从SIRT1/FoxO1通路介导的胰岛β细胞去分化角度部分阐明开郁清热方治疗2型糖尿病的作用机制

Type2 diabetes mellitus(T2DM) has been a critical illness that threatens human health. Recent studies indicate that the dedifferentiation of pancreatic β cells may be the key problem for the continuous islet dysfunction. FoxO1 has the function of inhibiting β cells dedifferentiation, while SIRT1 can enhance FoxO1 transcriptional activity through deacetylation. Our studies have shown that the main pathogenesis of T2DM is abdominal fullness and interior heat on the perspective of traditional Chinese medicine and Kaiyuqingre Formula is an effective prescription for the treatment of T2DM. Based on what has been reported that Kaiyuqingre Formula upregulated the level of SIRT1, we take the Ins2/GFP transgenic animal and β cell models of T2DM and the models with SIRT1interved by chemicals as objects , take cell lineage tracer as key technology to verify that whether Kaiyuqingre Formula may inhibit β cells dedifferentiation by upregulating the level of SIRT1 which may induce the deacetylation of FoxO1. Through the above research, we are looking forward to test the hypothesis that the down- regulation of SIRT1 reduces the activity of FoxO1 may be an important mechanism for the dedifferentiation of induced β cells in high glucose environment , while Kaiyuqingre Formula may inhibit the dedifferentiation of β cells by regulating the SIRT1/FoxO1 signaling pathway to protect the islet function. This research is expected to provide a new scientific basis for the prevention and treatment of T2DM with traditional Chinese medicine.

2型糖尿病（T2DM）是危害人类健康的重大疾病。新近研究表明胰岛β细胞去分化可能是T2DM胰岛功能持续减退的关键原因，FoxO1具有抑制β细胞去分化的作用，而SIRT1可通过去乙酰化增强FoxO1的转录活性。团队研究发现“中满内热”是T2DM的核心病机，开郁清热方是针对该病机治疗T2DM的有效方剂。本研究在前期证实开郁清热方可上调SIRT1表达的基础上，分别以Ins2/GFP转基因和化学试剂干预SIRT1表达的T2DM动物及β细胞模型为研究对象，运用细胞谱系示踪技术探讨开郁清热方是否通过上调SIRT1使FoxO1去乙酰化从而抑制β细胞去分化。通过上述研究，拟验证假说:SIRT1下调使FoxO1活性降低是高糖诱发β细胞去分化的重要机制，而开郁清热方可能通过调控SIRT1/FoxO1信号通路抑制β细胞去分化从而保护胰岛功能。本项目有望为中医药防治T2DM提供新的科学依据。

胰岛β细胞去分化是T2DM胰岛功能持续减退的关键原因，FOXO1具有抑制β细胞去分化的作用，而SIRT1可通过去乙酰化增强FOXO1的转录活性。团队前期研究显示开郁清热方是治疗T2DM的有效方剂，并可上调SIRT1的表达，因此提出假说:SIRT1下调使FOXO1活性降低是高糖诱发β细胞去分化的重要机制，而开郁清热方可能通过调控SIRT1/FoxO1信号通路抑制β细胞去分化延缓疾病。本研究以以DB/DB小鼠以及DIO小鼠构建的肥胖T2DM动物模型为研究对象，以SIRT1激动剂白藜芦醇及SGLT2抑制剂（抑制胰岛β细胞去分化）为阳性对照，研究结果发现开郁清热方可有效改善糖尿病小鼠胰岛功能、糖脂代谢，并改善炎症因子、氧化应激、瘦素以及脂联素水平，同时能够同时恢复β细胞标志性蛋白insluin、FOXO1、PDX1及NKX6.1表达，表明开郁清热方可有效的综合性的改善糖尿病，并抑制胰岛β细胞去分化。为进一步验证开郁清热方是否通过调控SIRT1/FOXO1通路抑制β细胞去分化，我们以高糖刺激的大鼠胰岛β细胞瘤细胞（RIN-m5F）为研究对象，以SIRT1激动剂白藜芦醇为阳性对照，以SIRT1沉默的细胞为阴性对照，发现高糖干预6天可显著降低RIN-m5F insulin、SIRT1及FOXO1表达水平，抑制PDX1及NKX6.1表达并上调OCT4表达，而开郁清热方含药血清及白藜芦醇可上调SIRT1及FOXO1表达，同时恢复insulin、PDX1及NKX6.1并抑制OCT4表达。沉默SIRT1后，RIN-m5F细胞insulin、FOXO1、PDX1、NKX6.1表达减少，干细胞标志性转录因子OCT4及ngn3表达增加，高糖刺激后，进一步恶化或变化不明显，而应用开郁清热方后，其表达也并未出现明显的改善。综上，本项目研究结果提示了在糖尿病早期，胰岛β细胞SIRT1的稳定表达对维持FOXO1及对抗去分化至关重要，而开郁清热方可以通过上调SIRT1/FOXO1对抗胰岛β细胞去分化。