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1.
Curr Issues Mol Biol ; 43(2): 818-830, 2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34449561

RESUMO

BACKGROUND: A high-cholesterol diet (HCD) induces vascular atherosclerosis through vascular inflammatory and immunological processes via TLRs. The aim of this study is to investigate the mRNA expression of TLRs and other noxious biomarkers expressing inflammation, fibrosis, apoptosis, and cardiac dysfunction in the rabbit myocardium during (a) high-cholesterol diet (HCD), (b) normal diet resumption and (c) fluvastatin or rosuvastatin treatment. METHODS: Forty-eight male rabbits were randomly divided into eight groups (n = 6/group). In the first experiment, three groups were fed with HCD for 1, 2 and 3 months. In the second experiment, three groups were fed with HCD for 3 months, followed by normal chow for 1 month and administration of fluvastatin or rosuvastatin for 1 month. Control groups were fed with normal chow for 90 and 120 days. The whole myocardium was removed; total RNA was isolated from acquired samples, and polymerase chain reaction, reverse transcription PCR and quantitative real-time PCR were performed. RESULTS: mRNA of TLRs 2, 3, 4 and 8; interleukin-6; TNF-a; metalloproteinase-2; tissue inhibitor of metalloproteinase-1; tumor protein 53; cysteinyl aspartate specific proteinase-3; and brain natriuretic peptide (BNP) increased in HCD. Statins but not resumption of a normal diet decreased levels of these biomarkers and increased levels of antifibrotic factors. CONCLUSIONS: HCD increases the levels of TLRs; inflammatory, fibrotic and apoptotic factors; and BNP in the rabbit myocardium. Atherogenic diets adversely affect the myocardium at a molecular level and are reversed by statins.


Assuntos
Colesterol na Dieta/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/tratamento farmacológico , Miocárdio/metabolismo , Receptores Toll-Like/metabolismo , Animais , Modelos Animais de Doenças , Fluvastatina/farmacologia , Hipercolesterolemia/etiologia , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Masculino , Miocárdio/patologia , Coelhos , Rosuvastatina Cálcica/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
2.
Biomed Pharmacother ; 139: 111668, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243630

RESUMO

Metabolic Syndrome (MetS) is a complex and multifactorial condition often characterised by obesity, hypertension, hyperlipidaemia, insulin resistance, glucose intolerance and fasting hyperglycaemia. Collectively, MetS can increase the risk of atherosclerotic-cardiovascular disease, which is the leading cause of death worldwide. However, no animal model currently exists to study MetS in the context of atherosclerosis. In this study we developed a pre-clinical mouse model that recapitulates the spectrum of MetS features while developing atherosclerosis. When BPHx mice were placed on a western type diet for 16 weeks, all the classical characteristics of MetS were observed. Comprehensive metabolic analyses and atherosclerotic imaging revealed BPHx mice to be obese and hypertensive, with elevated total plasma cholesterol and triglyceride levels, that accelerated atherosclerosis. Altogether, we demonstrate that the BPHx mouse has all the major components of MetS, and accelerates the development of atherosclerosis.


Assuntos
Aterosclerose/patologia , Dieta/efeitos adversos , Hipertensão/patologia , Síndrome Metabólica/patologia , Animais , Aterosclerose/sangue , Aterosclerose/metabolismo , Glicemia/metabolismo , Colesterol/sangue , Modelos Animais de Doenças , Feminino , Intolerância à Glucose/sangue , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Hipercolesterolemia/sangue , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Hiperglicemia/sangue , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Hiperlipidemias/sangue , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Hipertensão/sangue , Hipertensão/metabolismo , Resistência à Insulina/fisiologia , Síndrome Metabólica/sangue , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/sangue , Obesidade/metabolismo , Obesidade/patologia , Triglicerídeos/sangue
3.
Nat Commun ; 12(1): 2770, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986266

RESUMO

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.


