RESUMO
The objective was to investigate endocrine-disrupting effects of polar compounds from oxidized frying oil. Estrogenicity of polar compounds was tested with a rat uterotrophic bioassay. Dietary oxidized frying oil (containing 51% polar compounds) or polar compounds isolated from it were incorporated into feed (in lieu of fresh soybean oil) and fed to ovariectomized rats, with or without treatment with exogenous ethynyl estradiol. Exogenous estrogen restored uterine weight, and caused histological abnormalities (stratified epithelia and conglomerate glands) as well as proliferation of uterine epithelial cells. However, tamoxifen or polar compounds reduced these effects. Furthermore, tamoxifen or polar compounds down-regulated uterine mRNA expression of estrogen receptor (ER)-target genes, implicating reduced ER activity in this hypo-uterotrophic effect. Inhibition of ER signaling and mitosis by polar compounds were attributed to reduced MAPK and AKT activation, as well as a reduced ligand binding domain-transactivity of ERα/ß. We concluded polar compounds from frying oil are potential endocrine-disrupting chemicals, with implications for food and environmental safety.
Assuntos
Disruptores Endócrinos/toxicidade , Antagonistas de Estrogênios/toxicidade , Animais , Culinária , Dieta , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Feminino , Oxirredução , Ratos , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Óleo de Soja , Tamoxifeno/toxicidade , Útero/efeitos dos fármacos , Útero/metabolismo , Útero/patologiaRESUMO
BACKGROUND: Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in almost patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Activation of hepatic stellate cells (HSCs) is the prevailing process during hepatic fibrosis. Osthole is an active component majorly contained in the fruit of Cnidium monnieri (L.) Cusson. This present study investigated the therapeutic effects of osthole on rat liver fibrosis and HSC activation. RESULTS: We established the thioacetamide (TAA)-model of Sprague-Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and α-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects in inflammation-related genes and chemokines production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole attenuated TGF-ß1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly. Besides, osthole alleviated TGF-ß1- or ET-1-induced HSCs contractility. CONCLUSIONS: Our study demonstrated that osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats. In addition, osthole suppressed HSCs activation in vitro significantly.
Assuntos
Cumarínicos/farmacologia , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Actinas/metabolismo , Animais , Citocinas/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tioacetamida/toxicidadeRESUMO
BACKGROUND: Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu). METHODS: Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 (phox) subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-ß1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes. RESULTS: In vitro, Flu (1-20 µM) inhibited PA-induced free-radical production, gp91 (phox) expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats. CONCLUSIONS: We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.
Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Células Estreladas do Fígado/fisiologia , Hepatócitos/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Indóis/farmacologia , Cirrose Hepática/prevenção & controle , Comunicação Parácrina/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Animais , Colina/administração & dosagem , Colágeno Tipo I/genética , Meios de Cultivo Condicionados/farmacologia , Dieta , Ácidos Graxos Monoinsaturados/uso terapêutico , Fluvastatina , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Indóis/uso terapêutico , Molécula 1 de Adesão Intercelular/genética , Interleucina-6/genética , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Masculino , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) are involved in acute myocardial dysfunction by degrading several intracellular contractile proteins, including cardiac troponin I (cTnI). Here, we examined the temporal profiles of MMPs and cTnI in plasma and myocardial tissue in the acute stage of subarachnoid hemorrhage (SAH). MATERIALS AND METHODS: SAH was induced by the endovascular suture method in rats. Intracranial pressure and left ventricular (LV) function were recorded. Plasma cTnI and MMPs were measured at 0, 5, 15, 30, 60, 120, and 180 minutes after SAH. Myocardial cTnI and MMP activities were quantified at 30, 60 and 180 min after SAH from homogenized hearts. RESULTS: SAH-induced rats showed a marked decline in -LV dP/dt(max) (index of LV diastolic function). Plasma samples revealed a noticeable increase in cTnI and pro-MMP-9 activities over the course of 180 minutes. In myocardial tissue, there was a marked increase in pro-MMP-9, pro-MMP-2 activities and expression of activated MMP-2. Western blot analysis revealed a striking decrease in cTnI content and increase in cTnI degradation in myocardium. Simultaneous cTnI depletion and MMP-2 expression in myocardium was detected by immunohistochemistry as early as 30 minutes after SAH. MMPs correlated with -LV dP/dt(max) (% of baseline) both in plasma and in myocardial tissue. Furthermore, activated MMP-2 activity correlated positively with cTnI degradation in myocardium. CONCLUSIONS: Early activation of MMPs was observed in myocardium and plasma following SAH. Activated MMP-2 may regulate proteolytic cTnI and contribute to myocardium stunning injury in SAH rats.
Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Miocárdio/metabolismo , Hemorragia Subaracnóidea/metabolismo , Troponina I/metabolismo , Animais , Modelos Animais de Doenças , Pressão Intracraniana/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/fisiopatologia , Fatores de Tempo , Função Ventricular/fisiologiaRESUMO
The aim of this study was to investigate if armepavine (Arm, C19H23O3N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1-10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H2O2 production; and (iii) NF-κB, JunD and C/EBPß (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-ß (EBPß)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-ß1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPß pathways.
Assuntos
Benzilisoquinolinas/uso terapêutico , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Fitoterapia , Tioacetamida/efeitos adversos , Actinas/genética , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Alanina Transaminase/sangue , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Animais , Aspartato Aminotransferases/sangue , Benzilisoquinolinas/administração & dosagem , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Colágeno Tipo I/metabolismo , Citocinas/genética , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Imunofluorescência , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Peróxido de Hidrogênio/metabolismo , Cirrose Hepática/patologia , Masculino , Metalotioneína/genética , Metalotioneína/metabolismo , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Tioacetamida/administração & dosagem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Bronchiolitis obliterans organizing pneumonia (BOOP) is a chronic respiratory disease. Although the pathogenesis of BOOP is still incompletely understood, BOOP is responsive to steroids and has a good prognosis. In our five pigs with chronic postweaning multisystemic wasting syndrome (PMWS), typical BOOP lesions were revealed. All five porcine lungs showed typical intraluminal plugs, and porcine circovirus type 2 (PCV2) was identified. They also exhibited similar pathologic findings such as proliferation of type II pneumocytes and myofibroblasts (MFBs), extracellular collagen matrix (ECM) deposition, and fragmentation of elastic fibers. MFBs migration correlative molecules, for instance, gelatinase A, B and osteopontin, appeared strongly in the progressing marginal area of polypoid intraluminal plugs of fibrotic lesion. These molecules colocalized with the active MFBs. Both gelatinase activity and intercellular level of active MFBs were significantly increased (P < .05). Porcine chronic bronchopneumonia leads to BOOP and it is associated with PCV2 persistent infection. Swine BOOP demonstrates similar cellular constituents with human BOOP. Perhaps their molecular mechanisms of pathogenesis operate in a similar way. Thus we infer that the swine BOOP can be considered as a potential animal model for human BOOP associated with natural viral infection. Moreover, it is more convenient to obtain samples.
Assuntos
Infecções por Circoviridae/veterinária , Circovirus , Pneumonia em Organização Criptogênica/etiologia , Pneumonia em Organização Criptogênica/patologia , Doenças dos Suínos/virologia , Síndrome de Emaciação/veterinária , Actinas/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Movimento Celular , Infecções por Circoviridae/etiologia , Infecções por Circoviridae/patologia , Pneumonia em Organização Criptogênica/veterinária , Modelos Animais de Doenças , Humanos , Pulmão/enzimologia , Pulmão/patologia , Pulmão/virologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Osteopontina/metabolismo , Suínos , Doenças dos Suínos/patologia , Síndrome de Emaciação/complicações , Síndrome de Emaciação/patologiaRESUMO
Triptolide (C38H42O6N2, TP, a diterpene triepoxide derived from Tripterygium wilfordii Hook F.), is a potent immunosuppresive and antiinflammatory agent. The present study investigated whether TP exerted antihepatofibrotic effects in vitro and in vivo. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumor necrosis factor-α (TNF-α) or transforming growth factor (TGF)-ß1. The inhibitory effects of TP on the nuclear factor-κB (NFκB) signaling cascade and fibrosis markers, including α-smooth muscle actin (α-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats. The rats were randomly assigned to one of three groups: control rats, DMN rats receiving vehicle only and DMN rats receiving TP (20 µg/kg). Treatment was given by gavage twice daily for 3 weeks starting 1 week after the start of DMN administration. TP (5-100 nM) concentration-dependently inhibited the NFκB transcriptional activity induced by TNF-α, lipopolysaccharide and phorbol 12-myristate 13-acetate in HSC-T6 cells. In addition, TP also suppressed TNF-α and TGF-ß1-induced collagen deposition and α-SMA secretion in HSC-T6 cells. In vivo, TP treatment significantly reduced hepatic fibrosis scores, collagen contents, IL-6 and TNF-α levels, and the number of α-SMA and NFκB-positive cells in DMN rats. The results showed that TP exerted antifibrotic effects in both HSC-T6 cells and DMN rats.
