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7月份我们有一篇文章已在线发表,现可在pubmed上检索到,杂志为J Cardiovasc Pharmacol,影响影子:2.826,
标题:D4-F对THP-1巨噬细胞源性泡沫细胞ABCA1表达及胆固醇流出的影响
英文摘要:
ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in Apolipoprotein A-I (apoA-I) binding activity and promotes cellular cholesterol efflux. ApoA-I mimetic peptide D4-F has reported the similar ability to apoA-I. However, the detailed mechanisms of ABCA1 regulation by D4-F are not understood. In the present study, we investigated the effects of D4-F on ABCA1 expression and ABCA1-dependent cholesterol efflux and examined the role of Cdc42/cAMP/ PKA pathway on the regulation of ABCA1 by D4-F in THP-1 macrophage-derived foam cells. Results showed that D4-F stabilized ABCA1 protein and enhanced ABCA1-dependent cholesterol efflux but had no effect on ABCA1 mRNA expression. We also revealed that D4-F enhanced cAMP level and PKA activity and ABCA1 serine phosphorylation. SiRNA of PKA led to reduction of ABCA1 serine phosphorylation and ABCA1-mediated cholesterol efflux compensated by D4-F. PKA-specific activation by PKA agonist enhanced the up-regulation of ABCA1 serine phosphorylation and ABCA1-mediated cholesterol efflux by D4-F. However, ABCA1 expression did not change by treatment with PKA agonist or PKA-siRNA. We found that Secramine B of Cdc42 inhibitor reduced the cAMP level compensated by D4-F. These results provide evidence that D4-F enhances ABCA1 serine phosphorylation and ABCA1-dependent cholesterol efflux through Cdc42/cAMP/PKA pathway in THP-1 macrophage-derived foam cells.
中文摘要:
目的:本研究以THP-1巨噬细胞源性泡沫细胞为研究对象,采用多种实验方法,探讨ApoA-1模拟肽D4-F对ABCA1(ATP-binding cassette transporter A1, ABCA1)表达和胆固醇流出的影响及其可能的机制。
方法: THP-1单核细胞经PMA诱导贴壁后,加入oxLDL诱导其转化为泡沫细胞,经各种因素处理后,运用液体闪烁计数器检测细胞内胆固醇流出, 高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量, 用实时荧光定量PCR和Western印迹分析法分别检测ABCA1 mRNA与蛋白质的水平,并采用Western印迹分析法检测ABCA1磷酸化,采用酶联免疫检测环磷酸腺苷(cyclic AMP, cAMP),蛋白激酶A(protein kinase A, PKA),腺苷酸环化酶(adenyl cyclase, AC)和磷酸二酯酶(phosphodiesterase, PDE)水平。
结果: 实验结果显示,D4-F能明显降低ABCA1蛋白降解,增加细胞内胆固醇流出并减少细胞内胆固醇含量,但是对ABCA1 mRNA表达没有影响。D4-F能明显增加cAMP水平,激活PKA活性并增加ABCA1丝氨酸磷酸化。PKA-siRNA能使D4-F增加ABCA1丝氨酸磷酸化和ABCA1介导的胆固醇流出的作用减弱,而PKA激动剂能使D4-F增加ABCA1磷酸化和ABCA1介导的胆固醇流出更加明显。而PKA-siRNA和PKA激动剂对D4-F减少ABCA1蛋白质降解没有影响。D4-F能激活GTP-Cdc42活性,Cdc42抑制剂能降低cAMP水平。
结论:(1)D4-F可以减少ABCA1蛋白质降解。(2)D4-F可以通过Cdc42/cAMP/PKA途径增加ABCA1丝氨酸磷酸化和ABCA1介导的胆固醇流出。
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