A Study Of Effects Of Selexipag Upon ARDS And Associated Mechanisms

Background and purpose Body injuries caused by various pathogenic factors can induce inflammatory response.Excessive inflammatory responses cause damages to alveolar epithelial cells and pulmonary capillary endothelial cells,consequently giving rise to Acute Respiratory Distress Syndrome(ARDS)that features diffuse pulmonary interstitial edema and alveolar edema.Previous researches revealed that the imbalance between thromboxane A2(TXA2)and prostacyclin(PGI2)was closely associated with occurrence and development of ARDS.The concentration of PGI2 increases compensatively and reactively during ARDS so as to antagonize the adverse effect of TXA2.Both TXA2 and PGI2 last only a few minutes in the half life.The both two indicate highly unstable chemical properties and rapidly break down into TXB2 and 6-keto-PGF1α in vivo.PGI2 and its analogs,such as beraprost and iloprost,exhibited effects of anti-inflammation and endothelialdependent anti-edema in acute lung injury experiments.As a selective prostacyclin IP receptor agonist,the chemical structure of selexipag differs from that of PGI2 and its analogs.Selexipag demonstrates a longer half-life and less side effects.Recent studies have found that selexipag restrains inflammation,alleviating oxidative stress,protecting vascular endothelial cells,and resisting fibrosis.The first part of this study explored changes in plasma levels of Endothelial cell specific molecule-1(ESM-1),Thromboxane B2(TXB2)and 6-keto-PGF1α in ARDS patients and relevant clinical significance;the second part established a lipopolysaccharide(LPS)-induced ARDS model of mouse,based on which probed whether selexipag would indicate protective effect against ARDS and potential mechanisms as well;the third part investigated impacts of selexipag upon LPS-induced Human Umbilical Vein Endothelial Cells(HUVECs)and upon the signaling pathway comprising Suppressor of Cytokine Signaling3(SOCS3),Signal Transducer and Activator of Transcription3(STAT3),Ras homolog gene family member A(Rho A),and Rho Kinase 1(ROCK1).Method Part I The clinical data of 32 ARDS patients were enrolled,including gender,age,etiology,APACHE II within 24 hours after ARDS was established,EVLWI,WBC,serum Alb,D-dimer,PCT,and arterial blood gas analysis,and outcomes of patients were recorded(the research endpoint was set at the 28 th day after admission);plasma levels of ESM-1,TXB2,and 6-keto-PGF1α were detected among healthy persons and these above ARDS patients who were investigated within 24 hours after the diagnosis of ARDS was confirmed;factors associated with prognosis of these ARDS patients were analyzed and evaluated.Part II The mouse model of ARDS was produced by intraperitoneal injection of LPS.Mice were randomly assigned into four groups: the control group(Control),the LPS group(the model group),the LPS + prophylaxis of low-dose selexipag group,and the LPS + prophylaxis of high-dose selexipag group.All the four groups went through the following experiments:(1)Comparisons of the mortality of mice amid all the four groups;(2)Implementation of hematoxylin-eosin staining and lung injury score assessment upon mice.(3)Detection of lung leakage indicators in mice;(4)Measurement of inflammatory factors,referred to concentrations of IL-1 β,IL-6 and MCP-1 in lung tissue homogenate and alveolar lavage fluid of mice by ELISA;(5)Investigation of lung vascular endothelial injury and endothelial barrier function of mice,comprising expressions of lung E-selectin and VCAM-1 m RNA by the RT-PCR,expressions of VE-cadherin and ZO-1by the Western blotting;(6)Exploration of possible mechanisms through which selexipag protects ARDS mice,including level of cyclic Adenosine Monophosphate(c AMP)in lung tissue homogenate by ELISA,expressions of phosphorylated-protein kinase A(P-PKA),protein kinase A(PKA),and exchange protein directly activated by c AMP(Epac)1 by Western blotting.Part III LPS was used to induce HUVECs and effects of prophylaxis of selexipag on HUVECs were observed.The experiment in Part III included the following parts:(1)This part was to observe effects of selexipag over viability of HUVECs induced by LPS.HUVECs viability was detected by the MTT colorimetry.Different doses of LPS and selexipag were administered to stimulate HUVECs separately,then appropriate concentration ranges of LPS and selexipag for intervention were determined.