The Influence Of Poly ADP-ribose Polymerase 1 (PARP-1) On HMGB1 Localization And Secretion In RAW264.7 Cell

ObjectiveTo study the influence of poly ADP-ribose polymerase 1 (PARP-1) on HMGB1 localization and secretion in RAW264.7 cell, from the subucellular structures to observe HMGB1 from the nucleus to the cytoplasm translocation process, which provided a new therapeutic strategy for severe acute pancreatitis.MethodsRAW264.7 cells were treated with 100ng/ml LPS for 0h, 1h, 2h, 4h, 8h, 16h. The time pattern of PARP-1 activity was measured by Cell ELISA ; RAW264.7 cells were treated with 100ng/ml LPS (or using inhibitors of PARP-1 3-AB meanwhile, a final concentration of 10mmol/l) for 0h, 1h, 2h, 4h, 8h, 16h. HMGB1 temporal variations in the intracellular localization was observed under a fluorescence microscope by using immunofluorescence, of HMGB1 level in the of supernatant was detected by Western-blot; we constructed expression plasmid HMGB1-EGFP(wild type,WT) and HMGB1-EGFP(mutated type,MT)which glutamic acids 40,47,179 of NLS were point-mutated into alanine, transiently transfected into RAW264.7 cells, then transfected cells were treatment with 100ng/ml LPS, observed intracellular localization of HMGB1 under the fluorescence microscope.ResultsPARP-1 activated and tended to peak 4 hours after RAW264.7cells were induced with LPS, Subsequent activity decreased. HMGB1 translocated from the nucleus to the cytoplasm when RAW264.7cells were induced after 1 hour. HMGB1 level in the cytoplasm time-dependently increased. Mutation of glutamic located in HMGB1 NLS didn't reduce the HMGB1 translocation from the nucleus to the cytoplasm. The glutamine located in HMGB1 NLS didn't influence the HMGB1 translocation.ConclusionPoly ADP-ribose polymerase 1 (PARP-1) played a role in the process of the high mobility group protein 1 (HMGB1) translocation. PARP-1 regulated the secretion of HMGB1, which modified HMGB1 by ADP-ribosylation in posttranslation. The mutated glutamine located in HMGB1 NLS didn't influence the HMGB1 translocation. This indicated 3-AB could inhibit the ADP-ribosylation modification of HMGB1 by inhibiting activity of PARP-1, which can inhibit the translocation and secretion of HMGB1. The inhibitor of PARP-1 provided a new strategy for the therapy of inflammation which resulted from HMGB1.