| Objective: Obstructive sleep apnea(OSA)is one of the most common complications in patients with interstitial pulmonary disease(ILD).It is common in moderate and severe obstructive sleep apnea in patients with idiopathic pulmonary fibrosis(IPF).OSA is associated with increased mortality in patients with IPF.The relationship between IPF and OSA is complex,and the pathogenesis is still completely unclear.The purpose of this study is to study the pathological mechanism of disease progression and increased mortality in patients with OSA and IPF exacerbation through bioinformatics analysis,and then explore the role of PI3K/Akt autophagy pathway in A549 lung epithelioid cells through experiments,in order to further explore the theoretical basis for the diagnosis and treatment of OSA combined with IPF.Methods: The lung gene data were screened and downloaded from the NCBI public database,and the differentially expressed genes(DEGs)in the two sets of gene matrix data sets were identified with R software,and the two sets of DEGs were intersected for further analysis.Use the R package "cluster Profiler" to intersect the differentially expressed genes obtained above for GO and KEGG enrichment analysis,and study the biological functions related to the common DEGs in OSA and IPF.At the same time,the same high and low genes were selected for GSEA analysis.Use String online search tool to analyze protein-protein interaction(PPI)data.Then,the PPI network is analyzed and visualized using Cytoscape,and the first five central genes are determined according to the maximum cluster centrality(MCC)between the differentially expressed genes.After obtaining important modules,the module genes were enriched and analyzed again.In addition,at A549 lung epithelioid cells,cell hypoxia incubator and cobalt chloride(cocl2)were used to simulate nocturnal hypoxemia in patients with moderate and severe OSA,the transforming growth factor-β(TGF-β)were used established EMT cell model of pulmonary fibrosis,and the drug toxicity was tested with CCK-8.First,the cells in each group were divided into RA group(normal normoxic control group,A549 lung epithelioid cells were cultured in normal state for 3days),IH group(chronic intermittent hypoxia group,A549 lung epithelioid cells were given cocl2 200um/l or were incubated in a three-gas incubator with 1% oxygen for 4h/d for 3 days),TGF-β Group A(fibrosis EMT model normoxic group,A549 lung epithelioid cells were given 10 umol/l TGF-β Stimulation,normoxic culture for 3 days),TGF-β+IH group(fibrosis EMT model chronic intermittent hypoxia group,A549 lung epithelioid cells were given 10 umol/l TGF-β Stimulate and incubate with cocl2200um/l or in a three-gas incubator with 1% oxygen for 4h/d(continuous incubation for3 days),use CCK-8 to detect the cell activity of each group,immunoblotting and immunofluorescence to detect the HIF of each group-α、EMT-related protein vimentin and α-SMA,autophagy associated protein P62 and LC3Ⅱ/Ⅰ,as well as the expression of pathway protein p-AKT and its ratio to AKT,and the expression of inflammatory factor IL-6 in each group were detected by ELISA.Then they were divided into five groups: RA group,IH group,TGF-β+IH group,Ly+IH group(chronic intermittent hypoxia Ly294002 treatment group,A549 lung epithelioid cells were stimulated with5umol/l Ly294002,and incubated with cocl2 200umol/l or in a three-gas incubator with1% oxygen for 4h/d,continuous incubation for 3 days),Ly+TGF+IH group(chronic intermittent hypoxia Ly294002 treatment group,A549 lung epithelioid cells were given5umol/l Ly294002 and 10umol/l TGF-β Stimulate,and incubate with cocl2 200um/l or in a three-gas incubator with 1% oxygen for 4h/d,and incubate continuously for 3 days),then use CCK-8 to detect the cell activity of each group,and immunoblot to detect the protein expression of each group.Results: Compared with normal lung tissue,there are 233 DEGs in OSA group,of which 164 are up-regulated and 59 are down-regulated.IPF has 2426 DEGs,of which1494 are up-regulated and 932 are down-regulated.After crossing,there are common 21 DEGs in OSA and IPF,of which 14 are up-regulated and 7 are down-regulated.The common key genes of IPF and OSA are CD19,CD79 A,CD79B,COL1A1 and POU2AF1.The function of GO biological process is enriched in biological processes such as antigen receptor-mediated signal pathway,leukocyte proliferation,B cell receptor signal pathway,collagen fiber tissue,etc;The molecular function of GO is enriched in the structural components of extracellular matrix with tensile strength,the structural components of extracellular matrix,and the binding of platelet-derived growth factor;The function of GO cell components is enriched in the extracellular matrix containing collagen,collagen trimer,endoplasmic reticulum lumen,collagen trimer complex and other cell components.The enrichment results of KEGG pathway showed that the common differential genes of OSA and IPF were enriched in protein digestion and absorption,PI3K-AKT signal pathway,ECM receptor interaction and other signal pathways.After 3 days of transient hypoxia,compared with the normoxia control group,the morphological changes of A549 lung epithelioid cells in the transient hypoxia group were smaller.Compared with RA group,IH group cells HIF-α,Inflammatory factor IL-6,EMT-related protein vimentin and α-The expression of SMA,autophagy associated protein P62 and LC3Ⅱ/Ⅰ,and pathway protein p-AKT and its ratio to AKT were significantly increased(P<0.05).After Ly294003 inhibited PI3K/Akt signal pathway,the expression of autophagy related proteins LC3Ⅱ/Ⅰ and P62 in A549 lung epithelioid cells changed,the autophagy flow inhibited by hypoxia environment was restored,and the EMT related protein was decreased α-Expression of SMA and vimentin.Conclusion: OSA and IPF have a common pathogenic pathway and pathological mechanism,and their key pathogenic genes are CD19,CD79 A,CD79B,COL1A1,POU2AF1.Long-term nocturnal hypoxemia caused by OSA is an independent promoter of EMT in A549 lung epithelial cells,PI3K/Akt autophagy pathway plays an important role in it. |
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