RESUMO
High-fat diet (HFD)-induced obesity results in bone loss associated with an imbalanced gut microbiota and altered immune status. Probiotics are live microorganisms that are beneficial to the host and are important in maintaining bone health and gut homeostasis. In this study, the probiotic Lactobacillus coryniformis subsp. torquens (T3L) was isolated from traditional yak milk cheese produced in Lhasa and showed distinct acid and bile salt resistance as potential probiotics. Our data indicated that T3L not only reversed HFD-induced gut dysbiosis, as indicated by decreased Firmicutes-to-Bacteroidetes ratios but also reduced bone loss. The anti-obesity, microbiome-modulating, and bone-protective effects were transmissible via horizontal faeces transfer from T3L-treated mice to HFD-fed mice. The protective effects of T3L on bone mass were associated with regulatory T (Treg) cell-mediated inhibition of osteoclast differentiation. Our data indicate that T3L is a regulator of the gut microbiota and bone homeostasis in an animal model.
Assuntos
Microbioma Gastrointestinal , Resistência à Insulina , Probióticos , Animais , Camundongos , Camundongos Obesos , Resistência à Insulina/fisiologia , Obesidade , Dieta Hiperlipídica/efeitos adversos , Probióticos/farmacologia , Camundongos Endogâmicos C57BLRESUMO
AIM: 3-Phenyllactic acid (3-PLA) has been widely used in food and material industries. Three Lactobacillus crustorum strains have shown greater 3-PLA production ability in our previous study. The objectives of this study were to further improve 3-PLA yields in batch and continuous fermentation systems using of free-whole-cells of the three L. crustorum strains. MATERIALS AND RESULTS: The fermentation conditions of free-whole-cells of the three L. crustorum strains for 3-PLA production were optimized. Among these strains, L. crustorum NWAFU 1078 showed excellent reusability and significantly (P < 0·05) greater 3-PLA production ability than the other strains after 10th recycle. The strain possesses three l-lactate dehydrogenase and three d-lactate dehydrogenase catalysing 3-PLA production from phenylpyruvic acid (PPA). Under the optimal conditions, the strain produced 15·2 mmol l-1 3-PLA (76% PPA conversion rate) in a batch fermentation system and 6·5 mmol l-1 h-1 3-PLA (55% PPA conversion rate) in a continuous fermentation system using a 0·6 dilution rate. CONCLUSIONS: Free-whole-cells of L. crustorum NWAFU 1078 showed excellent reusability and higher 3-PLA yields under optimal biotransformation conditions in both batch and continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the possibility to use the free-whole-cells of L. crustorum NWAFU 1078 as a biocatalyst for effective production of 3-PLA.
Assuntos
Lactatos/metabolismo , Lactobacillus/metabolismo , Técnicas de Cultura Celular por Lotes , Biotransformação , Fermentação , Ácidos Fenilpirúvicos/metabolismoRESUMO
BACKGROUND AND PURPOSE: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. EXPERIMENTAL APPROACH: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. KEY RESULTS: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. CONCLUSIONS: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.
Assuntos
Canabidiol/farmacologia , Agonistas de Receptores de Canabinoides , Receptores Acoplados a Proteínas G/agonistas , Vasodilatação/efeitos dos fármacos , Animais , Benzoxazinas/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Canabidiol/análogos & derivados , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Tono Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Naftalenos/farmacologia , Fenilefrina/farmacologia , Cloreto de Potássio/farmacologia , Receptores de Canabinoides/genética , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Resorcinóis/farmacologiaRESUMO
The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression correlates with end-organ protection regardless of overt antihypertensive action.
