Gentamicin Ototoxicity Research Articles

Combinatorial protection of cochlear hair cells: not too little but not too much.

A number of drugs are toxic to the cochlear sensory cells known as hair cells (HCs), resulting in hearing loss. Treatment with survival-promoting growth factors, antioxidants, and inhibitors of cell death pathways or proteinases have been shown to reduce HC damage in in vivo and/or in vitro animal models. Conversely, translation to humans has often been disappointing. This may be due to the complexity of intracellular damage processes. We hypothesized that combining treatments targeting different cellular processes would be more effective. Using an in vitro model of gentamicin ototoxicity for murine cochlear hair cells, we screened all 56 possible combinations of inhibitors targeting five different cell damage mechanisms, plus the activator of one cell survival pathway, each of which have been shown to be singly effective in preventing HC loss in experimental studies. A high dose of gentamicin (200 μM) was used over three days in culture. All compounds were added at a dosage below that required for significant protection in the assay, and only this single dose was then employed. This was done so that we could more easily detect interactive, as opposed to additive, effects. Increasing protection of hair cells was observed as combinations of compounds were increased from two to four factors, although not all combinations were equally protective. The optimal combination of four compounds consisted of an anti-oxidant, an apoptosis inhibitor, an autophagy inhibitor and a protective growth factor. Increasing the number of factors to five or six resulted in decreased protection. The results support the hypothesis that targeting multiple cellular damage or survival pathways provides more an effective hair cell protection approach. The results help to identify critical interactions among the cellular processes that operate in gentamicin ototoxicity. They also suggest that inhibiting too many biological processes impairs functions critical to HC survival, resulting in decreased protection.

Regulation of the p53/SLC7A11/GPX4 Pathway by Gentamicin Induces Ferroptosis in HEI-OC1 Cells.

Gentamicin is a commonly used aminoglycoside antibiotic, with ototoxicity as a significant side effect. Ferroptosis, an iron-dependent form of cell death, has been implicated in a variety of disorders. Whether ferroptosis impacts gentamicin ototoxicity is not yet known. The current work used an in-vitro model to examine the influence of gentamicin-induced ferroptosis on cochlear hair cell damage and probable molecular biological pathways. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated with different concentrations of gentamicin for 24 hours, with or without ferrostatin-1 pretreatment, to observe gentamicin-induced ferroptosis. The role of p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling in gentamicin-induced ferroptosis was explored by pretreating cells with the p53 inhibitor pifithrin-α (PFT-α). We investigated the effect of gentamicin on cells by assessing cell viability. Cellular proteins were isolated and Western blots were performed to detect changes in the expression of p53, SLC7A11, and GPX4. Fluorescence staining was used to assess levels of reactive oxygen species. An enzymatic detection kit was used to detect glutathione, Fe, and malondialdehyde markers. Gentamicin reduced cell viability, glutathione content, and SLC7A11 and GPX4 protein levels, and increased levels of p53 protein, reactive oxygen species, malondialdehyde, and Fe. These effects were largely blocked by pretreatment with ferrostatin-1. Pretreatment with the p53 inhibitor PFT-α prevented the gentamicin-induced reduction in SLC7A11 and GPX4, which alleviated several features of ferroptosis including glutathione depletion, iron overload, and lipid peroxidation build-up. Gentamicin induces ferroptosis in the HEI-OC1 cell line, and the mechanism may be related to the p53/SLC7A11/GPX4 signaling pathway.

Hesperidin Alleviates Gentamicin Ototoxicity in Rats by Inhibiting Oxidative Stress, Inflammation and Apoptosis

Hesperidin Alleviates Gentamicin Ototoxicity in Rats by Inhibiting Oxidative Stress, Inflammation and Apoptosis

Therapeutic potential of salidroside in preserving rat cochlea organ of corti from gentamicin-induced injury through modulation of NRF2 signaling and GSK3β/NF-κB pathway.

