p300转录复合器在高糖记忆致微血管损伤中的作用及机制

Diabetic animals and patients continue to active multiple transcription factors and promote the mRNA and protein expression of effector molecules even after achieving glycemic control, eventually develop microvascular complications. This poorly understood "glycemic memory" phenomenon pose major challenges in treating diabetes. Although oxidative stress, advanced glycation end products, signal kinase activation and epigenetic modification have been implicated in the process, it is not known whether an unifying pathway exist or not. Recently, we and others have demonstrated transcriptional co-activator p300 indeed regulates profound transcription factor activation and gene expression via epigenetic changes such as chromatin histone acetylation. Thus, we hypothesized that high glucose→active various metabolic pathway→converge on p300, a transcriptional co-activator with histone acetytransferase activity→epigenetic changes in chromatin remodeling→transmission of epigenetic modification→persistent alteration of a large number of transcription factors and mRNA transcripts through p300 epigenetic modification after glucose normalization→leading to diabetic complication pheonotype. In this project, we initially determin whether hyperglycemia continue to cause increased p300 expression, and such an increase in p300 leads to functional alteration despite glucose normalization in vitro and in vivo. We then explore whether p300 can act as a integrator of diverse metabolic memory pathway. Next, to establish a pathogenetic role of p300, we assess a large number of transcription factors in the nuclear extracts by combination of recombinant adenovirus transfection and liquid chip approach. Then, we further exam whether the effect of p300 and transcription factors is mediated through binding to the promoters of specific transcripts altered by diabetes and high glucose. Hence, we perform ChIP assay using promoter specific primers. Finally, it is important to find out whether p300 indeed regulate production of specific transcripts which are important in mediating glycemic memory phenotyte. To assress this issue, we focus on specific vasoactive factors, cell homeostasis and extracellular matrix protein in animals or cells following p300 shRNA transfection, p300 mutant overexpression or treatment with p300 inhibitor. All in all, our project may provide direct evidence that activation of p300 is an important upstream event causing hyperglycemia-induced histone acetylation, transcription factor activation, effector protein expression in cells and in the microvascular tissues after glucose normalization. Hence, the specific inhibitor for p300 or its key transcription complex may have potential therapeutic targets to prevent diabetic microangiopathy.

糖尿病动物或患者即使血糖控制后，众多转录因子及效应蛋白仍持续表达，此即所谓"高糖记忆"现象。已证实生化机制和表观遗传参与该过程的发生，但是否有共同的发病机制尚不清楚。最近，我们和其他作者证实转录共刺激子p300可通过染色质组蛋白乙酰化(CHA)修饰参与转录因子激活和基因表达。因此我们假设高糖→代谢途径→汇聚于p300→p300发挥CHA作用→诱导转录因子参与的特定基因在血糖正常后持续开放→高糖记忆表型形成。本课题从表观遗传学角度，在整体和离体水平观察糖尿病诱导的p300 表达及CHA功能在血糖正常后的变化规律，试图发现p300可能系糖尿病各种记忆刺激的最终共同途径。并采用基因转移和液相芯片技术研究p300转录器的表达规律，高通量地筛选和鉴定转录因子谱和分析其下游特定基因启动子在血糖正常后的变化规律。本课题有助于深化高糖记忆发病机制认识，寻求p300转录器干预靶点，为高糖记忆防治探索新希望。

高糖记忆的分子机制尚未完全阐明，项目从表观遗传学入手，在我们前期证实心肌转录共刺激子p300可通过染色质组蛋白乙酰化(CHA )修饰调节心脏重构的基础上，首先在糖尿病动物、细胞层面，观察到高糖暴露后p300/SIRT表达变化，并证实即使血糖控制后，上述分子及CHA 修饰和效应蛋白仍持续表达，即p300/SIRT存在“高 糖记忆"现象。进而发现p300/SIRT介导的CHA修饰作用是高糖引起的氧化应激、蛋白非酶糖基化和蛋白激酶C激活等多种代谢记忆途径的汇聚焦点，并通过特定位点的单核苷酸多态性分析在糖尿病人群进行了验证，提出了p300/SIRT可能系糖尿病肾脏病各种记忆刺激的最终共同途径的观点。另外，项目还采用组学技术初步探讨了p300转录器的表达规律，高通量地筛选和鉴定转录谱在血糖正常后的变化规律。最后，我们在整体和离体水平证实漆黄素和利拉鲁肽可分别改善糖尿病诱导的p300/SIRT 表达，从而发挥肾脏保护和促血管新生作用。总之，本课题初步证实了高糖通过各种代谢途径，汇聚于p300/SIRT,发挥CHA 修饰,进而诱导特定基因在血糖正常后持续开放，促进高糖记忆表型的形成，此有助于深化高糖记忆发病机制认识，寻求p300转录器干预靶点，为高糖记忆防治探索新希望。