Role Of TLR4-NLRP3-GSDMD Pathway In Liver Injury Of Sepsis Aggravated By CD38 Deficiency

Objective:Sepsis is a serious infectious disease,which is caused by bacteria or bacterial products such as lipopolysaccharide（LPS）and other infectious factors.CD38,a multifunctional type II transmembrane protein,is a major ribose diphosphate cyclase in mammals.It is widely expressed on the surface of various immune cell membranes and participates in the host’s innate immune response to infection.It is closely related to toll like receptors（TLRs）,mitogen activated protein kinases（MAPK）and a variety of inflammatory signaling pathways such as pyroptosis.TLR4 is one of the important members of Toll like receptor family.TLR4 can activate NF-κB and MAPK signaling pathways after recognizing LPS,up regulate the expression of inflammatory cytokines,leading to septicemia,systemic inflammatory response syndrome and even death.In addition,recent studies have shown that NLRP3 inflammasome mediated pyroptosis plays an important role in sepsis related organ damage,mainly mediating the host immune response to microbial infection and cell damage.Our previous experimental results also showed that compared with WT mice,the expressions of TLR4 and inflammatory cytokines（TNF-α,IL-1β,i NOS,etc.）in CD38^-/-mice kidney were significantly increased in LPS induced septic acute kidney injury mice model,while in LPS induced CD38^-/-TLR4^mut septic mice,the expression of TLR4 and inflammatory cytokines（TNF-α,IL-1β,i NOS,etc.）decreased in varying degrees.At the same time,the pathological changes were significantly reduced.Therefore,we intend to further explore the role and potential mechanism of CD38 in E.coli-induced liver inflammation and pyroptosis.We speculate that CD38 deletion can aggravate liver injury in septic mice,and TLR4 mutation can reduce its injury effect.The purpose of this study was to elucidate the role and mechanism of CD38 in sepsis related-liver injury induced by E.coli in mice,and to explore the role of TLR4/NF-κB,MAPK and NLRP3 mediated pyroptosis signaling pathway in sepsis.The results of this study will broaden our understanding of the mechanism of CD38-TLR4 pathway regulating pyroptosis,clarify a new scheme to inhibit bacterial-induced liver injury in septicemia,and provide a new target for clinical treatment of septicemia.Methods:1)The standard strain of E.coli（ATCC25922）was resuscitated.After 24 hours of culture,the suspension was prepared and adjusted to the appropriate bacterial concentration by Macintosh turbidimeter.2)Male 8-week-old WT mice（C57BL/6）with 20±2g were intraperitoneally injected with 0.5m L PBS as control group and 0.5m L 3×10⁸cfu/m L E.coli as experimental group.Three hours later,the mice were anesthetized with ether and killed.The liver was dissected and blood centrifuged to collect serum.3)RT-q PCR was used to detect the m RNA expression of TNF-α,IL-1β,NLRP3,IL-18,IL-6 and i NOS in the liver of mice.Automatic biochemical analysis and ELISA were used to detect the content of AST and ALT in the serum.H&E staining was performed to observe the pathological changes,and bacteria culture,Gram staining were used to confirm the infection of E.coli in the liver.4)Male 8-week-old mice with 20±2g(WT,CD38^-/-and CD38^-/-TLR4^mut)were intraperitoneally injected with 0.5m L 3×10⁸cfu/m L E.coli.Three hours later,the mice were anesthetized with ether and killed,the liver was dissected and blood centrifuged to collect serum.5)The concentration of AST in serum was detected by automatic biochemical analysis three hours after E.coli infection.6)The concentration of ALT in serum was detected by ELISA three hours after E.coli infection.7)H&E staining was performed to compare the histopathological changes of liver in WT,CD38^-/-and CD38^-/-TLR4^mut mice infected with E.coli three hours later.8)Infected with E.coli three hours later,the expression of ERK1/2 and the distribution of ERK1/2 in the liver of WT,CD38^-/-and CD38^-/-TLR4^mut mice were detected by immunohistochemistry.9)The m RNA expression of TNF-α,IL-6,i NOS,Bax,NLRP3,ASC,caspase-1,IL-18,IL-1βand GSDMD were detected by RT-q PCR.10)The protein expression of TLR4,My D88,TRIF,NF-κB p65 and Phospho-NF-κB p65,ERK1/2 and Phospho-ERK1/2,inflammatory cytokines（TNF-α,IL-6,i NOS,Bax）and pyroptosis related molecules（NLRP3,ASC,pro caspase-1 and cleaved caspase-1,IL-18,IL-1β,GSDMD and its activation molecules GSDMD-C,GSDMD-N）in liver were detected by Western blot.Results:1)Intraperitoneal injection of 0.5m L 3×10⁸ cfu/m L E.coli could establish the model of septicemia related liver injury in mice.2)Compared with PBS group,E.coli could induce obvious pathological changes in liver tissue,including inflammatory cell infiltration,punctate necrosis,hepatocyte edema and hepatocyte proliferation.3)Compared with PBS group,E.coli stimulation significantly increased the concentration of AST（P<0.05）in serum and there is no significant difference of ALT.4)Compared with PBS group,the m RNA expression of pro-inflammatory cytokines