Assuntos
Diterpenos/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática Experimental/tratamento farmacológico , Fenantrenos/farmacologia , Actinas/metabolismo , Animais , Linhagem Celular , Colágeno/metabolismo , Dimetilnitrosamina/efeitos adversos , Compostos de Epóxi/farmacologia , Interleucina-6/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Experimental/patologia , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/farmacologia , Tripterygium/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The protective effect of combination therapy with valsartan and aliskiren against renal fibrosis remains to be defined. This study was undertaken to examine the protective effects of the combination of valsartan and aliskiren against renal fibrosis induced by unilateral ureteral obstruction (UUO). Combination therapy with valsartan (15 mg·kg(-1)·day(-1)) and aliskiren (10 mg·kg(-1)·day(-1)), valsartan monotherapy (30 mg·kg(-1)·day(-1)), and aliskiren monotherapy (20 mg·kg(-1)·day(-1)) all significantly ameliorated the increase in blood urea nitrogen and the degree of hydronephrosis determined by the increase in weight and length of the obstructed kidney. The dose titration study and blood pressure measurement confirmed that the combination therapy provided a greater benefit independent of the vasodilatory effect. There were no significant changes in serum levels of creatinine, sodium, and potassium in UUO rats and any treatment groups. Combination therapy also attenuated UUO-related increases in the scores of tubular dilatation, interstitial volume, interstitial collagen deposition, α-smooth muscle actin, the activation of ERK 1/2, the infiltration of monocytes/macrophages, the mRNA expression of snail-1, and transforming growth factor-ß1 to a greater extent compared with aliskiren or valsartan used alone. The mRNA expression of renin and the (pro)renin receptor significantly increased after UUO. Combination therapy and monotherapy of valsartan and aliskiren had a comparable enhancing effect on the mRNA expression of renin, whereas all these treatments did not affect the expression of the (pro)renin receptor. In conclusion, a direct renin inhibitor in conjunction with an angiotensin II receptor blocker exerts increased renal protection against renal fibrosis and inflammation during obstruction over either agent alone.
Assuntos
Amidas/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Fumaratos/uso terapêutico , Nefropatias/tratamento farmacológico , Nefropatias/etiologia , Tetrazóis/uso terapêutico , Obstrução Ureteral/complicações , Obstrução Ureteral/tratamento farmacológico , Valina/análogos & derivados , Actinas/biossíntese , Anatomia Transversal , Animais , Pressão Sanguínea/fisiologia , Western Blotting , Colágeno/metabolismo , Quimioterapia Combinada , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrose , Imuno-Histoquímica , Rim/patologia , Rim/fisiopatologia , Nefropatias/genética , Testes de Função Renal , Masculino , Infiltração de Neutrófilos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/biossíntese , Obstrução Ureteral/genética , Valina/uso terapêutico , ValsartanaRESUMO
Although Trypanosoma (Megatrypanum) theileri, a blood parasite of bovid species, is spread widely throughout the world, it has never been reported in Taiwan. When an anti-coagulated blood sample from febrile dairy cattle was directly smeared, no parasite was observed. However, a highly distinctive morphological feature of trypanosome appeared in baby hamster kidney (BHK) cell culture inoculated with non-thrown blood buffy coat. The different stages and typical ultrastructures of trypanosome were observed in our isolate. The isolate was subsequently identified as T. theileri by species-specific PCR assay (Tth625), 18S rDNA sequencing alignment and internal transcribed spacer of ribosomal genes (ITS) as a marker for molecular phylogenetic analysis. The first T. theileri isolate in Taiwan (TWTth1) could be periodically passaged in BHK cell culture for more than one year and retained good re-cryopreservation viability. The BHK culture method would be excellent for diagnostic isolation and maintenance long-term development of this parasite.
