RESUMO
We were intrigued to analyze donor eyes of two individuals without retinopathy even after 40 years of type 2 diabetes mellitus. Targeted molecular factors associated with angiogenesis and the key antioxidant enzymes in retinal tissue were analyzed. Accordingly PEDF, Adiponectin and Paraoxonase 2 showed augmented mRNA expression in both the retina with no significant change in VEGF expression. Vitreous showed increased PEDF protein in donor 1 and Adiponectin in donor 2 with no change in VEGF protein. This study highlights the profile of specific molecular factors that contribute to the non-development of diabetic retinopathy changes in these individuals.
Assuntos
Adiponectina/biossíntese , Arildialquilfosfatase/biossíntese , Diabetes Mellitus Tipo 2/diagnóstico , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica , Fatores de Crescimento Neural/biossíntese , Retina/patologia , Serpinas/biossíntese , Doadores de Tecidos , Adiponectina/genética , Idoso de 80 Anos ou mais , Arildialquilfosfatase/genética , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas do Olho/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural/genética , Estresse Oxidativo , RNA/genética , Retina/metabolismo , Doenças Retinianas , Serpinas/genéticaRESUMO
In addition to increased morbidity and mortality caused by respiratory and cardiovascular diseases, air pollution may also contribute to central nervous system (CNS) diseases. Traffic-related air pollution is a major contributor to global air pollution, and diesel exhaust (DE) is its most important component. DE contains more than 40 toxic air pollutants and is a major constituent of ambient particulate matter (PM), particularly of ultrafine-PM. Limited information suggests that exposure to DE may cause oxidative stress and neuroinflammation in the CNS. We hypothesized that males may be more susceptible than females to DE neurotoxicity, because of a lower level of expression of paraoxonase 2 (PON2), an intracellular anti-oxidant and anti-inflammatory enzyme. Acute exposure of C57BL/6 mice to DE (250-300µg/m3 for 6h) caused significant increases in lipid peroxidation and of pro-inflammatory cytokines (IL-1α, IL-1ß, IL-3, IL-6, TNF-α) in various brain regions (particularly olfactory bulb and hippocampus). In a number of cases the observed effects were more pronounced in male than in female mice. DE exposure also caused microglia activation, as measured by increased Iba1 (ionized calcium-binding adapter molecule 1) expression, and of TSPO (translocator protein) binding. Mice heterozygotes for the modifier subunit of glutamate cysteine ligase (the limiting enzyme in glutathione biosynthesis; Gclm+/- mice) appeared to be significantly more susceptible to DE-induced neuroinflammation than wild type mice. These findings indicate that acute exposure to DE causes neuroinflammation and oxidative stress in brain, and suggest that sex and genetic background may play important roles in modulating susceptibility to DE neurotoxicity.
Assuntos
Poluentes Atmosféricos/toxicidade , Química Encefálica/efeitos dos fármacos , Encéfalo/patologia , Inflamação/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Emissões de Veículos/toxicidade , Animais , Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/genética , Citocinas/biossíntese , Feminino , Variação Genética , Glutamato-Cisteína Ligase/biossíntese , Glutamato-Cisteína Ligase/genética , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndromes Neurotóxicas/patologia , Tamanho da Partícula , Material Particulado/toxicidade , Caracteres SexuaisRESUMO
Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.
