熟三七总皂苷调控胰高血糖素介导的cAMP/NIK/NF-κB2通路抑制肝糖生成的作用及机制研究

Hormone glucagon, as well as insulin, plays an important role in maintaining glucose homeostasis. The development and progression of diabetes mellitus type 2 is closely associated with glucagon dysfunction. Compared with crude notoginseng, steamed notoginseng contains novel rare ginsenosides produced by the stepwise deglycosylation and dehydration during processing. Steamed notoginseng shows significant anti-diabetic effects, which proved via glucagon regulation in vivo. Total ginsenosides can inhibit the activation of cAMP and NF-κB2 in liver, indicating the hypoglycemic effect may be related to the glucagon/cAMP /NIK/NF-κB2 pathway. Based on these previous works, this project is aimed to investigate the hypoglycemic effects of total ginsenosides and its glucagon dependent molecular mechanism. The major contents include：1) a series of glucagon-induced animal and cell models were applied to verify the effects of total ginsenosides on hyperglycemia and gluconeogenesis 2) to elucidate the intervention effect of ginsenosides on glucagon mediated cAMP/NIK/NF-κB2 signaling pathway; 3) to discover the potential interactions among the identical or different structural type ginsenosides. The results will reveal the mechanism of total ginsenosides in maintaining glucagon steady state, and provide new strategies for anti-diabetic drugs discovery.

胰高血糖素在维持血糖稳态中和胰岛素发挥同等重要作用。临床发现胰高血糖素调节紊乱与2型糖尿病的发生发展密切相关。申请人前期研究发现三七蒸制成熟三七过程中发生脱糖脱水反应，产生大量稀有皂苷，具有更强的降糖效果，尤其可降低急性胰高血糖素模型小鼠血糖的升高，熟三七总皂苷亦可抑制肝组织内cAMP生成、抑制炎症相关蛋白NF-κB2的表达。本项目提出假说：熟三七总皂苷可调控胰高血糖素/cAMP/ NIK/NF-κB2通路抑制肝糖生成，拟开展以下内容：1）采用胰高血糖素相关动物、细胞模型，评价熟三七总皂苷的体内外降糖作用及对糖异生的影响；2）结合基因沉默与过表达技术，阐明总皂苷对cAMP/NIK/NF-κB2通路的干预作用及对cAMP的调节机制；3）系统研究皂苷间的交互作用，发现熟三七中协同降糖皂苷组合。研究成果将揭示熟三七总皂苷通过调节胰高血糖素相关通路抑制肝糖生成的新机制，为降糖药物发现提供新思路。

本研究以胰高血糖素介导的肝糖异生为出发点，以新的分子作用机制及熟三七中皂苷成分的干预作用为核心，取得了两个重要科学发现：（1）从临床数据出发，利用长期高脂饲喂、短期高脂饲喂和胰高血糖素刺激等多种动物模型诱导小鼠肝糖异生的过度激活，采用基因沉默技术进行全身或肝脏特异性敲降，发现敲降p52能够明显改善小鼠肝糖异生的过度激活，证实了p52在胰高血糖素介导的肝糖异生中发挥着重要作用。在分子机制上，发现胰高血糖素可以直接激活p52并促进p52入核，p52抑制PDE4B转录从而使PDE4B表达下降，引起细胞内cAMP的堆积，通过PKA-CREB通路启动糖异生关键酶，放大胰高血糖素信号通路。熟三七皂苷Rb1能够抑制高脂饲喂和胰高血糖素直接刺激造成的糖异生过度激活，且部分依赖于抑制p52的活化发挥降糖作用；（2）发现熟三七中皂苷Rg1可以减轻高脂小鼠体重，降低空腹血糖，改善口服糖耐量与丙酮酸耐量，降低血清胰高血糖素含量。在机制上，Rg1可以促进肝组织Akt的磷酸化并与核转录因子FoxO1直接结合，下调糖异生关键酶G6Pase和PEPCK的表达，降低肝糖产生。进一步通过免疫共沉淀、激光共聚焦等方法，结合Akt siRNA、Akt抑制剂及活性位点突变，发现Rg1可以选择性激活Akt Ser473位点，抑制FoxO1入核及转录活性，进而降低肝糖输出；共发表标注该项目的SCI论文3篇，包括Nature Communications 1篇（IF=10），Theranostics 1篇（IF=8）、The American Journal of Chinese Medicine 1篇（IF＝3）。协助培养博士研究生1名，硕士研究生5名。