RESUMEN
AIMS/HYPOTHESIS: The mitochondrial chaperonin heat shock protein (HSP) 60 is indispensable in protein folding and the mitochondrial stress response; however, its role in nutrient metabolism remains uncertain. This study investigated the role of HSP60 in diet-induced non-alcoholic fatty liver disease (NAFLD). METHODS: We studied human biopsies from individuals with NAFLD, murine high-fat-diet (HFD; a diet with 60% energy from fat)-induced obesity (DIO), transgenic (Tg) mice overexpressing Hsp60 (Hsp60-Tg), and human HepG2 cells transfected with HSP60 cDNA or with HSP60 siRNA. Histomorphometry was used to assess hepatic steatosis, biochemistry kits were used to measure insulin resistance and glucose tolerance, and an automated home cage phenotyping system was used to assess energy expenditure. Body fat was assessed using MRI. Macrophage infiltration, the lipid oxidation marker 4-hydroxy-2-nonenal (4-HNE) and the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were detected using immunohistochemistry. Intracellular lipid droplets were evaluated by Nile red staining. Expression of HSP60, and markers of lipogenesis and fatty acid oxidation were quantified using RT-PCR and immunoblotting. Investigations were analysed using the two-way ANOVA test. RESULTS: Decreased HSP60 expression correlated with severe steatosis in human NAFLD biopsies and murine DIO. Hsp60-Tg mice developed less body fat, had reduced serum triglyceride levels, lower levels of insulin resistance and higher serum adiponectin levels than wild-type mice upon HFD feeding. Respiratory quotient profile indicated that fat in Hsp60-Tg mice may be metabolised to meet energy demands. Hsp60-Tg mice showed amelioration of HFD-mediated hepatic steatosis, M1/M2 macrophage dysregulation, and 4-HNE and 8-OHdG overproduction. Forced HSP60 expression reduced the mitochondrial unfolded protein response, while preserving mitochondrial respiratory complex activity and enhancing fatty acid oxidation. Furthermore, HSP60 knockdown enhanced intracellular lipid formation and loss of sirtuin 3 (SIRT3) signalling in HepG2 cells upon incubation with palmitic acid (PA). Forced HSP60 expression improved SIRT3 signalling and repressed PA-mediated intracellular lipid formation. SIRT3 inhibition compromised HSP60-induced promotion of AMP-activated protein kinase (AMPK) phosphorylation and peroxisome proliferator-activated receptor α (PPARα levels), while also decreasing levels of fatty acid oxidation markers. CONCLUSION/INTERPRETATION: Mitochondrial HSP60 promotes fatty acid oxidation while repressing mitochondrial stress and inflammation to ameliorate the development of NAFLD by preserving SIRT3 signalling. This study reveals the hepatoprotective effects of HSP60 and indicates that HSP60 could play a fundamental role in the development of therapeutics for NAFLD or type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico , Sirtuina 3 , Animales , Humanos , Ratones , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/metabolismo , Resistencia a la Insulina/genética , Metabolismo de los Lípidos , Lípidos , Hígado/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Sirtuina 3/genética , Sirtuina 3/metabolismoRESUMEN
Renal fibrosis is a hallmark of chronic and progressive renal diseases characterized by excessive fibroblast proliferation, extracellular matrix accumulation, and a loss of renal function, eventually leading to end-stage renal diseases. MicroRNA-26a-5p (miR-26a-5p) downregulation has been previously noted in the sera of unilateral ureteral occlusion (UUO)-injured mice, and exosome-mediated miR-26a-5p reportedly attenuated experimental pulmonary and cardiac fibrosis. This study evaluated the expression patterns of miR-26a in a human tissue microarray with kidney fibrosis and in tissues from a mouse model of UUO-induced renal fibrosis. Histologic analyses showed that miR-26a-5p was downregulated in human and mouse tissues with renal interstitial nephritis and fibrosis. Moreover, miR-26a-5p restoration by intravenous injection of a mimic agent prominently suppressed the expression of transforming growth factor ß1 (TGF-ß1) and its cognate receptors, the inflammatory transcription factor NF-κB, epithelial-mesenchymal transition, and inflammatory markers in UUO-injured kidney tissues. In vitro, miR-26a-5p mimic delivery significantly inhibited TGF-ß1-induced activation of cultured normal rat kidney NRK-49F cells, in terms of downregulation of TGF-ß1 receptors, restoration of the epithelial marker E-cadherin, and suppression of mesenchymal markers, including vimentin, fibronectin, and α-smooth muscle actin, as well as TGF-ß1/SMAD3 signaling activity. Our findings identified miR-26a-5p downregulation in kidney tissues with human interstitial nephritis and UUO-induced mouse kidney fibrosis. MiR-26a-5p restoration may exhibit an antifibrotic effect through the blockade of both TGF-ß and NF-κB signaling axes and is considered a novel therapeutic target for treating obstruction-induced renal fibrosis.
