RESUMEN
BACKGROUND: Obesity is associated with airway hyperresponsiveness and lung fibrosis, which may reduce the effectiveness of standard asthma treatment in individuals suffering from both conditions. Statins and proprotein convertase subtilisin/kexin-9 inhibitors not only reduce serum cholesterol, free fatty acids but also diminish renin-angiotensin system activity and exhibit anti-inflammatory effects. These mechanisms may play a role in mitigating lung pathologies associated with obesity. METHODS: Male C57BL/6 mice were induced to develop obesity through high-fat diet for 16 weeks. Conditional TGF-ß1 transgenic mice were fed a normal diet. These mice were given either atorvastatin or proprotein convertase subtilisin/kexin-9 inhibitor (alirocumab), and the impact on airway hyperresponsiveness and lung pathologies was assessed. RESULTS: High-fat diet-induced obesity enhanced airway hyperresponsiveness, lung fibrosis, macrophages in bronchoalveolar lavage fluid, and pro-inflammatory mediators in the lung. These lipid-lowering agents attenuated airway hyperresponsiveness, macrophages in BALF, lung fibrosis, serum leptin, free fatty acids, TGF-ß1, IL-1ß, IL-6, and IL-17a in the lung. Furthermore, the increased RAS, NLRP3 inflammasome, and cholecystokinin in lung tissue of obese mice were reduced with statin or alirocumab. These agents also suppressed the pro-inflammatory immune responses and lung fibrosis in TGF-ß1 over-expressed transgenic mice with normal diet. CONCLUSIONS: Lipid-lowering treatment has the potential to alleviate obesity-induced airway hyperresponsiveness and lung fibrosis by inhibiting the NLRP3 inflammasome, RAS and cholecystokinin activity.
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Dieta Alta en Grasa , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad , Fibrosis Pulmonar , Animales , Masculino , Dieta Alta en Grasa/efectos adversos , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Ratones , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Fibrosis Pulmonar/prevención & control , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/tratamiento farmacológico , Inhibidores de PCSK9 , Atorvastatina/farmacología , Atorvastatina/uso terapéutico , Ratones Obesos , Proproteína Convertasa 9/metabolismo , Proproteína Convertasa 9/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Hiperreactividad Bronquial/prevención & control , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/fisiopatología , Anticuerpos Monoclonales HumanizadosRESUMEN
House dust mite (HDM) allergens cause inflammatory responses and chronic allergic diseases such as bronchial asthma and atopic dermatitis. Here, we investigate the mechanism by which HDM induces C-C chemokine ligand 20 (CCL20) expression to promote chronic inflammation and airway remodeling in an HDM-induced bronchial asthma mouse model. We showed that HDM increased CCL20 levels via the Akt-ERK1/2-C/EBPß pathway. To investigate the role of CCL20 in chronic airway inflammation and remodeling, we made a mouse model of CCL20-induced bronchial asthma. Treatment of anti-CCL20Ab in this mouse model showed the reduced airway hyper-responsiveness and inflammatory cell infiltration into peribronchial region by neutralizing CCL20. In addition, CCL20 induced the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation through NLRP3 deubiquitination and transcriptional upregulation in BEAS-2B cells. As expected, anti-CCL20Ab markedly suppressed NLRP3 activation induced by CCL20. Moreover, HDM-induced CCL20 leads to epithelial-mesenchymal transition in the lung epithelium which appears to be an important regulator of airway remodeling in allergic asthma. We also found that anti-CCL20Ab attenuates airway inflammation and remodeling in an HDM-induced mouse model of bronchial asthma. Taken together, our results suggest that HDM-induced CCL20 is required for chronic inflammation that contributes airway remodeling in a mouse model of asthma.
