抑肝散治疗阿尔茨海默病的作用及机制探讨

Treatment and mechanism of Yigansan on Alzheimer's disease

目的探究抑肝散对阿尔茨海默病(AD)的治疗作用,通过网络药理学方法预测其机制,并进行实验验证。方法以D-半乳糖诱导AD小鼠模型,通过水迷宫和八臂迷宫实验考察抑肝散(400、800 mg·kg^(-1))对AD小鼠学习记忆功能的影响,并通过实时荧光定量PCR(qRT-PCR)法分析AD小鼠脑组织中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)的mRNA表达变化。通过中药系统药理学数据分析平台(TCMSP)对抑肝散的组方药材柴胡、甘草、川芎、当归、白术、茯苓和钩藤进行活性成分筛选,采用GeneCards等数据库获取AD疾病靶点,筛选其核心靶点,并对核心靶点进行基因本体(GO)和京都基因与基因组百科全书(KEGG)通路富集分析,分别获取核心靶点涉及的生物过程(BP)、分子功能(MF)和细胞组分(CC)以及KEGG信号通路。小鼠神经母细胞瘤Neuro-2a细胞和RAW264.7巨噬细胞分别与H_(2)O_(2)和抑肝散(3、6μg·mL^(-1))共培养,观察抑肝散对Neuro-2a细胞体外增殖和凋亡的影响,及对Neuro-2a细胞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)和线粒体膜电位(MMP)的影响;观察抑肝散对RAW264.7细胞TNF-α和IL-1β、IL-2、IL-6表达的影响,对网络药理学结果进行验证。结果抑肝散可缓解AD小鼠学习记忆功能损伤,通过网络药理学技术筛选得到抑肝散治疗AD的活性成分159个,度值前10位的化合物分别是槲皮素、山柰酚、异鼠李素、β-谷甾醇、豆甾醇、7-甲氧基异黄酮、芒柄花素、柚皮素、美迪紫檀素和甘草查尔酮A,筛选得到核心靶点227个,度值排名前10的核心靶点为丝氨酸/苏氨酸蛋白激酶1(AKT1)、肿瘤坏死因子(TNF)、白细胞介素6(IL6)、肿瘤蛋白P53(TP53)、白细胞介素1β(IL1B)、雌激素受体1(ESR1)、原癌基因(JUN)、前列腺素氧化环化酶2(PTGS2)、半胱氨酸蛋白酶3(CASP3)和信号转导和转录激活因子3(STAT3)。生物信息学分析发现抑肝散治疗AD与糖基化终末产物/糖基化终末产物受体和脂质和动脉粥样硬化等信号通路有关。动物实验结果显示,抑肝散可改善AD模型小鼠学习记忆功能损伤,下调AD小鼠脑组织中IL-1β、IL-2、IL-6、TNF-αmRNA表达;细胞实验结果显示,抑肝散显著缓解H_(2)O_(2)对Neuro-2a细胞的增殖抑制作用和凋亡诱导作用(P<0.05),降低Caspase-3、Caspase-8活性(P<0.05),降低细胞MDA和ROS水平(P<0.05),升高MMP及GSH水平;抑肝散对H_(2)O_(2)诱导的RAW264.7细胞TNF-α和IL-1β、IL-2、IL-6升高具有显著抑制作用(P<0.05)。结论抑肝散通过缓解学习记忆功能损伤和氧化损伤发挥治疗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 H_(2)O_(2)and hepatosuppress san(3 and 6μg·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-αin the brain tissues of AD mice.The results of cellular experiments showed that Yigansan significantly alleviated the proliferation inhibitory and apoptosis-inducing effects of H_(2)O_(2)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 H_(2)O_(2)-induced elevation of TNF-αand 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.