Assuntos
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Lipossomos/farmacologia , Pré-Albumina/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas , Pré-Albumina/genética , RNA Guia/genética , RNA Guia/metabolismo
4.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807969

RESUMO

Sitosterolemia is a lipid disorder characterized by the accumulation of dietary xenosterols in plasma and tissues caused by mutations in either ABCG5 or ABCG8. ABCG5 ABCG8 encodes a pair of ABC half transporters that form a heterodimer (G5G8), which then traffics to the surface of hepatocytes and enterocytes and promotes the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. We review the literature from the initial description of the disease, the discovery of its genetic basis, current therapy, and what has been learned from animal, cellular, and molecular investigations of the transporter in the twenty years since its discovery. The genomic era has revealed that there are far more carriers of loss of function mutations and likely pathogenic variants of ABCG5 ABCG8 than previously thought. The impact of these variants on G5G8 structure and activity are largely unknown. We propose a classification system for ABCG5 ABCG8 mutants based on previously published systems for diseases caused by defects in ABC transporters. This system establishes a framework for the comprehensive analysis of disease-associated variants and their impact on G5G8 structure-function.


Assuntos
Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Colesterol/metabolismo , Hipercolesterolemia , Enteropatias , Erros Inatos do Metabolismo Lipídico , Lipoproteínas , Mutação , Fitosteróis/efeitos adversos , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/história , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/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/história , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Enterócitos/metabolismo , Enterócitos/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , História do Século XXI , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/história , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Enteropatias/genética , Enteropatias/história , Enteropatias/metabolismo , Enteropatias/patologia , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/história , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Lipoproteínas/genética , Lipoproteínas/história , Lipoproteínas/metabolismo , Fitosteróis/genética , Fitosteróis/história , Fitosteróis/metabolismo
5.
Nat Commun ; 12(1): 2368, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888696

RESUMO

Endothelial cells play a key role in the regulation of disease. Defective regulation of endothelial cell homeostasis may cause mesenchymal activation of other endothelial cells or neighboring cell types, and in both cases contributes to organ fibrosis. Regulatory control of endothelial cell homeostasis is not well studied. Diabetes accelerates renal fibrosis in mice lacking the endothelial glucocorticoid receptor (GR), compared to control mice. Hypercholesterolemia further enhances severe renal fibrosis. The fibrogenic phenotype in the kidneys of diabetic mice lacking endothelial GR is associated with aberrant cytokine and chemokine reprogramming, augmented Wnt signaling and suppression of fatty acid oxidation. Both neutralization of IL-6 and Wnt inhibition improve kidney fibrosis by mitigating mesenchymal transition. Conditioned media from endothelial cells from diabetic mice lacking endothelial GR stimulate Wnt signaling-dependent epithelial-to-mesenchymal transition in tubular epithelial cells from diabetic controls. These data demonstrate that endothelial GR is an essential antifibrotic molecule in diabetes.


Assuntos
Nefropatias Diabéticas/patologia , Endotélio/patologia , Hipercolesterolemia/complicações , Túbulos Renais/patologia , Receptores de Glucocorticoides/deficiência , Adrenalectomia , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/etiologia , Células Endoteliais/patologia , Endotélio/citologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Ácidos Graxos/metabolismo , Fibrose , Glucocorticoides/metabolismo , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/etiologia , Hipercolesterolemia/patologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Túbulos Renais/citologia , Masculino , Camundongos , Camundongos Knockout para ApoE , Oxirredução , Receptores de Glucocorticoides/genética , Estreptozocina/administração & dosagem , Estreptozocina/toxicidade , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
6.
J Med Chem ; 64(8): 4396-4409, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33821652

RESUMO

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.


Assuntos
Endotélio/química , Análise Espectral Raman , Vasos Sanguíneos/química , Vasos Sanguíneos/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Endotélio/metabolismo , Endotélio/fisiopatologia , Humanos , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Mitocôndrias/química , Mitocôndrias/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo
7.
Eur J Med Chem ; 216: 113358, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33725656

RESUMO

Pancreatic triglyceride lipase (PTL) and Niemann-Pick C1-like 1 (NPC1L1) have been identified as attractive therapeutic targets for obesity and hypercholesteremia, respectively. Obesity and hypercholesteremia usually co-exist, however no dual-inhibitors against PTL and NPC1L1 were reported for the treatment of obesity patients with hypercholesteremia so far. In this work, molecular hybridization-based one-step modification screening identified a potent dual-inhibitor against PTL and NPC1L1. Compound P1-11 has IC50 values of 2.1 µM against PTL through covalent binding, as well as significantly reduces cholesterol absorption in a non-competitive inhibitory manner. Molecule docking and molecular dynamics studies revealed the reason of its activity to both PTL and NPC1L1. Moreover, the gene and protein expression levels of PTL and NPC1L1 were also determined respectively after the treatment of P1-11. Development of dual-inhibitors against PTL and NPC1L1 could provide novel treatment options for obesity patients with hypercholesteremia. The results of current research would great support the development of dual-inhibitors against PTL and NPC1L1.


