异菝葜皂苷元治疗阿尔茨海默病的组蛋白乙酰化调控机制研究

Smilagenin (SMI) is active ingredients of Yin tonic in Chinese traditional medicine, which can protect hippocampal neurons and improve learning and memory. It has also completed phase II clinical trials. Previous studies have shown that smilagenin exerts its neuroprotective effect through multiple pathways and targets, which related with upregulation of gene transcription. With the finding that smilagenin can regulate HDAC activity and expression, we believed that the important of histone acetylation in the benefits of smilagenin on alzheimer's disease. To test this hypothesis, follow-up study will focus on the following issues: 1) the effect of SMI on HDAC inhibition and histone acetylation; 2) To analyze the effect of SMI on AD related genes transcription including BDNF and Homer3 as well as the promoter acetylation level; 3) To study the seletive activation mechanism of smilagenin on target gene by luciferase assy and pull down followed by mass spectrometry;4）To explore the effect of smilagenin on the histone acetylation of BDNF and Homer3 and behaviour improvement in AD transgenic mouse model. Above mentioned researches can help to clarify the molecular mechanism of smilagenin in alzheimer's disease and eventully provide theoretical basis and potential therapeutic targets for AD.

知母中有效成分异菝葜皂苷元能够保护海马神经元，改善学习和记忆功能，并已完成国内II期临床试验。异菝葜皂苷元是通过多途径，多靶点的作用方式发挥其神经保护作用的，可能与其调节多基因转录表达相关。课题组前期发现异菝葜皂苷元能够调节组蛋白脱乙酰化酶（HDAC)的活性和表达，因此推测组蛋白乙酰化在异菝葜皂苷元治疗阿尔茨海默病种发挥重要调节作用。为此，本课题拟研究1）异菝葜皂苷元对HDAC的抑制活性及组蛋白乙酰化的影响；2）分析药物对特定靶基因mRNA表达及启动子区乙酰化水平的影响；3）通过荧光素酶活性分析和pull down结合质谱方法分析研究药物对基因的选择性激活机制；4）通过转基因AD模型研究药物对体内BDNF、Homer3基因组蛋白乙酰化的影响及相关行为学的改变。项目的成果将对阿尔茨海默病的发病机理及知母活性成分异菝葜皂苷元的分子药理机制提供较为明确的理论和实验依据。

异菝葜皂苷元（SMI）是一种从知母或天冬酰胺中提取的脂溶性甾体皂甙元。我们先前发现，在Aβ处理的SH-SY5Y细胞及原代神经元中，SMI显著增加脑源性神经营养因子（BDNF）转录。然而，SMI对BDNF的调控机制还有待进一步研究。本研究以H2O2损伤的SH-SY5Y细胞及N2a/APP695细胞为AD体外细胞研究模型，以APP/PS1双转基因小鼠为AD体内动物模型，研究SMI对AD模型的神经保护作用及其可能分子机制。体外实验中发现SMI可以显著升高拟AD细胞模型（H2O2损伤的SH-SY5Y细胞及N2a /APP695细胞）中下降的BDNFmRNA水平。SMI预处理后可以增加AD模型中H3乙酰化组蛋白（Acetylated-H3，H3AC）及H4乙酰化组蛋白（Acetylated-H4，H4AC）的水平，染色质免疫共沉淀证实SMI作用后可以升高细胞模型中BDNF基因转录子Ⅳ启动子处富集的H3AC，H4AC水平。APP/PS1转基因小鼠实验中，通过旷场实验，新物体识别，Y迷宫及水迷宫等行为学实验，我们观察到SMI可以增强APP/PS1小鼠的学习记忆能力，改善其认知功能障碍，并减少AD小鼠模型皮层和海马区域淀粉样斑块的沉积， SMI可以显著增加AD模型中P300的表达量， C646抑制p300基因降低了BDNF的乙酰化和蛋白水平，表明p300介导了SMI诱导的BDNF的稳定。综上所述，SMI通过抑制HDAC活性或增加p300蛋白的表达，促进BDNF基因启动子区的组蛋白乙酰化水平，进一步促进BDNF基因的表达，发挥对神经元的保护作用。这为SMI治疗阿尔茨海默病的研究提供了重要的理论基础。