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1.
Biol. Res ; 51: 34, 2018. tab, graf
Article in English | LILACS | ID: biblio-983938

ABSTRACT

BACKGROUND AND AIMS: Atherosclerotic cardiovascular disease is highly prevalent and its underlying pathogenesis involves dyslipidemia including pro-atherogenic high density lipoprotein (HDL) remodeling. Vitamins C and E have been proposed as atheroprotective agents for cardiovascular disease management. However, their effects and benefits on high density lipoprotein function and remodeling are unknown. In this study, we evaluated the role of vitamin C and E on non HDL lipoproteins as well as HDL function and remodeling, along with their effects on inflammation/ oxidation biomarkers and atherosclerosis in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice. METHODS AND RESULTS: Mice were pre-treated for 5 weeks before and during atherogenic diet feeding with vitamin C and E added to water and diet, respectively. Compared to a control group, combined vitamin C and E administration reduced serum total cholesterol and triglyceride levels by decreasing apo B-48-containing lipoproteins, remodeled HDL particles by reducing phospholipid as well as increasing PON1 and apo D content, and diminished PLTP activity and levels. Vitamin supplementation improved HDL antioxidant function and lowered serum TNF-α levels. Vitamin C and E combination attenuated atherogenesis and increased lifespan in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice. CONCLUSIONS: Vitamin C and E administration showed significant lipid metabolism regulating effects, including HDL remodeling and decreased levels of apoB-containing lipoproteins, in mice. In addition, this vitamin supplementation generated a cardioprotective effect in a murine model of severe and lethal atherosclerotic ischemic heart disease.


Subject(s)
Animals , Male , Female , Ascorbic Acid/pharmacology , Vitamin E/pharmacology , Myocardial Ischemia/prevention & control , Apolipoprotein B-48/drug effects , Hyperlipidemias/prevention & control , Lipoproteins, HDL/drug effects , Antioxidants/pharmacology , Reference Values , Coronary Artery Disease/prevention & control , Coronary Artery Disease/blood , Enzyme-Linked Immunosorbent Assay , Cardiotonic Agents/pharmacology , Immunoblotting , Reproducibility of Results , Cytokines/blood , Treatment Outcome , Myocardial Ischemia/blood , Dietary Supplements , Phospholipid Transfer Proteins/blood , Diet, Atherogenic , Scavenger Receptors, Class B/drug effects , Scavenger Receptors, Class B/blood , Lipid Metabolism/drug effects , Apolipoprotein B-48/blood , Hyperlipidemias/blood , Lipoproteins, HDL/blood , Mice, Inbred C57BL
2.
Braz. j. med. biol. res ; 50(11): e6389, 2017. tab, graf
Article in English | LILACS | ID: biblio-888946

ABSTRACT

The objective of this study was to observe the infection of human cytomegalovirus (HCMV) to human umbilical vein endothelial cells, and its effect on the expression of single-stranded DNA-binding protein (SSBP1) and on lipid metabolism in endothelial cells. We screened the differential expression of mRNAs after HCMV infection by suppression subtractive hybridization and the expression levels of SSBP1 mRNA and protein after HCMV infection by real-time PCR and western blot. After verification of successful infection by indirect immunofluorescent staining and RT-PCR, we found a differential expression of lipid metabolism-related genes including LDLR, SCARB, CETP, HMGCR, ApoB and LPL induced by HCMV infection. The expression levels of SSBP1 mRNA and protein after HCMV infection were significantly down-regulated. Furthermore, we found that upregulation of SSBP1 inhibited the expression of atherosclerosis-associated LDLR, SCARB, HMGCR, CETP as well as the accumulation of lipids in the cells. The results showed that the inhibition of SSBP1 by HCMV infection promotes lipid accumulation in the cells.


