白细胞介素18下调泡沫细胞ABCA1的表达及其机制

发布时间：2018-10-15 12:03

【摘要】：目的:白细胞介素18(IL-18)和白细胞介素12(IL-12)是具有多种生物学功能的炎症因子,近来发现两者在动脉粥样硬化(As)斑块中过量表达。本文观察IL-18和IL-12对THP-1巨噬细胞源性泡沫细胞ATP结合盒转运体A1(ABCA1)表达和胆固醇流出的影响,并探讨两者的协同效应及其调节ABCA1介导的胆固醇流出的相关机制。方法:THP-1巨噬细胞源性泡沫细胞经不同浓度IL-18(0,2,20,200 ng/ml)或IL-12(0,0.2,2,20 ng/ml)处理或者用2ng/ml IL-12和20 ng/ml IL-18共处理不同时间(0,6,12,24h);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,实时荧光定量PCR检测IL-18R、IL-12R、ABCA1和LXRαmRNA表达情况,Western印迹法检测IL-18R、ABCA1、LXRα及核内NF-κB p65蛋白质的表达水平。以IL-18R抑制剂IL-18结合蛋白(IL-18BP)和NF-κB抑制剂对甲苯磺酰-L-苯丙氨酸氯甲基甲酮(N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK)预处理细胞3h再加入IL-18/IL-12共处理细胞24小时,观察对ABCA1及其介导的胆固醇流出的影响。结果:IL-18和IL-12单独处理THP-1巨噬细胞源性泡沫细胞并不明显影响细胞总胆固醇、游离胆固醇、胆固醇酯含量及细胞内胆固醇流出,但IL-12预处理或IL-18/IL-12共处理后呈浓度和时间依赖性抑制ABCA1 mRNA及蛋白质的表达,核内NF-κB p65蛋白质的表达水平增加,用IL-18BP和TPCK预处理细胞后,IL-18/IL-12的这种作用被抑制。IL-12促进IL-18R表达,但LXRαmRNA和蛋白质的表达均不受IL-18/IL-12的影响。结论:IL-18/IL-12共刺激下调THP-1巨噬细胞源性泡沫细胞ABCA1的表达和细胞内胆固醇流出,促进细胞内脂质蓄积,IL-18R和NF-κB信号途径介导了IL-18对ABCA1的抑制作用,而IL-12通过促进泡沫细胞IL-18R表达参与了IL-18对ABCA1的抑制作用。
[Abstract]:Aim: interleukin-18 (IL-18) and interleukin-12 (IL-12) are inflammatory factors with many biological functions. Recently, it has been found that both of them are overexpressed in atherosclerotic (As) plaques. The effects of IL-18 and IL-12 on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux in THP-1 macrophage derived foam cells were studied. Methods: THP-1 macrophage derived foam cells were treated with different concentrations of IL-18 (0 ~ 220200 ng/ml) or IL-12 (0 ~ 0.2o ~ (22) ~ 20 ng/ml) or co-treated with 2ng/ml IL-12 and 20 ng/ml IL-18 for different time (0 ~ 61212 ~ 24 h), and cholesterol efflux was detected by liquid scintillation counter. The contents of total cholesterol, free cholesterol and cholesterol ester were analyzed by HPLC, the expression of IL-18R,IL-12R,ABCA1 and LXR 伪 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of IL-18R,ABCA1,LXR 伪 and NF- 魏 B p65 protein was detected by Western blot. IL-18R inhibitor IL-18 binding protein (IL-18BP) and NF- 魏 B inhibitor p-toluenesulfonyl-L-phenylalanine ketone (N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK) were pretreated with N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK) for 3 h and then co-treated with IL-18/IL-12 for 24 hours. The effects of N-p-Tosyl-L-phenylalanine chloro- methyl ketone, TPCK) on ABCA1 and cholesterol efflux were observed. Results: IL-18 and IL-12 alone did not affect the total cholesterol, free cholesterol, cholesterol ester content and intracellular cholesterol efflux of THP-1 macrophage derived foam cells. However, IL-12 pretreatment or co-treatment with IL-18/IL-12 inhibited the expression of ABCA1 mRNA and protein in a concentration-and time-dependent manner, and the expression of NF- 魏 B p65 increased in nucleus. After pretreatment with IL-18BP and TPCK, this effect of IL-18/IL-12 was inhibited. IL-12 promoted IL-18R expression. However, the expression of LXR 伪 mRNA and protein was not affected by IL-18/IL-12. Conclusion: IL-18/IL-12 costimulation down-regulates the expression of ABCA1 and intracellular cholesterol efflux in THP-1 macrophage derived foam cells, and promotes intracellular lipid accumulation. IL-18R and NF- 魏 B signaling pathway mediate the inhibitory effect of IL-18 on ABCA1. IL-12 plays an important role in the inhibition of ABCA1 by promoting the expression of IL-18R in foam cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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