| Research background:Lung injury is a pathological characteristic characterized by macrophage dominated inflammatory cell infiltration,epithelial integrity disruption,and barrier dysfunction.When the injury is severe,initiating a repair program based on fiber proliferation leads to fibroblast proliferation and collagen deposition,leading to irreversible fibrosis of the lung parenchyma.According to reports,there are many causes of lung injury,such as medication use,viral infections,various traumas,and long-term smoking.Chest pain,cough and progressive dyspnea are the main characteristics of lung injury and Pulmonary fibrosis,which may develop into respiratory distress or even shock in severe cases.A global system analysis shows that in 2017,544 million people worldwide suffered from chronic respiratory diseases and were listed as the third leading cause of death.Because of the global pandemic of COVID-19,the number of patients with lung injury has been too large to be counted.Due to its elusive pathogenic mechanism,there is currently no effective medication to treat or prevent it.So far,hormone therapy is commonly used in clinical practice,but the side effects are obvious such as gastrointestinal bleeding and femoral head necrosis,and the efficacy is limited.N-acetylglucosamine(NAG)is an acetylated derivative of glucose and a fundamental component of polysaccharides in biological cells.The molecular weight of NAG is only221.21 Da,which allows it to penetrate the cell membrane to achieve biological functions.Research has shown that NAG and its derivatives have numerous biological activities,such as antioxidant,anti-inflammatory,and anti-tumor properties.In particular,NAG shows a very strong therapeutic potential for Adenocarcinoma of the lung.It is worth noting that in a recent phase I clinical trial,NAG has been proved to significantly reduce the mortality of patients with COVID-19 and improve respiratory symptoms,but its underlying mechanism is still unclear.Objective:1、To evaluate the effect of N-acetylglucosamine(NAG)on lung function after Bleomycin(BLM)induced lung injury.2、Analyzing the morphological changes and collagen fiber deposition in lung tissue during BLM induced lung injury induced by NAG.3、Detect the changes in the number of macrophages in the lungs after BLM induced lung injury by NAG.4、Analyze the changes in gene expression during BLM induced lung injury induced by NAG,and reveal the potential pathogenesis of the disease.Methods:1.Construction of a mouse model of NAG protection against BLM induced lung injury:Constructing a lung injury model through tracheal instillation of BLM.The experiment was divided into three groups:Control,BLM,BLM+NAG.The lung function indexes of mice were measured by using the forced oscillatory plethysmography scientific respiratory equipment(Flexi Vent;SCIREQ Inc),such as the dynamic elasticity of the respiratory system elastance(Ers),the dynamic resistance of the respiratory system respiratory resistance(Rrs),the dynamic compliance of the respiratory system compliance(Crs),the forced vital capacity(FVC)and the main airway resistance newtonian resistance(Rn).To evaluate the effect of NAG on lung function in BLM-induced lung injury mice.2.At the tissue level,the lung tissue of mice was stained by H&E staining and Masson staining,and the pathology of lung tissue was observed.The effect of NAG on the protection of lung tissue morphology and the inhibition of pulmonary fibrosis in the lung injury model was evaluated by measuring the content of hydroxyproline in the lung of mice.3.At the cellular level,the proportion of macrophages in bronchoalveolar lavage fluid(BALF)and the relative expression of ARG1~+macrophages in mouse lung tissue were measured by immunofluorescence method to evaluate the effect of NAG on macrophages in lung injury model.4.At the cellular level,the expression of KRT8~+alveolar differentiation intermediate cells were detected by immunofluorescence staining of lung tissue.Detect the expression of PDGF Rβ~+fibroblasts by immunohistochemical staining of lung tissue.To evaluate the effect of NAG on the process of lung injury and then on pulmonary fibrosis.5.At the molecular level,transcriptome sequencing was used to analyze the expression of differential genes and the gene ontology(GO)analysis of reverse genes after NAG treatment in mice,to further explore the mechanism of NAG protecting lung from injury.Results:1.The lung function of mice was tested 28 days after BLM injury.Compared with