Assuntos
Anticolesterolemiantes/química , Lipase/antagonistas & inibidores , Proteínas de Membrana Transportadoras/metabolismo , Pâncreas/enzimologia , Anticolesterolemiantes/metabolismo , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Desenho de Fármacos , Ezetimiba/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/patologia , Lipase/metabolismo , Proteínas de Membrana Transportadoras/sangue , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Orlistate/química
8.
J Clin Endocrinol Metab ; 106(5): e2005-e2014, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33524107

RESUMO

PURPOSE: In resistance to thyroid hormone due to mutations in thyroid hormone receptor ß, peripheral tissues are variably refractory to the action of circulating thyroid hormones. We evaluated parameters contributing to atherosclerotic risk in this disorder. METHODS: We measured low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), nonesterified fatty acids (NEFA), intrahepatic lipid (IHL) and intramyocellular lipid (IMCL), Homeostasis-model assessment of insulin resistance (HOMA-IR), augmentation index (AIx) and pulse wave velocity (PWV), flow-mediated dilatation, and carotid intima-media thickness (cIMT) in an unselected, genetically confirmed cohort of adult RTHß patients (n = 27-77) and compared these with measurements in healthy subjects (up to n = 100) and thyrotoxic patients (n = 40). RESULTS: Resistance to thyroid hormone beta (RTHß) patients exhibited higher LDL-C (P = 0.008) and TG (P = 0.002) and lower HDL-C concentrations (P = 0.015 × 10-2) than control subjects, with LDL-C being higher than in thyrotoxic patients with comparable hyperthyroxinemia. Proprotein convertase subtilisin/kexin 9 (P = 0.002) and apolipoprotein B (P = 0.0009) levels were reduced in thyrotoxic patients but not lower in RTHß patients or control subjects. Intrahepatic lipid (P = 0.02 × 10-4), IMCL (P = 0.002), HOMA-IR (P = 0.01 × 10-2), and NEFA (P = 0.04 × 10-6) were significantly higher in RTHß patients than control subjects. Flow-mediated dilatation was increased (P = 0.04) but cIMT (P = 0.71), PWV P = 0.81), and AIx (P = 0.95) were unaltered in RTHß patients. CONCLUSIONS: We have documented mixed dyslipidemia with hepatic and IMCL accumulation in RTHß, suggesting that surveillance for these metabolic abnormalities is warranted. How they combine with enhanced endothelial function and unaltered vessel wall thickness and compliance to determine overall cardiometabolic risk in this disorder remains to be defined.


Assuntos
Dislipidemias/epidemiologia , Hipercolesterolemia/epidemiologia , Resistência à Insulina , Lipídeos/análise , Mutação , Receptores beta dos Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Adulto , Biomarcadores/análise , Espessura Intima-Media Carotídea , Estudos de Casos e Controles , Dislipidemias/metabolismo , Dislipidemias/patologia , Feminino , Seguimentos , Humanos , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Masculino , Prognóstico , Reino Unido/epidemiologia
9.
PLoS One ; 16(1): e0245481, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481866

