基于网络药理学探讨脉络宁治疗急性肾损伤的作用机制

Investigation on the mechanism of action of Mailuoning in the treatment of acute kidney injury using network pharmacology

摘要

目的：通过网络药理学方法，探讨脉络宁（mailuoning,MLN）对顺铂（cisplatin,CP）诱导的急性肾损伤（acute kidney injury,AKI）的保护作用，并分析其与抗凋亡途径相关的作用机制。方法：通过网络药理学筛选MLN活性成分及AKI相关靶点：基于TCMSP和ETCM 2.0数据库获取MLN活性成分，利用Swiss Target Prediction预测成分靶点，结合GeneCards筛选AKI疾病靶点，取交集后通过STRING数据库构建蛋白-蛋白互作（protein-protein interaction,PPI）网络，Cytoscape拓扑分析筛选核心靶点，并对其进行基因本体（gene ontology,GO）功能注释及京都基因与基因组百科全书（kyoto encyclopedia of genes and genomes,KEGG）通路富集分析。动物实验：40只小鼠随机分为溶剂对照、MLN毒性对照、CP模型及CP+MLN组。MLN组以15 mL/（kg·d）腹腔注射MLN连续10 d;CP模型与CP+MLN组于第7天单次腹腔注射CP(20 mg/kg)。CP干预3 d后检测血浆尿素氮（blood urea nitrogen,BUN）及肌酐（creatinine,Cre）水平，苏木精-伊红（hematoxylin-eosin,HE）染色评估肾组织病理损伤，Western blot检测肾组织中中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin,NGAL）及凋亡相关蛋白表达。结果：网络药理学筛选得到104种MLN活性成分及224个作用靶点，AKI靶点2 465个，交集靶点117个，其中17个被认定为核心靶点。通过KEGG和GO分析发现，凋亡相关信号转导通路可能是MLN防治AKI的重要作用通路。动物实验结果证实成功构建了CP诱导的小鼠AKI模型，与CP模型组相比，MLN处理明显降低了血浆中BUN和Cre水平（P<0.05），抑制了肾脏NGAL蛋白表达（P<0.05），同时缓解了肾组织的病理学损伤。此外，MLN明显降低了AKI模型小鼠肾组织中p-P53(ser 15)、cleaved caspase-3蛋白的表达及Bax/Bcl-2蛋白表达量的相对比值（P<0.05），同时上调蛋白激酶B(protein kinase B,AKT)磷酸化水平（P<0.05）。结论：MLN对CP诱导的小鼠AKI具有保护作用，其作用机制可能与其抗凋亡特性相关。

Abstract

Objective To investigate the protective effects of Mailuoning（MLN） against cisplatin（CP）-induced acute kidney injury （AKI） utilizing network pharmacology and to analyze the underlying mechanism of action related to anti-apoptotic pathways. Methods Active components of MLN and targets related to AKI were identified using network pharmacology. The active components of MLN were sourced from the TCMSP and ETCM 2.0 databases. The targets of components were predicted using the Swiss Target Prediction tool，and subsequently the targets related to AKI were retrieved from the GeneCards database to identify intersecting targets. A protein-protein interaction network was constructed using the STRING database，and a topological analysis was performed using Cytoscape to identify core targets. Gene Ontology（GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analyses were conducted on these core targets. For the animal experiments，forty mice were randomly assigned to four groups： solvent control，MLN toxicity control，CP model，and CP+MLN groups. The MLN group received intraperitoneal injections of MLN at a dose of 15 mL/（kg·d） for ten consecutive days. The CP model and CP+MLN groups were administered a single intraperitoneal injection of CP at 20 mg/kg on day 7. Three days after the CP treatment，plasma levels of blood urea nitrogen（BUN） and creatinine（Cre） were measured. The pathological injury of kidney tissues was assessed using hematoxylin-eosin staining. Western blot analysis was utilized to determine the expression of neutrophil gelatinase-associated lipocalin（NGAL） and apoptosis-related proteins in kidney tissues. Results A total of 104 active components of MLN and 224 targets were identified using network pharmacology，and 2 465 targets were identified to be related to AKI，resulting in 117 intersecting targets，of which，17 targets were classified as core targets. KEGG and GO analyses indicated that the apoptosis-related signal transduction pathway might be a crucial pathway through which MLN provided protective effects against AKI. The results of animal experiments confirmed the successful establishment of CP-induced AKI models in mice. Compared with the CP model group，MLN treatment significantly reduced plasma levels of BUN and Cre（P<0.05），inhibited NGAL protein expression in the kidneys（P<0.05），and improved the pathological injury observed in kidney tissues. Furthermore，MLN markedly reduced the expression levels of p-P53（ser 15） and cleaved caspase-3 proteins，as well as the Bax/Bcl-2 ratio in kidney tissues of AKI model mice（P<0.05），while upregulating protein kinase B phosphorylation levels（P<0.05）. Conclusion MLN demonstrates protective effects against CP-induced AKI in mice，potentially through mechanisms related to its anti-apoptotic properties.

Graphical abstract

关键词

网络药理学 / 脉络宁 / 顺铂 / 急性肾损伤 / 抗凋亡

Key words

network pharmacology / Mailuoning / cisplatin / acute kidney injury / anti-apoptosis

基金资助

广东省基础与应用基础研究基金资助项目(2022A1515010097)

广东医科大学附属医院高层次人才科研启动资助项目(GCC2020009)