Enhancing the Neuroprotective Synergy of Caffeine and ACSL4 Inhibition via Exosome-Mediated siRNA Delivery

Background: Ischemic stroke, a predominant cause of neurological impairment, involves complex ischemia/reperfusion (I/R) injury where ferroptosis—an iron-dependent cell death driven by lipid peroxidation—plays a critical role. The enzyme ACSL4 is a key facilitator of this process. While caffeine demonstrates neuroprotective potential by modulating adenosine receptors and ACSL4 activity, therapeutic targeting of ACSL4 via siRNA is hampered by the inherent instability and poor delivery efficiency of naked oligonucleotides. Methods: An in vitro hypoxia/reoxygenation (H/R) model was established using HT-22 neuronal cells. Bone marrow-derived mesenchymal stem cell (BMSC) exosomes were isolated, characterized via transmission electron microscopy and western blotting for specific markers (CD9, CD63, CD81), and loaded with siRNA targeting ACSL4 (exo-si-ACSL4). Cellular uptake of fluorescently labeled exosomes was confirmed. HT-22 cells subjected to H/R were treated with caffeine (500 µM), transfected with conventional si-ACSL4, or incubated with exo-si-ACSL4, both alone and in combination with caffeine. Assessments included cell viability (CCK-8), ACSL4 expression (RT-qPCR, western blot), inflammatory cytokines (ELISA), and ferroptosis indicators (iron, reactive oxygen species, malondialdehyde, glutathione, mitochondrial membrane potential). Results: BMSC-derived exosomes exhibited typical morphological and protein marker profiles and were effectively internalized by HT-22 cells. Exo-si-ACSL4 achieved superior transfection efficiency, reducing ACSL4 mRNA and protein levels by approximately 89% and 87%, respectively, which surpassed the knockdown efficacy of standard siRNA transfection. The combination of exo-si-ACSL4 and caffeine yielded a synergistic protective effect. This combined treatment significantly elevated cell viability, attenuated the release of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and robustly suppressed ferroptosis hallmarks compared to caffeine or either siRNA delivery method alone. Inhibition was evidenced by reduced lipid peroxidation, decreased intracellular iron and ROS, elevated GSH, and preserved mitochondrial membrane potential. Conclusions: This work substantiates that exosome-mediated delivery of si-ACSL4 markedly enhances the precision and efficacy of gene silencing. The synergistic interaction between this targeted molecular intervention and caffeine administration confers pronounced protection against H/R-induced neuronal damage, primarily through the potent attenuation of ferroptosis and inflammation. These findings propose a novel combinatory strategy, integrating a pharmacological agent with an advanced biomimetic delivery system, to overcome central limitations in current nucleic acid therapy for ischemic stroke.

Keywords

Cerebral ischemia/reperfusion injury; ferroptosis; acyl-CoA synthetase long-chain family member 4; extracellular vesicles; gene silencing, neuroprotection; combination therapy

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How to cite this paper

Enhancing the Neuroprotective Synergy of Caffeine and ACSL4 Inhibition via Exosome-Mediated siRNA Delivery

How to cite this paper: King Yiu (Kevin) Liao, Jun Zhou. (2025) Enhancing the Neuroprotective Synergy of Caffeine and ACSL4 Inhibition via Exosome-Mediated siRNA Delivery. Scientific Access, 1(2), 82-90.

DOI: http://dx.doi.org/10.26855/sa.2025.09.003

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Enhancing the Neuroprotective Synergy of Caffeine and ACSL4 Inhibition via Exosome-Mediated siRNA Delivery

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