Molecular Mechanism Of UCP2 In Regulating Vascular Endothelium In Sepsis-induced Acute Lung Injury

Purposes:Clinical part:To investigate the relationship between serum syndecan-1,sTM,and the predictive value of these biomarkers during sepsis and septic shock in the first part of the study.To explore the relationship between Serum UCP2 and Syndecan-1,VE-cadherin levels in patients with sepsis-induced acute lung injury,to further evaluate the diagnostic value of these biomarkers in patients with ALI and the prognostic value in patients with sepsis in the second part.Experimental part:To explore the Molecular Mechanism of UCP2 in Regulating vascular endothelium through mitochondrial dysfunction in sepsis-induced acute lung injury both in vivo and in vitro.Methods:Clinical part:105 patients admitted to the Department of Critical Care Medicine were enrolled in the first part of study.Enroll patients were divided into the infection(n=28),septic nonshock(n=31),and septic shock(n=46)groups;In the second part,68 patients with sepsis were enrolled in this study,then were divided into two groups,the sepsis-induced acute lung injury group(SALI,n=33)and the sepsis with non-acute lung injury(NSALI,n=35).Serum syndecan-1,soluble thrombomodulin(sTM),UCP2,VE-cadherin levels were measured by enzyme-linked immunosorbent assay,and clinical and laboratory data were recorded.Clinical parameters and standard laboratory test data were also recorded,and follow-up was performed on patients with sepsis.Experimental part:Firstly,UCP2 overexpression and UCP2-silenced human umbilical vein endothelial cells(HUVECs)model was constructed,used LPS induced HUVECs as an in vitro septic model,UCP2 overexpression in mice and UCP2 KO mice was constructed,then CLP-induced acute lung injury model was established.Indicators of inflammation,dysfunctional mitochondria,and vascular endothelial cell dysfunction were evaluated,examine the degree of damage in lung tissues of mice in each group.Meanwhile,protein-related transparency and signaling pathways were detected.Results:Clinical part:Part Ⅰ:The serum syndecan-1(102.84± 16.53 vs.76.06 ± 10.51 ng/ml,P<0.001),sTM(9.67 ± 3.38 vs.6.60 ± 1.44 ng/ml,P<0.001)level in the septic shock group significantly increased compared with the septic non-shock group,Serum Syndecan-1(76.06±10.51 vs.55.38 ± 12.34,P<0.001),sTM(6.60 ± 1.44 vs.5.23 ± 1.23 ng/ml,P<0.05)level in the non-shock group significantly increased compared with the infection group,Serum Syndecan-1(102.84 ± 16.53 vs.55.38±12.34ng/ml,P<0.001),sTM(9.67 ± 3.38 vs.5.23± 1.23 ng/ml,P<0.001)in the septic group significantly increased compared with the infection group.Additionally,serum Syndecan-1(rs=0.687,P<0.001),sTM(rs=0.6,P<0.01)levels were significantly positively correlated with the sequential organ failure assessment scores respectively.Syndecan-1,sTM had significant diagnostic value in diagnosing sepsis(Syndecan-1(AUC 0.95± 0.02,P<0.0001),sTM(AUC 0.87 ±0.04,P<0.0001))and septic shock(Syndecan-1(AUC 0.95 ± 0.02,P<0.001),sTM(AUC 0.65 ± 0.06,P=0.01)).Part Ⅱ:Serum UCP2(243.39±86.44 vs.190.97 ± 72.43 pg/ml,P=0.0084),Syndecan-1(97.12 ± 19.46 vs.73.50 ± 16.52 ng/ml,P<0.0001),VE-cadherin(1196.5±360.18 vs.1034.4 ± 158.39 ng/ml,P=0.018)levels within 24 h after ICU admission were significantly increased in the sepsis-induced acute lung injury(SALI)group compared with the sepsis non-acute lung injury(NSALI)group;serum UCP2(297.07± 84.63 vs.197.34 ± 71.18ng/ml,P<0.0001),Syndecan-1(109.76 ± 16.37 vs.79.10 ± 18.14ng/ml,P<0.0001),VE-cadherin(1326.60 ± 503.17 ng/ml vs.1062.57 ± 176.37,P=0.0021)were significantly increased in survival group compared with the non-survival group;serum UCP2 was moderately negatively correlated with Syndecan-1(rs=0.649,P<0.0001),VE-cadherin(rs=0.358,P=0.0408)in septic patients with acute lung injury.UCP2,Syndecan-1 can be used for early detection of sepsis acute lung injury(UCP2(AUC 0.662 ± 0.066,P=0.021),Syndecan-1(AUC0.825±0.053,P<0.0001))and prediction of sepsis(UCP2(AUC 0.826±0.060,P=0.0003),Syndecan-1(AUC 0.896 ±0.040,P<0.0001)).Experimental part:Silencing UCP2 augmented the LPS-induced inflammatory levels,increased mtROS generation and apoptosis,enhanced vascular permeability concomitant with the decrease in VE-cadherin and ZO2 expression in the HUVECs.over-expression of UCP2-protected cells against LPS-induced inflammatory,mitochondrial damage,cellular apoptosis,restrain vascular permeability concomitant with the increase in VE-cadherin and ZO1 expression in the HUVECs.Mitochondrial injury,inflammation,and cell apoptosis were aggravated in the lung tissue in the UCP2 knockout+LPS mice,however,those were ameliorated in the UCP2 overexpression mice.Overexpression of UCP2 also suppressed the relative expression of the signaling pathway proteins,NLRP3 and HMGB1,silencing of UCP2 activated the NLRP3and HMGB1 pathway.Conclusion:(1)The molecular mechanism of mitochondrial dysfunction mediated endothelial cell dysfunction during sepsis through UCP2-NLRP3;(2)Mitochondrial dysfunction mediated endothelial cell dysfunction in sepsis-induced acute lung injury partly via UCP2-NLRP3;(3)Upregulation of UCP2 could attenuate vascular endothelial cell dysfunction;(4)Serum syndecan-1 and sTM levels were associated with organ dysfunction severity in septic patients,and both were good predictors for early identification of sepsis;(5)The close correlation between UCP2 and Syndecan-1,VE-cadherin was found in septic patients with acute lung injury,indicating the close relationship between mitochondria and vascular endothelial dysfunction;(6)Serum UCP2,Syndecan-1 levels in septic patients have predictive value for acute lung injury and 28-day prognosis;(7)UCP2 as a therapeutic target through mitochondrial dysfunction mediates endothelial cell dysfunction in septic patients with acute lung injury.