基于内质网应激与自噬及其交互作用研究蒲黄总黄酮对游离脂肪酸诱导损伤胰岛β细胞影响的机制

Promoting survival of pancreatic β cells is extremely important for the prevention of β cells failure in type 2 diabetes. Chronic elevated free fatty acids (FFA) severely impair pancreatic β cells, which closely relates to FFA-induced endoplasmic reticulum stress and autophagy. According to Traditional Chinese Medicine, promoting blood circulation and removing blood stasis are a key principle for the treatment of type 2 diabetes. Previous studies have shown that Pollen Typhae total flavone (PTF), the extract from Pollen Typhae, a Chinese medicine which functions to promote blood circulation and to remove blood stasis, protected against FFA-induced impairment of pancreatic β cells, and promoted autophagy induced by FFA in pancreatic β cells, but the potential mechanisms keep unclear. The study aims to observe the effects of PTF on FFA-induced impairment of pancreatic β cells, and to clarify the mechanisms of PTF in regulating endoplasmic reticulum stress and autophagy induced by FFA through checking apoptosis by flow cytometry method, determing autophagic flux by electron microscopy, analyzing protein and gene expression of the molecules involving unfolded protein response pathway such as PERK, XBP1, CHOP, Bcl-2, Caspase-3 and signal pathways in mediating autophagy including Akt, mTOR, AMPK, JNK、LC3 and so on by Western blot and Real-time PCR, respectively, evaluating interaction between proteins by Co-immunoprecipitation, which will reveal the protective mechanisms of PTF against FFA-induced impairment of pancreatic β cells. The study will provide scientific evidence for the clinical application of pollen Typhae and developing new forms of PTF in preventing and treating type 2 diabetes.

促进胰岛β细胞存活对于防止2型糖尿病β细胞衰竭是极其重要的。长期高水平游离脂肪酸（FFA）严重损伤胰岛β细胞，这与其诱发的内质网应激与自噬密切相关。活血化瘀是治疗2型糖尿病的关键治则。前期研究表明，活血化瘀中药蒲黄提取物蒲黄总黄酮（PTF）能保护胰岛β细胞免受FFA损伤，并提高FFA诱导的自噬，但机制尚未清楚。本项目拟观察PTF对FFA诱导损伤胰岛β细胞的影响，并应用流式细胞术检测细胞凋亡，电镜分析自噬流，Western blot和Real-time PCR分别分析UPR途径PERK、XBP1、CHOP、Bcl-2、Caspase-3和自噬相关通路Akt、mTOR、AMPK、JNK、LC3等分子蛋白和基因表达，CO-IP分析蛋白相互作用，阐明PTF如何调控FFA诱发的内质网应激和自噬，从而揭示其保护胰岛β细胞免受FFA损伤的机制，为临床应用蒲黄和开发PTF新剂型防治2型糖尿病提供科学依据。

促进胰岛β细胞存活对于防止2型糖尿病β细胞衰竭是极其重要的。长期高水平游离脂肪酸（FFA）严重损伤胰岛β细胞，这与其诱发的内质网应激与自噬密切相关。活血化瘀是治疗2型糖尿病的关键治则。前期研究表明，活血化瘀中药蒲黄提取物蒲黄总黄酮（PTF）能保护胰岛β细胞免受FFA损伤。项目研究显示，PTF能够抑制FFA棕榈酸（PA）诱导损伤胰岛β细胞，虽然PTF不能影响PA诱导下胰岛β细胞PERK和eIF2α蛋白表达，但能抑制PERK和eIF2α蛋白磷酸化以及CHOP和XBP1蛋白表达，并促进Bcl-2蛋白表达，降低Bax和Cleaved caspase-3蛋白表达，进而抑制PA诱导胰岛β细胞凋亡。而且，除抑制内质网应激外，PTF还能提高PA诱导下胰岛β细胞AMPK活性、抑制mTOR磷酸化，以及促进Akt蛋白表达，并抑制活性氧（ROS)产生和JNK活性，进而促进PA诱导胰岛β细胞自噬和提高Bcl-2蛋白表达。研究结果揭示PTF能够抑制PA诱导胰岛β细胞内质网应激和促进PA诱导胰岛β细胞自噬，从而保护胰岛β细胞免受FFA损伤。项目研究从内质网应激及自噬角度阐明PTF保护胰岛β细胞免受FFA损伤的分子机制，为临床应用蒲黄和开发PTF新剂型防治2型糖尿病提供科学依据。