基于网络药理学和实验验证探究糖肾宝复方治疗糖尿病肾病的作用机制

The Mechanism Research of Tangshenbao Compound in Treating Diabetic Nephropathy Based on Network Pharmacology and Experimental Verification

网络药理学方法结合分子生物学实验探究糖肾宝复方(Tangshenbao compound,TSB)治疗糖尿病肾病(diabetic nephropathy, DN)的作用机制。使用TCMSP、Pubchem、Swiss Target Prediction数据库,结合相关文献,得到TSB相关活性成分与靶点;通过GeneCards、DisGeNET、OMIM数据库,获得TSB治疗DN候选靶点;利用在线作图软件绘制“TSB-DN”候选靶点Venn图,并结合Cytoscape 3.7.2软件进行GO和KEGG富集分析;然后使用PyMOL、AutoDock Tools和AutoDock Vina软件完成活性成分配体与靶蛋白晶体结构对接;再通过体外培养小鼠肾小球系膜细胞Mcs进行细胞实验验证,实时荧光定量PCR检测各组细胞中STAT3、PIK3CD、PIK3R1、MAPK8及关联性靶点Ets-1 mRNA表达水平,CCK-8法检测24 h后的细胞活性。结果发现,Swiss Target Prediction数据库合并处理后共获得TSB作用靶点835个,联合GeneCards、DisGeNET、OMIM等疾病数据库后共获得443个“TSB-DN”交集靶点,而PPI网络分析得出Degree前10靶点分别为SRC、PIK3R1、STAT3、PIK3CA等;GO、KEGG富集分析结果显示TSB治疗DN可能与蛋白磷酸化、细胞信号转导、基因表达正调控等功能相关;最后体外细胞实验表明,TSB复方能有效降低SV40-MES-13细胞中PIK3CD、PIK3R1、ETS-1的mRNA表达量,药物作用于细胞后能起到“抑制、镇静”的作用。结果表明,TSB复方可通过抑制PIK3R1、PIK3CD和MAPK8、STAT3、ETS-1等靶基因的mRNA表达,实现对PI3K-Akt、AGE-RAGE等信号通路的调控并发挥对DN的治疗作用。

The research investigated the mechanism of action of Tangshenbao compound(TSB)in treating diabetic nephropathy(DN)by combining network pharmacology methods with molecular biology experiments.The active ingredients and targets relat⁃ed to TSB were obtained using TCMSP,Pubchem,and Swiss Target Prediction databases,along with relevant literature.Candi⁃date targets for TSB in treating DN were obtained through GeneCards,DisGeNET,and OMIM databases.A Venn diagram of the"TSB-DN"candidate targets was drawn using online plotting software,followed by GO and KEGG enrichment analysis using Cytoscape 3.7.2 software. Subsequently, molecular docking of active ingredient ligands with target protein crystal structures was completed using PyMOL, AutoDock Tools, and AutoDock Vina software. Cell experiments were then conducted using Mcs me⁃ sangial cells cultured in vitro to validate the findings. Real-time fluorescence quantitative PCR was used to detect the mRNA ex⁃ pression levels of STAT3, PIK3CD, PIK3R1, MAPK8, and their associated targets in the cells of each group, while cell viabili⁃ ty after 24 hours was assessed using the CCK-8 method. Results showed that a total of 835 TSB target genes were obtained after processing the Swiss Target Prediction database. When combined with GeneCards, DisGeNET, OMIM, and other disease data⁃ bases, there were 443 intersecting target genes for “TSB-DN”. Protein-protein interaction (PPI) network analysis identified the top 10 degree targets as SRC, PIK3R1, STAT3 and PIK3CA. GO and KEGG enrichment analysis results indicated that TSB treatment for DN may be related to functions such as protein phosphorylation, cell signal transduction, and positive regulation of gene expression. Finally, cell experiments in vitro demonstrated that TSB could effectively reduce the mRNA expression levels of PIK3CD, PIK3R1, and ETS-1 in SV40-MES-13 cells, resulting in an “inhibitory, sedative” effect following drug action on the cells. The results indicated that the TSB compound can inhibit the mRNA expression of target genes such as PIK3R1, PIK3CD, MAPK8, STAT3, and ETS-1, thereby regulating the PI3K-Akt and AGE-RAGE signaling pathways and exerting a therapeutic effect on DN.