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1.
J Cardiovasc Pharmacol ; 75(4): 336-343, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31934911

RESUMO

OBJECTIVE: Our previous study showed that Coiled-Coil Domain Containing 80 (CCDC80) accelerates the development of atherosclerosis by decreasing lipoprotein lipase (LPL) expression and activity in apoE knockout mice. However, the regulatory mechanism for CCDC80 expression is unclear. This study was designed to evaluate whether noncoding RNAs involved the regulation of CCDC80 expression in vascular smooth muscle cells. METHODS AND RESULTS: Bioinformatics prediction and luciferase reporter gene results showed that miR-141-3p/200a-3p bound to the 3'UTR of CCDC80. Furthermore, miR-141-3p/200a-3p mimics decreased the expression of CCDC80 but increased LPL expression. Opposite results were observed with miR-141-3p/200a-3p inhibitors. We also found that lncRNA metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) interacted with the sequences of miR-141-3p/200a-3p and decreased their expression. RT-qPCR and western blotting results showed that MALAT1 overexpression increased CCDC80 expression and decreased LPL expression, while MALAT1 knockdown displayed an opposite phenotype. The effects of both MALAT1 overexpression and knockdown were blocked by miR-141-3p/200a-3p mimics or inhibitors. CONCLUSIONS: Thus, we demonstrated that lncRNA MALAT1 regulates CCDC80 and LPL expression through miR-141-3p/200a-3p.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , RNA Longo não Codificante/metabolismo , Sítios de Ligação , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica , Humanos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , MicroRNAs/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética
2.
Atherosclerosis ; 289: 143-161, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518965

RESUMO

BACKGROUND AND AIMS: Krüppel-like factor 14 (KLF14) is known to play a role in atherosclerosis, but the underlying mechanisms are still largely unknown. The aim of our study was to explore the effects of KLF14 on lipid metabolism and inflammatory response, providing a potential target for lowering the risk of atherosclerosis-causing disease. METHODS AND RESULTS: mRNA and protein levels of KLF14 were significantly decreased in oxidized low-density lipoprotein (oxLDL)-treated macrophages and in the atherosclerotic lesion area. Chromatin immunoprecipitation (ChIP) and luciferase reporter gene assays were used to confirm that KLF14 positively regulated miR-27a expression by binding to its promoter. We also found that KLF14 could restored appropriate cellular lipid homeostasis and inflammatory responses via negatively regulating lipoprotein lipase (LPL) expression in THP1-derived macrophages through miR-27a. In addition, gypenosides (GP), a KLF14 activator, delayed the development of atherosclerosis in apolipoprotein E deficient (apoE-/-) mice. CONCLUSIONS: KLF14 plays an antiatherogenic role via the miR-27a-dependent down-regulation of LPL and subsequent inhibition of proinflammatory cytokine secretion and lipid accumulation.


Assuntos
Aterosclerose/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Lipase Lipoproteica/metabolismo , MicroRNAs/metabolismo , Animais , Aterosclerose/patologia , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Gynostemma , Homeostase , Metabolismo dos Lipídeos , Lipídeos/química , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout para ApoE , Extratos Vegetais/farmacologia , Células RAW 264.7 , Transfecção
3.
Eur J Pharmacol ; 843: 177-189, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30439364

