作者：王丽娜，卢震钰，王颖，李媛，云春梅，孙德俊，高笑宇

单位：

1.014000 内蒙古自治区包头市，内蒙古科技大学包头医学院 2.010017 内蒙古自治区呼和浩特市，内蒙古自治区人民医院国家卫生健康委员会慢阻肺诊治重点实验室 内蒙古自治区呼吸疾病重点实验室 通信作者：高笑宇，E-mail：xiaoyugao2015@hotmail.com

Units：

1.Baotou Medical College, Science & Technology of Inner Mongolia University, Baotou 014000, China 2.Key Laboratory of National Health Commission for the Diagnosis & Treatment of COPD/Inner Mongolia Key Laboratory ofRespiratory Diseases, Inner Mongolia People's Hospital, Hohhot 010017, China Corresponding author: GAO Xiaoyu, E-mail: xiaoyugao2015@hotmail.com

关键词：

吸烟; 呼吸系统; 微生物群;

Keywords：

Smoking; Respiratory system; Microbiota

CLC：

DOI：

10.12114/j.issn.1008-5971.2022.00.008

Funds：

国家自然科学基金资助项目（81960013）；中国医学科学院中央级公益性科研院所基本科研业务费专项资金资助项目（2019PT350001）；中央引导地方科技发展资金项目（ZYZ20200486）；内蒙古自治区人民医院博士科研启动资金（2019BS08）；2017 年度内蒙古自治区卫生计生科研计划项目（201703001）

摘要：

背景随着高通量测序技术的发展,研究者们发现下呼吸道有大量微生物菌群定植。研究显示,吸烟对下呼吸道微生物菌群结构及组成有显著影响,且其变化可能与肺部疾病有关,但其具体机制尚不清楚。目的通过痰液微生物高通量测序技术分析吸烟对下呼吸道微生物菌群结构的影响,以预测其对机体生命活动的影响。方法选取2018年10月至2019年10月于内蒙古自治区人民医院健康体检中心招募的志愿者55例为研究对象。按照吸烟情况,将研究对象分为吸烟组(22例)和不吸烟组(33例)。采用高通量测序技术分析痰液微生物,分析吸烟对微生物丰度与菌群结构组成的影响、吸烟对微生物菌群结构的影响、差异微生物间的相关性及预测差异微生物的功能。结果吸烟组Shannon指数高于不吸烟组,Simpson指数低于不吸烟组(P <0.05)。Adonis检验及主成分分析(PCA)结果显示,吸烟组与不吸烟组β多样性指数比较,差异有统计学意义(P <0.05)。韦恩图分析结果显示,吸烟组和不吸烟组共有菌群1 146个,不吸烟组独有菌群1 080个,吸烟组独有菌群841个。在纲水平上,按照菌群相对丰度降序排列,差异菌群前5位分别是芽孢杆菌纲(Bacilli)、拟杆菌纲(Bacteroidia)、梭杆菌纲(Fusobacteria)、β-变形菌纲(Betaproteobacteria)、γ-变形菌纲(Gammaproteobacteria)。纲水平微生物在吸烟组中的优势菌群为:Bacilli、Bacteroidia、Fusobacteriia。在属水平上,按照菌群相对丰度降序排列,差异菌群前5位分别是链球菌属(Streptococcus)、普雷沃氏菌属(Prevotella)、卟啉单胞菌属(Porphyromonas)、拟普雷沃氏菌属(Alloprevotella)、奈瑟菌属(Neisseria)。属水平微生物在吸烟组中的优势菌群为:Streptococcus、Prevotella、Porphyromonas。吸烟组和不吸烟组下呼吸道共有1纲、2目、1科、8属及8种差异微生物。其中吸烟组增加的菌群为Selenomonadales目、巨球型菌属(Megasphaera)、Faucicola属、Sphaerochaeta属、Shuttleworthia属、Anaeroglobus属、巨球形菌(Megasphaera micronuciformis)、Spirochaeta canine oral taxon 379、未分类的普氏菌(Uncultured Prevotella)、Prevotella sp. oral clone FW035;不吸烟组增加的菌群为鞘脂杆菌纲(Sphingobacteriia)、鞘脂杆菌目(Sphingobacteriales)、噬几丁质科(Chitinophagaceae)、伯克霍尔德菌(Burkholderia)、弧菌属(Vibrionimonas)、罗尔斯通氏菌属(Ralstonia)、真口腔普雷沃氏菌(Prevotella veroralis)、Catonella sp. oral clone FL037、噬淀粉密螺旋体(Treponema amylovorum)、咽峡炎链球菌(Streptococcus anginosus)。吸烟组和不吸烟组的差异微生物(属水平)间均呈正相关(r≥0.6)。吸烟组和不吸烟组主要在物质合成、降解和运输相关通路上存在差异。吸烟组功能富集的生命活动包括:安沙霉素类生物合成、赖氨酸生物合成、戊糖和葡萄糖醛酸的相互转化、泛酸盐和辅酶A生物合成、蛋白运输等;不吸烟组主要富集的生命活动包括:甲苯降解、霍乱弧菌致病循环、柠檬烯和蒎烯降解、光合作用、二恶英降解等。结论吸烟能够增加下呼吸道微生物菌群相对丰度,影响微生物菌群结构组成,改变相关物质合成、降解和运输通路,吸烟暴露下下呼吸道微生物组的功能改变可能会影响宿主的内环境稳态,打破体内免疫系统的平衡,从而引发肺部疾病。