Assuntos
Bovinos/parasitologia , Trypanosoma/isolamento & purificação , Animais , Sangue/parasitologia , Cricetinae , Primers do DNA , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/genética , Indústria de Laticínios , Feminino , Rim/citologia , Rim/parasitologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Alinhamento de Sequência , Taiwan , Trypanosoma/genética , Trypanosoma/crescimento & desenvolvimento , Trypanosoma/ultraestruturaRESUMO
Activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrogenesis. armepavine (Arm, C19H23O3N), an active compound from Nelumbo nucifera, has been shown to exert immunosuppressive effects on T lymphocytes and on lupus nephritic mice. The aim of this study was to investigate whether Arm could exert anti-hepatic fibrogenic effects in vitro and in vivo. A cell line of rat HSCs (HSC-T6) was stimulated with tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) to evaluate the inhibitory effects of Arm. An in vivo therapeutic study was conducted in bile duct-ligated (BDL) rats. BDL rats were given Arm (3 or 10 mg/kg) by gavage twice daily for 3 weeks starting from the onset of BDL. Liver sections were taken for fibrosis scoring, immuno-fluorescence staining and quantitative real-time mRNA measurements. In vitro, Arm (1-10 microM) concentration-dependently attenuated TNF-alpha- and LPS-stimulated alpha-SMA protein expression and AP-1 activation by HSC-T6 cells without adverse cytotoxicity. Arm also suppressed TNF-alpha-induced collagen collagen deposition, NFkappaB activation and MAPK (p38, ERK1/2, and JNK) phosphorylations. In vivo, Arm treatment significantly reduced plasma AST and ALT levels, hepatic alpha-SMA expression and collagen contents, and fibrosis scores of BDL rats as compared with vehicle treatment. Moreover, Arm attenuated the mRNA expression levels of col 1alpha2, TGF-beta1, TIMP-1, ICAM-1, iNOS, and IL-6 genes, but up-regulated metallothionein genes. Our study results showed that Arm exerted both in vitro and in vivo antifibrotic effects in rats, possibly through anti-NF-kappaB activation pathways.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Benzilisoquinolinas/uso terapêutico , Células Estreladas do Fígado/efeitos dos fármacos , Imunossupressores/uso terapêutico , Cirrose Hepática Biliar/tratamento farmacológico , Cirrose Hepática Experimental/tratamento farmacológico , Fitoterapia , Actinas/biossíntese , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/toxicidade , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/toxicidade , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/ultraestrutura , Colágeno/biossíntese , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/ultraestrutura , Imunossupressores/farmacologia , Imunossupressores/toxicidade , Lipopolissacarídeos/farmacologia , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/patologia , Cirrose Hepática Experimental/sangue , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , NF-kappa B/metabolismo , Nelumbo/química , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Ventricular fibrillation (VF) during prolonged (>5 min) global ischemia (GI) could be due to repetitive endocardial focal discharges (REFDs). This hypothesis was tested in isolated rabbit hearts. METHODS AND RESULTS: With optical mapping, simultaneous endocardial (left ventricle, LV) and epicardial (both ventricles) activations during VF with prolonged GI were studied (protocol I, 8 hearts). Lugol solution was applied to the LV endocardium in additional 5 hearts after 5-min GI (protocol II). During prolonged GI, sustained VF (>30 s) was successfully induced in 7 protocol I hearts. The dominant frequency of summed optical signals at the LV endocardium was higher than at the epicardium (P<0.05). Mapping data showed that after 5-min GI, REFDs were present in >90% for recording time. There were 18 windows of optical recording showing spontaneous VF termination. In 10, once REFDs ceased, the VF episode terminated immediately. Electrical defibrillation was also performed on 3 hearts. Eight shocks showed early VF recurrence after successful defibrillation. REFDs were consistently involved in the initiation period of recurrence. In protocol II, Lugol subendocardial ablation diminished REFD genesis during re-induced VF. These VF episodes were all non-sustained. CONCLUSIONS: REFDs at the LV endocardium were important for both VF maintenance and post-shock recurrence during prolonged GI in this model.