Subject(s)
Humans , Cytomegalovirus Infections/metabolism , DNA-Binding Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/virology , Lipid Metabolism/physiology , Mitochondrial Proteins/metabolism , Atherosclerosis/metabolism , Atherosclerosis/virology , Cholesterol Ester Transfer Proteins/metabolism , Cholesterol/analysis , DNA-Binding Proteins/genetics , Down-Regulation , Hydroxymethylglutaryl CoA Reductases/metabolism , Lipid Metabolism/genetics , Mitochondrial Proteins/genetics , Receptors, LDL/metabolism , Scavenger Receptors, Class B/metabolism , Time Factors
3.
Rev. méd. Chile ; 143(9): 1097-1104, set. 2015. ilus, tab
Article in Spanish | LILACS | ID: lil-762679

ABSTRACT

Background: Plasma high density lipoproteins (HDL) are involved in reverse cholesterol transport mediated by the scavenger receptor class B type I (SR-BI). Nicotinic acid increases HDL cholesterol levels, even though its specific impact on SR-BI dependent-cellular cholesterol transport remains unknown. Aim: To determine the effect of nicotinic acid on HDL particle functionality in cholesterol efflux and uptake mediated by SR-BI in cultured cells in hypoalphalipoproteinemic patients. Material and Methods: In a pilot study, eight patients with low HDL (≤ 40 mg/dL) were treated with extended release nicotinic acid. HDL cholesterol and phospholipid levels, HDL2 and HDL3 fractions and HDL particle sizes were measured at baseline and post-therapy. Before and after nicotinic acid treatment, HDL particles were used for cholesterol transport studies in cells transfected with SR-BI. Results: Nicotinic acid treatment raised total HDL cholesterol and phospholipids, HDL2 levels as well as HDL particle size. Nicotinic acid significantly increased HDL cholesterol efflux and uptake capacity mediated by SR-BI in cultured cells. Conclusions: Nicotinic acid therapy increases SR-BI-dependent HDL cholesterol transport in cultured cells, establishing a new cellular mechanism by which this lipid-lowering drug appears to modulate HDL metabolism in patients with hypoalphalipoproteinemia.


Subject(s)
Aged , Female , Humans , Male , Middle Aged , Cholesterol, HDL/metabolism , Hypoalphalipoproteinemias/metabolism , Hypolipidemic Agents/pharmacology , Lipoproteins, HDL/metabolism , Niacin/pharmacology , Biological Transport , Cholesterol, HDL/drug effects , Pilot Projects , Phospholipids/blood , Scavenger Receptors, Class B/metabolism
4.
Acta Pharmaceutica Sinica ; (12): 440-446, 2015.
Article in Chinese | WPRIM | ID: wpr-251760

ABSTRACT

ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI/CLA-1) are the key proteins in reverse cholesterol transport (RCT). The high expression of ABCA1 and SR-BI/CLA-1 can decrease the danger of atherosclerosis. The purpose of the study is to find ABCA1 and CLA-1up-regulators for treating atherosclerosis by using cell-based high throughput screening models. Among 20 000 compounds screened, E0869 [1-(3, 4-dimethylphenyl)-1-oxopropan-2-yll4-((methylsulfonyl)methyl)benzoate] was found as the positive hit. The up-regulated activities of E0869 in ABCAl1-LUC and bCA-l1-LUC HepG2 cell were 160% and 175%, respectively. The EC50 values of E0869 in ABCAl1-LUC and CLA-l1-LUC HepG2 cell were 3.79 and 1.42 pμol- x ,(-1) respectively. E0869 could upregulate the mRNA and protein levels of ABCA1, SR-BI/CLA-1 and ABCGJ1genes in HepG2 and RAW264.7 cells by Real-Time Quantitative PCR and Western blotting analysis, but could not influence the expression of FAS, SREBP-l1 and CD36. Foam cell assay showed that E0869 could inhibit lipids accumulation in mouse peritoneal macrophages RAW264.7. Cholesterol efflux assay showed that E0869 could induce HDL-mediated cholesterol efflux in mouse peritoneal macrophages RAW264.7. In conclusion, E0869 could up-regulate ABCA1 and CLA-1 activity, and had good anti-atherosclerotic activity in vitro.


