EGF+GHRP6 combination therapy reprograms 223 proteins, boosts ROS detoxification, and activates HSF1 in acute ischemic stroke.

Acute ischemic stroke remains a leading cause of death and long-term disability, with limited therapeutic options beyond reperfusion. Current treatments often fail to fully mitigate secondary brain injury, which involves complex cascades of inflammation, oxidative stress, and apoptosis. Combined therapy with epidermal growth factor (EGF) and growth hormone-releasing peptide 6 (GHRP6) has shown promise, demonstrating neuroprotective effects in various brain ischemia models and preliminary safety/efficacy in clinical trials. This study aimed to uncover the specific molecular mechanisms by which this combination exerts its beneficial effects, addressing a critical gap in understanding its therapeutic potential.

Male Wistar rats were subjected to endothelin-1 (ET-1)-induced middle cerebral artery (MCA) occlusion to model acute ischemic stroke. Animals were randomly assigned to three groups (n=12 per group): EGF+GHRP6-treated ischemic, vehicle-treated ischemic, and sham-operated controls. Label-free quantitative proteomic analysis was performed on the ischemic penumbra region at 3 and 24 h post-treatment. Bioinformatics tools were then used for functional enrichment and pathway analysis of the differentially modulated proteins to identify key molecular changes.

Proteomic profiling successfully validated the ischemic model, identifying significant protein changes. EGF+GHRP6 co-administration differentially modulated 40 proteins at 3 h and a more extensive 223 proteins at 24 h post-treatment. Proteins involved in neurotransmitter transport were consistently overrepresented in the EGF+GHRP6-regulated proteome at both early and later time points. At 24 h post-treatment, the combination therapy significantly modulated proteins associated with reactive oxygen species (ROS) detoxification, heat shock factor 1 (HSF1) activation, and negative regulation of cellular hypoxia response. Additionally, anti-apoptotic and mitochondrial proteins were notably modulated within the ischemic penumbra, providing strong molecular support for the observed neuroprotective effects of EGF+GHRP6. This comprehensive proteomic signature indicates a broad, multi-target mechanism of action.

Proteins known to attenuate brain damage after stroke were up-regulated, while those promoting ischemic injury were down-regulated following the combined treatment.