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1.
Chem Biol Interact ; 312: 108817, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499053

RESUMO

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0-50 µM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 µM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 µM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 µM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 µM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis.


Assuntos
Aconitina/farmacologia , Androgênios/metabolismo , Regulação para Baixo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Testosterona/sangue , Testosterona/farmacologia
2.
Nutrients ; 11(9)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500172

RESUMO

A healthy dietary pattern and high quality nutrient intake reduce atherosclerotic cardiovascular disease risk. Red wine grape pomace (RWGP)-a rich natural source of dietary fiber and antioxidants-appears to be a potential functional food ingredient. The impact of a dietary supplementation with RWGP flour was evaluated in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice, a model of lethal ischemic heart disease. SR-B1 KO/ApoER61h/h mice were fed with atherogenic (high fat, cholesterol, and cholic acid, HFC) diet supplemented with: (a) 20% chow (HFC-Control), (b) 20% RWGP flour (HFC-RWGP), or (c) 10% chow/10% oat fiber (HFC-Fiber); and survival time was evaluated. In addition, SR-B1 KO/ApoER61h/h mice were fed for 7 or 14 days with HFC-Control or HFC-RWGP diets and plasma lipid levels, inflammation, oxidative damage, and antioxidant activity were measured. Atherosclerosis and myocardial damage were assessed by histology and magnetic resonance imaging, respectively. Supplementation with RWGP reduced premature death, changed TNF-α and IL-10 levels, and increased plasma antioxidant activity. Moreover, decreased atheromatous aortic and brachiocephalic plaque sizes and attenuated myocardial infarction and dysfunction were also observed. These results suggest that RWGP flour intake may be used as a non-pharmacological therapeutic approach, contributing to decreased progression of atherosclerosis, reduced coronary heart disease, and improved cardiovascular outcomes.


Assuntos
Antioxidantes/administração & dosagem , Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Suplementos Nutricionais , Frutas/química , Isquemia Miocárdica/prevenção & controle , Miocárdio/metabolismo , Estresse Oxidativo , Extratos Vegetais/administração & dosagem , Vitis/química , Ração Animal , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Biomarcadores/sangue , Dieta Aterogênica , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/sangue , Interleucina-10/sangue , Lipídeos/sangue , Masculino , Camundongos Knockout para ApoE , Isquemia Miocárdica/sangue , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Miocárdio/patologia , Extratos Vegetais/sangue , Extratos Vegetais/isolamento & purificação , Placa Aterosclerótica , Receptores Depuradores Classe B/deficiência , Receptores Depuradores Classe B/genética , Fator de Necrose Tumoral alfa/sangue
3.
Toxicol Lett ; 314: 53-62, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31319113

RESUMO

Benzyl butyl phthalate (BBP) is a widely used plasticizer and has raised public health concerns. Here, we report the effects of BBP on the testis development during rat puberty. BBP (0, 10, 100 or 1000 mg/kg) was gavaged to 35-day-old male Sprague Dawley rats for 21 days. The serum testosterone levels, Leydig cell number, the expressions of Leydig and Sertoli cell genes and proteins were measured. The in vitro effects on steroidogenesis and gene expression in immature Leydig cells were observed. BBP significantly increased serum testosterone level at 10 mg/kg but lowered its level at 1000 mg/kg without affecting serum luteinizing hormone and follicle-stimulating hormone levels. BBP increased Leydig cell number at all doses but inhibited steroidogenic capacity per Leydig cell at 1000 mg/kg. BBP significantly increased the ratio of phosphos-AKT2 (pAKT2)/AKT2, and phosphos-ERK1/2 (pERK1/2)/ERK1/2 in the testis. Mono-benzyl phthalate (the metabolite of BBP) inhibited steroidogenesis but BBP did not affect androgen production in immature Leydig cells in vitro. In conclusion, BBP non-linearly regulates Leydig cell development by increasing Leydig cell number but inhibiting steroidogenesis.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Plastificantes/toxicidade , Desenvolvimento Sexual/efeitos dos fármacos , Testosterona/biossíntese , Fatores Etários , Animais , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/patologia , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Testosterona/sangue
4.
Int J Mol Sci ; 20(15)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31344978