TNF-α（P<0.05）,IL-6（P<0.05）,i NOS（P<0.05）and pyroptosis related molecules NLRP3（P<0.01）,IL-1β（P<0.01）and IL-18（P<0.01）in liver of E.coli infected group were significantly up-regulated.5)The results of bacterial culture in liver showed that E.coli could invade liver by intraperitoneal injection.6)Compared with WT+E.coli group,CD38^-/-+E.coli group mice showed more severe pathological damage in liver,such as increased inflammatory cell infiltration,more punctate necrosis,cell edema and hepatocyte proliferation;compared with CD38^-/-+E.coli group,CD38^-/-TLR4^mut+E.coli group mice showed significantly alleviated liver injury.7)Compared with WT+E.coli group,the concentration of serum AST（P<0.01）in CD38^-/-+E.coli group was significantly increased;compared with CD38^-/-+E.coli group,the concentration of serum AST（P<0.05）in CD38^-/-TLR4^mut+E.coli group was significantly decreased;however,there was no significant difference of ALT among the three groups.8)Compared with WT+E.coli group,the m RNA expression of i NOS（P<0.01）,IL-6（P<0.05）and TNF-α（P<0.05）in the liver of CD38^-/-+E.coli group were significantly increased;compared with CD38^-/-+E.coli group,the m RNA expression of i NOS（P<0.01）and TNF-α（P<0.05）in the liver of CD38^-/-TLR4^mut+E.coli group were significantly decreased,and there was no significant difference of IL-6;there was no significant difference of Bax expression in each group.9)Compared with WT+E.coli group,the protein expression of i NOS（P<0.01）,IL-6（P<0.01）and Bax（P<0.01）in CD38^-/-+E.coli group were significantly increased;compared with CD38^-/-+E.coli group,the protein expression of i NOS（P<0.01）,IL-6（P<0.01）and Bax（P<0.01）in CD38^-/-TLR4^mut+E.coli group were significantly decreased;there was no significant difference for TNF-α.10)Compared with WT+PBS group,the protein expression of My D88（P<0.01）and Phospho-NF-κB p65（P<0.05）in liver of WT+E.coli group were significantly up-regulated,while the protein expression of TLR4 and TRIF had no significant difference;compared with WT+E.coli group,the expression of TLR4（P<0.01）,My D88（P<0.05）,TRIF（P<0.01）and Phospho-NF-κB p65（P<0.01）in liver of CD38^-/-+E.coli group were significantly up-regulated;while compared with CD38^-/-+E.coli group,the protein expression of TLR4（P<0.01）,My D88（P<0.01）,TRIF（P<0.01）and Phospho-NF-κB p65（P<0.01）in CD38^-/-TLR4^mut+E.coli group were significantly decreased,and the degree of decline of My D88 was higher than that of TRIF.11)The expression and phosphorylation of ERK1/2 in CD38^-/-+E.coli group were significantly increased,but no ERK1/2 phosphorylation expression was found in other groups.12)Compared with WT+PBS group,the m RNA expression of NLRP3（P<0.01）,caspase-1（P<0.05）,IL-1β（P<0.01）and IL-18（P<0.01）in liver of WT+E.coli group were significantly increased,while the m RNA expression of ASC was not significantly different;compared with WT+E.coli group,the m RNA expression of NLRP3（P<0.01）,ASC（P<0.01）,caspase-1（P<0.01）and IL-1β（P<0.01）in liver of CD38^-/-+E.coli group were significantly increased,and the m RNA expression of IL-18 was significantly increased but no significant difference;Compared with CD38^-/-+E.coli group,the m RNA expression of NLRP3（P<0.01）,ASC（P<0.01）,caspase-1（P<0.01）,IL-1β（P<0.01）and IL-18（P<0.01）in CD38^-/-TLR4^mut+E.coli group were significantly decreased.13)The protein expression of ASC（P<0.01）,cleaved caspase-1（P<0.05）,IL-1β（P<0.01）and IL-18（P<0.01）were increased in the liver of WT mice stimulated by E.coli;compared with WT+E.coli group,the protein expression of NLRP3（P<0.01）,cleaved caspase-1（P<0.01）and IL-1β（P<0.05）in CD38^-/-+E.coli group were significantly increased,while the protein expression of ASC were not significantly different;compared with CD38^-/-+E.coli group,the protein expression of NLRP3（P<0.01）,cleaved caspase-1（P<0.01）,IL-1β（P<0.01）and IL-18（P<0.01）in CD38^-/-TLR4^mut+E.coli group were significantly decreased,however,there was no significant difference in the expression of ASC.14)The m RNA expression of GSDMD in the liver of WT mice was significantly increased under the stimulation of E.coli,and CD38 deletion further increased the expression of GSDMD（5 times of WT）,but TLR4 mutation significantly reduced the expression of GSDMD in the liver of septic mice.15)In liver of CD38^-/-septic mice,GSDMD was significantly activated,GSDMD-C and GSDMD-N were highly expressed,and TLR4 mutation could significantly reduce the activation of GSDMD.Conclusions:1)Intraperitoneal injection of standard strain of Escherichia coli（ATCC25922）can cause septic liver injury in mice.2)CD38 deletion can activate TLR4-My D88-NF-κB signaling pathway and promote the expression of its downstream inflammatory cytokines,thus aggravating E.coli-induced liver injury in septicemia.3)TLR4 mutation can significantly inhibit the activation of ERK1/2 in the liver of CD38 deficient septic mice,thereby alleviating liver injury.4)NLRP3-GSDMD mediated pyroptosis is the main mechanism of aggravated liver injury in sepsis caused by CD38 deficiency,and in which TLR4 plays a key role.