A Preliminary Study On The Effect Of Plasma Exosomes From Mechanically Ventilated ARDS Rats On The Autophagy Level Of NRK-52E Cells

Background: Acute respiratory distress syndrome(ARDS)is an acute progressive respiratory failure.Its clinical features are respiratory distress and refractory hypoxemia.The incidence of ARDS in the intensive care unit is 10.4%,and the mortality rate is as high as 40%.Multiple organ failure is the main cause of death in patients with ARDS,including heart,liver,kidney,gastrointestinal tract and other organ failure,of which the kidney is the earliest organ to fail.According to statistics,the proportion of acute kidney failure(AKI)in patients with ARDS is about 44.3%.At present,mechanical ventilation is the main treatment for patients with ARDS.Some studies have shown that patients with ARDS mechanical ventilation are three times more likely to develop AKI than patients without mechanical ventilation.Research on the risk factors of AKI in critically ill patients found that ARDS and mechanical ventilation are two independent risk factors of AKI,but the mechanism of these two factors in AKI is not yet fully clear.Some studies believe that mechanically ventilated ARDS patients may release inflammatory factors and active mediators into the bloodstream and reach the kidneys through the systemic circulation to induce AKI.The renal proximal tubular epithelial cells are important targets for various stressors when AKI occurs.In the animal model of AKI,the autophagy level of proximal renal tubular epithelial cells is increased.Exosomes are small vesicles with a particle size of 30-200 nm.It is secreted by the cell to the outside and widely exists in body fluids.It can deliver its contents to target cells and affect cell function.It is the third mechanism of cell-to-cell communication.This study was to investigate the effect and mechanism of rat plasma exosomes on the autophagy level of proximal tubular epithelial cells in the background of ARDS mechanical ventilation.Objective: 1.To establish hydrochloric acid-induced ventilation model of ARDS rats,to extract and identify plasma-derived exosomes;2.To explore the effect and mechanism of ARDS rat-derived exosomes on NRK-52 E autophagy.Methods: 1.Infuse hydrochloric acid through the trachea and perform 6 hours of mechanical ventilation to establish a double-hit model of ARDS rats,set the saline group as a control.2.By measuring the arterial oxygenation index(P/F ratio),performing lung tissue pathological HE staining and lung injury score,the degree of lung tissue injury was evaluated.3.Using co-precipitation kit to extract rat plasma exosomes,using transmission electron microscopy,Nanoparticle Tracking Analysis(NTA),Western Blot(WB)to identify exosomes.4.In vitro test: Confocal microscope was used to detect the uptake of fluorescent dye-labeled exosomes by NRK-52E;after exosomes stimulated NRK-52 E cells,the autophagy staining kit method and ELISA detection of intracellular LC3 B protein method were used to evaluate the cells’ autophagy level.5.Sequencing of rat plasma exosomes: illumina high-throughput sequencing technology platform to sequence exosome mi RNAs and analyze the differences in the expression of mi RNAs in different groups of exosomes.Results: 1.After tracheal infusion of hydrochloric acid,the P / F ratio of rats decreased,and the histological lung injury score increased.2.Transmission electron microscopy results showed that rats plasma exosomes were in the form of double-layered vesicles;NTA results showed that the peak size of exosomes in rats plasma was: 137.5 nm;Western blot test can detect exosome specific marker protein CD81 And TSG101;3.The rat plasma exosomes can be taken up by NRK-52 E,thereby exerting its biological function on cells;4.The results of the autophagy staining showed that compared with the tamoxifen positive control group,the exosome stimulation group could not induce an increase in autophagy in NRK-52 E cells;ELISA results showed that the level of autophagy marker protein LC3 B in the exosome stimulation group did not increase significantly.5.HCl group VS saline group,246 mi RNAs were significantly different expression;saline mechanical ventilation group VS saline group,236 mi RNAs were significantly different expression;HCl mechanical ventilation group VS HCl group,202 mi RNAs were significantly different expression;HCl mechanical ventilation group VS saline mechanical ventilation group,225 mi RNAs were significantly different expression;HCl mechanical ventilation group VS saline group,105 mi RNAs were significantly different expression.Conclusion: It is confirmed that the plasma exosomes of ARDS rats can be taken up by NRK-52 E cells,but it cannot cause the increase of the autophagy level of NRK-52 E cells;ARDS rat plasma exosome mi RNA expression was significantly different compared to other groups.