脓毒症急性肺损伤小鼠肠肺菌群结构变化分析

Analysis on the structural changes of intestinal and pulmonary flora in mice with sepsis and acute lung injury

目的探讨脓毒症急性肺损伤小鼠血液、肺组织、粪便肠道微生态结构变化及关联。方法将12只健康雄性C57BL/6J小鼠按随机数字表法分为盲肠结扎穿孔术(CLP)组和假手术(Sham)组,每组6只。CLP组采用盲肠结扎穿刺法(cecal ligation punctual,CLP)制备脓毒症小鼠急性肺损伤模型;Sham组只开腹但不进行盲肠结扎穿孔。术后24 h取各组小鼠眼球血、肺组织、粪便。HE染色观察肺组织形态变化,16s核糖体RNA测序分析脓毒症小鼠各部位菌群微生态的结构变化并找出其中关联。结果(1)HE染色显示,CLP组小鼠肺泡腔有渗出、肺组织结构紊乱、伴随大量的炎性细胞浸润,肺组织病理评分较Sham组增高,差异有统计学意义(P<0.01)。(2)α多样性分析显示,Sham组与CLP组血液及粪便样本比较无统计学意义,肺组织样本中Ace指数、Chao指数、Simpson指数具有统计学意义(P<0.05)。(3)β多样性分析:Sham组与CLP组血液及粪便样本比较,组间差异均大于组内差异,分析Bray-curtis、Weighted、Unweighted指数具有统计学意义(P<0.05)。(4)在门水平,对比于Sham组,CLP组的变形菌门丰度逐渐增加,厚壁菌门及放线菌门丰度降低;在属水平,Sham组主要以不动杆菌属和杜氏杆菌属为主,而CLP组主要以大肠埃希菌和未分类的肠杆菌属为主。对比于粪便菌群,血液与肺组织菌群构成更为相似。结论脓毒症急性肺损伤小鼠血液、肺组织、以及肠道的菌群分布存在部分重合。

Objective To investigate the microecological structure changes and correlation in blood,lung tissue and fecal intestine of mice with sepsis and acute lung injury.Methods A total of 12 healthy male C57BL/6J mice were divided into the cecal ligation and perforation(CLP)group and sham operation(sham)group by random number table method,with six mice in each group.In the CLP group,acute lung injury model of sepsis mice was prepared by CLP method.In the sham group,only laparotomy but no perforation of cecal ligation was performed.Eye blood,lung tissue,and feces were collected from mice in each group 24 h after surgery.Lung tissue morphological changes were observed by HE staining,and 16s ribosome RNA sequencing was used to analyze the structural changes of microecology of the bacterial flora at each site in sepsis mice and find out the correlation.Results(1)HE staining showed that mice in the CLP group had exudation into the alveolar cavity of the lung,disordered lung tissue structure,accompanied by a large number of inflammatory cell infiltration,and the lung histopathological score was significantly higher than that in the sham group(P<0.01).(2)αdiversity analysis showed that there was no statistical significance in blood and fecal samples between the sham group and CLP group,while Ace index,Chao index and Simpson index in lung tissue samples were statistically significant(P<0.05).(3)βdiversity analysis showed that the differences in blood and fecal samples were greater between the sham group and CLP group than that within the group,and analysis of Bray Curtis,weighted,and unweighted indexes were statistically significant(P<0.05).(4)At the phylum level,compared with the sham group,the abundance of Proteobacteria gradually increased,and the abundance of Firmicutes and actinobacteria was decreased in the CLP group.At the genus level,the sham group was dominated by Acinetobacter and Duchenne,while the CLP group was dominated by Escherichia coli and unclassified Enterobacter.Blood flora was more similar to lung tissue flora composition as compared with fecal flora.Conclusions The distribution of bacterial flora in blood,lung tissue and intestine of sepsis mice with acute lung injury is partially overlapped.