Assuntos
Antioxidantes/metabolismo , Arildialquilfosfatase/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas Oncogênicas/biossíntese , Doença de Parkinson/genética , Animais , Apoptose/genética , Arildialquilfosfatase/genética , Sobrevivência Celular , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Oncogênicas/genética , Estresse Oxidativo , Doença de Parkinson/patologia , Proteína Desglicase DJ-1RESUMO
Post-translational modification (PTM) of proteins plays a crucial role in health and disease by affecting numerous aspects of protein structure, function, stability and subcellular localization. Protein S-nitrosylation is one type of PTM that involves the covalent modification of cysteine sulfhydryl groups with nitric oxide (NO) and has a regulatory impact similar to phosphorylation. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDL's antiatherogenic properties. The enzyme contains a free thiol group at Cys-284 which can also be modified covalently. As part of our effort to study the effect of PTMs on PON1 activities and properties and its implication for cardiovascular disease, we examined PON1's ability to undergo S-nitrosylation on its free Cys-284. Recombinant (re) PON1 was trans-S-nitrosylated by several NO donors, glutathione-NO (GSNO) was found to be the most effective. The S-nitrosylated rePON1 was analyzed by Q-TOF LC/MS and by Saville-Griess assay: the two analytical methods revealed closely similar results. rePON1 was also nitrosylated by nitrosylated human serum albumin (HSA-NO) via protein-protein trans-nitrosylation. HSA-NO transferred an NO group to rePON1 much more efficiently than GSNO with the formation of a higher than 70% rePON-NO when incubated with a 40-fold excess of a HSA-NO/HSA mixture. RePON1-NO was relatively stable: storage for 3days at 37°C resulted in only 25% decomposition. This is the first report of PON1's S-nitrosylation via GSNO and HSA-NO.
Assuntos
Arildialquilfosfatase/biossíntese , Doadores de Óxido Nítrico/metabolismo , Arildialquilfosfatase/metabolismo , Cromatografia Líquida , Humanos , Espectrometria de Massas , Fosforilação , S-Nitrosoglutationa/metabolismoRESUMO
Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and the detection of SCLCs at an early stage is necessary for successful therapy and for improving cancer survival rates. Fucosylation is one of the most common glycosylation-based modifications. Increased levels of fucosylation have been reported in a number of pathological conditions, including cancers. In this study, we aimed to identify and validate the aberrant and selective fucosylated glycoproteins in the sera of patients with SCLC. Fucosylated glycoproteins were enriched by the Aleuria aurantia lectin column after serum albumin and IgG depletion. In a narrowed down and comparative data analysis of both label-free proteomics and isobaric peptide-tagging chemistry iTRAQ approaches, the fucosylated glycoproteins were identified as up- or down-regulated in the sera of limited disease and extensive disease stage patients with SCLC. Verification was performed by multiple reaction monitoring-mass spectrometry to select reliable markers. Four fucosylated proteins, APCS, C9, SERPINA4, and PON1, were selected and subsequently validated by hybrid A. aurantia lectin ELISA (HLE) and Western blotting. Compared with Western blotting, the HLE analysis of these four proteins produced more optimal diagnostic values for SCLC. The PON1 protein levels were significantly reduced in the sera of patients with SCLC, whereas the fucosylation levels of PON1 were significantly increased. Fucosylated PON1 exhibited an area under curve of 0.91 for the extensive disease stage by HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The glycan structural analysis of PON1 by MS/MS identified a biantennary fucosylated glycan modification consisting of a core + 2HexNAc + 1Fuc at increased levels in the sera of patients with SCLC. In addition, the PON1 levels were decreased in the sera of the Lewis lung carcinoma lung cancer mouse model that we examined. Our data suggest that fucosylated protein biomarkers, such as PON1, and their fucosylation levels and patterns can serve as diagnostic and prognostic serological markers for SCLC.
Assuntos
Arildialquilfosfatase/sangue , Glicoproteínas/sangue , Proteômica , Carcinoma de Pequenas Células do Pulmão/genética , Adulto , Idoso , Arildialquilfosfatase/biossíntese , Biomarcadores Tumorais/sangue , Feminino , Regulação Neoplásica da Expressão Gênica , Glicosilação , Humanos , Lectinas/metabolismo , Masculino , Pessoa de Meia-Idade , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/patologia , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: Paraoxonase-2 (PON-2) is an intracellular antioxidant enzyme that can be modulated by polyphenols. The aim of this study was to verify whether yerba mate (Ilex paraguariensis), a plant species rich in phenolic compounds, modulates gene expression and the activity of PON-2 in macrophages in vitro and in monocytes from peripheral blood and monocyte-derived macrophages obtained after the ingestion of green or roasted yerba mate infusions by healthy subjects. METHODS: THP-1 macrophages were incubated with increasing amounts of yerba mate extracts or chlorogenic and caffeic acids (1-10 µmol/L). The in vivo effects of yerba mate or water (control) intakes were evaluated acutely (2 h after ingestion) and in the short term (after daily ingestion for 7 d) in 20 healthy women. RESULTS: In general, there was no difference between the two kinds of yerba mate studied. Yerba mate extracts or chlorogenic acid at 1 and 3 µmol/L increased PON-2 relative gene expression in THP-1 macrophages (P < 0.05), whereas higher concentrations (5 and 10 µmol/L) increased the activity only. Caffeic acid induced PON-2 activity only. The acute ingestion of yerba mate infusions increased relative gene expression and PON-2 activity in monocytes (P < 0.05), whereas the consumption of yerba mate for 7 d increased PON-2 relative gene expression (P < 0.05) and had a tendency to increase PON-2 activity in monocytes and monocyte-derived macrophages. CONCLUSION: It is suggested that green or roasted yerba mate modulates positively the mRNA relative expression and activity of the PON-2 enzyme in monocytes and macrophages, which may prevent cellular oxidative stress.