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Asma , Pyroglyphidae , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Asma/metabolismo , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Inflamación/complicaciones , Ligandos , Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Vitamin D (VitD) has pleiotropic effects. VitD deficiency is closely involved with obesity and may contribute to the development of lung fibrosis and aggravation of airway hyperresponsiveness (AHR). We evaluated the causal relationship between VitD deficiency and the lung pathologies associated with obesity. In vivo effects of VitD supplementation were analyzed using high-fat diet (HFD)-induced obese mice and TGF-ß1 (transforming growth factor-ß1) triple transgenic mice. Effects of VitD supplementation were also evaluated in both BEAS-2B and primary lung cells from the transgenic mice. Obese mice had decreased 25-OH VitD and VitD receptor expressions with increases of insulin resistance, renin and angiotensin-2 system (RAS) activity, and leptin. In addition, lung pathologies such as a modest increase in macrophages, enhanced TGF-ß1, IL-1ß, and IL-6 expression, lung fibrosis, and AHR were found. VitD supplementation to HFD-induced obese mice recovered these findings. TGF-ß1-overexpressing transgenic mice enhanced macrophages in BAL fluid, lung expression of RAS, epithelial-mesenchymal transition markers, AHR, and lung fibrosis. VitD supplementation also attenuated these findings in addition to the attenuation of the expressions of TGF-ß1, and phosphorylated Smad-2/3 in lung. Supplementing in vitro-stimulated BEAS-2B and primary lung cells with VitD inhibited TGF-ß1 expression, supporting the suppressive effect of VitD for TGF-ß1 expression. These results suggest that obesity leads to VitD deficiency and worsens insulin resistance while enhancing the expression of leptin, RAS, TGF-ß1, and proinflammatory cytokines. These changes may contribute to the development of lung fibrosis and AHR. VitD supplementation rescues these changes and may have therapeutic potential for asthma with obesity.
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Obesidad/complicaciones , Fibrosis Pulmonar/etiología , Hipersensibilidad Respiratoria/etiología , Deficiencia de Vitamina D/etiología , Animales , Biomarcadores/metabolismo , Peso Corporal/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Dieta Alta en Grasa , Suplementos Dietéticos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Inflamación/patología , Insulina/metabolismo , Leptina/sangre , Pulmón/metabolismo , Pulmón/patología , Masculino , Cloruro de Metacolina , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/sangre , Fibrosis Pulmonar/sangre , Receptores de Calcitriol/metabolismo , Renina/sangre , Sistema Renina-Angiotensina/efectos de los fármacos , Hipersensibilidad Respiratoria/sangre , Factor de Crecimiento Transformador beta1/metabolismo , Vitamina D/análogos & derivados , Vitamina D/sangre , Vitamina D/farmacología , Deficiencia de Vitamina D/sangreRESUMEN
BACKGROUND & AIMS: Transforming growth factor beta (TGF-ß) suppresses early stages of tumorigenesis, but also contributes to migration and metastasis of cancer cells. A large number of human tumors contain mutations that inactivate its receptors, or downstream proteins such as Smad transcription factors, indicating that the TGF-ß signaling pathway prevents tumor growth. We investigated the effects of TGF-ß inhibition on liver tumorigenesis in mice. METHODS: C57BL/6 mice received hydrodynamic tail-vein injections of transposons encoding HRASG12V and a short hairpin RNA (shRNA) to down-regulate p53, or those encoding HRASG12V and MYC, or those encoding HRASG12V and TAZS89A, to induce liver tumor formation; mice were also given injections of transposons encoding SMAD7 or shRNA against SMAD2, SMAD3, SMAD4, or SNAI1 (Snail), with or without ectopic expression of Snail. Survival times were compared, and livers were weighted and examined for tumors. Liver tumor tissues were analyzed by quantitative reverse-transcription PCR, RNA sequencing, immunoblots, and immunohistochemistry. We analyzed gene expression levels in human hepatocellular carcinoma samples deposited in The Cancer Genome Atlas. A cell proliferation assay was performed using human liver cancer cell lines (HepG2 and Huh7) stably expressing Snail or shRNA against Snail. RESULTS: TGF-ß inhibition via overexpression of SMAD7 (or knockdown of SMAD2, SMAD3, or SMAD4) consistently reduced formation and growth of liver tumors in mice that expressed activated RAS plus shRNA against p53, or in mice that expressed activated RAS and TAZ. TGF-ß signaling activated transcription of the Snail gene in liver tumors induced by HRASG12V and shRNA against p53, and by activated RAS and TAZ. Knockdown of Snail reduced liver tumor formation in both tumor models. Ectopic expression of Snail restored liver tumorigenesis suppressed by disruption of TGF-ß signaling. In human hepatocellular carcinoma, Snail expression correlated with TGF-ß activation. Ectopic expression of Snail increased cellular proliferation, whereas Snail knockdown led to reduced proliferation in human hepatocellular carcinoma cells. CONCLUSIONS: In analyses of transgenic mice, we found TGF-ß signaling to be required for formation of liver tumors upon expression of activated RAS and shRNA down-regulating p53, and upon expression of activated RAS and TAZ. Snail is the TGF-ß target that is required for hepatic tumorigenesis in these models.