RESUMO

The allicin pleiotropic effects, which include anti-inflammatory, anti-oxidant, anti-tumoral, and antibacterial actions, were well demonstrated and correlated with various molecular pathways. The immunostimulatory mechanism of allicin has not been elucidated; however, there is a possible cytokine stimulation from immunoglobulin release caused by allicin. In this study, when Wistar female rats and CD19+ lymphocytes were treated with three different doses of allicin, immunoglobulins, glutathione, and oxidative stress markers were assayed. Molecular docking was performed between S-allylmercaptoglutathione (GSSA)-a circulating form of allicin in in vivo systems formed by the allicin interaction with glutathione (GSH)-and scavenger receptors class A and B from macrophages, as well as CD19+ B lymphocytes. Our data demonstrated a humoral immunostimulatory effect of allicin in rats and direct stimulation of B lymphocytes by S-allyl-mercapto-glutathione, both correlated with decreased catalase (CAT) activity. The molecular docking revealed that S-allyl-mercapto-glutathione interacting with Colec12, MARCO (class A), and SCARB1 (class B) scavenger receptors in in vitro tests demonstrates a direct stimulation of immunoglobulin secretion by GSSA in CD19+ B lymphocytes. These data collectively indicate that GSSA stimulates immunoglobulin secretion by binding on scavenger receptors class B type 1 (SCARB1) from CD19+ B lymphocytes.


Assuntos
Colectinas/genética , Estresse Oxidativo/efeitos dos fármacos , Receptores Imunológicos/genética , Receptores Depuradores/genética , Receptores Depuradores Classe B/genética , Ácidos Sulfínicos/farmacologia , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Antígenos CD19/genética , Antígenos CD19/imunologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Catalase/genética , Glutationa/genética , Glutationa/imunologia , Humanos , Imunização , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Simulação de Acoplamento Molecular , Ratos , Ácidos Sulfínicos/imunologia
5.
Food Chem ; 300: 125232, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31352286

RESUMO

The mechanisms of main tomato carotenes (phytoene, phytofluene, lycopene and ß-carotene) intestinal absorption are still only partly understood. We thus compared carotene bioavailability in mice after gavage with carotene-rich oil-in-water emulsions. We also determined each carotene absorption profile along the duodenal-ileal axis of the intestine to identify their respective absorption sites and compared these profiles with the gene expression sites of their identified transporters, i.e. SR-BI and CD36. Our data show that phytofluene presented a significantly higher bioavailability compared to lycopene and ß-carotene (areas under the curve of 0.76 ±â€¯0.09 vs. 0.30 ±â€¯0.05, 0.09 ±â€¯0.05 and 0.08 ±â€¯0.01 µmol/L·h for phytofluene, phytoene, lycopene and ß-carotene, respectively). ß-Carotene was mostly converted in the proximal and median intestine. Phytoene and phytofluene accumulation tended to be more important in the distal intestine, which did not correlate with the proximal expression of both Scarb1 and CD36. Overall, these results highlight the high bioavailability of phytofluene.


Assuntos
Carotenoides/farmacocinética , Absorção Intestinal , Licopeno/farmacocinética , beta Caroteno/farmacocinética , Animais , Disponibilidade Biológica , Antígenos CD36/genética , Intestinos/efeitos dos fármacos , Lycopersicon esculentum/química , Masculino , Camundongos Endogâmicos C57BL , Período Pós-Prandial , Receptores Depuradores Classe B/genética
6.
Clin Biochem ; 73: 44-49, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31251897

RESUMO

BACKGROUND: Scavenger receptor class B type 1 (SR-BI) encoded by SCARB1 gene serves as a multifunctional HDL receptor, facilitating the uptake of cholesteryl esters from HDL to the liver. Recent studies have identified the association between the P376L missense mutation of the SCARB1 gene with increased serum HDL-Cholesterol level. However, the contribution of this variant to the development of cardiovascular disease (CVD) remains unclear. OBJECTIVE: We have investigated the association between the P376L polymorphism with the properties of HDL and CVD outcomes in a population sample recruited as part of the Mashhad-Stroke and Heart-Atherosclerotic-Disorders (MASHAD) cohort. METHODS: Six hundred and fifteen individuals who had a median follow-up period of 7 years were recruited as part of the MASHAD cohort. Anthropometric, biochemical parameters and HDL lipid peroxidation (HDLox) were assessed. Genotyping was performed using TaqMan-real-time-PCR based method. The association of P376L-rs74830766 with cardiovascular-risk-factors and CVD events were evaluated. RESULTS: Carriers of the P376L variant were significantly more likely than non-carriers to develop CVD using multivariate analyses adjusted for traditional CVD risk factors defined as: age, sex, BMI, presence of diabetes, or hypertension, positive smoking habit, and total cholesterol (OR: 3.75, 95%CI: 1.76-7.98, p = 0.001). In an adjusted model, there was a two fold increase in cardiovascular endpoints among individuals who were heterozygous for the P376L variant (hazard ratio, 2.08; 95% CI, 1.12-to 3.84, p = 0.02). Although there was no association between the presence of the P376L variant and HDL-C level, serum HDLox, measured as dysfunctional HDL, was 13% higher among carriers of the P376L variant than non-carriers. CONCLUSION: We have found that carriers of the P376L variant possessed higher HDLox and were at increased risk of CVD in a representative population-based cohort, as compared to non-carriers.