Assuntos
Arildialquilfosfatase/biossíntese , Bebidas , Indução Enzimática , Ilex paraguariensis/química , Macrófagos/enzimologia , Folhas de Planta/química , Adolescente , Adulto , Arildialquilfosfatase/sangue , Arildialquilfosfatase/genética , Brasil , Ácidos Cafeicos/metabolismo , Células Cultivadas , Ácido Clorogênico/metabolismo , Feminino , Manipulação de Alimentos , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/enzimologia , Monócitos/metabolismo , Concentração Osmolar , Extratos Vegetais/química , Extratos Vegetais/metabolismo , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.
Assuntos
Apoptose , Arildialquilfosfatase/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/biossíntese , Neoplasias/enzimologia , Superóxidos/metabolismo , Animais , Arildialquilfosfatase/genética , Citocromos c/genética , Citocromos c/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Células HEK293 , Humanos , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Regulação para Cima/genéticaRESUMO
Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme, synthesized in the liver and secreted into the blood. PON1 displays antioxidant properties and is involved in organophosphorous compounds and oxidized lipids degradation. Because of these beneficial effects, pharmacological regulation of PON1 appears to be highly relevant in toxicology and cardiology. Recent studies undertaken on the regulation of the PON1 promoter in our laboratory have identified resveratrol, through its activation of the Aryl hydrocarbon Receptor (AhR), as a putative inducer of PON1. We have tested a new modulator of AhR, (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile, and established that it is a more potent inducer of PON1 at the mRNA, protein and enzymatic activity as compared to resveratrol. It also acts by activating the AhR. However, in contrast with traditional AhR agonists, it does not induce cyp1A1 transcription. (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile is therefore a specific AhR modulator targeting PON1.
Assuntos
Acrilonitrila/análogos & derivados , Arildialquilfosfatase/biossíntese , Receptores de Hidrocarboneto Arílico/agonistas , Estilbenos/farmacologia , Acrilonitrila/química , Acrilonitrila/farmacologia , Arildialquilfosfatase/genética , Arildialquilfosfatase/metabolismo , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Estrutura Molecular , Resveratrol , Estilbenos/químicaRESUMO
The aims of this study were to characterize the expression of paraoxonase 2 (PON2) in mouse brain and to assess its antioxidant properties. PON2 levels were highest in the lung, intestine, heart and liver, and lower in the brain; in all tissues, PON2 expression was higher in female than in male mice. PON2 knockout [PON2(-/-)] mice did not express any PON2, as expected. In the brain, the highest levels of PON2 were found in the substantia nigra, the nucleus accumbens and the striatum, with lower levels in the cerebral cortex, hippocampus, cerebellum and brainstem. A similar regional distribution of PON2 activity (measured by dihydrocoumarin hydrolysis) was also found. PON3 was not detected in any brain area, while PON1 was expressed at very low levels, and did not show any regional difference. PON2 levels were higher in astrocytes than in neurons isolated from all brain regions, and were highest in cells from the striatum. PON2 activity and mRNA levels followed a similar pattern. Brain PON2 levels were highest around birth, and gradually declined. Subcellular distribution experiments indicated that PON2 is primarily expressed in microsomes and in mitochondria. The toxicity in neurons and astrocytes of agents known to cause oxidative stress (DMNQ and H(2)O(2)) was higher in cells from PON2(-/-) mice than in the same cells from wild-type mice, despite similar glutathione levels. These results indicate that PON2 is expressed in the brain, and that higher levels are found in dopaminergic regions such as the striatum, suggesting that this enzyme may provide protection against oxidative stress-mediated neurotoxicity.
