Fecal Bacteria Transplantation Improves Endotoxin-induced Acute Lung Injury By Regulating TLR4/NF-κB Signaling Pathway

Objective:Through the use of fecal bacteria transplantation（FMT）to interfere with lipopolysaccharide（LPS）-induced acute lung injury（ALI）in rats,observe the influence of intestinal microbes on lipopolysaccharide-induced acute lung injury in rats,and explore the effect of fecal bacteria transplantation in improving acute lung injury Pathological mechanism,fully explore the effect of fecal bacteria transplantation on Toll like rece-ptors 4（TLR4）/nuclear factor-kappa B（NF-κB）signaling pathway in acute lung injury.Methods:Fifteen adult healthy SD rats were selected,According to the random number table method,rats were divided into the following groups:control group（NS group,5 rats）,model group（LPS group,5 rats）and intervention group（FMT group,5rats）.LPS was injected intraperitoneally into LPS group and FMT group respectively to prepare ALI model.24 hours after injection,FMT group was given fecal bacteria solution for 2 consecutive days,while NS group and LPS group were given the same amount of 0.9%sodium chloride solution.24 hours after the intervention,all rats were killed and related items were analyzed:one of the lung tissues was stained with hematoxylin eosin（H-E）to observe the pathological changes of lung tissue,and the degree of lung injury was scored,The ratio of wet/dry weight（W/D）of lung was detected in another lung tissue;arterial blood was taken to measure arterial partial pressure of oxygen（Pa O₂）;the content of interleukin-10（IL-10）in serum was detected by enzyme-linked immunosorbent assay（ELISA）;Western blot was used to detect the content of IL-10 in serum The expression of TLR4/NF-κB signaling pathway related proteins in lung tissue was determined by Western blot（WB）;the expression of myeloid differentiation factor 88（My D88）in alveolar cells was detected by IHC;After collecting the fecal samples of rats,the DNA of microorganisms in feces was extracted.The variable regions V3and V4 of 16Sr DNA were selected for PCR amplification,and then high-throughput sequencing was performed on Illumina miseq platform.Results:The changes of lung W/D in each group:compared with NS group,the W/D value in LPS group increased significantly（P<0.05）,while the W/D value in FMT group decreased（P<0.05）.Results:compared with NS group,Pa O₂ in LPS group was significantly lower（P<0.05）,while Pa O₂ in FMT group was higher（P<0.05）.Lung pathomorphological analysis:compared with NS group,the pathological changes of ALI in LPS group were as follows:the alveolar wall structure was destroyed in different sizes,a large number of inflammatory cells gathered in the lung parenchyma and interstitial,pulmonary microvascular hemorrhage and thrombosis were formed,a large number of liquid exuded into the interstitial and alveolar cavity,and the pathological score of lung tissue was increased（P<0.05）;while in FMT group,the lung tissue injury was significantly alleviated,and the alveolar edema was increased Compared with LPS group,the pathological score of lung tissue decreased,P<0.05,and the difference was statistically significant.Analysis of serum inflammatory indexes:the concentration of IL-10 in LPS group was significantly lower than that in NS group（P<0.05）,while the concentration of IL-10 in rats after FMT was higher than that in LPS group（P<0.05）.TLR4/NF-κB signaling pathway in lung tissue:compared with NS group,the expressions of TLR4,P-NF-κBp65 and My D88 in LPS group were significantly up-regulated（P<0.05）,while the expressions of TLR4,P-NF-κBp65 and My D88 in FMT group were down regulated（P<0.05）.Changes of intestinal microflora:compared with NS group,LPS group showed disorder of normal microflora,structural disorder of intestinal microflora,decreased diversity and abundance,while FMT group gradually recovered and approached NS group.Conclusion 1.The ALI model of rats in each group was successfully established.FMT has a certain protective effect on the lung tissue of rats with ALI,and the pathological structure of the injured lung tissue was improved.2.The expression of key proteins TLR4,My D88,and P-NF-κB p65 in the TLR4/NF-κB cell signaling pathway in rat lung tissue was decreased by FMT intervention,which promoted the secretion of anti-inflammatory factor IL-10,and finally effectively inhibited the inflammatory state of rat lung tissue.3.FMT may improve lipopolysaccharide-induced ALI in rats by targeting TLR4/My D88/NF-κB cell signaling pathway and inhibiting immune inflammatory response in lung tissue.