Assuntos
Doenças Cardiovasculares , HDL-Colesterol/metabolismo , Polimorfismo Genético , Receptores Depuradores Classe B/genética , Adulto , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , HDL-Colesterol/genética , Feminino , Seguimentos , Humanos , Peroxidação de Lipídeos/genética , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Fatores de Risco , Receptores Depuradores Classe B/metabolismo
7.
Dev Comp Immunol ; 99: 103404, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31152761

RESUMO

Scavenger receptor (SR) class B (SR-B) is a transmembrane protein that belongs to the SR family with a wide range of functions in innate immunity. Here, an SR-B homologue, designated as AjSR-B, was cloned from the sea cucumber Apostichopus japonicus. AjSR-B comprised 2519 nucleotides with a 5'-untranslated region (UTR) of 153 bp, an open reading frame of 1581 bp encoding a 526 amino acid protein, and a 3'-UTR of 785 bp. SMART analysis indicated that AjSR-B has two transmembrane regions and a cluster determinant 36 domain. Multiple alignments and phylogenetic analysis supported that AjSR-B is a novel member of the SR-B protein family. Moreover, AjSR-B was constitutively expressed in all detected tissues, with the highest levels recorded in the intestine. Both were significantly induced in coelomocytes and the intestine after Vibrio splendidus challenge. Functionally, the recombinant rAjSR-B that corresponds to a large extracellular loop can bind pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan, and mannan, with a high binding affinity to LPS. Bacterial agglutination assay showed that rAjSR-B can agglutinate the four tested bacteria (Gram-negative and Gram-positive bacteria) with calcium dependence. However, the agglutination ability for Gram-negative bacteria completely disappeared in the presence of PAMPs but a weak ability to bind Gram-positive bacteria (Micrococcus luteus) was still exhibited, suggesting there might exist a competition between Gram-positive bacteria and PAMPs under same condition. Our current study indicated that AjSR-B is a PAMP that plays important roles in the innate immune process of sea cucumbers.


Assuntos
Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/imunologia , Stichopus/genética , Testes de Aglutinação , Sequência de Aminoácidos , Animais , Bactérias/imunologia , Sequência de Bases , Expressão Gênica/imunologia , Imunidade Inata/genética , Fases de Leitura Aberta , Padrões Moleculares Associados a Patógenos/metabolismo , Filogenia , Domínios Proteicos , Receptores de Reconhecimento de Padrão/química , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Receptores Depuradores Classe B/química , Alinhamento de Sequência , Stichopus/imunologia , Distribuição Tecidual
8.
EBioMedicine ; 43: 513-524, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31076347

