The Effect And Intervention Of NLRP3 Inflammasome In Infection-Induced Coagulation

Objective:Disseminated intravascular coagulation（DIC）is a common complication of serious infection.It is caused by uncontrolled activation of coagulation cascades.DIC is life-threatening and it has caused huge economic losses worldwide.However,the mechanism of infection-induced coagulation is not well understood.Previous research of our team demonstrated that noncanonical inflammasomes participate in infection-induced coagulation by control of Ca²⁺influx,thus trigger phosphatidylserine externalization and tissue factor activation.In addition to noncanonical inflammasomes,the role of other inflammasomes such as NLRP3,NLRP1,NLRP6,AIM2,pyrin,and NLRC4 in infection-induced coagulation is still unclear.Among these inflammasomes,the NLRP3inflammasome is the most extensively studied.The NLRP3 inflammasome was involved in many infectious and non-infectious inflammatory diseases,which is also reported its role in thrombosis-related diseases recently.However,whether the NLRP3 inflammasome is involved in infection-induced coagulation is still a mystery.We sellected series of transgenic mice lacking NLRP3 inflammasome components in a few mouse models to explore the role and mechanism of NLRP3 inflammasome in infection-induced coagulation.Meanwhile,we explored the possibility of targeting NLRP3 inflammasome to intervene infection-induced coagulation.On this basis,we screened extracts of traditional Chinese medicine that inhibit the NLRP3 inflammasome and evaluated its therapeutic effect on infection-induced coagulation,in order to further reveal the role of NLRP3inflammasome in infection-induced blood coagulation,and to provide convinced evidence for discovering traditional Chinese medicine extracts to alleviate infection-induced coagulation.Method:1 The function of NLRP3 inflammasome in infection-induced coagulation1.1 The role of the NLRP3 inflammasome in animal models of infection:We sellected wild-type mice,NLRP3^-/-mice and ASC^-/-mice in three serious infection models:two sepsis models:endotoxemia model,cecal ligation and puncture model（CLP）;and a common serious surgical infection model:acute pancreatitis model built with cerulein and LPS.After sacrifice,we detected coagulation indicators TAT,PAI-1,D-dimer and prothrombin time（PT）,as well as IL-1βand tissue factor（TF）;damages of tissues and fibrin IHC staining were observed.1.2 The effect of NLRP3 inflammasome on TF expression:Livers and lungs of wild-type mice,NLRP3^-/-mice and ASC^-/-mice in endotoxemia were taken TF IHC staining and further detected the transcription of TF by q PCR and the translation of TF by western blot.1.3 The mechanism of NLRP3 inflammasome on TF expression:All the wild-type mice,NLRP3^-/-mice and ASC^-/-mice were recruited to the correlation analysis of TAT,PAI-1 with IL-1β,as well as IL-1βand TF.2 The intervention of targeting the NLRP3 inflammasome in infection-induced coagulation2.1 The effect of MCC950 in infection-induced coagulation:In the endotoxemia model,wildtype mice were pretreated with MCC950,a classic NLRP3 inflammasome inhibitor.After sacrifice,coagulation indicators TAT,PAI-1,IL-1βand tissue factor were examined.2.2 Exploring the role of traditional Chinese medicine extracts in infection-induced coagulation2.2.1 Screening NLRP3 inflammasome inhibitors from traditional Chinese medicine extracts in vitro:Several common traditional Chinese medicine extracts were added to intervene the NLRP3 inflammasome agonist-stimulated mouse peritoneal macrophages（PM）,then IL-1βand LDH were detected to screen NLRP3 inflammasome inhibitors.2.2.2 The effect of PL on NLRP3 inflammasome activation in vitro:IL-1βand release of LDH were detected in supernatants from PM and human THP-1cells treated with different doses of PL and stimulated with NLRP3 inflammasome agonists.Immunoblots of cleaved Caspase-1 and cleaved IL-1βwere extracted from supernatants or cell lysates in those stimulated PM treated with different doses of PL.2.2.3 The effect of NLRP3 inflammasome inhibitor piperlongumine（PL）on infection-induced coagulation:We built the endotoxemia model in wildtype mice and pretreated with PL.Then we detected the coagulation indicators TAT,PAI-1,IL-1βand tissue factor.Lung injury was evaluated.2.2.4 Mechanism of PL inhibiting the NLRP3 inflammasome:Immunofluorescence microscopy analysis and Immunoblot analysis were performed to evaluate effect of PL on ASC speck formation in NLRP3inflammasome agonists-stimulated mouse peritoneal macrophages.Immunoprecipitation was performed to evaluate the interaction between NLRP3 and ASC or that between NLRP3 and NEK7.NLRP3oligomerization was detected by western blot.Result:1 The role of NLRP3 inflammasome in infection-induced coagulation1.1 NLRP3 or ASC deficiency attenuates infection-induced coagulation:In mice model of sepsis and acute pancreatitis,deletion of NLRP3 or ASC reduced coagulation indicators,IL-1βand TF.It also alleviated fibrin deposits in lungs and livers as well as the organ injuries.1.2 Deficiency of the NLRP3 inflammasome reduces TF expression:Results of IHC staining showed that the expression of TF in livers and lungs was markedly reduced in NLRP3 inflammasome-deficient mice.q PCR and WB suggested that the transcription and translation of TF of NLRP3 or ASC knockout mice were significantly blocked.1.3 The NLRP3 inflammasome contributes to infection-induced coagulation probably through IL-1βrelease-induced TF expression:With Pearson correlation analysis,TAT and PAI-1 were highly correlated to IL-1β;IL-1βwas significantly correlated with TF,suggesting that NLRP3inflammasome functions through IL-1βrelease-induced TF expression.2 Effect of intervening the NLRP3 inflammasome in infection-induced coagulation2.1 The NLRP3 inflammasome classic inhibitor MCC950 prevents infection-induced coagulation:In the endotoxemia model,mice pretreated with MCC950 presented lower TAT,PAI-1,IL-1βand TF levels in plasma.2.2 PL,a traditional Chinese medicine extracts,prevents infection-induced coagulation through inhibiting NLRP3 inflammasome.2.2.1 Screen NLRP3 inflammasome inhibitors from traditional Chinese medicine extracts in vitro:different traditional Chinese medicine extracts were added to the NLRP3 inflammasome agonist-stimulated mouse macrophages.Results showed that PL inhibited pyroptosis and the release of IL-1β,confirmed its inhibitory effect on the NLRP3inflammasome.2.2.2 PL inhibits NLRP3 inflammasome activation in vitro:PL inhibited pyroptosis and IL-1βin mouse macrophages and human THP-1cells stimulated by NLRP3 inflammasome agonists.PL also reduced cleaved Caspase-1 and cleaved IL-1β.2.2.3 PL attenuates endotoxemia-induced coagulation:In endotoxemia model,Pretreatment of PL reduced plasma TAT,PAI-1,IL-1βand TF.PL also attenuated lung injury.2.2.4 The mechanism of PL inhibiting the NLRP3 inflammasome activation:Immunofluorescence and WB results showed that PL significantly reduced ASC speck.Co-IP results suggested that PL inhibited the association between NLRP3 and NEK7,thus blocked the self-aggregation of NLRP3.Conclusions:1.The NLRP3 inflammasome contributes to infection-induced coagulation.2.The NLRP3 inflammasome promotes TF expression through IL-1βrelease.3.PL,extracts of traditional Chinese medicine,inhibits the NLRP3inflammasome and prevents infection-induced coagulation.4.PL inhibits the activation of the NLRP3 inflammasome by blocking the interaction between NLRP3 and NEK7.