Xingru Tao,* Meina Zhao,* Kai Gao,* Wei Zhang, Dong Xu, Wangting Li, Fei Mu, Rui Lin, Chao Guo, Ruili Li, Jingwen Wang

Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an, 710032, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Jingwen Wang, Email wangjingwen8021@163.com

Purpose: Wedelolactone (Wel), a furocoumarin compound extracted from the medicinal plant Eclipta prostrata L. has been shown to exhibit significant neuroprotective effects. However, the potential of Wel to improve ischemic stroke (IS) and the underlying mechanisms remain unclear.
Methods: The middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were established to evaluate the potential neuroprotective of Wel. Neurological function, brain infarct volume, brain swelling, and histopathological staining were analyzed to demonstrate the therapeutic efficacy of Wel. The occurrence of ferroptosis was confirmed by quantifying the levels of ROS and Fe2+, and by examining the ultrastructural features of cells. The binding affinity between Wel and HIF-1α was evaluated using Molecular docking. Immunofluorescence and Western blot analyses were conducted to explore the regulation of Wel on the HIF-1α/SLC7A11/GPX4 pathway. Finally, the expression of HIF-1α was down-regulated to verify whether Wel exerts neuroprotection by modulating HIF-1α to inhibit ferroptosis of cells.
Results: The results demonstrated that Wel effectively inhibit neuron ferroptosis after MCAO/R in a dose-time-dependent manner, thereby alleviating brain injury. Moreover, at the cellular level, Wel significantly reduced the accumulation of Fe2+ and ROS after OGD/R. Further investigation revealed that Wel might inhibit neuronal ferroptosis and improve IS by promoting the nuclear translocation and accumulation of HIF-1α, subsequently regulating the expression of downstream proteins SLC7A11 and GPX4. Notably, the inhibitory effect of Wel on ferroptosis was markedly attenuated by HIF- 1α siRNA in OGD/R PC12 cells.
Conclusion: Our discovery revealed that the Wel could alleviate IS by inhibiting oxidative damage and ferroptosis via HIF-1α/ SLC7A11/GPX4 signaling pathway. Wel may represent a promising active compound for anti-IS, while also providing novel insights into the elucidation of its molecular mechanisms and potential clinical implications.

Keywords: Wedelolactone, ischemic stroke, ferroptosis, HIF-1α/SLC7A11/GPX4 pathway