorbed through the intestine and mainly excreted in the urine. Of particular importance, COS and its derivatives have been demonstrated to possess several biological activities including anti-inflammation, immunostimulation, anti-tumor, anti-obesity, anti-hypertension, anti-Alzheimer's disease, tissue regeneration promotion, drug and DNA delivery enhancement, anti-microbial, anti-oxidation and calcium-absorption enhancement. The mechanisms of actions of COS have been found to involve the modulation of several important pathways including the suppression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) and the activation of AMP-activated protein kinase (AMPK). This review summarizes the current knowledge of the preparation methods, pharmacokinetic profiles, biological activities, potential therapeutic applications and safety profiles of COS and its derivatives. In addition, future research directions are discussed.
Assuntos
Quitosana/administração & dosagem , Desenho de Fármacos , Oligossacarídeos/administração & dosagem , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Quitosana/química , Quitosana/farmacocinética , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Oligossacarídeos/química , Oligossacarídeos/farmacocinética , SolubilidadeRESUMO
PURPOSE: This study investigated the protective effects of Riceberry bran extract (RBBE) on renal function, and the function and expression of renal organic anion transporter 3 (Oat3) in gentamicin-induced nephrotoxicity in rats and explored the mechanisms for its protective effects. MATERIAL AND METHODS: Male Sprague Dawley rats (n= 42) were divided into six groups to receive normal saline, gentamicin (100mg/kg), co-treatment of gentamicin and RBBE (at dose of 250, 500 and 1000mg/kg), and RBBE (at dose of 1000mg/kg) only, for consecutive fifteen days. Renal function, oxidative and antioxidative markers, the function and expression of Oat3 and histological changes in the kidney were evaluated. RESULTS: Elevation of BUN, serum creatinine levels and reduction in urine creatinine and creatinine clearance indicated decreased renal function in the gentamicin-treated rats. The decrease of [3H]ES uptake in the renal cortical slices of these rats, reflecting the attenuation of Oat3 transport function that was accompanied by decreased expression of Oat3. Moreover, increased MDA level and reduced superoxide dismutase (SOD) and glutathione (GSH) activities were found in gentamicin-treated rats compared to the control group. These changes were associated with the upregulated PKCα, Nrf-2, Keap 1, NQO-1 and HO-1 expressions in kidneys. RBBE treatment improved the renal function and Oat3 transport function and expression in gentamicin-treated rats. The oxidative status was also restored by RBBE treatment. CONCLUSION: RBBE protects kidney injury by its antioxidant effect, subsequently leading to modulation of the PKC/Nrf2 antioxidant defense pathway.
Assuntos
Gentamicinas , Rim , Fator 2 Relacionado a NF-E2 , Transportadores de Ânions Orgânicos , Oryza , Extratos Vegetais , Proteína Quinase C , Animais , Masculino , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Gentamicinas/efeitos adversos , Glutationa/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/fisiopatologia , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , Testes de Função Renal , Transportadores de Ânions Orgânicos/metabolismo , Fitoterapia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Proteína Quinase C/metabolismo , Ratos Sprague-Dawley , Sementes , Superóxido Dismutase/metabolismo , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
OBJECTIVES: Diabetes and exercise training have been shown to involve interleukin 6 (IL-6) signaling in muscle. However, the relationship between the actions of these two stimuli on muscle IL-6 and their downstream components is still unknown. Thus, the effect of endurance training on the key components of muscle IL-6 signaling transduction was investigated in a rat model of type 2 diabetes. METHODS: Diabetes was induced by streptozotocin (STZ) in male Wistar rats fed a high-fat diet, with normal rats acting as controls. The animals were left to conduct their normal activities or assigned to endurance training in a treadmill. At the end of 8 weeks, blood biochemical profiles, exercise performance, muscle oxidative capacity, glucose transporter 4 (GLUT4) protein distribution, and expressions of IL-6 and its downstream proteins were determined. RESULTS: Blood biochemical profiles of the diabetic rats were altered compared to normal rats, whereas endurance training improved blood chemistry and exercise performance. It also increased muscle oxidative capacity, and promoted GLUT4 subcellular localization to the membrane in muscles. Furthermore, protein expression of IL-6 receptor (IL-6Rα) was increased in both normal and diabetic rats after endurance training, but no significant changes in IL-6, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), or suppressor of cytokine signaling 3 (SOC3) were observed in muscles of normal and diabetic rats. CONCLUSIONS: IL-6 signaling pathway mediating muscle response to endurance training was conserved in type 2 diabetes. There was no link between training-induced IL-6 downstream targets in skeletal muscles and IL-6-induced type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Interleucina-6/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Transdução de Sinais/fisiologia , Animais , Transportador de Glucose Tipo 4/metabolismo , Masculino , Fosforilação , Ratos , Ratos Wistar , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismoRESUMO
Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca2+ influx via a mechanosensitive L-type Ca2+ channel, which subsequently raises intracellular Ca2+ and activates AMPK via Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca2+-channel-CaMKKß-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Sinalização do Cálcio , Fibroblastos/enzimologia , Membrana Sinovial/enzimologia , Animais , Canais de Cálcio Tipo L/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Ciclo-Oxigenase 2/metabolismo , Ativação Enzimática , Fibroblastos/patologia , Inflamação/enzimologia , Inflamação/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Coelhos , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Gentamicin is a commonly used aminoglycoside antibiotic. However, its therapeutic use is limited by its nephrotoxicity. The mechanisms of gentamicin-induced nephrotoxicity are principally from renal inflammation and oxidative stress. Since atorvastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, exerts lipid-lowering effects, antioxidant, anti-inflammatory as well as anti-apoptotic effects, this study aimed to investigate the protective effects of atorvastatin against gentamicin-induced nephrotoxicity. Male Sprague Dawley rats were used and nephrotoxicity was induced by intraperitoneal injection of gentamicin, 100 mg/kg/day, for 15 days. Atorvastatin, 10 mg/kg/day, was administered by orally gavage 30 min before gentamicin injection on day 1 to 15 (pretreatment) or on day 10 to15 (delayed treatment). For only atorvastatin treatment group, it was given on day 1 to 15. At the end of the experiment, kidney weight, blood urea nitrogen and serum creatinine as well as renal inflammation (NF-κB, TNFαR1, IL-6 and iNOS), renal fibrosis (TGFß1), ER stress (calpain, GRP78, CHOP, and caspase 12) and apoptotic markers (cleaved caspase-3, Bax, and Bcl-2) as well as TUNEL assay were determined. Gentamicin-induced nephrotoxicity was confirmed by marked elevations in serum urea and creatinine, kidney hypertrophy, renal inflammation, fibrosis, ER stress and apoptosis and attenuation of creatinine clearance. Atorvastatin pre and delayed treatment significantly improved renal function and decreased renal NF-κB, TNFαR1, IL-6, iNOS and TGFß1 expressions. They also attenuated calpain, GRP78, CHOP, caspase 12, Bax, and increased Bcl-2 expressions in gentamicin-treated rat. These results indicate that atorvastatin treatment could attenuate gentamicin-induced nephrotoxicity in rats, substantiated by the reduction of inflammation, ER stress and apoptosis. The effect of atorvastatin in protecting from renal damage induced by gentamicin seems to be more effective when it beginning given along with gentamicin or pretreatment.
Assuntos
Apoptose/efeitos dos fármacos , Atorvastatina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Gentamicinas/toxicidade , Substâncias Protetoras/farmacologia , Animais , Atorvastatina/uso terapêutico , Nitrogênio da Ureia Sanguínea , Caspase 12/metabolismo , Creatinina/sangue , Relação Dose-Resposta a Droga , Esquema de Medicação , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Interleucina-6/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , NF-kappa B/metabolismo , Nefrite/induzido quimicamente , Nefrite/patologia , Nefrite/prevenção & controle , Óxido Nítrico Sintase Tipo II/metabolismo , Substâncias Protetoras/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Synovial inflammation plays an important role in the early pathogenesis of osteoarthritis (OA). Chitosan oligosaccharide (COS) has been shown to activate AMPK and suppress inflammatory responses in intestinal epithelial cells. This study aimed to investigate the effect of COS on AMPK activation and synovial inflammation using both primary cultures of synoviocytes and a rabbit model of anterior cruciate ligament (ACL) transection-induced OA. COS induced AMPK activation in both rabbit and human synoviocytes. The mechanism of COS-induced AMPK activation involves an increase in the ADP/ATP ratio but not calcium/calmodulin-dependent protein kinase kinase beta (CaMKKß). Interestingly, COS suppressed the TNFα-induced iNOS and COX-2 expression via an AMPK-dependent mechanism in both rabbit and human synoviocytes. Importantly, oral administration of COS (10mg/kg/day) induced AMPK activation and alleviated signs of inflammation including COX-2 expression in the synovium of a rabbit ACL transection model. Taken together, our results indicate that COS suppresses synovial inflammation in vitro and in vivo via AMPK activation. COS may be useful in the prevention of OA.
