Colchicine Inhibits NLRP3 Involvement In Sepsis-associated Lung Injury Via Regulation Of Phosphorylating STAT3

Acute lung injury/acute respiratory distress syndrome(ALI/ARDS)is an acute respiratory failure caused by multiple factors and is a common respiratory critical illness in clinical practice.The mortality rate of ARDS continues to remain at a high level of approximately 40%due to the lack of effective drug therapy.The action of colchicine on microtubulin affects the assembly of inflammatory vesicles,the expression of multiple interleukins,and has been applied in various acute inflammatory diseases.Colchicine has been reported to have protective and therapeutic effects in lung injury induced by various diseases,but it is unknown whether colchicine also has a protective or therapeutic effect in acute lung injury caused by sepsis.Therefore,the aim of this study was to investigate the role of colchicine in alleviating sepsis-related ALI and to verify its therapeutic significance.In vivo experiments were used to investigate the effect of colchicine on the alleviation of oxidative stress and macrophage scorching in mice with sepsis-related ALI.Combined with raw letter analysis,in vivo and in vitro experiments were used to investigate the role and molecular mechanism of colchicine in inhibiting the activation of NLRP3(NOD-like receptor protein 3)inflammatory vesicles,oxidative stress and cell scorching in macrophages of sepsis-associated ALI mice.This study is divided into seven main parts as follows.Chapter 1 Colchicine alleviates sepsis-associatedlung injury in mice and its mechanism of actionObjective:OBJECTIVE:To verify whether colchicine has a therapeutic effect on sepsis-associated ALI and the mechanism of action.Methods:(1)intraperitoneal injection of LPS into mice to construct a sepsis-associated ALI model,and treatment with different concentrations of colchicine to assess survival and mortality;(2)detection of arterial blood gas to assess hypoxia in mice;(3)analysis of lung wet-to-dry ratio in mice by drying and weighing,and comparative assessment of the degree of relief of pulmonary edema;(4)detection of total protein in alveolar lavage fluid,HE staining of lung tissues to observe the remission of lung pathological damage;(5)ELISA assay of lung tissue homogenate supernatant and immunohistochemistry of lung tissue to observe the trend of lung inflammatory response;(6)statistical methods:data were expressed as mean ± standard deviation using Log-rank(Mantel-Cox)test,one-way-ANOVA and Tukey’s multiple comparisons test for analysis,test level α=0.05.(7)DHE fluorescence staining imaging and lung tissue homogenate supernatant assay were performed on the lung tissue of LPS mice to observe the changes of ROS,SOD and GSH-px levels after LPS and colchicine interventions;(8)The corresponding kits were tested to assess the serum TOS and TAS of mice,and to assess the overall oxidative stress level;(9)TUNEL staining was performed to observe apoptosis of alveolar macrophages in LPS mice;(10)WB detection of Bax and Bcl2 levels in lung tissue and observation of colchicine inhibition of apoptosis in lung tissue of ALI mice;(11)WB and tissue immunofluorescence staining were performed to detect lung tissue Cleaved-Gasdermin D,Gasdermin D,to observe that colchicine inhibited cell scorching in lung tissues of ALI mice;(12)Statistical methods:data were expressed as mean ± standard deviation and analyzed by one-way-ANOVA,two-way-ANOVA and Tukey’s multiple comparisons test,test level α=0.05.