The study shows that the synthetic peptide Herkinorin can protect human stem‑cell‑derived neurons from damage caused by lack of oxygen and glucose, but only at very low concentrations (0.1‑1 µM). It works by pulling the mu‑opioid receptor inside the cell, which seems to trigger anti‑death pathways, while higher doses (above 10 µM) actually kill the cells.

Hypoxic/ischemic brain injury remains a major clinical challenge, yet the cellular mechanisms linking oxygen-glucose deprivation/reperfusion (OGD/R) to opioid receptor regulation in human neurons are still not fully understood. The trafficking of μ-opioid receptors (MOR) and κ-opioid receptors (KOR) is a key regulator of neuronal survival under stress. Most studies to date in this field have employed rodent models. However, given the molecular and physiological differences between rodents and humans, this study employed human induced pluripotent stem cell (iPSC)-derived neurons to investigate opioid receptor trafficking during OGD/R, as well as the neuroprotective effects of Herkinorin. Human iPSC-derived neurons were subjected to 2 h of OGD followed by 24 h of reoxygenation. Cells were treated with Herkinorin (0.1, 0.5, or 1 μM) during OGD/R. Apoptosis was assessed using flow cytometry, while the localization of MOR and KOR in membrane and cytoplasmic fractions was analyzed using western blotting. Western blotting was also used to quantify the expression of apoptosis-related proteins Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), and cleaved Caspase-3. Statistical comparisons were performed using one-way ANOVA with Tukey's post hoc test or non-parametric equivalents. OGD/R significantly increased neuronal apoptosis, upregulated pro-apoptotic Bax and cleaved Caspase-3, and downregulated anti-apoptotic Bcl-2. These changes were accompanied by altered distribution of MOR, but not KOR localization, specifically decreasing cytoplasmic MOR while maintaining membrane levels. Herkinorin, particularly at 1 μM, induced redistribution of MOR from the plasma membrane to cytoplasm, consistent with receptor internalization; it also significantly reduced apoptosis in a concentration-dependent manner. Furthermore, treatment with Herkinorin reversed the OGD/R-induced molecular changes by decreasing the expression of Bax and cleaved Caspase-3, while increasing that of Bcl-2. KOR trafficking remained largely unchanged under all conditions. Importantly, Herkinorin concentrations above 10 μM reduced neuronal viability, indicating a narrow therapeutic window. Herkinorin exerts neuroprotective effects in human iPSC-derived neurons subjected to OGD/R, potentially by modulating MOR internalization and influencing mitochondrial-dependent apoptotic pathways. However, its efficacy is restricted to a limited dose range (0.1-1 μM), as higher concentrations are toxic. The receptor subtype-specific trafficking pattern observed in this study underscores the importance of human-relevant models for mechanistic and translational research on opioid receptors.