【摘要】：目的：ABCA1在促進(jìn)膽固醇逆向轉(zhuǎn)運(yùn)和抗動(dòng)脈粥樣硬化中發(fā)揮重要作用。EGCG是一種綠茶提取物，具有抗炎和調(diào)控脂代謝的藥理作用。以往的研究中發(fā)現(xiàn)SREBP-1c、apoB和LDLR等脂代謝相關(guān)蛋白參與了EGCG調(diào)控脂代謝及其抗炎作用。但是EGCG對(duì)ABCA1的作用尚不清楚。在我們的研究中，我們用TNF-α處理THP-1源性泡沫細(xì)胞，觀察EGCG預(yù)處理對(duì)ABCA1及其膽固醇流出影響及其機(jī)制。 方法：我們首先用PMA誘導(dǎo)THP-1細(xì)胞貼壁形成巨噬細(xì)胞，，加入OX-LDL形成泡沫細(xì)胞。在加入TNF-α (10ng/ml)前，分別用不同濃度的(0,10,20,40,80μg/mL)EGCG處理泡沫細(xì)胞，然后收集細(xì)胞進(jìn)行實(shí)驗(yàn)。膽固醇流出實(shí)驗(yàn)和油紅O染色觀察細(xì)胞內(nèi)膽固醇流出和脂質(zhì)蓄積情況。實(shí)時(shí)定量PCR觀察ABCA1和LXRα/β mRNA水平。熒光素酶報(bào)告基因分析ABCA1啟動(dòng)子活性。Western blot檢測(cè)ABCA1、LXRα/β、NF-κB p65, Nrf2, Keap1和IKKβ蛋白水平。EMSA檢測(cè)NF-κB和Nrf2的DNA結(jié)合活性。用siRNA轉(zhuǎn)染細(xì)胞沉默NF-κB和Nrf2. 結(jié)果：實(shí)驗(yàn)結(jié)果表明，TNF-α處理細(xì)胞能夠?qū)е翧BCA1mRNA表達(dá)水平啟動(dòng)子活性降低，以及膽固醇流出水平降低，而EGCG預(yù)處理能夠衰減TNF-α這一作用。EGCG通過(guò)抑制NF-κB途徑來(lái)衰減TNF-α對(duì)細(xì)胞毒性作用，而不是通過(guò)激活LXR途徑。在抑制NF-κB過(guò)程中，EGCG激活了Nrf2/Keap1途徑，一方面，解離的Nrf2進(jìn)入細(xì)胞核內(nèi)激活啟動(dòng)子區(qū)域含有ARE的相關(guān)基因，抑制NF-κB活化；另一方面，Keap1從復(fù)合體上解離后，直接與IKKβ結(jié)合，阻滯其下游對(duì)NF-κB的激活。 結(jié)論：在THP-1源性泡沫細(xì)胞中，EGCG通過(guò)激活Nrf2/Keap1途徑抑制TNF-α誘導(dǎo)的NF-κB激活，上調(diào)ABCA1的表達(dá)促進(jìn)細(xì)胞內(nèi)膽固醇流出。
[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- 偽, 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- 偽 (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 偽 / 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 偽 / 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- 偽 could decrease the promoter activity of ABCA1mRNA expression level and decrease the cholesterol efflux level. EGCG pretreatment can attenuate the effect of TNF- 偽. EGCG attenuates the cytotoxicity of TNF- 偽 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- 偽-induced NF- 魏 B activation by activating Nrf2/Keap1 pathway, and up-regulates the expression of ABCA1 to promote intracellular cholesterol efflux.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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