Subject(s)
ATP Binding Cassette Transporter 1 , Metabolism , Animals , Atherosclerosis , Drug Therapy , Biological Transport , Cholesterol , Hep G2 Cells , High-Throughput Screening Assays , Humans , Macrophages, Peritoneal , Mice , RNA, Messenger , Scavenger Receptors, Class B , Metabolism , Up-Regulation
5.
Acta Pharmaceutica Sinica ; (12): 1013-1018, 2014.
Article in Chinese | WPRIM | ID: wpr-299175

ABSTRACT

To explore the anti-atherosclerotic mechanism of estrogen and especially observe the effect of estradiol on the content of cholesterol in J774a.1 mouse mononuclear/macrophage-derived foam cells which were incubated with oxidized low-density lipoproteins (ox-LDL). J774a.1 mouse mononuclear/macrophages were incubated with ox-LDL or with both ox-LDL and estradiol (1, 0.1 or 0.01 micromol x L(-1)). Oil red O staining was used to observe the formation of foam cells, and cholesterol oxidase fluorometric was used to determine the content of cellular cholesterol content. Western blotting and RTFQ-PCR were used to observe the expressions of scavenger receptor class B type I (SR-B I ) in J774a.1 foam cells. Compared with the control cells, J774a.1 mouse mononuclear/macrophage-derived foam cells showed significantly increased contents of total cholesterol and cholesterol ester (P < 0.001) and decreased SR-B I mRNA expression (P < 0.01). Estradiol treatment significantly lowered the contents of total cholesterol and cholesterol ester (P < 0.05), and increased SR-B I protein and mRNA expression (P < 0.01) in the foam cells in a dose-dependent manner. Estradiol can inhibit the formation of mononuclear/macrophage-derived foam cells by decreasing the contents of total cholesterol and cholesterol ester and up-regulating the expression of SR-B I in the foam cells.


Subject(s)
Animals , Cell Line , Cholesterol , Metabolism , Cholesterol Esters , Metabolism , Estradiol , Pharmacology , Foam Cells , Cell Biology , Metabolism , Lipoproteins, LDL , Metabolism , Macrophages , Metabolism , Mice , Scavenger Receptors, Class B , Metabolism
6.
Biol. Res ; 46(2): 139-146, 2013. ilus, tab
Article in English | LILACS | ID: lil-683990

ABSTRACT

Studies of developmental effects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive effects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat offspring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg/kg body weight (bwyday, GEN 50 mg/kg bwday, GEN 400 mg/kg bwday, and two combinations of the two compounds (DEHP 250 mg/kg bwday + GEN 50 mg/kg bwday, DEHP 250 mg/kg bwday + GEN 400 mg/kg bwday). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coefficient, AGD / body weight1/3 י, serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bwday), GEN(50mg/kg bwday) or GEN(400mg/kg bwday) alone were not significantly different from the control group. When exposed to (DEHP 250mg/kg bwday +GEN 50mg/kg bwday) together during pregnancy and lactation, serum testosterone concentration, epididymis coefficient and Cypal17a1,Scarb1 m RNA expression significantly decreased compared to the control and GEN(50mg/kg bwday). When exposed to (DEHP 250mg/kg bwday +GEN 400mg/kg bwday) together during pregnancy and lactation, AGD / body weight1/3 י, serum testosterone concentration, testis and epididymis coefficient and Star, Cypal17a1 mRNA expression appeared significantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most significant alterations in the neonate, especially with GEN at high dose, although the effect of the DEHP-GEN mixture on adult offspring should be observed further.


Subject(s)
Animals , Female , Male , Pregnancy , Diethylhexyl Phthalate/toxicity , Endocrine Disruptors/toxicity , Genistein/toxicity , Genitalia, Male/drug effects , Lactation/drug effects , Phytoestrogens/toxicity , Plasticizers/toxicity , Cytochrome P-450 CYP11B2/genetics , Maternal Exposure/adverse effects , Phosphoproteins/genetics , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Scavenger Receptors, Class B/genetics , /genetics , Testis/drug effects
7.
Article in English | WPRIM | ID: wpr-88111

ABSTRACT

Hepatitis C virus (HCV) is a positive sense, single-stranded RNA virus in the Flaviviridae family. It causes acute hepatitis with a high propensity for chronic infection. Chronic HCV infection can progress to severe liver disease including cirrhosis and hepatocellular carcinoma. In the last decade, our basic understanding of HCV virology and life cycle has advanced greatly with the development of HCV cell culture and replication systems. Our ability to treat HCV infection has also been improved with the combined use of interferon, ribavirin and small molecule inhibitors of the virally encoded NS3/4A protease, although better therapeutic options are needed with greater antiviral efficacy and less toxicity. In this article, we review various aspects of HCV life cycle including viral attachment, entry, fusion, viral RNA translation, posttranslational processing, HCV replication, viral assembly and release. Each of these steps provides potential targets for novel antiviral therapeutics to cure HCV infection and prevent the adverse consequences of progressive liver disease.


