基于PI3K/AKT/DNMT1信号通路调控BACE1蛋白表达探讨化痰开窍法改善AD认知功能的作用机制

Traditional Chinese Medicine believes that the etiology and pathogenesis of dementia is closely related to phlegm. Phlegm can be produced by all viscera because of aging and infirmity. Moreover, Traditional Chinese Medicine has theories of "most diseases were caused by phlegm ", " treating phlegm is treating dementia ". Therefore, Traditional Chinese Medicine treatment of dementia is focused on eliminating phlegm for resuscitation. We choose Wendan decoction plus borneol, acorus calamus, radix curcumae as the prescription(WDTJW). Applying WDTJW-treated Alzheimer’s disease (AD) models to explore the mechanism of treating AD with the method of eliminating phlegm for resuscitation. The diagnostic and pathological features of AD are excessive deposition of beta-amyloid (Aβ) protein in the brain, meanwhile beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the key rate-limiting enzyme in Aβ production. Our previous research found that WDTJW treatment ameliorated cognitive impairment in AD mouse model, reduced Aβ deposition in the brain, decreased BACE1 protein level, and increased DNA methyltransferase 1 (DNMT1) protein level. Transcriptome RNA sequencing results suggested that WDTJW may affect the PI3K/AKT signaling pathway in the brain of AD mice model. This study aimed to further investigate whether the specific mechanism of WDTJW in improving AD cognitive impairment is associated with regulating the methylation level of BACE1 promoter through PI3K/AKT/DNMT1 signaling pathway, inhibiting the transcription of BACE1 and reducing the aggregation of Aβ in vivo and in vitro.

中医认为痴呆病因病机与“痰”邪密切相关，年老体虚，五脏虚衰皆可生痰。中医素有“百病兼痰”“治痰即治呆”等学说，故痴呆病的中医治疗重在化痰开窍。本课题选方温胆汤加冰片、石菖蒲、郁金（WDTJW）干预阿尔茨海默病（Alzheimer's disease, AD）体内外模型，探讨化痰开窍法治疗AD的作用机制。AD的诊断性病理特征是脑内β-淀粉样蛋白（β-Amyloid，Aβ）沉积，而BACE1为Aβ产生的关键限速酶。本课题前期研究发现WDTJW能改善AD模型小鼠认知功能障碍，减少脑内Aβ沉积，降低BACE1蛋白水平，提高DNMT1蛋白水平。转录组RNA测序结果提示WDTJW可能影响AD模型小鼠脑内PI3K/AKT信号通路。本课题旨在通过体内外实验探讨WDTJW是否通过调控PI3K/AKT/DNMT1信号通路上调BACE1启动子甲基化水平，抑制BACE1转录，减少Aβ聚集，从而改善AD认知功能。

中医认为痴呆病因病机与“痰”邪密切相关，年老体虚，五脏虚衰皆可生痰，故痴呆病的中医治疗重在化痰开窍。本课题选方温胆汤加冰片、石菖蒲、郁金（WDTJW）干预阿尔茨海默病（Alzheimer's disease, AD）体内外模型，探讨化痰开窍法治疗AD的作用机制。AD的诊断性病理特征是脑内β-淀粉样蛋白（β-Amyloid，Aβ）沉积，而BACE1为Aβ产生的关键限速酶。本课题体内研究模型采用脑内过表达Aβ的转基因小鼠APP/PS1，利用多种行为学方法（包括水迷宫、条件恐惧实验、新物体识别实验等）检测WDTJW对APP/PS1小鼠认知功能的影响，结果提示WDTJW能改善AD模型小鼠认知功能障碍；利用免疫荧光及免疫组化的方法检测WDTJW对APP/PS1小鼠脑内淀粉样斑块沉积的改善，结果提示WDTJW能减少APP/PS1小鼠脑内Aβ沉积；利用western-blot检测WDTJW对APP/PS1小鼠脑内APP代谢产物的表达影响，结果提示WDTJW可降低APP、BACE1蛋白水平，提高DNMT1蛋白水平。转录组RNA测序结果提示WDTJW可能影响AD模型小鼠脑内PI3K/AKT信号通路。进一步利用western-blot及甲基化PCR技术进行体内实验验证，体外实验采用含药血清处理swAPP HEK293细胞进行验证。结果提示WDTJW通过调控PI3K/AKT/DNMT1信号通路上调BACE1启动子甲基化水平，抑制BACE1转录，减少Aβ聚集，从而改善AD认知功能。本研究为WDTJW治疗阿尔茨海默病认知功能障碍提供了药理依据。ApoE在血液脂质代谢中起重要作用。“痰”邪的产生与脂质代谢异常密切相关，为探析“痰”邪在阿尔茨海默病中的病理生物基础，以载脂蛋白E (ApoE)为基础，结合分析阿尔茨海默病发病危险因素及临床常见指标，利用LASSO回归筛选危险因素，构建logistic回归预测模型，10折交叉进行区分度验证，Bootstrap法进行校准度验证，临床决策曲线评判预测模型的临床指导性，最后以列线图可视化呈现临床预测模型，构建阿尔兹海默病发病风险的临床预测模型。结果提示利用年龄、游离三碘甲状腺素、性别、ApoE构建的临床预测模型对患者进行个体化评估，可以预警阿尔兹海默病，开展早期预防干预，减缓该病的发生发展。

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