RESUMO

BACKGROUND: Chronic hepatic inflammation leads to liver fibrosis, which may progress to cirrhosis, a condition with high morbidity. Our aim was to assess the as yet unknown role of innate immunity protein CD5L in liver fibrosis. METHODS: CD5L was measured by ELISA in plasma samples from cirrhotic (n = 63) and hepatitis (n = 39) patients, and healthy controls (n = 7), by immunohistochemistry in cirrhotic tissue (n = 12), and by quantitative RT-PCR in mouse liver cell subsets isolated by cell sorting. Recombinant CD5L (rCD5L) was administered into a murine model of CCl4-induced fibrosis, and damage, fibrosis and hepatic immune cell infiltration, including the LyC6hi (pro-fibrotic)-LyC6low (pro-resolutive) monocyte ratio were determined. Moreover, rCD5L was added into primary human hepatic stellate cells to study transforming growth factor ß (TGFß) activation responses. FINDINGS: Cirrhotic patients showed elevated plasma CD5L concentrations as compared to patients with hepatitis and healthy controls (Mann-Whitney test p < 0·0001). Moreover, plasma CD5L correlated with disease progression, FIB4 fibrosis score (r:0·25, p < 0·0001) and tissue expression (r = 0·649; p = 0·022). Accordingly, CCl4-induced damage increased CD5L levels in total liver, particularly in hepatocytes and macrophages. rCD5L administration attenuated CCl4-induced injury and fibrosis as determined by reduced serum transaminase and collagen content. Moreover, rCD5L inhibited immune cell infiltration and promoted a phenotypic shift in monocytes from LyC6hi to LyC6low. Interestingly, rCD5L also had a direct effect on primary human hepatic stellate cells promoting SMAD7 expression, thus repressing TGFß signalling. INTERPRETATION: Our study identifies CD5L as a key pleiotropic inhibitor of chronic liver injury. FUND: Fundació Marató TV3, AGAUR and the ISCIII-EDRF.


Assuntos
Suscetibilidade a Doenças , Imunidade , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Receptores Depuradores Classe B/genética , Adulto , Idoso , Animais , Biomarcadores , Doença Hepática Induzida por Substâncias e Drogas/complicações , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Células Estreladas do Fígado/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Cirrose Hepática/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Receptores Depuradores Classe B/metabolismo , Adulto Jovem
9.
Int J Biol Macromol ; 134: 759-769, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31100394

RESUMO

Reverse cholesterol transport (RCT) has been demonstrated to reduce hyperlipidemia, and fucoidans are found to possess hypolipidemic effect. This study was designed to investigate the lipid-lowering effect of the fucoidan from the brown seaweed A. nodosum and whether it improves RCT-related genes expression in C57 BL/6J mice. Our results indicated that fucoidan A3 (100 mg/kg/day) intervention significantly reduced plasma total cholesterol (~23.2%), triglyceride (~48.7%) and fat pad index. This fucoidan significantly increased the mRNA expression of low-density lipoprotein receptor (LDLR), scavenger receptor B type 1 (SR-B1), cholesterol 7 alpha-hydroxylase A1 (CYP7A1), liver X receptor (LXR) ß, ATP-binding cassette transporter (ABC) A1 and sterol regulatory element-binding protein (SREBP) 1c, and decreased the expression of peroxisome proliferator-activated receptor (PPAR) γ, however, it had no effect on the expression of proprotein convertase subtilisin/kexin type 9, PPARα, LXRα, SREBP-2, ABCG1, ABCG8 and Niemann-Pick C1-like 1. These results demonstrated that this fucoidan improved lipid transfer from plasma to the liver by activating SR-B1 and LDLR, and up-regulated lipid metabolism by activating LXRß, ABCA1 and CYP7A1. In conclusion, this fucoidan lowers lipid by enhancing RCT-related genes expression, and it can be explored as a potential candidate for prevention or treatment of lipid disorders.


Assuntos
Ascophyllum/química , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperlipidemias/genética , Polissacarídeos/farmacologia , Alga Marinha/química , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Modelos Animais de Doenças , Hiperlipidemias/metabolismo , Hipolipemiantes/química , Hipolipemiantes/farmacologia , Intestino Delgado/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/química , RNA Mensageiro , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo
10.
BMC Pharmacol Toxicol ; 20(1): 32, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133074

RESUMO

BACKGROUND: It is an established fact that excess of glucocorticoids could cause the harmful effects, such as suppression on the male reproduction. Although glucocorticoids pharmacologically inhibit the Leydig cell function, their roles in Leydig cell development are unclear. Therefore, the present study was designed to investigate effects of synthetic glucocorticoid dexamethasone (DEX) on rat stem Leydig cell proliferation and differentiation. METHODS: Male Sprague-Dawley rats received a single intraperitoneal injection of 75 mg/kg EDS to eliminate Leydig cells and an in vitro culture system of the seminiferous tubules was established from Leydig cell-depleted testis. Using basal medium and Leydig cell differentiation-inducing medium (LIM) in the culture system, we examined the effects of DEX (0-100 nM) on the proliferation and differentiation of the stem Leydig cells in vitro, respectively. RESULTS: Results showed that LIM is a good agent to induce stem Leydig cell differentiation into Leydig cells that produce testosterone in vitro. DEX inhibited the differentiation of stem Leydig cells by reducing the expression levels of Cyp17a1 and Scarb1 and that NR3C1 antagonist RU38486 reversed the DEX-mediated effects. However, DEX are not involved with the proliferation of stem Leydig cells. CONCLUSIONS: DEX suppressed the differentiation of rat Leydig cells in vitro and glucocorticoid-induced effects acted through NR3C1. This suppression partially targets on Cyp17a1 and Scarb1 gene expression.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Dexametasona/toxicidade , Glucocorticoides/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos Sprague-Dawley , Receptores de Glucocorticoides/genética , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Células-Tronco/metabolismo , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo
11.
J Agric Food Chem ; 67(22): 6222-6231, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31117505

