The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

The inner ear, including the cochlea, used to be regarded as an immune-privileged site because of its immunologically isolated environment caused by the blood-labyrinthine barrier. Cochlear resident macrophages, which originate from the yolk sac or fetal liver during the embryonic stage and are maintained after birth, are distributed throughout various regions of the cochlear duct. Intriguingly, these cells are absent in the organ of Corti, where hair cells (HCs) and supporting cells (SCs) are located, except for a limited number of ionized calcium-binding adapter molecule 1 (Iba1)-positive cells. Instead, SCs exert glial functions varying from a quiescent to an emergency state. Notably, SCs acquire the nature of macrophages and begin to secrete inflammatory cytokines during viral infection in the organ of Corti, which is ostensibly unprotected owing to the lack of general resident macrophages. This review provides an overview of both positive and negative functions of SCs enabled to acquire macrophage phenotypes upon viral infection focusing on the signaling pathways that regulate these functions. The former function protects HCs from viral infection by inducting type I interferons, and the latter function induces HC death by necroptosis, leading to sensorineural hearing loss. Thus, SCs play contradictory roles as immune cells with acquired macrophage phenotypes; thereby, they are favorable and unfavorable to HCs, which play a pivotal role in hearing function.

Considerable evidence of reactive oxygen species (ROS) involvement in cochlear hair cell (HC) loss, leading to acquired sensorineural hearing loss (SNHL), were reported. Cochlear synaptopathy between HCs and spiral ganglion neurons has been gathering attention as a cochlear HC loss precursor not detectable by normal auditory evaluation. However, the molecular mechanisms linking ROS with HC loss, as well as the relationship between ROS and cochlear synaptopathy have not been elucidated. Here, we examined these linkages using NOX4-TG mice, which constitutively produce ROS without stimulation. mRNA levels of Piccolo 1, a major component of the synaptic ribbon (a specialized structure surrounded by synaptic vesicles in HCs), were decreased in postnatal day 6 NOX4-TG mice cochleae compared to those in WT mice; they were also decreased by noise exposure in 2-week-old WT cochleae. As noise exposure induces ROS production, this suggests that the synaptic ribbon is a target of ROS. The level of CtBP2, another synaptic ribbon component, was significantly lower in NOX4-TG cochleae of 1-month-old and 4-month-old mice compared to that in WT mice, although no significant differences were noted at 1.5- and 2-months. The decrease in CtBP2 plateaued in 4-month-old NOX4-TG, while it gradually decreased from 1 to 6 months in WT mice. Furthermore, CtBP2 level in 2-month-old NOX4-TG mice decreased significantly after exposure to cisplatin and noise compared to that in WT mice. These findings suggest that ROS lead to developmental delays and early degeneration of synaptic ribbons, which could be potential targets for novel therapeutics for ROS-induced SNHL.

Age-related hearing loss (ARHL) is associated with hair cell apoptosis, but the underlying mechanism of hair cell apoptosis remains unclear. Here, we investigated the expression profiles of long noncoding RNAs (lncRNAs) and mRNAs in an ARHL model created with C57BL/6 J mice using RNA sequencing and found that the expression of several lncRNAs was significantly correlated with apoptosis-associated mRNAs in the cochlear tissues of old mice compared to young mice. We found that lncRNA Mirg was upregulated in the cochlear tissues of old mice compared to young mice and its overexpression promoted apoptosis in House Ear Institute-Organ of Corti 1 (HEI-OC1). H2O2-induced oxidative stress increased HEI-OC1 cell apoptosis by upregulating lncRNA Mirg. Furthermore, the expression of lncRNA Mirg and Foxp1 showed the highest correlation coefficient in the cochlear tissues of old mice, and lncRNA Mirg promoted HEI-OC1 cell apoptosis by increasing Foxp1 expression. In conclusion, our findings suggest that lncRNA Mirg expression correlates with cell apoptosis-associated mRNAs in the ARHL model created using C57BL/6 J mice and that oxidative stress-induced lncRNA Mirg promotes HEI-OC1 cell apoptosis by increasing Foxp1 expression. These data suggest the potential therapeutic significance of targeting lncRNA Mirg/Foxp1 signaling in ARHL.

