背景:微血栓形成常见于脓毒症和COVID-19。紫雪散(ZXP)是一种传统的中草药配方,具有治疗微血管和感染性疾病的潜力。然而,ZXP在脓毒症相关血栓形成中的作用和机制尚不清楚.
目的:研究ZXP在脓毒症血栓形成中的治疗效果和潜在机制。
方法:用UPLC-QTOF-MS检查ZXP的成分。通过各种方法评估ZXP对脓毒症诱导的血栓形成的疗效:使用苏木精-伊红染色检查肝组织病理学,血小板计数由血细胞分析仪测定,酶联免疫吸附试验(ELISA)检测血清组织因子(TF)水平,血栓烷B2(TXB2),D-二聚体,和纤溶酶原激活物抑制剂-1(PAI-1)。中性粒细胞胞外陷阱(NETs)通过免疫荧光在肝组织中定位和表达,通过ELISA评估外周血中的NETs数量,测定血清中cf-DNA和MPO-DNA的含量。流式细胞术检测血小板P-选择素表达和血小板-中性粒细胞聚集,ELISA法测定血浆P-选择素表达。此外,使用STING激动剂研究了干扰素基因刺激因子(STING)信号通路在ZXP抗脓毒症血栓形成作用中的作用机制,蛋白质印迹实验,和免疫沉淀实验。
结果:UPLC-QTOF-MS鉴定出ZXP的40种化学成分。ZXP的管理导致肝血栓形成的显着改善,血小板计数,和TXB2,TF,脓毒症大鼠的PAI-1和D-二聚体。此外,ZXP抑制肝组织和外周血中的NETs形成。此外,ZXP降低了血小板和血浆中P-选择素的水平,以及血小板中性粒细胞聚集体的形成,从而抑制P-选择素介导的NETs释放。免疫沉淀和免疫荧光染色实验表明,ZXP通过抑制STING介导的血小板可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNAREs)复合物的组装来减弱P-选择素的分泌,最终阻止NETs形成的抑制。
结论:我们的研究表明,ZXP主要通过调节STING途径有效减轻血小板颗粒分泌,因此阻碍脓毒症中NET相关的血栓形成。这些发现为ZXP的开发和应用的未来研究提供了宝贵的见解。
BACKGROUND: Microthrombosis is commonly seen in sepsis and COVID-19. Zixue Powder (ZXP) is a traditional Chinese herbal formula with the potential to treat microvascular and infectious diseases. However, the role and mechanism of ZXP in sepsis-associated thrombosis remain unclear.
OBJECTIVE: Investigating the therapeutic effectiveness and underlying mechanisms of ZXP in septic thrombosis.
METHODS: ZXP\'s compositions were examined with UPLC-QTOF-MS. The efficacy of ZXP on sepsis-induced thrombosis was assessed through various methods: liver tissue pathology was examined using hematoxylin-eosin staining, platelet count was determined by a blood cell analyzer, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum tissue factor (TF), thromboxane B2 (TXB2), D-Dimer, and plasminogen activator inhibitor-1 (PAI-1). Neutrophil extracellular traps (NETs) were localized and expressed in liver tissues by immunofluorescence, and the number of NETs in peripheral blood was evaluated by ELISA, which measured the quantity of cf-DNA and MPO-DNA in serum. Platelet P-selectin expression and platelet-neutrophil aggregation were measured by flow cytometry, and plasma P-selectin expression was measured by ELISA. Furthermore, the mechanism of the stimulator of interferon genes (STING) signaling pathway in ZXP\'s anti-sepsis thrombosis effect was investigated using the STING agonist, Western blot experiments, and immunoprecipitation experiments.
RESULTS: UPLC-QTOF-MS identified 40 chemical compositions of ZXP. Administration of ZXP resulted in significant improvements in liver thrombosis, platelet counts, and levels of TXB2, TF, PAI-1, and D-Dimer in septic rats. Moreover, ZXP inhibited NETs formation in both liver tissue and peripheral blood. Additionally, ZXP decreased the levels of P-selectin in both platelets and plasma, as well as the formation of platelet-neutrophil aggregates, thereby suppressing P-selectin-mediated NETs release. Immunoprecipitation and immunofluorescence staining experiments revealed that ZXP attenuated P-selectin secretion by inhibiting STING-mediated assembly of platelet soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complex, ultimately preventing inhibition of NETs formation.
CONCLUSIONS: Our study showed that ZXP effectively mitigates platelet granule secretion primarily through modulation of the STING pathway, consequently impeding NET-associated thrombosis in sepsis. These findings offer valuable insights for future research on the development and application of ZXP.