Quercetin protects brain microvascular endothelial cells against hypoxia/reoxygenation-induced injury and ferroptosis by targeting the TCF3/ALOX15 axis: Implications for cerebral small vessel disease.

BACKGROUND: Cerebral small vessel disease (CSVD) is a leading cause of vascular cognitive impairment and stroke. Quercetin is a natural flavonoid with neuroprotective effects in various cerebrovascular disorders. However, its specific molecular targets and mechanisms in CSVD remain elusive. METHODS: Bioinformatic analysis identified arachidonate 15-lipoxygenase (ALOX15) as a potential ferroptosis-related target of quercetin in CSVD. The human cerebral microvascular endothelial cells (HCMEC/D3) subjected to hypoxia/reoxygenation (H/R) treatment were used for in vitro experiments. Cell viability, apoptosis, and angiogenesis were measured via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and tube formation assay, respectively. Ferroptosis markers, including Fe²⁺, superoxide dismutase (SOD), malondialdehyde (MDA), and lipid peroxidation were detected by commercial kits or flow cytometry. The expression of ALOX15, transcription factor 3 (TCF3), acyl-CoA synthetase long-chain family member 4 (ACSL4), and glutathione peroxidase 4 (GPX4) was assessed using western blot. Online prediction, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP) were performed to validate the TCF3-ALOX15 transcriptional regulation. A bilateral common carotid artery occlusion (BCAO) rat model of CSVD was established for in vivo validation. RESULTS: Quercetin improved H/R-induced HCMEC/D3 cell viability, promoted angiogenesis, and reduced apoptosis. Quercetin also attenuated ferroptosis, characterized by decreased Fe²⁺ accumulation, MDA and lipid peroxidation levels, and restoration of SOD activity and GPX4 expression while suppressing ACSL4 (all P < 0.05). TCF3 was identified as a transcriptional activator of ALOX15. TCF3 knockdown mimicked quercetin's protective effects, which were reversed by ALOX15 overexpression. Conversely, TCF3 overexpression counteracted quercetin-mediated protection. In BCAO rats, quercetin treatment ameliorated hippocampal neuronal damage, reduced brain edema, improved spatial learning and memory, and suppressed expression of TCF3/ALOX15 and ferroptosis markers (all P < 0.05). CONCLUSIONS: Quercetin protected against H/R-induced endothelial injury and ferroptosis in CSVD by targeting the TCF3/ALOX15 transcriptional axis, providing mechanistic insights into quercetin's cerebrovascular protective effects.