Assuntos
Quitosana/farmacologia , Inflamação/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oligossacarídeos/farmacologia , Sinoviócitos/efeitos dos fármacos , Animais , Ligamento Cruzado Anterior/efeitos dos fármacos , Ligamento Cruzado Anterior/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite/metabolismo , Osteoartrite/prevenção & controle , Coelhos , Sinoviócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cisplatin is widely used as a standard chemotherapy for solid tumors. The major adverse effect of cisplatin is nephrotoxicity in proximal tubular cells, via oxidative stress, DNA damage, cell apoptosis, and inflammation. The aim of this study was to investigate the pharmacological effect and mechanism of fibrate drugs on cisplatin-induced renal proximal tubular cell death. Cisplatin decreased cell viability of LLC-PK1 and HK-2 cells in a dose-dependent manner. Cisplatin-induced apoptosis was attenuated by co-treatment with fenofibrate while less so with clofibrate and bezafibrate. Fenofibrate's protective effect was not complimented by co-treatment with GW6471, a PPARα antagonist, indicating the protective effect occurred via a PPARα-independent mechanism. Treating cells with cisplatin induced reactive oxygen species (ROS), c-JUN N-terminal kinase (JNK), and p38 kinase (p38), but not extracellular signal-regulated kinase (ERK). Fenofibrate reversed cisplatin-induced JNK and p38 activation, but had no effect on ROS production. The findings suggest fenofibrate's protective effect on cisplatin-induced cytotoxicity is mediated by inhibition of JNK and p38. Moreover, fenofibrate did not alter cisplatin's antitumor effect on cancer cell lines including T84, SW-480, HepG2, and SK-LU-1 cells. Therefore, fenofibrate may be a candidate agent for further development as an adjuvant to cisplatin treatment.
Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cisplatino/toxicidade , Fenofibrato/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/patologia , Células LLC-PK1 , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação , Transdução de Sinais/efeitos dos fármacos , SuínosRESUMO
Cholera is caused by infection with Vibrio cholerae. This study aimed to investigate the pathophysiology of diarrhea caused by the V. cholerae O1 El Tor variant (EL), a major epidemic strain causing severe diarrhea in several regions. In the ligated ileal loop model of EL-induced diarrhea in the ICR mice, a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a calcium-activated chloride channel (CaCC) inhibitor similarly inhibited intestinal fluid secretion. In addition, barrier disruption and NF-κB-mediated inflammatory responses, e.g., iNOS and COX-2 expression, were observed in the infected ileal loops. Interestingly, intestinal fluid secretion and barrier disruption were suppressed by NF-κB and COX-2 inhibitors, whereas an iNOS inhibitor suppressed barrier disruption without affecting fluid secretion. Furthermore, EP2 and EP4 PGE2 receptor antagonists ameliorated the fluid secretion in the infected ileal loops. The amount of cholera toxin (CT) produced in the ileal loops by the EL was â¼2.4-fold of the classical biotype. The CT transcription inhibitor virstatin, a toll-like receptor-4 (TLR-4) antibody and a CT antibody suppressed the EL-induced intestinal fluid secretion, barrier disruption and COX-2 expression. The CT at levels detected during EL infection induced mild intestinal barrier disruption without inducing inflammatory responses in mouse intestine. Collectively, this study indicates that CT-induced intestinal barrier disruption and subsequent TLR-4-NF-κB-mediated COX-2 expression are involved in the pathogenesis of EL-induced diarrhea and represent promising novel therapeutic targets of cholera.