Asunto(s)
MicroARNs , Nefritis Intersticial , Obstrucción Ureteral , Animales , Humanos , Ratones , Ratas , Fibrosis , Riñón/metabolismo , MicroARNs/metabolismo , Nefritis Intersticial/metabolismo , Nefritis Intersticial/patología , FN-kappa B/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/metabolismoRESUMEN
Upper tract urothelial cancer (UTUC) is a less common disease in Western countries but has a high level of prevalence in Asian populations. Compared to bladder cancer, unique etiologic and genomic factors are involved in UTUC. Fibroblast growth factor receptor 3 (FGFR3) up-regulation has been proposed as a promising target for bladder cancer therapy. In this study, we aimed to profile the expression of FGFR3 in Asian and Caucasian UTUC tissues and to evaluate the in vitro therapeutic efficacy of small interference RNA (siRNA)-mediated FGFR3 silencing in UTUC treatment. The FGFR3 expression levels in renal pelvis tissues and microarray sections from Asian and Caucasian patients with UTUC, respectively, were measured via immunohistochemistry. The BFTC-909 and UM-UC-14 UTUC cell lines were used to examine the effects of FGFR3 silencing on proliferation, migration, epithelial-mesenchymal transition (EMT) marker expression, and signaling machinery. FGFR3 expression increased as the TNM stage increased in both Asian and Caucasian UTUC tumors, and no statistical difference was identified between the two groups. In vitro studies demonstrated that FGFR3 siRNA delivery significantly inhibited proliferation and migration and suppressed the expression of EMT markers and transcription factors in UTUC cells. Mechanistically, FGFR3 silencing alleviated the constitutive expression of RAS and the phosphorylation of MAPK signaling mediators, including ERK1/2 and JNK1/2. FGFR3 silencing elicited an apoptosis-inducing effect similar to that of FGFR inhibition. Conclusion: siRNA-targeted FGFR3 expression may impede the expansion and invasion of UTUC cells by alleviating the RAS/MAPK signaling pathway. The genetic interference of FGFR3 expression via siRNA in UTUC cells may constitute a useful therapeutic strategy.
Asunto(s)
Carcinoma de Células Transicionales , Neoplasias de la Vejiga Urinaria , Neoplasias Urológicas , Humanos , Neoplasias de la Vejiga Urinaria/patología , Carcinoma de Células Transicionales/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Neoplasias Urológicas/genética , ARN Interferente Pequeño/genéticaRESUMEN
Patients with advanced head and neck squamous cell carcinoma (HNSCC) usually show a dismal prognosis. It is this worthwhile to develop new, effective therapeutic regimens for these patients, such as molecular targeted therapy, which is promising as an alternative or combination treatment for HNSCC. The mammalian target of rapamycin (mTOR) pathway, which plays an important role in the carcinogenesis of HNSCC, is the most frequently activated, and is thus worthy of further investigation. In this study, two human HNSCC cell lines, FaDu and SAS, were evaluated for cell growth with trypan blue staining and tumor growth using an orthotopic xenograft model. The immunohistochemical expression of mTOR in the subcutaneous xenograft model and the inhibitory effects of docetaxel on the growth and state of activation of the PI3K/mTOR pathway were also evaluated and examined by colony formation and Western blot, respectively. Cell proliferation and migration were measured by water-soluble tetrazolium salt (WST-1) and OrisTM cell migration assay, respectively. Furthermore, the effects of rapamycin and BEZ235, a phosphatidylinositol 3-kinases (PI3K) and mTOR inhibitor in combination with docetaxel or CCL20 were evaluated in the FaDu and SAS cells. The results showed that the expression of mTOR was significantly higher in the SAS and FaDu xenograft models than in the control. Docetaxel treatment significantly suppressed HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. Additionally, when administered in a dose-dependent fashion, mTOR inhibitors inhibited the growth and migration of the HNSCC cells. This combination was synergistic with docetaxel, resulting in almost complete cell growth and migration arrest. In conclusion, docetaxel significantly inhibited HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. The synergistic and additive activity of mTOR inhibitors combined with docetaxel shows potential as a new treatment strategy for HNSCC.
