基于健脾化浊法的双蓣调脂汤调控HMGCR和NPC1L1蛋白减少胆固醇合成和吸收的作用机制

Hypercholesterolemia is the most major risk factor of cardiovascular and cerebrovascular diseases. It has been a troublesome clinical problem that it is difficult to reach the standard with anticholesteremic agents nowadays.Shuangyu Tiaozhi decoction is effective traditional Chinese medicine prescription for the treatment of hypercholesterolemia.Our previous research confirmed that Shuangyu Tiaozhi decoction could effectively reduce plasma cholesterol,but the molecular mechanism remains elusive.Cholesterol comes from liver biosynthesis and intestinal absorption. 3-hydroxyl methyl glutaric acyl coenzyme A reductase (HMGCR)is the rate-limiting enzyme in cholesterol synthesis.Niemann-Pick C1-Like1(NPC1L1) is the key protein of transportion in the process of cholesterol absorption.HMGCR and NPC1L1 are the molecular targets of cholsterol lowering agents such as statins and Ezetimibe.In order to explore the molecular mechanism,we will focus on the key targets using different methods containing in vitro and in vivo approaches.In vivo,we would like to set up the animal models as hypercholesterolemia.While in vitro,we will use the means such as lentiviral vector interferencing, blocking and activating the overexpression plasmid of these key molecules,Using Western blot、Real time PCR、EMSA、CHIP、Immunohistochemical、Luciferase to explore the role of diosgenin in the regulation of cholesterol synthesis and absorption mechanism and provide theoretical and experimental basis for clinical treatment.

高胆固醇血症是心脑血管疾病的主要原因，目前药物降脂达标率低。我们在前期临床和动物实验基础上，发现以健脾化浊为法组方的双蓣调脂汤有显著的降脂作用，但机制未明。体内胆固醇来源于肝脏的合成和小肠的吸收两方面，HMGCR是肝脏合成的限速酶，NPC1L1是小肠吸收的关键转运蛋白，二者对胆固醇的水平起关键作用。本课题拟以高胆固醇血症大鼠和原代细胞及细胞系为研究对象，以HMGCR和NPC1L1为主要靶点，从体内、体外两个层面，采取慢病毒干扰、阻断及过表达质粒激活关键分子的手段，应用蛋白印迹、实时定量荧光PCR、凝胶电泳试验、染色免疫共沉淀、免疫组化、荧光素酶等技术，从细胞、蛋白、基因不同水平探析双蓣调脂汤对胆固醇的调节机制。本课题将证实我们提出的“双蓣调脂汤通过抑制胆固醇的合成、减少胆固醇的吸收，多靶点干预治疗高胆固醇血症”的假说，为健脾化浊治疗大法提供实验依据，为高胆固醇血症的防治提供新思路和新靶点。

高胆固醇血症是心脑血管疾病的主要原因，目前药物降脂达标率低。我们在前期临床和动物实验基础上，发现以健脾化浊为法组方的双蓣调脂汤有显著的降脂作用，但机制未明。体内胆固醇来源于肝脏的合成和小肠的吸收两方面，HMGCR是胆固醇合成途径的限速酶，NPC1L1是小肠吸收的关键转运蛋白，二者对胆固醇的水平起关键作用。本项目以胆固醇合成途径的限速酶HMGCR和胆固醇吸收途径的关键转运蛋白NPC1L1为切入点进行了一系列的研究，主要结果如下：1.双蓣调脂汤可显著降低高胆固醇血症大鼠体重；2.双蓣调脂汤可显著降低高胆固醇血症大鼠血清以及肝脏胆固醇水平；3.双蓣调脂汤可显著改善高胆固醇血症大鼠脂肪肝的程度；4.双蓣调脂汤含药血清处理可显著降低肝细胞内胆固醇水平；5.双蓣调脂汤可显著抑制胆固醇合成限速酶HMGCR的表达；可抑制SREBP-2的表达，减弱SREBP-2与HMGCR启动子区SRE位点的结合，从而抑制HMGCR表达；6.双蓣调脂汤可明显抑制小肠关键转运蛋白NPC1L1的表达。本研究阐明了双蓣调脂汤抑制肝脏胆固醇合成和小肠吸收的分子机制，为双蓣调脂汤多靶点在降脂方面的应用提供了科学依据，为健脾化浊法治疗高胆固醇血症提供了新思路。