盐酸小檗碱调控SCD1表达改善肝脏脂肪沉积的机制研究

Berberine(BBR), a traditional Chinese herb, could decrease body weight and improve glucose and lipid metabolism and liver fat deposition,but the mechanisms have not been revealed fully. Stearoyl-CoA desaturase (SCD)1 converts saturated fatty acids to monounsaturated fatty acids. SCD1 knockout mice increases food intake, but protects against diet-induced obesity, hepatic steatosis, and metabolic abnormalities. Our previous studies showed that BBR could reduce the up-regulated expression of SCD1 mRNA to normal level, which was confirmed by western blot in the liver tissue of non-alcoholic fatty liver disease SD rat. Moreover, we found BBR could active the phosphorylation of Adenosine Monophosphate Activated Protein Kinase (AMPK), the upstream transcription factor of the Sterol Regulatory Element Binding Protein (SREBP)1 . SREBP1 has been verified modulating SCD1 expression in several studies. SREBP1 binding sites exist in SCD1 promoter. Therefore, we speculated to identify the AMPK - SREBP1 - SCD1 pathway whose dysfunction may be involved in the formation of fatty liver, and identify BBR protects against metabolic abnormalities by affacting this pathway. To verify this assumption, we plan to construct SCD1 and AMPK knock-down or overexpression animal and celluar model respectively，and establish SRE mutant of SCD1 promoter luciferase reporter gene, and four SRE repetitive sequences of reporter gene so that we can demonstrate berberine decreases SCD1 expression to ameliorate liver fat deposition via activation of AMPK phosphorylation and inhibition of SREBP1 activity.

盐酸小檗碱(BBR)有降低肝脏脂肪等多重代谢改善作用，但作用机制未完全阐明。硬脂酰辅酶A去饱和酶(SCD)1可催化饱和脂肪酸向单不饱和转化，SCD1敲除小鼠对高脂饮食诱导的脂肪肝等代谢异常产生明显的抵抗作用。我课题组前期研究显示BBR可使脂肪肝大鼠肝脏异常上调的SCD1表达下调至正常，并有促固醇调节原件结合蛋白(SREBP)1上游转录因子腺苷活化蛋白激酶(AMPK)磷酸化作用。SCD1启动子存在SREBP1结合位点，我们推测存在AMPK-SREBP1-SCD1通路，其功能异常对脂肪肝形成具有病理意义，而BBR可能作用于该通路发挥代谢保护作用。本项目拟构建SCD1、AMPK敲低或过表达动物细胞模型，建立SCD1启动子荧光素酶报告基因，构建缺失SRE突变体及SRE重复序列报告基因阐明该调节机制，探索BBR是否通过增加AMPK磷酸化，下调SREBP1来抑制SCD1表达，改善肝脏脂肪的作用新机制。

背景：小檗碱（BBR）是从中药黄连根部提取的一种天然化合物，在临床上已被证明能有效地缓解非酒精性肝脂肪变性。然而，这一作用背后的机制尚未完全阐明。.方法：检测脂肪酰辅酶A去饱和酶1（SCD1）在ob/ob小鼠肝脏的表达。检测小檗碱对高糖和棕榈酸处理的HepG2和AML12细胞的作用。油红O染色分析甘油三酯（TG）含量。采用实时定量聚合酶链反应（QPCR）和蛋白质印迹法（WB）分别检测相关基因的mRNA和蛋白表达。双荧光素酶报告基因实验测定BBR对SCD1启动子的活性。.结果：SCD1在ob/ob小鼠的肝脏中表达增加。不管体内或体外实验，BBR降低了肝脏TG的积累，降低了肝脏SCD1和其他与TG合成相关的基因的表达水平。在脂肪肝细胞中，SCD1敲低可以模拟BBR降低细胞TG沉积的作用，而SCD1的过表达减弱了BBR降低细胞TG沉积的作用。机制方面，BBR促进HepG2细胞和高脂饮食小鼠肝脏腺苷酸活化蛋白激酶（AMPK）和固醇调节元件结合蛋白-1c（SREBP-1c）的磷酸化。小檗碱通过AMPK-SREBP-1c通路的激活和SCD1启动子中的SRE区抑制SCD1表达。.结论及意义：BBR通过激活AMPK-SREBP-1c-SCD1通路，降低肝脏TG合成，从而减轻肝脏脂肪变性。该研究结果可为临床使用BBR治疗NAFLD提供部分理论依据。