A discovery-based proteomic approach of epidermal growth factor and growth hormone-releasing peptide-6 in a model of acute ischemic stroke

Combined therapy with epidermal growth factor (EGF) and growth hormone-releasing peptide 6 (GHRP6) has demonstrated neuroprotective effects in models of global and focal brain ischemia. Clinical studies in ischemic stroke patients have confirmed the safety and preliminary efficacy of this combined treatment. This study aimed to elucidate the molecular mechanisms underlying the effects of EGF+GHRP6 co-administration.

Male Wistar rats were subjected to endothelin-1 (ET-1)–induced middle cerebral artery (MCA) occlusion and randomly assigned to three experimental groups: EGF+GHRP6‑treated ischemic, vehicle‑treated ischemic, and sham-operated controls (n = 12 per group). Label-free quantitative proteomic analysis of the ischemic penumbra was performed at 3 and 24 h post-treatment. Functional enrichment and pathway analysis of differentially modulated proteins were performed using bioinformatics tools.

Proteomic profiling validated the ischemic model and revealed 40 and 223 proteins differentially modulated by EGF+GHRP6 at 3 and 24 h, respectively. Proteins involved in neurotransmitter transport were consistently overrepresented in the EGF+GHRP6-regulated proteome at both time points. At 24 h post-treatment, proteins associated with reactive oxygen species (ROS) detoxification, heat shock factor 1 (HSF1) activation, and negative regulation of cellular hypoxia response were significantly modulated. Additionally, anti-apoptotic and mitochondrial proteins were modulated in the ischemic penumbra, supporting the neuroprotective effects of EGF+GHRP6. Notably, proteins known to attenuate brain damage after stroke were up-regulated, while those promoting ischemic injury were down-regulated following the combined treatment.

These findings provide molecular evidence supporting the neuroprotective mechanism of action of EGF+GHRP6 co-administration and reinforce its potential as a therapeutic strategy for ischemic stroke.