Subject(s)
Tetraspanin 28/metabolism , Genome, Viral , Hepacivirus/genetics , Humans , RNA, Viral/metabolism , Scavenger Receptors, Class B/metabolism , Viral Envelope Proteins/chemistry , Viral Nonstructural Proteins/chemistry , Virus Assembly , Virus Internalization , Virus Replication
8.
Article in Chinese | WPRIM | ID: wpr-352333

ABSTRACT

<p><b>OBJECTIVE</b>To observe the effect of rosiglitazone on the content of cholesterol and expressions of Acy-coenzyme A: cholesterol acyltransferase 1 (ACAT-1) and scavenger receptor class B type I (SR-BI) in RAW264.7 macrophage-derived foam cells and explore the anti-atherosclerotic mechanism of rosiglitazone.</p><p><b>METHODS</b>RAW264.7 macrophages were incubated with oxidized low-density lipoproteins (ox-LDL) or with both ox-LDL and rosiglitazone (5, 10, or 20 µmol/L). Oil red O staining was used to observe the formation of foam cells, and cholesterol oxidase was used to determine the content of cellular cholesterol contents. Western blotting was used observe the expressions of ACAT-1 and SR-BI in RAW264.7 foam cells.</p><p><b>RESULTS</b>Compared with the control cells, RAW264.7 macrophage-derived foam cells showed significantly increased contents of total cholesterol and free cholesterol (P<0.01) and ACAT-1 expressions (P<0.05) with mildly increased SR-BI expression (P>0.05). Rosiglitazone treatments significantly lowered the contents of total cholesterol and free cholesterol (P<0.05), decreased the expression of ACAT-1 (P<0.05), and increased SR-BI expression (P<0.05) in the foam cells in a dose-dependent manner.</p><p><b>CONCLUSION</b>Rosiglitazone can decrease the contents of total and free cholesterol, down-regulate ACAT-1 expression and up-regulate SR-BI expression in the foam cells produce the anti-atherosclerotic effect.</p>


Subject(s)
Acetyl-CoA C-Acetyltransferase , Metabolism , Cell Line , Cholesterol , Metabolism , Foam Cells , Cell Biology , Metabolism , Humans , Scavenger Receptors, Class B , Metabolism , Thiazolidinediones , Pharmacology
9.
Article in Chinese | WPRIM | ID: wpr-306617

ABSTRACT

The present paper is to research the expression level of the mRNA of scavenger receptor class B type 1-receptor of high-density lipoprotein in endothelial cells after being treated by different shear stress. The second to fourth generations of the cultured human umbilical vein endothelial cells (HUVECs) were used in the experiment. The cells were divided into two groups. The first group was the control group which was not dealt with shear stress; the second group was the experimental group which concluded low shear stress group (4.2 dyne/cm2), moderate shear stress group (8.4 dyne/cm2) and high shear stress group (15 dyne/cm2). The load time was 1h, 2h, 4h and 8h, respectively. Harvesting the cells and extracting total RNA after being treated by different shear stresses, the expression level of the SR-B1 mRNA was detected by semi-quantitative RT-PCR technic. It was found that the expression of SR-B1 mRNA became weaker and weaker compared to the control group when it was stimulated continuously by the low shear stress, the lowest expression of SR-B1 mRNA appeared at 8h. In the moderate shear stress group, the expression of SR-B1 mRNA increased obviously. Compared to the control group, there was significant difference after being treated with 2h. In the high shear stress group, the expression of SR-B1 mRNA increased immediately when it was stimulated by the shear stress. And the expression of SR-B1 mRNA arrived peak value at 4h. Compared to the control group, there was significant difference after being treated for 1h. It was concluded that the harmful mechanism of the low shear stress is that it can increase the incidence of the atherosclerosis by reducing the reverse cholesterol transport and endothelial protection through decreasing the expression of the SR-B1. Otherwise, the high shear stress prevent the genesis of atherosclerosis by the contrary mechanism.


