| Background Decompression sickness(DCS),also called as caisson disease.DCS occurred when the surrounding pressure is reduced,the gas dissolved in tissues reforming bubbles.The travelling bubbles could lead to blood vessels blockage and endothelial cell damage.Moreover,as external substances,bubbles could cause platelet aggregation,resulting in widely thromboembolism and inflammatory response.The accompanying diseases would induce surrounding tissues and organs injuries,which could lead to respiratory disorders,nervous system dysfunction and even death.How to prevent and treat DCS has become an important issue concerning diving medical security,which would be the core of related research.Therefore,it’s extremely important to investigate the pathophysiological mechanism of DCS,which would evoke potential effective strategies against DCS in the early stage.The bubbles caused DCS were shown to include intravascular and extravascular ones.Data from previous studies had confirmed that bubbles could activate platelets and neutrophils,which would lead to subsequent pathophysiological reactions.Neutrophil Extracellular Traps(NETs)were shown to be secreted by activated neutrophils,which had been confirmed to induce endothelial cells injury and then disseminated intravascular coagulation and systematic inflammations.But it is still unclear whether NETs would participate in the pathophysiological process of DCS,which might provide potential strategies and targets for clinical intervention in the future.Data from previous study demonstrated that the adenosine receptor agonist dipyridamole could inhibit the production of NETs and thrombosis burden.In this study,we chose the novel selective A2 A receptor agonist regadenoson to investigate whether regadenoson could ameliorate vascular endothelial cell injury and embolism caused by secreted NETs,and then interrupt the pathological process of DCS,which would provide new targets and strategies for the treatment of DCS.There research could be summarized as the following two sections,1.To observe the effects of different doses of regadenoson on the morbidity and mortality of DCS rat models,which was explored by the abnormal symptomonset time,pathological changes of lung tissue sections and inflammation within the rats;2.Effect and mechanism of regadenoson against DCS by inhibiting the formation of NETs and subsequent endothelial cell injury of NETs.Methods 1.SD rats were randomized into Control,Model and Model+regadenoson groups.A special fast up-floating escape simulated animal experimental chamber was used to simulate in vitro environment.Then the following indicators were used to investigate the differences:(1)the morbidity and mortality;(2)changes of cardiac ultrasound parameters;(3)pathological examination and dry/wet weight ratio of lung tissue;(4)inflammatory factors in lung tissues;(5)levels of rat blood inflammatory factors and endothelial injury markers.2.The production of microbubbles in the decompression disease environment was simulated by pumping nitrogen to induce the release of NETs by neutrophils.The release of NETs at different blowing times(0min,10 min,20min,30 min,60min and 120min)was evaluated by CF-DNA assay.The optimum blowing time of induced NETs is found.NET production was further verified by neutrophil Cit H3 and MPO double fluorescence staining.The difference of neutrophil protein expression was determined by proteomic analysis after different treatment.Subsequently,Western blot and other methods were used to verify the inhibitory effect and molecular mechanism of PFDN6 in regulating neutrophil releasing NETs and regadenoson.3.The effects of NETs on endothelial cell growth,apoptosis and migration were evaluated by CCK8,immunofluorescence staining,Transwell assay,scratch assay and flow cytometry,respectively.The integrity of endothelial cell barrier formation was evaluated by trans-endothelial resistance and FD-4 fluorescence value to evaluate the damage of NETs to endothelial cell membrane.Result: 1.Compared with control group,NETs biomarkers were increased and the expression of NETs in lung tissues was increased in rats of rapid floating decompression disease model group.2.Regadenoson could reduce the morbidity of DCS model rats(67.5% vs 82.5%,P=0.0214).The left ventricular ejection fraction of regadenoson group was higher than that of model group in the initial 5-minute period(67.5% vs 82.5%,P=0.0214)However,with the passage of time,it soon decreased to the level with no significant difference from the model group.Rats in Regadenoson group showed lower pathological injury degree of lung tissue and reduced blood stasis in pulmonary circulation compared with rats in model group,together with lower m RNA expression of inflammatory factors in lung tissue and the levels of inflammatory factors and endothelial injury markers in blood.3.