3,5-Dicaffeoylquinic acid attenuates poststroke muscle atrophy through GPX4 dependent ferroptosis inhibition.

BACKGROUND

Ischemic stroke-induced sarcopenia (ISS) is a major contributor to poststroke motor dysfunction and severely impairs quality of life. Ferroptosis, an iron- and lipid peroxidation- dependent form of programmed cell death, has not been fully explored in ISS.

PURPOSE: To evaluate the natural compound 3,5-dicaffeoylquinic acid (3,5-DCQA) as a novel anti-ferroptosis agent that markedly attenuates ISS via glutathione peroxidase 4 (GPX4)-mediated ferroptosis inhibition.

METHODS

A permanent middle cerebral artery occlusion (pMCAO) rat model was established. Cerebral injury was assessed using laser speckle contrast imaging and Zea-Longa neurological scoring. The effect of 3,5-DCQA on muscle function was evaluated through behavioral analyses and histopathological staining. Molecular interactions were validated by combining GPX4 silencing via siRNA in L6 cells with RNA-seq and surface plasmon resonance (SPR). The involvement of the ferroptosis signaling pathway in ISS was examined using flow cytometry, immunofluorescence (IF), immunohistochemistry (IHC), and Western blotting (WB).

RESULTS

3,5-DCQA markedly improved muscle mass, strength, and motor performance. Mechanistically, it reduced ferrous iron and lipid peroxidation products, including malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and lipid peroxides, and downregulated muscle atrophy factors, muscle RING-finger protein-1 (MuRF1) and muscle atrophy F-box (MAFbx). In vitro, 3,5-DCQA decreased reactive oxygen species (ROS) and ferrous iron accumulation, inhibited acyl-CoA synthetase long-chain family member 4 (ACSL4), and restored GPX4 signaling and intracellular GSH, thereby alleviating erastin-induced ferroptosis in L6 myoblasts. Molecular docking and molecular dynamics simulations supported a stable interaction between 3,5-DCQA and GPX4, which was further confirmed by SPR with a KD value of 3.92 × 10-⁶ M. Moreover, 3,5-DCQA weakly bound to MuRF1. These findings confirm GPX4 as the key mediator, as GPX4 knockdown abolished its effects on ferroptosis, oxidative stress, and myogenic recovery.

CONCLUSIONS

3,5-DCQA mitigates poststroke muscle atrophy through GPX4-dependent ferroptosis suppression and redox restoration, supporting its therapeutic potential in ISS.