Luteolin attenuates oxidative stress in mice with cerebral ischemia-reperfusion by inhibiting Drp1 activation and promoting autophagy flux

Abstract

Objective To explore the mechanism by which luteolin alleviates oxidative stress in mice with cerebral ischemia-reperfusion. Methods We screened the antioxidants with potential therapeutic effect on cerebral ischemia-reperfusion injury using CMap dataset based on their targeted drug scores and verified the protective effect of luteolin in a mouse model of middle cerebral artery occlusion (MCAO). In a SH-SY5Y cell model of oxygen/glucose deprivation and reoxygenation (OGD/R), we examined the effects of different concentrations of luteolin (10 and 50 μmol/L) on intracellular reactive oxygen species (ROS) content and cell viability using MTT assay. Immunofluorescence assay and Western blotting were performed to detect the changes in autophagy flux and mitochondrial morphology in response to luteolin treatment to assess the capacity of the cells for ROS production and clearance. Results The high targeted drug score (96.86) of luteolin suggested its potential protective effect against cerebral ischemia-reperfusion injury. TTC staining showed that treatment with luteolin significantly reduced cerebral infarction area in MCAO mice (P < 0.05). The MTT value of OGD/R-induced SH-SY5Y cells was about 50% of that of control cells, and was significantly increased to 83% after treatment with 50 μmol/L luteolin (P < 0.05). Luteolin also dose-dependently reduced ROS production in SH-SY5Y cells with OGD/R (P < 0.05). Confocal laser scanning microscopy showed that the fluorescence intensity of the autophagosomes was increased by 64.6% (P < 0.05) and the co-localization of autophagosomes and lysosomes increased by 1.56 folds after treatment with 50 μmol/L luteolin (P < 0.05). Western blotting demonstrated that luteolin significantly inhibited the phosphorylation of Drp1 at Ser616 and its mitochondrial translocation (P < 0.05), and thereby improved the morphological changes of the mitochondria in OGD/R-induced SH-SY5Y cells (P < 0.05). Conclusion Luteolin has obvious protective effect against oxidative stress in mice with cerebral ischemia-reperfusion by promoting the degradation of mitophagosomes and autophagy flux to accelerate ROS clearance and by inhibiting the activity of Drp1 to protect mitochondrial morphology and reduce ROS production

Keywords

• luteolin
• cerebral ischemia-reperfusion
• mitochondria
• reactive oxygen species
• drp1
• autophagy flux