RESUMO
BACKGROUND: The aim of the present study was to assess the effect of seven days daidzein pretreatment in cecal ligation and puncture (CLP) model of sepsis. METHODS: We assessed the survival benefit of daidzein and its effect on lung injury in CLP-induced sepsis in mice and determined the bacterial load in peritoneal fluid, blood, and lung homogenates. Tumor necrosis factor α (TNF-α) and corticosterone levels were measured by enzyme-linked immunosorbent assay; relative mRNA expression was estimated by real-time polymerase chain reaction, and standard biochemical techniques were used to measure nitrite level, myeloperoxidase activity, and vascular permeability. RESULTS: Daidzein pretreatment for seven days at a dose of 1 mg/kg body weight subcutaneously increased the survival time of septic mice. Daidzein decreased the bacterial load in peritoneal fluid, blood, and lungs, reduced the tumor necrosis factor α and nitrite level in plasma, and partially suppressed lung injury by reducing vascular permeability and myeloperoxidase activity in septic mice. Further, it restored the relative mRNA expressions of inducible nitric oxide synthase, glucocorticoid receptor α, and glucocorticoid receptor ß genes in septic lungs were restored by daidzein pretreatment. CONCLUSIONS: Daidzein pretreatment for 7 d in sepsis increased the survival time in mice, which may be relate to decrease in bacterial load, anti-inflammatory effect, and protection from lung injury.
Assuntos
Isoflavonas/uso terapêutico , Fitoestrógenos/uso terapêutico , Sepse/tratamento farmacológico , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/prevenção & controle , Animais , Carga Bacteriana , Biomarcadores/metabolismo , Ceco/cirurgia , Corticosterona/metabolismo , Esquema de Medicação , Ensaio de Imunoadsorção Enzimática , Injeções Subcutâneas , Masculino , Camundongos , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Peroxidase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sepse/metabolismo , Sepse/microbiologia , Sepse/mortalidade , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The purpose of the present study was to characterize TRPV4 channels in the rat pulmonary artery and examine their role in endothelium-dependent relaxation. Tension, Real-Time polymerase chain reaction (Real-Time PCR) and Western blot experiments were conducted on left and right branches of the main pulmonary artery from male Wistar rats. TRPV4 channel agonist GSK1016790A (GSK) caused concentration-related robust relaxation (Emax 88.6±5.5%; pD2 8.7±0.2) of the endothelium-intact pulmonary artery. Endothelium-denudation nearly abolished the relaxation (Emax 5.6±1.3%) to GSK. TRPV4 channel selective antagonist HC067047 significantly attenuated GSK-induced relaxation (Emax 56.2±6.6% vs. control Emax 87.9±3.3%) in endothelium-intact vessels, but had no effect on either ACh-induced endothelium-dependent or SNP-induced endothelium-independent relaxations. GSK-induced relaxations were markedly inhibited either in the presence of NO synthase inhibitor L-NAME (Emax 8.5±2.7%) or sGC inhibitor ODQ (Emax 28.1±5.9%). A significant portion (Emax 30.2±4.4%) of endothelium-dependent relaxation still persisted in the combined presence of L-NAME and cyclooxygenase inhibitor indomethacin. This EDHF-mediated relaxation was sensitive to inhibition by 60mM K(+) depolarizing solution or K(+) channel blockers apamin (SKCa; KCa2.3) and TRAM-34 (IKCa; KCa3.1). GSK (10(-10)-10(-7)M) caused either modest decrease or increase in the basal tone of endothelium-intact or denuded rings, respectively. We found a greater abundance (>1.5 fold) of TRPV4 mRNA and protein expressions in endothelium-intact vs. denuded vessels, suggesting the presence of this channel in pulmonary endothelial and smooth muscle cells as well. The present study demonstrated that NO and EDHF significantly contributed to TRPV4 channel-mediated endothelium-dependent relaxation of the rat pulmonary artery.
