Salidroside pretreatment alleviates ferroptosis induced by myocardial ischemia/reperfusion through mitochondrial superoxide-dependent AMPK α 2 activation

Background: Ferroptosis, a form of regulated cell death (RCD) that relies on excessive reactive oxygen species (ROS) generation, Fe 2 + accumulation, abnormal lipid metabolism and is involved in various organ ischemia/ reperfusion (I/R) injury, expecially in myocardium. Mitochondria are the powerhouses of eukaryotic cells and essential in regulating multiple RCD. However, the links between mitochondria and ferroptosis are still poorly understood. Salidroside (Sal), a natural phenylpropanoid glycoside isolated from Rhodiola rosea , has multbioactivities. However, the effects and mechanism in alleviating ferroptosis caused by myocardial I/R injury remains unclear. Purpose: This study aimed to investigate whether pretreated with Sal could protect the myocardium against I/R damage and the underlying mechanisms. In particular, the relationship between Sal pretreatment, AMPK alpha 2 activity, mitochondria and ROS generation was explored. Study design and methods: Firstly, A/R or I/R injury models were employed in H9c2 cells and Sprague-Dawley rats. And then the anti-ferroptotic effects and mechanism of Sal pretreatment was detected using multirelevant indexes in H9c2 cells. Further, how does Sal pretreatment in AMPK alpha 2 phosphorylation was explored. Finally, these results were validated by I/R injury in rats. Results: Similar to Ferrostatin-1 (a ferroptosis inhibitor) and MitoTEMPO, a mitochondrial free radical scavenger, Sal pretreatment effectively alleviated Fe 2 + accumulation, redox disequilibrium and maintained mitochondrial energy production and function in I/R-induced myocardial injury, as demonstrated using multifunctional, enzymatic, and morphological indices. However, these effects were abolished by downregulation of AMPK alpha 2 using an adenovirus, both in vivo and in vitro . Moreover, the results also provided a non -canonical mechanism that, under mild mitochondrial ROS generation, Sal pretreatment upregulated and phosphorylated AMPK alpha 2, which enhanced mitochondrial complex I activity to activate innate adaptive responses and increase cellular tolerance to A/R injury. Conclusion: Overall, our work highlighted mitochondria are of great impotance in myocardial I/R-induced ferroptosis and demonstrated that Sal pretreatment activated AMPK alpha 2 against I/R injury, indicating that Sal could become a candidate phytochemical for the treatment of myocardial I/R injury.