RESUMO

BACKGROUND: Although accumulating evidence suggests a more extensive reduction of low-density lipoprotein cholesterol (LDL-C), it is unclear whether a higher statin dose is more effective and cost-effective in the Asian population. This study compared the efficacy, safety, and cost-effectiveness of atorvastatin 20 and 10 mg in high-risk Asian patients with hypercholesterolemia. METHODS: A 12-week, open-label, parallel, multicenter, Phase IV randomized controlled trial was conducted at ten hospitals in the Republic of Korea between October 2017 and May 2019. High-risk patients with hypercholesterolemia, defined according to 2015 Korean guidelines for dyslipidemia management, were eligible to participate. We randomly assigned 250 patients at risk of atherosclerotic cardiovascular disease to receive 20 mg (n = 124) or 10 mg (n = 126) of atorvastatin. The primary endpoint was the difference in the mean percentage change in LDL-C levels from baseline after 12 weeks. Cost-effectiveness was measured as an exploratory endpoint. RESULTS: LDL-C levels were reduced more significantly by atorvastatin 20 mg than by 10 mg after 12 weeks (42.4% vs. 33.5%, p < 0.0001). Significantly more patients achieved target LDL-C levels (<100 mg/dL for high-risk patients, <70 mg/dL for very high-risk patients) with atorvastatin 20 mg than with 10 mg (40.3% vs. 25.6%, p < 0.05). Apolipoprotein B decreased significantly with atorvastatin 20mg versus 10 mg (-36.2% vs. -29.9%, p < 0.05). Lipid ratios also showed greater improvement with atorvastatin 20 mg than with 10 mg (total cholesterol/high-density lipoprotein cholesterol ratio, -33.3% vs. -29.4%, p < 0.05; apolipoprotein B/apolipoprotein A1 ratio, -36.7% vs. -31.4%, p < 0.05). Atorvastatin 20 mg was more cost-effective than atorvastatin 10 mg in terms of both the average and incremental cost-effectiveness ratios. Safety and tolerability of atorvastatin 20 mg were comparable to those of atorvastatin 10 mg. CONCLUSION: In high-risk Asian patients with hypercholesterolemia, atorvastatin 20 mg was both efficacious in reducing LDL-C and cost-effective compared with atorvastatin 10 mg.


Assuntos
Anticolesterolemiantes/uso terapêutico , Grupo com Ancestrais do Continente Asiático/estatística & dados numéricos , Atorvastatina/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Feminino , Humanos , Hipercolesterolemia/epidemiologia , Hipercolesterolemia/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , República da Coreia/epidemiologia
10.
J Nutr Biochem ; 90: 108575, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33387610

RESUMO

Maternal hypercholesterolemia induces early onset of cardiovascular diseases in offspring; however, its underlying mechanism remains poorly understood. We hypothesized that maternal hypercholesterolemia increases offspring susceptibility to atherosclerosis in adulthood through developmental modifications of macrophages. Female apolipoprotein E (ApoE)-deficient mice were fed a Western-type diet (WD) or a control diet (CD) prior to and throughout gestation and lactation. The offspring were all fed a WD after weaning. Sixteen-week-old female offspring of WD-fed dams showed a significant increase in atherosclerotic lesions of the aorta and aortic root compared with those of CD-fed dams. This effect was associated with increased macrophage accumulation within lesions, macrophage inflammation and an increase in circulating Ly6Chigh monocyte and F4/80 macrophage counts. We further evidenced that in utero WD exposure promoted macrophage polarization toward the M1 phenotype by elevating M1 markers (Cd86, Inos/Nos2) without affecting M2 markers (Cd206, Arg1). Proinflammatory cytokine synthesis was also enhanced in response to LPS. Finally, maternal WD intake strongly inhibited the macrophage expression of Pparg and Lxra, which was associated with aberrant DNA methylation of Lxra promoter. Our findings demonstrate that maternal hypercholesterolemia exacerbates atherosclerosis, in part by altering the epigenetic state of the macrophage genome of the offspring, imprinting gene expression, and changing macrophage polarization, which ultimately contributes to plaque macrophage burden.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Aterosclerose/metabolismo , Hipercolesterolemia/metabolismo , Macrófagos/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Efeitos Tardios da Exposição Pré-Natal , Animais , Aorta/metabolismo , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Dieta Ocidental , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Fenótipo , Gravidez
11.
PLoS Genet ; 17(1): e1009285, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33513160

RESUMO

Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.


Assuntos
Aterosclerose/genética , Colesterol/genética , Lipoproteínas LDL/genética , Receptores de LDL/genética , Aterosclerose/patologia , Sistemas CRISPR-Cas/genética , Colesterol/metabolismo , Endocitose/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Lipoproteínas LDL/metabolismo , RNA Guia/genética
12.
Int J Biol Macromol ; 173: 66-78, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33482208

