Dihydromyricetin Attenuates Cerebral Ischemia Reperfusion Injury by Inhibiting SPHK1/mTOR Signaling and Targeting Ferroptosis

Jiangbo Xie,1,2 Tingting Zhang,3 Peichun Li,3 Dong Wang,2 Tao Liu,2 Shunliang Xu1 1Department of Neurology, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, People’s Republic of China; 2Department of Neurology, Weifang Traditional Chinese Hospital, Weifang, People’s Republic of China; 3Department of Rehabilitation Medicine, Weifang Traditional Chinese Hospital, Weifang, People’s Republic of ChinaCorrespondence: Shunliang Xu, Department of Neurology, The Second Hospital, Cheeloo College of Medicine, Shandong University, 247 Beiyuan Road, Jinan, Shandong, 250033, People’s Republic of China, Tel +86 15153169998, Email xushandaeryuan@163.comBackground: Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury.Methods: We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting.Results: DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR).Conclusion: This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.Graphical Abstract: Keywords: dihydromyricetin, ferroptosis, SPHK1, mTOR, cerebral ischemia reperfusion injury