运脾和络颗粒调控SIRT1-FoxO1-自噬通路抑制糖尿病脂肪异位沉积的研究

Ectopic lipid deposition induced by diabetic lipid metabolic disorders can lead to insulin resistance. In our previous study of using Yunpi Heluo Granules (YHG) to treat diabetes, we found that YHG could not only reduce lipid deposition in the pancreas, liver and muscles but regulate SIRT1.SIRT1-FoxO1-autophagy pathway is associated with diabetes . We therefore postulated that YHG might participate in ectopic lipid deposition via the SIRT1-FoxO1-autophagy pathway. The present study is designed to detect glucose, lipid metabolism and related gene changes by SIRT1 transgenic technology in a Zucker Diabetic Fatty (ZDF) Type 2 diabetic rat model receiving YHG treatment. Three cell models induced by high fat and high glucose will be constructed to find out differences in lipid deposition in different organs; cell proliferation, apoptosis and intracellular fat deposition will be observed in serum culture medium containing YHG; SIRT1-eukaryotic expression plasmids will be constructed to interfere with the expression of SIRT1 in pancreatic β cells by siRNA; and finally changes in fat deposition and related molecules will be measured, in an attempt to clarify the molecular mechanism of THG in inhibiting diabetes-induced ectopic fat deposition via the SIRT1-FoxO1-autophagy pathway.

糖尿病脂代谢紊乱造成的脂质异位沉积可导致胰岛素抵抗。项目组前期研究发现运脾和络颗粒可以减少脂质在胰腺、肝脏、肌肉的沉积，对SIRT1具有调控作用；SIRT1-FoxO1-自噬通路与糖尿病密切相关；我们推测运脾和络颗粒可能是通过调控SIRT1-FoxO1-自噬通路抑制脂质异位沉积。本项目拟应用SIRT1转基因技术作基因靶点探索；应用ZDF 2型糖尿病大鼠模型，检测运脾和络颗粒干预前后糖、脂代谢及相关分子变化；应用脂质组学技术分析不同器官脂肪沉积的差异和运脾和络颗粒作用的靶点。应用运脾和络颗粒含药血清干预高脂高糖刺激的β细胞等三种细胞；观察细胞增殖、凋亡和细胞内脂肪沉积情况；应用SIRT1真核表达质粒及siRNA技术干预β细胞SIRT1的表达；检测细胞脂质含量及相关分子的变化。通过上述研究，明确运脾和络颗粒是否通过SIRT1-FoxO1-自噬通路抑制糖尿病脂肪异位沉积。

本课题研究了运脾和络方通过调控肝脏、骨骼肌、胰腺组织的SIRT1-FoxO1 自噬信号通路改善2型糖尿病脂肪异位沉积的作用机制。研究表明：运脾和络方可有效治疗糖尿病大鼠的脂肪异位沉积，改善血糖和胰岛素抵抗。糖尿病模型组大鼠肝脏、骨骼肌、胰腺组织的SIRT1mRNA 和蛋白表达显著低于正常对照组，FoxO1mRNA 和蛋白表达要明显高于正常对照组；与糖尿病模型组对比，运脾和络方及SIRT1 过表达组大鼠肝脏、骨骼肌、胰腺组织的SIRT1mRNA 和蛋白表达显著增高，FoxO1 的mRNA 及蛋白表达显著降低。模型组HepG2肝细胞、骨骼肌细胞、胰腺细胞的SIRT1 mRNA 和蛋白表达明显低于对照组，而FoxO1mRNA 及蛋白表达明显高于对照组；运脾和络方可显著降低三种细胞内FoxO1 mRNA 及蛋白的表达，同时升高SIRT1 mRNA 及蛋白表达。此外，“鸟枪法”脂代谢组学研究结果显示，运脾和络方可通过多途径多靶点减轻2型糖尿病大鼠脂肪异位沉积。该研究首次阐明了运脾和络方通过调控肝脏、骨骼肌、胰腺组织的SIRT1-FoxO1 自噬信号通路改善2型糖尿病脂肪异位沉积和胰岛素抵抗。为SIRT1-FoxO1 自噬信号通路调控脂肪异位沉积的机制提供了研究基础，也为运脾和络方治疗糖尿病的调控机制奠定了基础。