Molecular Mechanism Of UCP2 In Regulating NLRP3 Inflammasome Pathway In Sepsis-Induced Myocardial Injury

Purposes:Clinical part:To investigate the correlation between serum UCP2(uncoupling protein 2)and NLRP3(nucleotide-binding domain,leucine-rich-repeat containing family,pyrin domain-containing 3)levels and their potential utilities as biomarkers in septic patients.Experimental part:To explore the underlying mechanism of the relationship between UCP2 and NLRP3 inflammasome medicated canonical pyroptosis pathway in cardiomyocytes during sepsis.Methods:Clinical part:For UCP2-Critically ill patients with diagnoses of sepsis-septic nonshock group(n=20)and septic shock group(n=53),and an ICU control group(n=15)were enrolled within 24h of entry into the ICU.For NLRP3-Patients were divided into five groups:healthy control(n=30),ICU control(n=22),infection(n=19),septic nonshock(n=33),and septic shock(n=83).Serum levels of UCP2 were measured by ELISA at ICU admission for all the groups and at ICU discharge/death for septic shock group.Serum NLRP3 levels were measured by ELISA upon enrollment.Clinical parameters and laboratorial tests(APACHE Ⅱ,SOFA,lactate,etc.)were also collected.Experimental part:Firsts to establish septic models in vitro and in vivo,AC 16 cells were cultured in the presence of LPS(lmg/L)and mice underwent the cecal ligation and puncture(CLP)procedure.Gain-and loss-of-function experiments(overexpression with plasmid and knock-down with siRNA in cells/the use of genipin to inhibit UCP2 protein in mice)were then performed to evaluate the mechanism of the interaction between UCP2 and NLRP3.Myocardial morphological injury,indicators of mitochondria injury,levels of NLRP3 inflammasome pathway-associated proteins(NLRP3/GSDMD/cleaved caspase-1),and levels of cytokines(IL-1β/IL-18)were assessed between the experimental groups.Results:Clinical part:Serum UCP2 concentrations on ICU admission were significantly increased in septic shock group and septic nonshock group,compared with control subjects(263.21±29.99 vs 115.96±32.99 vs 60.56± 10.05pg/mL,P<0.001).Concentrations of UCP2 performed better than other parameters(APACHE Ⅱ score,SOFA score,PCT;and WBC)in predicting the incidence of sepsis or septic shock on day of ICU admission,as reflected by AUC.On day of ICU admission,the AUC for UCP2 level associated with 28-day mortality was 0.704,higher than the AUC for SOFA and APACHE Ⅱ score.Patients with higher admission levels of UCP2(>246.52pg/mL)had significantly increased 28-day mortality compared with those with lower UCP2 levels(<246.52pg/mL).The NLRP3 level in the septic shock group was significantly higher(431.89,386.61-460.21pg/mL)than that in the healthy control group(23.24,9.38-49.73pg/mL),ICU control group(74.82,62.71-85.93pg/mL),infection group(114.34,99.21-122.56pg/mL),and septic nonshock group(136.99,128.80-146.98pg/mL;P<0.001 for all comparisons).The performance of serum NLRP3 level was not inferior to SOFA score for predicting the incidence of septic shock,as reflected by the AUC.Patients with higher NLRP3 serum levels(>147.72pg/mL)had significantly increased 30-day mortality.The patients with sepsis-induced cardiomyopathy had higher serum NLRP3 levels.Serum UCP2 and NLRP3 levels on admission of ICU(n=88)had a highly correlation(r=0.966,P<0.001).Experimental part:Expression of UCP2 increased after the LPS or CLP intervention in the cellular and animal models.Inhibition of UCP2 resulted in significantly increased activation of the NLRP3 inflammasome,as shown by alterations in the expression of NLRP3/GSDMD/cleaved caspase-1,the release of cytokines IL-1β/IL-18,and the pyroptosis rate of cells.Notably,upregulation of UCP2 in the cellular model blocked this aggravation.In addition,we demonstrated that silencing or inhibiting UCP2 resulted in a loss of mitochondrial membrane potential,enhanced ROS generation,decreased ATP synthesis,and reduced intracellular mtDNA copy numbers,indicating the protective effect of UCP2 on mitochondria.Conclusion:1.Upregulation of UCP2 could inhibit the activation of NLRP3 inflammasome via the protection of mitochondria,which effectively alleviates the organ injury(heart)and the inflammatory response during sepsis.2.The close correlation between UCP2 and NLRP3 from clinical to basic research,indicating the close relationship between mitochondria and NLRP3 inflammasome.3.The elevation of UCP2 during sepsis could be a compensatory reaction.4.UCP2 is the potential therapeutic target for sepsis,but the optimal intervention method and timepoint of UCP2 is needed further investigation.5.Serum UCP2 and NLRP3 levels could assist the diagnosis of sepsis/septic shock.6.Elevated serum UCP2 and NLRP3 levels were associated with the severity and organ function of disease-high serum UCP2 and NLRP3 levels predict poor outcome.