Salidroside (SAL) is a phenol glycoside compound found in plants of the Rhodiola genus which has natural antioxidant and free radical scavenging properties. SAL are able to protect against manganese-induced ototoxicity. However, the molecular mechanism by which SAL reduces levels of reactive oxygen species (ROS) is unclear. Here, we established an in vitro gentamicin (GM) ototoxicity model to observe the protective effect of SAL on GM-induced hair cells (HC) damage. Cochlear explants of postnatal day 4 rats were obtained and randomly divided into six groups: two model groups (treatment with 0.2 mM or 0.4 mM GM for 24 h); two 400 μmol/L SAL-pretreated groups pretreatment with SAL for 3 h followed by GM treatment (0.2 mM or 0.4 mM) for 24 h; 400 μmol/L SAL group (treatment with SAL for 24 h); control group (normal cultured cochlear explants). The protective effects of SAL on GM-induced HC damage, and on mRNA and protein levels of antioxidant enzymes were observed. HC loss occurred after 24 h of GM treatment. Pretreatment with SAL significantly reduced GM-induced OHC loss. In cochlear tissues, mRNA and protein levels of NRF2 and HO-1 were enhanced in the GM alone group compared with the SAL pretreatment GM treatment group. SAL may protect against GM-induced ototoxicity by regulating the antioxidant defense system of cochlear tissues; SAL can activate NRF2/HO-1 signaling, inhibit NF-κB activation, activate AKT, and increase inhibitory phosphorylation of GSK3β to decrease GSK3 activity, all of which exert antioxidant effects.

Cortisol Sensitizes Cochlear Hair Cells to Gentamicin Ototoxicity Via Endogenous Apoptotic Pathway.

The widespread use of aminoglycosides is a prevalent cause of sensorineural hearing loss. Patients receiving aminoglycosides usually have elevated levels of circulating stress hormones due to disease or physiological stress; however, whether the stress hormone cortisol impacts aminoglycoside-mediated injury of cochlear hair cells has not been fully investigated. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with or without cortisol pretreatment were exposed to gentamicin, we investigated the effect of cortisol pretreatment on gentamicin ototoxicity by assessing cell viability. Molecular pathogenesis was explored by detecting apoptosis and oxidative stress. Meanwhile, by inhibiting glucocorticoid receptors (GR) and mineralocorticoid receptors (MR), the potential roles of receptor types in cortisol-mediated sensitization were evaluated. Cortisol concentrations below 75 μmol/l did not affect cell viability. However, pretreatment with 50 μmol/l cortisol for 24 hours sensitized hair cells to gentamicin-induced apoptosis. Further mechanistic studies revealed that cortisol significantly increased hair cell apoptosis and oxidative stress, and altered apoptosis-related protein expressions induced by gentamicin. In addition, blockade of either GR or MR attenuated cortisol-induced hair cell sensitization to gentamicin toxicity. Cortisol pretreatment increased mammalian hair cell susceptibility to gentamicin toxicity. Sensitization was related to the activation of the intrinsic apoptotic pathway and excessive generation of reactive oxygen species. Cortisol may exacerbate aminoglycoside ototoxicity.

Hepatocyte growth factor mimetic confers protection from aminoglycoside-induced hair cell death in vitro

Loss of sensory hair cells from exposure to certain licit drugs, such as aminoglycoside antibiotics, can result in permanent hearing damage. Exogenous application of the neurotrophic molecule hepatocyte growth factor (HGF) promotes neuronal cell survival in a variety of contexts, including protecting hair cells from aminoglycoside ototoxicity. HGF itself is not an ideal therapeutic due to a short half-life and limited blood-brain barrier permeability. MM-201 is a chemically stable, blood-brain barrier permeable, synthetic HGF mimetic that serves as a functional ligand to activate the HGF receptor and its downstream signaling cascade. We previously demonstrated that MM-201 robustly protects zebrafish lateral line hair cells from aminoglycoside ototoxicity. Here, we examined the ability of MM-201 to protect mammalian sensory hair cells from aminoglycoside damage to further evaluate MM-201’s clinical potential. We found that MM-201 exhibited dose-dependent protection from neomycin and gentamicin ototoxicity in mature mouse utricular explants. MM-201’s protection was reduced following inhibition of mTOR, a downstream target of HGF receptor activation, implicating the activation of endogenous intracellular substrates by MM-201 as critical for the observed protection. We then asked if MM-201 altered the bactericidal properties of aminoglycosides. Using either plate or liquid growth assays we found that MM-201 did not alter the bactericidal efficacy of aminoglycoside antibiotics at therapeutically relevant concentrations. We therefore assessed the protective capacity of MM-201 in an in vivo mouse model of kanamycin ototoxicity. In contrast to our in vitro data, MM-201 did not attenuate kanamycin ototoxicity in vivo. Further, we found that MM-201 was ototoxic to mice across the dose range tested here. These data suggest species- and tissue-specific differences in otoprotective capacity. Next generation HGF mimetics are in clinical trials for neurodegenerative diseases and show excellent safety profiles, but neither preclinical studies nor clinical trials have examined hearing loss as a potential consequence of pharmaceutical HGF activation. Further research is needed to determine the consequences of systemic MM-201 application on the auditory system.