Assuntos
Biomarcadores/análise , Hipertensão/metabolismo , Osteopontina/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Ensaio de Imunoadsorção Enzimática , Hipertensão/fisiopatologia , Imuno-Histoquímica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Endogâmicos SHRRESUMO
This study tested the hypothesis that increased nitric oxide (NO) inactivation and concurrent peroxynitrite formation is responsible for endothelial dysfunction in the spontaneously hypertensive stroke-prone rat (SHRSP). In SHRSP, the aortic vasorelaxation to acetylcholine (ACh) was decreased (p < 0.05), but NO production was unchanged. Nitrotyrosine staining, a footprint of peroxynitrite (ONOO(-)) formation, was detected. Exposure of SHRSP to a high-salt, high-fat diet (SFD) further exacerbated hypertension and accelerated end-organ disease. A severe endothelial dysfunction [maximal ACh relaxation: 49.8 +/- 2.1 versus 94.5 +/- 1.8% in Wistar-Kyoto rats (WKY), p < 0.01], increased basal NO production (482 +/- 17 versus 356 +/- 21 nM, p < 0.01), decreased ACh-stimulated NO production (57 +/- 6 versus 112 +/- 6 nM, p < 0.01), extensive inducible NO synthase and nitrotyrosine staining, elevated nitrotyrosine content (21-fold increase over WKY), and a high percentage of cells with DNA damage were observed in the aortic tissues from these animals. Treatment of SHRSP on SFD with carvedilol restored ACh-induced vasorelaxation and NO production, inhibited nitrotyrosine formation, reduced vascular cell DNA damage, and reduced end-organ injury. These data demonstrate that endothelial dysfunction was caused by increased NO inactivation alone (SHRSP) or in combination with decreased NO production from endothelial NO synthase (SHRSP on SFD). Antioxidant treatment with carvedilol exerted significant vascular protective effects, attenuated end-organ damage, and decreased mortality under these conditions.
Assuntos
Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Acidente Vascular Cerebral/metabolismo , Tirosina/análogos & derivados , Acetilcolina/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Apoptose/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Carbazóis/farmacologia , Carvedilol , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Oxirredução , Propanolaminas/farmacologia , Ratos , Ratos Endogâmicos SHR , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/mortalidade , Tirosina/metabolismoAssuntos
Benzodiazepinas , Caspases/metabolismo , Fibrinolíticos/farmacologia , Coração/efeitos dos fármacos , Miocárdio/enzimologia , Oligopeptídeos/farmacologia , Piperidinas , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Animais , Caspase 3 , Células Cultivadas/efeitos dos fármacos , Cumarínicos/metabolismo , Ativação Enzimática , Fibrinolíticos/toxicidade , Miocárdio/citologia , Oligopeptídeos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Idoxifene (ID) is a tissue-selective estrogen receptor modulator (SERM). The pharmacological profile of ID in animal studies suggests that it behaves like an estrogen receptor (ER) agonist in bone and lipid metabolism while having negligible ER activity on the reproductive system. It is unknown whether ID retains the vascular protective effects of estrogen. METHODS AND RESULTS: In cultured vascular smooth muscle cells (VSMCs), ID inhibited platelet-derived growth factor-induced DNA synthesis and mitogenesis with IC(50) values of 20.4 and 27.5 nmol/L, respectively. Treatment with ID resulted in S-phase cell cycle arrest in serum-stimulated VSMCs. ID 1 to 100 nmol/L significantly protected endothelial cells from tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in vitro. Virgin Sprague-Dawley rats ovariectomized 1 week before the study were treated with ID (1 mg x kg(-1) x d(-1)) or vehicle by gavage for 3 days before balloon denudation in carotid artery. The SMC proliferation in injured vessels was determined by immunostaining for proliferating cell nuclear antigen (PCNA). The number of PCNA-positive SMCs was reduced by 69%, 82%, and 86% in the media at days 1, 3 and 7, respectively, and by 78% in the neointima at day 7 after injury in ID- versus vehicle-treated group (P:<0.01). ID significantly enhanced reendothelialization in the injured carotid arteries as determined by Evans blue stain and immunohistochemical analysis for von Willebrand factor. In the former assay, the reendothelialized area in injured vessels was 43% in ID-treated group versus 24% in the vehicle group (P:<0.05); in the latter assay, the numbers of von Willebrand factor-positive cells per cross section increased from 24. 8 (vehicle) to 60.5 (ID) (P:<0.01) at day 14 after injury. In addition, the production of nitric oxide from excised carotid arteries was significantly higher in ID-treated than the vehicle group (8.5 versus 2.7 nmol/g, P:<0.01). Finally, ID treatment reduced neointimal area and the ratio of intima to media by 45% and 40%, respectively (P:<0.01), at day 14 after balloon angioplasty. CONCLUSIONS: The results indicate that ID beneficially modulates the balloon denudation-induced vascular injury response. Inhibition of VSMC proliferation and acceleration of endothelial recovery likely mediate this protective effect of ID.