Asunto(s)
Quimiocina CCL20/metabolismo , Docetaxel/uso terapéutico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Docetaxel/farmacología , Humanos , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Rad51 is a protein which plays a vital role in DNA double-strand break repair and maintenance of telomeres. However, the underlying mechanism for its action in esophageal squamous cell carcinoma (ESCC) remains unclear. PATIENTS AND METHODS: Eighty-seven patients with ESCC were enrolled in this study. Expression of Rad51 in ESCC was determined by immunohistochemistry and correlated with clinicopathological variables by Chi square test. The role of Rad51 in patient survival was determined by Kaplan-Meier estimates. The effects of Rad51 knockdown and overexpression on esophageal cancer growth, migration, and invasion were examined using TE8, CE81T, and KYSE70 cells. The mechanisms involved were also analyzed. Nude mice models were used for assessment of tumor growth. RESULTS: Rad51 staining was predominantly observed in ESCC patients. ESCC patients with high Rad51 expression had significantly decreased survival (P < 0.001) combined with increased tumor size (P = 0.034) and lymph node metastasis (P = 0.039). Rad51 overexpression promoted, while its knockdown attenuated, esophageal cancer cell viability through cell cycle entry and migration/invasion via epithelial-mesenchymal transition. Moreover, Rad51 overexpression increased colony formation in vitro and tumor growth in vivo. In addition, high Rad51 expression increased cancer progression through the p38/Akt/Snail signaling pathway. CONCLUSIONS: This study indicates a new biological role for Rad51 in ESCC progression. Rad51 may serve as a potential prognostic biomarker and therapeutic target for ESCC patients.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Recombinasa Rad51/metabolismo , Transducción de Señal , Animales , Movimiento Celular , Proliferación Celular , Reparación del ADN , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Metástasis Linfática/genética , Metástasis Linfática/patología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Recombinasa Rad51/genéticaRESUMEN
Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.
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Autofagia , Colestasis/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Colestasis/patología , Citoprotección , Hepatocitos/citología , Humanos , Isoxazoles/farmacología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos ICR , Sustancias Protectoras/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Activación TranscripcionalRESUMEN
Transforming growth factor-ß (TGF-ß) plays a central role in hepatic fibrogenesis. This study investigated the function and mechanism of bone morphogenetic protein-2 (BMP-2) in regulation of hepatic fibrogenesis. BMP-2 expression in fibrotic liver was measured in human tissue microarray and mouse models of liver fibrosis induced by bile duct ligation surgery or carbon tetrachloride administration. Adenovirus-mediated BMP-2 gene delivery was used to test the prophylactic effect on liver fibrosis. Primary hepatic stellate cells (HSC), HSC-T6 and clone-9 cell lines were used to study the interplay between BMP-2 and TGF-ß1. Hepatic BMP-2 was localized in parenchymal hepatocytes and activated HSCs and significantly decreased in human and mouse fibrotic livers, showing an opposite pattern of hepatic TGF-ß1 contents. BMP-2 gene delivery alleviated the elevations of serum hepatic enzymes, cholangiocyte marker CK19, HSC activation markers, and liver fibrosis in both models. Mechanistically, exogenous TGF-ß1 dose dependently reduced BMP-2 expression, whereas BMP-2 significantly suppressed expression of TGF-ß and its cognate type I and II receptor peptides, as well as the induced Smad3 phosphorylation levels in primary mouse HSCs. Aside from its suppressive effects on cell proliferation and migration, BMP-2 treatment prominently attenuated the TGF-ß1-stimulated α-SMA and fibronectin expression, and reversed the TGF-ß1-modulated epithelial-to-mesenchymal transition marker expression in mouse HSCs. The mutual regulation between BMP-2 and TGF-ß1 signaling axes may constitute the anti-fibrogenic mechanism of BMP-2 in the pathogenesis of liver fibrosis. BMP-2 may potentially serve as a novel therapeutic target for treatment of liver fibrosis.
Asunto(s)
Proteína Morfogenética Ósea 2/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/farmacología , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/patología , Cirrosis Hepática/genética , Ratones , Ratas , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Up-regulation of nerve growth factor (NGF) in parenchymal hepatocytes with cholestatic injury has been previously demonstrated to exert hepatoprotective effects in an autocrine manner; however, the overall impact of NGF up-regulation remains elusive. This study aimed to profile the effects of exogenous NGF on cultured primary rat hepatocytes using transcriptome analysis. Total RNA was isolated from hepatocytes with and without 24â¯h of NGF exposure, and subjected to RNA enrichment by PCR and RNA sequencing procedures. Comparison of transcriptome profiles between control and NGF-stimulated hepatocytes demonstrated that NGF significantly up-regulated 10 genes and down-regulated 23 genes in hepatocytes. Subsequent KEGG pathway enrichment analysis indicated that NGF significantly affected the retinol metabolism pathway via increased retinol dehydrogenase 16 (RDH16) expression. In a mouse model of bile duct ligation-induced cholestatic liver injury, NGF supplementation significantly enhanced RDH16 expression, whereas administration of anti-NGF neutralizing antibodies prominently decreased RDH16 expression in cholestatic livers, supporting the positive role of NGF in the regulation of RDH16 in diseased livers. In vitro study further demonstrated that NGF triggered de novo synthesis of RDH16 in primary rat hepatocytes, mainly through an NF-κB signaling pathway. In conclusion, this study demonstrates the up-regulation of RDH16 by NGF in cultured rat hepatocytes and mouse cholestatic livers, and provides novel insights on the mechanistic role of NGF in the retinol metabolism of livers.
