Supplementary file 1_Evaluation of the in vitro otoprotective potential of 4-aminopyridine for cisplatin-induced ototoxicity.docx

BackgroundCisplatin, an effective chemotherapy, is a cause of ototoxicity. It enters inner ear cells through Organic Cation Transporter 2 (OCT2). Recently, we identified another cochlear transporter, Multidrug and Toxin Extrusion Protein 1 (MATE1); it allows cisplatin efflux in renal cells. OCT2 and MATE1 appear to work together in eliminating renal toxins. 4-aminopyridine (4-AP), a known blocker of voltage-dependent K+ channels, blocks OCT2 but not MATE1 in the kidney. We hypothesize that 4-AP could prevent cisplatin from entering cochlear cells, without inhibiting the efflux of cisplatin that may have already entered. This could enhance clearance, mitigating ototoxicity.
ObjectiveDetermine the otoprotective potential of 4-AP in vitro following cisplatin exposure.
MethodsMice cochlear explants were treated with cisplatin (20 μM), with or without 4-AP (500 μM) for 24 h, evaluated for caspase 3/7 activity with Cell Event®, visualized with confocal and phase contrast microscopy. Moreover, HeLa cells and cochlear explants were exposed to cisplatin (75 μM and 30 μM, respectively) with or without 4-AP in the explants and were evaluated for cisplatin-DNA adducts and cleaved caspase-3 immunohistochemistry.
ResultsFor cochlear explants exposed to cisplatin, Cell Event® staining occurred in the supporting cells, medial to the hair cells, and in the organ of Corti. No significant differences were found between cisplatin-exposed explants with or without 4-AP. In HeLa cells, consistent cellular staining for cisplatin-DNA adducts and cleaved caspase-3 was observed. However, this was not seen in cochlear explants, and there was no significant decrease in staining levels with 4-AP co-treatment.
ConclusionsIn this in vitro study, no otoprotective potential of 4-AP against cisplatin-induced ototoxicity was observed. While 4-AP blocks OCT2, it did not decrease cisplatin-DNA adducts in cochlear cells. Further research into OCT2 and MATE1 transport mechanisms is needed.

研究背景：顺铂（Cisplatin）是一种有效的化疗药物，同时也是耳毒性的诱因。它可通过有机阳离子转运体2（Organic Cation Transporter 2, OCT2）进入内耳细胞。本团队近期鉴定出另一种耳蜗转运体——多药及毒素外排蛋白1（Multidrug and Toxin Extrusion Protein 1, MATE1），该蛋白可在肾细胞中介导顺铂外排。OCT2与MATE1似乎可协同清除肾脏毒素。4-氨基吡啶（4-aminopyridine, 4-AP）是一种已知的电压门控钾离子通道阻滞剂，在肾脏中可阻断OCT2，但不影响MATE1的功能。我们提出假说：4-AP可阻止顺铂进入耳蜗细胞，同时不会抑制已进入细胞的顺铂外排，这或许能够增强顺铂的清除效率，从而减轻耳毒性。
研究目的：明确4-AP在顺铂暴露后的体外模型中发挥耳保护作用的潜力。
实验方法：将小鼠耳蜗外植体分别以20 μM顺铂单独处理，或联合500 μM 4-AP处理24小时，采用Cell Event®检测半胱天冬酶3/7（caspase 3/7）活性，通过共聚焦显微镜与相差显微镜成像进行观察。此外，分别用75 μM顺铂处理HeLa细胞、30 μM顺铂处理耳蜗外植体，分别联合或不联合4-AP，检测顺铂-DNA加合物（cisplatin-DNA adducts），并通过免疫组化检测剪切型半胱天冬酶-3（cleaved caspase-3）的表达。
实验结果：在顺铂暴露的耳蜗外植体中，Cell Event®染色见于毛细胞内侧的支持细胞以及柯蒂氏器（organ of Corti）。无论是否联合4-AP处理，顺铂暴露的外植体之间均未观察到显著差异。在HeLa细胞中，可观察到顺铂-DNA加合物与剪切型caspase-3的一致性细胞染色，但此类染色未出现在耳蜗外植体中，且4-AP共处理未使染色水平出现显著降低。
研究结论：本项体外研究未观察到4-AP对顺铂诱导的耳毒性具有耳保护潜力。尽管4-AP可阻断OCT2，但并未降低耳蜗细胞中的顺铂-DNA加合物水平。未来仍需针对OCT2与MATE1的转运机制开展进一步研究。