The Mechanism And Targeted Intervention Of HMGB1-caspase-11 Pathway In Promoting Disseminated Intravascular Coagulation In Sepsis

Objective: sepsis is a serious disease caused by infection and multiple organ dysfunction.It is easy to induce disseminated intravascular coagulation(DIC),which is a serious threat to human health.It is urgent to seek its pathogenesis and effective treatment.Previous studies of our research group showed that high mobility group protein B1(HMGB1)can mediate the transport of lipopolysaccharide(LPS)to activate caspase-11 containing cysteine in cells,form pores in the cell membrane through Nterminal-gasdermin D(GSDMD),and promote the activation of tissue factor(TF)by phosphatidylserine(PS),So as to activate the extrinsic coagulation pathway and finally induce DIC.During the onset of sepsis,a large number of extracellular vesicles(EV)released are rich in externalized PS on the surface,which can also activate the extrinsic coagulation pathway by activating TF.However,the mechanism is not clear at present.The purpose of this study was to explore the mechanism of DIC induced by sepsis and to seek the means of targeted intervention.Methods:(1)Overall animal experiments: adult C57BL/6 male mice were randomly divided into control group,simple drug group,model group and model + drug group.Glycyrrhizin and its clinical drugs were given to intervene in advance according to the groups to build endotoxemia model and cecal ligation and perforation(CLP)sepsis model.The survival of mice in each group was observed in 7 days,or the materials were taken 16-18 hours after the model was prepared,the pathological changes of liver and lung tissues were observed by H&E staining.The activation of caspase-11 and GSDMD in liver,lung and spleen tissues was detected by Western blot.The serum of interleukin-1α/β were detected by ELISA.The contents of thrombin antithrombin complex Ⅲ(TAT)and plasminogen activator inhibitor-1(PAI-1)were measured by automatic liver function analyzer,and the contents of alanine transaminase(ALT)and aspartate aminotransferase(AST)in serum were measured.The formation of thrombus in viscera and blood vessels of mice was observed by rotating disc confocal microscope in vivo.(2)In vitro cell experiments: primary peritoneal macrophages of wildtype,caspase-11,GSDMD,P2X7 R and IL-1R knockout mice were obtained and stimulated with LPS and CTB to induce cell death.After 16-18 hours,LDH content in cell supernatant was detected,and IL-1β in cell supernatant was detected by ELISA.The content of EV was detected by flow cytometry.Obtain the primary macrophage or human monocyte cell line THP-1 from the abdominal cavity of wild-type mice,give different doses of glycyrrhizin to interfere,add LPS and recombinant protein HMGB1 to co stimulate and induce cell death,collect the cell protein and supernatant 16-18 hours later,determine the toxicity and effective dose of glycyrrhizin through CCK8,and detect IL-1α,IL-1β in the supernatant by ELISA.The activation level of caspase-11/caspase-4,caspase-1,GSDMD and IL-1β were detected by western blot.(3)Molecular mechanism experiments: molecular docking was used to simulate the molecular docking between glycyrrhizin and HMGB1 and calculate the affinity.HMGB1 competition binding experiment was used to determine the inhibitory effect of glycyrrhizin on the binding of HMGB1 to LPS.Limulus amebocyte lysate test was used to detect whether glycyrrhizin reduced the content of intracellular LPS.Ortho ligation technique was used to determine whether glycyrrhizin inhibited the binding of intracellular LPS to caspase-11.Result:(1)The release of EV and the activation of TF depend on caspase-11 mediated cell death.Preventing cell death by glycine can inhibit TF and IL-1β.The intervention of caspase-11 knockout or glycine can reduce the contents of Tat and PAI-1 in serum of endotoxemia mice,and the elimination of macrophages can reduce the formation of thrombin and platelet aggregation in liver vessels of endotoxemia mice.(2)Glycyrrhizin and compound glycyrrhizin significantly reduce the levels of TAT,PAI-1,ALT,AST and IL-1β in serum of septic mice,and inhibit the cleavage of GSDMD in liver,lung and spleen,reduce the damage of organ and tissue structure,and improve the survival rate of mice.Glycyrrhizin could significantly inhibit the activation of caspase-11/caspase-4 and GSDMD under the stimulation of recombinant protein HMGB1+LPS,and reduce the release of LDH and cytokine IL-1α/β.(3)Glycyrrhizin is highly compatible with HMGB1 at the molecular level.It inhibits the binding of HMGB1 to LPS through competitive binding and reduces the binding of intracellular LPS to caspase-11.Conclusion: caspase-11 mediated cell death can induce the formation of disseminated intravascular coagulation in sepsis by releasing EV activated TF;glycyrrhizin and compound glycyrrhizin selectively inhibit HMGB1-caspase-11 mediated cell death,reduce lethal immune response and excessive coagulation activation in septic mice,and improve organ function;glycyrrhizin inhibits the intracellular transport of LPS by competitively binding HMGB1,thereby inhibiting the activation of caspase-11.