Subject(s)
Atherosclerosis , Cells, Cultured , Human Umbilical Vein Endothelial Cells , Cell Biology , Metabolism , Humans , RNA, Messenger , Genetics , Metabolism , Scavenger Receptors, Class B , Genetics , Metabolism , Stress, Mechanical
10.
Article in Chinese | WPRIM | ID: wpr-268764

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of FXR on scavenger receptor class B type I (SR-BI) expression.</p><p><b>METHODS</b>Human vascular endothelium Eahy926 cells were treated with FXR agonist androsterone, and the specific target gene of FXR SHP mRNA was detected by RT-PCR. SR-BI mRNA and protein were determined using RT-PCR, real-time PCR and Western blotting.</p><p><b>RESULTS</b>The level of SHP mRNA in Eahy926 cells increased after androsterone treatment at different concentrations for 24 h, demonstrating FXR activation in the cells. RT-PCR, real-time PCR and Western blotting detected increased SR-BI expression at both mRNA and protein levels after FXR activation.</p><p><b>CONCLUSION</b>FXR increases the expression of SR-BI in human vascular endothelium cells.</p>


Subject(s)
Androsterone , Pharmacology , Cell Line , Endothelial Cells , Metabolism , Humans , Receptors, Cytoplasmic and Nuclear , Metabolism , Scavenger Receptors, Class B , Metabolism
11.
Article in English | WPRIM | ID: wpr-167145

ABSTRACT

Atopic dermatitis (AD) is an inflammatory skin disorder that is both uncomfortable and distressing to patients, and its prevalence has been steadily increasing. It is obvious that the identification of efficient markers of AD in plasma would offer the possibility of effective diagnosis, prevention, and treatment strategies. In this study, a proteomic approach was used to analyze plasma glycoproteins from both children with AD and healthy child donors. Several protein spots showing significant quantitative changes in the AD patients were identified. Through sequential studies, it was confirmed that CD5L and ApoE were significantly up-regulated or down-regulated, respectively, in the plasma from AD patients compared with that from healthy donors. In addition, we suggest that the up-regulated CD5L in AD patients causes eosinophilia by inhibiting apoptosis or promoting the proliferation of eosinophils either in combination with or without IL-5. The glycoproteomic data in this study provides clues to understanding the mechanism of atopic alterations in plasma and suggests AD-related proteins can be used as candidate markers for AD.


Subject(s)
Apolipoproteins E/blood , Biomarkers/blood , Cell Line , Cell Proliferation , Child , Dermatitis, Atopic/metabolism , Eosinophilia/metabolism , Eosinophils/physiology , Female , Glycoproteins/blood , Humans , Interleukin-5/metabolism , Male , Proteomics , Scavenger Receptors, Class B/blood
12.
Article in Chinese | WPRIM | ID: wpr-813827

ABSTRACT

OBJECTIVE@#To explore whether oxidized low-density lipoprotein (ox-LDL) can stimulate the cholesterol efflux in fully differentiated 3T3-L1 cells and the possible mechanism.@*METHODS@#Fully differentiated 3T3-L1 cells were incubated in the medium containing various concentrations of ox-LDL ( 0 to 50 microg/mL) for 8 or 24 hours. 22(R)-Hydroxycholesterol (10 micromol/L) was exposed to preconditioned adipocytes with 25 microg/mL ox-LDL for 24 hours. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate ATP binding cassette transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI), and liver X receptor alpha (LXRalpha) mRNA expression. Cholesterol efflux mediated by apolipoprotein A-I (apoA-I) was determined using liquid scintillator.@*RESULTS@#Low levels (12.5-25 microg/mL) of ox-LDL could increase cholesterol efflux via the enhancement of ABCA1 pathway and SR-BI expression, whereas the higher concentration (50 microg/mL) could not. In adipocytes preincubated with 25 microg/mL ox-LDL for 24 hours, 22(R)-hydroxycholesterol could increase ABCA1 and LXRalpha mRNA and apoA-I-mediated cholesterol efflux, but had no effect on the SR-BI mRNA expression.@*CONCLUSION@#Low levels of ox-LDL may enhance the LXRalpha-ABCA1-apoA-I pathway in adipocytes, up-regulate SR-BI mRNA expression, and then increase the cholesterol efflux. This new effect of ox-LDL will not only make contribution to cholesterol homeostasis in adipocytes, but also be potentially atheroprotective.


