Fig 1: NOS3 reduces cisplatin‐induced ototoxicity in circadian rhythm‐disrupted mice. (A) Schematic diagram of the experimental design combining cisplatin chemotherapy with NOS3 treatment in mice with disrupted circadian rhythms. (B and C) Post‐treatment auditory brainstem response (ABR) and distortion‐product otoacoustic emission (DPOAE) thresholds in the Control, Cisplatin, and Cisplatin + NOS3 groups. (D) Representative immunofluorescence images of the apical, middle, and basal turns of the cochlea after treatment, stained for Myosin 7a (red, hair cells), Neurofilament (green, nerve fibers), and DAPI (blue, nuclei). Scale bars = 20 μm. (E) Representative immunofluorescence images of inner hair cell ribbon‐synapse regions in the three cochlear turns, labeled for Ctbp2 (red, presynaptic ribbons), Homer1 (green, postsynaptic densities), and DAPI (blue, nuclei). Scale bars = 10 μm. (F–I) Quantitative analysis of inner hair cells, outer hair cells, auditory nerve‐fiber terminals, and intact ribbon‐synapse density in the apical, middle, and basal turns after treatment. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Control (Light–Dark) group on all frequencies/turns (corrected for multiple comparisons); two‐way ANOVA with Bonferroni post hoc test, n = 6.
Fig 2: Circadian‐related Serotonin/Melatonin level Modulates Cisplatin Ototoxicity Susceptibility depended on NOS3–NO pathway. Circadian disruption upsets melatonin/serotonin balance in the inner ear. Melatonin protects against cisplatin‐induced ototoxicity, whereas serotonin worsens damage. This opposing regulation acts through NOS3 to modulate NO signaling and ferroptosis. Consequently, the melatonin/serotonin balance determines susceptibility to sensorineural hearing loss, revealing a mechanistic link between lifestyle‐related hormone dysregulation and cochlear vulnerability.
Fig 3: CREB acts downstream of the melatonin/serotonin and NOS3–NO pathways to suppress cisplatin‐induced ferroptosis. (A) Heatmap showing the phosphorylation levels of 55 proteins in cochlear explants comparing the NOS3 + Cisplatin group vs. the Cisplatin‐alone group (green: higher phosphorylation in NOS3 + Cisplatin; red: lower phosphorylation). (B) Schematic diagram of the NOS3/NO/cGMP/PKG signaling cascade. (C) Representative immunofluorescence images of cochlear explants after 48 h of treatment with cisplatin, NOS3 agonist, CW‐008, or 666‐15. Parvalbumin (green) labels hair cells; transferrin receptor (red) marks ferroptotic cells. Scale bars = 40 μm. (D) Quantitative analysis of Parvalbumin‐positive hair cells and Parvalbumin/transferrin receptor double‐positive ferroptotic cells based on the images in panel (C). Data are presented as mean ± SEM, n = 6.
Fig 4: Melatonin and serotonin mediate the regulation of cisplatin‐induced ototoxicity through the NOS3–NO pathway. (A) Venn diagram showing the overlap between differentially expressed genes in cochlear explants after cisplatin injury and high‐affinity target proteins predicted for melatonin and serotonin using SwissTargetPrediction. Nine cisplatin‐downregulated genes, including NOS3, were identified as potential binding targets for both ligands. (B) AutoDock modeling of the high‐affinity binding sites and binding affinities of melatonin (red) and serotonin (yellow) with the NOS3 protein. (C) qPCR analysis of nitric oxide (NO) signaling pathway‐related factors in cochlear explants after 24 h of culture in the indicated groups. Data are shown as Z‐scores. (D) Representative immunofluorescence images of the middle turn of cochlear basilar membrane explants cultured for 48 h. Parvalbumin (green) labels hair cells, showing colocalization with NOS3 (red) and active (phosphorylated) NOS3 (gray). Scale bars = 30 μm. (E) Tissue NO content in cochlear explants after 48 h of culture in the indicated groups. (F) Quantitative analysis of the mean fluorescence intensity of NOS3 and active (phosphorylated) NOS3 within the hair‐cell region from panel (D). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Control group; ### p < 0.001, #### p < 0.0001 vs. Cisplatin group; two‐way ANOVA with Bonferroni post hoc test, n = 6.
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