基于血清药物化学和网络药理学的知母-黄柏药对盐炙前后治疗2型糖尿病药效物质基础及作用机制分析  

Analysis of Pharmacodynamic Material Basis and Action Mechanism of Anemarrhenae Rhizoma-Phellodendri Chinensis Herb Pair in the Treatment of Type 2 Diabetes before and after Salt Processing Based on Serum Pharmacochemistry and Network Pharmacology

作　　者：孟祥祺 张春玲 贾耀霞 雷锦杰 夏阳淼 谭睿[2] 余凌英[1] MENG Xiangqi;ZHANG Chunling;JIA Yaoxia;LEI Jinjie;XIA Yangmiao;TAN Rui;YU Lingying(School of Pharmacy,Chengdu University of Traditional Chinese Medicine,State Key Laboratory of Southwestern Chinese Medicine Resources,Chengdu 610075,China;School of Life Science and Engineering,Southwest Jiaotong University,Chengdu 610031,China)

机构地区：[1]成都中医药大学药学院/西南特色中药资源国家重点实验室,四川成都610075 [2]西南交通大学生命科学与工程学院,四川成都610031

出　　处：《南京中医药大学学报》2024年第5期479-493,共15页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家中医药管理局中医药创新团队及人才支持计划(ZYYCXTD-C-202208);国家重点研发计划资助“中医药现代化”重点专项(2022YFC3500200,2022YFC3500201);国家中医药管理局高水平中医药重点学科建设项目(国中医药人教函〔2023〕85号);江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：目的基于液质联用技术对生知母-生黄柏、盐知母-盐黄柏药对水提液灌胃给药后大鼠入血成分分析,结合网络药理学预测盐炙在知母-黄柏药对治疗2型糖尿病的影响,并通过体外实验进行初步验证。方法大鼠连续灌胃给药生知母-生黄柏药对、盐知母-盐黄柏药对水提液2次,中间间隔1 h,末次给药60 min后腹主动脉取血,用甲醇沉淀蛋白法处理后复溶,采用色谱柱Shim-pack GIST C 18(4.6 mm×150 mm,5μm);流动相A相为0.1%甲酸水,B相为0.1%甲酸-乙腈;梯度洗脱,正、负离子全扫描模式,质量扫描范围m/z 100~1500。结合数据库二级谱图及文献,分析鉴定生知母-生黄柏、盐知母-盐黄柏药对的入血成分。检索2型糖尿病疾病靶点,对入血成分和疾病的交集靶点进行蛋白质互作网络分析、GO、KEGG通路富集分析,构建“入血成分-靶点”网络图,运用AutoDock软件对筛选出的核心成分和核心靶点进行分子对接验证。验证实验以HepG2细胞为实验对象,胰岛素联合高糖诱导胰岛素抵抗模型,CCK8法检验盐炙前后知母-黄柏药对对细胞增殖影响,Western blot检测PI3K-AKT信号通路相关蛋白的表达情况。结果生知母-生黄柏药对水提液大鼠血清中鉴定出15种原型成分,1个芒果苷代谢成分。盐知母-盐黄柏药对水提液大鼠血清中鉴定出17个原型成分,1个芒果苷代谢成分。盐炙后入血成分中芒果苷、小檗碱、3-异丁基戊二酸等成分含量较生品高。KEGG和GO结果显示,知母-黄柏药对治疗2型糖尿病可能与RNA聚合酶的转录调控、炎症反应、AGE-RAGE、PI3K-AKT、胰岛素抵抗等通路有关。细胞实验表明盐炙前后知母-黄柏药对可以上调p-PI3K/PI3K、p-AKT/AKT、GLUT4蛋白表达,且盐炙组效果优于生品组。结论初步阐释了知母-黄柏药对盐炙前后入血成分,阿魏酸、小檗碱、小檗红碱、芒果苷与mTOR、SIRT1、EGFR、PPARA等可能是知母-黄柏药对盐炙后增OBJECTIVE To analyze the serum chemical composition of rats after intragastric administration of water extract of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis based on liquid chromatography-mass spectrometry(LC-MS)technology,predict the effect of salt processing on the treatment of type 2 diabetes in Anemarrhenae Rhizoma-Pheellodendri Chinensis combined with network pharmacology,and preliminarily verify it through in vitro experiments.METHODS Rats were continuously intragastrically administered with crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair water extract twice,with an interval of 1 h.After 60 min of the last administration,the blood was taken from the abdominal aorta,and the protein was precipitated by methanol.After dissolution,the chromatographic column was Shim-pack GIST C 18(4.6 mm×150 mm,5μm);the mobile phase A was 0.1%formic acid water,and the mobile phase B was 0.1%formic acid-acetonitrile;gradient elution,positive and negative ion full scan mode,mass scan range 100-1500 m/z.Combined with the secondary spectrum of the database and literature,the blood components of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair were analyzed and identified.The disease targets of type 2 diabetes were retrieved,and the protein interaction network analysis,GO and KEGG pathway enrichment analysis were performed on the intersection targets of blood components and diseases.The“blood components-targets”network diagram was constructed,and the selected core components and core targets were verified by molecular docking using AutoDock software.In the verification experiment,HepG2 cells were used as the experimental object,and the insulin resistance model was induced by high insulin and high glucose.CCK8 method was used to test the effect of Rhizoma Anemarrhenae-Phellodend

关 键 词：知母-黄柏药对 盐炙 血清药物化学 2型糖尿病 网络药理学 分子对接 细胞验证 

分 类 号：R285.5[医药卫生—中药学]