白虎加人参汤治疗肥胖症合并2型糖尿病的潜在活性成分及作用机制分析

Analysis of Potential Active Ingredients and Mechanism of Baihu Jia Renshentang in Treatment of Obesity Complicated with Type 2 Diabetes Mellitus

目的:通过网络药理学结合体内实验验证,探讨白虎加人参汤治疗肥胖症合并2型糖尿病(T2DM)的潜在活性成分和作用靶点。方法:利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法(UPLC-Q-Exactive Orbitrap MS)分析鉴定白虎加人参汤物质基础。在ChEMBL、疗效药靶数据库(TTD)、YaTCM、DisGeNET和中药免疫肿瘤学(TCMIO)等数据库中查询活性成分作用的潜在靶点,并将这些靶点与疾病靶点取交集确定共同靶点。将共同靶点导入STRING数据库构建蛋白质-蛋白质相互作用(PPI)网络,通过cytoHubba插件确定hub基因,利用分子对接验证hub基因与白虎加人参汤中生物活性成分的结合能。同时,将共同靶点导入DAVID平台进行基因本体(GO)功能注释及京都基因和基因组百科全书(KEGG)通路富集分析。通过动物实验验证预测结果,将18只8周龄雄性骨骼肌胰岛素样生长因子-1受体功能缺失(MKR)小鼠高脂饮食饲养12周,制备肥胖症合并T2DM小鼠模型。随机将小鼠均分为模型组、二甲双胍组(0.2 g·kg^(-1))、白虎加人参汤组(以生药计27 g·kg^(-1)),另设6只同龄雄性FVB小鼠作为正常组,各组小鼠给予相应药物灌胃,正常组及模型组灌胃等量蒸馏水,1次/d,连续6周。给药结束后,检测各组小鼠的体质量、Lee's指数、空腹血糖(FBG)、口服糖耐量试验(OGTT),观察附睾白色脂肪组织病理形态,并采用实时荧光定量聚合酶链式反应(Real-time PCR)检测附睾白色脂肪组织中hub基因mRNA的表达水平。结果:共鉴定出白虎加人参汤200种化合物,其中64种生物活性成分反向匹配到384个靶点,与肥胖症合并T2DM相关的靶点共308个。hub基因包括丝裂原活化蛋白激酶1(MAPK1)、信号转导与转录激活因子3(STAT3)、MAPK3、白细胞介素(IL)-2、Janus酪氨酸蛋白激酶1(JAK1)、核转录因子-κB p65(RELA)、雌激素受体1(ESR1)、转录因子AP-1(JUN)、MAPK14和淋巴细胞特异性蛋白酪氨酸激酶(LCK)。GO功能注释结果显示,主要富集于细胞内的胞质、胞膜和胞核等成分,且与肽丝氨酸磷酸化、蛋白质磷酸化、炎症反应等重要生物过程密切相关。在KEGG富集分析中,代谢途径、脂质与动脉粥样硬化、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)和MAPK等信号通路被明显富集。分子对接结果显示,hub基因与白虎加人参汤中的槲皮素、异甘草素、桑黄素等10种生物活性成分具有稳定的结合关系。动物实验结果显示,白虎加人参汤能够显著降低肥胖症合并T2DM小鼠的体质量、Lee's指数和FBG水平(P<0.01),并能够改善附睾白色脂肪组织的病理变化,下调附睾白色脂肪组织中hub基因的mRNA表达(P<0.01)。结论:该研究通过网络药理学及动物实验验证确定了白虎加人参汤中槲皮素、异甘草素、桑黄素等10种潜在活性成分,可能通过调节代谢途径、脂质与动脉粥样硬化、PI3K/Akt和MAPK等信号通路治疗肥胖症合并T2DM,为其机制与临床研究提供了重要的线索和理论基础。

Objective:To investigate the potential active ingredients and targets of Baihu Jia Renshentang(BHJRST)for the treatment of obesity combined with type 2 diabetes mellitus(T2DM)by network pharmacology and in vivo experiments.Method:Ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry(UPLC-Q-Exactive Orbitrap MS)was used to analyze and identify the material basis of BHJRST.Subsequently,potential targets for the action of the active ingredients were queried in databases such as ChEMBL,Therapeutic Target Database(TTD),YaTCM,DisGeNET and Traditional Chinese Medicine on Immuno-Oncology(TCMIO),and the shared targets were identified by taking the intersection of these targets with disease targets.The shared targets were imported into the STRING database to construct a protein-protein interaction(PPI)network,the hub genes were identified by cytoHubba plug-in,and molecular docking was used to validate the binding energy of the hub genes to the bioactive ingredients in BHJRST.Meanwhile,the shared targets were imported into the DAVID platform for gene ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The predicted results were subsequently verified by animal experiments.Eighteen 8-weekold male skeletal muscle insulin-like growth factor-1 receptor dysfunction(MKR)mice were induced by a highfat diet for 12 weeks in order to prepare a mouse model of obesity combined with T2DM.The mice were randomly divided into the model group,metformin group(0.2 g·kg^(-1))and BHJRST group(27 g·kg^(-1) in raw material),and another 6 male FVB mice of the same age as the normal group.The mice in each group were were given the corresponding drugs by gavage,and the normal and model groups were given the same amount of distilled water by gavage,1 time/d for 6 consecutive weeks.At the end of administration,the body mass,Lee's index,fasting blood glucose(FBG),oral glucose tolerance tes(t OGTT)of mice in each group were examined,and the pathological morphology of the white adipose tissue of the epididymis was observed,and the expression of the mRNA of the hub genes in the white adipose tissue of the epididymis was detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Result:A total of 200 bioactive components of BHJRST were identified,of which 64 bioactive components were reverse-matched to 384 targets,and a total of 308 targets were associated with obesity combined with T2DM.Hub genes included mitogen-activated protein kinase 1(MAPK1),signal transducer and activator of transcription 3(STAT3),MAPK3,interleukin(IL)-2,Janus kinase 1(JAK1),nuclear transcription factor-κB p65(RELA),estrogen receptor 1(ESR1),transcription factor AP-1(JUN),MAPK14 and lymphocyte-specific protein tyrosine kinase(LCK).GO functional annotation showed that it was mainly enriched in cytoplasm,cell membrane and nucleus,and was closely related to important biological processes such as peptide serine phosphorylation,protein phosphorylation and inflammation.In KEGG enrichment analysis,metabolic pathway,lipid and atherosclerosis,phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)and MAPK signal pathways were significantly enriched.The molecular docking results showed that the hub genes had a stable binding relationship with 10 bioactive components,including quercetin,isoliquiritigenin,and morin,in BHJRST.The results of animal experiments showed that BHJRST could significantly reduce body mass,Lee's index and FBG levels(P<0.01)in mice with obesity combined with T2DM,improve the pathological changes of white adipose tissue,and down-regulate the the mRNA expression of the hub genes in white adipose tissue of the epididymis(P<0.01).Conclusion:In this study,10 potentially active components such as quercetin,isoliquiritigenin,and morin in BHJRST are identified through network pharmacology and animal experiments,and it is possible to treat obesity combined with T2DM by regulating lipid and atherosclerosis,phosphatidylinositol PI3K/Akt and MAPK signal pathways,which provides important clues and theoretical basis for the study of its mechanism and clinical application.