Puerarin alleviates cerebral ischemia/reperfusion (CIR)-induced neurocyte oxidative stress and apoptosis via DNA demethylation-mediated PI3K/Akt activation

Abstract

Background: Cerebral ischemia stroke (CIS) is a multifactorial and heterogeneous neurological disorder characterized by high mortality rate and longterm cognitive deficits. Puerarin (Pue) exhibits neuroprotective properties against cerebral ischemia/reperfusion (CIR) injury.

Methods: The DNA demethylation-mediated neuroprotective effects of Pue were investigated in vivo using TTC staining, H&E staining, and TUNEL assays. In vitro, cell viability assays, DCFH-DA staining, Annexin V-FITC/PI staining, and JC-1 staining were employed to examine the alleviation of oxidative stress and apoptosis. Molecular docking and microscale thermophoresis (MST) were employed to analyze Pue's binding affinity to DNA methyltransferases 3A and 3B (DNMT3A/B). Whole-genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) profiling identified Pue/DNMTs-regulated target genes, and dual-Luciferase reporter assay measured promoter activity of PI3K. The function of the PI3K/Akt pathway in CIR injury was verified using pharmacological inhibition and siRNA-mediated knockdown.

Results: Our findings demonstrate that Pue exerts neuroprotection against CIR injury by attenuating oxidative stress, apoptosis, and mitochondrial dysfunction through DNA demethylation. Mechanistically, Pue directly binds to DNMT3A/B, inhibiting their enzymatic activity and reducing global 5-methylcytosine (5mC) levels. Both knockdown of DNMT3A/B and pharmacological DNMT inhibition (SGI-1027) mimicked Pue's protective effects in OGD/R HT22 cells tMCAO rats. Integrated WGBS and RNA-seq analyses revealed that Pue treatment induced promoter demethylation and transcriptional upregulation of PI3K in hippocampi from tMCAO rats. Disruption of PI3K/Akt signaling, either with the pharmacological inhibitor (LY294002) or silencing PI3K, negated Pue-mediated neuroprotection in vitro and in vivo.

Conclusions: Pue inhibits DNMT3A/B activation, leading to demethylation of the PI3K promoter and subsequent activation of PI3K/Akt signaling, which mitigates CIR-induced oxidative stress and apoptosis. This study elucidates a novel epigenetic mechanism underlying Pue's neuroprotective effects and highlights its therapeutic potential for ischemic stroke treatment.

Keywords: Apoptosis; Cerebral ischemia/reperfusion (CIR) injury; DNA methylation; Hippocampus; Oxidative stress; Puerarin.