RESUMO

Recent studies showed that coiled-coil domain-containing 80 (CCDC80) has a positive link with atherosclerosis and that plasma CCDC80 levels are positively correlated with the levels of fasting plasma triglycerides (TG) in obese individuals. The underlying mechanisms, however, are unclear. Using Hematoxylin-eosin (H&E) and Oil Red O staining, we found that CCDC80 overexpression in vivo significantly increased plasma lipid contents, decreased the expression and activity of lipoprotein lipase (LPL), and accelerated the development of atherosclerosis. Conversely, knockdown of CCDC80 decreased plaque lesions area. In vitro, qRT-PCR and western blot results showed that CCDC80 overexpression significantly decreased, while CCDC80 knockdown increased, LPL expression in cultured vascular smooth muscle cells (VSMCs). Further, we found that CCDC80 reduced LPL expression via inhibiting the phosphorylation of extracellular regulated protein kinase 1/2 (ERK1/2) and also increased the methylation of LPL promoter via down-regulating Tet methylcytosine dioxygenase 2 (TET2). Our results also revealed that CCDC80 significantly down-regulated TET2 expression through decreasing the phosphorylation of ERK1/2. In addition, we found that CCDC80 decreased binding of TET2 to forkhead box O3 (FOXO3a) but had no effect on FOXO3a expression. On the other hand, and that FOXO3a was partially involved in TET2-regulated LPL expression. CCDC80 down-regulated ERK1/2 phosphorylation and decreased expression of TET2 and its interaction with FOXO3a, leading to a reduction of LPL expression and acceleration of atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipase Lipoproteica/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Metilação de DNA , Dioxigenases , Proteínas da Matriz Extracelular , Proteína Forkhead Box O3/metabolismo , Lipase Lipoproteica/genética , Masculino , Camundongos Knockout para ApoE , Músculo Liso Vascular/citologia , Fosforilação , Triglicerídeos/sangue
4.
Sci Rep ; 7(1): 12484, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970485

RESUMO

MRC-5 represents the most frequent human diploid cells (HDCs)-type cell substrate in the production of human viral vaccines. However, early-passage MRC-5 is diminishing and, due to both technical and ethical issues, it is extremely difficult to derive novel HDCs from fetal lung tissues, which are the common sources of HDCs. Our previous studies suggested that human umbilical cord may represent an alternative but convenient source of new HDCs. Here, we established a three-tiered cell banking system of a hUC-MSC line, designated previously as Cell Collection and Research Center-1 (CCRC-1). The full characterization indicated that the banked CCRC-1 cells were free from adventitious agents and remained non-tumorigenic. The CCRC-1 cells sustained its rapid proliferation even at passage 30 and were susceptible to the infection of a wide spectrum of viruses. Interestingly, the CCRC-1 cells showed much higher production of EV71 or Rubella viruses than MRC-5 and Vero cells when growing in serum-free medium. More importantly, the EV71 vaccine produced from CCRC-1 cells induced immunogenicity while eliciting no detectable toxicities in the tested mice. Collectively, these studies further supported that CCRC-1, and likely other hUC-MSCs as well, may serve as novel, safe and high-yielding HDCs for the production of human viral vaccines.


Assuntos
Infecções por Enterovirus/prevenção & controle , Células-Tronco Mesenquimais/virologia , Rubéola (Sarampo Alemão)/prevenção & controle , Vacinação , Vacinas Virais/biossíntese , Animais , Bancos de Espécimes Biológicos , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Meios de Cultura Livres de Soro/química , Diploide , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Feminino , Sangue Fetal/citologia , Humanos , Imunogenicidade da Vacina , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Rubéola (Sarampo Alemão)/imunologia , Rubéola (Sarampo Alemão)/virologia , Vírus da Rubéola/efeitos dos fármacos , Vírus da Rubéola/imunologia , Células Vero , Vacinas Virais/administração & dosagem
5.
Circ J ; 82(1): 28-38, 2017 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-28855441

RESUMO

BACKGROUND: Lipoprotein lipase (LPL) expressed in macrophages plays an important role in promoting the development of atherosclerosis or atherogenesis. MicroRNA-182 (miR-182) is involved in the regulation of lipid metabolism and inflammation. However, it remains unclear how miR-182 regulates LPL and atherogenesis.Methods and Results:Using bioinformatics analyses and a dual-luciferase reporter assay, we identified histone deacetylase 9 (HDAC9) as a target gene of miR-182. Moreover, miR-182 upregulated LPL expression by directly targetingHDAC9in THP-1 macrophages. Hematoxylin-eosin (H&E), Oil Red O and Masson's trichrome staining showed that apolipoprotein E (ApoE)-knockout (KO) mice treated with miR-182 exhibited more severe atherosclerotic plaques. Treatment with miR-182 increased CD68 and LPL expression in atherosclerotic lesions in ApoE-KO mice, as indicated by double immunofluorescence staining in the aortic sinus. Increased miR-182-induced increases in LPL expression in ApoE-KO mice was confirmed by real-time quantitative polymerase chain reaction and western blotting analyses. Treatment with miR-182 also increased plasma concentrations of proinflammatory cytokines and lipids in ApoE-KO mice. CONCLUSIONS: The results of the present study suggest that miR-182 upregulates LPL expression, promotes lipid accumulation in atherosclerotic lesions, and increases proinflammatory cytokine secretion, likely through targetingHDAC9, leading to an acceleration of atherogenesis in ApoE-KO mice.


