Therapeutic Effect Of Hemoglobin-based Oxygen Carrier On Bleomycin-induced Pulmonary Fibrosis In Mice

Objective: Mice with bleomycin（BLM）-induced pulmonary fibrosis were used as a model,and after successful modeling and infusion of hemoglobin oxygen carrier（HBOC）,the exercise tolerance of mice was observed,and the indices of HBOC on histopathology,electron microscopy,hypoxic probes,and inflammatory indexes of mice were analyzed.Transcriptome sequencing was used to investigate the potential regulatory mechanisms of HBOC on bleomycin-induced pulmonary fibrosis in mice.Methods: 120 male C57BL/6J mice of eight weeks.The mice were randomly divided into 4 groups.The groups were divided as follows: Blank control group（tracheal injection of saline,n=15 mice）;BLM group（tracheal injection of BLM（3.5 U/kg）,n=35 mice）;BLM-L（HBOC（2.5 g/d L）,n=35 mice）;BLM-L（HBOC（5g/d L）,n=35 animals）.The general conditions of mice such as body weight,mouse mobility,and mouse fur color,the behavior of mice and blood gas analysis were recorded and monitored daily during the modeling feeding.On the 22 nd day after modeling,the mice specimens were obtained for the following tests:（1）Weigh the body and wet weight of the mice’s lungs,and calculate the lung coefficient.（2）The right lower lung tissue was soaked in formalin and embedded in paraffin,and then stained with HE,Masson,TUNEL and hypoxic probe.（3）The right upper lobe was examined by making electron microscopic sections to observe the microstructure of the lung.（4）Obtained mouse bronchoalveolar lavage fluid to detect cytokines by flow cytometry.（5）Obtained lung tissues from mice by extracting RNA for transcriptome sequencing,screening for differential genes,enriching for differential gene-related pathways,and further exploring the potential mechanism of HBOC on IPF mice through analysis of the TF-miRNA-Gene triple interaction network.Results: A consistent level of reduction in the rate of body weight change in mice after BLM modeling was observed after successful modeling of pulmonary fibrosis,and the rate of body weight change in mice could be reduced by infusion of HBOC,which was statistically significant between BLM group and BLM-H group（P=0.0450）.There was no statistical difference between all four groups of mice in the fatigue rotameter.Time of treadmill,distance of treadmill and distance of treadmill × body weight were statistically different between BLM group and BLM-H group（P<0.05）.HBOC infusion significantly improved the lung coefficients of mice with pulmonary fibrosis.The results by pathological section staining,electron microscopic analysis and bronchoalveolar lavage showed that high concentrations of HBOC could reduce inflammation and fibrosis in pulmonary fibrosis mice to some extent.The results of hypoxic probing suggested that HBOC could reduce the degree of hypoxia in lung tissue to some extent.After screening out significant differentially expressed genes by transcriptome sequencing analysis,the pathways of differential genes and key modules were analyzed by metascape and cytoscape to screen out key genes,and the transcription factors and miRNAs of key genes were predicted to construct a TF-miRNA-Gene triple interaction network to further reveal the effect and potential mechanism of HBOC on BLM-induced of pulmonary fibrosis in mice.Conclusion: Thesis verified for the first time that HBOC infusion improved exercise tolerance and hypoxia in mice with BLM-induced pulmonary fibrosis,and to some extent reduced inflammation and fibrosis in mice with BLM-induced pulmonary fibrosis,and further revealed the potential regulatory mechanism of HBOC treatment of pulmonary fibrosis in mice by transcriptome sequencing.