RESUMO
Sickle cell disease (SCD) is caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, vasoocclusion, and intense hemolysis. P-selectin inhibition has been shown to prevent vasoocclusive events in patients with SCD; however, the chronic effect of P-selectin inhibition in SCD remains to be determined. Here, we used quantitative liver intravital microscopy in our recently generated P-selectin-deficient SCD mice to show that chronic P-selectin deficiency attenuates liver ischemia but fails to prevent hepatobiliary injury. Remarkably, we find that this failure in resolution of hepatobiliary injury in P-selectin-deficient SCD mice is associated with the increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in patients with SCD.
Assuntos
Anemia Falciforme/patologia , Isquemia/patologia , Fígado/irrigação sanguínea , Selectina-P/deficiência , Anemia Falciforme/fisiopatologia , Animais , Arteriopatias Oclusivas/etiologia , Arteriopatias Oclusivas/patologia , Senescência Celular , Células Epiteliais/patologia , Heme Oxigenase-1/análise , Hemólise , Fígado/patologia , Fígado/fisiopatologia , Proteínas de Membrana/análise , Camundongos , Camundongos Knockout , Modelos Animais , Selectina-P/genéticaRESUMO
PURPOSE: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. METHODS: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. RESULTS: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). CONCLUSION: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.
Assuntos
Apoptose/efeitos dos fármacos , Cinamatos/farmacologia , Glucosídeos Iridoides/farmacologia , Óxido Nítrico/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Testículo/irrigação sanguínea , Animais , Western Blotting , Heme Oxigenase-1/análise , Marcação In Situ das Extremidades Cortadas , Masculino , Malondialdeído/análise , NADP/análise , Peroxidase/análise , Distribuição Aleatória , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Reprodutibilidade dos Testes , Superóxido Dismutase/análise , Testículo/patologia , Xantina Oxidase/análiseRESUMO
Abstract Purpose: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. Methods: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. Results: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). Conclusion: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.
Assuntos
Animais , Masculino , Testículo/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Cinamatos/farmacologia , Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Glucosídeos Iridoides/farmacologia , Óxido Nítrico/biossíntese , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Distribuição Aleatória , Western Blotting , Ratos Sprague-Dawley , Peroxidase/análise , Marcação In Situ das Extremidades Cortadas , Heme Oxigenase-1/análise , Malondialdeído/análise , NADP/análiseRESUMO
PURPOSE: To investigate if oxymatrine pretreatment could ameliorate renal I/R injury induced in rats and explore the possible role of oxymatrine in Nrf2/HO-1 pathway. METHODS: Unilaterally nephrectomized rats were insulted by I/R in their left kidney. Twenty four rats were randomly divided into three groups: sham group, I/R + saline-treated group, I/R + OMT-treated group. Oxymatrine or vehicle solution was administered intraperitoneally injected 60 min before renal ischemia, respectively. Renal function, histology, makers of oxidative stress, cell apoptosis and Nrf2/HO-1 expressions were assessed. RESULTS: Oxymatrine pretreatment exhibited an improved renal functional recovery, alleviated histological injury and oxidative stress, inhibiting tubular apoptosis, and accompanied by upregulated the expression of Nrf2/HO-1 proteins. CONCLUSION: Oxymatrine may attenuate renal ischemia/reperfusion injury, and this renoprotective effect may be through activating the Nrf2/HO-1 pathway.
