Abstract: Objective To investigate the mechanism of prevention and treatment of presbycusis, human umbilical cord mesenchymal stem cells(hUC-MSCs)were used to delay the aging of mouse cochlea. Methods Stable human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)were isolated and cultured, followed by breeding C57BL/6J male mice of corresponding age. In the control group, mice received 0.2 mL of normal saline via tail vein injection using a microsyringeevery month starting at 3 months of age. The MSCs group received injections of fourth-generation hUC-MSCs at a concentration of 5×10⁶ cells/mL via tail vein every month starting at 3 months of age. Auditory brainstem response(ABR)threshold tests were performed at 3, 6, 10,17, and 21 months of age, with threshold data recorded. At 6, 10, and 21 months, the Corti's apparatus in the inner ear was dissected, and the basilar membrane was immunofluorescently stained. Morphological changes in cochlear outer hair cells and the number of zonal synapses were observed and documented using laser confocal microscopy. Serum levels of oxidative stress markers such as MDA, SOD, and GSH were measured and analyzed. Results The hUC-MSCs isolated and cultured in this study exhibited typical elongated, spindle-shaped adhesion growth with stable morphology after passage; flow cytometry analysis met the identification criteria for mesenchymal stem cells. Audiological results demonstrated progressive hearing decline in mice with increasing age, with high-frequency hearing impairment occurring earliest and gradually progressing to middle and low-frequency ranges. The ABR hearing thresholds in the MSCs group were significantly lower than those in the control group across all age groups(P<0.01), with significant differences observed at 32 kHz(6 months), as well as at Click frequency and 8/16 kHz(10 and 21 months, respectively)(P<0.05). Cochlear morphological findings revealed significantly lower total loss rates of outer hair cells in the MSCs group compared to the control group across all age groups(P<0.01), with significant differences observed in the basal, apical, and apical-medial turns at 6, 10, and 21 months(P<0.05). Synaptic banding analysis demonstrated that the MSCs group exhibited a higher density of synapses in the cochlear region at 16-22 kHz compared to the control group at 6 months(P<0.05), and increased synaptic density at medium-high frequencies of 4 kHz and 22 kHz at 21 months(P<0.05). Serum oxidative stress markers revealed no significant differences between the two groups at 6 months for MDA, SOD, or GSH levels; however, at 10 and 21 months, the hUC-MSCs group exhibited significantly lower malondialdehyde levels and significantly higher levels of superoxide dismutase and reduced glutathione(P<0.05). Conclusion Human umbilical cord mesenchymal stem cells(hUC-MSCs)can reduce the loss of outer hair cells and ribbon synapses in the inner ear of mice, as well as delay the aging of the basilar membrane in the cochlea. This suggests that hUC-MSCs effectively mitigate age-related hearing loss in mice and exhibit therapeutic potential for presbycusis.

Key words: Mesenchymal stem cells, Presbycusis, Aging, Hearing protection, Cochlear hair cells