Apigenin protects against cisplatin-induced ototoxicity. (A) Confocal image of a 5 dpf Tg (Brn3c:mGFP) zebrafish larva. The green fluorescent point circled by the yellow rectangle represents the anterior lateral line system (aLL), while the green fluorescent point circled by the red rectangle represents the posterior lateral line system (pLL). A schematic lateral view of a 5 dpf neuromast illustrating different cell types is shown below. (B) Schematic diagram illustrating the experimental workflow of zebrafish. (C) Live confocal imaging of Tg (Brn3c:mGFP) HCs (green) in L1 neuromast from larvae at 5 dpf. Zebrafish were exposed to varying concentrations of cisplatin with and without apigenin. Control animals received the vehicle alone (DMSO). (D) Quantification of the number of HCs in L1 neuromast after different treatments, represented as mean ± SEM (n = 10). (E) Peak velocity and (F) swimming distance of 5 dpf zebrafish larvae, indicative of auditory function, assessed by startle response. (G) Staining of hair cell damage with 2-(4-[dimethylamino]styryl)-N-ethylpyridinium iodide (DASPEI) showed that treatment with cisplatin decreased the number of hair cells in L1 neuromast. However, the co-treatment with 100 ?M apigenin protected reducing number of hair cell staining with DASPEI. (H) Average DASPEI stained area in L1 neuromast in different groups were shown NM: neuromast; API: apigenin; CP: cisplatin. Statistical significance indicated as ?, ??, ???and ???? above the bars (????P < 0.0001, ???P < 0.001, ??P < 0.01, ?P < 0.05). Scale bar equals 25 ?m. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Apigenin protects against cisplatin-induced ROS accumulation and cell death in neuromasts. (A) TUNEL assay (red) and corresponding DAPI staining (blue) were performed in zebrafish in the presence/absence of apigenin (100 ?M) and cisplatin (200 ?M). (B) Quantification of TUNEL-positive cells per neuromast for each treatment, presented as mean ± SEM. (C) ROS generation detected by MitoSOX staining, followed by fluorescence microscopy imaging. (D) Quantification of MitoSOX fluorescent intensity for each treatment, presented as mean ± SEM. Statistical significance indicated as ???P < 0.001, ????P < 0.0001. The scale bar equals 25 ?m. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Effect of apigenin on transcriptome profiles in response to cisplatin stimulation. (A) Venn diagram illustrating specific and overlapping gene expression among control, cisplatin, and apigenin + cisplatin groups. (B-C) Volcano plots depicting DEGs in various treatment groups, with each gene marked in red or blue indicating significant upregulation or downregulation, respectively. (D) GO analysis highlighting differences in DEGs between Control and cisplatin groups. (E) Top 20 KEGG pathways significantly affected after cisplatin treatment. (F) GO analysis comparing apigenin + cisplatin with cisplatin alone in DEGs. (H) Top 20 KEGG pathways significantly influenced following apigenin treatment. CP: cisplatin; API: apigenin; BP: Biological Process; CC: Cellular Component; MF: Molecular Function. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Apigenin mitigates activation of the apoptotic-related pathway. Real-time PCR analysis of the mRNA levels for gadd45ba, serpine1, pmaip1, nfkbiaa, cdkn1a, fosab, gadd45bb, and diabloa in zebrafish larvae treated with cisplatin or apigenin + cisplatin. Results were normalized to GAPDH expression and were presented as mean ± SEM from three independent experiments. CP: cisplatin; API: apigenin ???P < 0.001, ??P < 0.01, ?P < 0.05.

Overview of the molecular mechanisms of apigenin against cisplatin.