Designing a Prolonged Method of Therapeutic Delivery to Support Rehabilitation From Ototoxic Damage in a Schwann Cell Model.

The ototoxicity of gentamicin and cisplatin can be evaluated with a Schwann cell model to screen for otoprotective agents that can be encapsulated into poly (lactic-co-glycolic acid) (PLGA) microparticles for drug delivery to the inner ear. Aminoglycosides and cisplatin are widely prescribed but known to cause ototoxicity. There is strong evidence that compromise to Schwann cells ensheathing inner ear afferent neurons results in inner ear dysfunction mimicking drug-induced ototoxicity. There is a need for a model for ototoxic demyelination to screen medications for protective potential and to subsequently target and tune the delivery of any promising agents. RT4-D6P2T rat schwannoma cells were used as a Schwann cell model to assess gentamicin and cisplatin toxicity and to screen for protective agents. Cell viability was evaluated with the MTT cell proliferation assay. N -acetylcysteine (NAC) was encapsulated into a PLGA microparticle, and its elution profile was determined. The estimated 50% lethal concentration dose for gentamicin was 805.6 μM, which was 46-fold higher than that for cisplatin (17.5 μM). In several trials, cells dosed with NAC and cisplatin demonstrated a 22.6% ( p < 0.001) increase in cell viability when compared with cisplatin alone. However, this protective effect was not consistent across all trials. NAC was encapsulated into a PLGA microparticle and elution plateaued at 5 days. When dosed at their respective therapeutic ranges, cisplatin is more likely than gentamicin to induce damage to the Schwann cell model. Although NAC demonstrates an uncertain role in protecting against cisplatin-induced Schwann cell cytotoxicity, this study establishes a method to screen for other otoprotective medications to encapsulate into a tunable microparticle for localized drug delivery.

CDK2 regulates aminoglycoside-induced hair cell death through modulating c-Jun activity: Inhibiting CDK2 to preserve hearing.

Sensory hair cell death caused by the ototoxic side effects of many clinically used drugs leads to permanent sensorineural hearing loss in patients. Aminoglycoside antibiotics are widely used and well-known for their ototoxicity, but the molecular mechanisms of aminoglycoside-induced hair cell death are not well understood. This creates challenges in our attempts to alleviate or prevent such adverse side effects. Here, we report a regulatory role of CDK2 in aminoglycoside-induced hair cell death. Utilizing organotypic cultures of cochleae from neonatal mice, we show that blocking CDK2 activity by either pharmaceutical inhibition or by Cdk2 gene knockout protects hair cells against the ototoxicity of gentamicin—one of the most commonly used aminoglycoside antibiotics—by interfering with intrinsic programmed cell death processes. Specifically, we show that CDK2 inhibition delays the collapse of mitochondria and the activation of a caspase cascade. Furthermore, at the molecular level, inhibition of CDK2 activity influences proapoptotic JNK signaling by reducing the protein level of c-Jun and suppressing the gentamicin-induced upregulation of c-Jun target genes Jun and Bim. Our in vivo studies reveal that Cdk2 gene knockout animals are significantly less sensitive to gentamicin ototoxicity compared to wild-type littermates. Altogether, our work ascertains the non-cell cycle role of CDK2 in regulating aminoglycoside-induced hair cell apoptosis and sheds lights on new potential strategies for hearing protection against ototoxicity.

A Prospective Study on the Vestibular Toxicity of Gentamicin in a Clinical Setting.

