Data from the 2019 Global Burden of Disease report indicate that stroke persists as a major global health burden, ranking as the world’s second most common cause of mortality (accounting for 11.6% of all deaths) and the third primary contributor to combined death and disability (representing 5.7% of total disability-adjusted life years (DALYs) [1]. Large vessel occlusion (LVO) accounts for 15–30% of ischemic strokes and demonstrates disproportionately elevated morbidity and mortality burdens relative to other ischemic stroke subtypes [2]. Despite constituting the established standard of care for LVO-induced acute ischemic stroke, endovascular thrombectomy (EVT) is associated with death or permanent disability in approximately 50% of successfully reperfused patients [3, 4]. Improving post-EVT outcomes remains a critical clinical challenge.

Remote ischemic conditioning (RIC) entails applying transient ischemia-reperfusion cycles to distant tissues, conferring protection against subsequent prolonged ischemic insults in remote organs. A study has confirmed that RIC reduces stroke recurrence and improves patient prognosis [5]. Research has demonstrated that RIC exerts neuroprotective effects against cerebral ischemia-reperfusion injury, with these effects occurring in two distinct phases: an early phase (within 24 h after remote ischemic preconditioning) and a late phase (lasting up to 7 days or even 2 months after preconditioning) [6]. As a neuroprotective strategy, RIC has been explored in vascular recanalization therapies. The RIC-cerebral circulation time (CCT) trial demonstrated that short-term RIC significantly improved cerebral circulation time on the stenotic side, reduced contrast medium leakage, and lowered the incidence of cerebral hyperperfusion syndrome after carotid artery stenting in patients with severe internal carotid artery stenosis [7]. Nevertheless, the neuroprotective pathways of RIC during vascular recanalization are not fully elucidated.

The apelinergic system comprises apelin and Elabela (ELA) peptidergic ligands signaling through their cognate receptor Apelin receptor (APJ), a G protein-coupled receptor [8]. Apelin demonstrates selective neuroanatomical localization within the thalamus, frontal cortex, and hippocampus, modulated by key transcriptional regulators, including hypoxia-inducible factor 1$\alpha$ (HIF-1$\alpha$), signal transducer and activator of transcription 3 (STAT3), and activating transcription factor 4 (ATF4) [9, 10, 11]. Post-stroke neuronal injury results from interconnected pathological processes, including the induction of neuronal apoptosis, activation of the inflammatory cascade, disruption of immune homeostasis, and breakdown of the blood-brain barrier. Cerebral ischemia induces apoptotic programmed cell death, mediated through homeostatic balance between proapoptotic and antiapoptotic genetic pathways [12]. The apelinergic system critically mediates poststroke functional recovery through attenuation of neuronal apoptosis and neuroinflammation. Under hypoxic conditions, apelin/APJ expression is upregulated, potentially mediated by HIF-1$\alpha$, which binds to hypoxia-response elements (HREs) in the promoter regions of apelin/APJ genes, driving their transcriptional activation [13]. In murine models of ischemic stroke, apelin-13 suppresses proinflammatory cytokine transcription, significantly reducing tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and interleukin-1$\beta$ (IL-1$\beta$) mRNA levels. Apelin-13 treatment increases apelin/APJ and B-cell lymphoma 2 (Bcl-2) levels while reducing the activation of caspase-3, a key apoptosis-related gene, thereby enhancing cerebral blood flow restoration and promoting sustained functional recovery [14]. ELA, which is expressed predominantly in neurons surrounding cerebral infarct areas, suppresses ischemia-reperfusion (I/R)-induced ferroptosis and lipid peroxidation, mitigating infarct volume and neuronal degeneration [15]. Exogenous ELA administration has also been shown to alleviate direct neurotoxic effects under oxygen-glucose deprivation conditions [16]. In middle cerebral artery occlusion (MCAO) models, cerebrovascular endothelial cells exhibit marked increases in ELA and APJ expression. The ELA-APJ axis enhances angiogenic activity via the Yes-associated protein/transcriptional coactivator with Post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), Zonula occludens-1 (ZO-1) binding motif (PDZ-binding motif) (YAP/TAZ) pathway, promoting neovascularization and cerebral perfusion recovery and ultimately attenuating I/R injury [17]. Those findings collectively underscore the neuroprotective role of the apelinergic system in cerebral I/R injury. However, whether RIC, an emerging therapeutic intervention, mitigates apoptosis, inflammation, and reperfusion injury in ischemic stroke by modulating the apelinergic system, remains unexplored. In the present study, therefore, we hypothesizes that RIC reduces apoptosis, inflammatory responses, and reperfusion damage in MCAO rats through the regulation of the apelinergic system (Fig. 1). To test this hypothesis, we measured the cerebral levels of apelin, ELA, caspase-3, HIF-1$\alpha$, ATF4, STAT3, TNF-$\alpha$, and IL-1$\beta$ in MCAO rats to validate the neuroprotective mechanisms of RIC.

