RESUMO
Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating lipid and glucose metabolism. Ongoing drug discovery programs aim to develop dual PPARα/γ agonists devoid of the side effects of the marketed antidiabetic agents thiazolidinediones and the dual agonists glitazars. Recently, we described a new dual PPARα/γ ligand, LT175, with a partial agonist profile against PPARγ and interacting with a newly identified region of the PPARγ-ligand binding domain (1). Here we show that LT175 differentially activated PPARγ target genes involved in fatty acid esterification and storage in 3T3-L1-derived adipocytes. This resulted in a less severe lipid accumulation compared with that triggered by rosiglitazone, suggesting that LT175 may have a lower adipogenic activity. Consistent with this hypothesis, in vivo administration of LT175 to mice fed a high-fat diet decreased body weight, adipocyte size, and white adipose tissue mass, as assessed by magnetic resonance imaging. Furthermore, LT175 significantly reduced plasma glucose, insulin, non-esterified fatty acids, triglycerides, and cholesterol and increased circulating adiponectin and fibroblast growth factor 21 levels. Oral glucose and insulin tolerance tests showed that the compound improves glucose homeostasis and insulin sensitivity. Moreover, we demonstrate that the peculiar interaction of LT175 with PPARγ affected the recruitment of the coregulators cyclic-AMP response element-binding protein-binding protein and nuclear corepressor 1 (NCoR1), fundamentals for the PPARγ-mediated adipogenic program. In conclusion, our results describe a new PPAR ligand, modulating lipid and glucose metabolism with reduced adipogenic activity, that may be used as a model for a series of novel molecules with an improved pharmacological profile for the treatment of dyslipidemia and type 2 diabetes.
Assuntos
Adipogenia/efeitos dos fármacos , Compostos de Bifenilo/administração & dosagem , Hipoglicemiantes/farmacologia , Resistência à Insulina , Insulina/farmacologia , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/administração & dosagem , Células 3T3-L1 , Animais , Compostos de Bifenilo/metabolismo , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dislipidemias/tratamento farmacológico , Glucose/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/metabolismo , Insulina/sangue , Ligantes , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Correpressor 1 de Receptor Nuclear/metabolismo , PPAR alfa/metabolismo , PPAR gama/metabolismo , Fenilpropionatos/metabolismoRESUMO
This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here.
Assuntos
Camomila/química , Elastase de Leucócito/antagonistas & inibidores , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Extratos Vegetais/farmacologia , Adenocarcinoma/enzimologia , Anti-Inflamatórios/farmacologia , Linhagem Celular Tumoral , Flores/química , Humanos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Neoplasias Gástricas/enzimologia , Transcrição GênicaRESUMO
Neuroactive steroids act in the peripheral nervous system as physiological regulators and as protective agents for acquired or inherited peripheral neuropathy. In recent years, modulation of neuroactive steroids levels has been studied as a potential therapeutic approach to protect peripheral nerves from damage induced by diabetes. Nuclear receptors of the liver X receptor (LXR) family regulate adrenal steroidogenesis via their ability to control cholesterol homeostasis. Here we show that rat sciatic nerve expresses both LRXα and ß isoforms and that these receptors are functional. Activation of liver X receptors using a synthetic ligand results in increased levels of neurosteroids and protection of the sciatic nerve from neuropathy induced by diabetes. LXR ligand treatment of streptozotocin-treated rats increases expression in the sciatic nerve of steroidogenic acute regulatory protein (a molecule involved in the transfer of cholesterol into mitochondria), of the enzyme P450scc (responsible for conversion of cholesterol into pregnenolone), of 5α-reductase (an enzyme involved in the generation of neuroactive steroids) and of classical LXR targets involved in cholesterol efflux, such as ABCA1 and ABCG1. These effects were associated with increased levels of neuroactive steroids (e.g., pregnenolone, progesterone, dihydroprogesterone and 3α-diol) in the sciatic nerve, and with neuroprotective effects on thermal nociceptive activity, nerve conduction velocity, and Na(+), K(+)-ATPase activity. These results suggest that LXR activation may represent a new pharmacological avenue to increase local neuroactive steroid levels that exert neuroprotective effects in diabetic neuropathy.