Abstract：

【Abstract】　Background With the development of high-throughput sequencing technology, a large microfloracolonization of the lower respiratory tract has been found. Studies have shown that smoking has a significant effect on the structure andcomposition of the lower respiratory tract microflora and that changes may be associated with lung disease, but the exact mechanism isunclear. Objective To analyze the effect of smoking on microflora structure of lower respiratory tract by high-throughput sequencingof sputum microorganisms to predict its impact on the vital activity of the organism. Methods Fifty-five volunteers recruited at theHealth Examination Center of the Inner Mongolia People's Hospital from October 2018 to October 2019 were selected as the studysubjects. The study subjects were divided into smoking group (22 cases) and non-smoking group (33 cases) according to their smokingstatus. High-throughput sequencing technology was used to analyze sputum microorganisms, the effects of smoking on microbialabundance and flora structural composition, the effects of smoking on microbial flora structure and correlation between differentialmicroorganisms were analyzed, and the function of differential microorganisms was predicted. Results The Shannon index in thesmoking group was higher than that in the non-smoking group, and the Simpson index was lower than that in the non-smoking group(P< 0.05) . Adonis test and principal component analysis (PCA) showed that there was a statistically significant difference in the βdiversity index between the smoking group and the non-smoking group (P < 0.05) . The results of the Wayne plot analysis showedthat there were 1 146 floras in the smoking and non-smoking groups, 1 080 unique floras in the non-smoking group, and 841 uniquefloras in the smoking group. At class level, in descending order of relative abundance of the flora, the top 5 differential floras wereBacilli, Bacteroidia, Fusobacteria, Betaproteobacteria, andGammaproteobacteria. The dominant groups of microorganisms at theclass level in the smoking group were:Bacilli, Bacteroidia, andFusobacteriia. At the genus level, the top 5 differential groups indescending order of relative abundance were Streptococcus, Prevotella,Porphyromonas, Alloprevotella, Neisseria. The dominantgroups of genus-level microorganisms in the smoking group wereStreptococcus, prevotella, andPorphyromonas. There were oneclass, two orders, one family, eight genera and eight differential microorganisms in the lower respiratory tract of the smoking and nonsmoking groups. The increased groups in the smoking group were the orderSelenomonadales, the genera Megasphaera,Faucicola,Sphaerochaeta, Shuttleworthia, Anaeroglobus, Megasphaera micronuciformis, Spirochaeta canine oral taxon 379,UnculturedPrevotella, andPrevotella sp. oral clone FW035; the increased groups in the non-smoking group wereSphingobacterium class,Sphingobacterialesorder, Chitinophagaceaefamily,Burkholderia, Vibrionimonas genus,Ralstoniagenus,Prevotella veroralis,Catonella sp. oral clone FL037, Treponema amylovorum, and Streptococcus anginosus. There was a positive correlation betweenthe differential microorganisms (genus level) in both the smoking and non-smoking groups (r ≥ 0.6) . The smoking and nonsmoking groups differed mainly in the pathways related to substance synthesis, degradation and transportation. Life activitiesthat were functionally enriched in the smoking group included: biosynthesis of ansamycins and lysine, mutual transformation ofpentose and glucuronic acid, biosynthesis of pantothenate and coenzyme A, and protein export, etc.; life activities that were mainlyenriched in the non-smoking group included: toluene degradation, pathogenic cycle of V. cholerae, degradation of limonene andpinene, photosynthesis, and dioxin degradation, etc. Conclusion Smoking can increase the relative abundance of lower respiratorymicrobiota, affect the structural composition of microflora, and change the synthesis, degradation and transportation pathwaysof related substances, and functional changes of respiratory microbiome exposed to cigarette smoking may affect physiologicalhomeostasis of hosts, disrupt the balance of the immune system in vivo, and thus cause lung diseases.

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