基于网络药理学和分子对接探讨六味地黄丸异病同治老年性聋和阿尔茨海默病的作用机制

Study on one treatment for multiple diseases mechanism of Liuwei Dihuang Pills in treating presbycusis and Alzheimer's disease based on network pharmacology and molecular docking

目的借助网络药理学和分子对接的方法探讨六味地黄丸异病同治老年性聋和阿尔茨海默病(AD)的共同分子机制,并采用细胞实验进行验证。方法利用中药系统药理学数据库与分析平台(TCMSP)、成分靶点数据库(SwissADME)获取六味地黄丸有效成分,并结合基因和化学物质相互作用预测数据库(STITCH)获取作用靶点。利用人类基因综合数据库(Genecard)、人类疾病相关基因与突变位点信息数据库(DisGeNET)获取老年性聋和AD靶点。使用Cytoscape 3.7.1软件构建“中药-成分-靶点-疾病”网络,借助蛋白互作网络分析数据库(STRING)构建蛋白-蛋白相互作用(PPI)网络,获取核心靶点。利用富集分析数据平台(Metascape)对共有靶点进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。采用AutoDock Vina 1.1.2软件对核心靶点与相关成分进行分子对接。采用过氧化氢(H2O2)致PC12细胞氧化损伤模型,观察六味地黄丸的作用。采用细胞计数(CCK-8)法观察细胞存活率,活性氧(ROS)检测试剂盒检测ROS水平,酶联免疫吸附测定(ELISA)法检测肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平,Western blot法检测基质金属蛋白酶-9(MMP-9)、蛋白激酶B(AKT)、磷酸化-蛋白激酶B(p-AKT)蛋白表达。结果共获得六味地黄丸有效成分86种,主要来自薯蓣皂素、视黄醇、丹皮酚、软脂酸等化合物,六味地黄丸防治老年性聋和AD共有靶点49个,核心靶点为AKT1、TNF、IL-6、MMP-9等。GO分析表明这些共有靶点主要参与对氧化应激的反应、对脂多糖的反应等生物进程。KEGG通路分析显示,共有靶点主要涉及缺氧诱导因子-1(HIF-1)、核因子-κB(NF-κB)等信号通路。分子对接结果显示,TNF-α、IL-6、AKT1、MMP-9与相关成分均存在结合可能。细胞实验结果发现,六味地黄丸含药血清可以增强细胞存活率,降低细胞ROS、TNF-α、IL-6水平,增加MMP-9蛋白表达水平,降低p-AKT蛋白表达水平。结论六味地黄丸异病同治老年性聋和AD具有物质基础,其共同的分子机制,可能是通过薯蓣皂素、视黄醇、丹皮酚等多种成分,作用于AKT、TNF-α、IL-6、MMP-9等核心靶点,抑制炎症反应和氧化应激,减少神经元死亡。

Objective To explore the common molecular mechanism behind the effectiveness of Liuwei Dihuang Pills(LWDH)in treating presbycusis and Alzheimer's disease(AD)through the methodologies of network pharmacology and molecular docking,complemented by in vitro experimental validation.Methods Active compounds of LWDH were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and SwissADME.Interaction targets were obtained from search tool for interacting chemicals(STITCH),while disease-specific targets for presbycusis and AD were sourced from Genecard and DisGeNET,respectively.Cytoscape 3.7.1 facilitated the generation of a"herb-compound-target-disease"network,with the search tool for the retrieval of interacting genes/proteins(STRING)database constructing the protein-protein interaction(PPI)network to pinpoint core targets.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were conducted via Metascape.Core targets and related compounds underwent molecular docking using AutoDock Vina 1.1.2.An oxidative damage model in PC12 cells induced by H2O2 was used to assessed the therapeutic effects of LWDH.Cell viability was assessed using the cell counting kit-8(CCK-8)assay,while levels of reactive oxygen species(ROS)were measured with a ROS assay kit.Tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)levels were determined by enzyme-linked immunosorbent assay(ELISA).The expression of matrix metalloproteinase-9(MMP-9),protein kinase B(AKT),and phosphorylated protein kinase B(p-AKT)was analyzed by Western blot.Results Eighty-six active components of LWDH were identified,predominantly derived from diosgenin,retinol,paeonol,and palmitic acid and other compounds.Forty-nine common targets for treating presbycusis and AD were found,with AKT1,TNF,IL-6,and MMP-9 identified as core targets.The GO enrichment analysis indicated that these targets were primarily involved in biological processes such as oxidative stress response and lipopolysaccharide response.The KEGG pathway analysis showed that these targets were mainly involved in HIF-1 and NF-κB signaling pathways.The results of molecular docking confirmed the potential binding between core targets(TNF,IL-6,AKT,and MMP-9)and related compounds.In vitro experiments demonstrated that LWDH-containing serum could enhance PC12 cell viability,reduce ROS,TNF-α,and IL-6 levels,increase MMP-9 expression,and decrease p-AKT expression level.Conclusions The use of LWDH for presbycusis and AD treatment,as an example of one treatment for multiple diseases,has a molecular basis.Its common molecular mechanism may act on core targets such as AKT,TNF-α,IL-6,and MMP-9 through various components such as diosgenin,retinol and paeonol,inhibiting inflammatory responses and oxidative stress,thereby decreasing neuronal death.