Effect Of RELM ? Expression In Pulmonary Epithelial Cells Induced By Mechanical Stretching

Objective:The effects of mechanical stretching on the expression of RELM? in rat lung epithelial cells were investigated using cell mechanics and molecular biology methods.Methods:The experimental study is divided into two parts:Part one: Effect of RELM ? expression in pulmonary epithelial cells induced by mechanical stretching.RLE-6TN cultured in vitro was randomly divided into two groups: control group and mechanical stretching group.Control group was inoculated in Bio Flex 6 well.The cells in the plate were placed in the same incubator as the mechanical stretching group,but not connected with the Flexercell loading device;in the S group,the Bio Flex 6 well plate inoculated with RLE-6TN was attached to the Flexercell loading device for uniform mechanical stretching.According to the stretching time,the S group component is 4 subgroups,3S group(mechanical stretching lasts 3 hours),6S group(mechanical stretching lasts 6 hours),16 S group(mechanical stretching lasts 16 hours)and 24 S group(mechanical stretching lasts for 24 hours).Mechanical stretching was provided by Flexercell-4000 TTM cell flexible substrate loading system,stretched at set time,amplitude 20%,frequency 0.3 Hz,sine wave.After agglutination,the apoptotic rate,RELM ? and TNF-? expression of RLE-6TN cells were detected.The morphological changes of the cells before and after stretching were observed under electron microscope.The experimental data were statistically analyzed.Part two: Expression of RELM ? in rat lung epithelial cells transfected with adenovirus.Adenovirus-mediated RNA interference technique was used to transfect rat lung epithelial cells.The expressions of RELM ? and p-AKT were observed after transfection,the morphological changes and apoptosis of each group were observed.RLE-6TN were randomly divided into 4 groups,C group(control group,no stretching,no transfection),24 S group(only stretched for 24 h,no transfection),R group and N group.C group and 24 S group were treated with the first part,R group and N group.RLE-6TN was inoculated into Bio Flex 6-well culture plates,and Ad-Retnla sh RNA-GFP adenovirus and iso-gradient,same-volume Ad-GFP control were added respectively.The adenovirus was cultured in a thermostat with serum-free medium for 8 hours and then replaced with normal medium for further culture.After 48 hours,the culture plate was connected to the Flexercell-4000 TTM cell flexible substrate loading device with a stretching amplitude of 20% and a frequency of 0.3 Hz,sine wave,stretched for 24 hours.Total protein and cell supernatant were extracted.The expressions of RELM? and p-AKT protein were detected by Western Blot.The expressions of RELM? and TNF-? were detected by ELISA.Apoptosis was detected by flow cytometry.The morphological changes of the cells before and after stretching were observed under electron microscope.The experimental data were statistically analyzed.Results:1.Part one: Compared with the C group,the expression of RELM? and TNF-? in the S group was significantly up-regulated,and the apoptosis rate was increased.The difference was statistically significant(P<0.05).Electron microscopic observation showed that after 24 hours of continuous stretching,pulmonary epithelial cells were swollen,surface microvilli decreased,mitochondria swollen,volume enlarged and even vacuolated,mitochondrial cristae decreased,part of crest arranged disordered,fused or disappeared.The rough endoplasmic reticulum was dilated like a pool,and some rough endoplasmic reticulum was degranulated.The morphological damage of the cells was obvious.2.Part two: The results of post-transfection showed that the expression levels of RELM? and p-Akt in the R group were the lowest in the R,C,24 S,and N groups.Compared with the 24 S and N groups,the expression of RELM ?,TNF-? and p-Akt in the R group were significantly down-regulated,and the difference was statistically significant(P<0.05).Apoptosis rate was significantly decreased,and cell morphology damage was significantly improved,suggesting that down-regulation of RELM? expression may reduce the injury of lung epithelial cells induced by mechanical stretch,which may be related to the p-Akt pathway.Conclusion:1.Mechanical stretching can induce lung epithelial cell injury and high expression of RELM ? in rats.2.Adenovirus transfection could down-regulate the expression of RELM ? and p-AKT in rat lung epithelial cells,decrease the apoptosis rate and improve the morphological changes of lung epithelial cells induced by mechanical stretching.3.PI3 K / AKT signaling pathway is involved in the mechanical stretch-induced injury of rat lung epithelial cells,which is an important factor in this process.