SIRT2 Regulates HMGB1 Nucleoplasmic Shuttle And Degradation Via Deacetylation In Microglia

Objective High mobility group protein B1(HMGB1)is a class of nuclear DNA-binding proteins that are highly conserved in function and composition,and plays an important role in the pathogenesis of sepsis by acting as a damage-associated molecular pattern(DAMP)to regulate damage signals and stimulate immune responses during cell stress or death.The nucleo-cytoplasmic transfer of acetylated HMGB1 is the initial step for its release from activated immune cells into the extracellular system.SIRT2,as an NAD+ dependent deacetylase,can participate in the basic biological regulation of cells through the deacetylation process of various protein substrates.In the early stage of this experiment,SIRT2 was found to be the main factor regulating the occurrence and development of HMGB1 acetylation,but its process and biological effect mechanism remained unclear.Based on this,the interaction rule and biological effect mechanism between SIRT2 and HMGB1 were systematically discussed in this experiment.It provides potential targets and therapeutic approaches for the treatment or intervention of sepsis.Methods LPS was used to treat BV-2,and the inflammatory model was constructed in vitro.The regulatory relationship between SIRT2 and HMGB1 was determined by co-immunoprecipitation and immunofluorescence co-localization.Based on the results of pre-acetylation omics in the laboratory,wild SIRT2 and activity deletion plasmid,wild HMGB1 and lysine mutant plasmid at site 43 were constructed,and the effects of SIRT2 on the biological function of HMGB1 were determined by pulldown,immunofluorescence co-localization and diluciferase methods.In vivo experiments,wild-type mice and SIRT2 gene deletion(SIRT2-/-)mice were used to construct mice acute sepsis model by intraperitoneal injection of LPS,and hippocampal tissues of mice were collected and histological staining was used to observe hippocampal morphology.Western blot and quantitative PCR were used to detect the expressions of SIRT2,HMGB1 and related inflammatory factors in hippocampus.Results1,BV2 was treated with LPS to construct an in vitro inflammation model,and the regulatory relationship between SIRT2 and HMGB1 was clarified by immunoprecipitation and immunofluorescence co-localization;the results showed that SIRT2 and HMGB1 have a direct interaction;SIRT2 can interfere with HMGB1 translocation from the nucleus to the cytoplasm through deacetylation.2.Based on the results of pre-acetylation histology in the laboratory,SIRT2 wild and activity-deficient plasmids and HMGB1 wild and 43-site lysine mutant plasmids were constructed,and the biological function of SIRT2 on HMGB1 was clarified by pulldown,immunofluorescence co-localization and dual fluorophore enzymes,etc.The results showed that SIRT2 could interfere with HMGB1 through 43-site lysine The results showed that SIRT2 could deacetylate HMGB1 through lysine at site 43,and that SIRT2 interfered with the ubiquitin degradation of HMGB1 by up-regulating its acetylation modification in an enzyme activity-dependent manner.3.In the in vivo test,a mouse model of acute sepsis was constructed by intraperitoneal injection of LPS based on wild-type mice and SIRT2-/-mice,and hippocampal tissues were collected to observe the morphology of the hippocampal body by histological staining and Western blot and fluorescence quantitative PCR to detect SIRT2,HMGB1 and related inflammatory factors in the hippocampus,The expression of SIRT2,HMGB1 and related inflammatory factors in the hippocampus was measured by Western blot and fluorescence PCR.The results showed that SIRT2 deletion had no effect on the number of mature neurons,whereas a more severe neuroinflammatory phenomenon occurred in SIRT2-/-mice and was accompanied by a significant elevation of HMGB1 acetylation,which would also promote its K48 site-mediated ubiquitination degradation.Conclusion SIRT2 can deacetylate lysine site 43 of HMGB1 in a histidine 187 active site-dependent manner.The deacetylation function of SIRT2 interferes with the nucleo-cytoplasmic shuttle of HMGB1 and the degradation of polyubiquitin chain linked by lysine 48 site.SIRT2 gene deletion can aggravate the inflammatory response of nerve tissue and promote the occurrence and development of sepsis,but has no effect on the number of mature neurons.It was further proved that SIRT2 gene can affect the occurrence and development of sepsis.