The present study established a necroptosis model in vitro and investigated the role of HMGB1 in cell necroptosis. A combination of tumor necrosis factor-α and z-VAD-fmk was used to induce necroptosis in L929 cells with necroptosis inhibitor necrostatin-1 applied as an intervention. Flow cytometry and transmission electron microscopy (TEM) were used to measure cell necroptosis. Western blotting assay was applied to detect the expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL) and HMGB1. Co-immunoprecipitation (Co-IP) assay was used to confirm the interaction between HMGB1 and RIPK3. Our study demonstrated that HMGB1 migrated from the nucleus to the cytoplasm at the onset of necroptosis and was subsequently released passively to the extracellular matrix. Further experiments determined that the binding of HMGB1 with RIPK3 in the cytoplasm was loose during necroptosis. By contrast, when necroptosis was inhibited, the interaction in the cytoplasm was tight suggesting that this association between HMGB1 and RIPK3 might affect its occurrence. In conclusion, the transfer of HMGB1 from nucleus to cytoplasm, and its interaction with RIPK3 might be potentially involved in necroptosis.

本研究在体外建立了坏死性凋亡模型，并研究了HMGB1在细胞坏死性凋亡中的作用。肿瘤坏死因子-α和z-VAD-fmk的组合用于诱导L929细胞的坏死性凋亡，其中坏死性凋亡抑制剂necrostatin-1用作干预。流式细胞术和透射电子显微镜 (TEM) 用于测量细胞坏死。应用蛋白质印迹法检测受体相互作用的丝氨酸/苏氨酸蛋白激酶 3 (RIPK3)、混合谱系激酶结构域样假激酶 (MLK​​L) 和 HMGB1 的表达。共免疫沉淀 (Co-IP) 测定用于确认 HMGB1 和 RIPK3 之间的相互作用。我们的研究表明，HMGB1 在坏死性凋亡开始时从细胞核迁移到细胞质，随后被动释放到细胞外基质。进一步的实验确定 HMGB1 与 RIPK3 在细胞质中的结合在坏死期间是松散的。相比之下，当坏死性凋亡被抑制时，细胞质中的相互作用很紧密，这表明 HMGB1 和 RIPK3 之间的这种关联可能会影响其发生。总之，HMGB1 从细胞核向细胞质的转移及其与 RIPK3 的相互作用可能与坏死性凋亡有关。