Hypoxic/ischemic brain injury is a major cause of long-term neurological disability and mortality, with few effective therapies available. A persistent barrier to the development of treatments is the low success rate for translation of findings from preclinical studies to clinical therapies. While rodent models have provided valuable mechanistic insights, single-cell transcriptomic and spatial mapping studies have demonstrated substantial differences between rodent and human brains in terms of cortical cell-type composition, gene expression patterns, and laminar organization [1, 2]. Moreover, cross-species comparisons reveal that brain-region transcriptomic similarity is particularly limited in higher-order cognitive areas [2] that may influence injury responses and pharmacological outcomes. Neurochemical systems also differ markedly between species. Baseline levels of serotonin (5-HT), norepinephrine (NE), dopamine (DA), and acetylcholine (ACh) in the prefrontal cortex vary significantly between rodents and primates [3], potentially altering drug efficacy profiles. At the gene level, the µ opioid receptor gene (OPRM1) in rodents exhibits extensive alternative splicing, producing isoforms that differ from those in humans [4, 5]. These isoform-specific variations can influence ligand binding, signaling bias, and therapeutic effects, thereby limiting the direct extrapolation of rodent pharmacology to human contexts. G protein-coupled receptor (GPCR) signaling adds further complexity, as ligand-directed signaling bias and $\beta$-arrestin-dependent pathways display species-dependent differences [6]. For the µ opioid receptor (MOR), recruitment of $\beta$-arrestin and receptor trafficking are central to the ongoing debate about neuroprotection versus adverse effects such as tolerance and respiratory depression. Herkinorin, a semi-synthetic salvinorin A analog, is a MOR-selective agonist (~8-fold specificity over $\kappa$ opioid receptors (KOR), and ~98-fold over $\delta$ opioid receptor) with minimal recruitment of $\beta$-arrestin-2 [7, 8]. Its ability to induce MOR trafficking remains controversial, with some studies reporting negligible trafficking, while others suggest context-dependent trafficking [7, 9]. These unique pharmacological properties make Herkinorin an ideal tool to dissect the role of MOR trafficking in hypoxic/ischemic injury.

Human induced pluripotent stem cell (iPSC)-derived neurons offer a solution to such translational challenges. These cells retain a human genetic background, express human-specific receptor isoforms, and recapitulate patient-specific molecular phenotypes, showing high concordance with post-mortem brain molecular profiles [4, 10]. Our previous study showed that iPSC-derived neural cells express opioid receptors [11]. Therefore, iPSC-based neuronal models provide a physiologically relevant platform to investigate disease mechanisms and evaluate candidate therapeutics with greater translational potential.

In the current study, we hypothesized that Herkinorin exerts neuroprotective effects in human iPSC-derived neurons subjected to oxygen-glucose deprivation/reperfusion (OGD/R) by modulating MOR trafficking. To test this, we examined receptor trafficking patterns and apoptosis in iPSC-derived neurons under OGD/R, with and without Herkinorin treatment. This approach directly addresses the translational gap by combining a human-specific neuronal model with a mechanistic analysis of biased MOR agonism in hypoxic/ischemic injury.