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Oxidorreductasas de Alcohol/metabolismo , Hepatocitos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Animales , Colestasis/metabolismo , Perfilación de la Expresión Génica/métodos , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Masculino , Redes y Vías Metabólicas/fisiología , Ratones , Ratones Endogámicos ICR , FN-kappa B/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Elevation of free fatty acids (FFAs) is known to affect microvascular function and contribute to obesity-associated insulin resistance, hypertension, and microangiopathy. Proliferative and synthetic vascular smooth muscle cells (VSMCs) increase intimal thickness and destabilize atheromatous plaques. This study aimed to investigate whether saturated palmitic acid (PA) and monounsaturated oleic acid (OA) modulate autophagy activity, cell proliferation, and vascular tissue remodeling in an aortic VSMC cell line. Exposure to PA and OA suppressed growth of VSMCs without apoptotic induction, but enhanced autophagy flux with elevation of Beclin-1, Atg5, and LC3I/II. Cotreatment with autophagy inhibitors potentiated the FFA-suppressed VSMC growth and showed differential actions of PA and OA in autophagy flux retardation. Both FFAs upregulated lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) but only OA increased LDL uptake by VSMCs. Mechanistically, FFAs induced hyperphosphorylation of Akt, ERK1/2, JNK1/2, and p38 MAPK. All pathways, except OA-activated PI3K/Akt cascade, were involved in the LOX-1 upregulation, whereas blockade of PI3K/Akt and MEK/ERK cascades ameliorated the FFA-induced growth suppression on VSMCs. Moreover, both FFAs exhibited tissue remodeling effect through increasing MMP-2 and MMP-9 expression and their gelatinolytic activities, whereas high-dose OA significantly suppressed collagen type I expression. Conversely, siRNA-mediated LOX-1 knockdown significantly attenuated the OA-induced tissue remodeling effects in VSMCs. In conclusion, OA and PA enhance autophagy flux, suppress aortic VSMC proliferation, and exhibit vascular remodeling effect, thereby leading to the loss of VSMCs and interstitial ECM in vascular walls and eventually the instability of atheromatous plaques. J. Cell. Biochem. 118: 1249-1261, 2017. © 2016 Wiley Periodicals, Inc.
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Ácidos Grasos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Receptores Depuradores de Clase E/metabolismo , Regulación hacia Arriba , Animales , Aorta , Autofagia/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Músculo Liso Vascular/citología , Ácido Oléico/farmacología , Ácido Palmítico/farmacología , RatasRESUMEN
The involvement of microRNAs (miRNAs) in cancer development and their potential as prognostic biomarkers are becoming increasingly known. However, the signature of miRNAs and their regulatory roles in tumorigenesis of upper tract urothelial carcinoma (UTUC) remain to be elucidated. This study aimed to profile the miRNA expression pattern in UTUC tumor tissues and identify candidate miRNAs with prognostic and/or therapeutic functions. METHODS AND RESULTS: We collected 22 UTUC tissue and adjacent normal tissues samples from patients who underwent nephroureterectomy. The miRNAs signatures of three selected UTUC samples using next-generation sequencing showed that miR-30a-5p was significantly downregulated in UTUC tumors compared to adjacent normal tissues. The differentially-expressed miRNAs were specifically validated by quantitative real-time polymerase chain reaction. In addition, the miRNA expression signatures were analyzed with the transcriptome profile characterized by microarray. Further in vitro studies indicated that overexpression of miR-30a-5p significantly suppressed proliferation, migration, and epithelial-to-mesenchymal transition (EMT) in cultured BFTC-909 UTUC cells. As a potential target gene of miR-30a-5p in the tight junction pathway suggested by the pathway enrichment analysis, the reduced expression of tight junction protein claudin-5 in UTUC cells was demonstrated to be upregulated by miR-30a-5p genetic delivery. CONCLUSIONS: Taken together, our findings demonstrated that miR-30a-5p inhibits proliferation, metastasis, and EMT, and upregulates the expression of tight junction claudin-5 in UTUC cells. Thus, miR-30a-5p may provide a promising therapeutic strategy for UTUC treatment.
