摘要
目的观察粪菌移植(FMT)对脂多糖(LPS)诱导大鼠急性肺损伤(ALI)的影响,探讨其可能的作用机制,为急性肺损伤的治疗提供新的思路。方法成年健康雄性SPF级SD大鼠45只,按随机数字表法分为正常对照(NS)组、LPS组和FMT组,每组15只。采用腹腔注射LPS 5 mg/kg制ALI模型;FMT组于造模完成后灌胃粪菌液10 ml/kg(2次/d,连续2 d)进行干预。各组分别于干预结束后24 h、48 h、72 h随机处死5只大鼠,取肺组织行HE染色观察肺组织病理改变并对其进行病理学评分,取右肺检测肺湿/干重(W/D)比值,腹主动脉采血行PaO2分析,酶联免疫吸附试验(ELISA)检测支气管肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)、IL-1β、IL-6的含量,逆转录-聚合酶链反应(RT-PCR)检测肺组织中转化生长因子-β(TGF-β)、胞内信号转导蛋白Samds(Smad3、7)mRNA的表达,蛋白免疫印迹法(Western blot,WB)测定肺组织中ERK1/2、p-ERK1/2蛋白的表达量,收集大鼠粪便样品并提取DNA,对V3、V4区16S rDNA的PCR产物进行高通量测序,并对OTU为分类基础的微生物群落进行生物信息学分析。结果LPS组与同时间点NS组比较,肺组织肺泡间隔明显增宽,可见大量炎性细胞浸润,部分肺泡腔萎陷不张;FMT组较LPS组大鼠肺组织炎性细胞浸润、肺间质肿胀程度明显减轻,正常肺组织结构破坏少。与NS组比较,LPS组肺W/D比值、PaO2、BALF中TNF-α、IL-1β、IL-6的含量在干预后24 h明显升高,随后逐渐降低,但仍高于NS组(P<0.05);FMT组肺W/D比值、PaO2及BALF中TNF-α、IL-1β、IL-6含量明显少于同时间点LPS组(P<0.05)。与NS组比较,肺组织中TGF-β1、Smad3在各时间点的表达增多,Smad7的表达减少(P<0.05);FMT组TGF-β1、Smad3的表达明显低于与同时间点LPS组(P<0.05),Smad7的表达明显高于同时间点LPS组(P<0.05)。与同时间点NS组比较,LPS组p-ERK表达显著增加,其中第24小时表达量最高,第48和72小时逐渐降低(P<0.05);FMT组p-ERK表达显著低
Objective To observe the effects of fecal microbiota transplantation(FMT)on acute lung injury(ALI)induced by lipopolysaccharide(LPS)in rats and to investigate the possible mechanism in order to provide new thoughts for the treatment of acute lung injury.Methods Forty-five adult healthy male SPF level SD rats were randomly divided into three groups of normal saline(NS),LPS and FMT with 15 in each group.ALI was induced by intraperitoneal injection of rats with 5 mg/kg of LPS.The FMT group was given 10 ml/kg of fecal microbiota solution intervention(twice a day for two consecutive days)after ALI induction.Five rats in each group were randomly selected and sacrificed at 24 h,48 h and 72 h after intervention.Pathological changes in lung tissues were examined with hematoxylin and eosin(HE)staining and pathological scores were assessed.Right lung samples were weighed to measure wet/dry(W/D)ratios.Abdominal aorta blood samples were collected for PaO2 analysis.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin 6(IL-6)in bronchoalveolar lavage fluid(BALF)were detected by enzyme linked immunosorbent assay(ELISA).Expression of transforming growth factor-β(TGF-β)and intracellular signal transduction protein Smads(Smad3 and Smad7)at mRNA level was analyzed by reverse transcription-polymerase chain reaction(RT-PCR).Expression of ERK1/2 and p-ERK1/2 at protein level was detected by Western blot(WB).Rat fecal samples were collected to extract DNA and the PCR products of V3 and V4 regions were sequenced by Illumina MiSeq.Bioinformatics analysis on microbiota was conducted with Illumina MiSeq based on operational taxonomic unit(OTU)clustering.Results Compared with the NS group,the LPS group showed significantly widened alveolar septa,massive inflammatory cell infiltration and some alveolar collapse in lung tissues.Compared with the LPS group,the FMT group showed significantly alleviated inflammatory cell infiltration,reduced swelling in pulmonary interstitial tissues,and less damage to pulmonary
作者
李波
尹国芳
范贤明
Li Bo;Yin Guofang;Fan Xianming(Department of Respiratory and Critical Care MedicineⅡ,Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2019年第11期812-820,共9页
Chinese Journal of Microbiology and Immunology
基金
四川省科技厅-泸州市人民政府-泸州医学院联合科研专项资金计划项目(14ZC0048)
泸州市人民政府-西南医科大学科技战略合作项目(2019LZXNYDJ04)。
关键词
急性肺损伤
粪菌移植
高通量测序
脂多糖
Acute lung injury
Fecal microbiota transplantation
High-throughput Sequencing
Lipopolysaccharide