RESUMO

The anti-inflammatory effects and cellular transport mechanisms of all- E-astaxanthin and its 9Z- and 13Z-isomers were investigated in a Caco-2 cell monolayer model. All three astaxanthin isomers at 1.2 µM significantly reduced the TNF-α-induced secretion of IL-8 by 22-27%. Z-Astaxanthins, especially 9 Z-astaxanthin exhibited greater anti-inflammatory effect than all- E-astaxanthin by down-regulating pro-inflammatory cytokines COX-2 and TNF-α gene expression to 0.88 ± 0.01-fold and 0.83 ± 0.17-fold that of the negative control (NC), respectively. The anti-inflammatory effects of astaxanthin isomers were achieved via modulating the NF-κB signaling pathway as they down-regulated TNF-α-induced phosphorylation of IκBα from 5.3 ± 0.19-fold to 3.8 ± 0.33-4.5 ± 0.27-fold of NC. The scavenger receptor class B type I protein (SR-BI) was found to facilitate the cellular uptake of astaxanthin isomers. Its inhibitor (BLT-1) and antibody (Anti-SRBI) significantly reduced cellular uptake efficiency of all- E-astaxanthin (18.9% and 16.7%, respectively) and 13Z-astaxanthin (28.8% and 30.2%, respectively), but not of 9Z-astaxanthin. The molecular docking experiment showed that 13 Z-astaxanthin had significantly higher affinity with SR-BI (atomic contact energy: -420.31) than all- E-astaxanthin and 9 Z-astaxanthin, which at least partially supports the higher bioavailability of 13 Z-astaxanthin observed in vivo by others.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Receptores Depuradores Classe B/metabolismo , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Isomerismo , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Receptores Depuradores Classe B/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Xantofilas/química , Xantofilas/metabolismo
12.
Nucleic Acids Res ; 47(11): 5465-5479, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31034558

RESUMO

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.


Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleotídeos Antissenso/química , Organofosfonatos/química , Oligonucleotídeos Fosforotioatos/química , Células 3T3-L1 , Animais , Caspases/metabolismo , Linhagem Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células HeLa , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Fosforotioatos/administração & dosagem , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo
13.
J Biol Chem ; 294(22): 8732-8744, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-30996006

RESUMO

The bile acid (BA) nuclear receptor, farnesoid X receptor (FXR/NR1H4), maintains metabolic homeostasis by transcriptional control of numerous genes, including an intestinal hormone, fibroblast growth factor-19 (FGF19; FGF15 in mice). Besides activation by BAs, the gene-regulatory function of FXR is also modulated by hormone or nutrient signaling-induced post-translational modifications. Recently, phosphorylation at Tyr-67 by the FGF15/19 signaling-activated nonreceptor tyrosine kinase Src was shown to be important for FXR function in BA homeostasis. Here, we examined the role of this FXR phosphorylation in cholesterol regulation. In both hepatic FXR-knockout and FXR-knockdown mice, reconstitution of FXR expression up-regulated cholesterol transport genes for its biliary excretion, including scavenger receptor class B member 1 (Scarb1) and ABC subfamily G member 8 (Abcg5/8), decreased hepatic and plasma cholesterol levels, and increased biliary and fecal cholesterol levels. Of note, these sterol-lowering effects were blunted by substitution of Phe for Tyr-67 in FXR. Moreover, consistent with Src's role in phosphorylating FXR, Src knockdown impaired cholesterol regulation in mice. In hypercholesterolemic apolipoprotein E-deficient mice, expression of FXR, but not Y67F-FXR, ameliorated atherosclerosis, whereas Src down-regulation exacerbated it. Feeding or treatment with an FXR agonist induced Abcg5/8 and Scarb1 expression in WT, but not FGF15-knockout, mice. Furthermore, FGF19 treatment increased occupancy of FXR at Abcg5/8 and Scarb1, expression of these genes, and cholesterol efflux from hepatocytes. These FGF19-mediated effects were blunted by the Y67F-FXR substitution or Src down-regulation or inhibition. We conclude that phosphorylation of hepatic FXR by FGF15/19-induced Src maintains cholesterol homeostasis and protects against atherosclerosis.