Subject(s)
3T3-L1 Cells , ATP Binding Cassette Transporter 1 , Metabolism , Adipocytes , Metabolism , Animals , Cholesterol , Metabolism , Lipid Metabolism , Lipoproteins, LDL , Pharmacology , Liver X Receptors , Mice , Orphan Nuclear Receptors , Metabolism , Scavenger Receptors, Class B , Metabolism
13.
Article in Chinese | WPRIM | ID: wpr-283441

ABSTRACT

<p><b>OBJECTIVE</b>To study on the regulatory mechanism of lipid metabolism disorders in the blood fat of hyperlipemia rat model with Olive Antihyperlipidemia capsule, and do systematic observation on the functions of this medicine on low And high density lipoprotein receptor in rat liver gene expression, and then to clarify the mechanism of action of this medicine on treating hyperlipemia.</p><p><b>METHOD</b>To select SD rat as investigated subject. The hyperlipemia rat models were made with feeding high-fat forage and were randomly divided into six groups based on the total cholesterol level at the ratsfasting for 12 hours: group A, B, C, D, E and group F. The samples in the research were collected and analyzed the changes of LDLR/SR-B1 gene expression in rat's liver by RT-PCR.</p><p><b>RESULT</b>Olive Antihyperlipidemia capsule can markedly enhance LDLR/SR-B1 gene expression in rat's liver and finally accomplish the purpose of reducing blood fat. The experiment shows this medicine has the remarkable effect on hyperlipidemia and proved the theoretical system of treating hyperlipemia for curing the liver is correct.</p><p><b>CONCLUSION</b>Olive Antihyperlipidemia capsule has an applicable value on preventing the cause, enhance LDLR/SR-B1 gene expression in rat's liver and finally accomplish the purpose of reducing blood fat and development of hyperlipemia and its complications.</p>


Subject(s)
Animals , Capsules , Drugs, Chinese Herbal , Pharmacology , Gene Expression Regulation , Hyperlipidemias , Genetics , Pathology , Hypolipidemic Agents , Pharmacology , Lipoproteins, HDL , Genetics , Liver , Metabolism , Male , Olea , Chemistry , Plants, Medicinal , Chemistry , RNA, Messenger , Genetics , Random Allocation , Rats , Rats, Sprague-Dawley , Receptors, LDL , Genetics , Receptors, Lipoprotein , Genetics , Scavenger Receptors, Class B , Genetics
14.
Rev. chil. cardiol ; 26(4): 437-443, 2007. ilus, tab
Article in Spanish | LILACS | ID: lil-499074

ABSTRACT

Introducción: El receptor scavenger clase B tipo I (SR-BI) es un elemento clave en el metabolismo de las HDL, donde su expresión ejerce un importante efecto anti-aterogénico controlando la fase hepática del transporte reverso de colesterol. Así, el estudio de la modulación de la expresión de SR-BI permitiría el desarrollo de nuevas alternativas farmacológicas para el tratamiento de la ateroesclerosis. Objetivo: La meta de nuestro estudio fue determinar el efecto de la triiodotironina (T3) y el glucagón sobre el metabolismo del colesterol HDL y la expresión hepática de SR-BI en el ratón, evaluando simultáneamente su impacto sobre el colesterol total y lipoproteico plasmático y la secreción biliar de colesterol. Métodos: Se utilizaron ratones C57BL/6 tratados con T3 (30 nmol/kg/día) o glucagón (80 µg/día) más los respectivos grupos controles. Después del tratamiento, los animales se anestesiaron para recolección de bilis, plasma y tejido hepático. Los niveles totales de colesterol plasmático y biliar fueron medidos por métodos enzimáticos. El colesterol lipoproteico plasmático se evaluó por fraccionamiento cromatográfico del plasma y medición enzimática del colesterol en cada fracción. La expresión hepática de SR-BI se cuantificó mediante western blot. Resultados: El uso de T3 o glucagón disminuyeron significativamente el colesterol plasmático total y aumentaron el colesterol biliar con respecto al grupo control correspondiente. Las fracciones de colesterol VLDL, LDL y HDL disminuyeron en ambos grupos tratados, con un mayor efecto observado en la fracción HDL. La administración de ambas hormonas aumentaron significativamente los niveles hepáticos de SR-BI. Conclusión: Los resultados establecen que T3 y glucagón disminuyen el colesterol plasmático, predominantemente de tipo HDL, y aumentan la secreción de colesterol biliar en el ratón, probablemente como consecuencia del incremento en la expresión hepática...


