| Background:Sepsis is a life-threatening organ dysfunction distinguished by the dysregulated response to infection,which is the leading cause of global mortality and disability of critical patients.In addition,the exact pathogenesis of sepsis has not been fully understood,and there is bare of effective strategies have been exploited for sepsis treatment.Pyroptosis,a form of programmed cell death,is closely associated with the inflammatory response and directly affects the occurrence,progression,and outcome of inflammatory diseases such as sepsis.Moreover,high mobility group protein B1（HMGB1）is an important inflammatory mediator in sepsis,which plays a significant role in cellular necrosis.In this thesis,our results indicate a significant decrease of macrophages during the sepsis and inhibiting the cellular pyroptosis led to a recovery of the number of macrophages.This finding suggests that macrophage pyroptosis has a significant relevance with the sepsis development,and the specific mechanism still needs to be investigated.Purpose:The aim of this study is to investigate the functional role of HMGB1 in macrophages pyroptosis induced sepsis,which potentially provides a new idea for the clinical treatment of sepsis.Methods:1.To explore the sepsis mediated pyroptosis mode of macrophage and its role in sepsis:（1）To explore the effects of different cell death processes on the survival rate of septic mice:Mouse models of sepsis induced by lipopolysaccharide（LPS）,cecal ligation and puncture（CLP）were established respectively;the survival rate of septic mice was observed after treated with pyroptosis（disulfiram,DSF）,apoptosis（MX1013）,necroptosis（necrostatin-1,Nec-1）,and ferroptosis（ferrostatin,Fer-1）inhibitors;（2）To explore the effects of different pyroptosis pathways on the survival rate of septic mice:（1）Enzyme-linked immunosorbent assay（ELISA）was applied to detect the levels of inflammatory factors related to pyroptosis IL-1β,IL-18 and lactic dehydrogenase（LDH）of LPS,CLP models and patients with sepsis;（2）The survival of LPS and CLP models was observed after the treatment with a canonical pyroptosis inhibitor（VX-765）and a non-canonical pyroptosis inhibitor（lyso-phosphatidylcholine,LPC）;（3）To investigate the effects of non-canonical pyroptosis pathway of macrophages on the survival rate of septic mice:（1）Peritoneal macrophages of mice were exhausted by clodronate liposomes（Lipo）;LPS group,Lipo+LPS group and Lipo+LPS+LPC group were set,the survival of mice was observed and the survival curves were drawn;（2）Cleaved caspase-11 and N-GSDMD protein levels were detected in peripheral blood monocytes and peritoneal macrophages of LPS and CLP models by western blot.Cleaved caspase-4 and N-GSDMD protein levels were detected in peripheral blood monocytes of patients with sepsis by western blot.2.To explore the promoting mechanism of different sources and forms of HMGB1 in the non-canonical pyroptosis of macrophages:（1）To detect the HMGB1 binding to LPS in sepsis:（1）Plasma HMGB1 levels of LPS and CLP models and patients with sepsis were measured by ELISA;（2）The formation of HMGB1/LPS complex in plasma of LPS models was detected by mass spectrometry（MS）;（2）To explore the molecular mechanism of HMGB1 promoted the non-canonical pyroptosis of macrophages:（1）Macrophages were treated with LPS and 6×His-HMGB1,and the nuclear fraction of macrophages was separated to detect the levels of 6×His-HMGB1 in the cytoplasm and nucleus by western blot.The binding of HMGB1/LPS and caspase-11 was detected by co-immunoprecipitation（Co-IP）;（2）Macrophages were separated from Tlr4-/-mice,then treated with LPS and fully reduced HMGB1（fr HMGB1）to detect the levels of cleaved caspase-11 and N-GSDMD by western blot.Receptor for advanced glycation end products（RAGE）in macrophages was knocked down and treated with LPS and fr HMGB1 to detect the levels of cleaved caspase-11 and N-GSDMD by western blot;（3）To explore the effects of different sources and forms of HMGB1 on non-canonical pyroptosis of macrophages:（1）Cells in peritoneal lavage fluid from LPS model mice were separated