RESUMO
CONTEXT: Chloranthus serratus (Thunb.) Roem. et Schult. (Chloranthaceae) is an herb widely used as a folk medicine treating inflammatory diseases, although it is toxic. OBJECTIVE: To investigate hepatotoxicity and related mechanisms induced by ethanol extracts of different parts of C. serratus in rats. MATERIALS AND METHODS: Sprague Dawley rats were divided into control (Con), ethanol extract of roots (ER), stems (ES), and leaves (EL) groups, and acute oral toxicity studies were conducted. The rats received doses of 4.14, 3.20, and 1.16 g/kg/d extracts for 14 days, respectively. Liver index, liver function and oxidative stress biomarkers, liver pathology, ultrastructure, TNF-α, ICAM-1, and Nrf2/HO-1 proteins expression levels were determined. RESULTS: The LD50 of ER, ES, and EL were higher than 10.35, 8.05, and 2.90 g/kg/p.o., respectively. The liver indexes in the extract groups increased significantly. EL dramatically increased TP, GLB, AST, ALT, ALP, TBA, MDA, ICAM-1, and TNF-α levels (p < 0.01), and induced the most obvious pathological and ultrastructural changes. ES and EL obviously decreased the T-SOD, GSH, CAT, and CHOL levels. Nrf2 and HO-1 proteins expression was reduced significantly in ES (0.77 ± 0.06, 2.33 ± 0.20) and EL (0.23 ± 0.04, 2.14 ± 0.16) groups, and reduced slightly in ER (1.08 ± 0.10; 3.39 ± 0.21) group. DISCUSSION AND CONCLUSION: ES and EL induce stronger hepatotoxicity than ER through oxidative stress and the Nrf2/HO-1 pathway, and the root is a better medicinal part, which provides a basis for clinical research, safe applications, and reasonable development of C. serratus.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Medicamentos de Ervas Chinesas/toxicidade , Estresse Oxidativo , Animais , Doença Hepática Induzida por Substâncias e Drogas/mortalidade , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/análise , Heme Oxigenase (Desciclizante)/fisiologia , Molécula 1 de Adesão Intercelular/análise , Fígado/patologia , Masculino , Fator 2 Relacionado a NF-E2/fisiologia , Ratos , Ratos Sprague-Dawley , Aumento de Peso/efeitos dos fármacosRESUMO
Oxidative stress is well known to play a pivotal role in cerebral ischemia-reperfusion injury. The nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway has been considered a potential target for neuroprotection in stroke. 11-Keto-ß-boswellic acid (KBA) is a triterpenoid compound from extracts of Boswellia serrata. The aim of the present study was to determine whether KBA, a novel Nrf2 activator, can protect against cerebral ischemic injury. Middle cerebral artery occlusion (MCAO) was operated on male Sprague-Dawley rats. KBA (25 mg/kg) applied 1 h after reperfusion significantly reduced infarct volumes and apoptotic cells as well as increased neurologic scores at 48 h after reperfusion. Meanwhile, posttreatment with KBA significantly decreased malondialdehyde (MDA) levels, restored the superoxide dismutase (SOD) activity, and increased the protein Nrf2 and HO-1 expression in brain tissues. In primary cultured astrocytes, KBA increased the Nrf2 and HO-1 expression, which provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. But knockdown of Nrf2 or HO-1 attenuated the protective effect of KBA. In conclusion, these findings provide evidence that the neuroprotection of KBA against oxidative stress-induced ischemic injury involves the Nrf2/HO-1 pathway.
Assuntos
Antioxidantes/uso terapêutico , Heme Oxigenase (Desciclizante)/fisiologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fator 2 Relacionado a NF-E2/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fármacos Neuroprotetores/toxicidade , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/fisiologia , Triterpenos/toxicidade , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Boswellia/química , Encéfalo/patologia , Dano Encefálico Crônico/etiologia , Dano Encefálico Crônico/prevenção & controle , Hipóxia Celular , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Glucose/farmacologia , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase (Desciclizante)/genética , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Malondialdeído/análise , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Triterpenos/farmacologiaRESUMO
We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-ß (transforming growth factor ß) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.
