Vaspin通过抑制NF-κB信号途径上调THP-1巨噬细胞ABCA1的表达

发布时间：2018-08-19 06:15

【摘要】：目的:Vaspin(内脏脂肪组织来源的丝氨酸蛋白酶抑制物)被证实能对抗动脉粥样硬化发生发展,其具体作用机制尚未被完全认识。本研究主要探讨Vaspin能否通过抑制NF-κB途径上调THP-1巨噬细胞ATP结合盒转运体A1(ATP-binding cassette transporter A1,ABCA1)表达。方法:将人THP-1单核巨噬细胞诱导转变成为泡沫细胞,经不同浓度的Vaspin处理相同时间或同一浓度Vaspin(100ng/mL)处理不同时间(0,6,12,24,48h),用液体闪烁计数法检测细胞内胆固醇流出,油红O染色观察细胞内脂滴情况,高效液相色谱法测定细胞内总胆固醇、游离胆固醇及胆固醇酯含量;Western blot测定细胞内ABCA1蛋白含量,实时定量PCR检测ABCA1 mRNA水平。分别用miR-33a模拟肽(miR-33a mimic)和anti-miR-33a转染THP-1细胞对LPS作用下的细胞处理24h,再加入vaspin处理24h,检测细胞内ABCA1、miR-33a和SREBP mRNA表达水平。用NF-κB抑制剂预处理LPS作用下的THP-1细胞24h,再加或不加vaspin处理,检测细胞内NF-κB p65蛋白、ABCA1蛋白和mRNA表达、miR-33a mRNA表达、细胞胆固醇的流出情况。结果:Vaspin上调THP-1巨噬细胞ABCA1蛋白及mRNA水平呈时间和剂量依赖性。进一步实验证实vaspin可以抑制mi R-33am RNA及SREBP-2的表达,上调ABCA1mRNA的表达,促进胆固醇流出;NF-κB的特异性抑制剂PDTC处理和荷脂的THP-1细胞,vaspin可削弱NF-κB/miR-33a信号途径对ABCA1表达的抑制作用。结论:Vaspin可通过抑制NF-κB信号途径,下调miR-33a表达,进而上调THP-1巨噬细胞ABCA1蛋白及mRNA表达,并促进细胞内胆固醇流出。
[Abstract]:Objective: Vaspin (visceral adipose tissue derived serine protease inhibitor) has been proved to be able to prevent the development of atherosclerosis, and its mechanism has not been fully understood. The aim of this study was to investigate whether Vaspin can up-regulate the expression of ATP binding cassette transporter A1 (ATP-binding cassette transporter A1) in THP-1 macrophages by inhibiting NF- 魏 B pathway. Methods: human THP-1 mononuclear macrophages were induced to be foam cells and treated with different concentrations of Vaspin for the same time or at the same concentration of Vaspin (100ng/mL) for 48 h. Cholesterol efflux was detected by liquid scintillation counting. Lipid droplets were observed by oil red O staining, total cholesterol, free cholesterol and cholesterol ester were determined by HPLC, ABCA1 protein was detected by Western blot and ABCA1 mRNA was detected by real-time quantitative PCR. THP-1 cells were transfected with miR-33a mimic peptide (miR-33a mimic) and anti-miR-33a for 24 h, then treated with vaspin for 24 h. The expression levels of ABCA1 miR-33a and SREBP mRNA were detected. LPS cells were pretreated with NF- 魏 B inhibitor for 24 h, then treated with or without vaspin for 24 h. The expression of ABCA1 protein and mRNA protein of NF- 魏 B p65 was detected. The expression of miR-33a mRNA and cholesterol efflux were detected. Results Vaspin upregulated the levels of ABCA1 protein and mRNA in THP-1 macrophages in a time and dose-dependent manner. It was further confirmed that vaspin could inhibit the expression of mi R-33am RNA and SREBP-2, up-regulate the expression of ABCA1mRNA, promote the treatment of PDTC, a specific inhibitor of cholesterol efflux of NF- 魏 B, and inhibit the expression of ABCA1 by NF- 魏 B/miR-33a signaling pathway. Conclusion Vaspin can down-regulate the expression of miR-33a and up-regulate the expression of ABCA1 and mRNA in THP-1 macrophages by inhibiting NF- 魏 B signaling pathway, and promote intracellular cholesterol efflux.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R54

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