Protective Mechanism of Huperzine A in Sepsis-induced Endothelial Cell Damage Through Inhibition of NLRP3/Caspase-1/IL-1β Pathway Activation

Background Huperzine A (HupA), a natural alkaloid first isolated from Huperzia serrata , is an acetylcholinesterase inhibitor. It is characterized by high biological activity, relatively high lipophilicity, and small molecular size. Purpose The aim of this study was to demonstrate the protective effects of HupA on human umbilical vein endothelial cells (HUVECs) injured by sepsis serum and to explore its regulatory mechanisms on the NLRP3/Caspase-1/IL-1β pathway. Materials and Methods Serum samples were collected and prepared from sepsis patients and healthy individuals. HUVECs were cultured in vitro and randomly divided into the Blank group (normal culture), Control group (cultured with healthy serum), Sepsis group (cultured with sepsis serum), low-dose HupA group (LD-HupA group, cultured with sepsis serum + 1 × 10 −8 mol/L HupA), medium-dose HupA group (MD-HupA group, cultured with sepsis serum + 1 × 10 −6 mol/L HupA), and high-dose HupA group (HD-HupA group, cultured with sepsis serum + 1 × 10 −4 mol/L HupA). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis by flow cytometry, and reactive oxygen species (ROS) expression via immunofluorescence. Quantitative polymerase chain reaction (qPCR) measured mRNA levels of nuclear transcription factor kappa B (NF-κB), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and endothelial selectin (E-selectin). Western blot quantified protein levels of NLRP3, caspase-1, and IL-1β. Results There were no significant differences between the Blank and Control groups ( p > .05). The sepsis group showed reduced cell viability, increased apoptosis, higher ROS expression, and elevated mRNA levels of NF-κB, TNF-α, IL-6, ICAM-1, VCAM-1, and E-selectin, along with elevated protein levels of NLRP3, caspase-1, and IL-1β versus Control ( p < .05). In contrast, LD-HupA, MD-HupA, and HD-HupA groups had improved cell viability, reduced apoptosis, lower ROS expression, decreased mRNA levels of the aforementioned markers, and lower protein levels of NLRP3, caspase-1, and IL-1β versus the Sepsis group ( p < .05), showing dose-dependent effects. Conclusion HupA can protect against sepsis serum-induced endothelial cell damage by inhibiting NLRP3/caspase-1/IL-1β pathway activation.