TNF-α对HepG2细胞LXRα介导的胆固醇外流的影响及其分子机制

 目的:观察肿瘤坏死因子α（TNF-α）对肝X受体α（LXRα）启动子活性及其下游ATP结合盒转运蛋白（ABCA1和ABCG1）表达的影响，从而探讨TNF-α加速HepG2细胞内胆固醇积聚的分子机制。方法:构建LXRα基因的启动子表达载体,观察炎症因子TNF-α对LXRα启动子活性的影响，进一步将HepG2细胞分为对照组（control）、TNF-α组（20 μg/L）、高脂组（LDL,100 mg/L）及联合处理组（TNF-α 20 μg/L+ LDL 100 mg/L）。采用实时定量PCR和Western blotting检测LXRfα、ABCA1和 ABCG1的mRNA及蛋白的表达。［3H ］标记胆固醇,液体闪烁计数法检测胆固醇外流量。油红O染色和定量比色法分析细胞内胆固醇的含量。结果:成功构建LXRα启动子的萤火虫萤光素酶报告质粒，证明了该启动子有明显活性，TNF-α能明显抑制LXRα启动子的活性。进一步检测发现，和对照组相比，TNF-α下调了LXRα、ABCA1和ABCG1 mRNA与蛋白表达。高脂组胆固醇外流量增加，而TNF-α组胆固醇外流量明显降低。油红O染色显示，TNF-α使细胞内脂质染色明显增加。结论: TNF-α可通过抑制LXRα启动子活性从而抑制HepG2细胞内胆固醇外流导致肝细胞内脂质异常积聚。

TNF-α exacerbates cholesterol accumulation via down-regulating LXRα promoter activity in HepG2 cells

AIM: To investigate the exacerbating effect of tumor necrosis factor alpha (TNF-α) on lipid accumulation in HepG2 cells by inhibiting liver X receptor alpha (LXRα) signaling pathway. METHODS: Luciferase reporter plasmid driven by the LXRα promoter (pGL3-Basic-LXRα-P) was constructed and transfected into HepG2 cells to detect the LXRα promoter activity. HepG2 cells were incubated with serum-free medium (control), 20 μg/L TNF-α (TNF-α), 100 mg/L LDL (LDL) and 20  μg/L TNF-α plus 100 mg/L LDL (LDL+TNF-α), respectively. The effects of TNF-α on cholesterol accumulation were examined by oil red O staining and quantitative intracellular cholesterol assay. The expression of LXRα, ABCA1 and ABCG1 at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS: The pGL3-Basic-LXRα-P was constructed successfully. TNF-α decreased the activity of LXRα promoter in the absence or presence of LDL. Inflammatory stress inhibited the expression of LXRα, ABCA1and ABCG1 at mRNA and protein levels. The cholesterol efflux was increased after loading of LDL, while TNF-α decreased intracellular cholesterol efflux. The results of oil red O staining and quantitative intracellular cholesterol assay demonstrated that inflammatory stress increased cholesterol levels in HepG2 cells. CONCLUSION: TNF-α exacerbates the cholesterol accumulation in hepatic cells via inhibiting LXRα promoter activity and gene expression.