2. Academic Validation
  3. Rosmarinic Acid suppresses ferroptosis and confers neuroprotection in cerebral ischemia-reperfusion via direct KEAP1 inhibition and NRF2 activation

Rosmarinic Acid suppresses ferroptosis and confers neuroprotection in cerebral ischemia-reperfusion via direct KEAP1 inhibition and NRF2 activation

  • Free Radic Biol Med. 2025 Oct 28:S0891-5849(25)01322-X. doi: 10.1016/j.freeradbiomed.2025.10.291.
Lang Liu 1,  Jingyu Weng 2,  Lei Yue 3,  Yuanyuan Qin 4,  Jia Yu 5,  Yaqi Li 6,  Shuming Li 7,  Jiaofeng Wu 8,  Yuangui Yang 9,  Zhishu Tang 10,  Hongbo Xu 11
Affiliations

Affiliations

  • 1 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: 2012612942@qq.com.
  • 2 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: wengjy95@163.com.
  • 3 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: 2910984390@qq.com.
  • 4 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: 1959559860@qq.com.
  • 5 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: 2423843063@qq.com.
  • 6 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: 3395925486@qq.com.
  • 7 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: 1501034@sntcm.edu.cn.
  • 8 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: wujiaofeng@sntcm.edu.cn.
  • 9 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: ygyang2015@126.com.
  • 10 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China; Beijing University of Chinese Medicine,100105, Beijing, PR China. Electronic address: tzs6565@163.com.
  • 11 Shaanxi University of Chinese Medicine, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, 712083, Xianyang, PR China. Electronic address: xhb2005@sntcm.edu.cn.
PMID: 41167534 DOI: 10.1016/j.freeradbiomed.2025.10.291
Abstract

Cerebral ischemia-reperfusion injury exacerbates neuronal loss following acute ischemic stroke through oxidative stress and regulated cell-death pathways, notably Ferroptosis. Here, we investigated whether rosmarinic acid (RA), a natural polyphenol, confers neuroprotection by inhibiting Ferroptosis via activation of the Keap1-Nrf2 axis. In vivo, rats pretreated with RA (30 or 60 mg/kg, i.p.) for 14 days before middle cerebral artery occlusion/reperfusion exhibited dose-dependent improvements in neurological scores, reduced infarct volumes, preserved cerebral blood flow, and attenuated histopathological damage. In vitro, RA (25-50 μM) restored the viability of HT22 and PC12 cells subjected to oxygen-glucose deprivation/reperfusion, suppressed lipid Reactive Oxygen Species accumulation, iron overload, and mitochondrial ultrastructural collapse. Mechanistically, RA promoted nuclear factor erythroid 2-related factor 2 (NRF2) nuclear translocation, enhanced antioxidant response element -driven luciferase activity, and upregulated ferroptosis-defense genes (Gpx4, Slc7a11, and Gclm) at both mRNA and protein levels. Cycloheximide chase and ubiquitination assays, supported by molecular docking, molecular dynamics simulations, and surface plasmon resonance, demonstrated that RA directly binds Kelch-like ECH-associated protein 1 (KEAP1) to stabilize NRF2. A KEAP1 knockdown abolished RA's induction of NRF2 targets and its anti-ferroptotic effects. These findings establish RA as a targeted NRF2 activator and Ferroptosis inhibitor, offering a promising therapeutic strategy for limiting ischemia-reperfusion-induced neuronal injury.

Keywords

Ferroptosis; Ischemia-reperfusion injury; NRF2–KEAP1; Rosmarinic acid; Stroke.

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