Blockade of GPX4-mediated ferroptosis underlies the protective effect of Forsythiae Fructus water extract against poly(I:C)-induced inflammation

Ethnopharmacological relevance: Forsythiae Fructus is a widely used traditional Chinese medicine commonly employed in the treatment of inflammation-related diseases. However, its underlying anti-inflammatory mechanisms remain incompletely elucidated.

Aim of the study: This study aimed to evaluate the therapeutic effects of Forsythiae Fructus water extract (LQKL) against poly(I:C)-induced inflammation and to explore whether it acts by inhibiting Ferroptosis.

Materials and methods: The protective effects of LQKL were evaluated using poly(I:C)-induced inflammatory models in both RAW264.7 cells and zebrafish. Potential targets and pathways of LQKL against poly(I:C)-induced inflammation were initially predicted by network pharmacology analysis and subsequently validated through RT-qPCR, Western blot, immunofluorescence, siRNA transfection, and CRISPRi. Lipid peroxidation levels were assessed by measuring MDA content and ROS production using colorimetric assay and fluorescence microscopy, respectively. Furthermore, the key anti-inflammatory components in LQKL were identified through integrated approaches including molecular docking, DARTS, and CETSA assays.

Results: LQKL significantly attenuated poly(I:C)-induced inflammation in zebrafish, as evidenced by reduced neutrophil and macrophage infiltration and improved survival rate. It also downregulated the expressions of IL-6 and TNF-α in both poly(I:C)-stimulated zebrafish and RAW264.7 cells. Network pharmacology analysis indicated that LQKL may act through inhibition of the NF-κB signaling pathway which were verified by the decrease of p-NF-κB p65 and p-IκBα, as well as the nuclear translocation of NF-κB p65. Moreover, LQKL inhibited MDA content, suppressed the generation of ROS, restored GSH content and regulated the expressions of proteins related to Ferroptosis. Notably, GPX4 knockdown markedly diminished the anti-inflammatory effects of LQKL and its suppression of the NF-κB pathway. Furthermore, wogonin was identified as a key active component in LQKL that strongly binds to GPX4.

Conclusion: LQKL alleviates poly(I:C)-induced inflammation by attenuating Ferroptosis through GPX4 activation, leading to subsequent inhibition of the NF-κB pathway.

Ferroptosis; Forsythiae Fructus; GPX4; Inflammation; Poly(I:C).