Assuntos
Arildialquilfosfatase/fisiologia , Encéfalo/enzimologia , Fármacos Neuroprotetores/metabolismo , Animais , Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/metabolismo , Western Blotting , Encéfalo/fisiologia , Corpo Estriado/enzimologia , Corpo Estriado/fisiologia , Feminino , Glutationa/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/enzimologia , Núcleo Accumbens/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/enzimologia , Substância Negra/enzimologia , Substância Negra/fisiologiaRESUMO
1. Formaldehyde (FA) has been found to cause toxicity to neurons. However, its neurotoxic mechanisms have not yet been clarified. Increasing evidence has shown that oxidative damage is one of the most critical effects of formaldehyde exposure. Paraoxonase-1 (PON-1) is a pivotal endogenous anti-oxidant. Thus, we hypothesized that FA-mediated downregulation of PON1 is associated with its neurotoxicity. 2. In the present work, we used PC12 cells to study the neurotoxicity of FA and explore whether PON-1 is implicated in FA-induced neurotoxicity. 3. We found that FA has potent cytotoxic and apoptotic effects on PC12 cells. FA induces an accumulation of intracellular reactive oxygen species along with downregulation of Bcl-2 expression, as well as increased cytochrome c release. FA significantly suppressed the expression and activity of PON-1 in PC12 cells. Furthermore, H(2)S, an endogenous anti-oxidant gas, antagonizes FA-induced cytotoxicity as well as 2-hydroxyquinoline, a specific inhibitor of PON-1, which also induces cytotoxicity to PC12 cells. 4. The results of the present study provide, for the first time, evidence that the inhibitory effect on PON-1 expression and activity is involved in the neurotoxicity of FA, and suggest a promising role of PON-1 as a novel therapeutic strategy for FA-mediated toxicity.
Assuntos
Arildialquilfosfatase/metabolismo , Formaldeído/toxicidade , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/enzimologia , Síndromes Neurotóxicas/etiologia , Animais , Apoptose/efeitos dos fármacos , Arildialquilfosfatase/antagonistas & inibidores , Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/genética , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Citocromos c/genética , Citocromos c/metabolismo , Regulação para Baixo/genética , Formaldeído/efeitos adversos , Formaldeído/metabolismo , Sulfeto de Hidrogênio/farmacologia , Hidroxiquinolinas/farmacologia , Neurônios/enzimologia , Síndromes Neurotóxicas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Hipersensibilidade Respiratória/enzimologia , Hipersensibilidade Respiratória/metabolismo , Proteína de Morte Celular Associada a bcl/antagonistas & inibidores , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Human serum paraoxonase 1 (PON1), an arylesterase, is associated with high-density lipoprotein (HDL) and can inhibit the oxidative modification of low-density lipoprotein (LDL), implying that PON1 may prevent atherosclerosis. Berberine, a botanical alkaloid, lowers the cholesterol level in serum and is thought to display cardioprotective properties. However, the effect of berberine on PON1 gene expression remains unclear. Thus, we evaluated how berberine regulates PON1 gene expression. In human hepatoma HepG2 and Huh7 cells, the PON1 protein levels were increased by berberine in a dose- and time-dependent manner. Data from real time PCR analysis indicated that berberine could up-regulate PON1 expression at the transcriptional level. Additionally, treating HepG2 cells with berberine increased the levels of phosphorylated JNK and its downstream target c-Jun. The PON1 upstream region contained a consensus binding site for AP1, and the electrophoretic mobility shift assay and chromatin immunoprecipitation analysis indicated that the AP1 factors, especially c-Jun, bind to the upstream sequence of the PON1 promoter upon berberine treatment. Moreover, pretreatment with SP600125 (JNK inhibitor) or curcumin (AP-1 inhibitor) markedly attenuated the berberine-induced PON1 promoter activity and protein expression. This is the first study to suggest that JNK/c-Jun signalling pathway plays a crucial role in berberine-regulated PON1 transcription in human hepatoma cells. The induction of PON1 by berberine elucidates a potential mechanism through which berberine may protect against atherosclerosis.