Gentamicin is a widely used aminoglycoside with ototoxicity as a known adverse effect. Because of the difficulty in clinical recognition, the prevalence of gentamicin ototoxicity in practice is thought to be higher than reported. This study aimed to prospectively assess the effect of gentamicin on vestibular function and whether ototoxicity is underrecognized. Single-center, prospective, nonblinded trial. Inpatient tertiary hospital setting followed by vestibular outpatient clinic review. Forty-eight patients undergoing a urologic procedure were recruited, with 24 and 17 patients having one or two follow-up tests, respectively, after initial gentamicin administration. Single dose of gentamicin during a urologic procedure. Gains for the vestibuloocular reflex (VOR) were measured using the video head impulse test before receiving gentamicin and at two other timepoints after gentamicin. The gains in VOR were then compared with previous testing sessions to determine if there was a deterioration after gentamicin use. Before receiving gentamicin, the gains for horizontal VOR were measured for 48 patients. The gains were measured a second time for 24 patients at varying durations postgentamicin (1-56 d) and a third time for 17 patients (14-152 d) postgentamicin. The mean VOR gain for Timepoints 1, 2, and 3 were 0.72 ± 0.13, 0.75 ± 0.16, and 0.79 ± 0.18, respectively. Linear-mixed model with repeated-measure analysis revealed no significant difference in VOR gain between Timepoints 1 and 2 ( p = 0.19). There was no significant effect observed on mean VOR gain decrement after a single dose of gentamicin.

Heme Oxygenase-1 Protects Hair Cells From Gentamicin-Induced Death

Gentamicin ototoxicity can generate free radicals within the inner ear, leading to permanent damage to sensory hair cells (HCs) and eventually hearing loss. The following study examined the alterations of oxidative damage-related genes in the cochlea and important molecules responsible for oxidation following gentamicin injury in vitro. The RT2 Profiler polymerase chain reaction (PCR) array was used to screen candidate targets for treatment to prevent hearing loss caused by gentamicin. We found that during gentamicin-induced death in HCs, Heme oxygenase-1 (HO-1) had a high fold change in the HCs of the cochlea. Moreover, the use of CoPPIX to induce HO-1 inhibited gentamicin-induced HC death, while HO-1 inhibitors ZnPPIX after CoPPIX reversed this process. Furthermore, the inhibitors of NF-E2-related factor-2 (Nrf2) reduced the expression of HO-1 and inhibited the protective effect of HO-1 after gentamicin, thus suggesting that the Nrf2/HO-1 axis might regulate gentamicin-associated ototoxicity. We further demonstrated that induction of HO-1 up-regulated the expression of Nrf2 in both cochlear and HEI-OC1 cells. In summary, these findings indicated that HO-1 protects HCs from gentamicin by up-regulating its expression in HCs and interacting with Nrf2 to inhibit reactive oxygen species (ROS).

2,3,4',5‑Tetrahydroxystilbene‑2‑O‑β‑D‑glucoside ameliorates gentamicin‑induced ototoxicity by modulating autophagy via Sesn2/AMPK/mTOR signaling

Gentamicin is an important aminoglycoside antibiotic used in the treatment of gram-negative bacterial infections, but nephrotoxicity and ototoxicity reduce its utility. The autophagy pathway is involved in damage of auditory hair cells. With the aim of developing new strategies for attenuating gentamicin ototoxicity, the present study investigated the otoprotective mechanism of 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) in vitro using the mouse cochlear cell line UB/OC-2. MTT assay demonstrated that gentamicin reduced UB/OC-2 cell viability and western blotting showed that gentamicin upregulated autophagy-related proteins, such as Beclin, autophagy related 5 and LC3-II. THSG significantly attenuated gentamicin-induced cytotoxicity, clearly reduced LDH release observed by LDH assay and decreased the expression of autophagy-related proteins. Reverse-transcription-quantitative (RT-q) PCR and western blotting showed that THSG against gentamicin-induced autophagy via suppressing the expression of Sesn2, at both the mRNA and protein level and a possible involvement of AMP-activated protein kinase (AMPK)/mTOR signaling response. Collectively, the present study demonstrated that THSG decreased gentamicin-induced ototoxicity in UB/OC-2 cochlear cells via the autophagic signaling in regulating Sesn2/AMPK/mTOR pathway. These results suggested that THSG might be a new therapeutic agent with the potential to attenuate gentamicin ototoxicity.

