Effect Of Lyn Kinase On In Acute Respiratory Distress Syndrome (ARDS) Cell Model And Its Mechanism

Objective:To explore the intervention effect and mechanism of Lyn kinase on acute respiratory distress syndrome（ARDS）cell model,so as to find the new ideas and new methods for follow-up clinical prevention and treatment of ARDS.Methods:1.Human type II alveolar epithelial cells（A549 cells）were transfected with virus solution containing h LYN-e GFP lentivirus（constructed in our laboratory）by lentivirus transfection technique,and the A549 cell line with high expression of Lyn(Lyn^+/+-A549 cell line)was obtained for the experiment,and the untransfected A549 cell line（CTRL-A549 cell line）was used as control.2.Experimental groups:（1）Lyn^+/+-A549 cells and CTRL-A549cells were stimulated with 10μg/ml LPS for 4 hours,and the same dose of PBS stimulation was used as control.They were divided into LPS-Lyn^+/+group,LPS-CTRL group,PBS-Lyn^+/+group and PBS-CTRL group.3.Detection methods:（1）Flow cytometry was used to detect the apoptosis level of cells in each group after intervention with LPS or PBS for 4 hours.（2）ZO-1,Occludin,β-catenin,CHOP and Bi P were detected by immunofluorescence（IF）,and the results were observed by SP5 Leica confocal microscope.（3）The expression levels of Bcl-2,Caspase-3,CHOP,Bi P,p-PERK and p NF-κB were analysed by Western blot assay.Results:1.The A549 cell line with high expression of Lyn(Lyn^+/+-A549cell line)was successfully constructed.2.The results of flow cytometry showed that the level of apoptosis in PBS-CTRL group was similar to that in PBS-Lyn^+/+group（P>0.05）,apoptosis occurred in different degrees in LPS-CTRL group and LPS-Lyn^+/+group compared with PBS group,and the apoptosis rate in LPS-Lyn^+/+group was lower than that in LPS-CTRL group（P<0.05）.3.The results of Bcl-2 and Caspase-3 analysed by Western blot assay showed that there was no significant difference between PBS-CTRL group and PBS-Lyn^+/+group（P>0.05）,while the expression levels of Bcl-2 in LPS-CTRL group and LPS-Lyn^+/+group were notably lower than that in PBS groups（P<0.05）,the expression levels of Caspase-3 in LPS-CTRL group group were notably higher than that in PBS groups（P<0.05）,and there was no significant change in the expression levels of Caspase-3 between LPS-Lyn^+/+group and PBS groups（P>0.05）.4.The immunofluorescence intensity of ZO-1,Occludin andβ-catenin in LPS-Lyn^+/+group and LPS-CTRL group were lower than that in PBS groups,and the decrease in LPS-CTRL group was more significant than that in LPS-Lyn^+/+group（P<0.05）.5.The results of ZO-1,Occludin andβ-catenin analysed by Western blot assay showed that there was no significant difference between PBS-CTRL group and PBS-Lyn^+/+group（P>0.05）.The expression levels of ZO-1,Occludin andβ-catenin in LPS-CTRL group and LPS-Lyn^+/+group were notably lower than those in PBS groups,and the decrease in LPS-CTRL group was more significant than that in LPS-Lyn^+/+group（P<0.05）.6.The immunofluorescence intensity of CHOP and Bi P in LPS-CTRL group and LPS-Lyn^+/+group were notably higher than that in PBS group,and the immunofluorescence intensity of CHOP and Bi P in LPS-Lyn^+/+group were lower than that in LPS-CTRL group.7.The results of p-NF-k B analysed by Western blot assay showed that the expression levels of p-NF-k B in PBS-Lyn^+/+group was notably higher than that in PBS-CTRL group（P<0.05）;the protein expression levels of p-NF-k B in LPS-CTRL group and LPS-Lyn^+/+group were notably higher than those in PBS groups（P<0.05）;the change in LPS-CTRL group was more significant than that in LPS-Lyn^+/+group（P<0.05）.8.The results of CHOP and p PERK analysed by Western blot assay showed that there was no significant difference between PBS-CTRL group and PBS-Lyn^+/+group（P>0.05）,the expression levels of Bi P in PBS-CTRL group was notably higher than that in PBS-Lyn^+/+group（P<0.05）;the expression levels of CHOP,Bi P and p PERK in LPS-CTRL group and LPS-Lyn^+/+group were notably higher than those in PBS groups（P<0.05）;the change in LPS-CTRL group was more significant than that in LPS-Lyn^+/+group（P<0.05）Conclusion:1.High expression of Lyn kinase reduced apoptosis of A549cells in ARDS cell model;2.high expression of Lyn kinase reduced the destruction of intercellular tight junction complex in ARDS cell model and protected lung epithelial barrier function;3.high expression of Lyn kinase reduced the activity of NF-k B in ARDS cell model;4.high expression of Lyn kinase reduced the level of endoplasmic reticulum stress and apoptosis mediated by endoplasmic reticulum in ARDS cell model.5.Lyn kinase reduces apoptosis,protects pulmonary epithelial barrier function and reduces inflammation in ARDS,and its mechanism may be regulated by weakening the stress level of endoplasmic reticulum.