Assuntos
Benzoatos/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Epóxido Hidrolases/antagonistas & inibidores , Heme Oxigenase (Desciclizante)/fisiologia , Ureia/análogos & derivados , Actinas/metabolismo , Albuminúria/prevenção & controle , Animais , Benzoatos/antagonistas & inibidores , Benzoatos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Creatinina/urina , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/metabolismo , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Epóxido Hidrolases/metabolismo , Epóxido Hidrolases/fisiologia , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/fisiologia , Córtex Renal/metabolismo , Masculino , Proteínas de Membrana/urina , Metaloporfirinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ureia/antagonistas & inibidores , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
Carbon monoxide (CO) is produced during the catabolism of free haem, catalyzed by haem oxygenase (HO) enzymes, and its physiological roles include vasodilation, neurotransmission, inhibition of platelet aggregation and anti-proliferative effects on smooth muscle. In vivo preclinical studies have shown that exogenously administered quantities of CO may represent an effective treatment for conditions characterized by a dysregulated immune response. The carbon monoxide-releasing molecules (CORMs) represent a group of compounds capable of carrying and liberating controlled quantities of CO in the cellular systems. This review covers the physiological and anti-inflammatory properties of the HO/CO pathway in the central nervous system. It also discusses the effects of CORMs in preclinical models of inflammation. The accumulating data discussed herein support the possibility that CORMs may represent a novel class of drugs with disease-modifying properties in multiple sclerosis.
Assuntos
Boranos/uso terapêutico , Monóxido de Carbono/uso terapêutico , Carbonatos/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Compostos Organometálicos/uso terapêutico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Autoimunidade/efeitos dos fármacos , Boranos/administração & dosagem , Monóxido de Carbono/administração & dosagem , Monóxido de Carbono/metabolismo , Carbonatos/administração & dosagem , Cardiotônicos/administração & dosagem , Cardiotônicos/uso terapêutico , Citocinas/biossíntese , Avaliação Pré-Clínica de Medicamentos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Guanilato Ciclase/metabolismo , Heme/metabolismo , Heme Oxigenase (Desciclizante)/fisiologia , Heme Oxigenase-1/deficiência , Heme Oxigenase-1/fisiologia , Humanos , Inflamação/tratamento farmacológico , Esclerose Múltipla/imunologia , Neuroimunomodulação/efeitos dos fármacos , Neuroimunomodulação/fisiologia , Compostos Organometálicos/administração & dosagem , Oxirredução , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase Solúvel , Vasodilatadores/administração & dosagem , Vasodilatadores/uso terapêuticoRESUMO
OBJECTIVE: To cultivate human umbilical vein endothelial cells (HUVECs) in the serum of overfatigue rats with the intervention of Tongxinluo superfine powder (TXLSP). By examining the variation of the activity of JNK/c-Jun/HO-1 pathway, the possible mechanisms of vascular endothelial dysfunction under overfatigue conditions and the intervening effect of TXLSP were explored. METHODS: The HUVECs were randomly divided into the normal control group, the model group, the SP600125 (a specific antagonist of JNK) group, the TXLSP group and the TXLSP + SP600125 group. The content of carboyhemoglobin (COHb) and the leak rate of lactic dehydrogenase (LDH) in different groups were measured. The mRNA and protein expression of JNK, c-Jun, HO-1 and the phosphorylation level of c-Jun (P-c-Jun) were detected using Western blot and PCR methods. RESULTS: Compared with the normal control group, the COHb level in supernatant was increased significantly in the model group, and the expression of HO-1, JNK, c-Jun mRNA and corresponding proteins and P-c-Jun were also increased remarkably. The increases in these parameters were significantly decreased by SP600125. TXLSP showed remarkable up-regulation on the expression of JNK, c-Jun, P-c-Jun and HO-1 mRNA and their protein expression. Compared with the SP600125 group, the expressions of JNK, c-Jun, P-c-Jun and HO-1 mRNA and its protein in the TXLSP+SP600125 group were significantly increased at different time points (P<0.05, P<0.01). CONCLUSIONS: The vascular endothelial dysfunction under overfatigue conditions is related to the activity of the JNK/c-Jun/HO-1 pathway. One of the mechanisms of TXLSP in improving the vascular endothelial function is to adjust the activity of the JNK/c-Jun/HO-1 pathway at gene and protein levels.
