Effect of the peptide on Gen-induced hair cell loss. (A) Schematic illustration of lateral line neuromast hair cells. (B) Overview of the combined application of peptide YSQLENEFDR and Gen on zebrafish. (C) The hair cells were stained with YO-Pro-1 (scale bar = 10 μm). (D) Statistical analysis of the hair cell counts in each group (n = 12 per group). The number in the bar graph indicates the average number of hair cells in lateral line neuromast. Compared with the control group, ### p < 0.001; compared with the Gen-only-treated group, * p < 0.05 and *** p < 0.001. Gen, gentamicin.

Restorative effect of the peptide on defects in the ultrastructure of hair cells. The red arrow indicates the kinocilia of hair cells; the red asterisk indicates the stereocilia of hair cells.

Effects of the peptide on entry of Gen and function of the MET channel in hair cells. (A) Representative fluorescence images of GTTR in hair cells. (B) Statistical analysis of the fluorescence intensities of GTTR in hair cells (n = 10 per group; scale bar = 8 μm). Compared with the Gen-only-treated group, *** p < 0.001. (C) Representative fluorescence images of FM1-43 in hair cells. (D) Statistical analysis of the fluorescence intensities of FM1-43 in hair cells (n = 10 per group; scale bar = 50 μm). Gen, gentamicin; MET, mechanoelectrical transducer; GTTR, Gen–Texas Red.

Inhibition of the peptide on ROS accumulation in hair cells. ROS in hair cells were labeled by fluorescence (white arrows). (A) Zebrafish larva was not treated with Gen or the peptide. (B) Zebrafish larva was treated with only Gen. (C) Zebrafish larva was treated with both Gen and 10 μg/mL peptide. (D) Zebrafish larva was treated with both Gen and 15 μg/mL peptide. (E) Zebrafish larva was treated with both Gen and 20 μg/mL peptide (scale bar = 100 μm). ROS, reactive oxygen species; Gen, gentamicin.

Inhibition of the peptide on apoptosis in hair cells. Apoptotic hair cells were stained with red fluorescence (scale bar = 25 μm).

Effect of the peptide on mPTP in hair cells. (A) The state of mPTP in the hair cells is indicated by calcein (red arrows). When mPTP was closed, calcein was loaded onto the mitochondria, resulting in the presentation of green fluorescence. When mPTP was open, calcein flowed out of the mitochondria into the cytoplasm, resulting in the disappearance of green fluorescence (scale bar = 200 μm). (B) Quantitative assay of the neuromasts exhibiting green fluorescence (n = 10). Compared with the control group, ### p < 0.001; compared with the Gen-only-treated group, ** p < 0.01 and *** p < 0.001. mPTP, mitochondrial permeability transition pore.

Transcriptional alterations of genes. The amount of gene expression is exhibited as the relative expression. Compared with the control group, ## p < 0.01 and ### p < 0.001; compared with the Gen-only-treated group, * p < 0.05, ** p < 0.01, and *** p < 0.001.

The proposed mechanism underlying the protective effect of the peptide from Nac on hair cells. The peptide reduces the uptake of Gen, protects the mitochondria, improves the endogenous antioxidant system to restrain the excessive accumulation of ROS, and then exerts protective effects against AmAn-induced hair cell damage. The structural characteristics of peptides may contribute to this biological process. Nac, Neptunea arthritica cumingii; Gen, gentamicin; ROS, reactive oxygen species; AmAn, aminoglycoside antibiotics.