Introduction: The scavenger receptor class B type I (SR-BI) plays a key role in the metabolism of high-density lipoprotein (HDL) cholesterol. Its expression has an important anti-atherogenic effect by controlling the hepatic phase of the reverse cholesterol transport pathway in vivo. Thus, the study of the modulation of SR-BI expression may allow the development of new pharmacologic approaches for treatment of atherosclerotic cardiovascular disease. Objective: The goal of this study was to determine the effect of triiodothyronine (T3) and glucagon on HDL metabolism and hepatic expression of SR-BI in mice, evaluating also the impact in total and lipoprotein cholesterol as well as biliary cholesterol secretion. Methods: C57BL/6 mice were treated with T3 (30 nmol/kg/día) or glucagon (80 µg/día) in comparison to appropriate control groups. After treatment, bile, plasma and hepatic tissue were collected for analysis. Total plasma and biliary cholesterol levels were measured by enzymatic methods. Lipoprotein cholesterol was also measured enzymatically after chromatographic separation of plasma samples. The hepatic expression of SR-BI protein was quantified by western blotting. Results: The use of T3 or glucagon significantly decreased total plasma cholesterol levels and increased of biliary cholesterol concentrations compared to control groups. Levels of VLDL, LDL and HDL cholesterol were reduced in both treatment groups, with a more important effect observed in the HDL fraction. Both treatments increased hepatic SR-BI protein levels. Conclusions: These results show that T3 and glucagon decrease plasma cholesterol levels, particularly in HDL, and increase biliary cholesterol secretion in mice, probably as a consecuence of higher hepatic expression of SR-BI, which may have led to facilitated HDL cholesterol transport from plasma into bile.


Subject(s)
Animals , Mice , Cholesterol, HDL/metabolism , Glucagon/pharmacology , Liver/metabolism , Scavenger Receptors, Class B , Triiodothyronine/pharmacology , Blotting, Western , Bile/chemistry , Cholesterol, HDL/analysis , Cholesterol/analysis , Fluorescent Antibody Technique , Glucagon/administration & dosage , Liver , Receptors, Lipoprotein , Triiodothyronine/administration & dosage
16.
Article in English | WPRIM | ID: wpr-229756

ABSTRACT

<p><b>OBJECTIVE</b>To develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells.</p><p><b>METHODS</b>With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized.</p><p><b>RESULTS</b>Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries.</p><p><b>CONCLUSION</b>Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.</p>


Subject(s)
Animals , Baculoviridae , Genetics , Metabolism , Biological Assay , Carbocyanines , Metabolism , Cell Line, Tumor , Cholesterol, HDL , Metabolism , DNA, Complementary , Genetics , Metabolism , Fluorescent Dyes , Metabolism , Gene Expression , Humans , Lipoproteins, HDL , Genetics , Metabolism , Lipoproteins, LDL , Metabolism , Receptors, Lipoprotein , Genetics , Metabolism , Recombinant Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Scavenger Receptors, Class B , Genetics , Metabolism , Spodoptera , Genetics , Metabolism
17.
Article in Chinese | WPRIM | ID: wpr-231929