Assuntos
Fatores Biológicos/metabolismo , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Óxido Nítrico/metabolismo , Artéria Pulmonar/fisiologia , Canais de Cátion TRPV/metabolismo , Vasodilatação , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Masculino , Artéria Pulmonar/efeitos dos fármacos , Ratos , Ratos Wistar , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/análise , Canais de Cátion TRPV/antagonistas & inibidores , Vasodilatação/efeitos dos fármacosRESUMO
We investigated the effect of erythropoietin (EPO) posttreatment on survival time and vascular functions in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture. After 20 ± 2 hours of sepsis, thoracic aorta was isolated for assessing its reactivity to norepinephrine (NE) and acetylcholine (ACh). We also measured the tissue nitric oxide (NO) level, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), G protein-coupled receptor kinase 2 (GRK2), and α1D adrenoceptor messenger RNA (mRNA)/protein expression. In septic mice, EPO moderately improved the survival time from 19.68 ± 0.75 to 34.7 ± 3.2 hours. Sepsis significantly decreased the aortic contractile response to NE along with reduced α1D mRNA and protein expression. Erythropoietin significantly preserved the α1D receptor expression and restored NE-induced contractions to control levels in septic mice. Further, it attenuated the aortic α1D receptor desensitization in sepsis which was evident from reduced GRK2 mRNA expression. Accordingly, a selective GRK2 inhibitor markedly restored the contractile responses to NE in sepsis. Erythropoietin treatment attenuated iNOS mRNA expression and iNOS-induced overproduction of NO, but improved endothelium-dependent relaxation to ACh associated with increased eNOS mRNA expression. In conclusion, EPO seems to reverse sepsis-induced vasoplegia to NE through the preservation of α1D adrenoceptor mRNA/protein expression, inhibition of GRK2-mediated desensitization, and attenuation of NO overproduction in the mouse aorta.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Aorta Torácica/efeitos dos fármacos , Eritropoetina/farmacologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Norepinefrina/farmacologia , RNA Mensageiro/metabolismo , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Sepse/tratamento farmacológico , Vasoconstritores/farmacologia , Vasoplegia/prevenção & controle , Animais , Aorta Torácica/enzimologia , Aorta Torácica/fisiopatologia , Ceco/microbiologia , Ceco/cirurgia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/genética , Ligadura , Masculino , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Punções , RNA Mensageiro/genética , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Sepse/complicações , Sepse/enzimologia , Sepse/microbiologia , Sepse/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Vasoplegia/enzimologia , Vasoplegia/etiologia , Vasoplegia/genética , Vasoplegia/fisiopatologiaRESUMO
AIMS: The aim of the present study was to characterize TRPV4 channels in pregnant and nonpregnant mouse uterus and examine their functional role in spontaneous and agonist-induced contractions. MAIN METHODS: We used RT-PCR, Western blot and immunohistochemistry experiments to demonstrate the presence of TRPV4 mRNA and protein, respectively in both pregnant and nonpregnant mouse uterus. Tension experiments were conducted for functional characterization of the TRPV4 channels. KEY FINDINGS: TRPV4 mRNA and protein were detected in both pregnant and nonpregnant mouse uterus with distribution in both endometrium and myometrium. The TRPV4 channel agonist GSK1016790A (GSK) increased myometrial contraction in pregnant (Emax 336.8±21.35%; pD2 7.79±0.29) and nonpregnant (Emax 238±28.13%; pD2 7.61±0.57) animals. HC067047 (1µM), a selective blocker of the TRPV4 channel, antagonized the contractions to GSK in pregnant (Emax 171±18.26%; pD2 6.58±0.37) and nonpregnant (Emax 78.12±9.32%; pD2 7.54±0.9) uteri. Further, HC067047 (1µM) inhibited contractions induced by PGF2α in the pregnant (Emax 183.2±13.94%; pD2 7.01±0.30 versus control Emax 495.7±42.49%; pD2 7.12±0.24) and nonpregnant (Emax 105.3±7.10%; pD2 7.24±0.34 versus control Emax 232.5±12.27%; pD2 7.83±0.29) uteri. SIGNIFICANCE: TRPV4 channels are present in the pregnant and nonpregnant mouse uteri, and their activation by endogenous ligands like prostaglandin increases myometrial contractility. Thus, the TRPV4 channel can be an important target in reducing myometrial contractility in preterm labor.
