Necroptosis-Mediated eCIRP Release in Sepsis.
Necroptosis-Mediated eCIRP Release in Sepsis.
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DOI：
10.2147/jir.s370615
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发表时间：
2022
影响因子：
4.5
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中科院分区：
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细胞外冷诱导RNA结合蛋白(ECIRP)是一种内源性促炎介质，可加重炎症和脓毒症的损伤。ECIRP释放的机制尚未得到充分的探索。坏死性上睑下垂是一种细胞程序性死亡，依赖于混合谱系激酶结构域样假性激酶(MLKL)的激活，导致损伤相关分子模式的释放。我们假设，eCIRP通过坏死性下垂释放，并在脓毒症中加剧炎症。PAN-caspase抑制剂z-VAD(15μM)在脂多糖(1μg/mL)刺激前1h处理RAW264.7细胞。在加入内毒素的同时加入坏死抑素-1(NEC-1)(10μM)。24 h后用乳酸脱氢酶(LDH)法检测细胞毒作用。用Western印迹法检测培养上清液和细胞裂解液中的磷酸化MLKL(p-MLKL)水平。免疫荧光检测P-MLKL与细胞膜的相互作用。采用盲肠结扎穿孔(CLP)法建立C57BL/6小鼠脓毒症模型。给小鼠灌胃NEC-1(1 mg/kg)或DMSO。术后20h，用双抗体夹心法测定血清和腹腔液中eCIRP、肿瘤坏死因子-α和IL-6的水平。肺组织切片进行HE染色。我们发现，在RAW264.7细胞中，内毒素+z-VAD诱导坏死性下垂，表现为p-MLKL水平的增加和eCIRP的释放。NEC-1抑制脂多糖+z-VAD诱导的RAW264.7细胞p-MLKL活化和eCIRP释放。NEC-1还能抑制脓毒症小鼠血清和腹腔液中eCIRP、肿瘤坏死因子-α和IL-6的释放。我们使用计算模型预测了eCIRP和MLKL之间的瞬时相互作用，表明eCIRP可能通过p-MLKL形成的孔离开细胞。坏死性下垂是脓毒症时eCIRP释放的一种新机制。靶向坏死性下垂可能通过抑制eCIRP的释放而减轻脓毒症的炎症和损伤。
Extracellular cold-inducible RNA-binding protein (eCIRP) is an endogenous pro-inflammatory mediator that exacerbates injury in inflammation and sepsis. The mechanisms in which eCIRP is released have yet to be fully explored. Necroptosis is a programmed cell death that is dependent on the activation of mixed lineage kinase domain-like pseudo kinase (MLKL) which causes the release of damage-associated molecular patterns. We hypothesize that eCIRP is released through necroptosis and intensifies inflammation in sepsis. RAW264.7 cells were treated with pan-caspase inhibitor z-VAD (15 μM) 1 h before stimulation with LPS (1 μg/mL). Necroptosis inhibitor, Necrostatin-1 (Nec-1) (10 μM) was added to the cells with LPS simultaneously. After 24 h of LPS stimulation, cytotoxicity was determined by LDH assay. eCIRP levels in the culture supernatants and phospho-MLKL (p-MLKL) from cell lysates were assessed by Western blot. p-MLKL interaction with the cell membrane was visualized by immunofluorescence. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Mice were treated with Nec-1 (1 mg/kg) or DMSO. 20 h post-surgery, serum and peritoneal fluid levels of eCIRP, TNF-α and IL-6 were determined by ELISA. H&E staining of lung tissue sections was performed. We found that in RAW264.7 cells, LPS+z-VAD induces necroptosis as evidenced by an increase in p-MLKL levels and causes eCIRP release. Nec-1 reduces both p-MLKL activation and eCIRP release in LPS+z-VAD-treated RAW264.7 cells. Nec-1 also inhibits the release of eCIRP, TNF-α and IL-6 in the serum and peritoneal fluid in CLP-induced septic mice. We predicted a transient interaction between eCIRP and MLKL using a computational model, suggesting that eCIRP may exit the cell via the pores formed by p-MLKL. Necroptosis is a novel mechanism of eCIRP release in sepsis. Targeting necroptosis may ameliorate inflammation and injury in sepsis by inhibiting eCIRP release.