Assuntos
Endocárdio/fisiopatologia , Isquemia Miocárdica/complicações , Ramos Subendocárdicos/fisiopatologia , Fibrilação Ventricular/etiologia , Potenciais de Ação , Animais , Estimulação Cardíaca Artificial , Cardioversão Elétrica , Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Endocárdio/efeitos dos fármacos , Análise de Fourier , Técnicas In Vitro , Iodetos/farmacologia , Isquemia Miocárdica/fisiopatologia , Perfusão , Pericárdio/fisiopatologia , Ramos Subendocárdicos/efeitos dos fármacos , Coelhos , Recidiva , Processamento de Sinais Assistido por Computador , Fatores de Tempo , Fibrilação Ventricular/fisiopatologia , Fibrilação Ventricular/terapiaRESUMO
Nitric oxide (NO) or glutamate stimulation of dorsal facial area (DFA) increases blood flow in the common carotid artery (CCA), which supplies intra-and extra-cranial tissues. Nitrergic fibers and neurons as well as preganglionic cholinergic neurons are present in the DFA. We hypothesized the presence of nitrergic-glutamatergic fibers and preganglionic nitrergic-cholinergic neurons in the DFA that are involved in the regulation of CCA blood flow. In microdialysis studies, perfusion of the DFA with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) increased the glutamate concentration in the dialysate. This effect was abolished by co-perfusion of methylene blue (a guanylyl cyclase inhibitor). Intra-DFA injection of l-arginine (an NO precursor) or glutamate increased CCA blood flow. The l-arginine-induced flow increase was reduced by prior administration of NG-nitro-arginine methyl ester (l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor), d-2-amino-5-phosphonopentanoate (d-AP5, a competitive NMDA receptor antagonist), or glutamate diethylester (GDEE, a competitive AMPA receptor antagonist). The glutamate-induced blood flow increase was reduced by prior administration of l-NAME, 7-NI, or methylene blue. The induced increase in CCA blood flow, however, was not affected by endothelial NO synthase inhibitor. The findings indicate that NO-signal transduction within the DFA might cause glutamate release from presynaptic nitrergic-glutamatergic fibres and that the released glutamate activates NMDA/AMPA receptors on preganglionic nitrergic-cholinergic neurons in the nucleus to activate neuronal NO synthase and guanylyl cyclase in the neurons, leading to an increase in CCA blood flow. These findings may be important for developing therapeutic strategies for the diseases associated with CCA blood flow.
Assuntos
Artéria Carótida Primitiva/fisiologia , Nervo Facial/fisiologia , Ácido Glutâmico/fisiologia , Óxido Nítrico/fisiologia , Animais , Arginina/farmacologia , Artéria Carótida Primitiva/inervação , Gatos , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Feminino , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Indazóis/farmacologia , Masculino , Azul de Metileno/farmacologia , Microdiálise , Microinjeções , NG-Nitroarginina Metil Éster/farmacologia , Fibras Nervosas/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Fluxo Sanguíneo Regional/fisiologia , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
Sympathetic hyperactivation in many kinds of neurocardiogenic injury can result in obvious heart failure. We generated a vagotomized feline model in which sympathetic hyperactivation was induced by electrical stimulation of dorsal medulla (ESDM) of brain stem to investigate the relationship between disruption of extracellular collagen matrix (ECM) and activation of matrix metalloproteinases (MMPs) in myocardium in the sympathetic hyperactivity. Mean blood pressure, heart rate and plasma norepinephrine were all significantly increased from baseline to a peak at 5 min after ESDM. Echocardiographic study showed significant left ventricular dilatation and hypokinesia (ejection fraction: from 87.7 +/- 6.3% to 39.4 +/- 7.8%) from baseline to 180 mm after ESDM. Histopathological finding revealed significant overstretching or spring-like disappearance and disruption of ECM. MMP-2 expression was significantly increased in left ventricular myocardium as compared to sham. These results suggest that ESDM-induced sympathetic hyperactivity causes the expression of MMP-2 that disrupts myocardial ECM, contributing to the development of cardiac dysfunction.