Assuntos
Colesterol/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Hepatócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Quinases da Família src/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Ácidos e Sais Biliares/metabolismo , Colesterol/sangue , Regulação para Baixo , Fatores de Crescimento de Fibroblastos/deficiência , Fatores de Crescimento de Fibroblastos/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética
14.
Arch Biochem Biophys ; 666: 1-7, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30876920

RESUMO

BACKGROUND: The main functions of the skin are to protect against environmental insults and prevent water loss, which are performed by the complex lipid- and protein matrix present in the outermost layers of the epithelium. The lipidome of these outer layers is mainly composed of ceramides, fatty acids, and cholesterol, which regulates keratinocyte differentiation and skin barrier function. SR-B1 is a multifunctional scavenger receptor that is best known for facilitating uptake of cholesterol from HDL particles in the liver, but it is also expressed in the skin. OBJECTIVE: To determine the role of SR-B1 in keratinocyte differentiation. METHODS: We investigated the relationship between SR-B1 and keratinocyte differentiation using a physiologically relevant model, organotypic skin equivalents (SEs), wherein SR-B1 was knocked down via siRNA transfection. To assess effects of SR-B1 knockdown on keratinocyte differentiation, we performed hematoxylin/eosin staining, RT-PCR, western blotting, and immunohistochemistry. We also examined the effect of SR-B1 knockdown on lipid production by performing Oil Red O staining and thin layer chromatography. RESULTS: SR-B1 knockdown resulted in decreased lipid levels in SEs, specifically ceramides, and in decreased transcript levels of LDLR, PPAR-α and PPAR-γ, which are factors involved in regulating ceramide synthesis. In addition, filaggrin levels increased in SR-B1 KD tissues, but neither keratin 14 nor keratin 10 were affected. CONCLUSION: We conclude that one of the main functions of SR-B1 in the skin is to regulate ceramide levels and thereby maintain the barrier function of the skin, resulting in the protection of cutaneous tissues from outdoor insults.


Assuntos
Diferenciação Celular/fisiologia , Homeostase , Queratinócitos/citologia , Metabolismo dos Lipídeos , Receptores Depuradores Classe B/fisiologia , Pele/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Receptores Depuradores Classe B/genética , Pele/citologia
15.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823658

RESUMO

Melanoma is a skin tumor with a high tendency for metastasis and thus is one of the deadliest cancers worldwide. Here, we investigated the expression of the scavenger receptor class B type 1 (SR-BI), a high-density lipoprotein (HDL) receptor, and tested for its role in melanoma pigmentation as well as extracellular vesicle release. We first analyzed the expression of SR-BI in patient samples and found a strong correlation with MITF expression as well as with the melanin synthesis pathway. Hence, we asked whether SR-BI could also play a role for the secretory pathway in metastatic melanoma cells. Interestingly, gain- and loss-of-function of SR-BI revealed regulation of the proto-oncogene MET. In line, SR-BI knockdown reduced expression of the small GTPase RABB22A, the ESCRT-II protein VPS25, and SNAP25, a member of the SNARE complex. Accordingly, reduced overall extracellular vesicle generation was detected upon loss of SR-BI. In summary, SR-BI expression in human melanoma enhances the formation and transport of extracellular vesicles, thereby contributing to the metastatic phenotype. Therapeutic targeting of SR-BI would not only interfere with cholesterol uptake, but also with the secretory pathway, therefore suppressing a key hallmark of the metastatic program.


