背景:胆汁淤积性肝病引起局部和全身高凝,中性粒细胞胞外陷阱(NET)作为主要驱动因素。这些NETs与阻塞性黄疸患者肝功能下降有关。然而,NETs对胆汁淤积性肝病肝脏高凝的影响尚不清楚.
方法:我们利用胆管结扎来创建实验小鼠,并分析肝脏中的NETs形成。纤维蛋白沉积,组织因子表达,通过蛋白质印迹和免疫组织化学技术观察肝脏的炎症。LSEC与分离的NET孵育,我们使用凝血蛋白产生测定法和测量内皮通透性来检测内皮促凝血活性。在体内和体外环境中,DNaseI用于阐明NETs对肝内高凝状态的影响,肝毒性,LSEC,和巨噬细胞活化或损伤。
结果:胆管结扎小鼠肝组织中NETs水平显著升高,伴有中性粒细胞浸润,组织坏死,纤维蛋白沉积,和血栓形成倾向与假小鼠相比。值得注意的是,NET导致LSEC上的磷脂酰丝氨酸和组织因子暴露,增强凝血因子Xa和凝血酶的产生。增强的促凝血活性可以通过用DNaseI降解NETs来逆转。NET诱导的LSEC渗透率变化,以VE-钙粘蛋白表达和F-肌动蛋白回缩增加为特征,这可以通过DNaseI来拯救。同时,NET的形成与KC活化和炎症因子的形成有关。
结论:NET促进肝内凝血和炎症的激活,导致肝组织损伤。针对NET形成的策略可能为治疗胆汁淤积性肝病提供潜在的治疗方法。
BACKGROUND: Cholestatic liver diseases induce local and systemic hypercoagulation, with neutrophil extracellular traps (NETs) serving as major drivers. These NETs have been linked to decreased liver function in patients with obstructive jaundice. However, the impact of NETs on liver hypercoagulation in cholestatic liver disease remains unknown.
METHODS: We utilized bile duct ligation to create experimental mice and analyzed NETs formation in the liver. Fibrin deposition, tissue factor expression, and inflammation in the liver were visualized through western blot and immunohistochemical techniques. LSECs were incubated with isolated NETs, and we detected endothelial procoagulant activity using coagulation protein production assays and measuring endothelial permeability. In both in vivo and in vitro settings, DNase I was applied to clarify the effect of NETs on intrahepatic hypercoagulability, hepatotoxicity, LSEC, and macrophage activation or injury.
RESULTS: Bile duct ligation mice exhibited significantly increased levels of NETs in liver tissue, accompanied by neutrophil infiltration, tissue necrosis, fibrin deposition, and thrombophilia compared to sham mice. Notably, NETs resulted in phosphatidylserine and tissue factor exposure on LSEC, enhancing coagulation Factor Xa and thrombin production. The enhanced procoagulant activity could be reversed by degrading NETs with DNase I. Additionally, NETs-induced permeability changes in LSECs, characterized by increased VE-cadherin expression and F-actin retraction, which could be rescued by DNase I. Meanwhile, NET formation is associated with KC activation and the formation of inflammatory factors.
CONCLUSIONS: NETs promote intrahepatic activation of coagulation and inflammation, leading to liver tissue injury. Strategies targeting NET formation may offer a potential therapeutic approach for treating cholestatic liver disease.