Protecting the brain from stroke: Huoxue Rongluo formula (HXRLF) targets ferroptosis for neuroprotection.

Ethnopharmacological significance: Huoxue Rongluo Formula (HXRLF) is a traditional Chinese medicine prescription widely used to treat ischemic stroke (IS), based on the classical therapeutic principles of nourishing yin and activating blood circulation. Preliminary clinical studies have demonstrated its effectiveness in improving neurological function and prognosis. However, the precise mechanisms by which HXRLF confers neuroprotection, especially its role in modulating ferroptosis after cerebral ischemia-reperfusion injury (CIRI), remain unclear.

Aim of the study: This study aimed to investigate whether HXRLF ameliorates cerebral I/R injury by inhibiting neuronal ferroptosis through NR4A1-mediated activation of the xCT/GPX4 antioxidant axis.

Materials and methods: Active components of HXRLF were identified by UPLC-Q-TOF-MS. UPLC-Q-TOF-MS analysis revealed several representative active constituents of HXRLF, including catalpol, chebulic acid, didymin, armepavine, harpagoside, atractylenolide III, poncirin, and coclaurine, which together represent the diverse chemical basis underlying its pharmacological actions. Potential anti-ferroptosis targets were screened using network pharmacology and GEO transcriptomic data. A transient middle cerebral artery occlusion (tMCAO) model in mice and an OGD/R model in HT22 cells were used to evaluate the neuroprotective effects of HXRLF. Biochemical assays, immunofluorescence, transmission electron microscopy (TEM), and Western blotting were employed to detect ferroptosis-related indicators. The role of NR4A1 was further examined using its antagonist DIM-C-pPhOH, and the downstream involvement of ferroptosis was confirmed by the ferroptosis inhibitor Fer-1.

Results: HXRLF significantly improved neurobehavioral outcomes, reduced infarct volume, and alleviated pathological damage in tMCAO mice in a dose-dependent manner. Biochemical assays revealed that HXRLF reduced Fe²⁺ and MDA levels while increasing GSH content in brain tissue. HXRLF treatment also restored GPX4 expression and mitochondrial structure. In HT22 cells, HXRLF-containing serum improved cell viability, reversed lipid peroxidation, reduced iron accumulation, and restored mitochondrial membrane potential. Mechanistically, HXRLF upregulated NR4A1 and enhanced the expression of xCT and GPX4. Importantly, the neuroprotective and anti-ferroptotic effects of HXRLF were diminished by NR4A1 inhibition and partially rescued by Fer-1, confirming the involvement of the NR4A1-xCT-GPX4 axis in ferroptosis regulation.

Conclusion: HXRLF ameliorates cerebral I/R injury by inhibiting neuronal ferroptosis through NR4A1-mediated activation of the xCT/GPX4 antioxidant axis. These findings provide mechanistic insights into the neuroprotective effects of traditional Chinese medicine and offer a scientific foundation for developing ferroptosis-targeted therapies in ischemic stroke.