Assuntos
Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/prevenção & controle , Receptores Nucleares Órfãos/metabolismo , Esteroides/metabolismo , Esteroides/uso terapêutico , Análise de Variância , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Peso Corporal/efeitos dos fármacos , Cromatografia Líquida/métodos , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/etiologia , Ligantes , Receptores X do Fígado , Masculino , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Condução Nervosa/fisiologia , Limiar da Dor , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo , Nervo Isquiático/microbiologia , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Our previous observations have shown that neuroactive steroid levels in the brain are affected by acute experimental autoimmune encephalomyelitis (EAE) with sex and regional specificity (Giatti et al. 2010). To better understand the effect of EAE on neuroactive steroids, we have here assessed the levels of pregnenolone, progesterone and its derivatives (i.e. dihydroprogesterone, tetrahydroprogesterone and isopregnanolone), testosterone and its derivatives (dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol) in different CNS regions of male and female rats affected by chronic EAE. Data obtained by liquid chromatography tandem mass spectrometry revealed that chronic EAE results in sex and regional specific alterations in the levels of neuroactive steroids in the brain, which are in many cases different to those produced by acute EAE. The specific changes in neuroactive steroid levels after chronic EAE may be of relevance to design new possible therapeutic strategies for the disease.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Hormônios Esteroides Gonadais/metabolismo , Caracteres Sexuais , Animais , Doença Crônica , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Feminino , Hormônios Esteroides Gonadais/análise , Cobaias , Masculino , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/fisiopatologia , RatosRESUMO
Alterations in myelin membranes, as well as in the expression of myelin proteins have been reported in experimental models of diabetes. Data here reported show for the first time that the mRNA levels of two isoforms of myelin basic protein (MBP), 18.5 and 21.5 kDa, are decreased in the spinal cord of streptozotocin-treated rats and that treatment with a neuroactive steroid, such as progesterone (P), may counteract this effect. Interestingly, metabolism of progesterone into dihydroprogesterone (DHP) by the enzyme 5alpha-reductase seems to exert an important role in such an effect. As here demonstrated, 5alpha-reductase mRNA and DHP levels are reduced by diabetes in spinal cord, but treatment with P, is able to counteract these effects. Moreover, treatment with DHP is able to mimic the effect of P on MBP gene expression. Thus, the effects of P here observed are due to its enzymatic conversion into DHP. Because DHP, like P, interacts with P receptor (PR), the present results may suggest the importance to analyze the effects of PR modulators as tools of therapeutic strategies for diabetic complications occurring in nervous system.
Assuntos
20-alfa-Di-Hidroprogesterona/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Básica da Mielina/genética , Progesterona/farmacologia , Medula Espinal/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Animais , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/genética , Masculino , Proteína Básica da Mielina/biossíntese , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacosRESUMO
In vivo studies suggest that the phenolic component contributes to the anti-inflammatory and antiatherosclerotic actions of olive oil; however, the effects in circulating cells are not fully characterized. Monocytes play a key role in inflammation-based diseases by expressing several molecules, including metalloproteinases (MMPs). In the present study, we investigated the effects of olive oil phenolic extract and individual compounds on MMP-9 in THP-1 cells, a human monocyte-like cell line. Olive oil extract prevented the stimulation of MMP-9 expression and secretion in tumor necrosis factor alpha-treated THP-1 cells. Oleuropein aglycone, a typical olive oil phenol, was active at concentrations found in the extract, although other compounds probably contribute to the biological activity. We also found that the effect of the extract and individual compounds on MMP-9 is due to impaired nuclear factor-kappaB signaling. Our findings provide further evidence on the mechanisms by which olive oil reduces the inflammatory burden associated with disorders, such as atherosclerosis.
Assuntos
Metaloproteinase 9 da Matriz/genética , NF-kappa B/genética , Fenóis/farmacologia , Óleos de Plantas/química , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Primers do DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide , NF-kappa B/fisiologia , Azeite de Oliva , Fenóis/isolamento & purificação , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Incidence, progression and severity of the multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system (CNS) are affected in a sex-depending way. Physiological situations characterized by changes in sex steroid plasma levels, such as menstrual cycle, menopause or pregnancy, affect the disease course, suggesting that these molecules might exert a role in this disease. In order to understand better this possible relationship, we have here assessed the levels of neuroactive steroids present in different CNS regions of male and female rats affected by acute experimental autoimmune encephalomyelitis (EAE). In addition, we compared these levels with those present in plasma. Data obtained by liquid chromatography-tandem mass spectrometry indicate that the levels of neuroactive steroids show sex and regional differences in control and EAE nervous system and that a clear difference is also observed between CNS and plasma levels. In particular, among neuroactive steroids here considered, the levels of progesterone metabolites (i.e., dihydroprogesterone, tetrahydroprogesterone and isopregnanolone) and testosterone metabolites (i.e., dihydrotestosterone and 5alpha-androstane-3alpha17beta-diol), show sex dimorphic and region-specific changes in the CNS. Moreover, some changes observed in the CNS were not detected in plasma. These findings might represent an interesting background to design therapies and possibly sex-specific therapies for multiple sclerosis based on neuroactive steroids or synthetic ligands able to interact with classical and non-classical steroid receptors.