Assuntos
Proteínas Sanguíneas/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais/efeitos dos fármacos , Fadiga/sangue , Heme Oxigenase (Desciclizante)/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Animais , Células Cultivadas , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/administração & dosagem , Células Endoteliais/metabolismo , Fadiga/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Tamanho da Partícula , Pós/administração & dosagem , Pós/farmacologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Ratos Wistar , Soro/metabolismo , Soro/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismoRESUMO
Hyperglycemia-induced oxidative stress is a common phenomenon in diabetes. Since oxidative stress depletes adiponectin and insulin levels, we investigated whether an upregulated heme oxygenase (HO) system would attenuate the oxidative destruction of adiponectin/insulin and improve insulin sensitivity and glucose metabolism in streptozotocin (STZ)-induced type 1 diabetes. HO was upregulated with hemin (15 mg/kg ip) or inhibited with chromium mesoporphyrin (CrMP, 4 micromol/kg ip). Administering hemin to STZ-diabetic rats reduced hyperglycemia and improved glucose metabolism, whereas the HO inhibitor CrMP annulled the antidiabetic effects and/or exacerbated fasting/postprandial hyperglycemia. Interestingly, the antidiabetic effects of hemin lasted for 2 mo after termination of therapy and were accompanied by enhanced HO-1 and HO activity of the soleus muscle, along with potentiation of plasma antioxidants like bilirubin, ferritin, and superoxide dismutase, with corresponding elevation of the total antioxidant capacity. Importantly, hemin abated c-Jun NH2-terminal kinase (JNK), a substance known to inhibit insulin biosynthesis, and suppressed markers/mediators of oxidative stress including 8-isoprostane, nuclear-factor (NF)-kappaB, activating protein (AP)-1, and AP-2 of the soleus muscle. Furthermore, hemin therapy significantly attenuated pancreatic histopathological lesions including acinar cell necrosis, interstitial edema, vacuolization, fibrosis, and mononuclear cell infiltration. Correspondingly, hemin increased plasma insulin and potentiated agents implicated in insulin sensitization and insulin signaling such as adiponectin, adenosine monophosphate-activated protein kinase (AMPK), cAMP, cGMP, and glucose transporter (GLUT)4, a protein required for glucose uptake. These were accompanied by improved glucose tolerance [intraperitoneal glucose tolerance text (IPGTT)], decreased insulin intolerance [intraperitoneal insulin tolerance test (IPITT)], and reduced insulin resistance [homeostasis model assessment of insulin resistance (HOMA-IR) index], whereas CrMP nullified the hemin-dependent antidiabetic and insulin-sensitizing effects. In conclusion, by concomitantly enhancing insulin and paradoxically potentiating insulin sensitivity, this study unveils a novel, unique, and long-lasting antidiabetic characteristic of upregulating HO with hemin that could be exploited against insulin-resistant and insulin-dependent diabetes.
