House Ear Institute-Organ Of Corti 1 Cells Research Articles

Antioxidative Stress-Induced Destruction to Cochlear Cells Caused by Blind Antioxidant Therapy.

Verification that blind and excessive use of antioxidants leads to antioxidant stress which exacerbates cochlear cell damage. Basic research. The Third Affiliated Hospital of Sun Yat-Sen University. We compared and quantified hair cell-like house ear institute-organ of corti 1 (HEI-OC1) cell density, cell viability, and apoptosis caused by different concentrations of N-acetylcysteine(NAC) via Hoechst staining, Cell Counting Kit 8, Hoechst with propidium iodide staining, and Annexin V with propidium iodide (PI) staining. Apoptosis induced by high concentrations of M40403 and coenzyme Q10 in cochlear explants was analyzed and compared by cochlear dissection and activated caspase 3 labeling. With the increase of NAC concentration (0-1000 μmol/L), cell density decreased consequently and reached the lowest at 1000 μmol/L (****P ≤ .0001). Cell viability is also declining (**P < .01). The number of Annexin V-fluorescein isothiocyanate-labeled cells and PI-labeled cells increased with increasing NAC concentration after treatment of HEI-OC1 cells for 48 hours. The proportion of apoptotic cells also rose (*P < .05, **P < .01). Cochlear hair cells (HCs) treated with low concentrations of M40403 and coenzyme Q10 for 48 hours showed no damage. When the concentrations of M40403 and coenzyme Q10 were increased (concentrations＞30 μmol/L), HC damage began, followed by a dose-dependent increase in HC loss (*P < .001, **P < .0001). Activated caspase-3 was clearly apparent in cochlear explants treated with 50 μmol/L M40403 and coenzyme Q10 compared with cochlear explants without added M40403 and coenzyme Q10. These experimental results suggest that inappropriate application of antioxidants can cause severe damage to normal cochlear HCs.

20(S)-Ginsenoside Rh1 alleviates sevoflurane-induced ototoxicity by reducing oxidative stress levels.

Sevoflurane is an inhalational anesthetic widely used in pediatric surgery. However, animal studies have shown that multiple sevoflurane exposures during the neonatal period led to ototoxicity. 20(S)-Ginsenoside Rh1, a ginsenoside extract, protects against cisplatin-induced ototoxicity by scavenging free radicals. This study aimed to assess the effects of Rh1 on sevoflurane-induced ototoxicity. Neonatal cochlear explants and House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were cultured and randomly divided into three groups: the control group, the sevoflurane group and the Rh1 pretreatment group. We pretreated cochlear explants or HEI-OC1 cells with 100 μM Rh1 2 hours before performing sevoflurane exposure. Immunofluorescence was used to detect hair cells and spiral ganglion neurons. Cell Counting Kit-8 assay was used to determine cell viability. Annexin V-fluorescein isothiocyanate and propidium iodide were used to evaluate apoptosis. CellROX-Green and MitoSOX-Red probes were used to measure the amount of reactive oxygen species (ROS). Tetramethylrhodamine methyl ester labeling was used to examine mitochondrial membrane potential. Rh1 attenuated spiral ganglion neuron nerve fibers and synapses degeneration in cochlear explants after sevoflurane exposure. Rh1 significantly increased the viability of HEI-OC1 cells, reduced reactive oxygen species accumulation in HEI-OC1 cells, and prevented mitochondrial damage in HEI-OC1 cells after sevoflurane exposure. These findings suggest that Rh1 is a promising drug for preventing sevoflurane-induced ototoxicity.

Abnormal Cholesterol Metabolism and Lysosomal Dysfunction Induce Age-Related Hearing Loss by Inhibiting mTORC1-TFEB-Dependent Autophagy.