Asunto(s)
Claudina-5/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias Urológicas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Transcriptoma , Neoplasias Urológicas/metabolismo , Neoplasias Urológicas/patologíaRESUMEN
This study investigated the signaling mechanism underlying the anti-adhesive effect of all-trans retinoic acid (ATRA) on retinal pigment epithelial ARPE-19 cells. Adhesion kinetics with or without ATRA treatment were profiled by adhesion assay. Surface coating with type IV collagen, fibronectin, laminin, but not type I collagen, significantly enhanced adhesion and spreading of ARPE-19 cells, while ATRA at subtoxic doses (ranging from 10-7 to 10-6 M) profoundly suppressed the extracellular matrix-enhanced adhesion ability. Cell attachment on FN activated PI3K/Akt and MAPK cascades, whereas ATRA pretreatment blunted the early phosphorylation of Akt and MAPK signaling mediators including p38 MAPK, JNK1/2, and ERK1/2. Mechanistically, signaling blockade with selective kinase inhibitors demonstrated that all MAPK pathways were involved in the anti-adhesive effect of ATRA, whereas the PI3K inhibitor treatment significantly potentiated the ATRA-suppressed RPE cell adhesion. Moreover, ATRA treatment did not affect intracellular F-actin distribution, but remarkably reduced focal adhesion kinase (FAK) expression and its nuclear localization during ARPE-19 cell attachment. In conclusion, ATRA suppresses the adhering ability of ARPE-19 cells at least in part through MAPK and FAK pathways. Signaling blockade with PI3K inhibitor could be regarded as an alternative modality for treating proliferative vitreoretinopathy.
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Quinasa 1 de Adhesión Focal/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Tretinoina/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Humanos , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-ß1 (TGF-ß1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl4 induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-ß1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl4-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.
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Núcleo Celular/metabolismo , Proteína HMGB1/metabolismo , Células Estrelladas Hepáticas/patología , Hepatocitos/patología , Cirrosis Hepática/patología , Fragmentos de Péptidos/farmacología , Animales , Western Blotting , Tetracloruro de Carbono/toxicidad , Proliferación Celular , Células Cultivadas , Citoplasma/metabolismo , Proteína HMGB1/genética , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Peróxido de Hidrógeno/farmacología , Técnicas para Inmunoenzimas , Inmunoprecipitación , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Oxidantes/farmacología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Dopamine (DA) is commonly used to maintain the hemodynamic stability of brain-dead donors despite its controversial effects on organ functions. This study aimed at examining the hemodynamic effect of DA in a rat brain-dead model in vivo, alteration of hepatocyte integrity in liver grafts after ex vivo preservation, and changes in cultured clone-9 hepatocytes including cellular viability, cell cycle, apoptotic regulators, and lipopolysaccharide (LPS)-stimulated nuclear factor kappa B (NF-κB) signaling machinery. Although in vivo findings demonstrated enhanced portal venous blood flow and hepatic microcirculatory perfusion after DA infusion, no apparent advantage was noted in preserving hepatocyte integrity ex vivo. In vitro, prolonged exposure to high-dose DA reduced proliferation and induced G1 growth arrest of clone-9 hepatocytes with concomitant decreases in B cell lymphoma 2 (BCL2)/B cell lymphoma 2-associated X protein (BAX) and heat shock protein 70/BAX protein ratios and intracellular NF-κB p65. Moreover, DA pretreatment suppressed LPS-elicited inhibitor of κBα phosphorylation and subsequent NF-κB nuclear translocation, suggesting that DA may down-regulate NF-κB signaling, thereby reducing expression of antiapoptotic regulators, such as BCL2. In conclusion, despite augmentation of hepatic perfusion, DA infusion failed to preserve hepatocyte integrity both in vivo and ex vivo. In vitro findings demonstrated that high-dose DA may hamper the function of NF-κB signaling machinery and eventually undermine functional integrity of hepatocytes in liver grafts.