Analysis of potential protective effects of caffeic acid phenethyl ester against gentamicin ototoxicity: An experimental study

Objective(s):In this study, it is aimed to investigate the potential protective effect of caffeic acid phenethyl ester (CAPE) on ototoxicity caused by gentamicin in a rat model.Materials and Methods:Thirty Wistar albino rats were divided into 3 groups. Group I was selected as the control group. Gentamicin was administered intraperitoneally in group II, gentamicin and CAPE in group III. Audiological assessment was performed by the distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) measurements before and after treatment of each group. At the end of the study all rats were decapitated, cochlea was removed and electron microscopic examination was performed. Results:In group II post-treatment DPOAE levels were found to be lower than pretreatment DPOAE levels (P<0.05). However, in group III, there is no significant difference between pre- and post-treatment DPOAE levels (P>0.05). Except for Group I, ABR thresholds increased after the procedure and this increase was statistically significant (P<0.0001). According to histological examination by transmission electron microscopy, CAPE has a cellular protective effect against gentamicin ototoxicity. Conclusion:CAPE may ameliorate hearing deterioration caused by gentamicin ototoxicity and protect the cochlear cells from apoptosis due to the strong antioxidant effect.

Puerarin alleviates the ototoxicity of gentamicin by inhibiting the mitochondria‑dependent apoptosis pathway.

Gentamicin (GM) is a commonly used antibiotic, and ototoxicity is one of its side effects. Puerarin (PU) is an isoflavone in kudzu roots that exerts a number of pharmacological effects, including antioxidative and free radical scavenging effects. The present study investigated whether PU could protect against GM-induced ototoxicity in C57BL/6J mice and House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. C57BL/6J mice and HEI-OC1 cells were used to establish models of GM-induced ototoxicity in this study. Auditory brainstem responses were measured to assess hearing thresholds, and microscopy was used to observe the morphology of cochlear hair cells after fluorescent staining. Cell viability was examined with Cell Counting Kit-8 assays. To evaluate cell apoptosis and reactive oxygen species (ROS) production, TUNEL assays, reverse transcription-quantitative PCR, DCFH-DA staining, JC-1 staining and western blotting were performed. PU protected against GM-induced hearing damage in C57BL/6J mice. PU ameliorated the morphological changes of mouse cochlear hair cells and reduced the apoptosis rate of HEI-OC1 cells after GM-mediated damage. GM-induced ototoxicity may be closely related to the upregulation of p53 expression and the activation of endogenous mitochondrial apoptosis pathways, and PU could protect cochlear hair cells from GM-mediated damage by reducing the production of ROS and inhibiting the mitochondria-dependent apoptosis pathway.

Targeting Autophagy Induction as A possible Protective Mechanism by Verapamil Compared to Rapamycin (Sirolimus) Against Gentamicin -Induced Ototoxicity in Guinea Pigs

Background: Ototoxicity is a harmful feature of the cochlea or auditory nerve and sometimes vestibularapparatus. Gentamicin is known to cause irreversible bilateral ototoxicity. Autophagy induction hasbeen proposed as a target for prevention of gentamicin ototoxicity. Aim of the work: to investigate thepossible protective effect of autophagy induction by verapamil compared to rapamycin. Methods: Thisexperiment was conducted on 32 male guinea pigs. At the beginning each animal’s hearing status wasassessed using auditory brainstem response (ABR) audiometry. Then, they were divided into 4 equalgroups: Group 1: control group, Group 2: untreated gentamicin-induced ototoxicity group. Group 3:gentamicin-induced ototoxicity treated concomitantly with rapamycin. Group 4: gentamicin-inducedototoxicity treated concomitantly with verapamil. At the end of the experiment, ABR was repeatedthen the animals were sacrificed, and blood samples were obtained for assaying of reduced glutathioneand malondialdehyde levels. The left cochlea was processed for scanning electron microscope, whilethe right cochlea was processed for histopathology and LC3-II immunohistochemistry. Results:Verapamil revealed superiority compared to rapamycin proved by significant improvement in ABR,histopathological results, in addition to its antioxidant effect. Conclusion: verapamil could be suggestedas a potential therapeutic approach to decrease gentamicin ototoxicity.

Effects of Moringa Extract on Aminoglycoside-Induced Hair Cell Death and Organ of Corti Damage.