Cholesterol is a risk factor for age-related hearing loss (ARHL). However, the effect of cholesterol on the organ of Corti during the onset of ARHL is unclear. We established a mouse model for the ARHL group (24 months, n = 12) and a young group (6 months, n = 12). Auditory thresholds were measured in both groups using auditory brainstem response (ABR) at frequencies of 8, 16, and 32 kHz. Subsequently, mice were sacrificed and subjected to histological analyses, including transmission electron microscopy (TEM), H&E, Sudan Black B (SBB), and Filipin staining, as well as biochemical assays such as IHC, enzymatic analysis, and immunoblotting. Additionally, mRNA extracted from both young and aged cochlea underwent RNA sequencing. To identify the mechanism, in vitro studies utilizing HEI-OC1 cells were also performed. RNA sequencing showed a positive correlation with increased expression of genes related to metabolic diseases, cholesterol homeostasis, and target of rapamycin complex 1 (mTORC1) signaling in the ARHL group as compared to the younger group. In addition, ARHL tissues exhibited increased cholesterol and lipofuscin aggregates in the organ of Corti, lateral walls, and spiral ganglion neurons. Autophagic flux was inhibited by the accumulation of damaged lysosomes and autolysosomes. Subsequently, we observed a decrease in the level of transcription factor EB (TFEB) protein, which regulates lysosomal biosynthesis and autophagy, together with increased mTORC1 activity in ARHL tissues. These changes in TFEB and mTORC1 expression were observed in a cholesterol-dependent manner. Treatment of ARHL mice with atorvastatin, a cholesterol synthesis inhibitor, delayed hearing loss by reducing the cholesterol level and maintaining lysosomal function and autophagy by inhibiting mTORC1 and activating TFEB. The above findings were confirmed using stress-induced premature senescent House Ear Institute organ of Corti 1 (HEI-OC1) cells. The findings implicate cholesterol in the pathogenesis of ARHL. We propose that atorvastatin could prevent ARHL by maintaining lysosomal function and autophagy by inhibiting mTORC1 and activating TFEB during the aging process.

FAM134B-induced endoplasmic reticulum (ER)-phagy exacerbates cisplatin-insulted hair cell apoptosis ：Possible relation to excessive ER stress

AimsFAM134B, the initial endoplasmic reticulum (ER)-phagy receptor identified, facilitates ER-phagy during ER stress. The malfunction of FAM134B has been demonstrated to have a crucial role in the pathological mechanisms of diverse human ailments. However, the role of FAM134B-mediated ER-phagy in ototoxicity, particularly in cisplatin-induced ototoxicity, remains unclear. The present study endeavors to investigate whether FAM134B is expressed in House Ear Institute-Organ of Corti 1 (HEI-OC1) and C57BL/6 murine cochlear hair cells (HCs), and to explore its potential function in cisplatin-mediated ototoxicity, with the aim of discovering new insights that can mitigate or forestall the irreversible adverse effect of cisplatin. MethodsImmunofluorescence (IF) staining was used to test the expression pattern of FAM134B, levels of C/EBP-homologous protein (CHOP), autophagy, and co-localization ratio of lysosomes and ER. Western blotting was employed to measure changes in expression levels of FAM134B, LC3B, ER stress-related proteins, LAMP1 and apoptotic mediators. Cell apoptosis was examined using transferase dUTP nick end labeling (TUNEL) assay and flow cytometry. ResultsIn the present investigation, it was observed that FAM134B exhibited a diffuse expression pattern in the cytoplasm and nuclei of control HEI-OC1 cells. Following cisplatin administration, FAM134B was found to accumulate and form distinct dots around the nuclei, concomitant with increased levels of ER-phagy, ER stress, unfolded protein response (UPR), and cell apoptosis. Additionally, knockdown of FAM134B resulted in reduced ER-phagy, mitigated ER stress and UPR, and decreased apoptotic activity in HEI-OC1 cells following cisplatin exposure. ConclusionsCollectively, the findings of this study demonstrate that FAM134B-mediated ER-phagy enhances the susceptibility of HCs to ER stress and apoptosis in response to cisplatin-induced stress. This suggests a sequential progression of ER-phagy, ER stress and apoptosis following cisplatin stimulus, and implies the potential therapeutic benefit of inhibiting of FAM134B-mediated ER-phagy in the prevention of cisplatin-related ototoxicity.

Cinchonine and cinchonidine alleviate cisplatin-induced ototoxicity by regulating PI3K-AKT signaling.

Cinchonine (CN) and its isomer cinchonidine (CD), two of the common cinchona alkaloids, are wildly used as antimalarial drugs. However, the effects of CN and CD on the auditory system are unknown. Molecular docking and molecular dynamics (MD) simulation were used for predicting effective drugs. The CCK-8 assay was conducted for assessing cell viability in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. MitoSox Red staining revealed reactive oxygen species (ROS) amounts. TMRM staining was used to assess the mitochondrial membrane potential (ΔΨm). Immunofluorescence staining of myosin 7a was used to examine hair cells (HCs) in cisplatin-treated neonatal mouse cochlear explants, while TUJ-1 immunostaining was used for the detection of spiral ganglion neurons (SGNs). Cleaved caspase-3 and TUNEL immunostaining were utilized for apoptosis assessment. Immunoblot was carried out to detect PI3K-AKT signaling effectors. Pretreatment with CN or CD significantly increased cell viability and reduced mitochondrial dysfunction and ROS accumulation in cisplatin-treated HEI-OC1 cells. Immunofluorescent staining of cochlear explants showed that CN and CD attenuated cisplatin-induced damage to SGNs and HCs. Immunoblot revealed that CN and CD downregulated the expression of cleaved caspase-3 and activated PI3K-AKT signaling in cisplatin-injured HEI-OC1 cells. CD and CN can reduce ototoxicity caused by cisplatin and might help treat cisplatin-associated hearing loss.

Ferroptosis inhibition shields house ear institute-organ of corti 1 cells from free fatty acids-induced inflammatory injuries

Background Free fatty acids (FFAs) could induce inflammatory responses via various pathways. Ferroptosis is characterized by the accumulation of lipid peroxidation products and fatal reactive oxygen species derived from iron accumulation, which may be an upstream event in the inflammatory injuries. Objectives To investigate the involvement of ferroptosis during the FFAs-induced pathological hair cell inflammatory injuries and its underlying mechanisms. Material and Methods We utilized House Ear Institute-Organ of Corti 1 (HEI-OC1) cell line as an in vitro model. The palmitate acid (PA) was utilized as a substitute for FFA, with cotreatment with ferroptosis inducer RSL3 and ferroptosis inhibitor Fer-1. Cell viability, lactase dehydrogenase (LDH) release, the expressions of ferroptosis-related factors such as glutathione peroxidase-4 (GPX4), solute carrier family 7 member 11 (SLC7A11), as well as toll-like receptor 4 (TLR4), ferric ion and reactive oxygen species (ROS), and partial inflammatory cytokines were measured. Results PA treatment might induce ferroptosis in HEI-OC1 cells, manifested as decreased cell viability, upregulated LDH release, iron overload, and ROS accumulation. Several inflammatory cytokines including IL-1β, IL-6, IL-1β, IL-6, TNF-α, MCP-1, IL-13, IL-12 p40, CCL5, G-CSF, and GM-CSF were upregulated compared to the Ctr group, while GPX4 and SLC7A11 were downregulated. The expression of TLR4 in the inflammatory pathway was also upregulated. Besides, these changes were further exacerbated by RSL3 cotreatment and abolished by Fer-1 cotreatment. Conclusions and Significance Ferroptosis inhibition could alleviate the PA-induced inflammatory injuries via inactivation of TLR4 signaling pathway in HEI-OC1 cell line.

5,7-Dihydroxy-4-methylcoumarin modulates the JNK/FoxO1 signaling pathway to attenuate cisplatin-induced ototoxicity by suppressing oxidative stress and apoptosis in vitro

5,7-Dihydroxy-4-methylcoumarin (D4M) is attributed to free radical scavenging effects, with wide application for anti-oxidation. This work aimed to assess D4M's impact on cisplatin-induced ototoxicity. The cell viability was estimated with CCK-8 assay. Apoptosis was detected by the Annexin V-FITC and PI assay. The reactive oxygen species (ROS) level was determined by MitoSOX-Red and CellROX-Green probes. Mitochondrial membrane potential was analyzed with TMRM staining. Immunofluorescence was utilized for hair cells and spiral ganglion neuron detection. Apoptosis-associated proteins were assessed by cleaved caspase-3 and TUNEL staining. These results showed that D4M pretreatment protected hair cells from cisplatin-induced damage, increased cell viability, and decreased apoptosis in House Ear Institute-Organ of Corti1 (HEI-OC1) cells and neonatal mouse cochlear explants. D4M significantly inhibited cisplatin-induced mitochondrial apoptosis and reduced ROS accumulation. In addition, the protective effect of D4M on cisplatin-induced ototoxicity was also confirmed in cochlear hair cells and spiral ganglion neurons in neonatal mice. Mechanistic studies showed that D4M markedly downregulated p-JNK and elevated the expression ratio of p-FoxO1/FoxO1, thereby reducing cisplatin-induced caspase-dependent apoptosis. Meanwhile, D4M-related protection of HEI-OC1 cells was significantly blunted by JNK signaling induction with anisomycin. This study supports the possibility that D4M may be used as a new compound to prevent cisplatin-related hearing loss.