Assuntos
Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Heme Oxigenase (Desciclizante)/fisiologia , Resistência à Insulina , Estreptozocina , Animais , GMP Cíclico/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Avaliação Pré-Clínica de Medicamentos , Jejum/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hemina/farmacologia , Hemina/uso terapêutico , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Resistência à Insulina/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Transcrição AP-1/metabolismoRESUMO
Emerging evidence indicates that aldosterone causes oxidative stress by stimulating proinflammatory/oxidative mediators, including nuclear factor-kappaB, activating protein (AP-1), and c-Jun N-terminal kinase. Thus, in insulin-resistant type 2 diabetes (T2D), oxidative stress generated by hyperglycemia and aldosterone would potentiate the oxidative destruction of tissue and important regulators of glucose metabolism like adiponectin and insulin. Although heme oxygenase (HO)-1 is cytoprotective, its effects on T2D have not been fully characterized. Here we report an enduring antidiabetic effect of the HO inducer, hemin, on Zucker diabetic-fatty rat (ZDF), a model of insulin-resistant T2D. Chronically applied hemin to ZDF reduced and maintained significantly low fasting and postprandial hyperglycemia for 4 months after therapy. The antidiabetic effect was accompanied by enhanced HO activity, catalase, cyclic GMP, bilirubin, ferritin, total antioxidant capacity, and insulin. In contrast, reduced aldosterone alongside markers/mediators of oxidative stress, including 8-isoprostane, c-Jun N-terminal kinase, nuclear factor-kappaB, AP-1, and AP-2 were observed. Interestingly, in hemin-treated ZDF, inhibitory proteins of insulin-signaling, such as glycogen synthase kinase-3 and protein-tyrosine phosphatase-1B were reduced, whereas agents that promote insulin signaling including adiponectin, cAMP, AMP-activated protein kinase, aldolase-B, and glucose transporter-4 (GLUT4), were robustly increased. Correspondingly, hemin improved ip glucose tolerance, reduced insulin intolerance, and lowered insulin resistance (homeostasis model assessment of insulin resistance), and the inability of insulin to enhance GLUT4 was overturned. These results suggest that the suppression of hyperglycemia and aldosterone-induced oxidative stress alongside the potentiation of insulin-sensitizing pathways may account for the 4-month enduring antidiabetic effect. The synergistic interaction between the HO system, aldolase-B, adiponectin, AMP-activated protein kinase, and GLUT4 may be explored for novel strategies against postprandial/fasting hyperglycemia and insulin-resistant T2D.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Heme Oxigenase (Desciclizante)/fisiologia , Hemina/uso terapêutico , Hiperglicemia/prevenção & controle , Resistência à Insulina , Obesidade/metabolismo , Aldosterona/sangue , Animais , GMP Cíclico/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Avaliação Pré-Clínica de Medicamentos , Jejum/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hemina/farmacologia , Hiperglicemia/enzimologia , Hiperglicemia/metabolismo , Resistência à Insulina/fisiologia , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Obesidade/complicações , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Transdução de Sinais/fisiologiaRESUMO
Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. An 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827,600+/-129,000 vs. 294,500+/-57,510; p<0.05), neovessels measured by vital microscopy (21.91+/-1.05 vs. 12.77+/-1.55 mm; p<0.001) 4 days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.
Assuntos
Biliverdina/uso terapêutico , Neovascularização da Córnea/prevenção & controle , Ceratite/prevenção & controle , Animais , Córnea/enzimologia , Neovascularização da Córnea/enzimologia , Neovascularização da Córnea/patologia , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Feminino , Heme Oxigenase (Desciclizante)/deficiência , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase (Desciclizante)/fisiologia , Heme Oxigenase-1/metabolismo , Ceratite/enzimologia , Ceratite/patologia , Masculino , Camundongos , Camundongos Knockout , Peroxidase/metabolismoRESUMO
AIM: The aim of the present study was to explore whether heme oxygenase-1 (HO-1) is involved in the hyperthermia-provided protection of the small intestine from ischemia/reperfusion injury in rats. METHODS: Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion. Whole-body hyperthermia was induced in anesthetized rats by placement in a temperature-controlled water bath. Whole-body hyperthermia to a core temperature of 42-43 degrees C for 15 min was followed by passive cooling. We started the hyperthermic treatment 6 h before the vascular clamping. The severity of the mucosal injury was evaluated by several biochemical markers and histological findings. Hyperthermia-induced heat-shock proteins were detected by Western blotting. We also investigated the effect of zinc protoporphyrin IX (an HO-1 inhibitor) on the protective effect of hyperthermia. RESULTS: The rats, which were killed after ischemia/reperfusion, had severe intestinal inflammation. Hyperthermia significantly induced the production of Hsp70 and HO-1 in intestinal mucosa and significantly reduced ischemia/reperfusion-induced mucosal injury. The combination of zinc protoporphyrin IX with hyperthermia extinguished the protective effects of hyperthermia on ischemia/reperfusion injury. CONCLUSION: Hyperthermia protects against ischemia/reperfusion injury in rat small intestine through the expression of heat-shock proteins, especially HO-1.