Assuntos
Endotélio Vascular/lesões , Endotélio Vascular/patologia , Moduladores de Receptor Estrogênico/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Ferimentos não Penetrantes/patologia , Adulto , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/cirurgia , Estenose das Carótidas/metabolismo , Estenose das Carótidas/patologia , Estenose das Carótidas/prevenção & controle , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Ovariectomia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Fator de von Willebrand/metabolismoRESUMO
Estrogen is known to stimulate endothelial nitric oxide production and attenuate endothelial dysfunction after ischemia and reperfusion. However, estrogen therapy increases the risk of breast and endometrial cancer. The present study was designed to determine whether idoxifene, a selective estrogen receptor modulator without adverse effects on reproductive organs, may stimulate nitric oxide release and protect endothelial function. In U-46619 precontracted superior mesenteric arterial (SMA) segments isolated from ovariectomized rats, idoxifene and 17 beta-estradiol resulted in a comparable dose-dependent vasorelaxation (maximal relaxation: 75.3 +/- 4.9 and 71 +/- 4.7%, respectively). Treatment of the rings with N(omega)-nitro-L-arginine methyl ester completely blocked idoxifene- and 17 beta-estradiol-induced vasorelaxation. In vitro incubation of SMA rings with TNF alpha significantly reduced vasorelaxation to an endothelium-dependent vasodilator, acetylcholine (maximal relaxation: 73 +/- 3.7 versus 95 +/- 2.9% pre-TNF alpha, P <.01). Idoxifene, but surprisingly not 17 beta-estradiol, prevented TNF alpha-induced endothelial dysfunction (maximal relaxation: 86 +/- 2.6% in idoxifene-treated rings and 77 +/- 5.1% in 17beta-estrogen-treated rings). In vivo ischemia and reperfusion resulted in significant endothelial dysfunction as evidenced by decreased vasorelaxation to acetylcholine (maximal relaxation: 48 +/- 5.5 versus 92 +/- 3.9% in normal SMA rings), but a normal relaxation response to an endothelium-independent vasodilator, acidified NaNO(2) (95 +/- 3.2%). Treatment with idoxifene at either 1 or 2 mg/kg/day, or 17beta-estrogen at 1 mg/kg/day for 4 days significantly preserved endothelial function (P <.01 versus vehicle). Taken together, these results demonstrate that idoxifene is an endothelium-dependent vasodilator and exerts significant endothelial protective effects against TNF alpha- and ischemia-reperfusion-induced endothelial injury. These results suggest that selective estrogen receptor modulators have therapeutic potential in diseases where endothelial dysfunction plays an important role.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Óxido Nítrico/fisiologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Animais , Estradiol/sangue , Estradiol/farmacologia , Feminino , Técnicas In Vitro , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/fisiologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Óxido Nítrico/biossíntese , Óxido Nítrico/sangue , Ovariectomia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/fisiopatologia , Fator de Necrose Tumoral alfa/toxicidade , Vasodilatadores/farmacologiaRESUMO
Carvedilol, a selective alpha(1) and non-selective beta-adrenoceptor antagonist and antioxidant, has been shown to provide significant cardiac protection in animal models of myocardial ischemia. To further explore the mechanisms contributing to the efficacy of carvedilol cardioprotection, the effects of carvedilol on hemodynamic variables, infarct size and myeloperoxidase activity (an index of neutrophil accumulation) were compared with a beta(1) selective adrenoceptor antagonist, bisoprolol. Carvedilol (1 mg/kg) or bisoprolol (1 mg/kg) was given intravenously 5 min before reperfusion. In vehicle-treated rabbits, ischemia (45 min) and reperfusion (240 min) resulted in significant increases in left ventricular end diastolic pressure, large myocardial infarction (64.7+/-2.6% of area-at-risk) and a marked increase in myeloperoxidase activity (64+/-14 U/g protein in area-at-risk). Carvedilol treatment resulted in sustained reduction of the pressure-rate-index and significantly smaller infarcts (30+/-2.9, P<0.01 vs. vehicle) as well as decreased myeloperoxidase activity (26+/-11 U/g protein in area-at-risk, P<0.01 vs. vehicle). Administration of bisoprolol at 1 mg/kg resulted in a pressure-rate-index comparable to that of carvedilol and also decreased infarct size (48.4+/-2.5%, P<0.001 vs. vehicle, P<0.05 vs. carvedilol), although to a significantly lesser extent than that observed with carvedilol. Treatment with bisoprolol failed to reduce myeloperoxidase activity in the ischemic myocardial tissue. In addition, carvedilol, but not bisoprolol, markedly decreased cardiac membrane lipid peroxidation measured by thiobarbituric acid formation. Taken together, this study suggests that the superior cardioprotection of carvedilol over bisoprolol is possibly the result of carvedilol's antioxidant and anti-neutrophil effects, not its hemodynamic properties.