基于网络药理学和实验验证探讨小柴胡汤治疗阿尔茨海默病的作用机制  

Mechanism of Xiao Chaihutang in Treatment of Alzheimer’s Disease:Based on Network Pharmacology and in vitro Experimental Verification

作　　者：彭伟[1] 夏军 陈云慧[1,2] 淮文英 谢永美 田小平[1] 张天娥[1] PENG Wei;XIA Jun;CHEN Yun-hui;HUAI Wen-ying;XIE Yong-mei;TIAN Xiao-ping;ZHANG Tian-e(Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China;Sichuan University,Chengdu 610041,China)

机构地区：[1]成都中医药大学,成都611137 [2]四川大学,成都610041

出　　处：《中国实验方剂学杂志》2022年第5期169-177,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：成都中医药大学青年学者人才项目(QNXZ2019043);四川省科技厅国际合作与交流项目(2017HH0004);国家自然科学基金项目(81603537);四川省中医药管理局项目(2021MS464);四川省名中医工作室建设项目(川中医药办202120);成都中医药大学双一流建设项目。

摘　　要：目的:运用网络药理学结合生物信息学探讨小柴胡汤治疗阿尔茨海默病(AD)的潜在作用机制。方法:利用中药系统药理学数据库和分析平台(TCMSP)筛选小柴胡汤的可能活性化合物及其潜在治疗靶标,从基因表达综合数据库(GEO)中获取与AD相关的差异表达基因,取交集筛选出小柴胡汤可能的靶点;对交集靶点基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析;构建化合物—靶点网络、蛋白互作(PPI)网络,进行KEGG和GO富集分析。采用β淀粉样蛋白1-40(Aβ1-40)诱导PC12细胞制备AD细胞模型,以小柴胡汤干预处理后,细胞增殖与活性检测(CCK-8)法检测细胞活率,4’,6-二脒基-2-苯基吲哚(DAPI)染色观察细胞形态、细胞膜电位检测、流式细胞术检测细胞凋亡、细胞荧光免疫检测B细胞淋巴瘤-2(Bcl-2)/Bcl-2相关X蛋白(Bax)的表达,初步验证网络药理学预测结果。结果:小柴胡汤共收集到190个化学成分和41个AD相关靶点,包括丁子香萜,槲皮素,小檗碱,原卟啉,24-Ethylcholest-4-en-3-one,β-D-呋喃核糖苷等关键化合物及Sigma非阿片类细胞内受体1(SIGMAR1),细胞周期检测点激酶1(CHEK1),非受体型蛋白酪氨酸磷酸酶6(PTPN6),蛋白激酶C(PRKCH),核转录因子κB激酶亚单位β抑制蛋白(IKBKB),组织蛋白酶D(CTSD),半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3),Bax,Bcl-2样蛋白1(Bcl-2L1)等核心靶点。GO富集分析获得302个条目(P<0.05),主要与调控神经元细胞凋亡相关;KEGG通路富集获得相关通路73条(P<0.05),涉及的主要通路和生物过程包括细胞凋亡通路、脂质和动脉粥样硬化相关通路、癌症相关通路等。细胞实验表明,与空白组比较,模型组细胞活率降低(P<0.05),凋亡率升高(P<0.05)、线粒体膜电位降低,Bcl-2/Bax下调;与模型组比较,小柴胡汤组细胞活率升高(P<0.05),细胞凋亡率降低(P<0.05)、线粒体膜电位升高,Bcl-2/Bax上调,验证了预测结果的可靠性。结论Objective: To investigate the potential mechanism of Xiao Chaihutang(XCHT)in the treatment of Alzheimer’s disease(AD)based on network pharmacology and bioinformatics. Method: The active components of XCHT and corresponding targets were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and the differentially expressed genes related to AD were searched from Gene Expression Omnibus(GEO). Thereby,the common targets of XCHT and AD were yielded,followed by Gene Ontology(GO)term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of the common targets. The component-target network and protein– protein interaction(PPI)network were constructed. Furthermore,amyloid β-protein(Aβ)1-40 was used to induce AD in PC12 cells and then the AD cells were intervened with XCHT. Afterward,cell viability was detected by Cell Counting Kit-8(CCK-8)assay and cell morphology was observed based on 4’,6-diamidino-2-phenylindole(DAPI)staining. Cell membrane potential was determined and apoptosis was detected by flow cytometry,and cellular immunofluorescence detects the expression of B-cell lymphoma-2(Bcl-2)/Bcl-2-related X protein(Bax). Moreover, immunofluorescence assay was performed. Result: A total of 190 active components and 41 anti-AD targets of XCHT were screened out. The key components included mairin,quercetin,berberine,protoporphyrin,24-ethylcholest-4-en-3-one,and β-D-ribofuranoside,and the core targets were sigma non-opioid intracellular receptor 1(SIGMAR1),checkpoint kinase 1(CHEK1),protein tyrosine phosphatase non-receptor type 6(PTPN6),protein kinase C(PRKCH),inhibitor of nuclear factor kappa B kinase subunit beta(IKBKB),cathepsin D(CTSD),cysteine aspartate protease-3(Caspase-3),Bax,and Bcl-2-like protein 1(Bcl-2 L1). The anti-AD targets of XCHT were involved in 302 GO terms(P < 0.05),particularly the regulation of neuronal cell apoptosis,and 73 KEGG pathways(P<0.05). The major pathways and biological processes included the apoptosis

关 键 词：小柴胡汤 阿尔茨海默病(AD) 作用机制 网络药理学 

分 类 号：R2-0[医药卫生—中医学] R22