Exosomes Derived from microRNA-21 Overexpressing Neural Progenitor Cells Prevent Hearing Loss from Ischemia-Reperfusion Injury in Mice via Inhibiting the Inflammatory Process in the Cochlea.

Abstract

Both exosomes derived from neural progenitor cells (NPCs) can suppress inflammation. Whether exosomes derived from miR-21-transfected NPCs (miR-21-Exo) could be utilized to alleviate hearing loss is investigated. NPCs were transfected with lentiviral vectors overexpressing miR-21, and miR-21-Exo was purified. Morphology and exosome membrane markers were examined with nanoparticle tracking analysis, transmission electron microscopy, and Western blot. After incubation with different concentrations of miR-21-Exo, the viability of RAW 264.7 cells and the relative expressions of miR-21 and IL-10 were determined. The ischemia and reperfusion (I/R) model of C57BL/6 J mice was constructed, and the treatment benefit of miR-21-Exo was revealed by the auditory brainstem response (ABR) test. Immunofluorescence staining of caspase-3 and parvalbumin was used to detect apoptosis hair cells in the cochlea, and Western blot was utilized to detect the relative expressions of P53 and inflammatory cytokines in the cochlea. Isolated exosomes were confirmed by the size of 96 ± 25 nm, single membrane, and positive expression of CD9 and Tsg101. Upregulated miR-21 expression was detected in miR-21-transfected NPCs and miR-21-Exo. miR-21-Exo incubation demonstrated no cytotoxicity but upregulated miR-21 and IL-10 expressions in RAW 264.7 cells. The administration of miR-21-Exo inhibited the increased ABR threshold under 8, 16, and 32 kHz frequencies in cochlea-I/R injury mice and diminished the mean fluorescent intensity of caspase-3/parvalbumin. Moreover, miR-21-Exo treatment increased the IL-10 expression and prevented the increased TNF-α and IL-1β expressions in the cochlea of I/R mice both in mRNA and protein levels. Inner ear administration of miR-21-Exo effectively improved hearing damage caused by I/R.