基于“肠-肺轴”探讨肠道菌群与特发性肺纤维化的遗传因果关联及干预中药预测

Exploring genetic causal association between intestinal flora and idiopathic pulmonary fibrosis based on“gut-lung axis”and predicting intervention with traditional Chinese medicine

目的采用两样本、双向孟德尔随机化(Mendelianrandomization,MR)探索与特发性肺纤维化(idiopathicpulmonary fibrosis,IPF)存在因果关联的肠道菌群及潜在机制,并预测对其具有调控作用的中药,探析“肠-肺轴”生物学基础并提供IPF中医药治疗方案。方法利用MiBioGen和芬兰数据库获取肠道菌群和IPF的全基因组关联研究数据。以逆方差加权法为主要方法揭示肠道菌群与IPF的双向因果关联。采用Cochrane'sQ检验、留一法、MR-Egger-intercept和MR多效性残差及异质性检测法(mendelian randomizationpleiotropyRESidual sumandoutlier,MR-PRESSO)进行异质性检验、敏感性分析、水平多效性和离群值检验等质量控制。对单核苷酸多态性(singlenucleotidepolymorphisms,SNPs)临近基因进行蛋白质-蛋白质互作(protein-protein interaction,PPI)分析以筛选核心基因,对核心基因进行功能富集分析以探索肠道菌群介导IPF发生的潜在机制。使用比较毒理基因组学数据库(thecomparative toxicogenomicsdatabase,CTD)和Coremine数据库预测对核心基因具有潜在调控作用的化学成分与中药。统计预测中药的四气、五味、归经及功效。采用分子复合物检测模块(molecular complexdetection,MCODE)对预测中药进行聚类分析,并筛选核心中药,分子对接验证核心基因与中药成分的结合能力。结果正向MR分析发现牙孢杆菌纲(Bacilli)、厚壁菌纲(Negativicutes)、伯克霍尔德氏菌目(Burkholderiales)、Family XIII、活泼瘤胃球菌Ruminococcusgnavus、甲烷短杆菌属Methanobrevibacter和芬氏另枝菌Alistipesfinegoldi肠道菌群与IPF呈正相关,艾森伯格氏菌属Eisenbergiella与IPF呈负相关。SNPs间无异质性、水平多效性和离群值,且敏感性良好。反向MR分析提示上述菌群与IPF不存在双向因果关联。获得了156个SNPs临近基因,通过PPI筛选前50个基因作为核心基因。核心基因主要富集在细胞衰老、鼠肉瘤(rat sarcoma,Ras)、磷脂酰肌醇-3-羟激酶(phosphatidylinositol-3-hydroxykinase,PI3K)/蛋白激酶B(proteinkinaseB,Akt)等通路。通过CTD数据库获取到以榭皮素、山奈酚和脂多糖为代表的236个化学成分,基于化学成分使用Coremine数据库预测得到566味中药,四气以寒、温、平为主,五味以苦、甘、辛为主,归经以肝、肺经为主,脾、胃、肾经次之,功效以清热、补虚为主,兼以理气、活血、化痰止咳平喘等。MCODE将人参、生姜、姜黄、郁金确定为关键中药,分子对接显示上述中药成分与核心基因具有良好结合效能。结论基于MR发现8种肠道菌群与IPF存在因果关联,其机制可能与细胞衰老、RaS、PI3K/Akt等通路异常相关。预测中药主要体现益气、理气、化痰、活血及解毒等治法。丰富了“肠-肺轴”生物学基础,深化了IPF的病因学认识,并为中医药调控肠道菌群防治IPF提供了新的方向。

Objective To explore the intestinal flora causally associated with idiopathic pulmonary fibrosis(IPF)and its potential mechanism,and predict the traditional Chinese medicine(TCM)that have regulatory effects on it.By analyzing the biological basis of the“gut-lung axis”to provide the TCM regimen for IPF.Methods MiBioGen and Finggen databases were used to obtain the genomewide association studies data of intestinal flora and IPF.Inverse variance weighting was used as the main method to reveal the bidirectional causal effect of intestinal flora and IPF.Cochrane's Q test,leave-one-out method,MR-Egger-intercept and mendelian randomization pleiotropy RESidual sum and outlier(MR-PRESSO)were used for quality control of heterogeneity,sensitivity,level pleiotropy and outliers.Protein-protein interaction(PPI)analysis was performed on the nearest genes of single nucleotide polymorphisms(SNPs)to screen core genes.Functional enrichment analysis of core genes was performed to explore the potential mechanism of intestinal flora mediating IPF.Chemical components and Chinese herbs with potential regulatory effects on core genes were predicted using the CTD and Coremine databases.The four qi,five flavors,meridians and functions of the Chinese herbs were predicted.Molecular complex detection(MCODE)was to perform cluster analysis and screen the core traditional Chinese medicines TCMs.Molecular docking was to verify the binding energy between the core genes and the representative compounds in the key TCMs.Results MR analysis revealed Bacilli,Negativicutes,Burkholderiales,Family XIll,Ruminococcus gnavus,Methanobrevibacter and Alistipes finegoldi were positively correlated with IPF.Eisenbergiella was negatively correlated with IPF.Quality control suggested no heterogeneity,horizontal pleiotropy,and outliers among SNPs,and the sensitivity was good.Reverse MR analysis suggested that there was no bidirectional causal association between the above bacteria and IPF.A total of 156 SNPs-related genes were obtained,and the top 50 genes were selected as core genes by PPI.The core genes were mainly enriched in celluar senescence,rat sarcoma(Ras),phosphatidylinositol-3-hydroxykinase(PI3K)/protein kinase B(Akt)and other pathways.A total of 236 chemical components represented by quercetin,kaempferol,and lipopolysaccharide were obtained through the CTD database.A total of 566 herbs were obtained through the Coremine database.The four qi were mainly cold,warm and smooth,the five flavours were mainly bitter,sweet and spicy,the meridians were mainly liver and lung,followed by spleen,stomach and kidney,and their functions were mainly clearing heat and tonifying deficiency,regulate qi,promot blood circulation,resolve phlegm and relieve cough and asthma.MCODE identified Renshen(Ginseng Radix et Rhizoma),Shengjiang(Zingiberis Rhizoma Recens),Jianghuang(Curcumae Longae Rhizoma),Yujin(Curcumae Radix)as the key TCMs,and molecular docking showed that the above compounds in the key TCMs had good combination efficiency with the core genes.Conclusion Based on MR analysis,the eight kinds of intestinal flora had causal association with IPF,and the mechanisms were related to celluar senescence,Ras and PI3K/Akt pathway.The predicted TCMs mainly embodied the treatment of benefiting qi,regulating qi,resolving phlegm,promoting blood circulation and detoxifying.Our findings enrich the biological basis of the“gut-lung axis”,deepen the understanding of the etiology of IPF,and provide a new direction for the prevention and treatment of IPF by regulating intestinal flora with TCMs.