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Bisoprolol/farmacologia , Carbazóis/farmacologia , Fármacos Cardiovasculares/farmacologia , Propanolaminas/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Agonistas Adrenérgicos beta/farmacologia , Animais , Antioxidantes/farmacologia , Carvedilol , Creatina Quinase/sangue , Creatina Quinase/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Frequência Cardíaca/efeitos dos fármacos , Isoproterenol/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Lipídeos de Membrana/metabolismo , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Isquemia Miocárdica/prevenção & controle , Miocárdio/enzimologia , Miocárdio/patologia , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Coelhos , Traumatismo por Reperfusão/fisiopatologia , Pressão Ventricular/efeitos dos fármacosRESUMO
The extracellular signal-regulated kinase (ERK) pathway is activated by hypertrophic stimuli in cardiomyocytes. However, whether ERK plays an essential role or is implicated in all major components of cardiac hypertrophy remains controversial. Using a selective MEK inhibitor, U0126, and a selective Raf inhibitor, SB-386023, to block the ERK signaling pathway at two different levels and adenovirus-mediated transfection of dominant-negative Raf, we studied the role of ERK signaling in response of cultured rat cardiomyocytes to hypertrophic agonists, endothelin-1 (ET-1), and phenylephrine (PE). U0126 and SB-386023 blocked ET-1 and PE-induced ERK but not p38 and JNK activation in cardiomyocytes. Both compounds inhibited ET-1 and PE-induced protein synthesis and increased cell size, sarcomeric reorganization, and expression of beta-myosin heavy chain in myocytes with IC(50) values of 1-2 microm. Furthermore, both inhibitors significantly reduced ET-1- and PE-induced expression of atrial natriuretic factor. In cardiomyocytes transfected with a dominant-negative Raf, ET-1- and PE-induced increase in cell size, sarcomeric reorganization, and atrial natriuretic factor production were remarkably attenuated compared with the cells infected with an adenovirus-expressing green fluorescence protein. Taken together, our data strongly support the notion that the ERK signal pathway plays an essential role in ET-1- and PE-induced cardiomyocyte hypertrophy.
Assuntos
Cardiomegalia/enzimologia , Endotelina-1/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenilefrina/farmacologia , Animais , Sequência de Bases , Butadienos/farmacologia , Cardiomegalia/induzido quimicamente , Primers do DNA , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Carvedilol is a vasodilating beta-blocker and antioxidant approved for treatment of mild to moderate hypertension. angina, and congestive heart failure. Metoprolol is a beta1-selective adrenoceptor antagonist. When carvedilol and metoprolol were recently compared in clinical trials for heart failure, each showed beneficial beta-blocker effects such as improved symptoms, quality of life, exercise tolerance, and ejection fraction, with no between-group differences. When thiobarbituric acid reactive substance (TBARS) levels were measured in serum as an indirect marker of free radical activity, there were also no between-group differences. However, we had noted superior cardioprotection by carvedilol in comparison to metoprolol in ischemia and reperfusion models. We therefore examined antioxidant activity directly in cells and tissues. Here we show that in cultured rat cerebellar neurons, and in brain and heart membranes, carvedilol has far greater antioxidant activity than metoprolol, which is essentially inactive as an antioxidant in these model systems. The antioxidant activity of carvedilol could be explained by a greater degree of lipophilicity, as measured by its ClogP value of 3.841 as contrasted to a ClogP value of 1.346 for metoprolol. Alternatively, the molecular structure of carvedilol favors redox recycling, which the structure of metoprolol does not. Therefore, carvedilol could have additional pharmacologic effects that are favorable for long-term therapy.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Antioxidantes/farmacologia , Carbazóis/farmacologia , Metoprolol/farmacologia , Propanolaminas/farmacologia , Animais , Carvedilol , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Radicais Livres/química , Coração/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Three major mammalian mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)-induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/R+PD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury.