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Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica , Genes myc , Genes ras , Predisposición Genética a la Enfermedad , Células Hep G2 , Humanos , Hígado/patología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factores de Transcripción de la Familia Snail/genética , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p < 0.001), and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Notably, tumors from KRAS4AG12V mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene.
Asunto(s)
Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/patología , GTP Fosfohidrolasas/genética , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Apoptosis , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , GTP Fosfohidrolasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for hepatocellular carcinoma (HCC) and present in 80 to 90 % of patients with HCC. Current genetically engineered mouse models for HCC, however, generally do not feature liver fibrosis, which is a critical discrepancy between human HCC and murine models thereof. In this study, we developed a simple transgenic mouse model of HCC within the context of a fibrotic liver. METHODS: Employing hydrodynamic transfection (HT), coupled with the Sleeping Beauty (SB) transposon system, liver was stably transfected with transposons expressing cMyc and a short hairpin RNA down-regulating p53 (shp53). A chronic liver injury model, induced by hepatotoxic carbon tetrachloride (CCl4), was applied to the transgenic mice, allowing cells expressing cMyc plus shp53 to become malignant in the background of liver fibrosis. RESULTS: Livers harvested about 3 months after HT had excessive collagen deposition and activated hepatic stellate cells surrounding the tumors. Hepatocarcinogenesis was significantly accelerated in the fibrotic livers compared to those of the control, significantly decreasing the life span of the mice. The tumor incidence and average number of tumors per mouse were significantly higher in the group treated with CCl4 compared to the vehicle-treated control mice, following HT (p < 0.01). CONCLUSIONS: Considering the simplicity and efficiency in generating HCC for fibrotic livers, the transgenic HCC model has the potential to be effectively used in preclinical testing of HCC anticancer therapy and in studies of hepatocarcinogenesis in fibrotic livers.
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Carcinoma Hepatocelular/etiología , Cirrosis Hepática Experimental/complicaciones , Neoplasias Hepáticas Experimentales/etiología , Neoplasias Hepáticas/etiología , Animales , Tetracloruro de Carbono , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/metabolismo , Modelos Animales de Enfermedad , Genes myc/genética , Genes p53 , Células Estrelladas Hepáticas/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Ratones Transgénicos , ARN Interferente Pequeño , Transposasas/metabolismoRESUMEN
Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a tumor suppressor under a low-glucose condition.
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Proteínas Quinasas Activadas por AMP/metabolismo , Carbohidratos de la Dieta/administración & dosificación , Suplementos Dietéticos , Neoplasias Hepáticas Experimentales/prevención & control , Melanoma Experimental/prevención & control , Fitoquímicos/administración & dosificación , Sirtuina 1/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Carbohidratos de la Dieta/farmacología , Sinergismo Farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/mortalidad , Neoplasias Hepáticas Experimentales/patología , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/mortalidad , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Fitoquímicos/farmacocinética , Transducción de Señal , Sirtuina 1/genética , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: Liver fibrosis is an increasing health concern worldwide and a major risk factor for hepatocellular carcinoma (HCC). Although the involvement of Hedgehog signaling in hepatic fibrosis has been known for some time, the causative role of activated Hedgehog signaling in liver fibrosis has not been verified in vivo. METHODS: Using hydrodynamics-based transfection, a transgenic mouse model has been developed that expresses Sonic Hedgehog (SHH), a ligand for Hedgehog signaling, in the liver. Levels of hepatic fibrosis and fibrosis-related gene expression were assessed in the model. Hepatic expression of SHH was induced in a murine model for hepatocellular adenoma (HCA) and tumor development was subsequently investigated. RESULTS: The transgenic mice revealed SHH expression in 2-5% of hepatocytes. Secreted SHH activated Hedgehog signaling in numerous cells of various types in the tissues. Hepatic expression of SHH led to fibrosis, activation of hepatic stellate cells, and an upregulation of various fibrogenic genes. Liver injury and hepatocyte apoptosis were observed in SHH mice. Persistent expression of SHH for up to 13months failed to induce tumors in the liver; however, it promoted liver tumor development induced by other oncogenes. By employing a HCA model induced by P53(R172H) and KRAS(G12D), we found that the SHH expression promoted the transition from HCA to HCC. CONCLUSIONS: SHH expression in the liver induces liver fibrosis with concurrent activation of hepatic stellate cells and fibrogenic genes. It can also enhance hepatocarcinogenesis induced by other oncogenes.
