虫草素调控受体相互作用蛋白激酶1介导的凋亡改善缺氧/复氧诱导的人肾小管上皮细胞损伤

Regulation of receptor interacting protein kinase 1-mediated apoptosis by cordycepin attenuated hypoxia/reoxygenation-induced renal tubular epithelial cell injury

目的基于基因表达综合(gene expression omnibus,GEO)数据库中的基因芯片、网络药理学、分子对接和体外实验探讨虫草素治疗急性肾损伤(acute kidney injury,AKI)的作用机制。方法从GEO(GSE87025)、GeneCards 2个数据库获得AKI的疾病靶点。从TargetNet、BATMAN和GeneCards 3个数据库获得虫草素相关靶点。对AKI和虫草素相关靶点取并集,筛选出共同靶基因。进行基因本体论(gene ontology,GO)注释和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路富集分析后对虫草素及关键靶点进行分子对接。细胞实验设计对照组、缺氧/复氧组、缺氧/复氧+虫草素组对相关靶点进行实验验证。结果AKI和虫草素共同靶基因共12个,GO功能注释结果表明主要参与凋亡信号通路的负调控、坏死性凋亡过程等生物学过程,KEGG富集分析结果表明主要参与Toll样受体信号通路、坏死性凋亡、核苷酸结合寡聚化结构域(nucleotide-binding oligomerization domain,NOD)样受体信号通路、核因子κB(nuclear factor-κB,NF-κB)信号通路、肿瘤坏死因子(tumor necrosis factor,TNF)信号通路和细胞凋亡等常见信号通路。虫草素和受体相互作用蛋白激酶1(receptor-interacting protein kinase 1,RIPK1)分子对接的最优结合能为-7.1,表明两者有较强的结合活性。蛋白免疫印迹结果表明虫草素可下调RIPK1、Bcl-2相关X蛋白、凋亡指标胱天蛋白酶表达、上调B细胞淋巴瘤/白血病-2的表达,细胞免疫荧光结果表明虫草素可下调RIPK1表达。结论虫草素可通过调控RIPK1介导的凋亡缓解缺氧/复氧诱导的人肾小管上皮细胞损伤,以达到治疗AKI的效果。

Objective To explore the therapeutic mechanism of cordycepin for acute kidney injury(AKI)based upon gene chips in gene expression omnibus(GEO)database,network pharmacology,molecular docking and in vitro experiments.Methods Targeted genes of AKI were obtained from two databases,GEO(GSE87025)and GeneCards.Cordycepin-related targets were retrieved from three databases of TargetNet,BATMAN and GeneCards.The common targets of AKI and cordycepin were screened by taking the intersection of two sets.Molecular docking was performed for cordycepin and key targets after gene ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.Cell experiments were performed for control,hypoxia/reoxygenation and hypoxia/reoxygenation+cordycepin groups for experimental verifications of the relevant targets.Results There were 12 common targets of AKI and cordycepin.GO functional annotation results indicated that these targets participated predominantly in negative regulation of apoptotic signaling pathway and necroptosis process.KEGG enrichment analysis results showed that they played some roles in signaling pathways of Toll-like receptor,necroptosis,nucleotide-binding oligomerization domain(NOD)-like receptor,nuclear factor-κB(NF-kB),tumor necrosis factor(TNF)and cell apoptosis,etc.Optimal binding energy between cordycepin and receptor interacting protein kinase 1(RIPK1)in molecular docking was-7.1,denoting potent binding activity.Western blot indicated that cordycepin down-regulated the expressions of RIPK1,bcl-2 associated x protein(Bax)and caspase 3 and up-regulated B cell lymphoma/lewkmia-2(Bcl-2).Cell immunofluorescence results revealed that cordycepin down-regulated the expression of RIPK1.Conclusion Cordycepin may alleviate hypoxia/reoxygenation-induced renal tubular epithelial cell injury through regulating RIPK1-mediated apoptosis.Thus it achieves the therapeutic efficacy for AKI.