the Control group,the average values of Ers,Rrs,and Rn in the BLM group increased by 10.9%,53.0%,and 47.8%,respectively.The Crs and FVC decreased by 25%and 15.5%,which reflected the impairment of lung contractility and elasticity.However,after NAG treatment,the above indicators improved.Although there was no statistically significant difference in Rn parameters after NAG treatment compared to the BLM group,Rn tended to develop in the Control group.These results indicate that NAG improves lung function in BLM induced lung injury.2.H&E staining showed that the lung tissue structure of BLM injury group was seriously damaged,and the loose and regular alveolar cavity disappeared.In contrast,dense lung tissue is separated by bullae.However,the alveolar cavity in the NAG treatment group was relatively loose and there was no obvious structural damage area.In Masson’s staining,NAG treatment also reversed the changes of lung tissue morphology and collagen deposition.Collagen fibers(hydroxyproline is a unique amino acid)are deposited in an amount of fibrotic lung.Determination of hydroxyproline content can reflect the degree of fibrosis.In our experimental model,the content of hydroxyproline in the BLM injury group was twice higher than that in the Control group,and the NAG protection group significantly inhibited the increase of hydroxyproline caused by BLM treatment.In conclusion,we demonstrated that NAG has protective effect on BLM-induced lung injury.3.After 14 days of BLM injury,in order to explore the possible mechanism of lung protective effect of NAG,the number of macrophages in BALF was statistically analyzed.The number of macrophages in the BLM injury group was more than twice that in the Control group.The infiltration of macrophages in NAG treatment group was significantly reduced.This indicates that NAG plays a protective role in lung injury by influencing macrophages.The number of ARG1~+cells after NAG protection was significantly lower than that after BLM injury.These data show that NAG plays a role in lung protection by reducing the number of ARG1~+macrophages.4.KRT8~+alveolar differentiation intermediate(ADI)cells also tended to normalize after NAG administration.PDGF Rβ~+with excessive proliferation in fibrosis after NAG treatment.The number of fibroblasts decreased significantly and was close to normal.5.RNA seq analysis revealed that among the first 78 differentially expressed genes in this model compared to the BLM group,65 genes were downregulated and 13 genes were upregulated after NAG treatment.These differentially expressed genes included Col1a1(Collagen Type I Alpha 1 Chain),Col3a1(Collagen Type Ⅲ Alpha 1 Chain)and other genes related to collagen protein,as well as protease related genes such as Mmp2(Matrix Metallopeptidase 2)that degrade extracellular matrix.The reversed genes are associated with biological processes such as extracellular matrix degradation,collagen degradation,collagen assembly,and antiviral activity through GO analysis.Conclusion:NAG can improve lung function,improve lung tissue morphology,reduce the content of hydroxyproline in lung tissue,and reduce the degree of pulmonary fibrosis in lung injury mice.This study found that the mechanism of NAG’s lung protective effect in BLM induced lung injury models may be that NAG affects the trans-differentiation process of fibroblasts by reducing the number of ARG1~+macrophage cells and KRT8~+intermediate state cells in alveolar differentiation,thereby alleviating lung injury.Significance:The research and development of new drugs is closely related to the urgent needs of human health and the development of the pharmaceutical industry.It not only meets the needs of China’s economic development and people’s livelihood,but also serves as a key factor for China to achieve strategic heights in the international life sciences and biotechnology fields.NAG,as a naturally occurring monosaccharide in the exoskeleton of crustaceans,has various biological functions such as antioxidant and anti-inflammatory.This study for the first time confirms the protective mechanism of NAG on lung injury and fibrosis by affecting macrophages and the process of trans-differentiation,providing new directions and theoretical basis for making NAG a protective substance for improving lung health.The development and application of NAG related lung protection products will take a big step forward in human lung health and benefit people’s well-being. |
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