Preliminary discussion of high mobility group box-1 protein expression in tumor necrosis factor-α induced L929 cell necroptosis

Abstract

Objective Establish L929 cell necroptosis model, study the role of high mobility group box-1 protein (HMGB1)in necroptosis. Methods Build necroptosis model through the recombinant mouse 10 ng/ml tumor necrosis factor (TNF)-α, a pan-Caspase inhibitors z-VAD-FMK 10 μmol/L and necroptosis inhibitors NEC-1 30 μmol/L, detect the survival and propidium iodide (PI) positive by applying Annexin V-fluorescein isothiocyanate (Annexin V-FITC)/PI double staining, observe the morphology of cell death by electron microscopy, detect the necroptosis marker receptor-interacting proteinkinase 3/phosphorylation of mixed lineage kinase domain-like protein (RIP3/pMLKL) by Western blotting. Detect the HMGB1 of nucleus and cytoplasm in necropotosis by Western blotting, detect the extracellular HMGB1 by enzyme linked immunosorbent assay (ELISA). Utlize co-immunoprecipitation to detect the interaction between HMGB1 and RIP3 in necropotosis. Results PI positive increased significantly in the necroptosis group, the cell vitality decreased [(32.76±2.00)% vs. (16.16±3.00)%; (61.77±3.00)% vs. (78.63±3.50)%], after necroptosis inhibitor, the PI positive were reduced, vitality increased [(1.76±0.50)% vs. (32.76±2.00)%; (96.73±7.00)% vs. (61.77±3.00)%]. RIP3/pMLKL expression raised in the necroptosis group (t=71.085, P=0.000; t=58.314, P=0.000), after necroptosis inhibitor, RIP3/pMLKL decreased (t=-23.620, P=0.000; t=-36.616, P=0.000). HMGB1 expression within the nucleus is reduced, and increased in cytoplasm, and the release of HMGB1 increased when necroptosis occurred (t=-41.299, P=0.000; t=56.667, P=0.000; t=63.319, P=0.000), after necroptosis inhibitor, the release of HMGB1 reduced (t=-31.095, P=0.000). Intracellular HMGB1 combined with RIP3 is abate when necroptosis, while the binding force increase when necroptosis was inhibited (t=-20.201, P=0.000; t=18.529, P=0.000). Conclusion TNF-α and z-VAD-fmk could successfully induce L929 cell necroptosis model, HMGB1 shift from nuclear to cytoplasm during necroptosis, then passively release to extracellular to involve in the inflammatory reaction. HMGB1 within cytoplasm may participate in the necroptosis and its interaction with RIP3 could be negative to the regulation of necroptosis. Key words: High mobility group box-1 protein; Necroptosis; Receptor-interacting proteinkinase 3