Aspartame exacerbates cerebral ischemia–reperfusion injury via mitochondrial dysfunction and ERK/CREB1 pathway suppression

Aspartame, a widely used non-nutritive sweetener, has been associated with potential neurotoxicity. However, it remains unclear whether aspartame consumption can exacerbate cerebral ischemia–reperfusion injury (CIRI), a major contributor to stroke outcomes, and the underlying mechanisms are poorly understood. In vivo , male C57BL/6 J mice were given free access to 0.1% or 0.2% (w/v, equivalent to human acceptable daily intake level) aspartame, for 7 days, followed by bilateral common carotid artery occlusion (BCCAO) to induce CIRI. In vitro , hippocampal neural stem cells (NSCs) were subjected to oxygen–glucose deprivation (OGD) with aspartame. Cell injury, general and mitochondrial reactive oxygen species (ROS) burden, and mitochondrial function were assessed. Gene Ontology (GO), KEGG enrichment, and protein–protein interaction (PPI) network analyses were performed to identify potential targets. The ERK/CREB1 signaling pathway was evaluated by western blotting and pharmacological modulation. Aspartame significantly increased the infarct volume and aggravated neuronal damage in BCCAO-treated mice. In NSCs, aspartame, but not acesulfame or sucralose, selectively enhanced OGD-induced apoptosis, accompanied by mitochondrial depolarization and excessive ROS accumulation, while showing minimal effects under normoxia conditions. GO/KEGG and PPI analyses highlighted ERK/CREB1 as an important node in aspartame-induced neurotoxicity. Consistently, aspartame suppressed the phosphorylation of ERK1/2 and CREB1. The ERK activator LM22B-10 and mitochondria-targeted antioxidant Mito-TEMPO partially reversed mitochondrial dysfunction, apoptosis and ERK/CREB1 suppression. Additionally, aspartame increased the mRNA expression of pro-inflammatory cytokines (TNF- α , IL-1 β and IL-6) and increased NF-κB p65 phosphorylation and reduced the proportion of Tuj1-positive cells, which were mitigated by ERK activation. Aspartame exacerbates CIRI-associated injury in a stress-dependent manner, involving mitochondrial dysfunction, ROS accumulation, and ERK/CREB1 suppression. Future studies are warranted to explore the long-term neurobehavioral outcomes and validate these mechanisms in clinical scenarios. • Aspartame worsened cerebral ischemia–reperfusion injury in mice & cells, increasing infarct size, neuron loss, and cell death. • Aspartame increased oxidative stress & disrupted mitochondrial function under oxygen–glucose deprivation conditions in vitro. • Aspartame enhanced apoptosis, inflammation, & impaired neuron differentiation by suppressing ERK/CREB1 signaling. • Activating ERK or scavenging mitochondrial ROS reduced apoptosis & improved differentiation, suggesting therapeutic potential.