Assuntos
Colágeno/metabolismo , Metaloproteinases da Matriz/fisiologia , Bulbo/fisiologia , Miocárdio/metabolismo , Sistema Nervoso Simpático/fisiologia , Vagotomia , Animais , Gatos , Estimulação Elétrica , Modelos AnimaisRESUMO
Glutamate stimulation of the dorsal facial area, an area located dorsal to the facial nucleus, increases common carotid arterial blood flow. Nitrergic neurons are important in cardiovascular regulatory areas. We investigated whether the nitrergic neurons might be present and play a role in the dorsal facial area to regulate the arterial blood flow. Injections of L-arginine (an NO precursor) and sodium nitroprusside (an NO donor) into the area caused dose-dependent increases in the arterial blood flow. Injection of N(G)-nitro-arginine methyl ester (L-NAME, an NO synthase inhibitor) or methylene blue (a guanylate cyclase inhibitor) decreased the arterial blood flow. Nitrergic neurons and fibers were found in the dorsal facial area by histochemical staining of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase, a maker of NO synthase. In conclusion, nitrergic neurons are present in the dorsal facial area and appear to release NO tonically in stimulating the area to cause increase in common carotid arterial blood flow.
Assuntos
Artéria Carótida Primitiva/fisiologia , Bulbo/fisiologia , Neurônios Nitrérgicos/fisiologia , Animais , Arginina/farmacologia , Gatos , Feminino , Masculino , Bulbo/citologia , Microinjeções , NADP/metabolismo , NADPH Desidrogenase/antagonistas & inibidores , NADPH Desidrogenase/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Fluxo Sanguíneo RegionalRESUMO
Spinal muscular atrophy (SMA) is the most common genetic motoneuron degenerative disorder, but the mechanism(s) of motoneuron death is unclear. Previously, a direct interaction between tumor-suppressive TP53 protein and the SMA determinant gene product, survival motor neuron protein, was identified and therefore it has been suggested that a mechanism of TP53-dependent apoptosis plays an important role in motoneuron degeneration in SMA. We used our SMA model mice, generated by a combination of knockout and transgenic techniques, to decipher the role of TP53 protein in the motoneuron degeneration in SMA. We detected a significant increase of Trp53 expression in the spinal cord of SMA-like mice compared to their normal littermates. After crossing SMA-like mice with Trp53 knockout mice, the progeny Trp53-deficient SMA-like mice did not show milder disease severity or longer lifespan compared to SMA littermates with wild-type Trp53 genes. Our studies provide in vivo evidence indicating that Trp53-dependent apoptosis does not play a crucial role in motoneuron degeneration in SMA-like mice. European Journal of Human Genetics (2006) 14, 372-375. doi:10.1038/sj.ejhg.5201556; published online 4 January 2006.
Assuntos
Apoptose , Genes p53 , Atrofia Muscular Espinal/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genótipo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios Motores/patologia , Doenças Neurodegenerativas/patologia , Fenótipo , Medula Espinal/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: Amniotic fluid mesenchymal stem cells (MSCs) have the potential to differentiate into neuronal stem cells in vitro. We evaluated using amniotic fluid MSCs to support or enhance the ability of the injured sciatic nerve to cross a nerve gap. MATERIALS AND METHODS: We created a 5 mm nerve defect in Sprague Dawley rats. One group received therapy with MSCs embedded into woven oxidised regenerated cellulose gauze (Surgical; Ethicon, Somerville, NJ) and fibrin glue, while a control group received woven Surgicel and fibrin glue only. Evaluation methods included behavioural, electrophysiological and immunohistochemical studies. RESULTS: In gait analysis, the angle of the ankles in the treatment and control group were 46.4 degrees (standard deviation [SD]=15 degrees) and 36 degrees (SD=8.2 degrees), respectively, which was statistically significant (p=0.045). Five of 10 treated rats (50%) demonstrated partial foot movement, while none of the control group had any movement. The percentage amplitude of muscle compound action potential in the experimental group was 43% (SD=12.5%) compared to 29% (SD=8.8%) in the control group (p=0.038). The conduction latencies in the control and experimental groups was 2.5 ms (SD=0.45) and 1.7 ms (SD=0.47), respectively (p=0.005). Histological examination demonstrated that 70% of the treatment group achieved a maximum axon diameter percentage across the nerve gap of greater than 50%, compared with 0% in the control group. There were no differences in direction of fibre growth and fibrotic reaction between the two groups. CONCLUSION: Amniotic fluid MSC can augment growth of injured nerve across a nerve gap. This effect may be due to neurotrophic or induction effects of the MSC interacting with Schwann cells. Further study is required to determine the underlying mechanism of this effect.