Assuntos
Heme Oxigenase (Desciclizante)/fisiologia , Hipertermia Induzida , Traumatismo por Reperfusão/prevenção & controle , Animais , Quimiocinas CXC/análise , Indução Enzimática , Proteínas de Choque Térmico HSP70/biossíntese , Heme Oxigenase (Desciclizante)/biossíntese , Quinase I-kappa B/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/análise , Mucosa Intestinal/química , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Masculino , Peroxidase/metabolismo , Protoporfirinas/farmacologia , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
Although the brain generates NO and carbon monoxide (CO), it is unknown how these gases and their enzyme systems interact with each other to regulate cerebrovascular function. We examined whether CO produced by heme oxygenase (HO) modulates generation and action of constitutive NO in the rat pial microcirculation. Immunohistochemical analyses indicated that HO-2 occurred in neurons and arachnoid trabecular cells, where NO synthase 1 (NOS1) was detectable, and also in vascular endothelium-expressing NOS3, suggesting colocalization of CO- and NO-generating sites. Intravital microscopy using a closed cranial window preparation revealed that blockade of the HO activity by zinc protoporphyrin IX significantly dilates arterioles. This vasodilatation depended on local NOS activities and was abolished by CO supplementation, suggesting that the gas derived from HO-2 tonically regulates NO-mediated vasodilatory response. Bioimaging of NO by laser-confocal microfluorography of diaminofluorescein indicated detectable amounts of NO at the microvascular wall, the subdural mesothelial cells, and arachnoid trabecular cells, which express NOS in and around the pial microvasculature. On CO inhibition by the HO inhibitor, regional NO formation was augmented in these cells. Such a pattern of accelerated NO formation depended on NOS activities and was again attenuated by the local CO supplementation. Studies using cultured porcine aortic endothelial cells suggested that the inhibitory action of CO on NOS could result from the photo-reversible gas binding to the prosthetic heme. Collectively, CO derived from HO-2 appears to serve as a tonic vasoregulator antagonizing NO-mediated vasodilatation in the rat cerebral microcirculation.
Assuntos
Monóxido de Carbono/fisiologia , Circulação Cerebrovascular , Heme Oxigenase (Desciclizante)/fisiologia , Óxido Nítrico/fisiologia , Vasodilatação , Animais , Arteríolas/fisiologia , Encéfalo/metabolismo , Células Cultivadas , Artérias Cerebrais/fisiologia , Células Endoteliais/metabolismo , Heme Oxigenase (Desciclizante)/análise , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.
Assuntos
Endotélio Vascular/enzimologia , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase (Desciclizante)/fisiologia , Proteínas Musculares , Fator de Necrose Tumoral alfa/metabolismo , 6-Cetoprostaglandina F1 alfa/biossíntese , Apoptose , Western Blotting , Ciclo Celular , Células Cultivadas , Ciclo-Oxigenase 2 , DNA/metabolismo , DNA Complementar/metabolismo , Dinoprostona/biossíntese , Endotélio Vascular/imunologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Fase G1 , Heme Oxigenase-1 , Humanos , Inflamação , Isoenzimas/metabolismo , Proteínas de Membrana , Metaloporfirinas/farmacologia , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Retroviridae/genética , Transfecção , Regulação para CimaRESUMO
Carbon monoxide (CO) are produced by heme oxygenase (HO), and HO was detected in hypothalamus. However, the roles of CO produced in hypothalamus was not fully elucidated. So, we tested the effects of CO on body temperature because preoptic-anterior hypothalamus was known as the presumptive primary fever-producing site. CO-saturated aCSF (4 microl, i.c.v.) and hemin (10 microg, i.c.v.) elicited marked febrile response. Pretreatment with indomethacin completely inhibited CO- and hemin-induced fever. Zinc protoporphyrin-IX (10 microg, i.c.v.) or ODQ (50 microg, i.c.v.) partially reduced hemin-induced febrile response. Dibutyryl-cGMP (100 microg, i.c.v.) produced profound febrile response and this febrile response was attenuated by indomethacin. These results indicate that endogenous CO may have a role as a pyrogenic mediator in CNS and CO-mediated pyresis is dependent on prostaglandin production and partially on activation of soluble guanylate cyclase.