Assuntos
Alcaloides/farmacologia , Antioxidantes/farmacologia , Heme Oxigenase-1/metabolismo , Rim/irrigação sanguínea , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Quinolizinas/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Alcaloides/uso terapêutico , Animais , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Western Blotting , Modelos Animais de Doenças , Heme Oxigenase-1/análise , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Rim/patologia , Masculino , Fator 2 Relacionado a NF-E2/análise , Quinolizinas/uso terapêutico , Distribuição Aleatória , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do TratamentoRESUMO
PURPOSE: To investigate if oxymatrine pretreatment could ameliorate renal I/R injury induced in rats and explore the possible role of oxymatrine in Nrf2/HO-1 pathway. METHODS: Unilaterally nephrectomized rats were insulted by I/R in their left kidney. Twenty four rats were randomly divided into three groups: sham group, I/R + saline-treated group, I/R + OMT-treated group. Oxymatrine or vehicle solution was administered intraperitoneally injected 60 min before renal ischemia, respectively. Renal function, histology, makers of oxidative stress, cell apoptosis and Nrf2/HO-1 expressions were assessed. RESULTS: Oxymatrine pretreatment exhibited an improved renal functional recovery, alleviated histological injury and oxidative stress, inhibiting tubular apoptosis, and accompanied by upregulated the expression of Nrf2/HO-1 proteins. CONCLUSION: Oxymatrine may attenuate renal ischemia/reperfusion injury, and this renoprotective effect may be through activating the Nrf2/HO-1 pathway. .
Assuntos
Animais , Masculino , Alcaloides/farmacologia , Antioxidantes/farmacologia , Heme Oxigenase-1/metabolismo , Rim/irrigação sanguínea , /metabolismo , Estresse Oxidativo/efeitos dos fármacos , Quinolizinas/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Alcaloides/uso terapêutico , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Western Blotting , Modelos Animais de Doenças , Heme Oxigenase-1/análise , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Rim/patologia , /análise , Quinolizinas/uso terapêutico , Distribuição Aleatória , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Fatores de Tempo , Resultado do TratamentoRESUMO
O Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação do hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglogina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação ...
Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA ...
Assuntos
Heme Oxigenase-1/análise , Heme Oxigenase-1/antagonistas & inibidores , Heme/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Biliverdina , DNA , Eletroforese em Gel de Poliacrilamida , Análise Espectral/métodos , Immunoblotting/métodos , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Intravascular hemolysis may cause tissue injury directly or via a systemic inï¬ammatory response. Under physiological conditions, extracorpuscular hemoglobin (Hb) is bound by haptoglobin(Hp) and the complex internalized via the hemoglobin scavenger receptor CD163 on monocytes, prior to catabolism via heme-oxygenase-1 (HO-1). Recently, a novel subset of CD68(pos)CD163(high)HLA-DR(low) macrophages with high expression of HO-1 was recognized in hemorrhagic areas of atherosclerotic plaques, distinct from CD68(pos)CD163(low)HLA-DR(high) foam cell macrophages with low- HO-1 content. Considering the hemolytic insult during CPB, we hypothesized that an equivalent compensatory CD163(high)HLA-DR(low) phenotype will evolve in circulating CD14(pos) monocytes post surgery. METHODS: Twelve patients undergoing elective surgery with CPB were enrolled with informed consent.Whole-blood samples were collected in EDTA at predetermined time-points, pre- intra-, and postoperatively. Whole-blood was evaluated by three-color flow cytometry for expression of CD14, CD163, and HLA-DR; CD14(pos) cells were also permeabilized to detect intracellular HO-1 protein. Plasma [Hp-Hb] concentration was determined by ELISA. RESULTS: A striking phenotypic switch from CD163(low)HLA-DR(high) preoperatively to CD163(high)HLA-DR(low) postoperatively at 24 h was observed on circulating CD14(pos) monocytes. Intracellular HO-1 protein was also significantly up-regulated at 24 h after declamping. These phenotypic changes were preceded by peak Hb-Hp levels observed at 2 h. CONCLUSION: We have shown for the first time, a phenotypic commitment of monocytes towards a protective CD14(pos)CD163(high)HLA-DR(low) population with increased intracellular HO-1 occurring in the circulation during the recovery phase of CPB. These findings have implications for monitoring of anti-inflammatory interventions and linkage to clinical outcomes.
