Effect Of Regulating TLR9 Signaling Pathway Or Neutrophils Surface TLR9 Expression On Systemic Inflammatory Response Syndrome In Mice

Tissue damage or bacterial infection could induce systemic inflammatory response syndrome(SIRS),of which caused by infection is defined as sepsis.The high mortality of the disease has been a difficult clinical problem needing to be resolved.The inflammatory response mediated by Toll like receptor 9(TLR9)signaling pathway plays an important role in the development of SIRS.Using the SIRS-like mouse model induced by burn injury and sepsis mouse model induced by Escherichia coli(E.coli),our study observed the effect of TLR9 signaling pathway in the occurrence and development of the diseases,and intervened the inflammation of the mouse models by AAAG immunosuppressive oligodeoxynueleotide(AAAG ODN)or small molecular compound succinate.The results were as follows:1.Effect of regulating TLR9 signaling pathway on the development of burn induced SIRS in mice1.1 Establishment of burn induced SIRS-like mouse modelIn order to establish a TLR9 signaling pathway involved SIRS-like mouse model,we induced the systemic inflammatory response by burning the mouse skin,and observed the survival rate,the pathological changes of various organs,the copies of mt DNA in the plasma and the changes of TLR9 signaling regulated factors in local skin tissue after burn injury.Results showed that: 1)30% body surface area(TBSA)of the third-degree burns could cause 100% lethality of mice.2)The pathological changes of the kidney,liver,spleen and lung were all observed in the mice after burn,and there was obvious inflammatory cell infiltration and pulmonary hemorrhage in the lung tissue.3)The ratio of PMNs in the peripheral circulatory system of mice increased significantly after burn.4)The m RNA levels of TNF-? and IL-6 were significantly increased both in the skin tissue and white blood cells(WBCs)of mice after burn.5)The copies of mt DNA increased in the plasma of mice after burn,and the m RNA levels of TLR9,My D88,NF-?B and IRF5 in the skin tissue were significantly up-regulated.The results indicated that SIRS-like mouse model was successfully induced by the burn injury,and TLR9 signaling pathway was involved in the occurrence and development of SIRS.1.2 The regulating effect of AAAG ODN on inflammatory responses in SIRS-like mouse modelTo interfere with the development of SIRS,we injected immunosuppressive deoxynucleotide AAAG ODN into the abdominal cavity of SIRS-like mice,and observed the survival rate of mice,lung injury and the changes of cytokine TNF-? and IL-6.Results showed that: 1)Intraperitoneal injection of AAAG ODN could improve the survival rate of SIRS-like mice.2)AAAG ODN could reduce the lung injury in SIRS-like mice.3)AAAG ODN could reduce the m RNA levels of TNF-? and IL-6 in the skin tissue of SIRS-like mice.4)AAAG ODN could reduce the expression levels of TNF-? and IL-6 in WBCs of SIRS-like mice.The results suggested that AAAG ODN could interfere with the development of SIRS in model mice.1.3 The possible mechanism of the intervening effect of AAAG ODN on SIRS in model miceIn order to study the inhibition mechanism of AAAG ODN on SIRS,we detected the expression levels of TLR9,My D88,NF-?B and IRF5 of SIRS mice treated by AAAG ODN in the local damage skin tissue,and verified the TLR9 signaling pathway inhibition mechanism in vitro.Results showed that: 1)The expression level of IRF5,downstream of TLR9 signaling pathway,decreased after the intervention of AAAG ODN.2)IRF5 protein could bind directly with AAAG ODN.3)The IRF5 nuclear translocation induced by TLR9 agonist could be inhibited by AAAG ODN.The results suggested that the inhibition of AAAG ODN on SIRS was achieved by inhibiting the nuclear translocation of IRF5 which was the downstream of the TLR9 signaling pathway.2.Effect of regulating polymorphonuclear neutrophils surface TLR9 on sepsis in mice2.1 Effects of succinate on the expression of surface TLR9 on polymorphonuclear neutrophilsTo detect whether succinate could regulate the expression of surface TLR9(s TLR9)on polymorphonuclear neutrophils(PMNs)and their subsets,flow cytometry was used to analyze the expression of s TLR9 on PMNs and their subsets in white blood cells(WBCs)from normal mice treated with succinate in vivo and in vitro.Results showed that: 1)At 0.5 h post succinate injection,the expression of s TLR9 on PMNs and the ratio of s TLR9+cells in PMNs were both increased.2)At 6 h post succinate injection,the expression of s TLR9 on PMNs and the ratio of s TLR9+cells in PMNs were both decreased,while the ratio of PMNs in WBCs was significantly increased.3)In vitro,at 0.5 h post succinate stimulated WBCs,the expression of s TLR9 on PMNs and their subsets was upregulated,while 6 h after stimulation,the expression of s TLR9 on PMNs and their subsets was downregulated.The results suggested that the expression of s TLR9 on PMNs and their subsets was upregulated by short-term succinate stimulation,and downregulated by long-term stimulation.2.2 Effect of regulating the expression of s TLR9 on PMNs on the survival rate of sepsis-like mice and its possible mechanismTo investigate the effect of upregulation of s TLR9 expression on the survival of septic-like mice,we infected mice with E.coli at 0.5 h after injection of succinate.We observed the survival rate of septic-like mice and used flow cytometry to detect the expression of s TLR9 in PLCs and WBCs of septic-like mice within 8 h after infection.Results showed that: 1)The survival rate of septic-like mice could be improved by succinate.2)The expression of s TLR9 on PMNs and CD11b+ PMNs in PLCs and WBCs of septic-like mice could be upregulated by succinate.3)The ratio of s TLR9+CD11b+PMNs in PLCs of septic-like mice could be increased by succinate.4)The ratio of s TLR9+PMNs and s TLR9+CD11b+PMNs in WBCs of septic-like mice could be increased by succinate.5)The PMNs in PLCs and WBCs of septic-like mice could be reduced by succinate.The results suggested that upregulation of s TLR9 expression as well as the ratio of s TLR9+PMNs was associated with prolonged survival of sepsis mice.Summarily,we successfully established a burn-induced SIRS-like mouse model and found that: TLR9 signaling pathway was involved in the occurance and development of SIRS;inhibition of nuclear translocation of IRF5,a downstream molecule of TLR9 signaling pathway,could reduce the expression of pro-inflammatory cytokines in SIRS mice,prolonging their survival and reducing lung injury.In addition,we found that short-term succinate stimulation of WBCs in normal mice could upregulate the expression of s TLR9 on PMNs and their subsets.Besides,upregulation of s TLR9 expression as well as the ratio of s TLR9+PMNs was associated with the prolonged survival of sepsis-like mice.The study provided valuable experimental data for clinical rescue of SIRS patients.