| Forest musk deer(Moschus berezovskii,FMD)is a ruminant animal belonging to artiodactyla,musk deer family and musk deer genus.At present,FMD has been included in the national protection of animals.Duo to the overpoaching for musk and damaging the habitat of FMD,the number of wild FMD has declined precipitously.Though the captive program had been executed for more than 60 year,the captive population is still small for many reasons,especially the high susceptibility of FMD to pneumonia.Studies have shown that bacterial infection is the main cause of pneumonia in FMD,and the research on FMD diseases includes mainly isolation and identification of pathogens.The pathogenesis of FMD bacterial pneumonia is still unclear.Thus,there is an urgent need for further research in this field.Circulating mi RNA is an important biological molecule in fluid circulation that is secreted from the cells of pathological tissue.As a cellular communication mode,circulating mi RNA plays a role on information transfer from organs/tissue to the body fluid.A series of achievements have been made in the research of molecular etiology and treatment targets.FMD is an endangered and protected animal in China,just like other protected animals,it is forbidden to take samples of FMD lung.However,it is not conducive to the study of bacterial pneumonia of FMD without healthy control.In this study,the expression profile of circulating mi RNA in bacterial pneumonia FMD blood was detected by high-throughput sequencing,and the mi RNA related to bacterial pneumonia of FMD was screened out by combining with the constructed rat model of FMD pathogens mediated bacterial pneumonia.Finally,we subcultured the lung fibroblast cells of FMD,and the function of differentially expressed mi RNA in bacterial pneumonia was analysed by the FMD lung fibroblast cells.In this dissertation,the following experiments were conducted:(1)In this study,five FMD were dead with bacterial pneumonia.At autopsy,it was observed that the dead FMD lungs were severely swollen,covered with petechial hemorrhages,and surrounded by a yellow peptone-like exudate.The results of pathogen isolation and identification showed that the five FMD individuals died of bacterial pneumonia,and a pathogen was isolated from the lung of each of the dead FMD,including two Pseudomonas aeruginosa strains,one Klebsiella pneumoniae strain,one Streptococcus equinus strain and one Trueperella pyogenes strain,respectively.In the five FMD lungs,histopathological examination showed some similar pathological changes in each individual,mainly including the structure of normal alveolus was losted,fibrous tissue was increased,and the lung interstitium was filled with a large number of fibroblast cells,neutrophils and lymphocytes.Picro sirius red and Masson’s trichrome staining showed that the increased fibrin and collagen were found in lung tissue of FMD,implying that the five dead FMD were suffered from bacterial infection with pulmonary fibrosis.The results of circulating mi RNA analysis showed that the expression level of mi R-451-3p,mi R-142-5p,mi R-652,mi R-9-5p and mi R-206-3p were upregulated in the dead group,four mi RNAs,mi R-30 g,let-7f-5p,mi R-25-3p and mi R-27d-3p,were downregulated in the dead group.Besides,target gene prediction analysis showed that a total of 99 target genes were involved in ECM-receptor interaction and focal adhesion signaling pathways,which may be of particular importance in this study since these two pathways often associated with pulmonary fibrosis.Finally,the results of RT-q PCR showed that the trend of 5 differentially expressed mi RNAs(mi R-30 g,let-7f-5p,mi R-27-3p,mi R-25-3p and mi R-652)expression was consistent with the high-throughput sequencing data.(2)In order to further verify the function of the differentially expressed mi RNAs that associated with bacterial pneumonia in FMD,we constructed a rat model of bacterial pneumonia using FMD strains Klebsiella pneumoniae,Pseudomonas aeruginosa and Streptococcus equinus through nasal drop infection.The results showed that the lung weight ratio of rats was increased by infecting with three pathogens.The results of HE staining showed that the three pathogens caused the exfoliation of alveolar epithelial cells,the collapse of alveolar walls,the proliferation of fibrous tissue,and the exudation of fibroblasts,fibrocytes and fibro-like substances in the lung interstitium with infiltration of inflammatory cells.Masson staining and Sirius red staining showed that lung collagen and fibrin exudate increased in all test groups.Besides,the expression level of TGF-β1 and TNF-α were increased(p < 0.05)in the lung tissues of test group rats,indicating that pulmonary fibrosis occurred in test group rats.In the blood and lung of rat,RT-q PCR results showed consistent expression trends of let-7f-5p and mi R-652 with the FMD blood transcriptome analysis.Notably,the let-7f-5p expression level was significantly lower in the bacterial pneumonia FMD and rat(p < 0.01)and high expression level of the PI3K/Akt/COX2 signaling pathway genes m RNA(PIK3CA,PDK1,Akt1,IKBKA,NF-κB1 and COX2)and proteins found in the lung of the three test group rats,suggesting that there is a potential correlation between bacteria-induced pulmonary fibrosis and the PI3K/Akt/COX2 signaling pathway.