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Cardiotónicos/farmacología , Dopamina/farmacología , Hepatocitos/efectos de los fármacos , FN-kappa B/metabolismo , Animales , Apoptosis , Células Cultivadas , Hepatocitos/metabolismo , Técnicas In Vitro , Lipopolisacáridos , Hígado/efectos de los fármacos , Hígado/metabolismo , Circulación Hepática , Masculino , Preservación de Órganos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas Sprague-DawleyRESUMEN
Despite high in-hospital mortality associated with acute respiratory distress syndrome (ARDS), there is no effective therapeutic strategy. We tested the hypothesis that combined melatonin-mitochondria treatment ameliorates 100% oxygen-induced ARDS in rats. Adult male Sprague-Dawley rats (n = 40) were equally categorized into normal controls, ARDS, ARDS-melatonin, ARDS with intravenous liver-derived mitochondria (1500 µg per rat 6 hr after ARDS induction), and ARDS receiving combined melatonin-mitochondria. The results showed that 22 hr after ARDS induction, oxygen saturation (saO2 ) was lowest in the ARDS group and highest in normal controls, significantly lower in ARDS-melatonin and ARDS-mitochondria than in combined melatonin-mitochondria group, and significantly lower in ARDS-mitochondria than in ARDS-melatonin group. Conversely, right ventricular systolic blood pressure and lung weight showed an opposite pattern compared with saO2 among all groups (all P < 0.001). Histological integrity of alveolar sacs showed a pattern identical to saO2 , whereas lung crowding score exhibited an opposite pattern (all P < 0.001). Albumin level and inflammatory cells (MPO+, CD40+, CD11b/c+) from bronchoalveolar lavage fluid showed a pattern opposite to saO2 (all P < 0.001). Protein expression of indices of inflammation (MMP-9, TNF-α, NF-κB), oxidative stress (oxidized protein, NO-1, NOX-2, NOX-4), apoptosis (mitochondrial Bax, cleaved caspase-3, and PARP), fibrosis (Smad3, TGF-ß), mitochondrial damage (cytochrome C), and DNA damage (γ-H2AX+) exhibited an opposite pattern compared to saO2 in all groups, whereas protein (HO-1, NQO-1, GR, GPx) and cellular (HO-1+) expressions of antioxidants exhibited a progressively increased pattern from normal controls to ARDS combined melatonin-mitochondria group (all P < 0.001). In conclusion, combined melatonin-mitochondrial was superior to either treatment alone in attenuating ARDS in this rat model.
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Melatonina/uso terapéutico , Mitocondrias/metabolismo , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Animales , Western Blotting , Masculino , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Consumo de Oxígeno/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Because the molecular mechanisms of morphogenesis of the hepatic cord and sinus are unclear, we investigated the involvement of bone morphogenetic protein (BMP4) in hepatic sinusoid morphogenesis. METHODS: We used embryonic chicken livers, which develop rapidly, as our model, and investigated expression of BMP-related genes. BMP4 activity was manipulated by overexpressing BMP4 and its antagonist, noggin. RESULTS: During hepatic cord morphogenesis, BMP4 and its receptors are expressed in both peri-sinusoidal cells and hepatoblasts as the sinusoids form, whereas noggin is expressed transiently in peri-sinusoidal cells at early stages. Suppression of BMP activity with noggin overexpression disrupted normal hepatic sinusoid structure, leading to liver congestion, failure of fibronectin deposition, and markedly reduced numbers of peri-sinusoidal cells. However, overexpression of BMP did not change sinusoidal morphology but increased endothelial cell number. Noggin overexpression resulted in disrupted cord organization, and dilated sinusoidal space, eventually leading to increased apoptosis and failed hepatocyte differentiation. CONCLUSIONS: Our results show that proper BMP signaling mediates peri-sinusoidal cell-hepatoblast interactions during development; this is essential for hepatic cord organization among hepatoblasts, endothelium, and presumptive hepatic stellate cells.
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Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/metabolismo , Células Estrelladas Hepáticas/fisiología , Hígado/embriología , Hígado/metabolismo , Células Madre Mesenquimatosas/fisiología , Animales , Proteínas Portadoras/genética , Comunicación Celular , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Transducción de SeñalRESUMEN
Low back pain (LBP) is a leading cause of long-term disability globally. Intervertebral disk degeneration (IVDD) is mainly responsible for discogenic pain in LBP-affected young patients. There is no effective therapy to reverse disease severity and IVDD progression. This study investigates the effect of human peripheral blood-derived mononuclear cells (PBMCs) on pain relief and life quality improvement in IVDD patients. The enriched monocytes of the PBMCs could differentiate into CD14 and CD206 double-positive M2 macrophages in vitro. Preclinical evidence in rats showed that the transplanted PBMCs exhibited anti-inflammatory and moderate tissue-repair effects on controlling IVDD progress in the rat model. The PBMCs significantly steered the aggrecan and type II collagen expressions and attenuated the pro-inflammatory cytokines in the affected disk. Based on the animal results, 36 patients with chronic low back pain (CLBP) were included in clinical trials. The control group was conservative care only, and the experimental group was platelet-rich plasma (PRP) and PBMCs intradiscal injections. We first confirmed the single lumbar disk causing the discogenic pain by provocative discography or magnetic resonance imaging (MRI). Discogenic LBP participants received one intradiscal injection of autologous PBMCs and followed for 6 months. Our clinical trial showed that patients' LBP and disability were significantly ameliorated after the PBMCs transplantation rather than PRP. These preclinical and pilot clinical studies indicate that intradiscal injection of the enriched PBMCs might be a feasible and potential cell therapy to control pain and disability in IVDD patients.
