降糖消脂片保护胰岛β细胞的microRNAs调控机制研究

The progressive deterioration of beta-cell function and mass is one of the main reasons of the uncontrollability of disease progression and blood glucose in patients with type 2 diabetes. Elucidating the mechanism underlying the poor proliferative capacity of adult pancreatic β-cells is critical to regenerative therapeutic approaches for diabetes. The islet in T2DM is characterized by a deficit in β-cells, increased β-cell apoptosis, and extracellular amyloid deposits derived from islet amyloid polypeptide et al. The major factors for progressive loss of beta-cell function and mass are glucotoxicity, lipotoxicity, endoplasmic reticulum stress, oxidative stress, proinflammatory cytokines, leptin, and islet cell amyloid et al. Growing evidence indicates that microRNAs (miRNAs) involved in the regulation of the functions of pancreatic β-cells or insulin-target tissue in the context of diabetes. A large number of miRNAs have been implicated in normal pancreatic development and function. It is expected that aberrant miRNAs expression or mutations could result in β-cell pathology. Identification of miRNAs that were found to affect insulin transcription renders these miRNAs as potential therapeutic targets whereby insulin production can be regulated by modulating their expression accordingly. Our studies have shown that Jiangtang xiaozhi tablets could decrease the blood glucose level in the type 2 diabetes patients and animals, and increase the insulin level, as well as elevate the amount of β-cells and reduce the apoptosis of β-cells in type 2 diabetes animals. Based on the past studies, we put forward the hypothesis that protective effects of Jiangtang xiaozhi tablets on pancreaticβ-cells may be relevant to regulating miRNAs. This project aims to study the effects of Jiangtang xiaozhi tablets on the Goto-Kakizaki (GK) rat which is a well-studied non-obese spontaneous type 2 diabetes animal model characterized by impaired glucose-stimulated insulin secretion in the pancreatic β-cells. We aim to observe the blood glucose level and theβ-cells mass and apoptosis, identify miRNAs that are differentially-expressed in the pancreatic islets of the GK rats which are treated with Jiangtang xiaozhi tablets by using microarrays; Furthermore, we study the effects of serum carrying Jiangtang xiaozhi tablets on proliferation and apoptosis of islet cell tumor cell lines INS-1 exposed to high glucose, examine the miRNA expression and protein levels of its target gene. Then we use the antisense technology with which miRNA can be downexpressed or overexpressed in the INS-1 to assess the signal pathway of target miRNA. Our objective is to reveal the mechanism of Jiangtang xiaozhi tablets in protecting pancreaticβ-cells by regulating miRNAs. The significance of the subject is to further reveal the mechanism of traditional chinese medicine in the treatment of diabetes, and to explore the strategy of traditional chinese medicine on miRNAs regulation.

2型糖尿病患者胰岛β细胞功能进行性减退是疾病持续进展、血糖难以长期稳定控制的重要原因，改善或延缓胰岛β细胞功能进行性减退，促进β细胞增殖，减少凋亡，是糖尿病治疗的关键问题。多种microRNAs（miRNAs）在胰岛发育、β细胞分化、胰岛素分泌中扮演着重要角色。我们既往的研究表明，降糖消脂片能够有效降低2型糖尿病患者及动物模型血糖，升高胰岛素水平，增加胰岛数目，减少β细胞凋亡。本课题拟应用miRNAs芯片技术，研究降糖消脂片对2型糖尿病模型GK大鼠胰岛miRNAs差异表达的影响；探讨降糖消脂片含药血清对INS-1细胞增殖和凋亡的影响及对目标miRNA的作用，并应用反义寡核苷酸链技术进行基因沉默，揭示miRNAs调控保护胰岛β细胞的作用机制。以期证实假说"降糖消脂片对胰岛β细胞的保护作用与miRNAs调控有关"，进一步阐明中医药治疗糖尿病的作用机制，探索中医药对miRNAs调控的研究策略。

糖尿病是一种以慢性血糖水平增高为主要特征的代谢性疾病。其中胰岛β细胞功能进行性减退是疾病持续进展、肝脏中糖脂代谢紊乱、糖输出异常增多在2型糖尿病发生发展过程中起到重要作用。近年来，非编码RNA研究中，一些miRNA和lncRNA已被证实参与了糖异生、糖酵解以及糖原分解和胰岛素敏感等的调控，说明非编码RNA在糖尿病发生发展中的重要作用。本研究在前期研究的基础上，通过2型糖尿病模型大鼠模型，并应用高通量测序技术，对正常对照组、模型组和降糖消脂片新复方组大鼠肝脏进行转录组分析（mRNA、lncRNA和miRNAs），更全面系统的鉴定和筛选在肝脏糖脂代谢和糖尿病发生发展过程中起到关键作用的RNA分子，并从糖脂代谢信号通路和相关功能聚类等方面探索其在降糖消脂片新复方改善2型糖尿病大鼠胰岛素抵抗中的作用机制，进一步阐明中医药治疗糖尿病的作用机制，探索中医药对非编码RNA调控的研究策略。研究结果显示，降糖消脂片新复方可以通过调节和重构肝脏糖脂代谢，包括增加葡萄糖的摄取、下调肝糖合成，下调脂类的合成、氧化和转运等，从而有效改善肝细胞胰岛素抵抗，改善糖脂代谢，治疗2型糖尿病。