目的 探究抑肝散對阿爾茨海默?。ˋD）的治療作用，通過網(wǎng)絡藥理學方法預測其機制，并進行實驗驗證。方法 以D-半乳糖誘導AD小鼠模型，通過水迷宮和八臂迷宮實驗考察抑肝散（400、800 mg·kg^-1）對AD小鼠學習記憶功能的影響，并通過實時熒光定量PCR（qRT-PCR）法分析AD小鼠腦組織中腫瘤壞死因子-α（TNF-α）和白細胞介素-1β（IL-1β）、白細胞介素-2（IL-2）、白細胞介素-6（IL-6）的mRNA表達變化。通過中藥系統(tǒng)藥理學數(shù)據(jù)分析平臺（TCMSP）對抑肝散的組方藥材柴胡、甘草、川芎、當歸、白術、茯苓和鉤藤進行活性成分篩選，采用GeneCards等數(shù)據(jù)庫獲取AD疾病靶點，篩選其核心靶點，并對核心靶點進行基因本體（GO）和京都基因與基因組百科全書（KEGG）通路富集分析，分別獲取核心靶點涉及的生物過程（BP）、分子功能（MF）和細胞組分（CC）以及KEGG信號通路。小鼠神經(jīng)母細胞瘤Neuro-2a細胞和RAW264.7巨噬細胞分別與H₂O₂和抑肝散（3、6 μg·mL^-1）共培養(yǎng)，觀察抑肝散對Neuro-2a細胞體外增殖和凋亡的影響，及對Neuro-2a細胞Caspase-3和Caspase-8活性、活性氧（ROS）、谷胱甘肽（GSH）、丙二醛（MDA）、超氧化物歧化酶（SOD）和線粒體膜電位（MMP）的影響；觀察抑肝散對RAW264.7細胞TNF-α和IL-1β、IL-2、IL-6表達的影響，對網(wǎng)絡藥理學結果進行驗證。結果 抑肝散可緩解AD小鼠學習記憶功能損傷，通過網(wǎng)絡藥理學技術篩選得到抑肝散治療AD的活性成分159個，度值前10位的化合物分別是槲皮素、山柰酚、異鼠李素、β-谷甾醇、豆甾醇、7-甲氧基異黃酮、芒柄花素、柚皮素、美迪紫檀素和甘草查爾酮A，篩選得到核心靶點227個，度值排名前10的核心靶點為絲氨酸/蘇氨酸蛋白激酶1（AKT1）、腫瘤壞死因子（TNF）、白細胞介素6（IL6）、腫瘤蛋白P53（TP53）、白細胞介素1 β （ IL1B ）、雌激素受體1 （ ESR1）、原癌基因（ JUN ）、前列腺素氧化環(huán)化酶2 （ PTGS2）、半胱氨酸蛋白酶3 （CASP3）和信號轉導和轉錄激活因子3（STAT3）。生物信息學分析發(fā)現(xiàn)抑肝散治療AD與糖基化終末產(chǎn)物/糖基化終末產(chǎn)物受體和脂質和動脈粥樣硬化等信號通路有關。動物實驗結果顯示，抑肝散可改善AD模型小鼠學習記憶功能損傷，下調(diào)AD小鼠腦組織中IL-1β、IL-2、IL-6、TNF-α mRNA表達；細胞實驗結果顯示，抑肝散顯著緩解H₂O₂對Neuro-2a細胞的增殖抑制作用和凋亡誘導作用（P＜0.05），降低Caspase-3、Caspase-8活性（P＜0.05），降低細胞MDA和ROS水平（P＜0.05），升高MMP及GSH水平；抑肝散對H₂O₂誘導的RAW264.7細胞TNF-α和IL-1β、IL-2、IL-6升高具有顯著抑制作用（P＜0.05）。結論 抑肝散通過緩解學習記憶功能損傷和氧化損傷發(fā)揮治療AD作用。

Objective To explore the therapeutic effect of Yigansan on Alzheimer's disease (AD), to predict its mechanism through network pharmacology, and to carry out experimental verification. Methods The effects of Yigansan (400 and 800 mg·kg^-1) on the learning and memory function of AD mice were investigated in a D-galactose-induced AD mouse model, and the mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2) and interleukin-6 (IL-6) in the brain tissues of AD mice were analyzed by real-time quantitative PCR (qRT-PCR). Through the TCM Systematic Pharmacology Data Analysis Platform (TCMSP), the active ingredients of the herbal medicines Bupleuri Radix, Glycyrrhizae Radix et Rhizoma, Chuanxiong Rhizoma, Angelicae Sinensis Radix, Atractylodis Macrocephalae Rhizoma, Poria and Uncariae Ramulus cum Uncis were screened, and the AD targets were obtained by GeneCards and other databases, and the core targets were enriched by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The biological processes (BP), molecular function (MF) and cellular composition (CC) and KEGG signaling pathwayinvolved in the core targets were obtained, respectively. Mouse neuroblastoma Neuro-2a cells and RAW264.7 macrophages were co-cultured with H₂O₂ and hepatosuppress san (3 and 6 μg·mL^-1), respectively, to observe the effects of Yigansan on the proliferation and apoptosis of Neuro-2a cells in vitro, and the activity of Caspase-3 and Caspase-8, reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), effects on superoxide dismutase (SOD) and mitochondrial membrane potential (MMP). The effects of Yigansan on the expression of TNF-α, IL-1β, IL-2 and IL-6 in RAW264.7 cells were observed, and the network pharmacology results were verified. Results A total of 159 active ingredients were screened for the treatment of AD by network pharmacology technology, and the top 10 compounds were quercetin, kaempferol, isorhamnetin, β-sitosterol, stigmasterol, 7-methoxyisoflavone, mangopertin, naringenin, meditalpin, and licochalcone A, and 227 core targets were screened, and the top 10 core targets were serine/threonine kinase 1 (AKT1), tumor necrosis factor (TNF), interleukin 6 (IL6), tumor protein P53 (TP53), interleukin 1β (IL1B), estrogen receptor 1 (ESR), proto-oncogene (JUN), prostaglandin-endoperoxide synthase 2 (PTGS), caspase 3 (CASP3), signal transducer and activator of transcription 3 (STAT3). Bioinformatics analysis showed that the treatment of AD was related to glycosylation end products/glycation end product receptors and lipid and atherosclerosis signaling pathways. The results of animal experiments showed that Yigansan could improve the impairment of learning and memory function in AD model mice, and down-regulate the mRNA expression of IL-1β, IL-2, IL-6 and TNF-α in the brain tissues of AD mice. The results of cellular experiments showed that Yigansan significantly alleviated the proliferation inhibitory and apoptosis-inducing effects of H₂O₂ on Neuro-2a cells (P < 0.05), decreased the activities of Caspase-3 and Caspase-8 (P < 0.05), lowered the levels of cellular MDA and ROS (P < 0.05), and elevated the levels of MMP as well as GSH. Yigansan significantly suppressed the H₂O₂-induced elevation of TNF-α and IL-1β, IL-2 and IL-6 in RAW264.7 cells (P < 0.05). Conclusion Yigansan exerts therapeutic effects on AD by alleviating learning memory impairment and oxidative damage.

R285.5

天津市中醫(yī)藥重點領域科研項目（2022008）