RESUMO

Lactobacilli probiotics have been suggested to reduce cholesterol with low side effects to host. Bacteriocins and exopolysaccharides (EPSs) production are two meaningful examples of functional applications of lactobacilli in the food industry. Eight Lactobacillus strains were isolated from some Egyptian fermented food and tested for their probiotic properties. Analysis of the monosaccharide composition by thin layer chromatography showed the presence of glucose, galactose and unknown sugar. The main functional groups of EPSs were elucidated by Fourier-Transform Infrared Spectroscopy. Their fermentation cultures displayed powerful antioxidant activities extending from 97.5 to 99%, 40-75% for their EPSs and free cells, respectively, and exhibited in vitro cholesterol downgrading from 48 to 82% and 72 to 91% after 48 and 120 h, respectively. Their EPSs showed good anticancer activities against carcinoma cells with low IC50 values for HCT-116, PC-3 and HepG-2 cells. To the best of our knowledge, there have been no previous reports on the potential of Lactobacillus EPSs activity against PC-3. The selected strains, L. plantarum KU985433 and L. rhamnosus KU985436 produced two different bacteriocins as detected by gel permeation chromatography with good antimicrobial activities. In vivo study demonstrated that feeding Westar rats with fermented milk exhibited greater cholesterol, LDL and blood triglyceride reduction for both strains. Whereas, HDL was increased by about 43 and 38%, respectively, and the atherogenic indices decreased.


Assuntos
Anticolesterolemiantes/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Hipercolesterolemia/terapia , Polissacarídeos Bacterianos/farmacologia , Probióticos/farmacologia , Animais , Anticolesterolemiantes/isolamento & purificação , Antineoplásicos/isolamento & purificação , Antioxidantes/isolamento & purificação , Bacteriocinas , Sobrevivência Celular/efeitos dos fármacos , HDL-Colesterol/agonistas , HDL-Colesterol/metabolismo , LDL-Colesterol/antagonistas & inibidores , LDL-Colesterol/metabolismo , Modelos Animais de Doenças , Egito , Alimentos e Bebidas Fermentados/microbiologia , Células HCT116 , Células Hep G2 , Humanos , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Lactobacillus plantarum/química , Lactobacillus plantarum/metabolismo , Lactobacillus rhamnosus/química , Lactobacillus rhamnosus/metabolismo , Masculino , Células PC-3 , Polissacarídeos Bacterianos/isolamento & purificação , Probióticos/isolamento & purificação , Ratos , Ratos Wistar , Triglicerídeos/antagonistas & inibidores , Triglicerídeos/metabolismo
13.
Biomed Pharmacother ; 134: 111137, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33341055

RESUMO

Salidroside is a kind of phenylethanoid glycoside and widespread in the plants from Rhodiola and Ligustrum species. Our previous study has reported that salidroside can prevent atherosclerosis progression by ameliorating glyerolipid and glycerophospholipid metabolism in apoE-deficient (apoE-/-) mice. However, its effect on neutral lipids and underlying mechanism remains largely unclear. Here we investigated the molecular mechanism of salidroside action from the perspective of metabolic regulation by integrating metabonomics and transcriptomics pattern. The results showed that salidroside significantly reduced cholesterols, esterified cholesterols, fatty acids, unsaturated fatty acids and triacylclycerols biosynthesis in liver through down-regulating the genes expressions of sterol regulatory element-binding proteins (Srebf1 and Srebf2). The expressions of SREBPs targeted and downstream genes, such as the encoding genes of fatty acid synthase (Fasn), glycerol-3-phosphate acyltransferase (Gpam), stearoyl-CoA desaturase (Scd), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), and proprotein convertase subtilisin/kexin type 9 (Pcsk9), were also inhibited after salidroside administration. ATP citrate lyase gene (Acly) that encodes an important enzyme producing acetyl-CoA for cholesterol and fatty acid biosynthesis significantly decreased after treatment as well. Moreover, one of ketone body products, 3-hydroxybutyrate, was significantly up-regulated in drug-treated group, indicating that fatty acid degradation was accelerated by salidroside at the same time. Our findings identify salidroside as a regulator of lipid homeostasis in atherosclerotic mice, suggesting its potential to be an alternative medicine for lowering the risks of atherosclerosis-related diseases.


