粪菌移植对大鼠内毒素性急性肺损伤的作用及其机制研究

Involvement and mechanism of fecal microbiota transplantation on endotoxic acute lung injury in rats

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表达显著低于同时间点LPS组(P<0.05)。大便菌群测序结果揭示NS组含1362个OTU,LPS组含1443个OTU,FMT组含有1510个OTU,共享OTU为336个,共有的OTU所占百分比为26.4%。LPS组大鼠肠道菌群结构与正常大鼠肠道菌群结构具有显著不同,表现为较高的多样性指数,厚壁菌门/拟杆菌门比例增高、肠道菌群基因丰度降低,经过粪菌移植干预后FMT组肠道菌群结构接近正常对照组、厚壁菌门/拟杆菌门比例降低、菌群基因丰度增加(P<0.05)。结论粪菌移植可能通过调节肠道菌群并作用于TGF-β1/Smads/ERK通路,抑制免疫炎症反应,减少体内炎症因子的产生和释放,减轻肺泡上皮的损伤和异常修复,进而改善LPS诱导的大鼠内毒素性ALI。

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 structure.Compared with the NS group,the W/D lung ratio,PaO2 concentration and TNF-α,IL-1 and IL-6 levels in BALF in the LPS group significantly increased at 24 h after intervention and then gradually decreased,but still higher than those in the NS group(P<0.05).The W/D lung ratio,PaO2 concentration and TNF-α,IL-1 and IL-6 levels in BALF of the FMT group were significantly lower than those of the LPS group(P<0.05).Compared with the NS group,the expression of TGF-β1 and Smad3 at mRNA level in lung tissues increased at all time points in the LPS group,while the expression of Smad7 at mRNA level decreased(P<0.05).The expression of TGF-β1 and Smad3 at mRNA level in the FMT group were significantly lower than that in the LPS group(P<0.05),while the expression of Smad7 at mRNA level was higher(P<0.05).Compared with the NS group,p-ERK expression significantly increased in the LPS group with the highest expression at 24 h and gradually decreased at 48 h and 72 h(P<0.05).The expression of p-ERK in the FMT group was significantly lower than that in the LPS group(P<0.05).Results of the intestinal microbiota sequencing revealed 1362 OTU in the NS group,1443 OTU in the LPS group and 1510 OTU in the FMT group.There were 336 OTU shared by them accounting for 26.4%.The constitution of intestinal microbiota in the LPS group was significantly different with that of normal rats and characterized by high diversity with increased Firmicutes/Bacteroidetes ratio and decreased gene abundance of intestinal microbiota.After the intervention of fecal bacteria transplantation,the constitution of intestinal microbiota in the FMT group was similar to that of the NS group,showing decreased Firmicutes/Bacteroidetes ratio and increased gene abundance of the bacterial community(P<0.05).Conclusions FMT might inhibit immune inflammation,reduce the production and secretion of inflammatory markers in the body,alleviate alveolar epithelial damage and abnormal repair through regulating intestinal microbiota and TGF-β1/Smads/ERK pathway to improve the LPS-induced endotoxic ALI in rats.