The Study On The Regulatory Mechanism Of FOXN3 On Acute Lung Injury

BackgroundAcute lung injury（ALI）is a respiratory disease caused by infection or trauma.The injury of vascular endothelial cells and alveolar epithelial cells caused by ALI will induce diffuse pulmonary interstitial and alveolar edema,resulting in acute hypoxic respiratory insufficiency or respiratory failure.With the progression of this disease,ALI will gradually develop into acute respiratory distress syndrome（ARDS）,which is a life-threatening condition.FOXN3（Fork head box N3）is one of the most important transcription factors in the forkhead box protein family,which is associated with multiple inflammatory regulators.However,its role and mechanism in ALI-induced inflammatory response remains unclear.Objectives1.To explore the critical role of FOXN3 in regulattion of ALI;2.To reveal the specific molecular mechanism of FOXN3 in regulation of ALI;Methods1.To observe the expression of FOXN3 in ALI mouse model induced by LPS and S.aureus;C57BL/6 mice（8-9 weeks）were purchased from Charles River China,LPS（4 mg/kg）or S.aureus（2×10~8colony-forming units per mouse）were injected into mice by endotracheal intubation.The control group was injected with the same volume of PBS.After 4 days,lung tissue samples were harvested,and the expression of FOXN3 between ALI and control group were analyzed by q RT-PCR.On the other hand,IHC（Immunohistochemical staining）was performed to check the changes in protein expression of FOXN3 on histology.2.Adeno associated virus（AAV）was used to overexpress FOXN3 in mice to observe the effect of overexpression of FOXN3 on acute lung injury;AAV overexpressing FOXN3 and Vec control group were injected into the lung tissue of mice by intratracheal instillation.Three weeks later,the lung tissue was induced by LPS or S.aureus inflammatory injury and lung tissue samples were taken.The degree of inflammatory injury of lung tissue after FOXN3 overexpression were evaluated by H＆E and CD68 IHC staining;The expression of inflammatory factors in lung tissue was detected by q RT-PCR;The BALF（Bronchoalveolar Lavage Fluid）was collected for measuring the total protein concentration and cytokine content.All these experiments were to determine the effect of FOXN3 overexpression on ALI.3.To analyze the critical regulatory factors combined with FOXN3 by mass spectrometry;The plasmid of flag-tagged FOXN3 was transfected into HEK293T（Human Embryonic Kidney 293T）cells,FOXN3 was enriched by Co-IP（Co-Immunocoprecipitation）with anti-flag antibody,after Western Blot and Coomassie brilliant blue staining,mass spectrometry-based peptide segments analysis of the extra bands which was distinct with Ig G control group to identify the important regulatory factors binding to FOXN3 specifically.4.To explore the regulation of FOXN3 on NF-κB signaling pathway by Co-IP,Ch IP（Chromatin Immunoprecipitation assay,Ch IP）,q RT-PCR and ubiquitination experiment;Mass spectrometry analysis showed that FOXN3 may involved in the regulation of NF-κB signalling pathway by hnRNPU.In order to further prove this hypothesis,Co-IP assay was used to further confirm the interaction between FOXN3 and hnRNPU.Next,the effect of FOXN3 was observed by Ch IP or q RT-PCR with anti-p65 on the transcriptional activity of NF-κB and IκBαubiquitination experiment on the ubiquitination degradation after interference or overexpression of FOXN3.Results1.The ALI mouse model was successfully constructed by LPS or S.aureus,and the expression of FOXN3 was significantly decreased in the ALI model;2.The overexpression of FOXN3 in mouse lung tissue was carried out by AAV,,and the overexpression of FOXN3 significantly inhibited ALI induced inflammatory phenotype compared with wild-type（WT）mice,including alveolar edema,alveor wall thicken,inflammatory cells infiltration,alveolar permeability and expression of inflammatory factors as well;3.The inhibition of FOXN3 on the activation of NF-κB signalling pathway was confirmed by ubiquitination,Ch IP and q RT-PCR;4.The deletion mutants in different regions of hnRNPU were constructed,Co-IP assay was performed to verify that FOXN3 inhibited the ubiquitination degradation of IκBαby competitive binding with the intermediate region of hnRNPU,which further repressed the activation of NF-κB signalling pathway;Conclusion1.FOXN3 significantly inhibited the inflammation induced by ALI;2.FOXN3 inhibits the activation of NF-κB signalling pathway;3.FOXN3 inhibited the ubiquitination degration of IκBαand the activation of NF-κB signalling pathway by competitive binding with hnRNPU;...