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Temperatura Corporal/fisiologia , Monóxido de Carbono/fisiologia , Heme Oxigenase (Desciclizante)/fisiologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Temperatura Corporal/efeitos dos fármacos , Monóxido de Carbono/administração & dosagem , Monóxido de Carbono/farmacologia , Líquido Cefalorraquidiano/química , Dibutiril GMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Hemina/administração & dosagem , Hemina/farmacologia , Hipotálamo/fisiologia , Indometacina/farmacologia , Injeções Intraventriculares , Masculino , Oxidiazóis/farmacologia , Protoporfirinas/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Stress-induced downregulation of spermatogenesis remains poorly understood. This study examined the induction of heme oxygenase-1 (HO-1), a carbon monoxide-generating inducible enzyme, in modulation of spermatogenesis. Rats were exposed to cadmium chloride (CdCl(2)), a stressor causing oligozoospermia, and HO-1-induction was monitored by following HO isozyme expression. CdCl(2)-treated testes increased HO-1 activity and suppressed microsomal cytochromes P450, which are required for steroidogenesis. CdCl(2)-elicited HO-1 occurred mostly in Leydig cells and coincided with CO generation, as judged by bilirubin-IXalpha immunoreactivity. Under these circumstances, germ cells in peripheral regions of seminiferous tubules exhibited apoptosis; laser flow cytometry revealed that these apoptotic cells involve diploid and tetraploid germ cells, suggesting involvement of spermatogonia and primary spermatocytes in CdCl(2)-elicited apoptosis. Pretreatment with zinc protoporphyrin-IX, an HO inhibitor, but not copper protoporphyrin-IX, which does not block the enzyme, attenuated the CdCl(2)-induced apoptosis. Such antiapoptotic effects of zinc protoporphyrin-IX were repressed by supplementation of dichloromethane, a CO donor. Upon CdCl(2)-treatment, both Sertoli cells and the germ cells upregulated Fas ligand; this event was also suppressed by zinc protoporphyrin-IX and restored by dichloromethane. Thus, Leydig cells appear to use HO-1-derived CO to trigger apoptosis of premeiotic germ cells and thereby modulate spermatogenesis under conditions of stress.
Assuntos
Apoptose/fisiologia , Heme Oxigenase (Desciclizante)/fisiologia , Células Intersticiais do Testículo/enzimologia , Espermatogênese/fisiologia , Animais , Apoptose/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Monóxido de Carbono/metabolismo , Diploide , Indução Enzimática/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Imuno-Histoquímica , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/patologia , Masculino , Oligospermia/induzido quimicamente , Oligospermia/enzimologia , Oligospermia/patologia , Poliploidia , Ratos , Ratos Wistar , Espermatogênese/efeitos dos fármacos , Estresse Fisiológico/enzimologia , Estresse Fisiológico/patologia , Testículo/efeitos dos fármacos , Testículo/enzimologia , Testículo/patologiaRESUMO
Oxidant-mediated reperfusion injury of the gut is a major contributor of the systemic inflammatory response in hemorrhagic shock. Recent studies have suggested that heme-oxygenase-1 (HO-1) represents an endogenous protective mechanism against oxidant stress. We assessed whether HO-1 induction modulates the synthesis of tumor necrosis factor-alpha (TNF-alpha) in hemorrhagic shock. In rats submitted to hemorrhagic shock, pretreatment with hemoglobin (Hb) increased HO-1 mRNA expression in macrophages. This increased expression was associated with a decreased expression of TNF-alpha mRNA, as well as decreased plasma concentrations of TNF-alpha. These effects of Hb were reduced by the HO-1 inhibitor tin-protoporphyrin (Sn-PP 20 micromol/kg), while Sn-PP had no effect in the absence of Hb. In parallel, Hb pretreatment reduced pulmonary edema, vascular injury, and increased mesenteric blood flow, and these effects were reduced by Sn-PP. Thus, induction of HO-1 is protective in hemorrhagic shock, possibly through its antioxidant properties. Interventions that induce HO-1 may be beneficial in the treatment of shock states, leading to a reduced systemic inflammatory response.