Assuntos
Ponte Cardiopulmonar , Hemoglobinas/metabolismo , Monócitos/metabolismo , Antígenos CD/sangue , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/sangue , Antígenos de Diferenciação Mielomonocítica/imunologia , Citometria de Fluxo , Células Espumosas/química , Células Espumosas/imunologia , Células Espumosas/metabolismo , Antígenos HLA-DR/sangue , Antígenos HLA-DR/imunologia , Haptoglobinas/análise , Haptoglobinas/imunologia , Heme Oxigenase-1/análise , Humanos , Receptores de Lipopolissacarídeos/sangue , Receptores de Lipopolissacarídeos/imunologia , Monócitos/química , Monócitos/imunologia , Fenótipo , Período Pós-Operatório , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/imunologia , Regulação para CimaRESUMO
PURPOSE: To study the expression of heme-oxygenase-1 (HO-1), an enzyme induced by oxidative stress, in specimens obtained from an experimental model in rats that evaluated the role of gastric and duodenal reflux in esophageal carcinogenesis. METHODS: Esophageal specimens embedded in paraffin obtained from different experimental groups of rats were used for immunohistochemistry analysis of HO-1 expression. The rats had been divided into the following groups and were killed after 22 weeks: (1) cardioplasty to induce acid reflux; (2) esophagoduodenal anastomosis to induce duodenal reflux; (3) no treatment; (4) cardioplasty + diethylnitrosamine (DEN); (5) esophagoduodenal anastomosis + DEN; and (6) DEN. The study sample comprised 3 specimens from each group with the most severe histopathological lesions found on each study branch. RESULTS: The expression of HO-1 was seen only in rat specimens submitted to esophagoduodenal anastomosis (Groups 2 and 5), and the analysis of mean fluorescence intensity revealed a significant increase of HO-1 expression (4.8 and 4.6 fold, respectively) when compared with the control group (Group 3) (p<0.05). The main target for HO-1 induction was the inflammatory cells inside the tumor or in subepithelial areas. Rats exposed to gastric reflux had no HO-1 expression. CONCLUSION: Reflux esophagitis induced by reflux of duodenal contents, which provoked considerable oxidative stress, may play an important role in esophageal carcinogenesis. Acid reflux did not induce oxidative stress in this experimental model.
OBJETIVO: Estudar a expressão da HO-1 (enzima induzida pelo estresse) em diferentes peças esofágicas obtidas de um estudo experimental em ratos que avaliou o papel do refluxo gastroesofágico e duodeno esofágico na carcinogênese experimental. MÉTODOS: Blocos de parafina contendo peças de esôfago provenientes de um estudo experimental com ratos foram utilizados para verificar a expressão imunohistoquímica da HO-1. Os ratos haviam sido divididos nos seguintes grupos: (1) Cardioplastia com o objetivo de promover refluxo ácido, (2) Anastomose esofagoduodenal para indução de refluxo misto (ácido e biliar), (3) sem tratamento (controles), (4) cardioplastia + dietil-nitrosamina (DEN), (5) Anastomose esofagoduodenal + DEN, (6) DEN. Amostras contendo três peças de cada grupo com as lesões histopatológicas mais graves encontradas em cada braço do estudo foram escolhidas para avaliação da expressão imunoistoquímica da HO-1. RESULTADOS: A expressão da HO-1 foi observada somente nas peças de esôfago de ratos submetidos à anastomose esofagoduodenal (Grupos 2 e 5) e analise da intensidade média da fluorescência demonstrou uma diferença significativa na expressão da HO-1 nesses grupos quando comparada com o grupo controle (4,8 e 4,6 vezes respectivamente) (p<0,05). As células inflamatórias localizadas dentro dos tumores e nas regiões adjacentes ao epitélio foram as que mais intensamente expressaram a HO-1. Ratos expostos ao refluxo ácido (gástrico) apresentaram pouca ou nenhuma atividade da HO-1. CONCLUSÃO: Esofagite de refluxo induzida pelo refluxo com conteúdo duodenal provocou considerável estresse oxidativo, que parece exercer um papel importante na carcinogênese esofágica. O refluxo puramente ácido não foi capaz de induzir estresse oxidativo nesse modelo experimental