(5)the content of IL-6 and TNF-α in lung tissue was significantly reduced,and neutrophil chemotaxis was inhibited;(6)ROS content decreased after colchicine treatment,and the decrease in SOD and GSH-px content was alleviated;(7)TOS was inhibited,while TAS increased,and the oxidative stress index of TOS to TAS ratio decreased;(8)colchicine alleviated LPS mice macrophage apoptosis;(9)apoptosis-related protein Bax was inhibited and the level of anti-apoptotic protein Bcl2 rebounded;(10)colchicine down-regulated the level of Cleaved-Gasdermin D.Conclusions:(1)Colchicine reduced mortality,pulmonary edema,hypoxemia,pulmonary pathological damage and inflammatory response in mice with sepsis-associated ALI,showing that colchicine has protective or therapeutic effects on ALI.(2)Colchicine down-regulates ROS and TOS,up-regulates SOD,GSH-px and TAS,and inhibits LPS-induced oxidative stress.(3)Inhibit Bax and up-regulate Bcl2 to alleviate macrophage apoptosis.(4)Down-regulation of CleavedGasdermin D alleviates cell scorching.Chapter 2:Colchicine inhibits NLRP3 activation via regulation of STAT3 phosphorylation in sepsis-associated ALIObjective:To investigate the inhibitory effects of colchicine on NLRP3 inflammatory vesicle activation and STAT3 phosphorylation in sepsis-associated ALI lung tissues,as well as to verify the molecular mechanism that colchicine inhibition of LPS-induced NLRP3 activation in macrophages is accomplished by means of STAT3 phosphorylation.METHODS:(1)Tissue immunofluorescence staining,WB or qRT-PCR experiments were performed to observe the expression of NLRP3 and cleaved-caspase-1 in lung tissues;(2)ALI lung tissue homogenate supernatants were obtained and ELISA assays were performed to observe changes in IL-1β and IL-18 levels;(3)Prediction of colchicine target proteins in the SuperPRED database.intersection with sepsis-related GEO dataset and KEGG pathway enrichment analysis to further target the action targets,and autodock vina software to simulate the molecular docking of colchicine and target proteins;(4)WB and tissue immunofluorescence staining to observe the protein expression and phosphorylation levels of STAT3 in lung tissues of ALI mice;(5)corresponding kits to detect Ac-H3 and Ac-H4 levels;(6)LPS inflammation induction in macrophages,qRT-PCR for IL-1β,IL-18 and NLRP3 transcript levels in cell supernatant,WB for Cleaved-caspase-1,Cleaved-Gasdermin D,Gasdermin D,STAT3,P STAT3 and NLRP3 protein levels,and cellular immunofluorescence staining to assess NLRP3 and Gasdermin D expression;(7)observation of the number of positive cells for TUNEL staining to assess macrophage apoptosis;(8)overexpression of STAT3 in cells treated with LPS with colchicine,WB,qRT-PCR or immunofluorescence staining to detect Changes in the levels of Cleaved-caspase-1,Cleaved-Gasdermin D,STAT3,P-STAT3 NLRP3,IL-1β and IL-18 in macrophages,ChIP-qPCR assay to assess the enrichment of p-STAT3 in the NLRP3 promoter and to detect the p-STAT3 pull-down complex in a kit Ac-H3 and Ac-H4 levels;(9)Statistical methods:data were expressed as mean ± standard deviation and analyzed by one-way-ANOVA,two-way-ANOVA and Tukey’s multiple comparisons test with test level α=0.05.RESULTS:(1)Colchicine treatment significantly inhibited NLRP3 and caspase-1 activity in lung tissue of ALI mice;(2)LPS-induced elevated levels of IL-1β and IL-18 significantly decreased after colchicine action;(3)the predicted intersecting genes of colchicine target proteins with sepsis-related datasets were Ccrl,Ar,Cftr and Stat3.Further targeting STAT3 on the JAK/STAT pathway referred to by KEGG pathway enrichment;(4)Colchicine did not change the total protein content of STAT3,but down-regulated the phosphorylation level of STAT3;(5)Ac-H3 and Ac-H4 in lung tissue of ALI mice were inhibited by colchicine;(6)IL-1β,IL-18 in cell supernatants and Cleaved-caspase-1,Cleaved-Gasdermin D,p-STAT3,and NLRP3 were downregulated by colchicine;(7)the number of TUNEL-positive cells was significantly reduced;(8)Ac-H3 and Ac-H4 levels on the NLRP3 promoter increased with the enrichment of p-STAT3;(9)p STAT3 recruitment at the NLRP3 promoter was inhibited by colchicine.Conclusions:(1)Colchicine significantly inhibited NLRP3 activity in lung tissue