by differential adhesion method to acquire macrophages and non-macrophages,and their culture supernatants were collected respectively.DMEM（10%FBS）group,supernatants of macrophages group,supernatants of non-macrophages group,peritoneal lavage fluid（PLF）without LPS group,DMEM+LPS group,supernatants of macrophages+LPS group,supernatants of non-macrophages+LPS group,PLF without LPS+LPS group,PLF without LPS+LPS+HMGB1 Ab group were set,and the levels of cleaved caspase-11 and N-GSDMD were detected by western blot;（2）Heart,liver,lung,and kidney tissues of the LPS model mice were extracted and homogenized,LPS in the homogenate supernatant was removed.Macrophages were treated with the homogenate supernatant of these tissue cells with LPS.The levels of cleaved caspase-11 and N-GSDMD were detected by western blot.（3）HMGB1 in macrophages was knocked down,and the knocked-down and non-knocked-down macrophages were treated with LPS and fr HMGB1 to detect the levels of cleaved caspase-11 and N-GSDMD by western blot;（4）In a cell-free system,LPS-Biotin was incubated with fr HMGB1,disulfide HMGB1（ds HMGB1）,and terminally oxidized HMGB1（ox HMGB1）,respectively,then the binding of HMGB1and LPS was detected by western blot after streptavidin-agarose bead binding;Macrophages were treated with LPS and the three forms of HMGB1 to detect the levels of cleaved caspase-11 and N-GSDMD by western blot;3.To explore the effect and mechanism of A Box on non-canonical activation pathway mediated macrophage pyroptosis:（1）To explore the effect of A Box on septicemia mice:（1）The survival of septic mice was observed after the treatment with A Box and the survival curves were drawn;（2）The structure changes of cardiac,hepatic and pulmonary and the infiltration of inflammatory cells in LPS model mice were observed by hematoxylin-eosin staining（H（5）E）;（3）Plasma levels of IL-1β,IL-18 and LDH were detected by ELISA;（2）To explore the effects of A Box on macrophage pyroptosis via a non-canonical pathway:（1）The levels of cleaved caspase-11 and N-GSDMD proteins of peripheral blood monocytes and peritoneal macrophages in LPS and CLP mice models were detected by western blot;（2）Macrophages of mice were isolated and then treated with LPS,fr HMGB1 and A Box.The levels of N-GSDMD were detected by immunofluorescence（IF）.The levels of cleaved caspase-11 and N-GSDMD proteins were detected by western blot;（3）To explore the mechanism of A Box inhibiting macrophage non-canonical pyroptosis:（1）Macrophages were treated with LPS-Biotin and fr HMGB1.IF was used to detect the content of LPS in cytoplasm;（2）In a cell-free system,LPS-Biotin was incubated with fr HMGB1 and A Box with different dosages.Western Blot was used to detect the contents of HMGB1,and A Box bound to LPS after streptavidin-agarose bead binding.Results:1.Macrophage pyroptosis mediated by non-canonical activation pathway is closely associated with the sepsis induced high mortality.Inhibition of pyroptosis and apoptosis significantly improved the survival rate of mice in LPS and CLP models,higher survival rate was observed in the group treated with a pyroptosis inhibitor.These results suggest that pyroptosis was the main cause of death in septic mice.Plasma levels of IL-1β,IL-18,and LDH activity were significantly elevated in both LPS and CLP models of mice and septic patients.Inhibition of both canonical and non-canonical pathways of pyroptosis increased the survival rates of mice in LPS and CLP models,which the higher survival rate was observed in the group treated with a non-canonical pathway inhibitor.These results indicate that the non-canonical pathway-mediated pyroptosis was predominate in septic mice.Depletion of peritoneal macrophages in Lipo mice significantly improved the survival rate of mice in the LPS model.Compared to the LPS model mice with depleted peritoneal macrophages,the use of LPC did not further enhance the survival rate,suggesting that the non-canonical pyroptosis of peritoneal macrophages affected the survival rate in septic mice.Peripheral blood monocytes and peritoneal macrophages in LPS and