Shenfu injection ameliorates coagulopathy in sepsis-associated liver injury via dual inhibition of endothelial cell pyroptosis

BACKGROUND: Sepsis-associated liver injury (SALI) as a critical factor contributing to high mortality rates in patients with sepsis is characterized by significant coagulation disorders. Vascular endothelial cells (ECs) play a pivotal role in the coagulopathy in sepsis. Shenfu Injection (SFI), a traditional Chinese medicine, is widely used in sepsis management, yet its impact on EC activation and coagulopathy in SALI remains inadequately explored. OBJECTIVE: This study aims to elucidate the mechanisms by which SFI alleviates EC activation and coagulopathy associated with SALI and to dissect the contribution of individual compounds in SFI in regulating EC coagulation activation. METHODS: Plasma levels of coagulation- and liver injury-related biomarkers in septic patients treated with or without SFI were analyzed. A murine model of sepsis was built via intraperitoneal LPS injection for the study of SFI pharmacological effects in vivo. Human umbilical vein endothelial cells (HUVEC) stimulated with LPS were used as the in vitro model of sepsis-induced EC activation. Transcriptome sequencing was performed to investigate the molecular mechanisms of SFI in regulating EC activation. Transmission electron microscopy and confocal laser microscopy were applied for the evaluation of pyroptosis and LPS internalization in HUVEC. RESULTS: The clinical data showed that the coagulation markers and liver injury-related indicators in septic patients were significantly lower in SFI treated septic patients compared to that in non-SFI treated group. In the murine model, SFI administration alleviated liver damage and the inflammatory responses. The plasma levels of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) as well as the accumulation of fibrinogen in liver tissues were also reduced in septic mice treated with SFI. Mechanistically, SFI induced phosphorylation-dependent activation of AMPK, thereby leading to the dephosphorylation of RIPK2, which blocked NLRP3-mediated canonical pyroptosis. Additionally, SFI inhibited LPS internalization by blocking HMGB1/RAGE axis and suppressing the expression of LPS-binding protein (LBP), resulting in the inhibition of non-canonical pyroptosis. Furthermore, network pharmacology and molecular docking analysis highlighted ginsenoside Rb1 and ginsenoside Re in SFI for their strong binding affinity to AMPK and LBP, respectively, supporting the dual inhibitory effects of SFI on pyroptosis. CONCLUSION: Our findings revealed the significance of SFI for treating sepsis-associated coagulopathy and liver injury via dual inhibition of pyroptosis, providing theoretical basis for clinical application of SFI.