The age-related hearing loss allele (Cdh23ahl) of the cadherin 23 gene leads to a more severe hearing loss phenotype through additive effects with risk alleles for hearing loss. In this study, we genome edited the Cdh23ahl allele to the wild-type Cdh23+ allele in outbred ICR mice and inbred NOD/Shi mice established from ICR mice and investigated their effects on hearing phenotypes. Several hearing tests confirmed that ICR mice developed early onset high-frequency hearing loss and exhibited individual differences in hearing loss onset times. Severe loss of cochlear hair cells was also detected in the high-frequency areas in ICR mice. These phenotypes were rescued by genome editing the Cdh23ahl allele to Cdh23+, suggesting that abnormal hearing phenotypes develop because of the interaction of the Cdh23ahl and risk alleles in the genetic background of ICR mice. NOD/Shi mice developed more severe hearing loss and hair cell degeneration than ICR mice. Hearing loss was detected at 1 month old. Hair cell loss, including degeneration of cell bodies and stereocilia, was observed in all regions of the cochlea in NOD/Shi mice. Although these phenotypes were partially rescued by genome editing to the Cdh23+ allele, the phenotypes associated with high-frequency hearing were mostly unrecovered in NOD/Shi mice. These results strongly suggest that the genetic background of NOD/Shi mice contain a potential risk allele for the acceleration of early onset high-frequency hearing loss.

Noise-induced hearing loss (NIHL) is the second most common cause of sensorineural hearing loss, after age-related hearing loss, and affects approximately 5% of the world\'s population. NIHL is associated with substantial physical, mental, social, and economic impacts at the patient and societal levels. Stress and social isolation in patients\' workplace and personal lives contribute to quality-of-life decrements which may often go undetected. The pathophysiology of NIHL is multifactorial and complex, encompassing genetic and environmental factors with substantial occupational contributions. The diagnosis and screening of NIHL are conducted by reviewing a patient\'s history of noise exposure, audiograms, speech-in-noise test results, and measurements of distortion product otoacoustic emissions and auditory brainstem response. Essential aspects of decreasing the burden of NIHL are prevention and early detection, such as implementation of educational and screening programs in routine primary care and specialty clinics. Additionally, current research on the pharmacological treatment of NIHL includes anti-inflammatory, antioxidant, anti-excitatory, and anti-apoptotic agents. Although there have been substantial advances in understanding the pathophysiology of NIHL, there remain low levels of evidence for effective pharmacotherapeutic interventions. Future directions should include personalized prevention and targeted treatment strategies based on a holistic view of an individual\'s occupation, genetics, and pathology.

Teicoplanin is a glycopeptide antibiotic used to treat severe staphylococcal infections. It has been claimed that teicoplanin possesses ototoxic potential, although its toxic effects on cochlear hair cells (HCs) remain unknown. The TP53-induced glycolysis and apoptosis regulator (TIGAR) plays a crucial role in promoting cell survival. Prior research has demonstrated that TIGAR protects spiral ganglion neurons against cisplatin damage. However, the significance of TIGAR in damage to mammalian HCs has not yet been investigated. In this study, firstly, we discovered that teicoplanin caused dose-dependent cell death in vitro in both HEI-OC1 cells and cochlear HCs. Next, we discovered that HCs and HEI-OC1 cells treated with teicoplanin exhibited a dramatically decrease in TIGAR expression. To investigate the involvement of TIGAR in inner ear injury caused by teicoplanin, the expression of TIGAR was either upregulated via recombinant adenovirus or downregulated by shRNA in HEI-OC1 cells. Overexpression of TIGAR increased cell viability, decreased apoptosis, and decreased intracellular reactive oxygen species (ROS) level, whereas downregulation of TIGAR decreased cell viability, exacerbated apoptosis, and elevated ROS level following teicoplanin injury. Finally, antioxidant therapy with N-acetyl-L-cysteine decreased ROS level, prevented cell death, and restored p38/phosphorylation-p38 expression levels in HEI-OC1 cells injured by teicoplanin. This study demonstrates that TIGAR may be a promising novel target for the prevention of teicoplanin-induced ototoxicity.

