肝脏SOAT2酶调控游离胆固醇酯化及其影响胆固醇结石病发生的机制研究

Hepatic free cholesterol level is crucial in the regulation of cholesterol level in bile and influencing the formation of cholesterol gallstone. SOAT2 is the key enzyme to esterify free cholesterol into cholesteryl esters in hepatocytes. Our previous data showed that: 1) the SOAT2 activity in liver decreased in patients with gallstone disease, in accompany, free cholesterol level increased; 2) SOAT2 activity decreased in women compared with men; 3) SOAT2 activity increased in mice lack of estrogen receptor beta. Based on these findings, we hypothesize that decrease of hepatic SOAT2 activity inhibits esterification of free cholesterol. Excess free cholesterol, thus, may be secreted into bile, leading to supersaturation of biliary cholesterol and gallstone formation. The fact that activity of SOAT2 is lower in women may be one reason why gallstone disease is more prevalent in women. The gender difference of hepatic SOAT2 activity may be caused by the regulation of estrogen via estrogen receptor. In this project, we will analyze and compare the differences of hepatic SOAT2 activity, free cholesterol distribution in different liver extracts between patients with and without gallstone and analyze their association with biliary cholesterol content. With RNA interference, hepatic SOAT2 activity will be knocked down in mice fed with lithogenic diet. The hepatic cholesterol secretion into bile, biliary cholesterol saturation and incidence of gallstone will be compared in this model as well. With estrogen receptor beta knockout mice model, we will also study the regulation of hepatic SOAT2 activity by estrogen via estrogen receptor and its regulation of biliary cholesterol level and the influence on gallstone formation. The aim of this project is to clarify that decrease of hepatic SOAT2 activity and esterification of free cholesterol may increase biliary cholesterol content, and in turn, promote cholesterol gallstone formation.

肝脏游离胆固醇（FC）含量对调节胆汁胆固醇饱和度和影响胆结石发生具有重要作用。SOAT2是FC酯化的关键酶。我们在预实验和前期研究中发现：1）胆石病人肝脏SOAT2活性降低而FC升高；2）女性SOAT2活性较男性低；3）敲除小鼠雌激素受体(ER)beta，该酶活性升高。由此推测：肝脏SOAT2活性降低抑制FC酯化，过多FC分泌到胆汁促进胆石病发生；女性SOAT2活性低于男性，可能是其更易形成胆结石原因之一；这种性别差异可能与雌激素经ER参与的调控有关。为验证上述假设，我们将比较胆石组和对照组肝脏SOAT2活性及其与FC分布以及胆汁胆固醇含量关系；采用RNA干扰抑制小鼠肝脏SOAT2活性，观察对胆固醇分泌及成石性的影响；采用ERbeta敲除小鼠，观察雌激素对肝脏SOAT2活性、胆汁胆固醇含量以及成石性的影响。阐明“肝脏SOAT2活性降低，抑制FC酯化->增加胆汁胆固醇含量，促进成石”的机制。

本项目旨在阐明雌激素受体对于调控肝脏SOAT2表达性别差异的机制，并对后者在胆固醇代谢中的作用进行探索。采用雌激素受体alpha、beta敲除及双敲除小鼠，通过定量PCR,Western Blot以及代谢组学等方法进行研究。结果发现：1、小鼠肝脏SOAT2存在性别差异，与雌激素受体具有一定的关联。2、SOAT2调控胆固醇酯化进而影响胆固醇代谢。3、SOAT2活性调节脂肪酸代谢，进而肝脏脂肪肝的发生。4、OCPs类农药，在体内蓄积影响肝脏脂肪酸、胆汁酸代谢，并与胆石病相关。5、肠道菌群-肝脏FMOT3途径代谢物TMAO影响肝脏胆固醇分泌，具有促进胆石病发生的作用。结论：本课题阐明了SOAT2的表达调控机制，并对其调节肝脏胆固醇代谢的作用提供了进一步的实验依据。