Assuntos
Âmnio/transplante , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa/fisiologia , Neuropatia Ciática/cirurgia , Transplante de Células-Tronco/métodos , Âmnio/citologia , Animais , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Ratos , Ratos Sprague-Dawley , Neuropatia Ciática/patologiaRESUMO
BACKGROUND: Intravenous norepinephrine (NE) at a dose of 1-6 microg/kg/minute can induce increased extracellular matrix (ECM) and hypertrophic cardiomyopathy. This study aimed to investigate the effects of a higher dose of NE on cardiac remodeling. METHODS: After intraperitoneal urethane-chloralose anesthesia, 7 cats (3.03 +/- 0.58 kg) received intravenous infusion of NE 30 microg/kg/minute for 3 hours. Aortic blood pressure and heart rate (HR) were measured by polygraphy at 0, 5, 15, 30, 60, 90, 120, and 180 minutes. Left ventricular size and ejection fraction (EF) were measured by M-mode echocardiography before and after NE administration. Histopathology was performed by hematoxylin-eosin, silver impregnation, and Sirius red staining. Activity of matrix metalloproteinases (MMP) in the left ventricle was measured by zymography. RESULTS: Mean blood pressure (mmHg) increased from 139 +/- 20 to 198 +/- 19, 187 +/- 23, and 166 +/- 16 at 15, 30, and 60 minutes, respectively, during NE infusion. HR (beats/minute) decreased from 214 +/- 10 to 158 +/- 28 at 15 minutes and then recovered gradually. The left ventricles showed significant dilatation (end-diastolic diameter: from 1.20 +/- 0.18 to 1.58 +/- 0.23cm, p=0.003; end-systolic diameter: from 0.62 +/- 0.23 to 1.35 +/- 0.29cm, p=0.002) and hypokinesia (EF: from 86.2 +/- 5.2 to 33.1 +/- 16.5%, p < 0.001). Histopathology revealed that left ventricular myocytes were elongated, wavy, and fragmented, while collagen fibers were overstretched, straightened, and disrupted. MMP-9 activity was significantly elevated (p = 0.003 vs. control), while MMP-2 activity was unchanged. CONCLUSION: High-dose NE increases MMP-9 activity and causes ECM disruption, left ventricular dilatation and dysfunction.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Norepinefrina/toxicidade , Disfunção Ventricular Esquerda/induzido quimicamente , Animais , Pressão Sanguínea/efeitos dos fármacos , Gatos , Dilatação Patológica , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Miocárdio/enzimologia , Miocárdio/patologiaRESUMO
The present study aimed to establish embryonic stem (ES) cell lines, i.e., ntES cells, using rabbit blastocyst stage embryos cloned by somatic cell nuclear transfer. First, we investigated the development of cloned rabbit embryos reconstructed with normal fibroblasts and fibroblasts transfected with enhanced green fluorescence protein (eGFP). Blastocyst rates were 27.4% and 23.9%, respectively, for the embryos reconstructed with normal fibroblasts and fibroblasts transfected with eGFP compared with that from the parthenogenetic group (43.1%). One ntES cell line was established from embryos reconstructed with eGFP-transfected fibroblasts (1 of 17, 5.9%), and three ntES cell lines were derived from those with normal fibroblasts (3 of 17, 17.6%). All the ntES cell lines retained alkaline phosphatase activity and expressed ES cell-specific markers SSEA-4, Oct-4, TRA-1-60, and TRA-1-81. The pluripotency was further confirmed by reverse transcription-polymerase chain reaction analyses of Oct-4, Nanog, and Sox-2 expressions in ntES cell lines. The differentiation capacity of ntES cells was also examined in vitro and in vivo, by which these ntES cell lines were able to differentiate into all three germ layers through embryoid bodies and teratomas. In conclusion, it is apparent that the efficiency of ntES cells derived using eGFP-transfected donor cells is lower than that with nontransfected, normal fibroblasts donor cells. Similar to those from parthenogenetic embryos, all ntES cell lines derived from cloned rabbit embryos are able to express pluripotency markers and retain their capability to differentiate into various cell lineages both in vitro and in vivo.