CLP model mice exhibited significantly elevated expression levels of N-GSDMD and cleaved caspase-11,while septic patients showed significantly elevated expression levels of N-GSDMD and cleaved caspase-4 in peripheral blood monocytes.These results indicate that monocytes/macrophages experienced a non-canonical pathway pyroptosis in LPS and CLP model mice and septic patients.2.fr HMGB1 released from the tissue cells binding to LPS through RAGE,activated caspase-11,which further induced macrophage pyroptosis in sepsis mice.Plasma levels of HMGB1 were significantly increased in LPS and CLP models,as well as in septic patients.Mass spectrometry（MS）detected multiple HMGB1peptides in the plasma of LPS models,indicating the formation of the complexed HMGB1/LPS structures.Treatment of peritoneal macrophages with LPS and 6×His-HMGB1 resulted in a detection of 6×His-HMGB1 in the cytoplasm of macrophages,and caspase-11 was detected.These findings demonstrate that HMGB1 combined with LPS in the cytoplasm and activated the caspase-11 in a complex formation.The knockdown of RAGE prevented non-canonical pyroptosis of macrophages with LPS and HMGB1 treatment,while Tlr4-/-macrophages treated with LPS and HMGB1showed similar level of pyroptosis-related protein compared with the control group.These results indicate that HMGB1-mediated pyroptosis depended on RAGE rather than TLR4.It was suggested that the binding of HMGB1 to LPS facilitated the LPS delivery to the cytoplasm via RAGE,and binding with caspase-11 promoted the macrophage pyroptosis.In the presence of LPS,PLF from LPS models significantly enhanced non-canonical pyroptosis in macrophages,while neutralizing antibodies against HMGB1 inhibited the pyroptosis occurrence.These results indicate that HMGB1 derived from PLF contributed to pyroptosis.Peritoneal macrophage culture supernatant from LPS models did not promote non-canonical pyroptosis in macrophages,indicating that HMGB1 derived from peritoneal macrophages did not cause the pyroptosis.However,the homogenate supernatant from the heart,liver,lung,and kidney in LPS models LPS promoted the non-canonical pathway-mediated pyroptosis in macrophages,indicating that tissue-derived HMGB1 induced the pyroptosis.Pyroptosis-related protein levels remained elevated in macrophages transfected with sh HMGB1 interference lentivirus after LPS and HMGB1 treatment,suggesting that HMGB1 within macrophages did not affect caspase-11-mediated non-canonical pyroptosis.Protein pull-down assay results demonstrate that only fr HMGB1could bind to LPS and promoted caspase-11-mediated non-canonical pyroptosis in macrophages.3.A Box protects septic mice by competitively inhibiting the binding of HMGB1to LPS,and further inhibits the non-canonical pyroptosis of macrophages.In vivo experiments suggest that A Box significantly improved the survival rate of mice with sepsis,alleviated inflammatory cell infiltration and tissue injury,reduced the level of plasma pyroptosis related factors such as IL-1β,IL-18,and LDH,and inhibited the non-canonical pyroptosis of monocytes/macrophages.In vitro experiments confirm that A Box inhibited the non-canonical pyroptosis of macrophages mediated by caspase-11.These results illustrate that HMBG1 A Box has the ability to improve the survival rate of septic mice by inhibiting non-canonical pyroptosis of macrophages.IF images indicate that A Box reduced the LPS content in cytoplasm.Pull-down results show that A Box inhibited the binding of HMBG1 to LPS by competitively binding with LPS,and the dose-dependent tendency was observed of the binding ability of A Box to LPS.Conclusions:1.The sepsis induced non-canonical pyroptosis of macrophages was the main cause of death in septic mice.2.The fr HMGB1 released from the tissue cells in sepsis process can combine with LPS to promote the non-canonical pyroptosis of macrophages.3.A Box inhibited the non-canonical pyroptosis of macrophages by competitively inhibiting the binding of HMGB1 to LPS,thereby improved the survival rate of sepsis mice. |
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