ABSTRACT

<p><b>OBJECTIVE</b>To establish a new drug screening model based on transcriptional regulation of human high density lipoprotein (HDL) receptor gene CD36 and LIMPII analogous-1 (CLA-1) for discovering up-regulator of this receptor.</p><p><b>METHODS</b>The upstream regulatory sequence of CLA-1 was obtained by polymerase chain reaction. A recombinant reporter plasmid pGL3-CLAP was constructed by inserting the regulatory sequence upstream of luciferase gene of pGL3-Basic. Human hepatoma cell line BEL-7402 was transfected with pGL3-CLAP. Samples were detected by testing luciferase activity of transfected BEL-7402 cells in microtiter wells.</p><p><b>RESULTS</b>The drug screening model was established and optimized. Significant difference was present between pGL3-CLAP and pGL3-Basic transfected BEL-7402 cells (P< 0.001), and coefficient of variation was less than 10%. After primary and secondary screening, 1 compounds and 3 fermentation extracts had up-regulating activities.</p><p><b>CONCLUSION</b>This new drug screening model may be efficiently used to screen up-regulators of human HDL receptor expression, which might become lead compounds for new anti-atherosclerosis drugs.</p>


Subject(s)
CD36 Antigens , Cholesterol Esters , Metabolism , Drug Evaluation, Preclinical , Methods , Gene Expression Regulation , Humans , Hypolipidemic Agents , Pharmacology , Lipoproteins, HDL , Genetics , Metabolism , RNA, Messenger , Metabolism , RNA-Binding Proteins , Receptors, Immunologic , Genetics , Receptors, Lipoprotein , Genetics , Receptors, Scavenger , Scavenger Receptors, Class B , Transcription, Genetic , Up-Regulation
18.
Article in English | WPRIM | ID: wpr-148813

ABSTRACT

Both hydropathy plot and in vitro translation results predict the topology of SR-BI; the receptor is an integral membrane protein of 509 amino acids, consisting of a short cytoplasmic N-terminus of 9 amino acids followed by a first transmembrane domain of 22 amino acids, the extracellular domain of 408 amino acids, the second transmembrane domain of 22 amino acids, and the cytoplasmic C-terminus of 47 amino acids. The immunoblot of rBBMV in the presence or absence of pAb589 peptide antigen (the C-terminal 22 amino acid residues of SR-BI) confirmed that the bands at apparent molecular weight of 140 and 210 kDa are SR-BI related protein which might be multimeric forms of SR-BI. 125I apo A-I overlay analysis showed that SR-BI can bind to its ligand, apo A-I, only when it is thoroughly matured - glycosylated and dimerized. The antibody which was generated against extracellular domain of SR-BI (pAb230) not only prevented 125I-labeled apo A-I from binding to 140 kDa band but also inhibited the esterified cholesterol uptake of rabbit BBMV with its IC50 value of 40 microgram/ml of IgG. In contrast, the antibody generated against the C-terminal domain of SR-BI (pAb589) did not show any effect either on cholesterol uptake of rabbit BBMV or 125I-labeled apo A-I binding to 140 kDa band. Overall results show that the ligand binding site of SR-BI in rabbit BBMV is located in extracellular domain, and SR-BI is only functional when it is part of dimeric forms which rationalize the previously found cooperative nature of the binding interaction and maybe a fundamental finding towards the so far poorly understood mechanism of SR-BI function.


Subject(s)
Amino Acid Sequence , Animals , CD36 Antigens/metabolism , Apolipoprotein A-I/metabolism , Binding Sites/physiology , Blotting, Western , Caco-2 Cells , Cholesterol Esters/metabolism , Humans , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Iodine Radioisotopes , Membrane Proteins/metabolism , Microvilli/metabolism , Molecular Sequence Data , Rabbits , Receptors, Immunologic , Receptors, Lipoprotein/metabolism , Receptors, Scavenger , Scavenger Receptors, Class B , Surface Properties
19.
Biol. Res ; 33(2): 97-103, 2000.
Article in English | LILACS | ID: lil-443673

ABSTRACT

Scavenger receptors were discovered as cell surface proteins capable of binding and internalization of modified lipoproteins. These receptors exhibit a broad ligand binding specificity including potential physiological and pathophysiological ligands other than modified lipoproteins. Different classes of scavenger receptors have been identified, and their relevance in normal and pathological conditions is under investigation. Recent in vitro and in vivo studies strongly support the role of class A and class B scavenger receptors in lipid transport and atherogenesis.


Subject(s)
Animals , Humans , Arteriosclerosis/metabolism , Lipoproteins, LDL/metabolism , Receptors, Immunologic/metabolism , Biological Transport , Membrane Proteins/metabolism , Receptors, Scavenger , Scavenger Receptors, Class A , Scavenger Receptors, Class B
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