(1)The content of CF-DNA in supernatant was 20.71±3.4,37.02±5.55,68.3±10.48,146.78±22.99,209.41±34.64 and 233.93±23.95 respectively,when nitrogen was injected at 0min,10 min,20min,30 min,60min and 120 min.Statistical analysis showed that neutrophils secreted NETs significantly when nitrogen was injected for 60 min,compared with 10 min,20min and 30 min.Moreover,there was no significant difference in the value of CF-DNA when nitrogen injection time was 60 min and 120min(P > 0.05).(2)Cit H3 and MPO fluorescence staining showed stronger Cit H3 fluorescence staining and MPO fluorescence staining in the NETs group compared with the Control group,indicating that bubbles generated by micro-pumping nitrogen injection for 60 min can induce neutrophils to secrete NETs.(3)Proteomic analysis showed that regadenoson significantly up-regulated the expression of PNFD6 proteinb(17.67±0.12)compared to NETs group(16.67±0.25),indicating that regadenoson inhibit the NETs derived from neutrophil by up-regulating PNFD6 expression.(4)Western blot results showed Compared to the NETs group,the expression levels of PDFN6,A2 AR and p-CREB were significantly increased with regadenoson intervention(P < 0.05),indicating that regadenoson might inhibit the formation of NETs by activating A2 A receptor,activating c AMP and then promoting PFDN6 protein expression.4.(1)CD31 immunohistochemical staining results showed that compared with the control group,there were significantly more cell components in the vascular lumen of the lung tissues of rats in the model group,while the blood cell components in the vascular lumen of the lung tissues of rats in the regadenoson intervention group were significantly reduced,suggesting less embolism.(2)Flow cytometry showed that there were fewer apoptotic cells in Control group,NETs+REG group and NETs+SC79 group,and the statistical values of apoptotic cells in the above groups were 2.3±0.13,11.16±2.09 and 6.61±1.76,respectively,while there were more apoptotic cells in the NETs group,and the statistical values were 48.73±8.28.Endothelial cell apoptosis was significantly increased after NETs treatment,(P<0.001),while the apoptosis rate of endothelial cells was significantly decreased in the treatment of regadenoson(P<0.001)compared with the NETs group,the number of apoptotic cells in the NETs+SC79 group was significantly decreased(P<0.001).(3)Western blot results showed that the expression levels of P-Akt,P-PI3 K and Claudin-1 in endothelial cells were significantly down-regulated by NETs(P < 0.05),while the expression levels of P-Akt,P-PI3 K and Claudin-1 were significantly upregulated by SC79(P < 0.05).Meanwhile,regadenoson also up-regulated the expression of these proteins(P < 0.05),indicating that NETs induced endothelial cell apoptosis by inhibiting the PI3K/AKT signaling pathway and the expression of membrane protein Claudin-1,while regadenoson inhibited the formation of NETs and blocked the inhibition of The AKT signaling pathway related protein(PI3K,P-Akt)by NETs.(4)Immunofluorescence results showed that the expression of ROS and NO was significantly increased after NETs treatment(P < 0.001),indicating that NETs induced increased intracellular reactive oxygen species expression,resulting in damage to endothelial cells.However,both SC79 and regadenoson intervention significantly reduced the expression of reactive oxygen species in endothelial cells(P < 0.001),indicating that activation of AKT signaling pathway can reduce the high expression of reactive oxygen species in endothelial cells induced by NETs.(5)NETs could disrupt the transverse and longitudinal migration of endothelial cells,while the migration of endothelial cells was significantly recovered after regadenoson and SC79 were applied(P < 0.05).The migration area of scratch experiment increased from 24.33±2.52 to 37.67±4.16 and 51.67±3.06,respectively.In transwell assay,the number of migrated cells increased from 242.33±39.4 to 392±74.65 and 345.67±36.3,respectively.(6)The transmembrane impedance and membrane permeability of endothelial cells were significantly decreased after NETs treatment,while the transmembrane impedance and membrane permeability of endothelial cells were significantly increased after regadenoson and SC79 treatment,respectively(P < 0.05).Conclusion NETs are involved in the pathology of rapid upward decompression sickness and mediate endothelial damage,a key link in decompression sickness pathology,through the AKT signaling pathway.Regadenoson inhibited neutrophils from secreting NETs by upregulating the PFDN6 protein,thereby reducing endothelial injury and thrombosis. |
PDF Full Text Request