基于网络药理学和实验验证探讨白虎加人参汤治疗2型糖尿病的分子机制

Study on the Molecular Mechanism of Baihu and Renshen Decoction in the Treatment of Type 2 Diabetes Based on Network Pharmacology and Experimental Verification

目的 通过网络药理学方法全面系统筛选白虎加人参汤治疗2型糖尿病(T2DM)的治疗靶点及相关信号通路,并通过动物实验进一步明确其作用机制。方法 应用TCMSP、TCMID和PubChem数据库获取白虎加人参汤的化学活性成分及相关靶点信息,在GEO数据库获得影响T2DM的差异基因,并与白虎人参汤靶点交集获取共同靶点;运用STRING V11.0数据库进行蛋白互作分析并获取蛋白互作网络图及核心靶点;通过DAVID 6.8分析平台,获得GO功能注释结果及KEGG通路富集结果,分析通路相关基因及功能,并通过实验进行验证。选取MKR小鼠24只,随机分为模型组、低剂量白虎加人参汤组、高剂量白虎加人参汤组,各8只,另选取8只同龄同性别FVB小鼠作为对照组。各组给药4周后,测定小鼠空腹血糖(FBG),采用酶联免疫吸附测定(ELISA)检测小鼠血清中白细胞介素-6(IL-6)、IL-1β、肿瘤坏死因子-α(TNF-α)水平,蛋白免疫印迹法(Western blot)检测PI3K、AKT、NF-κB的蛋白表达,实时荧光定量聚合酶链式反应(RTqPCR)检测PI3K、AKT、NF-κB的mRNA相对表达量。结果 筛选得到白虎加人参汤中101种潜在成分和70个共同靶点,成分-靶点网络图显示白虎加人参汤可能通过VEGFA、FYN、AR、ABL1等蛋白靶点,经由磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)信号通路、丝裂原活化蛋白激酶(MAPK)等信号通路发挥治疗T2DM的作用。动物实验结果显示,与模型组比较,白虎加人参汤降低MKR小鼠空腹血糖及血清中IL-6、IL-1β及TNF-α水平(P<0.05,P<0.01),下调PI3K/AKT信号通路上的PI3K、AKT、NF-κB蛋白表达及其mRNA相对表达量(P<0.01)。结论 白虎加人参汤可以通过多成分、多靶点、多通路的作用效应治疗T2DM,实验证实了白虎加人参汤能够下调PI3K/AKT通路相关蛋白表达及mRNA相对表达量,降低MKR小鼠血糖和血清炎症因子水平,验证了网络药理学结果,为深入研究白虎加人参汤治疗T2DM提供了依据。

Objective To explore the targets and relevant signaling pathways of Baihu and Renshen Decoction in the treatment of type 2 diabetes through network pharmacology,and to apply animal experiments to further define its mechanism of action.Methods The TCMSP database,TCMID database and PubChem database were used to filter the chemical composition and target of Baihu and Renshen Decoction.The differential genes that affect type II diabetes was searched in the GEO database,and intersect with the related target of Baihu and Renshen Decoction to obtain common target.Protein interaction network was drawn through the String database,and the key targets were selected according to their degrees.Through David 6.8 analysis platform,go function annotation results and KEGG pathway enrichment results were obtained,and pathway related genes and functions were analyzed and verified by experiments.Twenty-four MKR mice were randomly divided into model group,low-dose Baihu and Renshen Decoction group and high-dose Baihu and Renshen Decoction group with 8 mice in each group. Eight FVB mice of the same age were selected as controlgroup. After 4 weeks of administration, fasting blood glucose (FBG), was measured. The levels of interleukin-6 (IL-6),IL-1β and tumor necrosis factor-α (TNF-α) in serum were detected by ELISA method. The protein expressions of PI3K,AKT and NF-κB were detected by Western blot, and the relative mRNA expressions of PI3K, AKT and NF-κB weredetected by RT-qPCR. Results 101 chemical components of Baihu and Renshen Decoction and 70 common targets wereobtained. The network analysis indicated that Baihu and Renshen Decoction can treat T2DM by regulating VEGFA,FYN and AR targets, involving phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway, mitogenactivatedprotein kinase (MAPK) signaling pathway. The results of animal experiment showed that compared with themodel group, Baihu and Renshen Decoction decreased fasting blood glucose and serum IL-6, IL-1β and TNF-α levelsin MKR mice (P<0.05, P<0.01), and down-regulated PI3K, AKT, NF- κB protein expression and mRNA relativeexpression in PI3K/ AKT signal pathway (P<0.01). Conclusion Baihu and Renshen Decoction can treat type 2 diabetesthrough the effects of multi-components, multi-targets and multi-pathways. The experiment confirmed that Baihu andRenshen Decoction can down-regulate the expression of proteins related to PI3K/AKT pathway and the relativeexpression of mRNA, and reduce the levels of blood glucose and serum inflammatory factors in MKR mice, whichverifies the results of network pharmacology and provides a basis for further study of Baihu and Renshen Decoction inthe treatment of type 2 diabetes.