Assuntos
Anticolesterolemiantes/farmacologia , Aterosclerose/prevenção & controle , Colesterol/biossíntese , Glucosídeos/farmacologia , Hipercolesterolemia/prevenção & controle , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fenóis/farmacologia , Ácido 3-Hidroxibutírico/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Glicogênio/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Lipidômica , Lipogênese/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Espectroscopia de Prótons por Ressonância Magnética
14.
Toxicol Appl Pharmacol ; 412: 115388, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33383043

RESUMO

Phytosterol diosgenin (DG) exhibits cholesterol-lowering properties. Few studies focused on the underlying mechanism of DG attenuation of hypercholesterolemia by promoting cholesterol metabolism. To investigate the roles of SRB1/CES-1/CYP7A1/FXR pathways in accelerating cholesterol elimination and alleviating hypercholesterolemia, a rat model of hypercholesterolemia was induced by providing a high-fat diet (HFD). Experimental rat models were randomly divided into a normal control (Con) group, HFD group, low-dose DG (LDG) group (150 mg/kg/d), high-dose DG (HDG) group (300 mg/kg) and Simvastatin (Sim) group (4 mg/kg/d). Body weights, serum and hepatic lipid parameters of rats were tested. The expression levels of scavenger receptor class B type I (SRB1), carboxylesterase-1 (CES-1), cholesterol7α- hydroxylase (CYP7A1), and farnesoid X receptor (FXR) were determined. The results showed that DG reduced weight and lowered lipid levels in HFD-fed rats. Pathological morphology analyses revealed that DG notably improved hepatic steatosis and intestinal structure. Further studies showed the increased hepatic SRB1, CES-1, CYP7A1 and inhibited FXR-mediated signaling in DG-fed rats, which contributing to the decrease of hepatic cholesterol. DG also increased intestinal SRB1 and CES-1, inhibiting cholesterol absorption and promoting RCT. The expression levels of these receptors in the HDG group were higher than LDG and Sim groups. These data suggested that DG accelerated reverse cholesterol transport (RCT) and enhanced cholesterol elimination via SRB1/CES-1/CYP7A1/FXR pathway, and DG might be a new candidate for the alleviation of hypercholesterolemia.


Assuntos
Anticolesterolemiantes/farmacologia , Hidrolases de Éster Carboxílico/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Colesterol/sangue , Diosgenina/farmacologia , Hipercolesterolemia/prevenção & controle , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Depuradores Classe B/metabolismo , Animais , Biomarcadores/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/sangue , Hipercolesterolemia/enzimologia , Hipercolesterolemia/patologia , Fígado/enzimologia , Fígado/patologia , Masculino , Ratos Sprague-Dawley , Transdução de Sinais , Sinvastatina/farmacologia
15.
PLoS One ; 15(11): e0238407, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33237915

RESUMO

Calcific aortic valve disease (CAVD) is a deadly disease that is rising in prevalence due to population aging. While the disease is complex and poorly understood, one well-documented driver of valvulopathy is serotonin agonism. Both serotonin overexpression, as seen with carcinoid tumors and drug-related agonism, such as with Fenfluramine use, are linked with various diseases of the valves. Thus, the objective of this study was to determine if genetic ablation or pharmacological antagonism of the 5-HT2B serotonin receptor (gene: Htr2b) could improve the hemodynamic and histological progression of calcific aortic valve disease. Htr2b mutant mice were crossed with Notch1+/- mice, an established small animal model of CAVD, to determine if genetic ablation affects CAVD progression. To assess the effect of pharmacological inhibition on CAVD progression, Notch1+/- mice were treated with the 5-HT2B receptor antagonist SB204741. Mice were analyzed using echocardiography, histology, immunofluorescence, and real-time quantitative polymerase chain reaction. Htr2b mutant mice showed lower aortic valve peak velocity and mean pressure gradient-classical hemodynamic indicators of aortic valve stenosis-without concurrent left ventricle change. 5-HT2B receptor antagonism, however, did not affect hemodynamic progression. Leaflet thickness, collagen density, and CAVD-associated transcriptional markers were not significantly different in any group. This study reveals that genetic ablation of Htr2b attenuates hemodynamic development of CAVD in the Notch1+/- mice, but pharmacological antagonism may require high doses or long-term treatment to slow progression.


