新生期DEHP暴露诱导大鼠胰岛β细胞损伤的分子机制研究

Diabetes mellitus, a disease mainly characterized by hyperglycemia, has been rising at epidemic rates over the last several decades. Increasing epidemiological and experimental studies suggesting that environmental pollutants play an important part in the elevated incidence of type 2 diabetes. Di(2-ethylhexyl) phthalate (DEHP) is one of the most widespread plasticizers to impart ?exibility of polyvinyl chloride (PVC), which is commonly used in the manufacture of many daily products. It has been reported that exposure to DEHP is highly associated with the increased risk of glucometabolic disorders and DEHP is thus suggested as a new risk factor for diabetes. However，the specific molecular mechanisms are mostly unclear. Loss of β-cells function is central to the development and progression of diabetes. β-cells are particulartly vulnerable to oxidative stress damage because of lower antioxidant capacity. Sustained oxidative stress induced by chemicals would disrupt insulin secretion and finally result in β-cells failture. More importantly, β-cells are more sensitive to chemicals exposure during vital developmental periods, so the present study genenated a rat model to determine whether neonatal exposure to DEHP disrupts the whole body glucose homeostasis and induces β-cells dysfunction, and if so, whether these adverse effects are mediated through oxidative stress regulated signal pathway. We firstly tested the effects of neonatal exposure to DEHP on glucose homeostasis and pancreatic β-cells function in the rat. Then ROS generation, mitochondrial funtion and endoplasmic reticulum stress related molecules were examined in DEHP exposed β-cells in both vivo and vitro. We also investigated the potential protective effects of the antioxidant N-acetylcysteine on DEHP-induced β-cells damage in vitro to further explore the underlying mechanisms that oxidative stress was involved in DEHP induced β-cells dysfunction. Taken together, this study would address mechanism of DEHP induced β-cells dysfunction at cellular and molecular levels, and furthermore, creat new insights into the association of DEHP exposure and the onset of diabetes mellitus.

DEHP是一种高性能的增塑剂，广泛用于塑料制品的生产。流行病学调查和动物实验指出，DEHP 暴露与血糖代谢紊乱存在密切联系，但作用机制研究报道较少。胰岛β细胞是分泌胰岛素的唯一细胞，也是氧化应激的重要靶点，其功能缺陷是糖尿病发生发展的中心环节。新生期是胰岛塑形的关键时期，对外源化合物异常敏感，所以本项目拟建立新生期DEHP直接暴露的大鼠模型，观察DEHP暴露后机体血糖稳态和β细胞胰岛素分泌功能的变化；测定β细胞的氧化水平；阐明β细胞损伤过程中内质网应激和线粒体凋亡通路的作用；最终揭示氧化应激、线粒体损伤、内质网应激与β细胞凋亡的内在关联。在此基础上，进一步通过体外细胞实验，结合活性氧清除剂拮抗处理，从细胞和分子水平阐明DEHP诱导β细胞凋亡的分子机制。本项目有望为DEHP致糖尿病的发病机制研究开辟一个新领域。

随着经济的发展，糖尿病的发病率日益增加，逐渐成为21世纪危害人体健康的主要疾病。尽管遗传、无节制的高热量饮食以及久坐不动的生活方式是糖尿病发病率急剧增高的主要诱因，近年来的研究发现，环境内分泌干扰物暴露也是不可忽视的危险因素。本项目选取邻苯二甲酸二（2-乙基）己酯（DEHP）和双酚A（BPA）两种典型的内分泌干扰物为研究对象，探讨其致胰岛β细胞功能损伤的风险和分子机制。首先，研究DEHP暴露对INS-1细胞的活力、胰岛素分泌功能、氧化应激水平、Nrf2介导的抗氧化功能、内质网Ca2+稳态以及内质网应激关键通路基因和蛋白表达的影响。结果表明：DEHP暴露导致INS-1细胞活力显著降低，葡萄糖刺激的胰岛素释放功能受损，凋亡增加，具有剂量依赖性。而且，DEHP暴露还导致INS-1细胞ROS生成增多，Nrf2介导的抗氧化作用失调，内质网Ca2+耗竭，内质网分子伴侣GRP78和GRP94的表达增加以及PERK和eIF2α磷酸化，进而上调ATF4和CHOP的转录和翻译水平，最终促发细胞凋亡。其次，项目对BPA暴露后INS-1细胞的活力，胰岛素分泌功能，线粒体功能等指标进行测定分析。结果表明：BPA暴露导致INS-1细胞活力降低，葡萄糖刺激的胰岛素分泌功能受损，具有剂量依赖性。此外，BPA暴露导致INS-1细胞线粒体功能障碍和凋亡，主要表现为：线粒体肿胀、空泡化，质量减小，ATP含量显著降低，线粒体膜电位降低，细胞色素c释放增加以及Bcl-2和Caspase家族蛋白表达异常。综上所述，DEHP和BPA均可诱导胰岛β细胞功能受损及凋亡，但二者作用机制不同。DEHP暴露主要通过激活β细胞内质网非折叠蛋白响应信号通路PERK-eIF2a-ATF4，促进内质网Ca2+耗竭，启动细胞凋亡程序；BPA暴露则主要通过作用于β细胞线粒体呼吸链，引起线粒体功能障碍，促发细胞凋亡。以上研究成果有望为环境内分泌干扰物暴露致糖尿病的机制研究提供新的思路与线索，有助于提出针对性的预防措施。项目已正式发表3篇SCI研究论文，1篇国内研究综述；培养博士研究生2名；课题负责人和主要参与人均由中级职称晋升为高级职称。项目超额完成研究计划，达到预期的研究目标。