Momordica charantia small extracellular vesicles mitigate neuronal ferroptosis by inhibition of GPX4 ubiquitination in ischemic stroke

Abstract

Background

Ischemic stroke (IS) is a prevalent cerebrovascular disorder associated with high rates of mortality and long-term disability. Neuronal ferroptosis, characterized by lipid peroxidation, plays a pivotal role in the pathogenesis of IS-related neurological dysfunction.

Purpose

This study aimed to elucidate the neuroprotective role and mechanisms of small extracellular vesicles (sEVs) derived from Momordica charantia (MC-sEVs) in alleviating neuronal ferroptosis after IS.

Methods

MC-sEVs were isolated using gradient centrifugation and ultracentrifugation and characterized by particle size analysis, morphology observation, and exosomal marker proteins. Small RNA sequencing and lipidomic analysis were also performed to analyze the components of MC-sEVs. Using behavioral tests and a rat model of middle cerebral artery occlusion, the neuroprotective and anti-ferroptosis role of MC-sEVs was assessed. The oxygen-glucose deprivation/reoxygenation (OGD/R) induced HT22 cell model was used to evaluate the anti-ferroptosis effect of MC-sEVs in vitro. Utilizing bioinformatics analysis and molecular docking prediction, Momordica charantia-derived miR-5813b and its downstream target gene, TRIM62, were identified, which was further confirmed using a dual-luciferase reporter gene assay. TRIM62-mediated GPX4 ubiquitination was detected by co-immunoprecipitation. The miR5813b mimic and AAV-hsyn-EGFP-miR5813b were both constructed, and their neuroprotective roles were observed in vitro and in vivo. The biosafety of intravenous injection of MC-sEVs was also evaluated.

Results

we characterized MC-sEVs which resemble mammalian EVs and are enriched in small non-coding RNAs and lipids. Systemic administration of MC-sEVs significantly attenuated neuronal ferroptosis and promoted neurological recovery in the rat model of transient middle cerebral artery occlusion. Mechanistically, we identified miR-5813b, a plant-specific microRNA encapsulated within MC-sEVs, as a direct regulator of TRIM62, an E3 ubiquitin ligase that predominantly facilitates K48-linked ubiquitination of GPX4. Knockdown of TRIM62 or transfection with miR-5813b mimics markedly inhibited neuronal ferroptosis in vitro, while inhibition of miR-5813b abrogated the anti-ferroptotic effects of MC-sEVs. Furthermore, overexpression of AAV-hsyn-miR-5813b enhanced post-stroke neurological function in vivo. Importantly, intravenous administration of MC-sEVs demonstrated excellent biosafety.

Conclusions

These findings reveal that MC-sEVs derived miR-5813b effectively modulated GPX4 ubiquitination to counteract neuronal ferroptosis after IS. This study highlights MC-sEVs as a promising and biocompatible therapeutic platform for the treatment of IS.