基于网络药理学及实验验证探讨新加消渴方保护胰岛β细胞作用机制

Discussion on Mechanism of Xinjia Xiaoke Prescription in Treatment of PancreaticβCell Dysfunction Based on Network Pharmacology and Experimental Validation

目的采用网络药理学和实验验证方法探讨新加消渴方干预2型糖尿病、保护胰岛β细胞功能的可能机制。方法通过TCMSP、ETCM、化学专业数据库结合文献报道,筛选新加消渴方活性成分并预测其潜在靶点,建立靶点数据库;通过GeneCards、OMIM、DrugBank、TTD数据库筛选2型糖尿病、胰岛β细胞功能相关靶点;将药物靶点与疾病靶点取交集,采用Cytoscape3.8.2构建“中药-成分-作用靶点”网络并进行拓扑分析,通过STRING数据库构建潜在靶点蛋白相互作用(PPI)网络,将PPI信息导入Cytoscape3.8.2进行核心靶点提取,运用DAVID数据库对潜在作用靶点进行富集分析。采用高脂高糖饲料联合2%链脲佐菌素-柠檬酸缓冲液35 mg/kg腹腔注射建立2型糖尿病大鼠模型,将30只成模大鼠随机分为模型组、新加消渴方组、格列本脲组,每组10只,另取10只大鼠作为正常组。新加消渴方组大鼠予新加消渴方17 g/(kg•d)灌胃,格列本脲组予格列本脲混悬液0.23 mg/(kg•d)灌胃,模型组及正常组予1.9 mL生理盐水灌胃,连续5周。检测空腹血糖(FBG)、空腹胰岛素(FINS)、糖化血清蛋白(GSP)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6),计算胰岛素分泌指数(HOMA-β),HE染色观察胰岛病理形态学改变,检测胰腺组织胰岛素及TNF信号通路关键蛋白TNF-α、IL-6表达。结果筛选出新加消渴方有效成分272个,靶点709个,其中与2型糖尿病、胰岛β细胞功能交集靶点417个。分析得到PTGS2、PTGS1、GABRA1、CHRM1等关键靶点,槲皮素、大豆苷元、山柰酚、木犀草素等核心成分。KEGG通路富集分析结果涉及TNF信号通路、PI3K-Akt信号通路、凋亡等信号通路。与正常组比较,模型组大鼠FBG、GSP、TNF-α、IL-6升高,FINS、HOMA-β降低,胰腺组织TNF-α、IL-6蛋白表达升高(P<0.01);HE染色显示胰岛内细胞数量明显减少,形状不规则,间质增多,部分胰岛组织结构甚至完全萎缩、崩解。与模型组比较,新加消渴方组各项指标均明显改善(P<0.01);HE染色显示胰岛内细胞数量明显增多,胰岛呈完整团块状分布,且胰岛内细胞质较丰富。结论新加消渴方通过多成分、多靶点、多途径共同干预2型糖尿病,保护胰岛β细胞功能,可为其临床应用提供依据。

Objective To explore the possible mechanism of Xinjia Xiaoke Prescription intervening in type 2 diabetes mellitus and protecting pancreaticβcell function based on network pharmacology and experimental verification method.Methods TCMSP,ETCM,Chemistry Database combined with literature reports were used to retrieve the active components of Xinjia Xiaoke Prescription,and its potential targets were predicted and a target database was established.The related targets of type 2 diabetes mellitus and pancreaticβcell dysfunction were obtained by GeneCards,OMIM,DrugBank and TTD databases;taking the intersection of drugs targets and disease targets,Cytoscape 3.8.2 was used to construct the network of“Chinese materia medica-component-target”,and topological analysis was performed.The potential target protein-protein interaction(PPI)network was constructed through the STRING database,and the PPI information was imported into Cytoscape 3.8.2 for core target extraction.The DAVID database was used for enrichment analysis of potential targets.High-fat and high-sugar feed combined with intraperitoneal injection of 2%streptozotocin-citrate buffer 35 mg/kg were used to establish the type 2 diabetes mellitus rat model.Totally 30 successfully modeled rats were randomly divided into model group,Xinjia Xiaoke Prescription group,Glibenclamide group,with 10 rats in each group;and another 10 SD rats were set as the normal group.Xinjia Xiaoke Prescription group were given 17 g/(kg•d)Xinjia Xiaoke Prescription by gavage;the Glibenclamide group was given Glibenclamide suspension 0.23 mg/(kg•d)by gavage;the model group and the normal group were administered with 1.9 mL normal saline by gavage.Five weeks later,fasting blood glucose(FBG),fasting insulin(FINS),glycosylated serum protein(GSP),TNF-α,IL-6 were detected and HOMA-βwas calculated;HE staining was used to observe the pathological changes of pancreatic islets;the protein expressions of insulin and TNF-α,IL-6 in the pancreatic tissue were detected.Results Totally 272 effective components and 709 targets were screened out,among which,417 targets intersected with type 2 diabetes mellitus and pancreaticβcell function.The analysis obtained key targets such as PTGS2,PTGS1,GABRA1,CHRM1,and core components such as quercetin,daidzein,kaempferol,and luteolin.The results of KEGG enrichment analysis involved TNF signaling pathway,PI3K-Akt signaling pathway,apoptosis and other signaling pathways.Compared with the normal group,FBG,GSP,TNF-αand IL-6 increased,FINS and HOMA-βdecreased in the model group,and the protein expressions of TNF-αand IL-6 in the pancreatic tissue increased(P<0.01);HE staining showed that the number of cells in the islets was significantly reduced,the shape was irregular,the interstitium increased,and some of the islet tissue structures even completely atrophied and disintegrated.Compared with the model group,all indicators in the Xinjia Xiaoke Prescription group were significantly improved(P<0.01);HE staining showed that the number of cells in the islets significantly increased,the islets were distributed in a complete mass,and the cytoplasm in the islets was richer.Conclusion Xinjia Xiaoke Prescription can intervene in type 2 diabetes mellitus and protect the function of pancreaticβcells through multi-component,multi-target and multi-way,which can provide a basis for the clinical application of Xinjia Xiaoke Prescription.