基于体内过程和点击化学的小檗碱体内降糖活性形式及机制研究

Coptidis Rhizoma, as well as its compound prescription, have been used for thousands of years a traditional Chinese herb for the treatment and management diabetes mellitus. Modern pharmacological studies have shown that berberine is the main active ingredient involved with the lipid- and glucose-lowering effects of Coptidis Rhizoma. However, pharmacokinetic studies have shown that berberine has low blood concentration after oral administration and a low bioavailability, thus, it is difficult to explain its clinical efficacy. Previous studies have shown that berberine and its metabolites could reduce the content of triglyceride and improve insulin resistance of adipocytes. Although the mechanisms of berberine are not clear, these results indicate that the in vivo metabolites of berberine are closely related with its hypoglycemic effect. In the present study, insulin resistant models will be used to investigate the real active forms of berberine in vivo. Subsequently, we will identify target proteins of berberine as well as its metabolites by clicking chemistry and genechip technology. The project will fully elucidate the real active forms and the mechanism of berberine in vivo through the study of drug metabolism. The study will provide a reference for the study of the pharmacodynamic material basis of other traditional Chinese medicine with low bioavailability and reputable curative effect.

中药黄连及其复方治疗糖尿病已有千年历史。现代药理学研究证实小檗碱是中药黄连发挥治疗糖尿病的主要活性成分。然而，药代动力学研究发现，口服小檗碱的血药浓度和生物利用度很低，以此难以解释其临床功效。申请人前期研究发现小檗碱原型及其代谢产物均可以降低肝细胞内甘油三酯含量及改善脂肪细胞胰岛素抵抗的作用，提示小檗碱体内代谢转化产物与其降糖效果具有密切关系，然而其体内具体作用机制尚不清楚。本课题在上述研究工作的基础上，拟以小檗碱及其代谢产物为研究对象，利用糖尿病模型筛选出有效降糖的形式，并通过点击化学结合基因芯片技术深入探讨小檗碱原型及其代谢产物作用靶点及机制。本课题从药物体内代谢角度阐明小檗碱在体内真正发挥降糖作用的形式及其作用靶点，对于其他生物利用低、疗效确切的中药药效物质基础的研究具有示范作用。

小檗碱（BBR）是黄连中的主要有效成分，近年来其在治疗糖尿病方面受到了广泛的关注。课题组前期研究发现BBR在体内可以产生多个代谢产物，如药根碱（M1）、非洲防己碱（M2）、小檗红碱（M3）以及去亚甲基小檗碱（M4）等，且一些代谢产物具有明显的降脂活性，提示这些体内代谢产物可能是BBR发挥药理作用的真正物质。本项目在前期工作基础上，分别采用高浓度葡萄糖和棕榈酸刺激人肝癌HepG2细胞和L6大鼠成肌细胞建立胰岛素抵抗模型，考察了BBR及其代谢产物的降糖活性，并对其作用机制进行初步地探究。结果显示：BBR、M1、M2和M3均具有明显的降糖活性。本研究采用Western blot和Real-Time PCR法分别考察了待测化合物对葡萄糖转运、糖原合成关键蛋白和糖异生相关蛋白的影响。结果显示，M2可能通过影响GLUT2蛋白水平促进HepG2细胞摄取葡萄糖。BBR和M1可以通过抑制GSK3β的表达增加糖原合成。BBR、M1、M2和M3均可以抑制糖异生关键基因G6Pase 和PEPCK的mRNA表达。接着，本研究深入研究了作用机制，发现BBR及其活性代谢产物可能通过激活PI3K/AKT信号通路，及上调FAM3A和FAM3C表达，进而发挥改善胰岛素抵抗的作用。另外，本研究采用基因芯片技术检测BBR及其代谢产物的作用机制，发现cAMP、AMPK和胰岛素信号通路、亚油酸代谢途径等可能涉及到BBR及其代谢产物的降糖作用。