Dihydromyricetin attenuates cerebral ischemia-reperfusion injury associated with modulation of sirtuin 1-forkhead box O3 signaling and mitophagy.

Background

Ischemic stroke remains associated with substantial neurological disability, and effective strategies for cerebral ischemia-reperfusion injury (CIRI) are limited. Dihydromyricetin (DHM) has shown neuroprotective properties in experimental neurological disorders, but its effects and associated molecular mechanisms in CIRI remain incompletely understood. This study therefore evaluated the protective effects of DHM pretreatment and examined the signaling events associated with these effects in experimental CIRI.

Methods

A mouse model of middle cerebral artery occlusion followed by reperfusion and an HT-22 cell model of oxygen-glucose deprivation followed by reoxygenation were used to evaluate the effects of DHM. In vivo outcomes included infarct volume, histopathological injury, neurological deficits, and recognition memory. In vitro outcomes included cell viability, lactate dehydrogenase release, apoptosis, oxidative stress, adenosine triphosphate content, and mitochondrial morphology. Network pharmacology was used to nominate candidate regulatory targets. Sirtuin 1 (SIRT1) expression and deacetylase activity, forkhead box O3 (FOXO3) acetylation and subcellular localization, and mitophagy-related changes were assessed by immunoblotting, nuclear/cytoplasmic fractionation, immunofluorescence, and co-immunoprecipitation. The selective SIRT1 inhibitor EX527 and small interfering RNA-mediated SIRT1 knockdown were used to examine SIRT1 involvement, whereas bafilomycin A1 and Parkin knockdown were used to assess mitophagic flux and the functional contribution of Parkin. A cellular thermal shift assay was used to test whether DHM produced a detectable change in SIRT1 thermal stability.

Results

DHM pretreatment reduced infarct volume, improved neurological scores and recognition memory, and ameliorated histopathological injury in mice subjected to middle cerebral artery occlusion/reperfusion. In HT-22 cells subjected to oxygen-glucose deprivation/reoxygenation, DHM improved cell viability and adenosine triphosphate content, reduced oxidative stress and apoptosis, and preserved mitochondrial morphology. These effects were accompanied by increased SIRT1 expression and deacetylase activity, reduced FOXO3 acetylation, increased nuclear FOXO3, and changes in phosphatase and tensin homolog-induced kinase 1/Parkin and mitophagy-related markers consistent with enhanced mitophagic flux. EX527 and SIRT1 knockdown attenuated several molecular and protective effects of DHM, and Parkin knockdown weakened DHM-associated improvements in cellular outcomes. The cellular thermal shift assay did not detect a DHM-induced shift in SIRT1 thermal stability.

Conclusion

In the tested experimental models, DHM attenuated experimental CIRI, and these effects were associated with increased SIRT1 activity, FOXO3 deacetylation and nuclear accumulation, and Parkin-mediated mitophagy. Post-ischemic treatment studies and direct molecular validation are required to establish therapeutic relevance and the precise causal relationships among these events.