Moringa extract, a naturally occurring anti-oxidant, protects against aminoglycoside-induced hair cell death and hearing loss within the organ of Corti. Reactive oxygen species (ROS) arise primarily in the mitochondria and have been implicated in aminoglycoside-induced ototoxicity. Mitochondrial dysfunction results in loss of membrane potential, release of caspases, and cell apoptosis. Moringa extract has not previously been examined as a protective agent for aminoglycoside-induced ototoxicity. Putative otoprotective effects of moringa extract were investigated in an organotypic model using murine organ of Corti explants subjected to gentamicin-induced ototoxicity. Assays evaluated hair cell loss, cytochrome oxidase expression, mitochondrial membrane potential integrity, and caspase activity. In vitro application of moringa conferred significant protection from gentamicin-induced hair cell loss at dosages from 25 to 300 μg/mL, with dosages above 100 μg/mL conferring near complete protection. Assays demonstrated moringa extract suppression of ROS, preservation of cytochrome oxidase activity, and reduction in caspase production. Moringa extract demonstrated potent antioxidant properties with significant protection against gentamicin ototoxicity in cochlear explants.

Intratympanic Lipopolysaccharide Elevates Systemic Fluorescent Gentamicin Uptake in the Cochlea.

Objectives/HypothesisLipopolysaccharide (LPS), a key component of bacterial endotoxins, activates macrophages and triggers the release of inflammatory cytokines in mammalian tissues. Recent studies have shown that intratympanic injection of LPS simulates acute otitis media (AOM) and results in morphological and functional changes in the inner ear. Here we established an AOM mouse model with LPS to investigate the uptake of ototoxic gentamicin in the inner ear, and elucidated the underlying mechanism by focusing on cochlear inflammation as a result of AOM.Study DesignPreclinical rodent animal model.MethodsFluorescently tagged gentamicin (GTTR) was systemically administered to mice with AOM. Iba1‐positive macrophage morphology and inner ear cytokine profile were evaluated by immunofluorescence technique and a mouse cytokine array kit, respectively.ResultsWe observed characteristic symptoms of AOM in the LPS‐treated ears with elevated hearing thresholds indicating a conductive hearing loss. More importantly, the LPS‐induced AOM activated cochlear inflammatory responses, manifested by macrophage infiltration, particularly in the organ of Corti and the spiral ligament, in addition to the up‐regulation of proinflammatory cytokines. Meanwhile, GTTR uptake in the stria vascularis and sensory hair cells from all the LPS‐treated ears was significantly enhanced at 24, 48, and 72‐hour post‐treatment, as the most prominent enhancement was observed in the 48‐hour group.ConclusionIn summary, this study suggests that the pathological cochlea is more susceptible to ototoxic drugs, including aminoglycosides, and justified the clinical concern of aminoglycoside ototoxicity in the AOM treatment. Laryngoscope, 131:E2573–E2582, 2021

XIAP inhibits gentamicin-induced hair cell damage and ototoxicity through the caspase-3/9 pathway

Gentamicin (GM), an aminoglycoside antibiotic, is one commonly used clinical drugs with ototoxic side effects. One of the most principal mechanisms of its ototoxicity is that GM can activate caspase-mediated cell death pathways in the cochlea. Since the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) has been reported to directly bind to activated caspase protein and inhibit their activities, we hypothesized that it might protect cochlea hair cells from GM ototoxicity. To evaluate this hypothesis, postnatal day 2–3 (P2-3) transgenic (TG) mice, in which XIAP gene is over-expressed under a pure C57BL/6J genetic background was constructed. We first extracted the cochlea tissue of normal mice and treated them with different concentrations of GM, and the number of hair cells were observed to determine the concentration of GM used in subsequent experiments. Next, we used Western Blot experiment to examine the effect of GM on XIAP protein expression in normal mouse cochlea, and then Western Blot and RT-PCR experiments were used to identify the transgenic mice. Finally, immunofluorescence assays were used to detect the effect of GM on the expression of caspase protein and verify the protective effect of XIAP. We found that GM at a concentration of 0.5 mM significantly affected the function of cochlea hair cells, up-regulating the expression of cleaved-caspase-3 and cleaved-caspase-9 protein but down-regulating XIAP protein. In the cochlea tissues of TG mice, this effect of GM was suppressed, and the destruction of hair cells was significantly reduced, and the cleaved-caspase-3 and cleaved-caspase-9 proteins were significantly suppressed. These results suggested that XIAP reduces GM-induced ototoxicity and caspase-3/9 pathway is associated with this process.

