【摘要】 目的 基于血清药物化学及蛋白芯片技术探讨加味六君子汤治疗慢性萎缩性胃炎的作用机制。方法（1）将Wistar大鼠随机分成空白对照组、加味六君子汤组(43.36 g·kg^-1)，每日灌胃给药2次，持续7 d，制备加味六君子汤含药血清及空白血清。基于血清药物化学方法，采用超高效液相色谱-四极杆飞行时间质谱（UHPLC-Q-TOF-MS/MS）分析技术，并结合文献报道和数据库信息，确认加味六君子汤的入血成分。（2）使用PharmMapper网站进行入血成分作用靶点预测；利用GeneCards、OMIM、PharmGkb、TTD和DrugBank数据库筛选慢性萎缩性胃炎疾病相关靶点；将上述靶点输入韦恩图制作平台，所得交集靶点即为加味六君子汤治疗慢性萎缩性胃炎的潜在作用靶点。通过STRING数据平台对潜在作用靶点进行蛋白互作（PPI）网络构建及核心靶点筛选。通过Cytoscape软件导入加味六君子汤入血成分、潜在作用靶点，构建“入血成分-靶点”网络，筛选核心成分。通过DAVID数据库对潜在作用靶点进行GO功能及KEGG通路富集分析。利用AutoDock Vina软件对核心成分和核心靶点进行分子对接验证。（3）采用CCK-8法检测GES-1细胞活性，筛选1-甲基-3-硝基-1-亚硝基胍（MNNG）最合适的造模浓度及加味六君子汤含药血清的最佳给药浓度。采用40μmol·L^-1 MNNG干预GES-1细胞，构建慢性萎缩性胃炎细胞模型，同时以20%含药血清干预24 h后，进行CSP100 plus磷酸化抗体芯片检测。结果 （1）共鉴定出加味六君子汤入血成分23种。得到入血成分对应的作用靶点424个，慢性萎缩性胃炎疾病相关靶点1 028个，取交集得到加味六君子汤治疗慢性萎缩性胃炎的潜在作用靶点109个。筛选得到加味六君子汤治疗慢性萎缩性胃炎的核心靶点：TP53、IL6、TNF、SRC、EGFR、AKT1、CTNNB1、MMP9、CASP3、MAPK8，以及核心成分：甘草酸、DL-精氨酸、芸香柚皮苷、大豆素、茯苓酸G、人参皂苷Rg2、人参皂苷Ro、香风草甙、芹糖异甘草苷、人参皂苷Rb1。潜在作用靶点涉及的生物过程主要有对氧化应激的反应、脂多糖反应、细胞来源分子反应、凋亡信号通路等；KEGG通路主要涉及病毒、癌症、炎症等方面，主要通路包括PI3K/AKT信号通路、TNF信号通路、IL-17信号通路、MAPK信号通路等。核心成分与核心靶点的100组分子对接结果中，结合能≤-5 kcal·mol^-1的对接组合有93组，结合能≤-7 kcal·mol^-1的对接组合有73组。（2）与模型组比较，经含药血清干预后，共有49个磷酸化抗体和46个非磷酸化抗体发生了明显变化。其中，在PI3K/AKT信号通路中有18个磷酸化抗体和11个非磷酸化抗体发生了显著变化；在MAPK信号通路中有13个磷酸化抗体和17个非磷酸化抗体发生了显著变化。结论 加味六君子汤可能通过甘草酸、人参皂苷Rg2、大豆素等入血成分，调控PI3K/AKT、MAPK关键信号通路，发挥治疗慢性萎缩性胃炎并延缓其向胃癌转变的作用。更多还原

【Abstract】 Objective To explore the mechanism of Modified Liujunzi Decoction in the treatment of chronic atrophic gastritis based on serum pharmacochemistry and protein chip technology.Methods （1）Wistar rats were randomly divided into blank control group and Modified Liujunzi Decoction group (43.36 g·kg^-1),intragastric administration was given two times a day for consecutive 7 days to prepare Modified Liujunzi Decoction containing serum and blank serum.Based on the serum pharmacochemistry method,the ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry （UHPLC-Q-TOF-MS/MS） technology combined with literature reports and database information was used to confirm the constituents absorbed into blood of Modified Liujunzi Decoction.（2） The targets of the constituents absorbed into blood were predicted using Pharm Mapper website.Disease targets related to chronic atrophic gastritis were screened using Gene Cards,OMIM,Pharm Gkb,TDD,and Drug Bank databases.The above targets were input into the Venn diagram production platform,and the obtained intersection targets were the potential targets of Modified Liujunzi Decoction in the treatment of chronic atrophic gastritis.Protein-protein interaction （PPI） network construction and core target screening of potential targets were performed by STRING data platform.The constituents absorbed into blood and potential targets of Modified Liujunzi Decoction were introduced by Cytoscape software,and the‘constituents absorbed into blood-targets’network was constructed to screen the core components.GO function and KEGG signaling pathway enrichment analysis of potential targets were performed by DAVID database.Auto Dock Vina software was used to verify the molecular docking of core components and core targets.（3） The CCK-8method was used to detect the activity of GES-1 cells,and the most suitable modeling concentration of 1-methyl-3-nitro-1-nitrosoguanidine （MNNG） and the optimal administration concentration of Modified Liujunzi Decoction containing serum were screened.GES-1 cells were treated with 40μmol·L^-1 MNNG to construct a cell model of chronic atrophic gastritis.At the same time,after 24 hours of intervention with 20%drug-containing serum,CSP100 plus phosphorylated antibody chip detection was performed.Results （1） A total of 23 constituents absorbed into blood of Modified Liujunzi Decoction were identified.A total of 424 targets corresponding to constituents absorbed into blood and1 028 targets related to chronic atrophic gastritis were obtained,and 109 potential targets of Modified Liujunzi Decoction in the treatment of chronic atrophic gastritis were obtained.The core targets of Modified Liujunzi Decoction in the treatment of chronic atrophic gastritis were screened:TP53,IL6,TNF,SRC,EGFR,AKT1,CTNNB1,MMP9,CASP3,MAPK8,and core components:glycyrrhizic acid,DL-arginine,narirutin,daidzein,pachymic acid G,ginsenoside Rg2,ginsenoside Ro,vanillin,apioside,ginsenoside Rb1.The biological processes involved in the potential targets mainly include the response to oxidative stress,lipopolysaccharide response,cell-derived molecular response,apoptosis signaling pathway,etc.The KEGG pathway mainly involves viruses,cancer,inflammation,etc.The main pathways include PI3K/AKT signaling pathway,TNF signaling pathway,IL-17 signaling pathway,MAPK signaling pathway,etc.Among the 100 groups of molecular docking results of core components and core targets,there were 93 groups of docking combinations with binding energy≤-5 kcal·mol^-1 and 73 groups of docking combinations with binding energy≤-7 kcal·mol^-1.（2） Compared with the model group,a total of 49 phosphorylated antibodies and 46 non-phosphorylated antibodies changed significantly after medicated serum intervention.Among them,18 phosphorylated antibodies and 11 non-phosphorylated antibodies were significantly changed in the PI3K/AKT signaling pathway;in the MAPK signaling pathway,13 phosphorylated antibodies and 17 non-phosphorylated antibodies were significantly changed.Conclusion Modified Liujunzi Decoction may regulate the key signaling pathways of PI3K/AKT and MAPK through constituents absorbed into blood such as glycyrrhizic acid,ginsenoside Rg2 and daidzein,and play a role in treating chronic atrophic gastritis and delaying its transformation to gastric cancer.更多还原