MitoQ as novel agent for protecting against aminoglycoside ototoxicity

描述(申请人提供)：氨基糖苷类(AG)，如庆大霉素，是全球普遍使用的抗生素，因为它们相对便宜，对严重的细菌感染非常有效，而且受处方销售的限制较少。不幸的是，这些药物即使在临床指南内使用，也会导致听力损失和平衡障碍。氨基糖苷类药物引起的听力损失(AIHL)严重影响了患者的生活质量，这可能是患者违背医生建议停止抗生素治疗的主要原因。开发降低AIHL的治疗方法不仅可以保护听力，还可以提高患者对导致听力损失的抗生素治疗的依从性。活性氧(ROS)的产生是AIHL的主要机制，抗氧化剂可以减少这些缺陷。由于线粒体是ROS的主要来源，我们推测针对线粒体的抗氧化剂在预防AIHL方面更有效。在我们的初步研究中，我们发现了一种新型的线粒体靶向抗氧化剂CoQ10的衍生物MitoQ，它可以减弱庆大霉素诱导的HEI-OC1细胞凋亡，并且不影响庆大霉素的抗生素疗效。我们还发现，口服MitoQ可以减轻庆大霉素诱导的豚鼠模型耳蜗毛细胞损伤和听力损失。在拟议的研究中，我们将进一步研究MitoQ在预防AG诱导的豚鼠细胞死亡和听力损失方面的有效性，并将确定是否选择性抑制 线粒体ROS加MitoQ可减轻庆大霉素引起的氧化损伤和线粒体功能障碍。听觉脑干反应(ABR)和失真产物耳声发射(DPOAEs)将被用来评估接受庆大霉素治疗的豚鼠的听觉功能，包括补充和不补充MitoQ。生化评估将包括测量凋亡标记物(bax、cytc、caspase3、caspase9)和caspase非依赖性途径(AIF、endog、PARP1)的基因和/或蛋白表达，以及使用Western blotting、qPCR、免疫组织化学和商业试剂盒测量一些氧化标记物的积累，包括MDA或4-HNE、蛋白质羰基和DNA损伤。荧光和扫描电子显微镜也将被用来评估药物诱导的耳蜗毛细胞的破坏，以及MitoQ是否可以减轻这种损害。线粒体分析将包括耳蜗中抗氧化防御的基因和蛋白表达(例如，MnSOD)，以及测量线粒体蛋白质的水平(例如，ND6C、CytC1)。

DESCRIPTION (provided by applicant): Aminoglycosides (AGs) such as gentamicin are commonly used antibiotics worldwide because they are relatively less expensive, highly effective against serious bacterial infections and less regulated by prescription only sale. Unfortunately, these drugs can cause both hearing loss and balance disorders, even when used within clinical guidelines. Aminoglycoside-induced hearing loss (AIHL) significantly compromises quality of life for patients, and it can be a primary reason patients discontinue antibiotic treatment against medical advice. Development of therapies that reduce AIHL would not only protect hearing, but might also improve patient adherence with antibiotic treatments that result in hearing loss. Generation of reactive oxygen species (ROS) is a primary mechanism involved in AIHL, and that antioxidants reduce these deficits. Since mitochondria are the major source of ROS, we hypothesize that antioxidants targeted to mitochondria are more effective in preventing AIHL. In our preliminary study, we showed that MitoQ, a novel mitochondria-targeted antioxidant derivative of CoQ10, attenuates gentamicin- induced apoptosis in HEI-OC1 cells and does not compromise gentamicin antibiotic efficacy. We also found that oral supplementation of MitoQ attenuated the gentamicin-induced cochlear hair cell damage and hearing loss in a guinea pig model. In the proposed study, we will further investigate the efficacy of MitoQ in preventing AG-induced cell death and hearing loss in guinea pigs and we will determine if selective inhibition of mitochondrial ROS with MitoQ will attenuate oxidative damage and mitochondrial dysfunction induced by gentamicin treatments. Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAEs) will be used to assess auditory function of guinea pigs treated with gentamicin, with and without MitoQ supplementation. Biochemical assessments will include measuring gene and/or protein expression of markers of apoptotic (Bax, cyt c, caspase 3, caspase 9) and caspase- independent (AIF, endoG, PARP1) pathways, as well as measuring the accumulation of a number of oxidative markers including MDA or 4-HNE, protein carbonyls, and DNA damage using Western blotting, qPCR, immunohistochemistry, and commercially available kits. Fluorescent and scanning electron microscopy will also be used to assess drug-induced destruction of cochlear hair cells and if MitoQ attenuates this damage. Mitochondrial assays will include gene and protein expression of antioxidant defenses in the cochlea (eg, MnSOD) and measuring levels of mitochondrial proteins (e.g., ND6C, cyt c1).