Over recent decades, animal models have played a crucial role in neuroscience, particularly for in vitro and in vivo modeling of disease. These models have been instrumental in elucidating various pathophysiological events that might otherwise have remained unknown. However, the biological and genetic discrepancies between species have been a significant barrier in the development of effective treatments for many neurological and neurodegenerative diseases. Approximately 90% of drugs that show efficacy in animal models ultimately fail in human clinical trials [15]. The utilization of human tissues, derived mainly from autopsies or fetal specimens, for cell culture models is limited by the scarcity of these resources and the challenges in scaling up primary cultures. Human iPSC-based models offer efficient platforms for elucidating pathological mechanisms and drug screening in inflammatory diseases. iPSC-derived neurons and glial cells are critical for probing the molecular mechanisms underlying hypoxic or ischemic injury and for exploring potential treatments [16, 17]. The integration of human iPSC-derived brain organoids with cell culture methods to assess the effects of OGD/R is a promising approach for stroke research [18]. Luminescent neurospheres derived from human iPSCs are particularly suited for high-throughput screening aimed at discovering new treatments for acute stroke [19]. Furthermore, brain organoids based on human iPSCs serve as a novel research platform for anti-stroke drug development, and the use of iPSC-induced human cortical organoids to model OGD enables detailed investigation of the mechanisms underlying cerebral ischemia [20, 21]. De Paola et al. [21] developed a novel human 3D self-assembled iPSC-derived model (human cortical organism, hCO) to examine ischemic effects caused by OGD. These authors found that hCOs exhibited neuronal death and a reduction in neuronal network complexity after being subjected to OGD. Although OGD is commonly utilized to simulate hypoxic or ischemic injury, existing studies report considerable variability in the severity of injury [22]. Previous OGD research predominantly utilized primary rodent neuronal cells or genetically altered cell lines in non-physiological states, such as the SH-SY5Y cell line which is known for its genetic abnormalities. Hence, there is a pressing need to develop non-neoplastic, in vitro human cell-based models. In 2020, Juntunen et al. [23] established the first in vitro human stroke model using neurons differentiated from human iPSCs. The lack of a standardized OGD protocol to accurately replicate hypoxic or ischemic injury in vitro is also noteworthy. Experimental OGD durations have varied from 1 to 24 h, sometimes followed by a reperfusion period, while oxygen levels in hypoxic conditions have ranged between 0% and 8% [23]. Our earlier study demonstrated that neurons derived from human iPSCs are capable of expressing µ-opioid receptors [11]. Using human iPSC-derived neurons to model OGD/R, the present study assessed the effects of Herkinorin on apoptosis-related proteins and the distribution of MOR. We found that OGD/R increased the expression of pro-apoptotic Bax and cleaved Caspase-3, while decreasing that of anti-apoptotic Bcl-2. Moreover, Herkinorin at 1 µM reversed these changes. Herkinorin was also observed to alter MOR localization, reduce membrane-associated MOR, and increase cytoplasmic MOR in a concentration-dependent manner. Our data support previous evidence that MOR activation confers neuroprotection by modulating mitochondrial apoptotic pathways [24], while extending these findings to human iPSC-derived neurons. This model has greater physiological relevance than rodent neurons or immortalized cell lines [16, 17, 23, 25]. The Herkinorin-induced reduction in Bax and cleaved Caspase-3 expression, coupled with increased Bcl-2, suggests a shift toward an anti-apoptotic profile, consistent with MOR-mediated pro-survival signaling. In the current study we limited the experimental concentration range of Herkinorin to 0.1–1 µM, as higher concentrations ($\ge$10 µM) showed measurable cytotoxicity in preliminary MTT assays. This approach ensured that any observed neuroprotective effects were not confounded by overt toxicity. Nevertheless, it remains possible that concentrations $>$1 µM could elicit stronger MOR redistribution or neuroprotective effects under certain conditions. Future studies should systematically explore the 1–10 µM range while implementing strategies to mitigate potential toxicity, such as shorter exposure durations, pulse dosing, or co-treatment with cytoprotective agents to more fully define the therapeutic window for Herkinorin in human neurons.

A notable point of contention in the literature is whether Herkinorin induces MOR internalization. While some studies have reported no $\beta$-arrestin recruitment or receptor trafficking [7, 9], we observed a redistribution under ischemia-like conditions. Potential explanations for this discordance include differences in model systems (endogenous MOR in human neurons vs. overexpressed receptors in heterologous cells), detection methods (biochemical fractionation vs. microscopy/$\beta$-arrestin assays), and experimental context (hypoxia/reoxygenation vs. normoxia). Hypoxic stress may sensitize MOR to ligand-induced internalization, with this factor being absent in normoxic studies.

The observed reciprocal changes in membrane and cytoplasmic MOR suggest internalization, which could enable protective intracellular signaling. However, we did not directly measure $\beta$-arrestin engagement or downstream pathways, and hence this remains speculative. Live-cell imaging and the use of pathway-specific inhibitors are needed to elucidate the exact mechanism. Limitations include the immature phenotype of iPSC-derived neurons, lack of temporal profiling of receptor trafficking, and the absence of detailed KOR analysis [26, 27]. It would also be helpful to include a well-characterized MOR agonist, such as DAMGO, as a positive control for receptor trafficking and anti-apoptotic signaling. This would strengthen the mechanistic interpretation of the effects of Herkinorin by providing a benchmark for MOR-dependent responses. However, this was not feasible here due to the scope and timeline of our study. Future studies should address these gaps by incorporating mechanistic assays to validate MOR trafficking pathways.