Assuntos
Aterosclerose/induzido quimicamente , Lipase Lipoproteica/efeitos dos fármacos , MicroRNAs/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Animais , Biologia Computacional , Citocinas/efeitos dos fármacos , Células HEK293 , Histona Desacetilases , Humanos , Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos , Camundongos , Camundongos Knockout para ApoE , Células THP-1
6.
PLoS One ; 11(6): e0157085, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27257686

RESUMO

Atherosclerotic lesions are lipometabolic disorder characterized by chronic progressive inflammation in arterial walls. Previous studies have shown that macrophage-derived lipoprotein lipase (LPL) might be a key factor that promotes atherosclerosis by accelerating lipid accumulation and proinflammatory cytokine secretion. Increasing evidence indicates that microRNA-27 (miR-27) has beneficial effects on lipid metabolism and inflammatory response. However, it has not been fully understood whether miR-27 affects the expression of LPL and subsequent development of atherosclerosis in apolipoprotein E knockout (apoE KO) mice. To address these questions and its potential mechanisms, oxidized low-density lipoprotein (ox-LDL)-treated THP-1 macrophages were transfected with the miR-27 mimics/inhibitors and apoE KO mice fed high-fat diet were given a tail vein injection with miR-27 agomir/antagomir, followed by exploring the potential roles of miR-27. MiR-27 agomir significantly down-regulated LPL expression in aorta and peritoneal macrophages by western blot and real-time PCR analyses. We performed LPL activity assay in the culture media and found that miR-27 reduced LPL activity. ELISA showed that miR-27 reduced inflammatory response as analyzed in vitro and in vivo experiments. Our results showed that miR-27 had an inhibitory effect on the levels of lipid both in plasma and in peritoneal macrophages of apoE KO mice as examined by HPLC. Consistently, miR-27 suppressed the expression of scavenger receptors associated with lipid uptake in ox-LDL-treated THP-1 macrophages. In addition, transfection with LPL siRNA inhibited the miR-27 inhibitor-induced lipid accumulation and proinflammatory cytokines secretion in ox-LDL-treated THP-1 macrophages. Finally, systemic treatment revealed that miR-27 decreased aortic plaque size and lipid content in apoE KO mice. The present results provide evidence that a novel antiatherogenic role of miR-27 was closely related to reducing lipid accumulation and inflammatory response via downregulation of LPL gene expression, suggesting a potential strategy to the diagnosis and treatment of atherosclerosis.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Inflamação/metabolismo , Lipase Lipoproteica/farmacocinética , MicroRNAs/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/sangue , Aterosclerose/genética , Linhagem Celular , Quimiocina CCL2/sangue , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/sangue , Inflamação/genética , Interleucina-1beta/sangue , Interleucina-6/sangue , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipase Lipoproteica/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Células RAW 264.7 , Reação em Cadeia da Polimerase em Tempo Real , Receptores Depuradores/metabolismo , Fator de Necrose Tumoral alfa/sangue
7.
Biochem Biophys Res Commun ; 472(3): 410-7, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-26546816

