特发性肺纤维化特征基因和靶向中药的探索与验证

Exploration and validation of characteristic genes and targeted traditional Chinese medicines for idiopathic pulmonary fibrosis

目的 探索特发性肺纤维化(idiopathic pulmonary fibrosis, IPF)的特征基因及靶向中药预测,为疾病机制研究及新药研发提供思路。方法 通过GEO数据库获取4个IPF相关芯片,挖掘差异表达基因(differentially expressed genes,DEGs)后进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析。利用机器学习方法从DEGs中筛选特征基因并做外部芯片验证,再进行免疫浸润分析,探索特征基因与免疫浸润的相关性。然后通过动物实验验证IPF的特征基因。基于特征基因在Coremine和TCMSP数据库中预测靶向中药及活性成分,通过Cytoscape 3.7.2构建网络图,并进行活性成分与IPF特征基因的分子对接验证。结果 在271个IPF的DEGs中共筛选出11个特征基因,外部芯片验证提示CDH3、MAOA、FNDC1、ITGBL1在IPF组呈高表达,而EPB41L5、FAM167A、MS4A15、SCARA3、CRTAC1、CCK、TSPAN7呈低表达,且受试者工作特征(receiver operator characteristic,ROC)曲线提示特征基因的准确性良好。免疫浸润提示特征基因可能与活化的记忆性CD4+T细胞和调节性T细胞等免疫细胞的浸润存在相关性。富集分析提示含胶原蛋白的细胞外基质、趋化因子信号通路和糖尿病并发症中的晚期糖基化终产物-晚期糖基化终产物受体(advanced glycation end products-receptor for advanced glycosylation end products,AGE-RAGE)信号通路等可能与IPF的发生发展密切相关。动物实验显示,IPF组小鼠肺组织CDH3、FNDC1、ITGBL1和MAOA的m RNA及蛋白表达水平均高于对照组,而SCARA3 m RNA及蛋白表达水平反之,差异具有统计学意义(P<0.05)。预测地骨皮、太子参与西红花等补虚药和活血药对IPF具有干预作用,符合中医对IPF“虚”和“瘀”的病机认识;槲皮素、β-谷甾醇与山柰酚等活性成分对IPF具有干预作用。分子对接显示中药活性成分与IPF特征基因具有较好的结合能力。结论 CDH3、MAOA、FNDC1、ITGBL1和SCARA3是IPF的潜在特征基因,地骨皮、太子参与西红花等是治疗IPF的潜在中药。

Objective To provide ideas for disease mechanism research and novel therapeutic development,the characteristic genes and herbs of idiopathic pulmonary fibrosis(IPF)were explored.Methods Differentially expressed genes(DEGs)were mined using 4 microarrays linked to IPF that were acquired from the GEO database.Next,gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analyses were carried out.Characteristic genes from DEGs were screened using a machine learning method,and its external validation was also carried out.Additionally,an analysis was done on the relationship between characteristic genes and immune infiltration.An experiment on animals was done to confirm the presence of characteristic genes.In the meantime,Cytoscape 3.7.2 was used to build the networks of herbs and their active ingredients based on predictions made in the TCMSP and Coremine databases.Ultimately,molecular docking validation was carried out to confirm the combining ability between characteristic genes and active ingredients.Results A total of 11 characteristic genes were obtained from 271 DEGs in IPF.External validation showed that whereas CDH3,MAOA,FNDC1 and ITGBL1 were highly expressed in IPF,EPB41L5,FAM167A,MS4A15,SCARA3,CRTAC1,CCK and TSPAN7 were lowly expressed.ROC curves indicated that characteristic genes have good accuracy.The infiltration of immune cells,such as T cells CD4 memory activated and T cells regulatory,may also be correlated with the characteristic genes.The development of IPF may be intimately linked to collagen-containing extracellular matrix,chemokine signaling pathway and AGE-RAGE signaling pathway in diabetic complications,according to enrichment analyses.Animal experiment suggested the mRNA and protein levels of CDH3,FNDC1,ITGBL1 and MAOA were higher in IPF group than those in control group,while the SCARA3 mRNA and protein expression levels were opposite,and the differences were statistically significant(P<0.05).Herbs that were predicted to be deficiency tonics and blood activators were Digupi(Lycii Cortex),Taizishen(Pseudostellariae Radix)and Xihonghua(Croci Stigma).These findings aligned with the understanding of traditional Chinese medicine on the pathogenesis of IPF as“deficiency and blood stasis”in IPF held by Traditional Chinese Medicine.Quercetin,beta-sitosterol and kaempferol were their active ingredients.Lastly,molecular docking indicated that the active ingredients exhibited strong binding capacity with characteristic genes.Conclusion CDH3,MAOA,FNDC1,ITGBL1 and SCARA3 are potential characteristic genes of IPF.Lycii Cortex,Pseudostellariae Radix and Croci Stigma are potentially important herbs for the treatment of IPF.