Assuntos
Arildialquilfosfatase/biossíntese , Berberina/farmacologia , Hepatócitos/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Arildialquilfosfatase/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de Sinais , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Regulação para CimaRESUMO
There is increasing evidence that the HDL-associated enzyme paraoxonase 1 (PON1) may have a protective function in the atherosclerotic process. An enhancement of PON1 activity by dietary factors including flavonoids is therefore of interest. Quercetin, a flavonol frequently present in fruits and vegetables has been shown to induce PON1 in cultured liver cells, but the in vivo efficacy of a dietary quercetin supplementation has yet not been evaluated. To this end, we fed laboratory mice quercetin-enriched diets with quercetin concentrations ranging from 0.05 to 2 mg/g diet for 6 weeks and determined the expression of the hepatic PON1 gene and its protein levels. Since we could establish a moderate but significant induction of PON1 mRNA levels by dietary quercetin in mice, we aimed to proof whether healthy human volunteers, given graded supplementary quercetin (50, 100 or 150 mg/day) for two weeks, would respond with likewise enhanced plasma paraoxonase activities. However, PON1 activity towards phenylacetate and paraoxon was not changed following quercetin supplementation in humans. Differences between mice and humans regarding the PON1 inducing activity of quercetin may be related to differences in quercetin metabolism. In mice, unlike in humans, a large proportion of quercetin is methylated to isorhamnetin which exhibits, according to our reporter gene data in cultured liver cells, a potent PON1 inducing activity.
Assuntos
Arildialquilfosfatase/metabolismo , Quercetina/farmacologia , Adulto , Animais , Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/genética , Método Duplo-Cego , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quercetina/administração & dosagem , Quercetina/metabolismo , Especificidade da Espécie , Células Tumorais Cultivadas , Adulto JovemRESUMO
PON3 is a member of the paraoxonase gene family that includes PON1 and PON2. For example, PON3 and PON1 share approximately 60% identity at the amino acid level. Recent studies have demonstrated that PON3 is present in human and rabbit HDL but not in mouse HDL. Mouse PON3 appears to be cell-associated and is expressed in a wide range of tissues such as liver, adipose, macrophage, and the artery wall. In vitro studies have shown that PON3 can prevent LDL oxidation and destroy bacterial quorum-sensing molecules. Previous studies also showed that human PON3 transgenic mice were protected from obesity and atherosclerosis in both the C57BL/6 J wild-type and LDLR knockout genetic background. Administration of adenovirus expressing the human PON3 gene into apoE -/- mice also decreased atherosclerotic lesion formation. In order to further understand the functions of PON3 in physiology and disease, we performed in situ hybridization analysis to examine Pon3 gene expression patterns in newborn and adult mice, in various tissues, including atherosclerotic lesions of apoE -/- mice. Our results show relatively high levels of Pon3 mRNA labeling in the adrenal gland, submaxillary gland, lung, liver, adipose, pancreas, large intestine, and other tissues of newborn mice. In the adult mouse, Pon3 mRNA levels were much lower in the corresponding tissues as mentioned above for the newborn mouse. Sections of the aortic root from the hearts of both wild-type and apoE -/- mice displayed moderate levels of Pon3 mRNA labeling. Pon3 mRNA was also detected in the atherosclerotic lesion areas at the aortic root of apoE -/- hearts. Our data revealed that mouse Pon3 is expressed in a wide range of tissues, and that its expression is temporally controlled.
