Study On The Mechanism Of Lipoxin A4 In Alleviating Lipopolysaccharide-induced Acute Lung Injury

Objective: It is currently known that lipoxin A4(LXA4)can relieve lipopolysaccharide(LPS)-induced acute lung injury(ALI)and acute respiratory distress syndrome(ARDS),however,the exact mechanism that LXA4 mediates which candidate gene to promote the opening of sodium channels(ENaC)in lung epithelial cells is not clear.This study mainly explores which candidate gene LXA4 mediates the expression of ENaC subunits and what kind of signal pathway does LXA4 use to achieve its regulation of ENaC.Methods: 1.Establishment of ALI lung epithelial cell model.LPS was applied to A549 cells(adenocarcinomic human alveolar basal epithelial cells)culture medium to establish an ALI lung epithelial cell model,and the changes in the transcription level of ENaC subunits of A549 cells were measured and recorded by q RT-PCR(quantitative real-time PCR).2.Observation of the effect of LXA4 on the transcription level of ENaC subunits in A549 cells.LXA4 was applied to A549 cells culture medium,and the transcription level of ENaC subunits was measured and observed by q RT-PCR.3.To observe the effect of LXA4 on the ENaC subunit transcription level of the established ALI lung epithelial cell model.LXA4 was added to the culture medium of A549 cells that already contained LPS,and the changes of the transcription level of ENaC subunits in A549 cells were measured and observed by q RT-PCR.4.Transcriptome sequencing analysis was performed on the treated A549 cells.By comparing m RNA expression profiles between A549 cells groups,we explored potential candidate genes mediated by LXA4 exerting its therapeutic effect on ALI.5.q RT-PCR was used to determine the dynamic changes of the m RNA from its potential candidate genes,in order to find possible candidate genes.6.q RT-PCR was used to determine and observe the changes of the candidate gene transcription level in the A549 cells group after LPS、LXA4、LXA4 + LPS treatment.7.Applied different concentrations of LXA4 to the culture medium of A549 cells treated with LPS,and then determined and observed the relationship between the m RNA level of candidate genes and the concentration of LXA4 by q RT-PCR.The relationship between protein levels of the candidate gene and concentration of LXA4 were analyzed by Western blot.8.Western blot was used to determine and observe the change trend of the protein level of the candidate gene of A549 cells which was treated with LXA4 at different time intervals(0,1,2,4,6,8h).9.Add LXA4 receptor(ALX)inhibitor(BOC-2)to the culture medium of A549 cells treated with LPS+ LXA4,then determined and analyzed the relationship between candidate gene and ENaC protein levels with BOC-2 by Western blot.10.A549 cells were treated with si RNA of the candidate gene,and then the effect of candidate genes on the survival rate of A549 cells was measured by CCK-8(Cell Counting Kit-8).The expression of ENaC subunits was measured and analyzed by Western blot.11.Using PI3K(phosphoinositide-3-kinase)inhibitor(LY294002)to determine whether the PI3 K signaling pathway was involved in regulating the expression of the candidate genes induced by LXA4.Results: 1.LPS inhibited the m RNA expression of ENaC subunit in A549 cells,while LXA4 can enhance the expression.LXA4 can also antagonize the inhibition of LPS on the m RNA expression of ENaC subunit in A549 cells,which indicateed that LXA4 can effectively alleviate ALI caused by LPS.2.The transcriptome sequencing analysis of the treated A549 cells revealed that the potential candidate genes were PLIN2,ANGPTL4,LRP1,and NDRG1,and NDRG1 was the most likely one.3.LPS could inhibit the m RNA expression of NDRG1,while LXA4 could antagonize this inhibition and enhanced the m RNA expression of NDRG1.4.The m RNA expression level of NDRG1 increased with the concentration of LXA4,and the protein expression level of NDRG1 was positively correlated with the concentration of LXA4.In addition,LXA4 also induced the expression of NDRG1 protein in a time-dependent manner.5.BOC-2 could inhibit the protein expression of NDRG1 and ENaC induced by LXA4.6.The si RNA of NDRG1 could not only inhibit the expression of ENaC subunits,but also reduce the survival rate of A549 cells.7.LY294002 not only inhibited the expression of NDRG1 and ENaC subunits,but also inhibited the phosphorylation level of SGK1,which revealed that the signal pathway of NDRG1 expression was the PI3 K signaling pathway.Conclusions: LXA4 mediated the expression of NDRG1 via the PI3 K signaling pathway,which enhanced the expression of ENaC in A549 cells and promoted the opening of sodium ion channels(ENaC)in lung epithelial cells.Furthermore,LXA4 promoted the elimination of water molecules in lung epithelial cells and reduced pulmonary edema,which achieved the relief and treatment of ALI and ARDS or other respiratory emergencies.The results of this study will provide help for the clinical treatment of patients with acute respiratory diseases such as ALI/ARDS,and provide theoretical support for the research and development of new drugs for the treatment of such diseases.