Herbacetin Attenuates Oxidative Stress via Activating Nrf2/HO-1 Signaling Pathway in RAW 264.7 Cells and Caenorhabditis elegans

Flavonoids have long been used as food additives due to their antioxidant properties. To discover bioactive natural flavonoids, the antioxidant capabilities of a range of natural flavonoids were investigated in RAW 264.7 cells and Caenorhabditis elegans induced by H₂O₂. Chemical antioxidant activity of a range of flavonoids was performed by DPPH and ABTS⁺ radicals scavenging assays. Then, the cellular antioxidant activity assays were employed to detect the antioxidant mechanisms by flow cytometry and qRT-PCR analyses. The underlying antioxidant mechanisms were explored by western blotting, small interfering RNA (siRNA), molecular docking, and cellular thermal shift assay (CETSA). Next, the antioxidant potential of active compounds was verified in H₂O₂-induced Caenorhabditis elegans. Our studies showed herbacetin exhibited the most pronounced DPPH and ABTS ⁺ radicals scavenging capacity among all the tested flavonoids. And herbacetin also showed improved antioxidant activity against H₂O₂-induced oxidative stress in RAW 264.7 cells compared to quercetin and Trolox. The structure-activity relationship (SAR) analysis indicated that hydroxyl groups at the 7′ and 8′ positions on the A-ring of herbacetin played a crucial role in inhibiting H₂O₂-induced cellular oxidative stress. Interestingly, herbacetin’s activation of HO-1 was closely associated with its binding to Keap1. Notably, in vivo assays demonstrated that herbacetin protected Caenorhabditis elegans from H₂O₂-induced oxidative stress. This study screens and elucidates the excellent antioxidant capacity of herbacetin both in vitro and in vivo. These findings suggest that herbacetin holds promise as a natural agent for antioxidant therapy.