OBJECTIVE: This study was conducted to elucidate vitexin's protective effects and underlying mechanism in ameliorating cerebral ischemia/reperfusion injury (CIRI) through regulation of mitophagy.
METHODS: Focal CIRI in mice was induced using the middle cerebral artery occlusion and reperfusion method. 2,3,5-triphenyltetrazolium chloride staining was performed for the evaluation of cerebral infarction. Neurological deficits and brain tissue damage were assessed by neurological deficit scores and hematoxylin-eosin staining, respectively. HT22 cells underwent oxygen-glucose deprivation/reoxygenation (OGD/R) exposure to develop an in vitro model. Prior to OGD/R, we pretreated the HT22 cells with vitexin, the mitophagy inhibitor (Mdivi-1), or the SIRT1 inhibitor (EX-527). Determination of cell viability and apoptosis were carried out through the cell counting kit-8 assay and flow cytometry, respectively. JC-1 fluorescence staining and MitoSOX™ Red staining were respectively performed for assessing mitochondrial membrane potential (MMP) and detecting levels of mitochondrial reactive oxygen species (mtROS). Expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), microtubule-associated protein 1 A/1B-light chain 3 (LC3), sequestosome-1 (p62), PTEN-induced kinase 1 (PINK1), Parkin, as well as silent information regulator two 1 (SIRT1) was determined via Western blot.
RESULTS: Vitexin was found to significantly alleviate CIRI in mice and mitigate HT22 cell injury due to OGD/R exposure, as confirmed by our in vivo and in vitro experiments, accompanied by activation of mitophagy and the SIRT1/PINK1/Parkin pathway. The OGD/R+Vitexin+Mdivi-1 group (versus the OGD/R+Vitexin group) displayed decreased cell viability, increased apoptosis, a reduced Bcl-2/Bax ratio, diminished MMP, elevated mtROS levels, downregulated PINK1, LC3-II, and Parkin expression, and upregulated p62 expression. Similarly, the OGD/R+Vitexin+EX-527 group showed reduced cell viability, increased apoptosis, a decreased Bcl-2/Bax ratio, decreased MMP, elevated mtROS levels, downregulated SIRT1, PINK1, LC3-II, and Parkin expression, and upregulated p62 expression.
CONCLUSION: Vitexin ameliorates CIRI by activating mitophagy via the SIRT1/PINK1/Parkin pathway.
