Department of General Surgery, The Third Xiangya Hospital of Central South University, Changsha, Hunan, China.
Department of Physiology, School of Basic Medical Science, Central South University, Changsha, Hunan, China.
Thromb Res. 2022 Jun;214:8-15. doi: 10.1016/j.thromres.2022.04.001. Epub 2022 Apr 8.
Excessive activation of the coagulation cascades leads to life-threatening disseminated intravascular coagulation (DIC) in sepsis. Two recent studies by our group and others have both demonstrated the noncanonical inflammasome is pivotal for the endotoxin or gram-negative bacterial-induced coagulation. Based on this, we further evaluated the function of the NLRP3 inflammasome, the most studied inflammasome, in endotoxin-induced coagulation.
We established an endotoxin-induced coagulation model by intraperitoneal injection of sublethal doses of LPS in mice. Mice were sacrificed 8 h after injection and blood was collected for thrombin-antithrombin (TAT), plasminogen activator inhibitor-1 (PAI-1), prothrombin time (PT), D-dimer, IL-1β and tissue factor (TF) measurements by commercial ELISA. Lungs and livers were examined via HE staining images to determine injury scores and immunohistochemistry for TF expression and fibrin deposits. The role of NLRP3 activation was evaluated in wild-type (WT), Nlrp3, Asc (apoptosis-associated speck-like protein containing a CARD), Caspase-11 mice and 30 min after treatment with MCC950, a potent inhibitor of NLRP3. Western blotting and Q-PCR were performed to assess TF expression in the lungs and livers. To uncover the different effects of NLRP3 and Caspase-11, we also compared the time-dependent IL-1β release in LPS-treated Nlrp3 and Caspase-11 mice. Correlation analysis of TAT, PAI-1 were estimated the relationship of coagulation and release of IL-1β, as well as IL-1β and TF.
Inhibition of NLRP3 by MCC950 as well as NLRP3 or ASC deficiency decreased TAT, PAI-1, PT, D-dimer, and TF levels in blood and impaired the thrombus formation and fibrin deposition, as well as declined expression of TF in the liver and lung in endotoxin-induced coagulation but not caspase-11 deficiency. Impressively, IL-1β release is increased in LPS-treated Caspase-11 mice, but not in Nlrp3 mice. Moreover, the correlation analysis is indicated that downstream of the NLRP3 inflammasome, IL-1β expression, is positively correlated with TAT, PAI-1 and TF in blood circulation.
The NLRP3 inflammasome contributes to endotoxin-induced coagulation by promoting TF expression at least in part through the induction of IL-1β release. These findings broadened our understanding of the mechanism of coagulation and implicated a possible therapeutic strategy for preventing coagulation in sepsis.
在脓毒症中，凝血级联的过度激活会导致危及生命的弥漫性血管内凝血（DIC）。我们小组和其他小组的两项最近的研究都表明，非经典的炎症小体对于内毒素或革兰氏阴性菌诱导的凝血至关重要。基于此，我们进一步评估了最具研究意义的炎症小体 NLRP3 在内毒素诱导的凝血中的功能。
我们通过腹腔内注射亚致死剂量的 LPS 在小鼠中建立内毒素诱导的凝血模型。注射后 8 小时处死小鼠，收集血液用于通过商业 ELISA 测量凝血酶-抗凝血酶（TAT）、纤溶酶原激活物抑制剂-1（PAI-1）、凝血酶原时间（PT）、D-二聚体、IL-1β和组织因子（TF）。通过 HE 染色图像检查肺和肝，以确定损伤评分和 TF 表达和纤维蛋白沉积的免疫组织化学。在野生型（WT）、Nlrp3、Asc（含 CARD 的凋亡相关斑点样蛋白）、Caspase-11 小鼠和用强效 NLRP3 抑制剂 MCC950 处理 30 分钟后，评估 NLRP3 激活的作用。进行 Western blot 和 Q-PCR 以评估 TF 在肺和肝中的表达。为了揭示 NLRP3 和 Caspase-11 的不同作用，我们还比较了 LPS 处理的 Nlrp3 和 Caspase-11 小鼠中 IL-1β释放的时间依赖性。TAT、PAI-1 的相关性分析估计了凝血与 IL-1β释放以及 IL-1β和 TF 之间的关系。
MCC950 抑制 NLRP3 以及 NLRP3 或 ASC 缺乏降低了内毒素诱导的凝血中的 TAT、PAI-1、PT、D-二聚体和 TF 水平，并损害了血栓形成和纤维蛋白沉积，以及降低了内毒素诱导的凝血中的 TF 在肝脏和肺部的表达，但不降低 Caspase-11 缺乏的表达。令人印象深刻的是，IL-1β的释放增加在 LPS 处理的 Caspase-11 小鼠中，但不在 Nlrp3 小鼠中。此外，相关性分析表明，NLRP3 炎症小体下游的 IL-1β表达与血液中的 TAT、PAI-1 和 TF 呈正相关。
NLRP3 炎症小体通过促进 TF 表达至少部分通过诱导 IL-1β释放来促进内毒素诱导的凝血。这些发现拓宽了我们对凝血机制的理解，并暗示了一种可能的治疗策略，以防止脓毒症中的凝血。