of ALI mice,which in turn inhibited downstream caspase-1 activity and secretion of IL-1β and IL-18,showing that colchicine has an inhibitory effect on the lung inflammatory response in ALI mice;(2)Colchicine inhibited STAT3 phosphorylation.The effect of colchicine on STAT3 was achieved through the inhibition of STAT3 phosphorylation;(3)Colchicine inhibited Ac-H3 and Ac-H4 and down-regulated IL-1β,IL-18 and cellular Cleaved-caspase-1,Cleaved-Gasdermin D,P-STAT3,NLRP3,indicating that colchicine inhibited STAT3 phosphorylation through the inhibition of STAT3 phosphorylation thereby decreasing the recruitment of phosphorylated STAT3 at NLRP3,leading to the inhibition of NLRP3 and thus affecting the activation of inflammatory vesicles.Chapter 3:Phosphorylated STAT3 recruitment to EP300 forms a complex that activates NLRP3 inflammatory vesicles in macrophagesObjective:To investigate the activation of NLRP3 inflammatory vesicles in macrophages by phosphorylated STAT3 via recruitment of EP300 to form complexes.Methods:(1)The binding peak of EP300 at the promoter of NLRP3 was queried in the ChIP-seq database,and Cluspro2.0 software realized the simulated molecular docking of STAT3 and EP300;(2)Co-IP experiments were performed to explore the binding relationship between phosphorylated STAT3 and EP300 under the action of LPS and colchicine;(3)WB detection of changes in EP300 levels;(4)ChIP-qPCR was applied to detect the enrichment of EP300 at the promoter of NLRP3 and its changes with STAT3 phosphorylation;(5)macrophages overexpressing STAT3,the activator of EP300,CTB treatment,treated with LPS and colchicine,were detected by WB,immunofluorescence or RT-qPCR for EP300,p STAT3,NLRP3,cleaved-caspase-1,Cleaved-Gasdermin D Gasdermin D,IL-1β and IL-18 transcription or expression,the levels of Ac-H3 and Ac-H4 were detected by the kit,and the number of positive cells for TUNEL staining was observed for macrophage apoptosis;(6)Statistical methods:data were expressed as mean ± standard deviation and analyzed by one-way-ANOVA,two-way-ANOVA and Tukey’s multiple comparisons test with test levelα=0.05.Results:(1)EP300 in lung tissue had a significant binding peak at the promoter of NLRP3 in the ChIP-seq database,and STAT3 was able to stably bind the EP300 essential subunit during molecular simulation docking in Cluspro2.0 software.;(2)The enrichment of EP300 in the complexes pulled down by phosphorylated STAT3 was significantly higher after LPS treatment in the Co-IP experiments of AM,and colchicine inhibited the binding of phosphorylated STAT3 to EP300;(3)EP300 was significantly increased by LPS induction and this change was unaffected by colchicine treatment.(4)In ChIP-qPCR using EP300 antibody,there was a significant enrichment of EP300 at the promoter of NLRP3;(5)EP300 activation by the activator of EP300,CTB,after treatment of AM.EP300 activation was also able to promote NLRP3 expression in the absence of p-STAT3.expression of cytokines IL-1β and IL-18 in AM supernatant and cleaved-caspase-1 and Cleaved-Gasdermin D in AM were increased;(6)TUNEL-positive cell levels were also increased in EP300 activation of NLRP3 was significantly increased.(7)Statistical methods:data were expressed as mean±standard deviation and analyzed by one-way-ANOVA,two-way-ANOVA and Tukey’s multiple comparisons test with test level α=0.05.Conclusion:(1)phosphorylated STAT3 activates NLRP3 inflammatory vesicles in macrophages by recruiting EP300 to form a complex;(2)colchicine inhibits the inflammatory response by inhibiting the binding of phosphorylated STAT3 to EP300 and thereby downregulating NLRP3 vesicle levels.Chapter 4:ConclusionColchicine inhibition of STAT3 phosphorylation affects the regulation of NLRP3 promoter histone acetylation by phosphorylated STAT3 forming a complex with EP300 inhibiting NLRP3 inflammatory vesicle activation to alleviate sepsis-induced ALI.