Cisplatin, which is effective for the treatment of solid tumors, also can induce cochlear hair cell damage. Therefore, this study was intended to explore how Hippo/YAP signaling pathway affects the cochlear hair cell injury by regulating ferroptosis. After cisplatin induction, or LAT1-IN-1 (YAP activator) and verteporfin (YAP inhibitor) treatment or transfection, the viability of HEI-OC1 cells was detected by cell counting kit-8 (CCK-8) assay. The iron level and the levels of oxidative stress markers (ROS, MDA and 4-HNE) were analyzed by iron assay kit, reactive oxygen species (ROS), malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) assay kits, respectively. The expression of ferritin light chain (FTL) in HEI-OC1 cells was detected by immunofluorescence and protein expressions of yes associated protein (YAP,) phosphorylated YAP (p-YAP), transferrin receptor (TFRC), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) in HEI-OC1 cells were detected by western blot. The transcription of FTL and TFRC by YAP1 was verified by dual-luciferase reporter assay. The transfection efficiency of small interfering RNA (si-RNA) specific to FTL (siRNA-FTL) and TFRC (siRNA-TFRC) was confirmed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). As a result, cisplatin inhibited the viability of HEI-OC1 cells by increasing free Fe2+ level and decreasing FTL level. LAT1-IN-1 promoted the viability of cisplatin-induced HEI-OC1 cells by suppressing oxidative stress level, free Fe2+ level, ferroptosis and increasing FTL level, while the effect of verteporfin was the opposite. YAP1 transcriptionally regulated the expression of FTL and TFRC. Inhibition of FTL suppressed the viability of cisplatin-induced HEI-OC1 cells by increasing oxidative stress level, free Fe2+ level, ferroptosis and decreasing FTL level, while the effect of TFRC inhibition was the opposite. In conclusion, YAP1 ameliorated cochlear hair cell injury by upregulating FTL and TFRC to suppress ferroptosis.

This study aimed to investigate the protective effects of PPARγ/CPT-1 regulation on cisplatin-induced cochlear hair cell injury. The viability, apoptosis and mitochondrial membrane potential of cisplatin-induced HEI-OC1 cells were determined by CCK-8 assay, TUNEL and JC-1 staining, respectively. The oxidative stress and lipid metabolism were detected by the assay kits of MDA, ROS, SOD, CAT, TG and FFA. The transfection efficiency of overexpression (OV)-PPARG and OV-CPT1A was examined by RT-qPCR and the expressions of apoptosis- and lipid metabolism-related proteins were detected by western blot. As a result, cisplatin with varying concentrations (5, 10, 30 μM) suppressed the viability, promoted the apoptosis and hindered the mitochondrial function of HEI-OC1 cells, accompanied with up-regulated expressions of Bax and cleaved caspase-3 and down-regulated expression of Bcl-2. The oxidative stress was aggravated and lipid metabolism was inhibited by cisplatin (5, 10, 30 μM) induction, evidenced by the increased levels of MDA, ROS, TG, FFA and the decreased levels of SOD and CAT. Overexpression of PPARG or CPT1A could improve the viability, mitochondrial function, lipid metabolism and suppress the oxidative stress and apoptosis of cisplatin-induced HEI-OC1 cells. In conclusion, up-regulation of PPARG or CPT1A ameliorated cochlear hair cell injury by improving cellular lipid metabolism and inhibiting oxidative stress.

Substantial evidence suggests that pyroptosis is involved in renal, cerebral, and myocardial ischemia-reperfusion injury. However, whether pyroptosis is involved in ischemia-reperfusion injury of cochlear hair cells has not been explored. In this study, we examined the effects of melatonin on the oxygen-glucose deprivation/reperfusion (OGD/R) of hair cell-like House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear hair cells in vitro to mimic cochlear ischemia-reperfusion injury in vivo. We found that melatonin treatment protected the HEI-OC1 and cochlear hair cells against OGD/R-induced cell pyroptosis and reduced the expression level of ROS in these cells. However, these effects were completely abolished by the application of luzindole (a non-selective melatonin receptor blocker) and largely offset by the use of ML385 (an nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor). These findings suggest that melatonin alleviates OGD/R-induced pyroptosis of the hair cell-like HEI-OC1 cells and cochlear hair cells via the melatonin receptor 1A (MT-1) and melatonin receptor 1B (MT-2)/Nrf2 (NFE2L2)/ROS/NLRP3 pathway, which may provide credible evidence for melatonin being used as a potential drug for the treatment of idiopathic sudden sensorineural hearing loss in the future.