Assuntos
Contração Muscular/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Contração Uterina/efeitos dos fármacos , Útero/metabolismo , Animais , Western Blotting , Células Cultivadas , Feminino , Técnicas Imunoenzimáticas , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Gravidez , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Útero/citologia , Útero/efeitos dos fármacosRESUMO
We have recently reported that pre-treatment, but not the post-treatment with atorvastatin showed survival benefit and improved hemodynamic functions in cecal ligation and puncture (CLP) model of sepsis in mice. Here we examined whether combined treatment with atorvastatin and imipenem after onset of sepsis can prolong survival and improve vascular functions. At 6 and 18h after sepsis induction, treatment with atorvastatin plus imipenem, atorvastatin or imipenem alone or placebo was initiated. Ex vivo experiments were done on mouse aorta to examine the vascular reactivity to nor-adrenaline and acetylcholine and mRNA expressions of α1D AR, GRK2 and eNOS. Atorvastatin plus imipenem extended the survival time to 56.00±4.62h from 20.00±1.66h observed in CLP mice. The survival time with atorvastatin or imipenem alone was 20.50±1.89h and 27.00±4.09h, respectively. The combined treatment reversed the hyporeactivity to nor-adrenaline through preservation of α1D AR mRNA/protein expression and reversal of α1D AR desensitization mediated by GRK2/Gßγ pathway. The treatment also restored endothelium-dependent relaxation to ACh through restoration of aortic eNOS mRNA expression and NO availability. In conclusion, combined treatment with atorvastatin and imipenem exhibited survival benefit and improved vascular functions in septic mice.
Assuntos
Atorvastatina/administração & dosagem , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Imipenem/administração & dosagem , Sepse/tratamento farmacológico , Animais , Antibacterianos/administração & dosagem , Quimioterapia Combinada , Endotélio Vascular/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Sepse/sangue , Sepse/mortalidade , Taxa de Sobrevida/tendênciasRESUMO
Impaired cardiac ß-adrenoceptor signaling is an important cause of sepsis-induced myocardial depression in man and experimental animals. We examined the effect of atorvastatin (ATR) pretreatment on myocardial ß1-adrenoceptor (ß1-AR) expressions and post-receptor signaling in a mouse model of sepsis (cecal ligation and puncture [CLP]). After 20 ± 2 h of surgery, hearts were isolated for the measurement of left ventricular functions (left ventricular developed pressure, dp/dt(max) and dp/dt(min)) using Langendorff setup. Western blot was used to determine ß1-AR and G protein-coupled receptor kinase 2 protein expressions. Real-time polymerase chain reaction was done to determine ß1-AR mRNA expression. Atorvastatin prevented sepsis-induced decrease in left ventricular functions, such as left ventricular developed pressure (CLP 75.90 ± 0.53 vs. ATR 100.24 ± 1.64 mmHg), dp/dtmax (CLP 3,742 ± 71 vs. ATR 4,291 ± 88 mmHg/s), and dp/dt(min) (CLP -1,010 ± 24 vs. ATR -1,346 ± 84 mmHg/s). Associated with functional impairments, sepsis decreased both myocardial ß1-AR protein and mRNA expressions by 52% ± 9% and 62% ± 7%, respectively. However, ATR treatment of CLP mice (ATR) preserved ß1-AR protein (96% ± 11%) and mRNA (88% ± 14%) expressions comparable to sham-operated level. Furthermore, it not only attenuated sepsis-induced decrease in basal cardiac adenosine 3',5'-cyclic monophosphate content (CLP 1.30 ± 0.27 vs. ATR 6.30 ± 0.67 pmol/mg protein), but also prevented its refractoriness to dobutamine stimulation (CLP 1.72 ± 0.27 vs. ATR 10.83 ± 1.37 pmol/mg protein). Atorvastatin also inhibited sepsis-induced increase in cardiac G protein-coupled receptor kinase 2 protein expression (CLP 1.73 ± 0.18-fold vs. ATR 1.10 ± 0.18-fold), protein kinase A activity (CLP 1.12 ± 0.14 vs. ATR 0.66 ± 0.08 U/mg protein) and plasma catecholamines (CLP 138 ± 22 vs. ATR 59 ± 2 pg/mL). In conclusion, ATR seems to improve left ventricular functions in vitro through the preservation of ß(1)-AR signaling in sepsis.