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Degeneración del Disco Intervertebral , Disco Intervertebral , Dolor de la Región Lumbar , Humanos , Animales , Ratas , Degeneración del Disco Intervertebral/terapia , Disco Intervertebral/patología , Dolor de la Región Lumbar/tratamiento farmacológico , Dolor de la Región Lumbar/etiología , Inyecciones/efectos adversos , Antiinflamatorios/farmacología , Resultado del TratamientoRESUMEN
Statin therapy is known to down-regulate inflammatory activities in atheromatous tissues of animals. The aims of this study were to examine the regulatory role of interleukin-18 (IL-18) in the connexin 43 (Cx43) and the proliferation of cultured aortic smooth muscle cells (SMCs) as well as to elucidate the underlying therapeutic mechanism of simvastatin. Vytorin therapy significantly alleviated high-cholesterol diet-induced hypercholesterolemia, suppressed neointimal hyperplasia, macrophage infiltration, and Cx43 and IL-18 expression in rabbit aortic walls. In vitro study using an aortic SMC line showed that IL-18 up-regulated constitutive Cx43 expression and potentiated tumor necrosis factor-α (TNF-α)-triggered Akt and MAPK signaling pathways. Simvastatin treatment alone reduced constitutive Cx43 levels and prevented the TNF-α-induced IL-18 up-regulation. Mechanistic investigation using kinase-specific inhibitors showed that simvastatin pretreatment attenuated TNF-α-elicited Akt and ERK1/2 phosphorylation, whereas PI3K and all MAPK activities were also implied in the additive effect of TNF-α and IL-18 on Cx43 up-regulation. Proliferation assay indicated that IL-18 stimulated SMC proliferation and synergized the TNF-α-stimulated cell proliferation. Likewise, simvastatin treatment suppressed the SMC over-proliferation induced not only by TNF-α alone, but also by simultaneous treatment with TNF-α and IL-18. The suppression of simvastatin in SMC proliferation was not mediated through mitochondrial related pro-apoptogenesis under both scenarios. In conclusion, simvastatin attenuates the additive effects of TNF-α and IL-18 on Cx43 up-regulation and over-proliferation of aortic SMCs, mainly through the blockade of Akt signaling pathway. These findings may fortify the rationale underlying the atheroprotective mechanism of statin therapy.
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Aorta/patología , Conexina 43/metabolismo , Interleucina-18/farmacología , Miocitos del Músculo Liso/metabolismo , Simvastatina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Azetidinas , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dieta Alta en Grasa , Regulación hacia Abajo/efectos de los fármacos , Combinación de Medicamentos , Sinergismo Farmacológico , Combinación Ezetimiba y Simvastatina , Hipercolesterolemia/patología , Macrófagos/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Neointima/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND AIMS: We tested the hypothesis that sitagliptin is capable of increasing blood flow in the rat critical limb ischemia (CLI) model by enhancement of angiogenesis. METHODS: Adipose tissue from adult-male Fischer 344 rats (n = 6) were cultured in endothelial progenitor cell culture medium for 14 d with (25 µmol/L) or without sitagliptin. CLI was induced by ligation of the left femoral artery. Rats (n = 32) were equally separated into four groups: untreated controls (group 1), sitagliptin (4 mg/kg per day; group 2), CLI (group 3) and CLI with sitagliptin (group 4). RESULTS: In vitro, 7 and 14 d after cell culture, endothelial progenitor cell biomarkers assessed by flow cytometry (Sca-1/CD31+, CXCR4+, c-kit+ and CD34+ cells) and Western blot (vascular endothelial growth factor, CXCR4 and stromal-derived factor [SDF]-1α) were remarkably higher in group 4 than in the other groups (all P < 0.01). In vivo, 2 and 14 d after the CLI procedure, circulating angiogenic cell (Sca-1/CD31+, Sca-1+ and CD31+) numbers were significantly higher in group 4 than in the other groups (all P < 0.001). Additionally, the messenger RNA and protein expression of angiogenic biomarkers (CXCR4, SDF-1α and vascular endothelial growth factor), immunofluorescent staining of angiogenic cells (CXCR4+, SDF-1α+, CD31+, von Willebrand factor + cells) and immunohistochemical staining of small vessel numbers in the ischemic area were significantly higher in group 4 than in the other groups (all P < 0.01). Furthermore, laser Doppler showed that the ratio of ischemic/normal blood flow was remarkably higher group 4 than in group 3 by days 14 and 28 after the CLI procedure (all P < 0.01). CONCLUSIONS: Sitagliptin therapy enhances circulating angiogenic cell numbers, angiogenesis and blood flow in the CLI area.
