目的 整合網(wǎng)絡(luò)藥理學(xué)、分子對接技術(shù)和實驗驗證，系統(tǒng)探討青娥丸治療阿爾茨海默?。ˋD）的潛在活性成分及其作用機制。方法 經(jīng)過ETCM數(shù)據(jù)庫、TCMSP數(shù)據(jù)庫及文獻(xiàn)搜索通過SWISS ADME平臺和Swiss Target Prediction數(shù)據(jù)庫篩選出青娥丸主要活性成分及靶點，通過GeneCards數(shù)據(jù)庫和OMIM數(shù)據(jù)庫獲取AD靶點；取交集靶點后用STRING數(shù)據(jù)庫構(gòu)建青娥丸治療AD潛在靶點的蛋白質(zhì)–蛋白質(zhì)相互作用（PPI）。運用Cytoscape 3.9.1軟件篩選核心靶點；采用DAVID數(shù)據(jù)庫對交集靶點進行基因本體（GO）功能富集分析及京都基因與基因組百科全書（KEGG）通路富集分析。使用PyMol軟件和AutoDock1.5.7軟件進行分子對接驗證。建立HT22細(xì)胞模型，采用CCK-8法檢測細(xì)胞活力，通過Western blotting檢測磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）通路和細(xì)胞凋亡的關(guān)鍵作用靶點蛋白表達(dá)水平。結(jié)果 共得到青娥丸活性成分119個，靶點900個；疾病靶點1 409個。PPI網(wǎng)絡(luò)獲得核心靶點Akt1、非受體酪氨酸激酶（SRC）、熱休克蛋白90α家族A類成員1（HSP90AA1）、腫瘤壞死因子（TNF）和表皮生長因子受體（EGFR）等。GO和KEGG富集分析獲得1 532個GO功能注釋，192條信號通路，主要涉及PI3K/Akt通路、細(xì)胞凋亡等。分子對接結(jié)果顯示，青娥丸中的主要活性成分與關(guān)鍵靶點均有強的結(jié)合能力，對接結(jié)合能均低于−6.0 kcal/mol。細(xì)胞實驗表明，與對照組比較，青娥丸可以改善Aβ25-35誘導(dǎo)的HT22細(xì)胞活力（P＜0.01），提高Aβ25-35誘導(dǎo)的HT22細(xì)胞中p-Akt、p-PI3K和Bcl-2蛋白的表達(dá)，抑制Bax蛋白的表達(dá)（P＜0.05）。結(jié)論 青娥丸可能通過調(diào)節(jié)PI3K/Akt信號通路和細(xì)胞凋亡保護神經(jīng)元，延緩病理進程治療AD，為青娥丸的臨床應(yīng)用和AD治療的研究提供依據(jù)。

Objective Integrate network pharmacology, molecular docking technology and experimental verification to systematically explore the potential active ingredients and mechanisms of Qing’e Pills in treating Alzheimer′s disease (AD).Methods The main active components and targets of Qing’e Pills were screened using the ETCM database, TCMSP database, and literature search via the SWISS ADME platform and Swiss Target Prediction database. AD-related targets were obtained from the Gene Cards database and OMIM database. Intersection targets were identified, and the STRING database was used to construct a protein-protein interaction (PPI) network of potential targets for Qing’e Pills in treating AD. Core targets were screened using Cytoscape 3.9.1 software. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the intersection targets were performed using the DAVID database. Molecular docking validation was conducted using PyMol software and Auto Dock 1.5.7 software. An HT22 cell model was established, and cell viability was assessed using the CCK8 assay. Western blotting was used to detect the expression levels of key target proteins in the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway and apoptosis.Results A total of 119 active components and 900 targets of Qing′e Pills were identified, along with 1 409 disease-related targets. The PPI network revealed core targets such as Akt1, non-receptor tyrosine kinase (SRC), heat shock protein 90 alpha family class A member 1 (HSP90AA1), tumor necrosis factor (TNF), and epidermal growth factor receptor (EGFR). GO and KEGG enrichment analysis yielded 1 532 GO functional annotations and 192 signaling pathways, primarily involving the PI3K/Akt pathway and apoptosis. Molecular docking results demonstrated that the main active components of Qing′e Pills exhibited strong binding affinities to the key targets, with docking binding energies all below −6.0 kcal/mol. Cell experiments showed that, compared to the control group, Qing’e Pills significantly improved Aβ25-35-induced HT22 cell viability (P < 0.01), increased the expression of p-Akt, p-PI3K, and Bcl2 proteins in Aβ25-35-induced HT22 cells, and inhibited the expression of Bax protein (P < 0.05).Conclusion Qing’e Pills may protect neurons and delay the pathological process of AD by regulating the PI3K/Akt signaling pathway and apoptosis, which provides a basis for the clinical application of Qing′e Pills and the research of AD treatment.

R285

中國博士后（54批）科學(xué)基金項目（2013M541427）