Assuntos
Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/genética , Animais , Regulação da Expressão Gênica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Miocárdio/metabolismo , RNA Complementar/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Distribuição TecidualRESUMO
OBJECTIVE: The present study investigates the role of paraoxonase 2 (PON2) in the attenuation of macrophage triglycerides (TG) biosynthesis, and oxidative stress, under diabetic conditions. METHODS: Peritoneal macrophages (MPM) from PON2-deficient and from C57BL/6 control mice were harvested and cultured under normal (5mM) or high glucose concentration (30mM), and evaluated for cellular TG metabolism as well as for their oxidative stress. RESULTS: In PON2-deficient MPM vs. control MPM, under diabetic conditions (high glucose concentration), we observed substantial increment in TG accumulation (3 fold), TG biosynthesis (2.6 fold) and microsomal diacylglycerol acyltransferase1 (DGAT1) activity (+60%). Furthermore, in these cells we have demonstrated increased oxidative stress, as expressed by significant increment in cellular oxidative stress (+25%), macrophage-mediated LDL oxidation (+41%) and expression of the receptor for advanced glycation end products - RAGE (+18%). Apocynin, an NADPH-oxidase inhibitor, abolished the increment in MPM TG accumulation, MPM TG biosynthesis, and microsomal DGAT1 activity, as a result of PON2-deficiency, under diabetic conditions. CONCLUSION: We conclude that PON2 has a significant protective role against macrophage triglyceride accumulation, macrophage TG biosynthesis, microsomal DGAT1 activity and macrophage oxidative stress, under high glucose concentrations. We suggest that this protective effect may be mediated by PON2 through the attenuation of NADPH-oxidase activity. The use of appropriate means to increase macrophage PON2 expression can lead to attenuation in macrophage TG accumulation and in cellular oxidative stress, under diabetic conditions, and thus may contribute to the decrement in macrophage atherogenicity and foam cell formation, attenuating the development of vascular complications in diabetes mellitus.
Assuntos
Arildialquilfosfatase/genética , Arildialquilfosfatase/fisiologia , Diacilglicerol O-Aciltransferase/metabolismo , Glucose/metabolismo , Macrófagos/metabolismo , NADPH Oxidases/metabolismo , Triglicerídeos/metabolismo , Animais , Arildialquilfosfatase/biossíntese , Colesterol/metabolismo , Diabetes Mellitus Experimental , Camundongos , Camundongos Endogâmicos C57BL , Microssomos/metabolismo , Estresse Oxidativo , Oxigênio/química , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: Serum paraoxonase-1 (PON1) expression is regulated by polyphenols, shown to activate the peroxisome proliferator-activated receptor gamma (PPARgamma). Pomegranate juice (PJ) is a polyphenol-rich fruit. Because promoter sequence of PON1 gene indicates that it could be regulated by nuclear receptors, we investigated the effect of PJ polyphenols on PON1 gene expression in HuH7 hepatocytes. METHODS AND RESULTS: PON1 protein or mRNA expression, determined by immunocytochemistry, or quantitative PCR, respectively, as well as PON1 gene promoter activation, was significantly increased in hepatocytes incubated with PJ or with its major polyphenols punicalagin, or gallic acid (GA). Ellagic acid (EA) elicited only modest stimulatory effect. Accordingly, PJ, punicalagin, GA, and less so EA, dose-dependently increased cell-associated and hepatocyte-secreted PON1 arylesterase activity. Functionally, the secreted PON1 exhibited biological activity by protecting LDL and HDL from oxidation. Finally, PJ polyphenols upregulated the hepatocyte PON1 expression, at least in part, via the intracellular signaling cascade PPARgamma-PKA-cAMP. CONCLUSIONS: This study shows for the first time that PJ polyphenols have a specific transcriptional role in hepatocyte PON1 expression upregulation, and its secretion to the medium. We conclude that the anti-atherogenic characteristics of PJ polyphenols are modulated, at least in part, via hepatocyte PON1 upregulation and its subsequent release to the medium.
