参麦饮改善糖尿病肾病的网络药理学研究及实验验证

Network Pharmacology and Experimental Study on the Mechanism of Shenmai Decoction on Improving Diabetic Nephropathy

目的:利用网络药理学技术结合动物实验验证,探讨参麦饮改善糖尿病肾病(Diabetic Nephropathy,DN)可能的作用靶点与作用机制。方法:利用中药系统药理学分析平台数据库(TCMSP)、中医百科全书平台(ETCM)收集参麦饮中红参、麦冬的化学成分。通过整合药物靶标数据库(TTD)、Drugbank数据库、疾病基因与突变位点数据库(DisGeNET)筛选出DN相关的靶标建立靶点信息。运用蛋白质相互作用网络数据库(STRING)平台构建靶蛋白互作网络(PPI),将PPI网络导入Cytoscape软件进行网络拓扑结构分析,筛选出关键靶点,利用功能注释生物信息学分析平台(DAVID)数据库对关键靶点进行基因本体论(GO)富集分析及京都基因与基因组百科全书(KEGG)通路富集分析;运用Cytoscape 3.2.1软件构建化合物-靶点-通路网络。造模糖尿病肾病大鼠模型,参麦饮干预6 w后检测大鼠的血糖(FBG)、24 h微量白蛋白(mAlb)、血尿素氮(BUN)、血肌酐(Scr)。肾组织使用蛋白免疫印迹法(Western blot)验证预测信号通路。结果:利用网络合并及网络拓扑结构分析得到关键靶点15个,并对关键靶点进行基因GO功能分析及KEGG通路富集分析,预测出Akt1、VEGFA、MAPK3等为关键靶点,VEGF信号通路、TNF信号通路、PI3K/Akt信号通路等为关键通路。与正常对照组比较,模型对照组大鼠空腹FBG、mAlb、Scr、BUN、NF-κB蛋白表达水平显著升高(P<0.05或P<0.01),Akt蛋白表达水平明显下调(P<0.01);与模型对照组比较,参麦饮1.1 g/kg组、5.4 g/kg组的大鼠空腹FBG、mAlb、Scr、BUN值明显降低,NF-κB蛋白表达明显下调(P<0.05或P<0.01),Akt蛋白表达水平明显上调或有上调趋势(P<0.05)。结论:参麦饮具有多成分、多靶点、多途径的作用特点,能够改善糖尿病肾病的血糖、肾脏指标等,对肾脏有显著的保护作用,并对预测出的PI3K/Akt信号通路有一定的调控作用,为后续临床改善糖尿病肾病提供新的思路。

Objective:To screen the possible active targets and explore the mechanism of Shenmai Decoction treating diabetic nephropathy by network pharmacology combined with animal experiment.Methods:TCMSP and ETCM were used to retrieve the chemical constituents of Ginseng Radix et Rhizoma Rubra and Ophiopogonis Radix in Shenmai Decoction.TTD,Drugbank,DisGeNET were used to screen DN related targets to establish the targets information.The PPI network was constructed by STRING platform and was imported into into the Cytoscape software.Network topology Analysis was used to select key targets,DAVID database was used to perform GO function enrichment analysis and KEGG pathway enrichment analysis of key target proteins.Cytoscape 3.2.1 software was used to build compound-target-pathway network.After diabetic nephropathy rats were established,they were administered with Shenmai Decoction for 6 weeks,then the levels of fasting blood glucose(FBG),microalbuminuria(mAlb),blood urea nitrogen(BUN)and serum creatinine(Scr)were measured.Western blot was used on kidney tissues to predict the signal pathway.Results:15 key targets were obtained by network merger and topological analysis,and were analyzed by gene GO function and KEGG pathway enrichment.The results showed that Akt1、VEGFA、MAPK3,etc.,were key targets,and key signaling pathways were VEGF、TNFg pathway、PI3 K/Akt,etc.Compared with the control group,the expressions of FBG、mAlb、Scr、BUN、NF-κB protein were significantly increased(P<0.05 or P<0.01),the expression of Akt protein was significantly decreased(P<0.01)in the model group.Compared with the model group,the expressions of FBG、mAlb、Scr、BUN、NF-κB proteins were significantly decreased or has the trend of increased in Shenmai Decoction groups(1.1 g/kg and 5.4 g/kg)(P<0.05 or P<0.01),the expression of Akt protein was significantly increased(P<0.05 or P<0.01).Conclusion:Shenmai Decoction has the characteristics of multi-component,multi-target and multi-channel synergistic effects,which may improve the blood glucose and nephropathy index,to protect the nephropathy significantly.It also has the regulation effect on PI3 K-Akt signaling pathway,which provides new ideas for the following clinical treatment of diabetic nephropathy.