Fig. 1.

Schematic of how the ELA-apelin-APJ system mediates RIC neuroprotection (Blue arrows: neuronal damage processes; Green arrows: potential neuroprotective processes elicited by RIC). ELA, Elabela; APJ, apelin receptor; RIC, remote ischemic conditioning; STAT3, signal transducer and activator of transcription 3; HIF-1$\alpha$, hypoxia-inducible factor 1$\alpha$; ATF4, activating transcription factor 4; TNF-$\alpha$,tumor necrosis factor-$\alpha$; IL-1$\beta$, interleukin-1$\beta$; ER, endoplasmic reticulum.

Using MCAO modeling, this study defined the neuroprotective mechanisms of RIC in cerebral ischemia-reperfusion. The findings demonstrated that (1) RIC treatment significantly reduced the cerebral infarct volume and alleviated neuronal damage in MCAO-induced I/R rats. (2) RIC attenuated the inflammatory response, cellular apoptosis, and reperfusion injury in MCAO-induced I/R rats, possibly through the apelinergic system.

EVT is a therapeutic approach that utilizes endovascular interventional techniques to directly remove thrombi obstructing cerebral arteries, thereby restoring blood flow. Its core objectives are rapid revascularization, reducing disability, and lowering of mortality rates. Clinical-trial data indicated that EVT is safe and effective for anterior circulation stroke patients with low alberta stroke program early CT score (ASPECTS) scores within 6 h of stroke onset [18]. However, successful revascularization does not fully correlate with favorable clinical outcomes, and improving prognoses for patients undergoing endovascular therapy remains a major focus for clinicians [19].

RIC, a low-cost, accessible, safe, noninvasive neuroprotective strategy, has been explored in combination with revascularization therapies. In 2018, Zhao et al. [20] demonstrated that RIC application in acute ischemic stroke patients undergoing EVT was safe and feasible. The RIC-CCT study further confirmed that short-term RIC significantly improved cerebral circulation time on the stenotic side, reduced contrast agent extravasation, and decreased the incidence of cerebral hyperperfusion syndrome after carotid artery stenting in patients with severe internal carotid artery stenosis [7]. Nevertheless, the neuroprotective mechanisms of RIC in revascularization therapy have remained unclear. The present study aimed to investigate the neuroprotective mechanisms of RIC during ischemia-reperfusion using an MCAO ischemia-reperfusion model. To align with clinical practice, RIC treatment was administered post-reperfusion in MCAO rats.

Previous randomized controlled trials have demonstrated that pharmacotherapy combined with RIC, administered after acute cerebral infarction, can reduce cerebral infarct volume, improve functional outcomes, prevent stroke recurrence, and attenuate cognitive impairment [21]. In MCAO animal models, RIC has been shown to mitigate infarct size and ameliorate neurological deficits [22, 23], which is consistent with the findings of our present study.

The pathophysiology of ischemic brain injury involves the activation of detrimental signaling cascades, including hypoxia, endoplasmic reticulum stress, glucose deprivation, inflammatory responses, and apoptotic pathways [24]. A study has suggested that the ELA-apelin-APJ system is implicated in ischemic stroke, critically protecting against ischemia-induced apoptosis and neurological deficits, highlighting its therapeutic potential [25]. Research has demonstrated that apelin-13 protects against oxidative damage in ischemic stroke via the AMP-activated protein kinase/glycogen synthase kinase-3 beta/nuclear factor erythroid 2-related factor 2 (AMPK/GSK-3$\beta$/Nrf2) pathway [26]. Additionally, different apelin isoforms inhibit neuronal apoptosis through distinct mechanisms [27, 28]. In rat models, apelin also modulates inflammatory responses by suppressing proinflammatory mediators including IL-1$\beta$, TNF-$\alpha$, and intercellular adhesion molecule 1 (ICAM-1) [29]. Notably, RIC shares neuroprotective mechanisms with the ELA-apelin-APJ system, as RIC mitigates neuronal injury via anti-apoptotic, anti-inflammatory, and antioxidant pathways [30]. These findings led us to hypothesize that RIC alleviates inflammation, apoptosis, and reperfusion injury in MCAO rats by modulating the apelinergic system.

