Mesenchymal stem cell-derived exosomes ameliorate gentamicin-induced vestibular hair cell injury by regulating the SNARE pathway and enhancing autophagy.

OBJECTIVE: To investigate the delivery efficiency of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-EXOs) via intratympanic injection into vestibular end organs, evaluate their protective effects against gentamicin-induced vestibular dysfunction and hearing loss on gentamicin-induced vestibular dysfunction and hearing loss, and explore their regulatory mechanisms on hair cell apoptosis and autophagy. METHODS: Exosome characteristics were identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot. PKH26 labeling was used to trace their distribution in the vestibule. SD rats were randomly divided into four groups: control group, gentamicin group (GEN group), gentamicin + exosome group (GEN + EXO group), and gentamicin + dexamethasone group (GEN + DEX group). On day 6 after administration, vestibular function was assessed via open-field test and beam balance test. On day 7, high-frequency hearing (32 kHz) was detected by auditory brainstem response (ABR). The quantity and structural changes of hair cells were analyzed by immunofluorescence staining and scanning electron microscopy. Proteomics was used to analyze differentially expressed proteins in vestibular tissues treated with dexamethasone or hucMSC-EXOs. The regulatory effects on Caspase-3 (apoptosis) and LC3 (autophagy) were validated by immunofluorescence. RESULTS: hucMSC-EXOs administered via intratympanic injection were found to target the utricle, saccule, and crista ampullaris. Behavioral studies showed that the GEN + EXO group exhibited significant suppression of gentamicin-induced reduction in total movement distance (p < 0.05) and movement speed (p < 0.05, superior to the GEN + DEX group), with a 60.5% reduction in beam balance test passage time (p < 0.05). ABR results revealed that the auditory threshold at 32 kHz in the GEN + EXO group was 18.3 dB SPL lower than that in the injury group (p < 0.01), with no statistical difference compared to the GEN + DEX group. Hair cell counting showed significant protective effects of exosomes in reducing hair cell loss in the utricular striola (+ 25%), saccular striola (+ 44%), and central crista ampullaris (+ 44%), with partial regions outperforming the GEN + DEX group (e.g., 109% repair rate in the saccular striola). Scanning electron microscopy confirmed that exosomes reduced hair cell cilia loss and structural vacuolization. Proteomics showed significant activation of the complement and coagulation cascades in the GEN group, while the GEN + EXO group enhanced autophagy via enriching the vesicle transport-related SNARE interaction pathway and improved energy metabolism and oxidative stress by regulating metabolic pathways. Immunofluorescence demonstrated that exosomes significantly reduced the expression of the apoptosis marker Caspase-3 (p < 0.01) and enhanced the activity of the autophagy marker LC3 (p < 0.05). CONCLUSIONS: hucMSC-EXOs delivered via intratympanic injection effectively target vestibular sensory epithelia, significantly suppressing gentamicin-induced balance dysfunction and high-frequency hearing loss, with superior efficacy in partial indicators (e.g., movement speed, saccular hair cell repair) compared to dexamethasone. hucMSC-EXOs exert protective effects through a dual mechanism of "anti-apoptosis and pro-autophagy," providing a novel therapeutic strategy for vestibular hair cell injury.