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Miembro Posterior/efectos de los fármacos , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Pirazinas/farmacología , Triazoles/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/fisiopatología , Animales , Arterias/efectos de los fármacos , Arterias/metabolismo , Arterias/fisiología , Biomarcadores/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Miembro Posterior/metabolismo , Miembro Posterior/fisiología , Isquemia/metabolismo , Isquemia/fisiopatología , Masculino , Ratas , Ratas Endogámicas F344 , Flujo Sanguíneo Regional/efectos de los fármacos , Flujo Sanguíneo Regional/fisiología , Fosfato de Sitagliptina , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Madre/fisiologíaRESUMEN
BACKGROUND: Bile duct ligation (BDL) is a commonly used cholestatic liver disease (CLD) model. We recently found that L-arginine levels were significantly raised by melatonin in young rats with BDL. We hypothesized that protein kinase C-α (PKC-α) is involved in the increases of L-arginine in melatonin-treated BDL rats. In addition, we tested whether melatonin prevents nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced reactive oxygen species (ROS) production, in rats with BDL, through PKC. METHODS: Four groups of young male rats were studied: shams (n = 6), untreated BDL rats (n = 9), melatonin-treated shams (n = 6, M), and melatonin-treated BDL rats (n = 6, BDL + M). Melatonin-treated rats received daily melatonin 1 mg/kg/d via i.p. injection. All surviving rats were killed 14 d after surgery. RESULTS: Melatonin prevented BDL-induced mortality and kidney injury. Melatonin additionally increased L-arginine concentrations in BDL liver, which is correlated with decreased PKC-α translocation. Next, melatonin increased L-arginine levels in BDL kidneys, which was correlated with decreased renal levels of arginase II. In the BDL kidney, melatonin decreased PKC-ß translocation, reduced p47phox translocation, and diminished NADPH-dependent superoxide production. CONCLUSION: Melatonin inhibits PKC-α to increase cationic amino acid transporter-1 (CAT-1)-mediated L-arginine uptake in BDL liver, whereas it inhibits PKC-ß to reduce NADPH-dependent superoxide production.
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Arginina/metabolismo , Colestasis Extrahepática/tratamiento farmacológico , Conducto Colédoco/cirugía , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Melatonina/farmacología , NADPH Oxidasas/metabolismo , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Arginasa/metabolismo , Arginina/análogos & derivados , Arginina/sangre , Transporte Biológico , Transportador de Aminoácidos Catiónicos 1/metabolismo , Colestasis Extrahepática/sangre , Colestasis Extrahepática/enzimología , Colestasis Extrahepática/etiología , Modelos Animales de Enfermedad , Femenino , Inyecciones Intraperitoneales , Riñón/enzimología , Ligadura , Hígado/enzimología , Masculino , Melatonina/administración & dosificación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C beta , Proteína Quinasa C-alfa/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/administración & dosificación , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Expression of toll-like receptor-4 (TLR4) on tumor cells is known to mediate innate immune responses that influence tumor cell growth and migration. This study aimed to characterize TLR4 expression and elucidate its functional significance in human hepatoblastoma (HB) cells. PROCEDURE: Immunohistochemistry (IHC) was used to determine TLR4 expression level and its distribution pattern in HB liver tissues. Transcripts of tumor necrosis factor (TNF)-α, interleukin (IL)-8, matrix metalloproteinase (MMP)-2, MMP-13, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 in HB HepG2 cells with lipopolysacharide (LPS) treatment were measured by quantitative PCR. Soluble cytokines and peptides in conditioned media were measured by ELISA. MMP-2 activity was determined by using gelatin zymography. Cell motility and invasiveness was determined using wound healing migration and Matrigel invasion assays, respectively. RESULTS: TLR4 IHC staining demonstrated that TLR4 overexpression in HB liver tissues dramatically vanished after chemotherapy. In vitro study using an HB cell line, HepG2, showed that TLR4 agonist, LPS, significantly decreased transcripts of IL-8 and TNF-α, but did not affect MMP-13 mRNA level. By contrast, LPS only down-regulated IL-8 production and MMP-2 gelatinolytic activity. The latter might be in part due to the increased levels of MMP-2/TIMP-2 complex in conditioned media, thus leading to the decreased motility and invasiveness of HepG2 cells. CONCLUSIONS: HB cells overexpress TLR4, whereas TLR4 agonistic treatment inhibits migration and invasion of HB HepG2 cells. These findings suggest that TLR4 signaling pathway is a potential therapeutic target for control of HB tumor progression.