RESUMO

Angiopoietin-like 4 (Angptl4), a secreted protein, is an important regulator to irreversibly inhibit lipoprotein lipase (LPL) activity. Macrophage LPL contributes to foam cell formation via a so-called"molecular bridge" between lipoproteins and receptors on cell surface. It has been reported that macrophage ANGPTL4 suppresses LPL activity, foam cell formation and inflammatory gene expression to reduce atherosclerosis development. Recently, some studies demonstrated that microRNA-134 is upregulated in atherosclerotic macrophages. Here we demonstrate that miR-134 directly binds to 3'UTR of ANGPTL4 mRNA to suppression the expression of ANGPTL4. To investigate the potential roles of macrophage miR-134, THP-1 macrophages were transfected with miR-134 mimics or inhibitors. Our results showed that LPL activity and protein were dramatically increased. We also found that miR-134 activated LPL-mediated lipid accumulation. Collectively, our findings indicate that miR-134 may regulate lipid accumulation and proinfiammatory cytokine secretion in macrophages by targeting the ANGPTL4 gene. Our results have also suggested a promising and potential therapeutic target for atherosclerosis.


Assuntos
Angiopoietinas/imunologia , Inflamação/imunologia , Metabolismo dos Lipídeos/imunologia , Lipase Lipoproteica/imunologia , Macrófagos/imunologia , MicroRNAs/imunologia , Proteína 4 Semelhante a Angiopoietina , Linhagem Celular , Ativação Enzimática , Humanos , Macrófagos/enzimologia , Transdução de Sinais/imunologia
8.
Clin Chim Acta ; 453: 107-13, 2016 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-26683354

RESUMO

Cardiovascular diseases, such as atherosclerosis and hypertension, are the major cause of mortality and morbidity in the world. Adropin was first discovered in 2008 by Kumar and his coworkers. Adropin, encoded by the Energy Homeostasis Associated gene, is expressed in many tissues and organs, such as pancreatic tissue, liver, brain, kidney, endocardium, myocardium, and epicardium. In this review, we have summarized recent data suggesting the roles of adropin in several major cardiovascular diseases. Increasing evidence suggests that adropin is a potential regulator of cardiovascular functions and plays a protective role in the pathogenesis and development of cardiovascular diseases. However, further studies are needed to elucidate the specific mechanisms underlying the association between adropin and cardiovascular diseases.


Assuntos
Proteínas Sanguíneas/metabolismo , Doenças Cardiovasculares/metabolismo , Peptídeos/metabolismo , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Doenças Cardiovasculares/genética , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/química , Peptídeos/genética
9.
Biochimie ; 119: 192-203, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26542288

RESUMO

BACKGROUND: Atherosclerosis is a major cause of coronary artery disease, which is characterized by cellular lipid accumulation. Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism. Studies have shown that macrophage-derived LPL exhibits proatherogenic properties, and plays a major role in lipid accumulation in macrophages. Evidence suggests that oxidative stress can effectively enhance macrophage LPL production. Betulinic acid (BA) is a pentacyclic lupane triterpene with a potent antioxidant activity. In this study, we investigated whether BA affects the expression of macrophage LPL and how it regulates cellular lipid accumulation. METHODS AND RESULTS: We revealed that BA downregulated H2O2-simulated macrophage LPL protein, mRNA levels and its activity in both concentration- and time-dependent manners. Furthermore, BA decreased LPL-involved total cholesterol and triglyceride levels in macrophages. In addition, cellular lipid staining by Oil Red O showed that BA decreased cellular lipid droplet deposition. Next, we confirmed that pretreatment with BA decreased H2O2-induced production of intracellular reactive oxygen species in a concentration-dependent manner. Further studies demonstrated that BA inhibited H2O2-induced membrane translocation of PKC, phosphorylation of ERK1/2 and c-Fos. Finally, the induction of LPL production and activity by H2O2 was abolished by BA, inhibition of PKC or ERK or depletion c-Fos, respectively. CONCLUSIONS: BA, through its role of antioxidant activity, attenuated macrophage-derived LPL expression and activity induced by oxidative stress, and effectively reduced cellular lipid accumulation, likely through inhibition of the pathways involving PKC, ERK and c-Fos. These effects of BA may contribute to its mitigation of atherosclerosis and help develop BA as a therapeutic compound in treatment of atherosclerosis.


