Salvia miltiorrhiza Bge. processed with porcine cardiac blood inhibited GLRX5-mediated ferroptosis alleviating cerebral ischemia-reperfusion injury.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is a destructive adverse reaction of ischemic stroke, leading to high disability and mortality rates. Salvia miltiorrhiza Bge. (Danshen, DS) processed with porcine cardiac blood (PCB-DS), a characteristic processed product, has promising anti-ischemic effects. However, the underlying mechanism of PCB-DS against CIRI remains unclear.

PURPOSE: Ferroptosis is demonstrated to be involved in CIRI. The aim of this study was to explore the molecular mechanism underlying PCB-DS inhibited GLRX5-mediated ferroptosis alleviating CIRI, which was different from DS.

METHODS: Quality evaluation of PCB-DS and DS was conducted by UPLC. Pharmacological activities of PCB-DS and DS against CIRI were compared using neurobehavioral scores, infarct volume, proinflammatory factors, and pathological examinations. Proteomics was employed to explore the potential specific mechanism of PCB-DS against CIRI, which was different from DS. Based on the differential protein GLRX5, ferroptosis-related iron, GSH, MDA, SOD, ROS, liperfluo, and mitochondrial morphology were analyzed. Then, the proteins of GLRX5-mediated iron-starvation response and SLC7A11/GPX4 were analyzed. Finally, OGD/R-induced SH-SY5Y cells upon GLRX5 silencing were constructed to demonstrate that PCB-DS improved CIRI by GLRX5-mediated ferroptosis.

RESULTS: PCB-DS better alleviated CIRI through decreasing neurological score, reducing the infarct volume, and suppressing the release of inflammatory cytokines than DS. Proteomics suggested that PCB-DS may ameliorate CIRI by inhibiting GLRX5-mediated ferroptosis, which was different from DS. PCB-DS reversed the abnormal mitochondrial morphology, iron, GSH, MDA, SOD, ROS, and liperfluo to inhibit ferroptosis in vitro and in vivo. PCB-DS directly activated GLRX5 suppressing the iron-starvation response and downregulated the SLC7A11/GPX4 signaling pathway to inhibit ferroptosis. Finally, silencing GLRX5 activated the iron-starvation response in SH-SY5Y cells and PCB-DS unimproved OGD/R injury upon GLRX5 silencing.

CONCLUSION: Different from DS, PCB-DS suppressed ferroptosis to alleviate CIRI through inhibiting GLRX5-mediated iron-starvation response. These findings give a comprehensive understanding of the molecular mechanism underlying the effect of PCB-DS against CIRI and provide evidence to assess the product in clinical studies.