Orientin alleviates severe inflammation via regulating macrophage glycolysis and immune function in sepsis

Sepsis is a heterogeneous syndrome triggered by a dysregulated host response to infection, with glycolysis playing a vital role in maintaining macrophage immune function, which is critical for immune homeostasis and host survival during severe sepsis. Targeting glycolytic enzymes may offer effective strategies to mitigate macrophage-mediated inflammatory responses during sepsis. This study investigated the anti-inflammatory and metabolic modulatory effects of Orientin (Ori) in murine models of endotoxemia and sepsis, with a particular focus on its interaction with the glycolytic enzyme phosphofructokinase liver type (PFKL). Ori was administered at varying dosages in vivo, while in vitro experiments involved lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs) and RAW264.7 cells. Inflammatory responses were assessed using Western blot, Enzyme-Linked Immunosorbent Assay (ELISA), and immunofluorescence, while glycolytic activity was evaluated through lactate production, glucose uptake, and extracellular acidification rate (ECAR). Cellular Thermal Shift Assay (CETSA) and molecular docking confirmed the direct binding between Ori and PFKL, and further analyses using network pharmacology and PFKL overexpression elucidated the enzyme's role in mediating Ori's effects. Ori significantly improved survival, reduced lung injury, and suppressed cytokine release in septic mice, while in vitro it attenuated LPS-induced inflammatory cytokine expression and glycolysis. Notably, macrophage-specific PFKL overexpression abrogated Ori's protective effects. These findings demonstrate that Ori alleviates sepsis-induced inflammation and metabolic dysfunction by directly targeting PFKL, highlighting its potential as a novel therapeutic candidate for sepsis.