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Proteínas Hedgehog/fisiología , Cirrosis Hepática Experimental/etiología , Neoplasias Hepáticas Experimentales/etiología , Animales , Apoptosis , Transición Epitelial-Mesenquimal , Proteínas Hedgehog/análisis , Cirrosis Hepática Experimental/patología , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
AIM: Hepatocellular carcinoma (HCC), one of the most common malignancies in adults displays aberrant miRNA expression during its pathogenesis. We assessed expression of miRNA in surgically resected human HCC of an early stage and murine HCC with a high malignancy in order to find miRNA overexpressed in HCC regardless of tumor stage and underlying etiology. Further, the role of the deregulated miRNA in HCC pathogenesis was investigated. METHODS: miRNA were isolated from HCC tissues and surrounding non-tumorous tissues from HCC patients and a murine transgenic model of HCC. A quantitative reverse transcription polymerase chain reaction was performed to determine expression levels of miRNA. Human HCC cell lines stably expressing individual miRNA were generated to investigate the biological function of overexpressed miRNA. RESULTS: We found that levels of miR-221, -181b-1, -155-5p, -25 and -17-5p were significantly upregulated in both human and murine HCC regardless of tumor stage, underlying etiology or the presence of fibrosis. Using HCC cell lines stably expressing respective miRNA, we found that miR-221 increased the proliferation of hepatoma cells, while miR-17-5p induced cell migration. CONCLUSION: We identified miRNA that are consistently upregulated in HCC. The overexpressed miRNA could potentially be used as a bona fide biomarker for HCC.
RESUMEN
Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).
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Proteínas Fluorescentes Verdes/metabolismo , Neoplasias Hepáticas/patología , Luciferasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Luciferasas/genética , Mediciones Luminiscentes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Sistemas de Lectura Abierta/genética , Imagen Óptica , Proteínas Proto-Oncogénicas p21(ras)/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Piel/metabolismo , Piel/patología , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
UNLABELLED: We sought to determine the hepatic fibrosis-reversal effects upon simultaneous administration of lithospermate B (LAB), an anti-oxidant, and nivocasan, a caspase inhibitor, to rats compared with each compound alone. Liver fibrosis was induced in Sprague-Dawley rats by thioacetamide (TAA). Rats were treated with TAA and then given LAB and (or) nivocasan. Fibrotic areas were evaluated quantitatively by computerized morphometry. Apoptosis was assessed using a TUNEL assay, and immunohistochemical staining for malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4HNE) was performed to assess oxidative stress levels. Real-time quantitative PCR was used to quantify expression of fibrosis-related genes. The degree of hepatic fibrosis was significantly reduced in rats treated with LAB and nivocasan compared to either treatment alone (P < 0.001). Treatment with each compound significantly decreased expression of fibrosis-related genes, such as type I collagen α1 (col1α1), α-SMA and TGF-ß1 (P < 0.05). Co-treatment with LAB and nivocasan further reduced col1α1 expression compared to treatment with either compound. A TUNEL assay revealed that hepatocyte apoptosis was significantly decreased in the group treated with nivocasan compared to other groups (P < 0.01). Immunohistochemistry showed a decrease in MDA and 4HNE, reflecting amelioration of oxidative stress, when LAB or LAB+nivocasan was administered compared to nivocasan alone (P < 0.01). Nivocasan was found to inhibit caspase-1, -3, -7, -9 and gliotoxin-induced death of rat-derived hepatic stellate cells was inhibited by nivocasan administration without overexpression of α-SMA. CONCLUSIONS: Co-incidental administration of LAB and nivocasan suppressed oxidative stress and apoptosis, resulting in enhanced reversal of hepatic fibrosis in rat.