Assuntos
Animais , Ratos , Carcinoma/induzido quimicamente , Neoplasias Esofágicas/induzido quimicamente , Esofagite/induzido quimicamente , Refluxo Gastroesofágico/complicações , Heme Oxigenase-1/análise , Biomarcadores/análise , Carcinógenos , Dietilnitrosamina , Modelos Animais de Doenças , Esôfago/enzimologia , Estresse Oxidativo , Ratos WistarRESUMO
PURPOSE: To study the expression of heme-oxygenase-1 (HO-1), an enzyme induced by oxidative stress, in specimens obtained from an experimental model in rats that evaluated the role of gastric and duodenal reflux in esophageal carcinogenesis. METHODS: Esophageal specimens embedded in paraffin obtained from different experimental groups of rats were used for immunohistochemistry analysis of HO-1 expression. The rats had been divided into the following groups and were killed after 22 weeks: (1) cardioplasty to induce acid reflux; (2) esophagoduodenal anastomosis to induce duodenal reflux; (3) no treatment; (4) cardioplasty + diethylnitrosamine (DEN); (5) esophagoduodenal anastomosis + DEN; and (6) DEN. The study sample comprised 3 specimens from each group with the most severe histopathological lesions found on each study branch. RESULTS: The expression of HO-1 was seen only in rat specimens submitted to esophagoduodenal anastomosis (Groups 2 and 5), and the analysis of mean fluorescence intensity revealed a significant increase of HO-1 expression (4.8 and 4.6 fold, respectively) when compared with the control group (Group 3) (p<0.05). The main target for HO-1 induction was the inflammatory cells inside the tumor or in subepithelial areas. Rats exposed to gastric reflux had no HO-1 expression. CONCLUSION: Reflux esophagitis induced by reflux of duodenal contents, which provoked considerable oxidative stress, may play an important role in esophageal carcinogenesis. Acid reflux did not induce oxidative stress in this experimental model.
Assuntos
Carcinoma/induzido quimicamente , Neoplasias Esofágicas/induzido quimicamente , Esofagite/induzido quimicamente , Refluxo Gastroesofágico/complicações , Heme Oxigenase-1/análise , Animais , Biomarcadores/análise , Carcinógenos , Dietilnitrosamina , Modelos Animais de Doenças , Esôfago/enzimologia , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
AIMS: To study the nitric oxide (NO) and carbon monoxide roles in the regulation of the pulmonary circulation in lowland and highland newborn sheep and llamas. METHODS AND RESULTS: We used neonatal sheep (Ovis aries) and llamas (Lama glama) whose gestation and delivery took place at low (580 m) or high (3600 m) altitude. In vivo, we measured the cardiopulmonary function basally and with a NO synthase (NOS) blockade and calculated the production of carbon monoxide by the lung. In vitro, we determined NOS and soluble guanylate cyclase (sGC) expression, NOS activity, and haemoxygenase (HO) expression in the lung. Pulmonary arterial pressure was elevated at high altitude in sheep but not in llamas. Sheep at high altitude relative to sea level had significantly greater total lung NOS activity and eNOS protein, but reduced sGC and HO expression and carbon monoxide production. In contrast, llamas showed no difference in NO function between altitudes, but a pronounced increase in pulmonary carbon monoxide production and HO expression at high altitude. CONCLUSIONS: In the llama, enhanced pulmonary carbon monoxide, rather than NO, protects against pulmonary hypertension in the newborn period at high altitude. This shift in pulmonary dilator strategy from NO to carbon monoxide has not been previously described, and it may give insight into new treatments for excessive pulmonary vasoconstriction.