Assuntos
Antioxidantes/farmacologia , Repressão Enzimática/efeitos dos fármacos , Lipase Lipoproteica/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Peróxido de Hidrogênio/toxicidade , Hipolipemiantes/farmacologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Oxidantes/toxicidade , Triterpenos Pentacíclicos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7 , Interferência de RNA , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Ácido Betulínico
10.
PLoS One ; 10(9): e0138788, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26397958

RESUMO

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE-/-) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE-/- mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE-/- mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1ß (IL-1ß)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion.


Assuntos
Apolipoproteínas E/genética , Aterosclerose/enzimologia , Lipase Lipoproteica/genética , MicroRNAs/genética , Animais , Aorta/enzimologia , Aorta/patologia , Antígenos CD36/metabolismo , Citocinas/sangue , Repressão Enzimática , Metabolismo dos Lipídeos , Lipase Lipoproteica/metabolismo , Macrófagos Peritoneais/enzimologia , Masculino , Camundongos Knockout , MicroRNAs/metabolismo , Interferência de RNA
11.
Biochimie ; 106: 81-90, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25149060

RESUMO

BACKGROUND: Accumulating evidence suggests that microRNA-590 (miR-590) has protective effects on cardiovascular diseases, but the mechanism is unknown. Interestingly, previous studies from our laboratory and others have shown that macrophage-derived lipoprotein lipase (LPL) might accelerate atherosclerosis by promoting lipid accumulation and inflammatory response. However, the regulation of LPL at the post-transcriptional level by microRNAs has not been fully understood. In this study, we explored whether miR-590 affects the expression of LPL and its potential subsequent effects on lipid accumulation and pro-inflammatory cytokine secretion in human THP-1 macrophages. METHODS AND RESULTS: Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-590 directly inhibited LPL protein and mRNA expression by targeting LPL 3'UTR. LPL Activity Assays showed that miR-590 reduced LPL activity in the culture media. Oil Red O staining and high-performance liquid chromatography assays showed that miR-590 had inhibitory effects on the lipid accumulation in human THP-1 macrophages. We also illustrated that miR-590 alleviated pro-inflammatory cytokine secretion in human THP-1 macrophages as measured by ELISA. With the method of small interfering RNA, we found that LPL siRNA can inhibit the miR-590 inhibitor-induced increase in lipid accumulation and secretion of pro-inflammatory cytokines in oxLDL-treated human THP-1 macrophages. CONCLUSIONS: MiR-590 attenuates lipid accumulation and pro-inflammatory cytokine secretion by targeting LPL gene in human THP-1 macrophages. Therefore, targeting miR-590 may offer a promising strategy to treat atherosclerotic cardiovascular diseases.


Assuntos
Citocinas/metabolismo , Lipídeos/análise , Lipase Lipoproteica/genética , Macrófagos/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Expressão Gênica , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Lipase Lipoproteica/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico
12.
Biochem Biophys Res Commun ; 443(2): 428-34, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24309104

RESUMO

Atherosclerosis is a lipid disorder disease characterized by chronic blood vessel wall inflammation driven by the subendothelial accumulation of macrophages. Studies have shown that lipoprotein lipase (LPL) participates in lipid metabolism, but it is not yet known whether post-transcriptional regulation of LPL gene expression by microRNAs (miRNAs) occurs in vivo. Here, we tested that miR-467b provides protection against atherosclerosis by regulating the target gene LPL which leads to reductions in LPL expression, lipid accumulation, progression of atherosclerosis and production of inflammatory cytokines in apolipoprotein E knockout (apoE(-/-)) mice. Treatment of apoE(-/-) mice with intra-peritoneal injection of miR-467b agomir led to decreased blood plasma levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1ß and monocyte chemotactic protein-1 (MCP-1). Using Western blots and real time PCR, we determined that LPL expression in aorta and abdominal cavity macrophages were significantly down-regulated in the miR-467b agomir group. Furthermore, systemic treatment with miR-467b antagomir accelerated the progression of atherosclerosis in the aorta of apoE(-/-) mice. The present study showed that miR-467b protects apoE(-/-) mice from atherosclerosis by reducing lipid accumulation and inflammatory cytokine secretion via downregulation of LPL expression. Therefore, targeting miR-467b may offer a promising strategy to treat atherosclerotic vascular disease.


