EGCG对THP-1源性泡沫细胞ABCA1的影响及其机制

发布时间：2019-03-20 17:01

【摘要】：目的：ABCA1在促进胆固醇逆向转运和抗动脉粥样硬化中发挥重要作用。EGCG是一种绿茶提取物，具有抗炎和调控脂代谢的药理作用。以往的研究中发现SREBP-1c、apoB和LDLR等脂代谢相关蛋白参与了EGCG调控脂代谢及其抗炎作用。但是EGCG对ABCA1的作用尚不清楚。在我们的研究中，我们用TNF-α处理THP-1源性泡沫细胞，观察EGCG预处理对ABCA1及其胆固醇流出影响及其机制。方法：我们首先用PMA诱导THP-1细胞贴壁形成巨噬细胞，，加入OX-LDL形成泡沫细胞。在加入TNF-α (10ng/ml)前，分别用不同浓度的(0,10,20,40,80μg/mL)EGCG处理泡沫细胞，然后收集细胞进行实验。胆固醇流出实验和油红O染色观察细胞内胆固醇流出和脂质蓄积情况。实时定量PCR观察ABCA1和LXRα/β mRNA水平。荧光素酶报告基因分析ABCA1启动子活性。Western blot检测ABCA1、LXRα/β、NF-κB p65, Nrf2, Keap1和IKKβ蛋白水平。EMSA检测NF-κB和Nrf2的DNA结合活性。用siRNA转染细胞沉默NF-κB和Nrf2. 结果：实验结果表明，TNF-α处理细胞能够导致ABCA1mRNA表达水平启动子活性降低，以及胆固醇流出水平降低，而EGCG预处理能够衰减TNF-α这一作用。EGCG通过抑制NF-κB途径来衰减TNF-α对细胞毒性作用，而不是通过激活LXR途径。在抑制NF-κB过程中，EGCG激活了Nrf2/Keap1途径，一方面，解离的Nrf2进入细胞核内激活启动子区域含有ARE的相关基因，抑制NF-κB活化；另一方面，Keap1从复合体上解离后，直接与IKKβ结合，阻滞其下游对NF-κB的激活。结论：在THP-1源性泡沫细胞中，EGCG通过激活Nrf2/Keap1途径抑制TNF-α诱导的NF-κB激活，上调ABCA1的表达促进细胞内胆固醇流出。
[Abstract]:Aim: ABCA1 plays an important role in promoting cholesterol reverse transport and anti-atherosclerosis. EGCG is a green tea extract with anti-inflammation and regulation of lipid metabolism. Previous studies have found that lipid metabolism-related proteins such as SREBP-1c,apoB and LDLR are involved in the regulation of lipid metabolism and its anti-inflammatory effects by EGCG. But the effect of EGCG on ABCA1 is unclear. In our study, we treated THP- 1-derived foam cells with TNF- 伪, and observed the effect and mechanism of EGCG pretreatment on ABCA1 and cholesterol efflux. Methods: we first induced THP-1 cells to adhere to the wall to form macrophages with PMA, and then added OX-LDL to form foam cells. Foam cells were treated with (0,10,20,40,80 渭 g / mL) EGCG) of (0,10,20,40,80 渭 g / mL) EGCG) respectively before TNF- 伪 (10ng/ml) was added, and then the cells were collected for experiment. Cholesterol efflux test and oil red O staining were used to observe intracellular cholesterol efflux and lipid accumulation. The levels of ABCA1 and LXR 伪 / 尾 mRNA were measured by real-time quantitative PCR. The promoter activity of ABCA1 was analyzed by luciferase reporter gene analysis. The levels of ABCA1,LXR 伪 / 尾, NF- 魏 B p65, Nrf2, Keap1 and IKK 尾 proteins were detected by Western blot, and the DNA binding activities of NF- 魏 B and Nrf2 were detected by EMSA. Cell silencing of NF- 魏 B and Nrf2. by siRNA transfection Results: the results showed that TNF- 伪 could decrease the promoter activity of ABCA1mRNA expression level and decrease the cholesterol efflux level. EGCG pretreatment can attenuate the effect of TNF- 伪. EGCG attenuates the cytotoxicity of TNF- 伪 by inhibiting the NF- 魏 B pathway, rather than activating the LXR pathway. In the process of inhibiting NF- 魏 B, EGCG activates the Nrf2/Keap1 pathway. On the one hand, the dissociated Nrf2 enters the activation promoter region of nucleus and contains the related gene of ARE, which inhibits the activation of NF- 魏 B; On the other hand, when Keap1 is dissociated from the complex, it binds directly to IKK 尾 and blocks its downstream activation of NF- 魏 B. Conclusion: in THP- 1-derived foam cells, EGCG inhibits TNF- 伪-induced NF- 魏 B activation by activating Nrf2/Keap1 pathway, and up-regulates the expression of ABCA1 to 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】：2012
【分类号】：R363

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