数据挖掘并验证人参总皂苷改善酒精性肝炎的机制

Data Mining and Experimental Validation of Total Ginsenosides Ameliorating Alcoholic Hepatitis

摘要:

目的 通过数据分析和实验验证探究人参总皂苷改善酒精性肝炎（AH）的新靶点和中药，为AH的临床治疗提供新方向。方法 该研究前期从GEO数据库筛选GSE28619作为分析对象，选择GSE83148和GSE103580作为验证集。基于limma包和加权基因共表达网络分析方法（WGCNA）识别AH相关差异基因和模块基因；通过“Venny”软件取交集基因。构建蛋白质-蛋白质相互作用（PPI）网络，并进行富集分析，再进一步筛选核心基因评估其诊断价值。经过数据集验证后，综合分析出AH新潜在靶点并预测中药。对靶点和中药有效成分进行分子对接，并通过实验验证。将48只SD大鼠随机分出8只为空白组，剩下进行乙醇灌胃造模成功后随机均分为人参总皂苷低（10 mg·kg^-1）、中（20 mg·kg^-1）、高（40 mg·kg^-1）剂量组、模型组和美他多辛组（117 mg·kg^-1），空白组以等量生理盐水灌胃。灌胃3周后取血清测定血清天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）水平；取肝组织进行苏木素-伊红（HE）染色、蛋白免疫印迹法（Western blot）和实时荧光定量聚合酶链式反应（Real-time PCR）检测肝组织潜在靶点蛋白和基因表达水平。结果 数据分析预测出潜在的原癌基因（FOS）、Ⅰ型胶原蛋白α2链基因（COL1A2），实验验证显示给药后肝脏损伤程度均较造模后减轻；血清AST、ALT水平在给药后降低；Western blot和Real-time PCR结果显示给药后FOS蛋白和基因表达水平均明显增高，COL1A2则相反。结论 人参总皂苷能通过FOS和COL1A2改善AH。

Abstract:

Objective To explore new targets and herbal medicines of total ginsenosides in ameliorating alcoholic hepatitis （AH） by data mining and experimental validation and to provide new directions for the clinical treatment of AH.Method GSE28619 was selected as the test set from the GEO database and GSE83148 and GSE103580 were selected as the validation sets. The limma package and weighted gene co-expression network analysis （WGCNA） were employed to identify the AH-related differentially expressed genes and modular genes， and Venny was used to extract the common genes. The protein-protein interaction （PPI） network was constructed and the enrichment analysis was carried out. The hub genes were further screened and evaluated for their diagnostic value. After validation with the datasets， new potential targets of AH and traditional Chinese medicine were predicted. Molecular docking between the targets and active ingredients of traditional Chinese medicine was performed， and the results were validated by experiments. Eight out of 48 SD rats were randomly selected into a blank group and received an equal amount of normal saline. The rest rats were subjected to modeling with ethanol by gavage and then randomized into low- （10 mg·kg^-1）， medium- （20 mg·kg^-1）， and high-dose （40 mg·kg^-1） total ginsenosides， model， and positive control （metadoxine， 117 mg·kg^-1） groups. After 3 weeks of gavage， serum samples were collected for the measurement of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） levels， and liver samples were collected for hematoxylin-eosin （HE） staining. Western blot and Real-time PCR were employed to determine the protein and mRNA levels， respectively， of potential targets in the liver tissue.Result Data mining predicted the potential genes： Proto-oncogene FOS and collagen type Ⅰ alpha 2 （COL1A2）. Experimental validation showed that the liver injury was alleviated after drug administration compared with that after modeling. The serum AST and ALT levels were reduced after drug administration. The protein and mRNA levels of FOS were significantly up-regulated， while those of COL1A2 were down-regulated after drug administration.Conclusion Total ginsenosides ameliorate HA via FOS and COL1A2.