吐温80抑制肠道胆固醇吸收的作用和机制研究

Hyperlipidemia is the major case of atherosclerosis and atheroslerosis-associated conditioins. Intestinal cholesterol(Ch) absorption is an important determinant of whole-body cholesterol homeostasis and is directly correlated with plasma total cholesterol(TC) and LDL-C concentration. Inhibition of intestinal Ch absorption is an attractive and effective method to reduce serum TC and LDL-C levels. The first Ch absorption inhibitor-ezetimibe successfully proven this point, and thus set off the upsurge of such drug research. Our pre-experiments showed that tween 80 may lower plasma TC and LDL-C selectively and effectively, but it must be administrated before the high fat diet, suggesting that tween 80 lower serum cholesterol by inhibiting intestinal Ch absorption. Ch absorption process is complex. Firstly, Ch will be solubilized by forming micelles with bile salts, phosphatidylcholine, and dietary lipids and their digestion products. Micelles diffuse through the aqueous lumenal contents to the brush border, where NPC1L1(Niemann-pick C1-like 1 protein) uptake Ch from the micelles into enterocytes, whereas ACAT2 (acyl-CoA:cholesterol acyltransferase 2) converts Ch to cholesteryl ester (CE), which will be packed into chylomicrons. Subsequently, chylomicrons were transferred into blood through the lymphatic system. ABCG5/8 (ATP-binding cassette transporters subfamily G members 5 and 8) returns the unabsorbed free Ch left in the enterocytes to the lumen for excretion. Ch is mainly eliminated from the body via conversion to bile acids, intestinal bile acid absorption is an essential step in the enterohepatic circulation, ASBT (ATP-binding cassette transporter) has been shown to mediate approximately 75% of the bile acids recycled in the human body. Consequently, the inhibition of ASBT activity reduces the amount of circulating bile acids, leading to the stimulation of their synthesis and a reduction in cholesterol levels. In this project, We will detect the inhibitory effect of tween 80 on intestinal Ch absorption using fecal dual-isotope ratio method. We will detect if tween 80 could reduces micellar Ch solubility in bile salts micelles using a gel filtration chormatography and spectrophotometry. RT-PCR and western-blot will be used to detect the RNA and protien expression of NPC1L1, ABCG5/8, ACAT2 and ASBT in hypercholesterolemia mice after administration of tween 80, the proteins changed the most dramaticaly may be the targets of tween 80, we will further study the effect of tween 80 on those proteins function using the caco-2 cell model. Which protein would be the target of tween 80 will be analyzed by combing all of the data especially from expression and function experiments. we will also evaluate if the hypocholesterol action of tween 80 could mediate an anti-atherosclerotic effect in apoE-/- mice.

高胆固醇血症是动脉粥样硬化及相关疾病的主要危险因素，抑制肠道胆固醇吸收是降低血浆胆固醇的重要手段,首个胆固醇吸收抑制剂- - 依折麦布的成功证明了这一点，由此掀起此类药物研究热潮。本课题组预实验显示：吐温80可选择性显著降低血浆总胆固醇和LDL-C，但须高脂饲料前给药才有效，提示"吐温80是通过抑制肠道胆固醇吸收而降脂"。肠道胆固醇吸收涉及多个环节及关键蛋白：胆汁酸助溶、小肠胆固醇摄取载体NPC1L1、泵出载体ABCG5/8、胆固醇酯化酶ACAT2、胆汁酸重吸收载体ASBT等。本课题将围绕这些环节，采用凝胶过滤+酶法定量结合的方法检测吐温80对胆汁酸助溶作用的影响，高脂动物模型观察其对各蛋白质表达的影响，并对其中影响明显的蛋白质，采用caco2细胞进行蛋白质功能实验，综合数据分析吐温80的作用靶点，应用ApoE-/-小鼠动脉粥样硬化模型进一步评价吐温80的降脂作用对动脉粥样硬化的拮抗作用。

项目是在前期预实验基础上进行的，其结果表明：吐温80可选择性显著降低血浆总胆固醇和LDL-C，但需要高脂饲料前给药方才有效，提示“吐温80是通过抑制肠道胆固醇吸收而降低血脂”的。为了进一步探讨吐温80抑制肠道胆固醇吸收机制，项目进行了一系列深入研究：其中吐温80对高脂血症大鼠血脂、肝脂、肝胆固醇、肝胆汁酸水平及肝脏组织病理学损伤的影响的实验结果不仅进一步印证吐温80可选择性降低血浆总胆固醇和LDL-C，而且还表明吐温80可降低血浆谷丙转氨酶、谷草转氨酶，降低肝脏总脂质、总胆固醇、游离胆固醇和胆固醇酯的水平，减轻肝组织病理学损伤，但是对肝脏总胆汁酸的水平没有影响。吐温80对肠道胆固醇吸收的影响实验结果表明吐温80可较模型组显著升高给予高脂饮食后大鼠每日胆固醇排泄总量、百克体重胆固醇排泄量，升高每日胆固醇排泄量与每日胆固醇摄入量的比值，吐温80还可升高每日粪便中胆汁酸排泄量、每百克体重每日粪便胆汁酸排出量。吐温80对肠道运动的影响的实验表明，吐温80可加速肠道内容物向下游运输，提高肠内碳末的推进距离和推进率。吐温80可逆转高脂饮食对肠道NPC1L1、ABCG8、ASBT的影响，使这几种转运体的表达水平维持至接近正常的水平，还可对抗高脂饲料对大鼠肝脏HMG-CoA、Cyp7a1，LDL受体，LPL、CETP、NPC1L1等的影响。项目所获得数据为胆固醇抑制肠道胆固醇吸收的机制提供一些可能的解释，首先吐温80可促进肠道运动，促进肠道胆固醇排出；再次，吐温80对肠道胆固醇吸收的关键性转运体的表达水平有影响，对肝脏中参与胆固醇代谢的酶、受体、转运体等的表达水平有影响，为进一步准确定位吐温80的靶点提供线索。