Assuntos
Arildialquilfosfatase/biossíntese , Ácido Elágico/farmacologia , Ácido Gálico/farmacologia , Hepatócitos/enzimologia , Taninos Hidrolisáveis/farmacologia , Lythraceae , PPAR gama/fisiologia , Regulação para Cima , Células Cultivadas , HumanosRESUMO
OBJECTIVE: The parasympathetic nervous system regulates inflammation in peripheral tissues through a pathway termed the "cholinergic anti-inflammatory reflex" (CAIR). Mice deficient in the alpha 7 nicotinic acetylcholine receptor (alpha7(-/-)) have an impaired CAIR due to decreased signaling through this pathway. The purpose of this study was to determine if the increased inflammation in alpha7(-/-) mice is associated with enhanced serum and macrophage atherogenicity. METHODS: We measured serum markers of inflammation and oxidative stress, and macrophage atherogenicity in mouse peritoneal macrophages harvested from alpha7(-/-) mice on the background of C57BL/6 mice, as well as on the background of the atherosclerotic Apolipoprotein E-deficient (ApoE(-/-)) mice. RESULTS: alpha7-Deficiency had no significant effects on serum cholesterol, or on markers of serum oxidative stress (TBARS and paraoxonase1 activities). However, alpha7-deficiency significantly increased serum CRP and IL-6 (p<0.05) levels in atherosclerotic mice, confirming an anti-inflammatory role for the alpha7 receptor. Macrophage cholesterol mass was increased by 25% in both normal and atherosclerotic mice in the absence of the alpha7 receptor (p<0.05). This was accompanied by conditional increases in oxidized LDL uptake and in macrophage total peroxide levels. Furthermore, alpha7-deficiency reduced macrophage paraoxonase2 mRNA and activity by 50-100% in normal and atherosclerotic mice (p<0.05 for each), indicating a reduction in macrophage anti-oxidant capacity in the alpha7(-/-) mice. CONCLUSION: The above results suggest an anti-atherogenic role for the macrophage alpha7nAchr, through a mechanism that involves attenuated macrophage oxidative stress and decreased uptake of oxidized LDL.
Assuntos
Arildialquilfosfatase/biossíntese , Aterosclerose/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Receptores Nicotínicos/fisiologia , Animais , Aterosclerose/etiologia , Colesterol/sangue , Inflamação/complicações , Inflamação/enzimologia , Inflamação/metabolismo , Macrófagos/enzimologia , Camundongos , Camundongos Knockout , Estresse Oxidativo , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Reactive oxygen species (ROS) play an important role as mediators of pulmonary damage in mineral dust-induced diseases. Studies carried out to date have largely focused on silica-induced production of ROS by lung phagocytes. In this study we investigated the hypothesis that crystalline silica Min-U-Sil 5 can induce elevations in intracellular ROS in human bronchial epithelial cells BEAS-2B, via an indirect mechanism that involves ROS-inducing intracellular factors, through a reduction of antiglycation (glyoxalase enzymes) and antioxidant (paraoxonase 1 and glutathione-S-transferases) enzymatic defenses. The results show that crystalline silica Min-U-Sil 5 causes a significant reduction in the efficiency of antiglycation and antioxidant enzymatic defenses, paralleled by an early and extensive ROS generation, thus preventing the cells from an efficient scavenging action, and eliciting oxidative damage. These results confirm the importance of ROS in development of crystalline silica-induced oxidative stress and emphasize the pivotal role of antiglycation/antioxidant and detoxifying systems in determining the level of protection from free radicals-induced injury for cells exposed to crystalline silica Min-U-Sil 5.