Assuntos
Apolipoproteínas E/genética , Aterosclerose/imunologia , Citocinas/imunologia , Inflamação/imunologia , Metabolismo dos Lipídeos/imunologia , Lipase Lipoproteica/imunologia , MicroRNAs/farmacologia , Animais , Aterosclerose/prevenção & controle , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/prevenção & controle , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipase Lipoproteica/biossíntese , Masculino , Camundongos , Camundongos Knockout , Resultado do Tratamento
13.
Cell Biochem Biophys ; 67(3): 1239-48, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23695786

RESUMO

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the ß-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic ß-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it's activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Interferon gama/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antineoplásicos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Interferon gama/farmacologia , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
14.
Biochimie ; 94(12): 2749-55, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22963823

RESUMO

LPL (lipoprotein lipase) is a rate-limiting enzyme involved in the hydrolysis of triglycerides. Previous studies have shown that microRNA (miR)-467b regulates hepatic LPL expression and plays a role in the progression of steatosis or abnormal lipid retention in obese mice. Macrophage-derived LPL has been shown to promote atherosclerosis. However, if miR-476b influences macrophage LPL expression and the subsequent effects are unknown. Here, we utilized oxLDL-treatment RAW 264.7 macrophages that were transfected with miR-467b mimics or inhibitors to investigate the potential roles of macrophage miR-476b. We found that miR-467b significantly decreased lipid accumulation and IL-6, IL-1ß, TNF-α and MCP-1 secretions. Furthermore, our studies suggested an additional explanation for the regulatory mechanism of miR-467b on its functional target, LPL in RAW 264.7 macrophages. Thus, our findings indicate that miR-467b may regulate lipid accumulation and proinflammatory cytokine secretion in oxLDL-stimulated RAW 264.7 macrophages by targeting the LPL gene.


Assuntos
Citocinas/metabolismo , Lipídeos/análise , Lipase Lipoproteica/genética , Macrófagos/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Quimiocina CCL2/metabolismo , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipase Lipoproteica/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , MicroRNAs/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Fator de Necrose Tumoral alfa/metabolismo
15.
J Pharm Pharmacol ; 64(2): 293-301, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22221106

RESUMO

OBJECTIVES: The aim of the study was to elucidate the possible role and mechanism of NO-1886 (ibrolipim, a lipoprotein lipase activator) in ameliorating insulin resistance induced by high palmitate. METHODS: HepG2 cells were cultured in RPMI 1640 medium and were treated with palmitate to induce insulin resistance. Free fatty acids (FFAs), glucose, glycogen, cell viability and mRNA and protein levels were analysed separately. KEY FINDINGS: We found that HepG2 cells treated with 0.5 mm palmitate for 48 h led to a significant decrease of insulin-induced glucose consumption (from 2.89 ± 0.85 mm in the control to 0.57 ± 0.44 mm in palmitate). Insulin resistance (IR) of HepG2 cells was induced by 0.5 mm palmitate for 48 h. NO-1886 stimulated glucose consumption, glycogen synthesis and FFA absorption in insulin-resistant HepG2 cells. Maximum stimulation effects were observed with 10 µm NO-1886 for 24 h. Compared with the dimethyl sulfoxide-treated group, 2.5 µm NO-1886 or higher could induce the mRNA expression of lipoprotein lipase. Meanwhile, NO-1886 increased the protein content of P-GSK-3ßser(9) and decreased the protein level of GSK-3ß in insulin-resistant HepG2 cells, but NO-1886 didn't change the protein levels of PI3-Kp85 and Akt2. CONCLUSION: Lipoprotein lipase activator NO-1886 could increase glycogen synthesis in HepG2 cells and could ameliorate the insulin resistance, which was associated with GSK-3 signalling.