Assuntos
Encéfalo/metabolismo , Encefalomielite Autoimune Experimental/sangue , Esclerose Múltipla/sangue , Neurotransmissores/sangue , Caracteres Sexuais , 20-alfa-Di-Hidroprogesterona/sangue , Doença Aguda , Androstano-3,17-diol/sangue , Animais , Encéfalo/anatomia & histologia , Encéfalo/fisiopatologia , Cromatografia Líquida , Di-Hidrotestosterona/sangue , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Masculino , Espectrometria de Massas , Esclerose Múltipla/fisiopatologia , Neurotransmissores/análise , Pregnanolona/sangue , Progesterona/metabolismo , Ratos , Testosterona/metabolismoRESUMO
Neuropathy and encephalopathy represent important complications of diabetes. Recent observations obtained in experimental models have suggested that, in male rats, neuroactive steroids are protective agents and that their levels in peripheral (PNS) and central (CNS) nervous system are strongly affected by the disease. It is interesting to highlight that incidence, progression and severity of diabetic neuropathy and diabetic encephalopathy are different in the two sexes. Consequently, it is important to determine the changes in neuroactive steroid levels in the PNS and the CNS of both males and females. To this aim, we have evaluated the levels of neuroactive steroids such as, pregnenolone, progesterone and its metabolites, testosterone and its metabolites, and dehydroepiandrosterone in different CNS regions (i.e., cerebral cortex, cerebellum and spinal cord) and in the sciatic nerve of control and diabetic (i.e., induced by streptozotocin) male and female rats. Data obtained by liquid chromatography-tandem mass spectrometry indicate that the levels of neuroactive steroids show sex and regional differences in control animals. Streptozotocin-induced diabetes resulted in a strong general decrease in neuroactive steroid levels, in both the PNS and the CNS. In addition, the effects of diabetes on neuroactive steroid levels also show sex and regional differences. These findings may have strong implications for the development of new sex-oriented therapies for the treatment of diabetic neuropathy and diabetic encephalopathy, based on the use of neuroactive steroids.
Assuntos
Encefalopatias/metabolismo , Diabetes Mellitus Experimental/metabolismo , Neuropatias Diabéticas/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Fármacos Neuroprotetores/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/patologia , Encefalopatias/prevenção & controle , Estudos de Casos e Controles , Desidroepiandrosterona/metabolismo , Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/patologia , Neuropatias Diabéticas/prevenção & controle , Ciclo Estral/metabolismo , Feminino , Masculino , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/patologia , Pregnenolona/metabolismo , Progesterona/metabolismo , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais , Fatores Sexuais , Medula Espinal/metabolismo , Medula Espinal/patologia , Testosterona/metabolismoRESUMO
AIM OF THE STUDY: Sun-dried rind of the immature fruit of Punica granatum L. (Punicaceae) (Pg) is presently used as a herbal formulation (OMARIA) in Orissa, India, for the therapy and prophylaxis of malaria. The aims of this study were (i) to assess in vitro the antiplasmodial activity of the methanolic extract, of a tannin enriched fraction and of compounds/metabolites of the antimalarial plant, (ii) to estimate the curative efficacy of the Pg extracts and (iii) to explore the mechanism of action of the antiplasmodial compounds. Urolithins, the ellagitannin metabolites, were also investigated for antiplasmodial activity. MATERIALS AND METHODS: Chloroquine-susceptible (D10) and -resistant (W2) strains of Pf were used for in vitro studies and the rodent malaria model Plasmodium berghei-BALB/c mice was used for in vivo assessments. Recombinant plasmepsins 2 and 4 were used to investigate the interference of Pg compounds with the metabolism of haemoglobin by malaria parasites. RESULTS: The Pg methanolic extract (Pg-MeOH) inhibited parasite growth in vitro with a IC(50) of 4.5 and 2.8 microg/ml, for D10 and W2 strain, respectively. The activity was found to be associated to the fraction enriched with tannins (Pg-FET, IC(50) 2.9 and 1.5 microg/ml) in which punicalagins (29.1%), punicalins, ellagic acid (13.4%) and its glycoside could be identified. Plasmepsin 2 was inhibited by Pg-MeOH extract and by Pg-FET (IC(50) 7.3 and 3.0 microg/ml), which could partly explain the antiparasitic effect. On the contrary, urolithins were inactive. Both Pg-MeOH extract and Pg-FET did not show any in vivo efficacy in the murine model. CONCLUSIONS: The in vitro studies support the use of Pg as antimalarial remedy. Possible explanations for the negative in vivo results are discussed.