Theophylline alleviates gentamicin-induced cytotoxicity to sensory hair cells by maintaining HDAC2 expression

Sensorineural hearing loss is a health problem with global prevalence. Aminoglycoside antibiotics, for instance gentamicin, may cause ototoxicity in mammals as a result of apoptosis and elevated oxidative stress in cochlear hair cells. Our study aimed to examine the potential effects of theophylline, an HDAC2 agonist, on gentamicin-induced cytotoxicity to sensory hair cells. Mouse cochlear explants and HEI-OC1 cells were in vitro cultured and challenged by gentamicin to induce ototoxicity, with or without theophylline. Cochlear hair cells were evaluated by fluorescent microscopy, and their mechanotransduction was assessed by electrophysiology. Expression levels of HDAC2 and apoptosis pathway factors were also evaluated following gentamicin and theophylline treatments. The functional role of HDAC2 in this setting was investigated by siRNA targeted silencing. Theophylline protected cochlear hair cells from ototoxicity induced by gentamicin, in terms of preserving cochlear structure and mechanotransduction ability, and preventing the activation of the intrinsic apoptosis pathway dose-dependently. HDAC2 expression was downregulated by gentamicin, which could be restored by theophylline. HDAC2 silencing in HEI-OC1 cells negated the beneficial effect of theophylline against gentamicin-induced growth defect and apoptosis activation. Theophylline protects sensory hair cells from gentamicin ototoxicity by maintaining HDAC2 expression. Our study thereby discovers a critical role of HDAC2 in gentamicin-induced ototoxicity, which could shine light on potential therapeutic options for treatment against sensorineural hearing loss.

Icariin Treatment Protects Against Gentamicin-Induced Ototoxicity via Activation of the AMPK-SIRT3 Pathway.

Ototoxicity is a serious health problem that greatly affects millions of people worldwide. This condition is caused by the entry of aminoglycosides into auditory hair cells, subsequently inducing reactive oxygen species (ROS) production and accumulation. Several strategies have been adopted to overcome irreversible ROS-induced hair cell loss in mammals. In recent years, icariin, a major active component of the traditional herb Epimedium, has been widely studied and revealed to have antioxidant, anti-inflammatory, and anti-apoptotic properties. In this study, we found that icariin pretreatment improved the survival rate of gentamicin-treated House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants. Icariin remarkably suppressed HEI-OC1 cell apoptosis and inhibited ROS production in cells. Notably, icariin upregulated PGC-1α (SIRT3 promoter) and SIRT3 expression in HEI-OC1 cells. In addition, SIRT3 inhibition significantly attenuated the anti-apoptotic effect of icariin. We also found that icariin can increase AMPK phosphorylation. Further studies showed that inhibition of SIRT3 activity had no significant effect on AMPK phosphorylation. Furthermore, the AMPK inhibitor compound C significantly suppressed SIRT3 expression, meaning that AMPK, as an upstream molecule, regulates SIRT3 expression. Meanwhile, inhibition of AMPK activity significantly reduced the protective effect of icariin on gentamicin ototoxicity. Based on these results, icariin exerts its protective effect on gentamicin-induced ototoxicity via activation of the AMPK-SIRT3 signaling pathway, thus providing a new strategy for treating ototoxicity caused by aminoglycoside antibiotics.

Dissociating antibacterial from ototoxic effects of gentamicin C-subtypes

Gentamicin is a potent broad-spectrum aminoglycoside antibiotic whose use is hampered by ototoxic side-effects. Hospital gentamicin is a mixture of five gentamicin C-subtypes and several impurities of various ranges of nonexact concentrations. We developed a purification strategy enabling assaying of individual C-subtypes and impurities for ototoxicity and antimicrobial activity. We found that C-subtypes displayed broad and potent in vitro antimicrobial activities comparable to the hospital gentamicin mixture. In contrast, they showed different degrees of ototoxicity in cochlear explants, with gentamicin C2b being the least and gentamicin C2 the most ototoxic. Structure-activity relationships identified sites in the C4'-C6' region on ring I that reduced ototoxicity while preserving antimicrobial activity, thus identifying targets for future drug design and mechanisms for hair cell toxicity. Structure-activity relationship data suggested and electrophysiological data showed that the C-subtypes both bind and permeate the hair cell mechanotransducer channel, with the stronger the binding the less ototoxic the compound. Finally, both individual and reformulated mixtures of C-subtypes demonstrated decreased ototoxicity while maintaining antimicrobial activity, thereby serving as a proof-of-concept of drug reformulation to minimizing ototoxicity of gentamicin in patients.