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.