Necroptosis represents a newly defined form of regulated necrosis and participates in various human inflammatory diseases. It remains unclear whether necroptosis is presented in heatstroke-induced lung injury. We show that heat stress(HS) triggered an significant upregulation of receptor-interacting protein 1 (RIP1) and mixed lineage kinase domain-like protein (MLKL) expression in a time-dependent manner, without a significant change of receptor-interacting protein 3 (RIP3). Furthermore, co-immunoprecipitation assays showed that RIP1 binds to RIP3 to form the necrosome in heat stress-induced PMVECs. In vitro, necrostatin-1 (Nec-1) pre-treatment reduced heat stress-induced PMVECs necroptosis, which also inhibited HMGB1 translocation from the nucleus into the cytoplasm. Similarly, inhibition for ERK (PD98059), NF-κB (BAY11-7082) and c-Jun (c-Jun peptide), respectively, also suppressed the HMGB1 cytoplasm translocation. Furthermore, siRNA-mediated RIP1/RIP3 knockdown negatively regulated the release of HMGB1 in HS-induced necroptosis through the ERK, NF-κB, and c-Jun signaling pathways. Our study reveals that HS induces RIP1/RIP3-dependent necroptosis through the MAPK, NF-κB, and c-Jun signaling pathways in PMVECs.

坏死性凋亡代表了一种新定义的调节性坏死形式，并参与多种人类炎症性疾病。目前尚不清楚中暑引起的肺损伤是否存在坏死性凋亡。我们发现热应激（HS）以时间依赖性方式引发受体相互作用蛋白1（RIP1）和混合谱系激酶结构域样蛋白（MLKL）表达的显着上调，而受体相互作用蛋白3没有显着变化（安息吧3）。此外，免疫共沉淀分析表明，RIP1 与 RIP3 结合，在热应激诱导的 PMVEC 中形成坏死体。在体外，necrostatin-1 (Nec-1) 预处理减少了热应激诱导的 PMVEC 坏死性凋亡，同时也抑制了 HMGB1 从细胞核易位到细胞质。同样，分别抑制 ERK (PD98059)、NF-κB (BAY11-7082) 和 c-Jun（c-Jun 肽）也抑制了 HMGB1 细胞质易位。此外，siRNA介导的RIP1/RIP3敲低通过ERK、NF-κB和c-Jun信号通路负向调节HS诱导的坏死性凋亡中HMGB1的释放。我们的研究表明，HS 通过 PMVEC 中的 MAPK、NF-κB 和 c-Jun 信号通路诱导 RIP1/RIP3 依赖性坏死性凋亡。