Herkinorin, a derivative of salvinorin A and a potent agonist of MOR and KOR, may not cause the significant tolerance or dependence that is typically associated with traditional opioids. Recent research suggests that Herkinorin produces a dose-dependent, antinociceptive effect in a rat pain model, indicating its potential as a novel analgesic with a lower risk of dependence or tolerance [28]. However, research on the neuroprotective effects of Herkinorin is limited, with only a few studies hinting at such properties [14, 29]. Further investigations are needed to better understand the neuroprotective capabilities of Herkinorin and the associated mechanisms. Our study revealed distinct alterations in the distribution of MOR between membrane and cytoplasmic fractions under OGD/R, particularly after Herkinorin treatment. OGD/R alone did not significantly affect membrane MOR, but 1 µM Herkinorin reduced the membrane expression of MOR while increasing cytoplasmic levels. This reciprocal change suggests ligand-induced internalization, whereby MOR translocates from the membrane to the cytoplasm, potentially engaging protective intracellular signaling. Alternatively, OGD/R may promote cytoplasmic MOR degradation, with Herkinorin preventing this process, or enhancing redistribution. These dose-dependent effects were most pronounced at 1 µM and warrant further investigation using live-cell trafficking assays and pathway-specific analyses. However, it is important to emphasize the narrow therapeutic window observed in our study. At concentrations above 10 µM, Herkinorin reduced cell viability. Thus, the observed neuroprotection is confined to a limited concentration range (0.1–1 µM), which may pose challenges for clinical translation. Overall, our results indicate that OGD/R and Herkinorin treatments lead to a redistribution of MOR between the cytoplasm and membrane in neuronal cells. This may influence mitochondrial function and thereby affect cell survival. Conversely, some studies have found that Herkinorin does not induce $\beta$-arrestin recruitment or receptor trafficking [7, 9]. Such discrepancies in research findings are not uncommon and may be due to differences in experimental design, methodology, conditions, and the cell lines used. Technical variations or experimental conditions could also contribute to these differences. Differences in extraction methods for membrane and cytoplasmic proteins during Western blotting may cause inconsistencies in protein recovery, thus affecting expression levels. Therefore, additional research is essential to further elucidate the mechanism of action of Herkinorin and its effects on opioid receptor trafficking. Well-designed and controlled studies are required to ensure the accuracy and reproducibility of results.

In the present study, we observed that OGD/R failed to induce detectable KOR internalization in human iPSC-derived neurons, even though MOR redistribution was evident following Herkinorin treatment. This finding may be explained by several factors. First, differences between models could play a key role. Previous observations of OGD-induced KOR internalization were mostly in rodent or immortalized neuronal cell lines (e.g., Neuro2A cells stably expressing tagged KOR) in which receptor expression levels, membrane composition, and intracellular trafficking machinery differ substantially from those of human iPSC-derived neurons [30]. Second, KOR internalization is known to require specific $\beta$-arrestin- and Akt-dependent signaling pathways [30, 31]. The activity of these pathways under ischemia-like conditions may vary between cell types, potentially accounting for the absence of KOR trafficking in our system. Finally, the pharmacological profile of Herkinorin provides another possible explanation. Despite its structural similarity to salvinorin A (a potent KOR agonist), Herkinorin displays approximately 8-fold selectivity for MOR over KOR, with markedly reduced efficacy for KOR [32]. Such selectivity may explain the lack of detectable KOR redistribution in our OGD/R model, where MOR activation likely predominates in mediating the observed neuroprotection. Nonetheless, it remains possible that KOR contributes to protective signaling via non-internalizing, G protein–biased pathways. This warrants further investigation using KOR-selective ligands and pathway-specific assays.

In summary, Herkinorin reduced OGD/R-induced apoptosis and modulated MOR distribution in human iPSC-derived neurons. Future work should focus on strategies to expand its therapeutic range, such as structural modification of Herkinorin analogs, controlled delivery systems, or combination therapies designed to mitigate toxicity while preserving efficacy. These findings highlight the potential of physiologically relevant human neuronal models for studying opioid receptor biology under ischemia-like conditions. They also underscore the need to consider experimental context when interpreting conflicting data on membrane trafficking. The future use of physiological iPSC-derived neural cells to simulate hypoxic or ischemic injury in the context of OGD should lead to further valuable insights in this field.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

XC and GW designed the study. ZJ, YY, and XL developed culture cells and performed immunofluorescence, flow cytometry, and Western blot analyses. Data were analyzed by ZJ and XL. The manuscript was written by ZJ and XC. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

Not applicable.

This work was supported by Beijing Natural Science Foundation-7212019.

The authors declare no conflict of interest.