Assuntos
Benzamidas/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogenólise/efeitos dos fármacos , Resistência à Insulina/fisiologia , Ativadores de Lipase de Lipoproteínas/farmacologia , Compostos Organofosforados/farmacologia , Palmitatos/metabolismo , Benzamidas/química , Células Cultivadas , Glicogênio Sintase Quinase 3 beta , Células Hep G2 , Humanos , Ativadores de Lipase de Lipoproteínas/química , Compostos Organofosforados/química , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Estatística como Assunto
16.
Acta Pharmacol Sin ; 31(10): 1343-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20871621

RESUMO

AIM: To determine the effects and potential mechanisms of ibrolipim on ATP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1) expression from human macrophage foam cells, which may play a critical role in atherogenesis. METHODS: Human THP-1 cells pre-incubated with ox-LDL served as foam cell models. Specific mRNA was quantified using real-time RT-PCR and protein expression using Western blotting. Cellular cholesterol handling was studied using cholesterol efflux experiments and high performance liquid chromatography assays. RESULTS: Ibrolipim 5 and 50 µmol/L significantly increased cholesterol efflux from THP-1 macrophage-derived foam cells to apoA-I or HDL. Moreover, it upregulated the expression of ABCA1 and ABCG1. In addition, LXRα was also upregulated by the ibrolipim treatment. In addition, LXRα small interfering RNA completely abolished the promotion effect that was induced by ibrolipim. CONCLUSION: Ibrolipim increased ABCA1 and ABCG1 expression and promoted cholesterol efflux, which was mediated by the LXRα signaling pathway.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzamidas/farmacologia , Células Espumosas/efeitos dos fármacos , Ativadores de Lipase de Lipoproteínas/farmacologia , Compostos Organofosforados/farmacologia , Receptores Nucleares Órfãos/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transporte Biológico , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/metabolismo , Células Espumosas/metabolismo , Regulação da Expressão Gênica , Humanos , Receptores X do Fígado , Receptores Nucleares Órfãos/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Regulação para Cima
17.
Clin Exp Pharmacol Physiol ; 36(9): e32-9, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19473196

RESUMO

1. MicroRNAs (miRNAs) play essential roles in many biological processes. It is known that aberrant miRNA expression contributes to some pathological conditions. However, it is not known whether miRNAs play any role in the development of insulin resistance in adipocytes, a key pathophysiological link between obesity and diabetes. 2. To investigate the function of miRNAs in the development of insulin resistance, using miRNA microarray analysis we compared miRNA expression profiles between normal insulinsensitive 3T3-L1 adipocytes and 3T3-L1 adipocytes rendered insulin resistant following treatment with high glucose (25mmol/L) and high insulin (1 mol/L). Furthermore, adipocytes were transfected with specific antisense oligonucleotides against miRNA-320 (anti-miR-320 oligo) and the effects on the development of insulin resistance were evaluated. 3. We identified 50 upregulated and 29 downregulated miRNAs in insulin-resistant (IR) adipocytes, including a 50-fold increase in miRNA-320 (miR-320) expression. Using bioinformatic techniques, the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) was found to be a potential target of miR-320. In experiments with anti-miR-320 oligo, insulin sensitivity was increased in IR adipocytes, as evidenced by increases in p85 expression, phosphorylation of Akt and the protein expression of the glucose transporter GLUT-4, as well as insulin-stimulated glucose uptake. These beneficial effects of anti-miR-320 oligo were observed only in IR adipocytes and not in normal adipocytes. 4. In conclusion, the miRNA profile changes in IR adipocytes compared with normal 3T3-L1 adipocytes. Anti-miR-320 oligo was found to regulate insulin resistance in adipocytes by improving insulin­PI3-K signalling pathways. The findings provide information regarding a potentially new therapeutic strategy to control insulin resistance.


Assuntos
Adipócitos/metabolismo , Perfilação da Expressão Gênica , Resistência à Insulina/genética , Insulina/metabolismo , MicroRNAs/metabolismo , Células 3T3-L1 , Adipogenia/genética , Animais , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Biologia Computacional , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , Transfecção
18.
Acta Pharmacol Sin ; 27(2): 151-7, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16412263