The relationship between RIC and the ELA-apelin-APJ system in the field of ischemic stroke remains unexplored. A previous study has demonstrated that in renal I/R injury models, RIC can mitigate ischemia-induced renal damage by modulating apelin expression [31]. Our research has demonstrated that in the MCAO model, after treatment with RIC, the expression of apelin and ELA is increased, the volume of cerebral infarction is reduced, and neuronal damage is alleviated. This indicates that RIC can alleviate brain injury caused by ischemia by regulating the expression of apelin and ELA. Our results further demonstrated that in the MCAO+RIC group, RIC significantly upregulated STAT3 and ELA expression, along with apelin levels, while concurrently downregulating the expression of caspase-3, TNF-$\alpha$, IL-1$\beta$, phosphorylated STAT3 (p-STAT3), HIF-1$\alpha$, and ATF4. These molecular alterations exhibited significant linear correlations. Collectively, these data indicated that RIC ameliorates neuronal injury and attenuates apoptosis and neuroinflammation through coordinated mechanisms, including the correction of ischemic hypoxia (reflected by HIF-1$\alpha$ reduction), alleviation of endoplasmic reticulum stress (indicated by ATF4 decrease), and suppression of the inflammatory response (evidenced by diminished p-STAT3, TNF-$\alpha$, and IL-1$\beta$). It is important to note that our findings suggest that the ELA-apelin-APJ system serves as a key upstream mediator of these multifaceted neuroprotective effects conferred by RIC.

A key limitation of this study is the absence of pharmacological interventions using activators or inhibitors targeting the ELA-apelin-APJ system. This finding precludes definitive conclusions regarding whether RIC alleviates neuronal apoptosis and inflammation in the MCAO-induced ischemia-reperfusion model via a specific pathway or through synergistic interactions of multiple mechanisms. Future studies will incorporate these tools to dissect the precise contributions of individual signaling components within the ELA-apelin-APJ axis and clarify the mechanistic hierarchy underlying RIC-mediated neuroprotection.

The focus of the present study was to explore the neuroprotective effects of RIC and the possible mechanisms underlying its effect after reperfusion injury in ischemic stroke. These findings support the hypothesis that RIC alleviates neuronal apoptosis and inflammation in an MCAO-induced ischemia/reperfusion model by modulating the ELA-apelin-APJ system. These findings suggest that the mechanism by which RIC alleviates ischemic stroke may involve the modulation of the ELA-apelin-APJ system.

RIC, Remote ischemic conditioning; MCAO, middle cerebral artery occlusion; WB, Western blotting; HE, Hematoxylin-eosin; LVO, large vessel occlusion; EVT, Endovascular thrombectomy; HIF-1$\alpha$, hypoxia-inducible factor 1$\alpha$; STAT3, signal transducer and activator of transcription 3; ATF4, activating transcription factor 4; HREs, hypoxia-response elements; I/R, ischemia-reperfusion; SD, Sprague-Dawley; SPF, specific-pathogen-free; NDSs, neurological deficit scores; IL-1$\beta$, interleukin-1$\beta$; RCTs, randomized controlled trials; ER, endoplasmic reticulum.

The data that support the findings of this study are available in the supplementary material of this article.

JS: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—review and editing, Supervision, Project administration. FZ: Conceptualization, Methodology, Validation, Formal analysis, Data curation, Resources, Writing—review and editing, Supervision, Project administration, Funding acquisition. YW: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—original draft, Writing—review and editing, Visualization. XC: Validation, Formal analysis, Investigation, Data curation. ZP: Validation, Formal analysis, Investigation. XW: Validation, Formal analysis, Investigation. SG: Validation, Formal analysis, Investigation. CS: Validation, Formal analysis, Investigation. JR: Data curation. All authors contributed to critical revision of the manuscript for important intellectual content. 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.

All animal experiments received approval from the Institutional Animal Care and Use Committee of Jiangxi Zhonghong Boyuan Biotechnology Co., Ltd. (approval number: LL-202403080006) and conducted in compliance with China’s national laboratory animal regulations: the Regulations on the Administration of Laboratory Animals (State Science and Technology Commission) and Implementation Rules for Medical Laboratory Animal Administration (Ministry of Health). Experimental procedures strictly adhered to the ARRIVE reporting guidelines.

This study was supported by the Taiyuan Bureau of Science and Technology, Science, Technology, and Innovation Program of the National Regional Medical Center (No. 202227).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/JIN39897.