Melatonin Alleviates the Oxygen-Glucose Deprivation/Reperfusion-Induced Pyroptosis of HEI-OC1 Cells and Cochlear Hair Cells via MT-1,2/Nrf2 (NFE2L2)/ROS/NLRP3 Pathway.

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.

Kinesin spindle protein inhibitor exacerbates cisplatin-induced hair cell damage

There is emerging evidence indicating that Kinesin family, plays vital roles in influencing the growth of axons, interference with the progression of tumor. However, the function of Kinesin member in the auditory organs remains unknown. SB743921, a kinesin spindle protein (KSP) inhibitor, was applied in mouse organ of Corti and House Ear Institute-Organ of Corti 1 (HEI-OC1) cell line to examine the role of KSP in auditory system with and without cisplatin damage. Cell Counting Kit-8 (CCK-8) and Lactase dehydrogenase (LDH) release assay were conducted to evaluate cell activity and toxicity. Pretreatment with SB743921 increased the sensitivity of HEI-OC1 cells to cisplatin ototoxicity through promoting cell apoptosis and deteriorating superoxide generation mediated damage from cisplatin. SB743921 also enhanced cisplatin induced hair cell damage in explants of mouse cochleae in vitro. Furthermore, the combined N-acetylcysteine (NAC) treatment with cisplatin or with cisplatin and SB743921 both completely rescued the reduced number of hair cells impaired by cisplatin, confirming the strengthening function of superoxide accumulation by SB743921 after cisplatin treatment. Inhibition of kinesin spindle protein enhanced the susceptibility of hair cells to cisplatin induced damage in mouse cochlear explants and HEI-OC1 cells, indicating that kinesin spindle protein might be an unprecedented target to weaken the ototoxicity of platinum medicaments.

Salubrinal Protects Against Cisplatin-Induced Cochlear Hair Cell Endoplasmic Reticulum Stress by Regulating Eukaryotic Translation Initiation Factor 2α Signalling.

ObjectiveCisplatin is a broad-spectrum anti-tumour drug commonly used in clinical practice. However, its ototoxicity greatly limits its clinical application, and no effective method is available to prevent this effect. Endoplasmic reticulum stress (ERS) is reportedly involved in cisplatin ototoxicity, but the exact mechanism remains unclear. Therefore, this study aimed to investigate the role of eukaryotic translation initiation factor 2α (eIF2α) signalling and its dephosphorylation inhibitor salubrinal in cisplatin ototoxicity.MethodsWe evaluated whether salubrinal could protect against cisplatin-induced damage in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and mouse cochlear explants. By knocking down eIF2α, we elucidated the vital role of eIF2α in cisplatin-induced damage in HEI-OC1 cells. Whole-mount immunofluorescent staining and confocal microscopy of mouse cochlear explants and HEI-OC1 cells were performed to analyse cisplatin-induced damage in cochlear hair cells and the auditory cell line.ResultsData suggested salubrinal attenuated cisplatin-induced hair cell injury by inhibiting apoptosis. In addition, salubrinal significantly reduced ERS levels in hair cells via eIF2α signalling, while eIF2α knockdown inhibited the protective effect of salubrinal.SignificanceSalubrinal and eIF2α signalling play a role in protecting against cisplatin-induced ototoxicity, and pharmacological inhibition of eIF2α-mediated ERS is a potential treatment for cisplatin-induced damage in the cochlea and HEI-OC1 cells.