(3)To further verify the targeting relationship between let-7f-5p and PIK3 CA gene,we first analyzed the conservation of let-7f-5p among different species and its complementary relationship with PIK3 CA 3’UTR.The conservative analysis of let-7f-5p showed that let-7f-5p sequence was highly conserved across human,rat,cow,rabbit and FMD,and the let-7f-5p mature sequence was located on the 5’ arm of the stem ring structure of pre-let-7f-5p in the human,rat and FMD species.Besides,the seed sequence of let-7f-5p complementary to the 3’UTR of PIK3 CA m RNA among many species,suggesting that the let-7f-5p may participate in post-transcriptional regulation of PIK3 CA gene.Besides,we successfully constructed the double luciferase reporter plasmid based on the target gene PIK3 CA wild-type(PIK3CA-WT)and mutant-type(PIK3CA-MUT).In the PIK3 CA wild-type recombinant plasmid group,double luciferase reporter gene assay showed that overexpression of let-7f-5p significantly decreased luciferase expression compared with transfection with let-7f-5p inhibitor(p < 0.05).While in the PIK3 CA mutant recombinant plasmid group,the expression of luciferase was not affected by overexpression or suppression of let-7f-5p.The result further confirmed the targeting relationship between let-7f-5p and PIK3 CA gene.(4)In order to further explore the effect of let-7f-5p on PI3K/Akt/COX2 signaling pathway in vitro,we constructed the lung fibroblast cell line of FMD.The FMD lung cells were isolated from lung tissues of one dead FMD by mechanical separation and trypsin cold digestion method.The cells were purified by differential adhesion method and then subcultured,and a series of biological characteristics were determined.The results showed that ciliary wobble could be observed in the collected lung tissue during trypsin treatment,and the primary cultured cells were mainly fibroblast-like cells and epithelial-like cells.During growth,the lung cells showed typical fibrous and long spindle shape,with strong refraction,full cytoplasm and clear nuclear cytoplasm.Microbial culture,DAPI staining and transmission electron microscopy showed that there was no microbial contamination in the lung fibroblast cells.The growth curve of the lung fibroblast cells was presented a typical "S" shape.The rabbit anti-vimentin and rabbit anti-cytokeratin immunofluorescence staining showed that the FMD lung fibroblast cells were positive for the expression of vimentin and negative for the expression of cytokeratin,which further confirmed that the cultured cells were lung fibroblast cells.Karyotype analysis showed that 58 chromosomes were found in the FMD lung fibroblast cells,suggesting that there was no chromosome aneuploidy.Furthermore,the expression of PI3K/Akt/COX2 signaling pathway genes(PIK3CA,Akt1,PDK1,IKBKA,NF-κB1 and COX2)were down-regulated in the FMD lung fibroblast cells after transfection of let-7f-5p.In addition,we construct an adeno-associated virus vector(rp AAV-CMV-e GFP-pri-let-7f-5p)that over-expressing let-7f-5p based on the FMD pri-let-7f-5p sequence,and recombinant adeno-associated virus(r AAV-let-7f-5p)was achieved.Finally,the expression level of let-7f-5p was increased after infection with r AAV-let-7f-5p,and the PI3K/Akt/COX2 signaling pathway was inhibited.These results suggest that let-7f-5p can inhibit the activity of PI3K/Akt/COX2 signaling pathway,and then participate in the pulmonary fibrosis disease that mediated by lung pathogens in FMD.In conclusion,we successfully identified that let-7f-5p and PI3K/Akt/COX2 signaling pathway related to FMD bacterial pneumonia through the analysis of mi RNA in FMD blood,rat blood and lung.Of note,the targeting relationship between let-7f-5p and PIK3 CA gene was verified by the double luciferase reporter gene experiment.Let-7f-5p could inhibit the activity of PI3K/Akt/COX2 signaling pathway through suppression of PIK3 CA expression in FMD lung fibroblast cells based on the overexpression of let-7f-5p.This study laid an important foundation for the study of pathogenesis of bacterial pneumonia in FMD. |
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