Assuntos
Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Receptores Depuradores Classe B/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Receptores Depuradores Classe B/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
16.
Biochem J ; 476(6): 951-963, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30837308

RESUMO

High-density lipoproteins (HDLs) facilitate reverse cholesterol transport, a process in which HDL removes cholesterol from circulation and carries it to the liver for biliary excretion. Reverse cholesterol transport is also facilitated by HDL's high-affinity receptor, scavenger receptor-BI (SR-BI), by mechanisms that are not fully understood. To improve our understanding of SR-BI function, we previously solved the NMR (nuclear magnetic resonance) structure of a peptide encompassing amino acids 405-475 of SR-BI. This segment of SR-BI, that includes the functionally critical C-terminal transmembrane domain and part of the extracellular domain, also contains four conserved proline (Pro) residues. We hypothesized that these proline residues support SR-BI in a conformation that allows for efficient cholesterol transport. To test this, we generated individual Pro-to-alanine mutations in full-length SR-BI and transiently expressed the mutant receptors in COS-7 cells to measure the effects on SR-BI-mediated cholesterol transport functions. Our findings reveal that HDL cell association and uptake of HDL-cholesteryl esters are impaired by mutation of Pro-412, Pro-438, or the transmembrane proline kink residue (Pro-459). In addition, SR-BI-mediated cholesterol efflux and membrane cholesterol distribution are impaired by mutation of Pro-412 or Pro-438, indicating that these residues are essential for a fully functional SR-BI receptor. Furthermore, we demonstrate that Pro-408 is necessary for proper SR-BI expression, but mutation of Pro-408 does not cause SR-BI to become misfolded or rapidly degraded by the proteasome or the lysosome. We conclude that key proline residues play an important role in SR-BI function by allowing for the efficient transport of cholesterol between cells and HDL.


Assuntos
Colesterol/química , Colesterol/metabolismo , Receptores Depuradores Classe B/química , Receptores Depuradores Classe B/metabolismo , Substituição de Aminoácidos , Animais , Transporte Biológico Ativo/fisiologia , Células COS , Colesterol/genética , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Receptores Depuradores Classe B/genética
17.
Eur J Pharmacol ; 853: 111-120, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30902657

RESUMO

Farnesoid X receptor (FXR) agonists play important regulatory roles in bile acid, lipid and glucose metabolism in vitro and in vivo. Thus, FXR agonists exhibit potential therapeutic effects on metabolism-related diseases that are associated with extrahepatic manifestations induced by hepatitis C virus (HCV) infection. This study investigated the effect and mechanism of FXR agonist GW4064 against HCV in vitro to explore the potential application of FXR agonists. Results showed that GW4064 and other FXR agonists have potent antiviral activity against HCV in Huh7.5 cells. GW4064 down-regulated the expression of scavenger receptor class B type I protein via FXR and thereby indirectly inhibited HCV entry into cells, leading to interruption of HCV life cycle. GW4064 also exhibited synergistic anti-HCV effect with known direct-acting antiviral agents (DAAs) used in the clinic and remained sensitive to DAA-resistant HCV mutations. Therefore, FXR agonists are also a kind of antiviral agent, and might be helpful in treatment of HCV-induced hepatic and extrahepatic manifestations.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Isoxazóis/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Depuradores Classe B/genética , Internalização do Vírus/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Sinergismo Farmacológico , Hepacivirus/genética , Humanos , Mutação , RNA Viral/biossíntese , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Virais/metabolismo
18.
Food Funct ; 10(2): 1049-1061, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30706921

RESUMO

The present study was designed to explore the neuroprotective effects of docosahexaenoic acid (DHA) and/or vitamin E (VE) in vitro. The PC12 cells were pretreated with DHA and/or VE for 4 h, followed by 50 µmol L-1 Aß25-35 treatments for another 48 h. The cells were then collected and used for the measurements of oxidative stress parameters. Real time-PCR and western blot were applied to measure fatty acid transporters, Nrf2 and its downstream antioxidant targets' gene and protein expression. Our results indicated that the Aß25-35 treatment inhibited cellular growth, increased intracellular ROS generation and decreased the mitochondrial membrane potential. The Aß25-35 treatment decreased the total antioxidant capacity (T-AOC), whereas it increased the MDA levels in neuron cells. Pretreatment of cells with VE or DHA could antagonize the Aß25-35-mediated cell growth inhibition and mitochondrial membrane potential decline. Activation of the Nrf2 signaling pathway and regulation of CD36, SRB1 and FABP5 expression were observed in DHA- and DHA + VE-pretreated cells. Our results indicated a synergistic effect of DHA and VE in antagonizing the oxidative damage caused by Aß25-35 in the PC12 cells. The results of the present study will shed light on the application of nutritional intervention for DHA and VE in preventing neuronal damage-related diseases.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Ácidos Docosa-Hexaenoicos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Vitamina E/farmacologia , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/administração & dosagem , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vitamina E/administração & dosagem
19.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(4): 443-451, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30633988