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Antioxidantes/administración & dosificación , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas/administración & dosificación , Medicamentos Herbarios Chinos/administración & dosificación , Cirrosis Hepática/tratamiento farmacológico , Animales , Caspasas/genética , Caspasas/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Quimioterapia Combinada , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/fisiopatología , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Liver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. Here, we report a simple methodology by which oncogenic cooperation between cancer-related genes can be efficiently investigated in the liver. We developed various non-germline transgenic mouse models using hydrodynamics-based transfection which express HrasG12V, SmoM2, and a short-hairpin RNA down-regulating p53 (shp53) individually or in combination in the liver. In this transgenic system, firefly luciferase was co-expressed with the oncogenes as a reporter, allowing tumor growth in the liver to be monitored over time without an invasive procedure. Very strong bioluminescence imaging (BLI) signals were observed at 4 weeks post-hydrodynamic injection (PHI) in mice co-expressing HrasG12V and shp53, while only background signals were detected in other double or single transgenic groups until 30 weeks PHI. Consistent with the BLI data, tumors were observed in the HrasG12V plus shp53 group at 4 weeks PHI, while other transgenic groups failed to exhibit a hyperplastic nodule at 30 weeks PHI. In the HrasG12V plus shp53 transgenic group, BLI signals were well-correlated with actual tumor growth in the liver, confirming the versatility of BLI-based monitoring of tumor growth in this organ. The methodology described here is expected to accelerate and facilitate in vivo studies of the hepatocarcinogenic potential of cancer-related genes by means of oncogenic cooperation.
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Carcinoma Hepatocelular/metabolismo , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Imagen de Cuerpo Entero , Animales , Hidrodinámica , Neoplasias Hepáticas/metabolismo , Luciferasas , Luminiscencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Células 3T3 NIH , Oncogenes , Plásmidos , Factores de Tiempo , TransfecciónRESUMEN
Magnesium lithospermate B (MLB) is one of the major active components of Salvia miltiorrhizae. The anti-oxidative effects of Salvia miltiorrhizae have been previously reported. The aim of this study was to investigate the effect of purified MLB on hepatic fibrosis in rats and on the fibrogenic responses in hepatic stellate cells (HSCs). Hepatic fibrosis was induced in rats by intraperitoneal thioacetamide (TAA) injections over a period of 8 or 12 weeks. MLB was orally administered daily by gavage tube. Serum AST and ALT levels in TAA+ MLB group were significantly lower than those in TAA only group at week 8. Hepatic fibrosis was significantly attenuated in TAA+MLB group than in TAA only group at week 8 or 12. Activation of HSCs was also decreased in TAA+MLB group as compared to TAA only group. Hepatic mRNA expression of α-smooth muscle actin (α-SMA), TGF-ß1, and collagen α1(I) was significantly decreased in TAA+MLB group as compared to TAA only group. Incubation with HSCs and MLB (>or=100 µM) for up to 48 h showed no cytotoxicity. MLB suppressed PDGF-induced HSC proliferation. MLB inhibited NF-ΚB transcriptional activation and monocyte chemotactic protein 1 (MCP-1) production in HSCs. MLB strongly suppressed H(2)O(2)-induced reactive oxygen species (ROS) generation in HSCs, and MLB inhibited type I collagen secretion in HSCs. We concluded that MLB has potent antifibrotic effect in TAA-treated cirrhotic rats, and inhibits fibrogenic responses in HSCs. These data suggest that MLB has potential as a novel therapy for hepatic fibrosis.
