The Mechanism And Significance Of PARP-1Impacting HMGB1Release Of LPS-induced Macrophage

Part I The influence of PARP-1on LPS-induced release and translocation of HMGB1from nucleus to cytoplasmObjective:To study the time pattern of PARP-1activity change in LPS-induced RAW264.7cells and the influence of PARP-1on the HMGB1secretion, to investigate the role of PARP-1in the inflammatory response.Methods:Cell ELISA methods were used to detect the time pattern of PARP-1activity change in RAW264.7cells LPS stimulated; Western blot assays were used to detect the content change of HMGB1in culture media supernatant, cytoplasm and nucleus of RAW264.7cells stimulated with LPS (100ng/ml) plus DMSO and LPS plus3-AB (10mM); Constructing PARP-1siRNA plasmid and the contrast sc siRNA plasmid, transient transfection into RAW264.7cells, which were stimulated with100ng/ml of LPS, using western blot assays to detect the content change of HMGB1in culture media supernatant, cytoplasm and nucleus.Results:The activity of PARP-1in RAW264.7cells was increased after the stimulation of LPS,which reached a peak at4h.4,8,16,24h after stimulation with LPS plus DMSO, the contents of HMGB1in cell culture supernatant were increased;2,4,8h after stimulation, the contents of HMGB1in the cytoplasm were increased, but decreased in nucleus, above mentioned reactions were inhibited when stimulated with LPS plus3-AB or silenced PARP-1expression through transfection of PARP-1siRNA.Conclusion:the activity of PARP-1in RAW264.7cells was increased after stimulated with LPS. restraining PARP-1activity or silencing its expression can reduce HMGB1to shift from nucleus to cytoplasm and its extracellular release. PARP-1participated in regulation of the translocation and release of HMGB1in LPS-induced cells. Part Ⅱ PARP-1mediated the LPS-induced nucleoplasm shift of HMGB1through promoting its acetylationObjective:To study the influence of PARP-1on the acetylation modification of HMGB1, and the activity of acetyltransferase and deacetylase in LPS-induced RAW264.7cells, to investigate the role of PARP-1in the inflammatory response.Methods:we used3-AB to inhibit the activity of PARP-1and silenced gene expression of PARP-1, then purify HMGB1through the immune precipitation methods, and then used western blot methods to detect acetylation modification and ADP-ribosylation modification of HMGB1, used colorimetric assay for detection of the HAT and HDAC activity in RAW264.7cells.Results:The acetylation levels of HMGB1in RAW264.7cells increased after LPS stimulation. Restraining PARP-1activity or silencing PARP-1expression can reduce acetylation levels of HMGB1, inhibit the HAT activity and increase HDAC activity.Conclusion:PARP-1can promote the acetylation of HMGB1by increasing the intracellular HAT activity, mediate the nucleoplasm shift of HMGB1. Part III the influence of the PAR modification point mutations in the NLS of HMGB1on its translocation from nucleus to cytoplasm.Objective:To study the influence of the PAR modification point mutations in the NLS of HMGB1on its nucleoplasm shift, to explore the role of PARP-1in the inflammatory response.Methods:We used3-AB to inhibit the activity of PARP-1and silenced gene expression of PARP-1, then purify HMGB1through the immune precipitation methods, and then used western blot methods to detect ADP-ribosylation modification of HMGB1. We mutationed the PAR modification sites in the NLS of HMGB1, RAW264.7cells were transfected with the HMGB1-GFP fusion protein we constructed, and then stimulated the cells with LPS, and inhibited PARP-1activity with3-AB. Western blot methods were used to detect HMGB1expression in cytoplasm and nucleus, and directly observed the expression of HMGB1in the nucleus and cytoplasm under a fluorescence microscope.Results:The ribosylation levels of HMGB1in RAW264.7cells increased after LPS stimulation. Restraining PARP-1activity or silencing PARP-1expression can reduce ribosylation levels of HMGB1. The PAR modification point mutations in the NLS of HMGB1inhibited the translocation of HMGB1to the cytoplasm. After the PAR modification point mutations in the NLS of HMGB1, inhibiting the activity of PARP-1had no effect on the shift of HMGB1from nucleus to cytoplasm.Conclusion:The PAR modification point mutations in the NLS has a predominance in the regulation of HMGB1translocation from nucleus to cytoplasm. Part IV The influence of PARP-1on LPS-induced mouse sepsisObjective:To investigate the impact of PAPR-1on the serum HMGB1content of sepsis in mice, as well as on the survival rate of sepsis.Methods:sixty male SPF Kunming mice were randomly divided into4groups:control group (PBS group), the solvent control group (LPS+DMSO group),3-AB intervention group (LPS+3-AB group), and AZD2281intervention group (LPS+AZD2281group)(n=15). The mice were given intraperitoneal injection of LPS to produce sepsis model. The control group was given intraperitoneal injection of PBS, and the remaining three groups were given DMSO,3-AB, and AZD2281respectively30min before intraperitoneal injection of LPS to make sepsis model. Three mice in each group were sacrificed8h after the completion of injection. Heart puncture to obtain blood sampling, serum was separated after centrifugation, western blot methods were used to test serum HMGB1levels, and the remaining mice were observed120h, record the time of death and number.Results:The HMGB1levels in serum increased after LPS stimulation, the serum HMGB1levels in LPS+3-AB group and LPS+AZD2281group was significantly lower than LPS+DMSO group8h after stimulation, the difference was significant (P<0.05). LPS induced sepsis lead to a significant lethal effect, the survival time of mice significantly prolonged after treatment with3-AB and AZD2281, no deaths observed during the period in the PBS control group. The survival time was significantly shorter in the LPS+DMSO group than the LPS+3-AB group and LPS+AZD2281group.Conclusion:The inhibition of PARP-1activity can reduce serum HMGB1levels in sepsis mice, and improve survival.