Assuntos
Modelos Animais de Doenças , Heme Oxigenase (Desciclizante)/fisiologia , Heme Oxigenase (Desciclizante)/uso terapêutico , Intestinos/irrigação sanguínea , Traumatismo por Reperfusão/complicações , Choque Hemorrágico/enzimologia , Choque Hemorrágico/imunologia , Síndrome de Resposta Inflamatória Sistêmica/enzimologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Avaliação Pré-Clínica de Medicamentos , Hidratação/métodos , Regulação da Expressão Gênica/fisiologia , Hemodinâmica , Inflamação , Macrófagos Peritoneais/enzimologia , Masculino , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Wistar , Ressuscitação/métodos , Choque Hemorrágico/etiologia , Choque Hemorrágico/fisiopatologia , Choque Hemorrágico/terapia , Circulação Esplâncnica/fisiologia , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Síndrome de Resposta Inflamatória Sistêmica/terapia , Fator de Necrose Tumoral alfa/análiseRESUMO
The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.
Assuntos
Proteínas de Bactérias/genética , Heme Oxigenase (Desciclizante)/fisiologia , Heme/metabolismo , Neisseria meningitidis/enzimologia , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Biliverdina/metabolismo , Teste de Complementação Genética , Heme Oxigenase (Desciclizante)/genética , Peróxido de Hidrogênio/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Mutação , Pseudomonas putida/enzimologia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Preliminary studies showed that the inducible form of heme oxygenase (HO-1) was induced and played a protective role in the process of inflammation. The present study investigated the possible role of HO-1 in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. We measured HO-1 activity in TNBS-induced colitis in rats and analyzed the severity of colitis along with altered HO activity by assessing lesion area and myeloperoxidase activity. HO-1 mRNA and protein expressions were determined at different time points after TNBS induction. Free radical production and inducible nitric oxide synthase (iNOS), which participate in oxidative injury, were also assayed. HO activity and HO-1 gene expression increased markedly after TNBS induction. Administration with tin mesoporphyrin (SnMP), a HO inhibitor, potentiated the colonic damage along with a reduction in HO-1 activity. Furthermore, the reduction of HO-1 expression by SnMP also enhanced reactive oxygen species and iNOS expression, both of which were dramatically increased after the TNBS enema. L-Arginine pretreatment further aggravated the injurious action of SnMP. Our results indicate that HO-1 plays a protective role in the colonic damage induced by the TNBS enema, and the preventive effects probably result from decreased free radical production and inhibition of iNOS expression in colonic tissues.
Assuntos
Colite Ulcerativa/enzimologia , Heme Oxigenase (Desciclizante)/fisiologia , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Inibidores Enzimáticos/farmacologia , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/imunologia , Heme Oxigenase-1 , Imuno-Histoquímica , Medições Luminescentes , Masculino , Metaloporfirinas/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Peroxidase/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Ácido TrinitrobenzenossulfônicoRESUMO
Although it is considered that L-Glutamine (L-Gln) supplementation improves gut morphology and survival in animal models such as radiation and drug-induced enterocolitis, the mechanisms underlying are far from being established. Recently, Gln has been reported to give protection against stress in in vitro intestinal epithelial cell lines through the induction of heat shock proteins (HSPs). This study is designed to examine whether L-Gln may induce cytoprotective molecules such as heme oxygenase-1/HSP32 (HO-1) and reduced glutathione (GSH) in in vivo intestinal tissues, and to clarify whether these molecules may play a role in warm ischemia and reperfusion (I/R) injury. We measured the releases of serotonin and tumor necrosis factor-alpha (TNF-alpha), and graft survival as viability assays following reperfusion of warm ischemically injured intestinal grafts. The substantial expression of HO-1 after L-Gln administration was observed in villous epithelial cells, crypts and muscular layers, and peaked at 6 h, while that of the control group pretreated with lactated Ringer (LR) solution was observed throughout tissues to be slightly similar to those of fresh untreated tissues. Tissue GSH contents slightly increased 24 h after administration and were less reduced through the periods of I/R than those of the LR group. Releases of serotonin and TNF-alpha in L-Gln group were attenuated during the brief periods of warm ischemia, compared with those in the LR group. A significant graft survival rate was also observed between both groups (6/6 of L-Gln group vs. 1/6 of LR group; p < 0.05). In conclusion, the protective effects of L-Gln in small intestines against warm I/R injury were considered to be in part mediated by up-regulation of molecules such as HO-1 and GSH via cellular antioxidant activity. Thus, L-Gln pretreatment may represent an innovative approach to the prevention of complex I/R injury.