MicroRNA Signature and Cellular Characterization of Undifferentiated and Differentiated House Ear Institute-Organ of Corti 1 (HEI-OC1) Cells

MicroRNAs (miRNAs) regulate gene expressions and control a wide variety of cellular functions. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells are widely used to screen ototoxic drugs and to investigate cellular and genetic alterations in response to various conditions. HEI-OC1 cells are almost exclusively studied under permissive conditions that promote cell replication at the expense of differentiation. Many researchers suggest that permissive culture condition findings are relevant to understanding human hearing disorders. The mature human cochlea however consists of differentiated cells and lacks proliferative capacity. This study therefore aimed to compare the miRNA profiles and cellular characteristics of HEI-OC1 cells cultured under permissive (P-HEI-OC1) and non-permissive (NP-HEI-OC1) conditions. A significant increase in the level of expression of tubulin β1 class VI (Tubb1), e-cadherin (Cdh1), espin (Espn), and SRY (sex determining region Y)-box2 (Sox2) mRNAs was identified in non-permissive cells compared with permissive cells (P < 0.05, Kruskal–Wallis H test, 2-sided). miR-200 family, miR-34b/c, and miR-449a/b functionally related cluster miRNAs, rodent-specific maternally imprinted gene Sfmbt2 intron 10th cluster miRNAs (-466a/ -467a), and miR-17 family were significantly (P < 0.05, Welch’s t-test, 2-tailed) differentially expressed in non-permissive cells when compared with permissive cells. Putative target genes were significantly predominantly enriched in mitogen-activated protein kinase (MAPK), epidermal growth factor family of receptor tyrosine kinases (ErbB), and Ras signaling pathways in non-permissive cells compared with permissive cells. This distinct miRNA signature of differentiated HEI-OC1 cells could help in understanding miRNA-mediated cellular responses in the adult cochlea.

Urolithin A attenuates auditory cell senescence by activating mitophagy

Aging of sensory organs is associated with a decline in mitochondrial function and the accumulation of dysfunctional mitochondria. Impaired mitophagy blocks the turnover of dysfunctional mitochondria and leads to their accumulation. Urolithin A (UA) induces mitophagy in various mammalian cells. This study was aimed at investigating the effect of the mitophagy activator, UA, on premature senescent auditory cells. The levels of cellular senescence-associated p53 and p21 significantly increased in H2O2-induced senescent House Ear Institute‐Organ of Corti 1 (HEI-OC1) cells and cochlear explants. However, the levels of mitophagy-related molecules significantly decreased. UA significantly decreased the expression of senescence-associated p53 and p21, and increased the expression of mitophagy-related proteins, in H2O2-induced senescent cells and cochlear explants. The percentage of β-galactosidase-stained senescent cells also reduced in H2O2-treated cells and cochlear explants upon UA pre-treatment. The formation of mitophagosomes and mitophagolysosomes was restored upon UA pre-treatment of H2O2-induced senescent cells. The knockdown of mitophagy-related genes (Parkin and Bnip3) resulted in annulment of UA-induced anti-senescent activity. UA significantly increased the ATP content, mitochondrial DNA (mtDNA) integrity, and mitochondrial membrane potential in senescent HEI-OC1 cells. These findings indicate that UA counteracted mitophagy decline and prevented premature senescence in auditory cells. Hence, UA administration might be a promising strategy for preventing mitochondrial dysfunction in patients with age-related hearing loss.

NRF2/HO-1 pathway activation by ATF3 in a noise-induced hearing loss murine model