Assuntos
Brônquios/efeitos dos fármacos , Brônquios/enzimologia , Pneumopatias/induzido quimicamente , Pneumopatias/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Dióxido de Silício/toxicidade , Arildialquilfosfatase/antagonistas & inibidores , Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/genética , Arildialquilfosfatase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Lactoilglutationa Liase/antagonistas & inibidores , Lactoilglutationa Liase/biossíntese , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Aldeído Pirúvico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/biossíntese , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
OBJECTIVE: Macrophage foam cells are characterized by increased oxidative stress. Macrophage urokinase plasminogen activator (uPA) was shown to contribute to atherosclerosis progression. We hypothesized that uPA atherogenicity is related to its ability to increase macrophage oxidative stress. Increased macrophage oxidative stress in turn was shown to enhance PON2 expression. In the present study we investigated the effect of uPA on macrophage PON2 expression in relation to cellular oxidative stress. METHODS AND RESULTS: uPA increased PON2 expression in THP-1 macrophages in a dose-dependent manner. This effect required uPA/uPAR interaction and was abolished by cell treatment with antioxidants. uPA increased macrophage oxidative stress, measured by increased lipid peroxides, reactive oxygen species formation, superoxide anion release, and cell-mediated LDL oxidation. These effects were related to uPA-mediated activation of NADPH oxidase, and could not be reproduced in mouse peritoneal macrophages (MPM) harvested from p47(phox)-/- mice, suggesting a causal relationship between NADPH oxidase activation and the effects of uPA on macrophage oxidative stress and PON2 expression. Finally, MPM from PON2(-/-) mice were more susceptible to uPA-induced cellular oxidative stress than wild-type MPM, suggesting that PON2 protects against uPA-stimulated macrophage oxidative stress. CONCLUSIONS: Upregulation of macrophage PON2 may provide a compensatory protective mechanism against uPA-stimulation of macrophage oxidative stress during atherogenesis.
Assuntos
Arildialquilfosfatase/metabolismo , Macrófagos/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Acetofenonas/farmacologia , Animais , Antioxidantes/farmacologia , Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/genética , Aterosclerose/enzimologia , Aterosclerose/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Indução Enzimática , Humanos , Peroxidação de Lipídeos , Lipoproteínas LDL/metabolismo , Lythraceae , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
Advanced atherosclerotic lesions are characterized by a progressive increase in the unesterified cholesterol (UC) content and a decrease in its cholesteryl ester (CE) content. In the present study, we examined mechanisms involved in the effect of UC and CE on the expression of paraoxonase 2 (PON2) in macrophages. J774A.1 macrophages were enriched with CE or UC by incubation for 14-48 h with 50 microg acetylated low-density lipoprotein in the absence or presence of the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor 58035 (50 microg/ml), respectively. Macrophage PON2 mRNA expression, protein abundance and activity were increased only in the UC-enriched cells. In UC-enriched cells, inhibition of phosphatidylinositol 3-kinase (PI(3)K; using wortmannin or LY294002) attenuated the increase in PON2 mRNA expression by 50%, compared to untreated cells. In addition, we evidenced an increased phosphorylation of Akt in UC-enriched cells. Thus, we conclude from our data that macrophage PON2 expression is upregulated in UC-enriched macrophages through activation of the PI(3)K signal pathway.
Assuntos
Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/genética , Colesterol/farmacologia , Macrófagos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Animais , Western Blotting , LDL-Colesterol/química , LDL-Colesterol/isolamento & purificação , LDL-Colesterol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Macrófagos/efeitos dos fármacos , Camundongos , Microscopia Confocal , Fosfatidilinositol 3-Quinases/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Highly active antiretroviral therapy in Human Immunodeficiency Virus (HIV) has been associated with lipodystrophy, insulin resistance and atherosclerosis. We investigated the effects of rosiglitazone or metformin on fasting and postprandial inflammatory and antioxidant variables in HIV-infected males with lipodystrophy. Thirty-one patients were randomly assigned to receive either rosiglitazone (4 mg twice daily) or metformin (1 g twice daily) for 26 weeks. At baseline and after treatment, standardized 10-h oral fat loading tests were performed. Before treatment, inflammatory variables remained unchanged but there was a postprandial decrease in high density lipoprotein (HDL)-cholesterol and paraoxonase (PON1) activity. Rosiglitazone and metformin reduced homeostasis model assessment index (HOMA) similarly (-34% and -37%, respectively, P<0.05 for each). Both treatments increased fasting and postprandial PON1 activity and decreased postprandial monocyte chemoattractant protein 1 (MCP-1) concentrations. However, plasma C-reactive protein (CRP) and Interleukin-6 (IL-6) concentration did not change throughout the study. To decrease insulin resistance results in a higher anti-oxidant and consequent lower pro-inflammatory action of HDL. This may confer protection against accelerated atherosclerosis in these patients.