Assuntos
Antimaláricos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Elágico/farmacologia , Taninos Hidrolisáveis/farmacologia , Lythraceae/química , Malária/tratamento farmacológico , Extratos Vegetais/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antimaláricos/uso terapêutico , Modelos Animais de Doenças , Ácido Elágico/uso terapêutico , Frutas , Hemoglobinas/metabolismo , Taninos Hidrolisáveis/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacosRESUMO
Mechanisms underlying the spasmolytic activity of chamomile still remain unclear. Inhibition of cAMP- and cGMP-phosphodiesterases (PDE) is one of the mechanisms operated by spasmolytic drugs. In this study, the effect of chamomile on PDE was investigated. Human platelet cAMP-PDE and recombinant PDE5A1 were assayed in the presence of infusions prepared from sifted flowers and capitula. LC-ESI-MS/MS analysis showed different compositions in infusions made with sifted flowers and capitula. Chamomile inhibited cAMP-PDE activity (IC50 = 17.9-40.5 microg/mL), while cGMP-PDE5 was less affected (-15% at 50 microg/mL). Among the individual compounds tested, only flavonoids showed an inhibitory effect (IC50 = 1.3-14.9 microM), contributing to around 39% of the infusion inhibition; other compounds responsible for cAMP-PDE inhibition still remain unknown. Although experimental evidence supporting the use of chamomile for gastrointestinal minor spasms dates back to the fifties, cAMP-PDE inhibition as a likely mechanism underlying the spasmolytic activity is reported for the first time.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Matricaria/metabolismo , Parassimpatolíticos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Preparações de Plantas/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Células COS , Chlorocebus aethiops , Flavonoides/farmacologia , Flores/química , Flores/metabolismo , Humanos , Matricaria/química , Parassimpatolíticos/química , Inibidores de Fosfodiesterase/química , Preparações de Plantas/químicaRESUMO
The aim of the present study was to confirm that olive oil phenols reduce human platelet aggregability and to verify the hypothesis that cAMP- and cGMP- phosphodiesterases (PDE) could be one of the targets of the biological effect. Four extracts from oils characterized by a high phenol content (HPE), and low phenol levels (LPE) were prepared and analyzed qualitatively and quantitatively by HPLC-UV and electrospray ionization-MS/MS. Human washed platelets stimulated with thrombin were used for the aggregation assay. Human platelet cAMP-PDE and recombinant PDE5A1 were used as enzyme source. Platelet aggregation and enzyme activity were assayed in the presence of HPE, LPE and individual phenols. The phenol content of HPE ranged between 250 and 500 mg/kg, whereas the LPE content was 46 mg/kg. The compounds identified were hydroxytyrosol (HT), tyrosol (TY), oleuropein aglycone (OleA) and the flavonoids quercetin (QU), luteolin (LU) and apigenin (AP). OleA was the most abundant phenol (range 23.3 to 37.7 %) and LU was the most abundant flavonoid in the extracts. Oil extracts inhibited platelet aggregation with an 50% inhibitory concentration interval of 1.23-11.2 microg/ml. The inhibitory effect of individual compounds (10 microm) including homovanillyl alcohol (HVA) followed this order: OleA>LU>HT = TY = QU = HVA, while AP was inactive. All the extracts inhibited cAMP-PDE, while no significant inhibition of PDE5A1 (50 microg/ml) was observed. All the flavonoids and OleA inhibited cAMP-PDE, whereas HT, TY, HVA (100 microm) were inactive. Olive oil extracts and part of its phenolic constituents inhibit platelet aggregation; cAMP-PDE inhibition is one mechanism through which olive oil phenols inhibit platelet aggregation.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Fenóis/farmacologia , Óleos de Plantas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , 3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Plaquetas/enzimologia , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Azeite de Oliva , Fenóis/análise , Extratos Vegetais/farmacologia , Óleos de Plantas/químicaRESUMO
The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol), 17alpha- and 17beta-estradiol (17alpha-E and 17beta-E) by liquid chromatography and tandem mass spectrometry (LC-MS/MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17alpha-E in cerebellum, 17alpha-E, 17beta-E, DHP and THP in cerebral cortex, and 17alpha-E, 17beta-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3alpha-diol in cerebral cortex, of PROG, DHT and 3alpha-diol in spinal cord and of PREG, DHP, THP, T, DHT and 3alpha-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC-MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy.