Multi-scale discovery of herbacetin as a macrophage-targeted SGK1-STAT1 signaling inhibitor alleviating lung inflammation and barrier dysfunction

Introduction: Acute lung injury (ALI) is characterized by dysregulated macrophage-epithelial interactions, leading to barrier disruption and hyperinflammation. Broad-spectrum anti-inflammatory therapies are vital for managing ALI induced by diverse pathogens, yet effective interventions remain limited. Objectives: The study aimed to identify novel therapeutic natural products for ALI and elucidate their mechanisms of action, using a zebrafish-based drug screening platform followed by multi-scale validation. Methods: An ALI-like zebrafish model was established by morpholino knockdown, followed by high-content screening of a natural compound library. In vitro, a macrophage-epithelium crosstalk model was used to assess the effect of the positive hit on barrier function. Mechanistic studies employed immunoprecipitation-mass spectrometry, co-IP and protein-protein docking. In vivo validation included zebrafish swim bladder (functional analog of mammalian lungs) injury model and three murine ALI models (LPS-, spike protein-, and live coronavirus-induced). Pathological damages were evaluated via H&E staining. QPCR, ELISA, western blot, IHC and IF were used to quantify molecular alterations. Results: By knocking down the ptpn6 gene, an ALI-like zebrafish model with disrupted swim bladder formation was established. High-content screening of a natural compound library combined with structure-activity optimization identified herbacetin (HBT), a polyhydroxylated flavone, as the most potent ant-inflammatory candidate. HBT restored macrophage-induced lung epithelial barrier dysfunction in vitro by selectively inhibiting macrophage activation rather than directly affecting epithelial cells. Mechanistically, HBT-mediated SGK1 inhibition suppresses STAT1 phosphorylation and downstream inflammatory responses in lung barrier protection. HBT consistently preserved pulmonary barrier function in all the multi-scale models. Conclusion: Collectively, our findings not only highlight HBT’s therapeutic potential for ALI but also identify the SGK1-STAT1 signaling as a novel target for macrophage-driven inflammatory diseases, offering a promising strategy for ALI intervention.