Assuntos
Clonagem de Organismos/veterinária , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/fisiologia , Coelhos/embriologia , Animais , Blastocisto , CariótipoRESUMO
Although serum leptin concentrations are reported by several studies to increase in patients with liver cirrhosis, the mechanisms underpinning this increase remain unclear. Circulating tumor necrosis factor alpha (TNF-alpha) concentrations are also recognized to increase in liver cirrhosis. Furthermore, TNF-alpha administration to rodents results in increased expression and secretion of leptin from adipose tissue in a manner dependent on type 1 TNF-alpha receptor (TNF-RI). The present study was undertaken to examine adipose leptin expression and to explore potential relationships between leptin expression and TNF-alpha in subjects with liver cirrhosis. Liver cirrhosis was induced in male Sprague-Dawley rats by dimethylnitrosamine (DMN) administration or by common bile duct ligation (BDL). Ad libitum and pair-fed animals constituted controls. Serum leptin and TNF-alpha concentrations were determined by immunoassay. Gene expression was determined by the reverse transcription-polymerase chain reaction, and protein levels were measured by Western blotting. Serum leptin values after adjustment of body fat mass in DMN-treated rats were significantly higher than in pair-fed or ad libitum groups. Leptin mRNA and protein levels in epididymal fat in DMN rats increased by 1.8-fold and 2.3-fold, respectively, as compared with ad libitum controls, and by 4-fold and 6-fold, respectively, as compared with the pair-fed group. Epididymal TNF-alpha and membranous TNF-RI (mTNF-RI) concentrations were both 2.3 times higher in DMN rats than in ad libitum controls but did not differ between ad libitum and pair-fed groups. Adipose leptin protein levels correlated directly with TNF-alpha and mTNF-RI concentrations in combined DMN, ad libitum, and pair-fed rats (r=0.64 and r=0.49, respectively; P<.05). In BDL-treated rats, however, serum and adipose leptin concentrations were identical to those in ad libitum controls despite 2.1-fold and 2.4-fold increase in epididymal TNF-alpha and mTNF-RI, respectively. TNF-alpha administration to fasting control animals increased serum and adipose leptin concentrations significantly. The observed TNF-alpha-associated leptin up-regulation in DMN-induced, but not in BDL-induced, cirrhotic rats is consistent with distinctly different roles for TNF-alpha in rats with nonbiliary, as opposed to biliary, cirrhosis.
Assuntos
Ductos Biliares/cirurgia , Dimetilnitrosamina , Leptina/genética , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Fator de Necrose Tumoral alfa/análise , Tecido Adiposo/química , Animais , Bilirrubina/sangue , Western Blotting , Ingestão de Alimentos/efeitos dos fármacos , Epididimo/química , Expressão Gênica , Imunoensaio , Leptina/análise , Leptina/farmacologia , Ligadura , Fígado/enzimologia , Cirrose Hepática/sangue , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Many kinds of brain lesions cause sympathetic hyperexcitation and myocardial damage. A novel animal model was developed for the correlation of sympathetic excitation with ventricular dysfunction and myocardial damage. Six cats (3.23+/-0.26 kg) under intraperitoneal urethane-chloralose anesthesia were artificially ventilated and bilaterally vagotomized. They underwent an electrical stimulation of unilateral dorsal medulla for 180 min (monopolar square-wave pulses, 10 Hz, 10 V, 0.5 ms). Mean blood pressure, heart rate plasma concentration of norepinephrine and left ventricular size and ejection fraction were measured at 0, 5, 15, 30, 60, 90, 120 and 180 min. Mean blood pressure (mm Hg), heart rate (beats/min) and norepinephrine (pg/ml) increased abruptly from 128+/-15, 203+/-22 and 353+/-123 to 234+/-26, 240+/-13 and 4727+/-2159 at 5 min after electrical stimulation (all p<0.01). The left ventricles showed significant dilatation (end-diastolic diameter: from 1.35+/-0.13 to 1.84+/-0.21 cm, p=0.006; end-systolic diameter: from 0.65+/-0.20 to 1.54+/-0.24 cm, p=0.002) and hypokinesia (ejection fraction: from 88.9+/-6.4% to 37.3+/-8.7%, p<0.001). Cardiac pathology revealed myocardial hemorrhage, cardiomyocyte apoptosis and coagulative myocytolysis (contraction band necrosis), characterized by sarcoplasmic coagulation, granulation and disruption. In conclusions, the present experiment develops a novel animal model in which stimulation of the pressor area in the dorsal medulla in vagotomized cats produces sympathetic hyperexcitation accompanied with myocardial dysfunction and damage. This model may be applicable for studying protective effect of drugs on myocardial dysfunction and damage caused by sympathetic hyperexcitation occurring in brain diseases.