Assuntos
Valva Aórtica/patologia , Colesterol/metabolismo , Hemodinâmica/genética , Receptor Notch1/genética , Receptor 5-HT2B de Serotonina/genética , Animais , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Calcinose/genética , Calcinose/patologia , Dieta , Modelos Animais de Doenças , Progressão da Doença , Ecocardiografia/métodos , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/patologia , Hemodinâmica/fisiologia , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Hiperlipidemias/genética , Hiperlipidemias/patologia , Camundongos
16.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228116

RESUMO

Statins are a class of drugs used to lower low-density lipoprotein cholesterol and are amongst the most prescribed medications worldwide. Most statins work as a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), but statin intolerance from pleiotropic effects have been proposed to arise from non-specific binding due to poor enzyme-ligand sensitivity. Yet, research into the physicochemical properties of statins, and their interactions with off-target sites, has not progressed much over the past few decades. Here, we present a concise perspective on the role of statins in lowering serum cholesterol levels, and how their reported interactions with phospholipid membranes offer a crucial insight into the mechanism of some of the more commonly observed pleiotropic effects of statin administration. Lipophilicity, which governs hepatoselectivity, is directly related to the molecular structure of statins, which dictates interaction with and transport through membranes. The structure of statins is therefore a clinically important consideration in the treatment of hypercholesterolaemia. This review integrates the recent biophysical studies of statins with the literature on the physiological effects and provides new insights into the mechanistic cause of statin pleiotropy, and prospective means of understanding the cholesterol-independent effects of statins.


Assuntos
Anticolesterolemiantes/uso terapêutico , Membrana Celular/efeitos dos fármacos , LDL-Colesterol/antagonistas & inibidores , Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Anticolesterolemiantes/síntese química , Anticolesterolemiantes/metabolismo , Transporte Biológico , Biotransformação , Membrana Celular/química , Membrana Celular/metabolismo , LDL-Colesterol/biossíntese , LDL-Colesterol/sangue , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/síntese química , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica
17.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228147

RESUMO

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.


Assuntos
Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Ésteres do Colesterol/metabolismo , Colesterol/metabolismo , Ácido Cólico/metabolismo , Lipoproteínas/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 5 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/química , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Transporte Biológico , Colesterol/química , Ésteres do Colesterol/química , Ácido Cólico/química , Expressão Gênica , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Enteropatias/genética , Enteropatias/metabolismo , Enteropatias/patologia , Cinética , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Lipoproteínas/química , Lipoproteínas/genética , Modelos Moleculares , Mutação , Fitosteróis/efeitos adversos , Fitosteróis/genética , Fitosteróis/metabolismo , Pichia/química , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
18.
Sci Rep ; 10(1): 16601, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024178

RESUMO

Many novel therapies to treat myocardial infarction (MI), yielding promising results in animal models, nowadays failed in clinical trials for several reasons. The most used animal MI model is based on permanent ligation of the left anterior descending (LAD) coronary artery in healthy mice resulting in transmural MI, while in clinical practice reperfusion is usually accomplished by primary percutaneous coronary interventions (PCI) limiting myocardial damage and inducing myocardial ischemia-reperfusion (MI-R) injury. To evaluate a more similar murine MI model we compared MI-R injury to unreperfused MI in hypercholesterolemic apolipoprotein (APO)E*3-Leiden mice regarding effects on cardiac function, left ventricular (LV) remodeling and inflammation. Both MI-R and MI resulted in significant LV dilation and impaired cardiac function after 3 weeks. Although LV dilation, displayed by end-diastolic (EDV) and end-systolic volumes (ESV), and infarct size (IS) were restricted following MI-R compared to MI (respectively by 27.6% for EDV, 39.5% ESV, 36.0% IS), cardiac function was not preserved. LV-wall thinning was limited with non-transmural LV fibrosis in the MI-R group (66.7%). Two days after inducing myocardial ischemia, local leucocyte infiltration in the infarct area was decreased following MI-R compared to MI (36.6%), whereas systemic circulating monocytes were increased in both groups compared to sham (130.0% following MI-R and 120.0% after MI). Both MI-R and MI models against the background of a hypercholesterolemic phenotype appear validated experimental models, however reduced infarct size, restricted LV remodeling as well as a different distributed inflammatory response following MI-R resemble the contemporary clinical outcome regarding primary PCI more accurately which potentially provides better predictive value of experimental therapies in successive clinical trials.


