GSTP Inhibits The Nucleation And Release Of HMGB1 To Improve The Molecular Mechanism Of The Survival Rate Of Septic Mice

Background:Sepsis is a severe complication of infection,burn,trauma,shock and it has a high mortality rate.Recent evidence indicates that high mobility group protein B1(HMGB1)is a late mediator of sepsis,and HMGB1-targeting strategies are effective to human sepsis.Macrophages and monocytes actively release HMGB1 after challenged by different stimulators,such as lipopolysaccharide(LPS),TNF-α or interleukin-1(IL-1).Activation of cells results in the redistribution of HMGB1 from the nucleus to cytoplasm,followed by HMGB1 is secreted via an unconventional protein secretion pathway.Once released into extracellular,HMGB1 stimulates massive production of inflammatory mediators(e.g.TNF-α,IL-1,IL-6),which further expand the inflammatory response.In mammalian cells,glutathione S-transferases P(GSTP)is the most widely distributed and abundant one in Glutathione S-transferases(GSTs)family.In a previous report,we found that GSTP inhibited LPS-induced excessive TNF-α,IL-1β and NO production.Meanwhile,mice treated with recombinant GSTP protein,could remarkably reduce the mortality rate of endotoxic shock.Methods:In this study,to investigate the role of endogenous GSTP in septic mice,we constructed the CLP model of sepsis and compared the mortality rate,acute lung injury level and HMGB1 release level of Gstp-/-and Gstp+/+septic mice.We further used ELISA,western blot,nuclear extract preparation,co-immunoprecipitation,immunofluorescence microscopy,mass spectrometry and a variety of molecular biology methods to clarified the intermolecular interaction between GSTP,HMGB1 and classic protein kinase C(cPKC)in LPS-induced monocyte and macrophage.Results:1.Gstp-/-mice displayed a higher mortality rate,acute lung injury level and HMGB1 release level than Gstp+/+mice in CLP model and administration of GSTP protein can significantly improve the survival rate of septic mice.2.Over-expressed GSTP could translocate to nucleus and inhibit lipopolysaccharide(LPS)-induced HMGB1 release in macrophages owing to interaction with HMGB1.3.LPS elevated the phosphorylation,especially the Ser184 of GSTP,which was crucial to the nucleus translocation of GSTP.4.cPKC was the upstream kinase of GSTP Ser184 and the activation of cPKC was essential for the nuclear translocation of GSTP in LPS-induced RAW264.7 cells.Conclusion:Both endogenous and exogenous GSTP protect against excessive inflammation in the animal septic model.Phosphorylated GSTP translocates to the nucleus to inhibit LPS-induced HMGB1 cytoplasmic translocation and release in monocyte and macrophage,which improves the survival rate of septic mice.This study elucidate the intermolecular interaction between GSTP,HMGB1 and cPKC three in LPS stimulated cells and provided a theoretical basis and clinical reference for the treatment of sepsis.