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Antioxidantes/administración & dosificación , Medicamentos Herbarios Chinos/administración & dosificación , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática Experimental/tratamiento farmacológico , Hígado/efectos de los fármacos , Actinas/genética , Actinas/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibrosis/prevención & control , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/fisiopatología , Masculino , FN-kappa B/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Salvia miltiorrhiza/inmunología , Tioacetamida/administración & dosificación , Activación Transcripcional/efectos de los fármacosRESUMEN
Radiation therapy (RT) has been emerging as one of the palliative treatments for locally advanced hepatocellular carcinoma (HCC). However, hepatic toxicity is a major obstacle in radiotherapy for HCC. The purpose of this study is to identify proteins indicating radiation-induced hepatic toxicity in cirrhotic rats, which can be used as possible biomarkers. Liver cirrhosis was induced in Wistar rats with thioacetamide (TAA) 0.3 g/L in drinking water for 9 weeks. The development of liver cirrhosis was observed histologically. Radiation hepatic injury was induced by treating 1/3 of the liver with 10 Gy single dose radiation. To find out commonly expressed proteins, liver tissue and serum were analyzed using two-dimensional electrophoresis and quadrupole time of flight mass spectrometry. Identified proteins were validated using western blotting. Histological examination showed that the degree of hepatic fibrosis increased by radiation in liver cirrhosis. It was associated with a decrease in the proliferation of cell nuclear antigen and an increase of apoptosis. The proteomic analysis of liver tissue and serum identified 60 proteins which showed significant change in expression between the TAA-alone and TAA-plus-radiation groups. Among these, an increase of heparanase precursor and decrease of hepatocyte growth factor were shown commonly in liver tissue and serum following radiation. Hepatic fibrosis increased following radiation in cirrhotic rats. These proteins might be useful in detecting and monitoring radiation-induced hepatic injury.
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Cirrosis Hepática Experimental/complicaciones , Cirrosis Hepática Experimental/metabolismo , Proteínas/metabolismo , Traumatismos Experimentales por Radiación/complicaciones , Traumatismos Experimentales por Radiación/metabolismo , Animales , Biomarcadores/metabolismo , Precursores Enzimáticos/metabolismo , Glucuronidasa/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Hígado/lesiones , Hígado/metabolismo , Hígado/efectos de la radiación , Cirrosis Hepática Experimental/patología , Masculino , Proteómica , Traumatismos Experimentales por Radiación/patología , Tolerancia a Radiación , Ratas , Ratas WistarRESUMEN
BACKGROUND/AIMS: Hepatitis B virus (HBV) induces inflammatory signaling leading to progressive liver damage. Polymorphisms of the toll-like receptor (TLR) 2 (Arg677Trp, Arg753Gln) and TLR4 (Asp299Gly, Thr399Ile) genes, which are important components of innate immunity against viral infection, have recently been described. We evaluated the association between TLR2 and TLR4 polymorphisms and the development of liver cirrhosis in Koreans with chronic HBV infection. METHODOLOGY: This study enrolled 456 Koreans with chronic HBV infection between December 2004 and October 2007; 242 with chronic hepatitis B (group I) and 214 with liver cirrhosis (group II). TLR2 and TLR4 polymorphisms were determined using direct sequencing. RESULTS: Mean age differed significantly between groups (group I, 34.8 +/- 11.4 years; group II 51.0 +/- 8.9 years; p < 0.001), whereas the proportion of males (80.2% vs. 73.4%, respectively; p = 0.085) and hepatitis B e antigen-positive patients (40.7% vs. 43.6%, respectively; p = 0.575) did not. The serum alanine aminotransferase level was significantly higher in group I (87.9 +/- 138.5 IU/L) than in group II (56.6 +/- 70.7 IU/L, p = 0.003). However, the TLR2 Arg677Trp and Arg753Gln and TLR4 Asp299Gly and Thr399Ile mutant alleles were not detected in any patients. CONCLUSIONS: The TLR2 Arg677Trp, Arg753Gln and TLR4 Asp299Gly, Thr399Ile mutant alleles were not detected in any patient, suggesting that they are very rare in the Korean population. Our results do not permit any conclusion regarding their role in the development of liver cirrhosis.