Assuntos
Apolipoproteína E3 , Hipercolesterolemia/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Remodelação Ventricular , Animais , Modelos Animais de Doenças , Feminino , Ventrículos do Coração/patologia , Hipercolesterolemia/patologia , Inflamação , Leucócitos/patologia , Camundongos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/terapia , Intervenção Coronária Percutânea
19.
Int J Mol Sci ; 21(19)2020 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-32977437

RESUMO

BACKGROUND: We recently developed novel matrix metalloproteinase-2 (MMP-2) inhibitor small molecules for cardioprotection against ischemia/reperfusion injury and validated their efficacy in ischemia/reperfusion injury in cardiac myocytes. The aim of the present study was to test our lead compounds for cardioprotection in vivo in a rat model of acute myocardial infarction (AMI) in the presence or absence of hypercholesterolemia, one of the major comorbidities affecting cardioprotection. METHODS: Normocholesterolemic adult male Wistar rats were subjected to 30 min of coronary occlusion followed by 120 min of reperfusion to induce AMI. MMP inhibitors (MMPI)-1154 and -1260 at 0.3, 1, and 3 µmol/kg, MMPI-1248 at 1, 3, and 10 µmol/kg were administered at the 25th min of ischemia intravenously. In separate groups, hypercholesterolemia was induced by a 12-week diet (2% cholesterol, 0.25% cholic acid), then the rats were subjected to the same AMI protocol and single doses of the MMPIs that showed the most efficacy in normocholesterolemic animals were tested in the hypercholesterolemic animals. Infarct size/area at risk was assessed at the end of reperfusion in all groups by standard Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining, and myocardial microvascular obstruction (MVO) was determined by thioflavine-S staining. RESULTS: MMPI-1154 at 1 µmol/kg, MMPI-1260 at 3 µmol/kg and ischemic preconditioning (IPC) as the positive control reduced infarct size significantly; however, this effect was not seen in hypercholesterolemic animals. MVO in hypercholesterolemic animals decreased by IPC only. CONCLUSIONS: This is the first demonstration that MMPI-1154 and MMPI-1260 showed a dose-dependent infarct size reduction in an in vivo rat AMI model; however, single doses that showed the most efficacy in normocholesterolemic animals were abolished by hypercholesterolemia. The further development of these promising cardioprotective MMPIs should be continued with different dose ranges in the study of hypercholesterolemia and other comorbidities.


Assuntos
Cardiotônicos , Hipercolesterolemia/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Cardiotônicos/química , Cardiotônicos/farmacologia , Modelos Animais de Doenças , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Masculino , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Wistar
20.
J Cell Mol Med ; 24(18): 11024-11029, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32767644

RESUMO

The chronopharmacology refers to the utilization of physiological circadian rhythms to optimize the administration time of drugs, thus increasing their efficacy and safety, or reducing adverse effects. Simvastatin is one of the most widely prescribed drugs for the treatment of hypercholesterolaemia, hyperlipidemia and coronary artery disease. There are conflicting statements regarding the timing of simvastatin administration, and convincing experimental evidence remains unavailable. Thus, we aimed to examine whether different administration times would influence the efficacy of simvastatin. High-fat diet-fed mice were treated with simvastatin at zeitgeber time 1 (ZT1) or ZT13, respectively, for nine weeks. Simvastatin showed robust anti-hypercholesterolaemia and anti-hyperlipidemia effects on these obese mice, regardless of administration time. However, simvastatin administrated at ZT13, compared to ZT1, was more functional for decreasing serum levels of total cholesterol, triglycerides, non-esterified free fatty acids and LDL cholesterol, as well as improving liver pathological characteristics. In terms of possible mechanisms, we found that simvastatin did not alter the expression of hepatic circadian clock gene in vivo, although it failed to change the period, phase and amplitude of oscillation patterns in Per2::Luc U2OS and Bmal1::Luc U2OS cells in vitro. In contrast, simvastatin regulated the expression of Hmgcr, Mdr1 and Slco2b1 in a circadian manner, which potentially contributed to the chronopharmacological function of the drug. Taken together, we provide solid evidence to suggest that different administration times affect the lipid-lowering effects of simvastatin.


Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Hiperlipidemias/tratamento farmacológico , Sinvastatina/farmacocinética , Animais , Cronofarmacocinética , Relógios Circadianos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/biossíntese , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Dieta Hiperlipídica/efeitos adversos , Cronoterapia Farmacológica , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Obesos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Sinvastatina/administração & dosagem , Sinvastatina/uso terapêutico
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