Assuntos
Apoptose/fisiologia , Coração/fisiopatologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Interleukin-1beta (IL-1beta) is a pleiotropic cytokine capable of inducing smooth muscle activation and leukocyte recruitment in restenosis and atherosclerosis. Our present study investigated the expression of IL-1beta, IL-1 receptor antagonist (IL-1ra), and IL-1 receptor (IL-1RI and IL-1RII) mRNA in carotid artery after balloon angioplasty using semiquantitative reverse transcription/polymerase chain reaction (RT/PCR) and Northern analysis. Time course studies revealed that IL-1beta mRNA was rapidly induced at 6 h (30-fold increase over control, P < 0.001) post balloon injury and diminished to basal levels at 24 h. The increased expression of IL-1ra mRNA was delayed, reaching a peak at 24 h (400-fold increase, P < 0.001) and sustained up to 14 days. The expression of IL-1RII mRNA was remarkably similar to that of IL-1beta, whereas the IL-1RI (the signaling receptor) mRNA expression was delayed (significantly induced at day 1; 14.2-fold increase, P < 0.01) post balloon injury. Immunohistochemical studies revealed a strong induction of IL-1beta in the area with actively proliferating and migrating smooth muscle cells (i.e., in the inner medial layer at day 1 and in neointima at day 14 after balloon injury). The differential but concomitant expression of IL-1beta, IL-1ra, IL-1RI, and IL-1RII mRNAs after balloon angioplasty suggests that each of these IL-1 system components may play a distinct role in neointima formation.
Assuntos
Angioplastia com Balão , Artérias Carótidas/metabolismo , Interleucina-1/biossíntese , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/biossíntese , Animais , Northern Blotting , Imuno-Histoquímica , Interleucina-1/genética , Cinética , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para CimaRESUMO
Monocyte chemotactic protein-3 (MCP-3) is a CC chemokine that functions in chemoattraction and activation of monocytes, T lymphocytes, eosinophils, basophils, natural killer cells and dendritic cells. The activation of the target cells by MCP-3 is via specific chemokine receptors CCR2 and CCR3, of which CCR2 is shared with MCP-1. MCP-1 and CCR2 have been implicated in vascular diseases including atherosclerosis and restenosis, that are known to be involved in inflammation (accumulation of T lymphocytes and monocytes) and smooth muscle cell (SMC) activation (proliferation, migration and matrix deposition). To investigate a potential role of MCP-3 in vascular injury, the present work examined its mRNA expression in rat aortic SMCs stimulated with various inflammatory stimuli including LPS, TNF-alpha, IL-1beta, IFN-gamma and TGF-beta. A time- and concentration-dependant induction of MCP-3 mRNA in SMCs was observed by means of Northern analysis. A strikingly similar expression profile was observed for MCP-3 and MCP-1 mRNA in SMCs. Furthermore, MCP-3 mRNA expression was induced in rat carotid artery after balloon angioplasty. A significant induction in MCP-3 mRNA was observed in the carotid artery at 6 h (41-fold increase over control, P<0.001), 1 day (13-fold increase, P<0.001) and 3 days (6-fold increase, P<0.01) after balloon angioplasty as quantitated by reverse transcription and polymerase chain reaction. These data provide evidence for the cytokine-induced expression of MCP-3 in SMCs and in carotid artery after balloon angioplasty, suggesting a potential role of MCP-3 in the pathogenesis of restenosis and atherosclerosis.
Assuntos
Angioplastia com Balão , Artérias Carótidas/metabolismo , Citocinas , Proteínas Quimioatraentes de Monócitos/biossíntese , Músculo Liso Vascular/metabolismo , Animais , Aorta , Células Cultivadas , Quimiocina CCL7 , Relação Dose-Resposta a Droga , Substâncias de Crescimento/farmacologia , Interferon gama/farmacologia , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Proteínas Quimioatraentes de Monócitos/genética , Músculo Liso Vascular/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The realization that apoptosis is genetically programmed raises the exciting prospect that modulating apoptosis may provide novel approaches for treatment of cardiovascular diseases in which apoptosis has been demonstrated. Low molecular weight inhibitors of caspases and mitogen-activated protein kinases have been evaluated, with promising results in a variety of cardiovascular apoptotic models.
