Daphnetin alleviates influenza pneumonia by enhancing chaperone-mediated autophagy of KEAP1 through Heat shock cognate 71 kDa protein.

BACKGROUND

Inflammatory injury due to viral pneumonia (e.g., influenza A virus [IAV]) is a major cause of death in severe respiratory diseases. Daphnetin, a natural coumarin, has therapeutic potential against viral pneumonia; however, its direct target and mechanism of action remain unclear.

PURPOSE: Experimental study on the therapeutic effect and mechanism of daphnetin in viral pneumonia.

METHODS

We integrated limited proteolysis-mass spectrometry, surface plasmon resonance, and molecular docking with cellular experiments and murine IAV models. Target validation was performed via siRNA knockdown and pathway inhibitors. Molecular expression and signaling were assessed using flow cytometry and western blotting.

RESULTS

Daphnetin directly bound to Heat shock cognate 71 kDa protein (HSC70), enhancing its interaction with kelch-like ECH-associated protein 1 (KEAP1) to activate chaperone-mediated autophagy (CMA) and KEAP1 lysosomal degradation, thereby sustaining Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) antioxidant pathway activation. This mitigated mitochondrial oxidative stress and mitochondrial DNA (mtDNA) leakage, inhibited cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) inflammation, restored macrophage efferocytosis, and reduced pulmonary inflammatory injury in mice.

CONCLUSION

This study identified the HSC70-CMA-NRF2 axis as the key mechanism underlying the modulation of macrophage function by daphnetin, providing a theoretical basis for its development as a targeted immunometabolic regulator of viral pneumonia.