NR2B基因组蛋白乙酰化调控与酒依赖及戒断的关系研究

Although alcohol dependence is a severe public health problem all over the word, the biological mechanism is unknown. The epigenetic regulation gradually has become a hot topic in the research field. NMDA receptor especially NR2B, plays an important role in the development and the change of behavioral presentations of alcohol dependence, but the results of its epigenetic regulation research are not unanimous. On the basis of previous studies, we will establish models of rats with alcohol dependence to study the relationship between craving, withdrawal as well as cognitive impairment and NR2B expression, histone acetylation in NR2B in prefrontal cortex, hippocampal, vental tegmental area at different time. Second, the research will explore the activity of histone deacetylase and its expression change at different time, and find the key histone deacetylase regulating histone acetylation in NR2B. Finally, we will regulate the histone acetylation with histone deacetylase inhibition and molecular intervention to verify the relationship of histone acetylation in NR2B and NR2B mRNA and behavioral presentations. The study will reveal that histone acetylation in NR2B is one of the regulation mechanisms of alcohol dependence and withdrawal.

酒依赖为全球严重公共卫生问题，有关神经生物学机制未明。现有资料表明表观遗传修饰调控逐渐成为酒依赖机制研究热点。既往研究提示NMDA受体尤其是NR2B在酒依赖形成及临床表征演变中起重要作用，但NR2B基因表达的表观修饰调控研究结果不一。本项目将在既往研究工作基础上，以酒依赖大鼠为实验模型，评定酒依赖及戒断行为表征演变（渴求、戒断、认知损害等）与模型大鼠前额叶皮质、海马、腹侧被盖区NR2B基因转录水平以及NR2B基因乙酰化水平时序性变化的关系；进而探讨酒依赖及戒断不同时点组蛋白去乙酰化酶活性及表达水平是否发生改变以及哪些组蛋白去乙酰化酶对NR2B基因乙酰化修饰起到关键作用；再后采用组蛋白乙酰化药物调控及分子调控实验来验证NR2B基因乙酰化水平与NR2B基因转录水平及行为表征的关系。本项目预期结果将有助于揭示组蛋白乙酰化调控NR2B基因转录水平是酒依赖及戒断行为表征演变的调控机制之一。

本研究以酒依赖大鼠模型为研究对象，利用酒精戒断评分和条件位置偏爱评定大鼠酒依赖及戒断行为表征演变（渴求、戒断、认知损害等），利用RT-PCR和免疫印迹技术检测模型大鼠海马区NR2B基因转录水平和蛋白水平，利用染色质共沉淀技术检测 NR2B基因组蛋白H3K9乙酰化水平，并进一步分析型大鼠戒断综合征、海马区NR2B表达水平及NR2B基因启动子区组蛋白H3K9乙酰化表达水平的相关性。研究结果表明在模型大鼠戒断不同节点和饮酒过程中海马脑区NR2B mRNA、蛋白和启动子区H3K9乙酰化表达水平均呈上调趋势，初步证实了酒依赖大鼠及戒断不同时点NR2B基因mRNA及其启动子区组蛋白乙酰化存在一致时序性变化。本研究进一步通过对酒依赖大鼠模型进行丁酸钠干预，抑制组蛋白去乙酰化酶，结果显示丁酸钠处理后加重了酒精诱导的大鼠觅药行为，且显著提高了大鼠海马NR2B蛋白、NR2BmRNA及NR2B基因启动子区H3K9乙酰化表达。本项目推测NR2B基因启动子区H3K9乙酰化可能通过增加酒精诱导的NR2B基因表达参与了酒精CPP形成的过程。本研究结果初步验证了组蛋白H3K9乙酰化调控NR2B基因转录水平可能是酒依赖及戒断行为表征演变的调控机制之一，并为预防和治疗酒精依赖的提供新靶点和新思路。