Assuntos
Glutamina/farmacologia , Heme Oxigenase (Desciclizante)/fisiologia , Intestino Delgado/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Animais , Indução Enzimática , Sobrevivência de Enxerto/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase-1 , Intestino Delgado/transplante , Masculino , Ratos , Serotonina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Endogenous carbon monoxide (CO) shares with nitric oxide (NO) a role as a putative neural messenger in the brain. Both gases are believed to modulate CNS function via an increase in cytoplasmic cGMP concentrations secondary to the activation of soluble guanylate cyclase (sGC). Recently CO and NO were proposed as a possible mediator of febrile response in hypothalamus. NO has been reported to activate both the constitutive and inducible isoform of the cyclooxygenase (COX). Thus, we investigated whether CO arising from heme catabolism by heme oxygenase (HO) is involved in the febrile response via the activation of COX in the hypothalamus. PGE2 which is a final mediator of febrile response released from primary cultured hypothalamic cells was taken as a marker of COX activity. PGE2 concentration was measured with EIA kits. Exogenous CO (CO-saturated medium) and hemin (a substrate and potent inducer of HO) evoked an increase in PGE2 release from hypothalamic cells, and these effects were blocked by methylene blue (an inhibitor of sGC). And membrane permeable cGMP analogue, dibutyryl-cGMP elicited significant increases in PGE2 release. These results suggest that there may be a functional link between HO and COX enzymatic activities. The gaseous product of hemin through the HO pathway, CO, might play a role through the modulation of the COX activity in the hypothalamus.
Assuntos
Heme Oxigenase (Desciclizante)/fisiologia , Hipotálamo/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Monóxido de Carbono/fisiologia , Células Cultivadas , Dinoprostona/metabolismo , Hemina/farmacologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
In a rat model of free flap transfer local heat shock-priming is able to prevent ischemia/reperfusion-induced inflammatory response by expression of heat shock protein-32.
Assuntos
Heme Oxigenase (Desciclizante)/fisiologia , Hipertermia Induzida , Retalhos Cirúrgicos/imunologia , Síndrome de Resposta Inflamatória Sistêmica/prevenção & controle , Animais , Transplante Ósseo/imunologia , Adesão Celular/imunologia , Endotélio Vascular/imunologia , Heme Oxigenase-1 , Leucócitos/imunologia , Microcirculação/imunologia , Ratos , Ratos Wistar , Síndrome de Resposta Inflamatória Sistêmica/imunologiaRESUMO
Heme oxygenase 2 (HO-2), which synthesizes carbon monoxide (CO), has been localized by immunohistochemistry to endothelial cells and adventitial nerves of blood vessels. HO-2 is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Enzyme studies demonstrated that tin protoporphyrin-9 is a selective inhibitor of HO with approximately 10-fold selectivity for HO over endothelial nitric oxide synthase (NOS) and soluble guanylyl cyclase. Inhibition of HO activity by tin protoporphyrin 9 reverses the component of endothelial-derived relaxation of porcine distal pulmonary arteries not reversed by an inhibitor of NOS. Thus, CO, like NO, may have endothelial-derived relaxing activity. The similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiologic roles for these two mediators.