(2)The HUVECs were divided into four groups: the Control group(control group),the LPS group(model group),the LPS + prophylaxis of low-dose selexipag group,and the LPS + prophylaxis of high-dose selexipag group.Firstly,the permeability of the HUVECs of each group was investigated by Transwell chamber and FITC-dextran.Secondly,levels of IL-1β and IL-6 in supernatant of the HUVECs and the intracellular level of c AMP inside the HUVECs in every group were detected by ELISA.Thirdly,expressions of VE-cadherin,β-catenin,ZO-1 and claudin-1 proteins in the HUVECs in each group were examined by immunofluorescence and western blotting.(3)The part was to survey possible mechanisms through which selexpag provides protection to the HUVECs induced by LPS.Experimental groupings include the Control group(control group),the LPS group(model group),the LPS + selexipag group,LPS + selexipag + ESI-09(Epac inhibitor)group or the LPS + selexipag + H89(PKA inhibition Agent)group.Western blotting was administered to investigate expressions of Epac1,PKA,SOCS3/STAT3 and Rho A/ROCK1 proteins in the HUVECs in each group.Results Part I The levels of TXB2,6-keto-PGF1α,ESM-1 and TXB2/6-keto-PGF1α in ARDS patients were evidently higher than that of healthy persons with statistically significant differences.Among 32 patients with ARDS,19 survived and 13 died.No significant differences were detected between the dead patients and the survival ones with respect to gender and age.Both the APACHEⅡ score and the EVLWI in the dead patients were significantly higher than that in the survival ones.Comparisons over levels of WBC,Alb,TXB2,and 6keto-PGF1α between the two kinds of patients revealed no significant differences.The levels of PCT,ESM-1,D-dimer as well as the ratio of TXB2/6-keto-PGF1α in the dead patients were significantly higher than that in the survival ones.The ratio of ESM-1 was positively correlated with the level of TXB2/6-keto-PGF1α and EVLWI seperately.Part II Prophylactic administration of selexipag reduced mice mortality,alleviating lung pathological damage and lung tissue leakage showed by LPS-induced ARDS mice,decreasing levels of LPS-induced inflammatory factors,including IL-1β,IL-6,and MCP-1,cutting down expressions of LPS-induced adhesion molecules,like E-selectin and VCAM-1 m RNA,increasing expressions of VE-cadherin and ZO-1 protein,raising the level of c AMP in lung tissue,and up-regulating expressions of lung p-PKA and Epac1.Part III 10μg/ml or above LPS decreased the proliferation of HUVECs.However,10μg/ml or 20μg/ml of selexipag increased the viability of the HUVECs that had been depressed by LPS.The permeability of the HUVECs significantly increased 6 hours after LPS administration,but the precondition with selexipag reduced the enhanced permeability of the HUVECs.Selexipag inhibited secretions of inflammatory factors,like IL-6 and IL-1β,from the HUVECs induced by LPS,but promoted expressions of HUVECs connexin VE-cadherin,β-catenin,ZO-1,and Claudin-1,which had been inhibited by LPS.LPS induced HUVECs to increasingly express SCOC3,p-STAT3,Rho A-GTP,and ROCK1,while to decrease levels of c-AMP,Epac1,and p-PKA.The precondition with selexipag up-regulated levels of c AMP,Epac1,SOCS3,and p-PKA,while down-regulated expressions of p-STAT3,Rho A-GTP,and ROCK1 protein.ESI-09,an Epac inhibitor,partially reversed the up-regulation of SOCS3 and down-regulation of STAT3 by selexipag,and indicated no effects on the expressions of Rho A and ROCK1 protein.H89,a PKA inhibitor,only in part reversed the down-regulations of Rho A-GTP and ROCK1 by selexipag,and slightly inhibited the up-regulation of SOCS3 and down-regulation of p-STAT3 by selexipag.Conclusion 1.The levels of the APACHEⅡ score,EVLWI,D-dimer,PCT,ESM-1 and TXB2/6–keto-PGF1α were significantly higher in the dead patients with ARDS than that in the survival ones.The ratio of ESM-1 was positively correlated with the level of TXB2/6-keto-PGF1α and EVLWI seperately.2.Selexipag may increase the levels of lung c AMP and activate phosphorylated PKA protein and Epac1 protein in the downstream,to restrict inflammation,to reduce damages to pulmonary vascular endothelial cells,to enhance the barrier function of pulmonary capillary endothelial cells,and eventually to alleviate lung injuries of ARDS mice induced by LPS.3.Selexipag may remit inflammatory responses,enhance vascular barrier function,and protect the HUVECs injured by LPS through the signaling pathways of Epac1/SOCS3/STAT3 and PKA/Rho A/ROCK1.