The Role Of High Mobility Group Protein In Endotoxin Tolerance And Its Mechanism

Background:High mobility group box protein1(HMGB1), as a non-histone proteins located in nucleus. It could be released and plays a key role in the intracellular signaling pathways involved in the development of inflammation Exogenous recombinant HMGB1preconditioning could induced endotoxin tolerance. This process, termed endotoxin tolerance, is characterized by decreased production of proinflammatory cytokines such as TNF-α but not anit-inflammatory cytokines such as IL-10. Endotoxin tolerance certain controls the development of the inflammatory response, but also is a key contributor to both second infection and mortality in sepsis patients. The complex effects of low-dose LPS preconditioning on immune signaling mechanisms are not fully understood until now. We do not know the neither certain process of endotoxin tolerance nor whether there is a promoter during this process.Objective:Endogenous HMGB1signaling mechanisms during low-dose lipopolysaccharide (LPS)-induced endotoxin tolerance and its mechanism were investigated.Methods:Chapter1:Mice were preconditioned with recombinant HMGB1or heat-treated recombinant HMGB1for1h, then injected with lethal-dose LPS. ELISA and WB measured TNF-a levels in serums and IRAK-M levels in hepatic tissues3h after the final treatments. Chapter2:Mice were preconditioned with low-dose LPS for24h, then injected with lethal-dose LPS. WB and ELISA measured HMGB1levels in hepatic tissues and serum3h after the final treatments. RAW264.7cells were preconditioned with low-dose LPS for18h, and then treated with lethal-dose LPS. The locations of HMGB1were observer by immunofluorescence4h after the final treatments; Chapter3:Mice were preconditioned with low-dose LPS for24h, followed by treatment with three consecutive injections of sodium butyrate at0.5,4and10h, respectively, and then injected with lethal-dose LPS. WB and ELISA measured HMGB1levels in hepatic tissues and TNF-α level in serums3h after the final treatments. TNF-α mRNA in hepatic tissues were measured by RT-PCR1h after the final treatments. Chapter4:Mice were preconditioned with low-dose LPS for24h, followed by treatment with three consecutive injections of anti-HMGBl neutralizing antibody at2,12and22h, respectively, then injected with lethal-dose LPS. Serum levels of TNF-α and hepatic levels of IRAK-M were measured by ELISA and WB3h after the final treatments. WB measured nuclear levels of NF-κB in hepatic tissue1h after the final treatments, and its ability to couple with DNA were measured by EMS A3h after the final treatments.Results:Chapter1:Recombinant HMGB1preconditioning induce endotoxin tolerance by increasing the expression of IRAK-M. LPS contamination does not play a role in tolerance induced by recombinant HMGB1in these experiments. Chapter2: low-dose LPS preconditioning enhance expression and release of endogenous HMGB1. Chapter3:Sodium butyrate inhibited endotoxin tolerance induced by low-dose LPS. Chapter4:Neutralizing endogenous HMGB1with anti-HMGB1antibodies following low-dose LPS preconditioning altered endotoxin tolerance by reducing hepatic IRAK-M expression, and partially restoring nuclear factor κB (NF-κB).Conclusions:Increased IRAK-M and decreased NF-κB activity in endotoxin tolerance is associated with endogenous HMGB1expression after low-dose LPS preconditioning.