Multiple organ perfusion is impaired in sepsis. Clinical studies suggest that persistent perfusion disturbances are prognostic of fatal outcome in sepsis. Pyroptosis occurs upon activation of caspases and their subsequent cleavage of Gsdmd, resulting in Gsdmd-N that form membrane pores to induce cell death in sepsis. In addition, Gsdmd^-/- mice are protected from a lethal dose of lipopolysaccharide (LPS). However, how Gsdmd-mediated pyroptosis occurs in endothelial cells and leads to impaired perfusion remain unexplored in endotoxemia. We used transgenic mice with ablation of Gsdmd and determined that mice lacking Gsdmd exhibited reduced breakdown of endothelial barrier, improved organ perfusion, as well as increased survival in endotoxemia. Phospholipase Cγ1 (PLCγ1) contributed to Gsdmd-mediated endothelial pyroptosis in a calcium-dependent fashion without affecting Gsdmd-N production. Cytosolic Calcium signaling promoted Gsdmd-N translocating to the plasma membrane enhancing endothelial pyroptosis induced by LPS. We used adeno-associated virus (AAV9) vectors carrying a short hairpin RNA (shRNA) against murine PLCγ1 mRNA under control of the tie1 core promoter (AAV-tie1-sh-PLCγ1) to uniquely downregulate PLCγ1 expression in the endothelial cells. Here, we showed that unique inhibition of endothelial PLCγ1 attenuated breakdown of endothelial barrier, reduced vascular leakage , and improved perfusion disturbances. Moreover, unique downregulate endothelial PLCγ1 expression markedly decreased mortality of mice in endotoxemia. Thus, we establish that endothelial injury as an important trigger of fatal outcome in endotoxemia. Additionally, these findings suggest that interfering with Gsdmd and PLCγ1-calcium pathway may represent a new treatment strategy for critically ill patients sustaining endotoxemia.

中文翻译：

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Gsdmd-N的磷脂酶Cγ1/钙依赖性膜定位可驱动内皮细胞的热解，从而导致脂多糖诱导的致命结果。

败血症会损害多器官灌注。临床研究表明，持续性灌注障碍是败血症致命预后的预后。在胱天蛋白酶激活及其随后的Gsdmd裂解后发生细胞凋亡，导致Gsdmd-N形成膜孔以诱导败血症中的细胞死亡。另外，Gsdmd ^-/-保护小鼠免于致死剂量的脂多糖（LPS）。然而，内毒素血症尚不清楚Gsdmd介导的细胞凋亡如何在内皮细胞中发生并导致灌注受损。我们使用消融Gsdmd的转基因小鼠，并确定缺乏Gsdmd的小鼠表现出减少的内皮屏障破坏，改善的器官灌注以及内毒素血症的存活率。磷脂酶Cγ1（PLCγ1）以钙依赖的方式促进了Gsdmd介导的内皮细胞凋亡，而不影响Gsdmd-N的产生。胞质钙信号传导促进Gsdmd-N易位至质膜，增强LPS诱导的内皮细胞凋亡。我们使用在tie1核心启动子（AAV-tie1-sh-PLCγ1）的控制下携带针对鼠类PLCγ1mRNA的短发夹RNA（shRNA）的腺相关病毒（AAV9）载体来唯一下调内皮细胞中PLCγ1的表达。在这里，我们显示出对内皮PLCγ1的独特抑制作用会减弱内皮屏障的破坏，减少血管渗漏并改善灌注障碍。此外，独特的下调内皮细胞PLCγ1的表达显着降低了小鼠内毒素血症的死亡率。因此，我们确定内皮损伤是内毒素血症致命结果的重要触发因素。此外，这些发现表明，干扰Gsdmd和PLCγ1-钙途径可能代表维持内毒素血症的重症患者的新治疗策略。