RESUMO

AIM: To investigate the protective effect of high density lipoprotein 3 (HDL3) on oxidized low density lipoprotein (ox-LDL)-induced apoptosis in RAW264.7 cells. METHODS: RAW264.7 cells were exposed to 50 mg/L ox-LDL for various durations up to 48 h, and apoptosis was detected using Hoechst 33258 staining and flow cytometric analysis. Total cholesterol levels were detected by high performance liquid chromatography, cholesterol efflux was determined by Tritium labeling, and the cellular lipid droplets were assayed by oil red O staining. RESULTS: Treatment with 50 mg/L ox-LDL for 12, 24, and 48 h increased the apoptotic rate of RAW264.7 cells in a time-dependent manner. The peak apoptotic rate (47.7%) was observed after 48 h incubation. HDL3 at various concentrations (50 mg/L, 100 mg/L, and 200 mg/L) inhibited the ox-LDL (50 mg/L for 48 h)-mediated apoptosis that was accompanied by an increased rate of intracellular cholesterol efflux, and decreased total cholesterol levels in cells in a concentration-dependent manner. Blockage of cholesterol efflux by brefeldin decreased the protective effect of HDL3 on ox-LDL-induced apoptosis. Increase of the cholesterol efflux effected by another cholesterol acceptor,beta-cyclodextrin, led to a dramatic decrease in the apoptotic rate of cells. CONCLUSION: HDL3 antagonizes ox-LDL-induced apoptosis in RAW264.7 cells, through reducing the accumulation of toxic cholesterol.


Assuntos
Apoptose/efeitos dos fármacos , Colesterol/metabolismo , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/antagonistas & inibidores , Animais , Brefeldina A/farmacologia , Linhagem Celular , Lipoproteínas HDL3 , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Oxirredução , beta-Ciclodextrinas/farmacologia
19.
Zhonghua Xue Ye Xue Za Zhi ; 25(10): 579-82, 2004 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-15634588

RESUMO

OBJECTIVE: To investigate the apoptosis-related genes and protein expression patterns in relation to classical Hodgkin lymphomas (CHL) and the origin of H/RS cell. METHODS: Sixty-two cases of CHL were retrieved from Shanxi Tumor Hospital files. An ABC method was used to detect the expression of bcl-2, CD3, CD20, CD30, CD15 and CD10, a double immunohistochemical method to study the H/RS cells P53 expression, a double immunohistochemical ABC-DNA end labeling technique to detect the apoptosis, a double immunohistochemical ABC- in situ hybridization technique to detect the expression of kappa mRNA and lambda mRNA, and a multiple mark techniques to detect the distribution of background non-neoplastic T and B cells. RESULT: Of 62 CHL, 14 (22.58%) were p53 positive and 35 (56.45%) bcl-2 positive. Apoptosis was found in the background non-neoplastic cells in all of the cases, but in H/RS cells in only 10 of 62 cases. There was a significant reverse correlation between bcl-2 expression and apoptosis in H/RS cells (P = 0.02). CD30 positive H/RS cells were observed in all cases, whereas CD15 positive in only 41 cases, and CD20 positive in 8 cases. None was positive for CD3, MPO, bcl-6, CD10, kappa RNA and lambda RNA in H/RS cells. The H/RS cells were surrounded by non-neoplastic T cells looked like a rosette and the outer periphery was B cells. CONCLUSIONS: The H/RS cell of classical Hodgkin lymphoma has a great variety of B lineage markers. The characteristic distributions of T, B and H/RS cells may serve as a reference for the diagnosis. Multiple marker technique is able to highlight the critical cells, and facilitate the study of H/RS cells. Abnormal expression of P53 may not play a major role in CHL. Over expression of bcl-2 may be linked to blockage of apoptosis in CHL.


Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Doença de Hodgkin/patologia , Células de Reed-Sternberg/patologia , Adolescente , Adulto , Idoso , Antígenos CD20/genética , Antígenos CD20/metabolismo , Biomarcadores Tumorais/genética , Complexo CD3/genética , Complexo CD3/metabolismo , Criança , Pré-Escolar , Doença de Hodgkin/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-1/genética , Antígeno Ki-1/metabolismo , Antígenos CD15/genética , Antígenos CD15/metabolismo , Pessoa de Meia-Idade , Neprilisina/genética , Neprilisina/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células de Reed-Sternberg/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Adulto Jovem
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