男性骨质疏松症患者肠道菌群组成及基因功能

Composition and gene function of intestinal microbiota in male osteoporotic patients

目的探讨男性骨质疏松症患者肠道菌群结构和基因功能改变与骨密度之间的关系。方法选取2019年3至12月西安交通大学附属红会医院就诊的50岁及以上初诊男性骨质疏松症患者14例、低骨量者26例及骨量正常者21例,收集研究对象的粪便样本提取肠道微生物DNA,进行16S rRNA V3~V4区和宏基因组扩增,使用Illumina平台进行高通量测序,对测序结果进行物种注释、菌群结构差异和基因功能分析,并观察其与骨密度之间的相关性。结果大多数肠道菌群在3组人群中共同存在,在门、纲、目、科、属、种各水平均存在一定的差别。相对于骨量正常组人群,Leuconostocaceae科、Weissella属和Lactobacillus gasseri种是骨质疏松症组患者肠道内丰度显著升高的关键差异菌群;而Cardiobacteriales目、unidentified Cardiobacteriales科和Ignatzschineria属是骨量正常组人群肠道内丰度显著升高的菌群。相关性分析显示,Selenomonadales和Lactobacillus salivarius相对丰度与骨密度之间具有一定的相关性(r=0.326,P<0.05;r=-0.288,P<0.05)。肠道宏基因组功能注释分析显示,在基因功能注释与分级注释(Kyoto Encyclopedia of Genes and Genomes functional database annotations,KEGG)级水平,细胞过程、环境信息加工和新陈代谢功能区域与骨密度呈正相关;在KEGG 2级水平,碳水化合物代谢和信号转导功能与骨密度呈正相关;在KEGG 3级水平,淀粉和蔗糖代谢、二组分系统注释功能与骨密度呈正相关;基因功能信号转导、蔗糖和淀粉代谢、二组分系统对于鉴别骨质疏松症患者具有较好的敏感度和特异性。结论16S rRNA和宏基因组测序分析有助于明确50岁及以上男性骨质疏松症患者的肠道菌群结构和基因功能的变化,为研究肠道菌群与骨代谢的关系提供理论依据。

Objective To explore the composition and gene functional alterations of intestinal microbiota in older male osteoporotic patients,and the relationship between intestinal microbiota and bone mineral density(BMD).Methods Subjects were recruited from March 2019 to December 2019 on their first visits to Honghui Hospital,including 14 patients with osteoporosis(OP),26 patients with osteopenia(ON),and 21 controls with normal BMD(NC).Fresh fecal samples were collected for DNA extraction,16 S rRNA and metagenomic sequencing.Species annotation,intestinal microbiota composition,and gene functions were analyzed based on the sequencing data.Their correlations with BMD were also assessed.Results The intestinal microbio to profile was similar in all subjects,with certain differences in phylum,class,order,family,genus,and species levels.Compared with normal BMD subjects,relative abundances of Leuconostocaceae,Weissella and Lactobacillus gasseri were greatly increased in OP patients,while Cardiobacteriale,unidentified Cardiobacteriales and Ignatzschineria were decreased significantly(P<0.05).Moreover,the relative abundances of Selenomonadales and Lactobacillus salivarius were correlated with BMD(r=0.326,P<0.05;r=-0.288,P<0.05).Metagenomic sequencing analysis demonstrated that cellular processes,environmental information processing and metabolism were annotated in Kyoto Encyclopedia of Genes and Genomes functional database annotations(KEGG)level 1 and positively correlated with BMD.In level 2,carbohydrate metabolism and signal transduction were annotated and correlated positively with BMD,while starch and sucrose metabolism and two component system were positively correlated with BMD in level 3 after annotation.Gene functional analysis indicated that signal transduction,starch and sucrose metabolism and two component system could identify OP patients with preferable sensitivity and specificity.Conclusion 16 S rRNA and metagenomic sequencing has great potential in intestinal microbiota analysis,which could provide theoretical foundation for the correlations between intestinal microbiota and bone metabolism in older OP men.