Impact Of Fecal Microbiota Transplantation On TGF-?1/Smads/ERK Signaling Pathway Of Acute Lung Injury In Rats

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:Fifteen adult healthy male SPF level SD rats were randomly divided into normal saline?NS group?,LPS group,and FMT group,and each group consisted of 5 rats.LPS group and FMT group were intraperitoneally injected with LPS 5mg/kg to replicate the ALI model.FMT group was administered to fecal microbiota solution?10 ml/kg?by gavage 24h after completion of modeling,while NS group and LPS group were administered to the same amount of NS,twice a day for two days.Five rats in each group were sacrificed 24 hours after the intervention.Left lung tissue was examined after hematoxylin and eosin?HE?staining and the pathology score was observed.The right lung was weighed to measure lung wet/dry weight ratio?W/D?,the abdominal aorta blood was collected for Partial arterial oxygen pressure?PaO₂?analysis,tumor necrosis factor-??TNF-??,interleukin-1??IL-1??,interleukin 6?IL-6?in serum and bronchoalveolar lavage fluid?BALF?were determined by enzyme linked immunosorbent assay?ELISA?.The mRNA expressions of transforming growth factor-?1?TGF-?1?,intracellular signal transduction proteins Smads?Smad3,Smad7?in lung tissues were determined by reverse transcription-polymerase chain reaction?RT-PCR?.The expressions of TGF-?1,Smad3,and Smad7 in lung tissues were determined by immunohistochemical?IHC?.Western Blot?WB?was used to detect the expression of extracellular regulated kinases1/2?ERK1/2?and phosphorylation ERK?p-ERK?protein in lung tissues.The fecal samples and deoxyribonucleic acid?DNA?of rats were extracted,and the polymerase chain reaction?PCR?products of V3 and V4were sequenced by metagenomic high throughput.Informatics analysis on microbiota was conducted with Illumina MiSeq based on information of Operational Taxonomic Unit?OUT?peak degree.Results:Compared with the NS group,the lung tissue of the LPS group showed significantly widened alveolar septa,a large number of inflammatory cell infiltration,and partial alveolar collapse.Compared with the LPS group,the FMT group showed less inflammatory cell infiltration and interstitial lung exudates with significantly reduced damage of normal alveolar structure.Compared with the NS group,the PaO₂ in the LPS group was significantly reduced?P<0.05?,and the PaO₂ in the FMT group was higher than that of the LPS group?P<0.05?.The levels of lung W/D,TNF-?,IL-1?,and IL-6 in serum and BALF in the LPS group were significantly higher than those in the NS group?all P<0.05?.FMT group lung W/D,TNF-?,IL-1?,and IL-6 in serum and BALF was significantly less than that in the LPS group?all P<0.05?.Compared with the NS group,the expressions of TGF-?1,Smad3,and p-ERK in lung tissues of LPS group were significantly increased?all P<0.05?,while the expression of Smad7 decreased?P<0.05?.The expressions of TGF-?1,Smad3,and p-ERK in the FMT group were significantly lower than those in the LPS group?all P<0.05?,and the expression of Smad7 was significantly higher than that in the LPS group?P<0.05?.The results of intestinal microbiota sequencing revealed 1342 OTU in the NS group,1216 OTU in the LPS group,1567 OTU in the FMT group,and 593 OTU were shared,with the percentage of Shared OTU being 37.3%.The structure of intestinal microbiota between the LPS group and NS group has a significant difference.Characterized by lower diversity index,Firmicutes/Bacteroidetes decreased proportionally and the gene abundance of intestinal microbiota decreased.Compared with the LPS group,the structure of intestinal microbiota in the FMT group was close to the NS group,the ratio of Firmicutes/Bacteroidetes increased,the diversity index and the gene abundance of the bacterial community increased.Conclusion:FMT might act on the TGF-?1/Smads/ERK pathway by regulating intestinal microbiota,inhibiting immune inflammation,reducing the production of inflammatory markers in the body and release,and reducing alveolar epithelial damage and repair,thereby improving the endotoxic ALI in rats induced by LPS.