Assuntos
Apoptose , Doenças Cardiovasculares/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Doenças Cardiovasculares/patologia , Inibidores Enzimáticos/farmacologia , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in apoptotic cell death. The role of p38 MAPK in myocardial injury caused by ischemia/reperfusion, an extreme stress to the heart, is unknown. METHODS AND RESULTS: Studies were performed with isolated, Langendorff-perfused rabbit hearts. Ischemia alone caused a moderate but transient increase in p38 MAPK activity (3.5-fold increase, P<0.05 versus basal). Ischemia followed by reperfusion further activated p38 MAPK, and the maximal level of activation (6.3-fold, P<0.01) was reached 10 minutes after reperfusion. Administration of SB 203580, a p38 MAPK inhibitor, decreased myocardial apoptosis (14.7+/-3.2% versus 30.6+/-3.5% in vehicle, P<0.01) and improved postischemic cardiac function. The cardioprotective effects of SB 203580 were closely related to its inhibition of p38 MAPK. Administering SB 203580 before ischemia and during reperfusion completely inhibited p38 MAPK activation and exerted the most cardioprotective effects. In contrast, administering SB 203580 10 minutes after reperfusion (a time point when maximal MAPK activation had already been achieved) failed to convey significant cardioprotection. Moreover, inhibition of p38 MAPK attenuated myocardial necrosis after a prolonged reperfusion. CONCLUSIONS: These results demonstrate that p38 MAPK plays a pivotal role in the signal transduction pathway mediating postischemic myocardial apoptosis and that inhibiting p38 MAPK may attenuate reperfusion injury.
Assuntos
Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Animais , Apoptose/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Necrose , Piridinas/farmacologia , Coelhos , Fatores de Tempo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Clinical studies conducted with carvedilol suggest that beta-adrenoceptor antagonism is an effective therapeutic approach to the treatment of heart failure. However, many beta-adrenoceptor antagonists are weak partial agonists and possess significant intrinsic sympathomimetic activity (ISA), which may be problematic in the treatment of heart failure. In the present study, the ISAs of bucindolol, xamoterol, bisoprolol, and carvedilol were evaluated and compared in normal rats [Sprague-Dawley (SD)], in rats with confirmed heart failure [spontaneously hypertensive heart failure (SHHF)], and in isolated neonatal rat cardiomyocytes. At equieffective beta1-adrenolytic doses, the administration of xamoterol and bucindolol produced a prolonged, equieffective, and dose-related increase in heart rate in both pithed SD rats (ED50 = 5 and 40 microgram/kg, respectively) and SHHF rats (ED50 = 6 and 30 microgram/kg, respectively). The maximum effect of both compounds in SHHF rats was approximately 50% of that observed in SD rats. In contrast, carvedilol and bisoprolol had no significant effect on resting heart rate in the pithed SD or SHHF rat. The maximum increase in heart rate elicited by xamoterol and bucindolol was inhibited by treatment with propranolol, carvedilol, and betaxolol (beta1-adrenoceptor antagonist) but not by ICI 118551 (beta2-adrenoceptor antagonist) in neonatal rat. When the beta-adrenoceptor-mediated cAMP response was examined in cardiomyocytes, an identical partial agonist/antagonist response profile was observed for all compounds, demonstrating a strong correlation with the in vivo results. In contrast, GTP-sensitive ligand binding and tissue adenylate cyclase activity were not sensitive methods for detecting beta-adrenoceptor partial agonist activity in the heart. In summary, xamoterol and bucindolol, but not carvedilol and bisoprolol, exhibited direct beta1-adrenoceptor-mediated ISA in normal and heart failure rats.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Animais , Animais Recém-Nascidos , Bisoprolol/farmacologia , Carbazóis/farmacologia , Carvedilol , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Estado de Descerebração , Relação Dose-Resposta a Droga , Insuficiência Cardíaca/metabolismo , Iodocianopindolol , Ligantes , Masculino , Miocárdio/metabolismo , Propanolaminas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Xamoterol/farmacologiaRESUMO
TL1 is a recently discovered novel member of the tumor necrosis factor (TNF) cytokine family. TL1 is abundantly expressed in endothelial cells, but its function is not known. The present study was undertaken to explore whether TL1 induces apoptosis in endothelial cells and, if so, to explore its mechanism of action. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to TL1 showed morphological (including ultrastructural) and biochemical features characteristic of apoptosis. TL1-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 72 ng/ml). The effect of TL1 was not inhibited by soluble TNF receptors 1 or 2. TL1 up-regulated Fas expression in BPAEC at 8 and 24 h after treatment, and significantly activated stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (p38 MAPK). The peak activities of SAPK and p38 MAPK in TL1-treated BPAEC were increased by 9- and 4-fold, respectively. TL1-induced apoptosis in the BPAEC was reduced by expression of a dominant-interfering mutant of c-Jun (62.8%, p < 0.05) or by a specific p38 inhibitor, SB203580 (1-10 microM) dose-dependently. TL1 also activated caspases in BPAEC, and TL1-induced apoptosis in BPAEC was significantly attenuated by the caspase inhibitor, ZVAD-fluromethyl-ketone. The major component activated by TL1 in BPAEC was caspase-3, which was based on substrate specificity and immunocytochemical analysis. These findings suggest that TL1 may act as an autocrine factor to induce apoptosis in endothelial cells via activation of multiple signaling pathways, including stress protein kinases as well as certain caspases.
