联合网络药理学和转录组学探讨参芪扶正注射液改善顺铂诱导的急性肾损伤的分子机制

Exploration of Molecular Mechanisms of Shenqi Fuzheng Injection in Improving Cisplatin-Induced Acute Kidney Injury by Combining Network Pharmacology and Transcriptomics

目的:运用网络药理学和转录组学方法,探究参芪扶正注射液改善顺铂诱导的急性肾损伤(AKI)的潜在作用靶点,为进一步指导临床提供实验依据。方法:(1)网络药理学分析:在本草组鉴数据库(HERB)、中药系统药理学数据库与分析平台(TCMSP)、中草药化合物蛋白质靶标数据库(HIT)、化合物靶点预测平台(Swiss Target Predictio)、有机小分子生物活性数据库(PubChem)、蛋白质分析数据库(UniProt)、蛋白质相互作用(PPI)关系数据库(STRING)中检索得到参芪扶正注射液主要成分的潜在靶点;使用在线人类孟德尔遗传数据库(OMIM)、人类基因综合数据库(GeneCards)、治疗靶点数据库(TTD)检索获得AKI的潜在靶点;利用STRING数据库和Cytoscape 3.9.1软件构建参芪扶正注射液主要成分和AKI共同潜在靶点的PPI网络图并进行网络拓扑分析;使用R语言软件进行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。(2)动物实验和转录组学分析:建立4T1乳腺癌荷瘤小鼠模型,分为4组(模型组、参芪扶正注射液组、顺铂组、参芪扶正注射液联合顺铂组),给药4周后取血检测肌酐(CRE)与尿素氮(BUN),取肾脏组织进行转录组学测序分析,运用实时荧光定量聚合酶链式反应(qRT-PCR)法进行实验验证。结果:通过网络药理学数据库和Cytoscape 3.9.1软件预测出65个参芪扶正注射液主要成分和AKI的共同潜在靶点。PPI网络图和共同潜在靶点网络图表明,参芪扶正注射液的主要成分和AKI与65个共同潜在靶点之间存在着多种作用关系。GO功能富集分析和KEGG通路富集分析结果表明,参芪扶正注射液改善AKI的作用与氧化和抗氧化、炎性和免疫反应以及T细胞受体信号通路、缺氧诱导因子-1信号通路等途径密切相关。顺铂组血清CRE、BUN水平均高于模型组(P<0.05)。参芪扶正注射液组血清CRE水平低于模型组(P<0.05)。参芪扶正注射液组血清CRE、BUN水平均低于顺铂组(P<0.05)。参芪扶正注射液联合顺铂组血清CRE、BUN水平均低于顺铂组(P<0.05)。参芪扶正注射液联合顺铂组血清CRE水平高于参芪扶正注射液组(P<0.05)。参芪扶正注射液联合顺铂组血清BUN水平与参芪扶正注射液组比较,差异无统计学意义(P>0.05)。4组小鼠肾脏组织HE染色结果表明,参芪扶正注射液改善了顺铂诱导的肾组织损伤。结合网络药理学和转录组学分析,得到9个共同潜在靶点,其中参芪扶正注射液下调基因包括黄嘌呤脱氢酶、白细胞介素10、E-钙黏蛋白、干扰素因子-γ、雌激素受体α基因、前列腺素内过氧化物合酶1,上调基因包括过氧化物酶体增殖物激活受体-γ、白细胞介素6、CD4^(+)T细胞。结论:参芪扶正注射液能够通过影响氧化应激、炎性与免疫反应来改善顺铂诱导的AKI,且具有多途径、多靶点的优势。

Objective:Using network pharmacology and transcriptomics methods,this paper explores the potential targets of Shenqi Fuzheng Injection in improving cisplatin-induced acute kidney injury(AKI),providing experimental basis for further clinical guidance.Methods:①Network pharmacology analysis:The HERB Database(HERB),Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),Herbal Ingredients'Targets(HIT),Swiss Target Prediction,PubChem BioAssay Database(PubChem),UniProt Knowledgebase(UniProt),and Protein-protein Interaction(PPI)Database(STRING)were applied to obtain potential targets for the main components of Shenqi Fuzheng Injection.The Online Mendelian Inheritance in Man(OMIM),Human Gene Database(GeneCards),and Therapeutic Target Database(TTD)were used to obtain potential targets for AKI.Using STRING database and Cytoscape 3.9.1 software,a PPI network diagram for common potential targets of the main components of Shenqi Fuzheng Injection and AKI was constructed and network topology analysis was performed.By using R language software,the gene ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed.②Animal experiment transcriptomics analysis:The 4T1 tumor-bearing mouse model of breast cancer were established and divided into four groups(the model group,the Shenqi Fuzheng Injection group,the cisplatin group,the Shenqi Fuzheng Injection combined with cisplatin group).After four weeks of administration,blood was taken to detect creatinine(CRE)and urea nitrogen(BUN),and kidney tissue was taken for transcriptome sequencing analysis.Real time quantitative polymerase chain reaction(qRT-PCR)was used for experimental verification.Results:A total of 65 common potential targets of the main components of Shenqi Fuzheng Injection and AKI were predicted through a network pharmacology database and Cytoscape 3.9.1 software.The PPI network diagram and common potential target network diagram indicated that there are multiple functional relationships between the main components of Shenqi Fuzheng Injection and AKI with 65 common potential targets.The results of GO functional enrichment analysis and KEGG pathway enrichment analysis indicated that the improvement of AKI by Shenqi Fuzheng Injection is closely related to oxidative and antioxidant,inflammatory and immune responses,as well as T cell receptor signaling pathways and hypoxia inducible factor-1 signaling pathways.The CRE and BUN levels in serum in the cisplatin group were higher than those in the model group(P<0.05).The CRE level in the Shenqi Fuzheng Injection group was significantly lower than that in the model group(P<0.05).The levels of CRE,BUN in serum in the cisplatin group were higher than those in the model group(P<0.05).The CRE level in serum in the Shenqi Fuzheng Injection group was lower than that in the model group(P<0.05).The levels of CRE,BUN in serum in the Shenqi Fuzheng Injection group were lower than those in the cisplatin group(P<0.05).The levels of CRE,BUN in serum in the Shenqi Fuzheng Injection combined with cisplatin group were lower than those in the cisplatin group(P<0.05).The CRE level in serum in the Shenqi Fuzheng Injection combined with cisplatin group was higher than that in the Shenqi Fuzheng Injection group(P<0.05).There was no significant difference in the BUN levels in serum between the Shenqi Fuzheng Injection combined with cisplatin group and the Shenqi Fuzheng Injection group(P>0.05).The HE staining results of kidney tissue in the four groups of mice showed that Shenqi Fuzheng Injection improved cisplatin-induced kidney injury.Combining network pharmacology and transcriptomics analysis,nine common potential targets were identified,among which the down-regulated genes of Shenqi Fuzheng Injection included xanthine dehydrogenase,interleukin-10,E-cadherin,and interferon-γ,estrogen receptorα,prostaglandin endoperoxide synthase 1,and up-regulated genes included peroxisome proliferator-activated receptors-γ,interleukin-6,and CD4^(+)T cells.Conclusion:Shenqi Fuzheng Injection can improve cisplatin-induced AKI by affecting oxidative stress,inflammation,and immune response,and has the advantages of multiple pathways and targets.