Phillyrin ameliorates sepsis via targeting microRNA-203a-mediated caspase-4/caspase-11/caspase-B downregulation to suppress endothelial pyroptosis

Background: Sepsis, driven by dysregulated host inflammation, remains a leading cause of global mortality and lacks sufficiently effective therapies. Phillyrin (PHN), a lignan glycoside from Forsythia suspensa (Thunb.) Vahl (Oleaceae), exhibits anti-inflammatory and antimicrobial properties. However, its molecular mechanism in sepsis remains poorly understood. Objective: To delineate the molecular pathways by which PHN mitigates sepsis. Methods: Reverse transcription-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, cytotoxicity assessments, and calcein acetoxymethyl ester/propidium iodide staining were employed to evaluate the protective effects of PHN against lipopolysaccharide (LPS)-mediated pyroptosis in human pulmonary microvascular endothelial cells (HPMECs). Network pharmacology and integrative bioinformatics were used to identify candidate regulatory axes. Findings were then validated across multiple models: HPMEC-differentiated human promyelocytic acute leukemia cell co-cultures systems, Tg(mpx:GFP) zebrafish, and BALB/c mice. Validation techniques comprised luciferase reporters, pharmacological modulators, and morpholino knockdown. Results: PHN significantly attenuated LPS-induced inflammatory pyroptosis in HPMECs. Evidence included reduced proinflammatory cytokine production, decreased lactate dehydrogenase leakage, and fewer pyroptotic cells. Computational analyses identified the microRNA (miR)-203a-caspase-4 (CASP4) axis as a primary mediator of PHN's anti-septic activity. In co-culture systems, PHN suppressed cytoplasmic LPS-triggered pyroptosis through miR-203a-dependent CASP4 downregulation, thereby reducing inflammatory cytokine secretion and neutrophil recruitment. In LPS-challenged zebrafish, PHN upregulated miR-203a to suppress caspase-B (CASPB), diminishing cytokine expression and neutrophil migration while improving survival. In murine sepsis models induced by LPS or cecal ligation and puncture, PHN modulated the miR-203a-caspase-11 (CASP11) axis to confer multiple therapeutic benefits. These included improved survival rates, stabilized body temperature, reduced bronchoalveolar lavage protein levels and neutrophil infiltration, attenuated multi-organ injury, and decreased systemic cytokine levels. Conclusion: These data identify the miR-203a-CASP4/11/B axis as a critical mediator of endothelial pyroptosis in sepsis. PHN attenuates sepsis by upregulating miR-203a to inhibit CASP4/11/B-dependent pyroptosis. Therefore, PHN warrants further investigation as a potential therapeutic agent for sepsis.