Assuntos
Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspases/metabolismo , Endotélio Vascular/fisiologia , Proteínas Quinases Ativadas por Mitógeno , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Antígenos CD/metabolismo , Caspase 3 , Bovinos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Artéria Pulmonar , Piridinas/farmacologia , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Regulação para Cima , Receptor fas/biossíntese , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The aim of this study was to investigate the effect of SB 211475, a metabolite of carvedilol with weak alpha1-adrenoceptor antagonism and antioxidant effect, on myocardial reperfusion injury and infarct size in anesthetized rabbits. The rabbits were subjected to 60 min of regional myocardial ischemia and 180 min of reperfusion. SB 211475 was administered either as 0.3, 1.0 or 3.0 mg/kg and compared to vehicle and carvedilol (1 mg/kg) treated animals. The lowest dose of SB 211475 (0.3 mg/kg) did not reduce infarct size compared to vehicle, whereas SB 211475 1.0 or 3.0 mg/kg reduced infarct size significantly compared to vehicle (41.2 +/- 2.2% and 40.5 +/- 2.8% vs. 59.1 +/- 3.9%, p < 0.05). Carvedilol reduced infarct size significantly more than SB 211475 1.0 and 3.0 mg/kg (28.8 +/- 3.9% vs. 41.2 +/- 2.2% and 40.5 +/- 2.7%, p < 0.05). Carvedilol and SB 211475 1.0 and 3.0 mg/kg reduced myeloperoxidase activity to the same extent, indicative of reduced inflammation. Rate-pressure product did not differ between doses of SB 211475. In conclusion, SB 211475 in the two highest doses reduced infarct size by protecting from reperfusion injury, possibly by reduced neutrophil accumulation. The superior cardiac protective effect of carvedilol over SB 211475 are most likely due to its adrenergic pharmacology including non-selective beta- and alpha1-adrenoceptor antagonism.
Assuntos
Antagonistas Adrenérgicos alfa/uso terapêutico , Antioxidantes/uso terapêutico , Carbazóis/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Propanolaminas/uso terapêutico , Animais , Antioxidantes/administração & dosagem , Carbazóis/administração & dosagem , Carvedilol , Relação Dose-Resposta a Droga , Hemodinâmica/efeitos dos fármacos , Masculino , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Peroxidase/metabolismo , Propanolaminas/administração & dosagem , CoelhosRESUMO
Focal cerebral ischemia elicits local inflammatory reaction as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain. Interferon-inducible protein-10 (IP-10) is a member of the C-X-C chemokine family that possesses potent chemoattractant actions for monocytes, T cells, and smooth muscle cells. To investigate a potential role of IP-10 in focal stroke, we studied the temporal expression of IP-10 mRNA after occlusion of the middle cerebral artery in rat by means of northern analysis. IP-10 mRNA expression after focal stroke demonstrated a unique biphasic profile, with a marked increase early at 3 h (4.9-fold over control; p < 0.01), a peak level at 6 h (14.5-fold; p < 0.001) after occlusion of the middle cerebral artery, and a second wave induction 10-15 days after ischemic injury (7.2- and 9.3-fold increase for 10 and 15 days, respectively; p < 0.001). In situ hybridization confirmed the induced expression of IP-10 mRNA and revealed its spatial distribution after focal stroke. Immunohistochemical studies demonstrated the expression of IP-10 peptide in neurons (3-12 h) and astroglial cells (6 h to 15 days) of the ischemic zone. To explore further the potential role of IP-10 in focal stroke, we demonstrated a dose-dependent chemotactic action of IP-10 on C6 glial cells and enhanced attachment of rat cerebellar granule neurons. Taken together, the data suggest that ischemia induces IP-10, which may play a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neuron attachment/sprouting after focal stroke.