RESUMO

OBJECTIVE: Since cholesterol is the sole precursor for glucocorticoid synthesis, it is hypothesized that genetic defects in proteins that impact the cellular cholesterol pool may underlie glucocorticoid insufficiency in humans. In the current study, we specifically focused on the cholesterol efflux mediator ATP-binding cassette transporter G1 (ABCG1) as gene candidate. METHODS: The adrenal transcriptional response to fasting stress was measured in wild-type mice to identify putative novel gene candidates. Subsequently, the adrenal glucocorticoid function was compared between ABCG1 knockout mice and wild-type controls. RESULTS: Overnight food deprivation induced a change in relative mRNA expression levels of cholesterol metabolism-related proteins previously linked to steroidogenesis, i.e. scavenger receptor class B type I (+149%; P < 0.001), LDL receptor (-70%; P < 0.001) and apolipoprotein E (-41%; P < 0.01). Strikingly, ABCG1 transcript levels were also markedly decreased (-61%; P < 0.05). In contrast to our hypothesis that decreasing cholesterol efflux would increase the adrenal cholesterol pool and enhance glucocorticoid output, ABCG1 knockout mice as compared to wild-type mice exhibited a reduced ability to secrete corticosterone in response to an ACTH challenge (two-way ANOVA: P < 0.001 for genotype) or fasting stress. As a result, glucocorticoid target gene expression levels in liver and hypothalamus were reduced and blood lymphocyte concentrations and spleen weights increased in ABCG1 knockout mice under fasting stress conditions. This was paralleled by a 48% reduction in adrenal cholesteryl ester stores and stimulation of adrenal NPC intracellular cholesterol transporter 2 (+37%; P < 0.05) and apolipoprotein E (+59%; P < 0.01) mRNA expression. CONCLUSION: ABCG1 deficiency is associated with mild glucocorticoid insufficiency in mice.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/deficiência , Apolipoproteínas E/genética , Glucocorticoides/deficiência , Receptores de LDL/genética , Receptores Depuradores Classe B/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Privação de Alimentos , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
20.
Herz ; 44(7): 644-650, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29516118

RESUMO

BACKGROUND: The scavenger receptor class B type I (SCARB1) gene plays an important role in high-density lipoprotein cholesterol (HDL-C) metabolism and may be involved in the pathogenesis of coronary artery disease (CAD). The rs5888 (C/T) single-nucleotide polymorphism (SNP) in the SCARB1 gene is functional, and we therefore examined the association between rs5888 and CAD. METHODS: The rs5888 genotypes were analyzed in 287 Chinese patients with CAD and 367 controls via the high-resolution melting curve (HRM) method. Allele frequency and genotype distribution were compared. The levels of plasma triglyceride (TG), total cholesterol (TC), HDL-C, and low-density lipoprotein-cholesterol (LDL-C) were also compared between the groups with different genotypes. RESULTS: The proportion of subjects with TT and CT genotypes in the control group was significantly higher than that in the CAD group (50.95% vs. 32.75%, p < 0.001). The frequency of individuals with T alleles in the control group was significantly higher than that in the CAD group (28.75% vs. 17.25%, p < 0.001). In the combined population (CAD and control groups), the HDL-C concentration in individuals with the TT genotype was significantly higher than in those with the CT genotype (1.81 ± 1.29 vs. 1.42 ± 0.56, p = 0.026) or in those with the CC genotype (1.81 ± 1.29 vs. 1.42 ± 0.72, p = 0.021). CONCLUSION: The results of this study suggest that the rs5888 SNP in the SCARB1 gene is associated with CAD; furthermore, the TT genotype is associated with a higher HDL-C concentration.


Assuntos
Doença da Artéria Coronariana , Frequência do Gene , Receptores Depuradores Classe B , Grupo com Ancestrais do Continente Asiático , China , Doença da Artéria Coronariana/genética , Humanos , Polimorfismo de Nucleotídeo Único , Receptores Depuradores Classe B/genética
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