BackgroundExcessive oxidative stress of the inner ear as a result of high, intense noise exposure is regarded as a major mechanism underlying the development of noise-induced hearing loss (NIHL). The present study was designed to explore the effect and mechanism of activated transcription factor 3 (ATF3) in reduction/oxidation homeostasis of NIHL. MethodIn vitro and in vivo assays were performed to investigate the functional role of ATF3 in the inner ear. Mice hearing was measured using auditory brainstem response. ATF3 short hairpin RNA (shRNA) was transfected into House Ear Institute-Organ of Corti 1 (HEI-OC1) cells to decrease ATF3 expression. Western blotting and quantitative real-time polymerase chain reaction (RT-qPCR) were performed to quantify ATF3, NRF2, HO-1 and NQO1 expression. Glutathione (GSH) assay was performed to detect intracellular GSH levels. ATF3 immunofluorescence analysis was carried out in cochlear cryosectioned samples and HEI-OC1 cells to localize ATF3 expression. Cell counting kit 8 assay and flow cytometry were performed to analyze cell viability. ResultATF3 was upregulated in noise-exposed cochleae and HEI-OC1 cells treated with H2O2. NRF2 is a key factor regulated by ATF3. NRF2, HO-1, NQO1, and GSH expression was significantly downregulated in shATF3 HEI-OC1 cells. ATF3 silencing promoted reactive oxygen species accumulation and increased apoptosis and necrosis with H2O2 stimulus. ConclusionATF3 functions as an antioxidative factor by activating the NRF2/HO-1 pathway.

Agmatine Alleviates Cisplatin-Induced Ototoxicity by Activating PI3K/AKT Signaling Pathway.

Cisplatin-induced ototoxicity can be partially attributed to excessive reactive oxygen species (ROS) production, and agmatine is well-known for the activation of the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway to inhibit ROS production. Whether agmatine could be used to alleviate cisplatin-induced ototoxicity is investigated. Cisplatin-exposed House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants showed increased ROS production detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining and decreased cell viability detected by Cell Counting Kit-8 (CCK-8) or Myosin 7a staining, which could be reversed by the agmatine pretreatment. Cisplatin intraperitoneally injected C57BL/6 mice demonstrated damaged auditory function as indicated by distortion products otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) assays, and trans-tympanically administrated agmatine in the left ears could partly prevent the auditory function loss. Mechanistically, downregulated B-cell lymphoma 2 (Bcl-2) expression, upregulated Bcl2-associated x (Bax) expression, and diminished p-PI3K and p-AKT expression were detected in cisplatin-exposed HEI-OC1 cells and cochlear explants, which could be prevented by the pretreatment with agmatine. Our investigation demonstrates that agmatine pretreatment could alleviate cisplatin-induced ototoxicity with the activation of PI3K/AKT signaling pathway.

Downregulation of Cav3.1 T-type Calcium Channel Expression in Age-related Hearing Loss Model.

Age-related hearing loss (AHL), characterized by degeneration of cochlea structures, is the most common sensory disorder among the elderly worldwide. The calcium channel is considered to contribute to normal hearing. However, the role of the T-type voltage-activated calcium channel, Cav3.1, remains unclear in AHL. Here, we investigate the age-related change of Cav3.1 expression in the cochlea and D-gal-induced senescent HEI-OC1 cells. Cochleae from C57BL/6 mice at 2 months and 12 months of age were assessed. Senescence in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells was induced by D-gal treatment. The immunofluorescence technique was employed to investigate the distribution of Cav3.1 in vivo and in vitro. Quantitative assessment was achieved by Western blotting and real-time PCR. In comparison with 2-month-old animals, 12-month old C57BL/6 mice exhibited great loss of hair cells and elevated auditory brainstem threshold. The Cav3.1 was located in hair cells, spiral ganglion cells, lateral walls, and the expression of Cav3.1 protein and mRNA decreased in the aged cochleae. D-gal-induced senescence assay confirmed the down-regulation of Cav3.1 expression in senescent HEI-OC1 cells. Our results show that age-related down-regulated expression of Cav3.1 in the cochleae is associated with AHL and may contribute to the pathogenesis of AHL.

Melatonin protects cochlear hair cells from nicotine-induced injury through inhibiting apoptosis, inflammation, oxidative stress and endoplasmic reticulum stress.

Hearing loss positively links with cigarette smoking. However, the involved mechanism and treatment strategies are largely unrevealed. This study aimed to investigate the damaging effect of nicotine on cochlear hair cells, reveal the underlying mechanism and evaluate the therapeutic effect of melatonin on nicotine-induced injury. The results showed that nicotine induced cytotoxicity of House Ear Institute-Organ of Corti 1 (HEI-OC1) cochlear hair cells in a dose-dependent manner (0, 2.5, 5, 10, 20, 40 and 80 μM). Functional investigations showed that nicotine (10 μM) stimulation dramatically promoted apoptosis, inflammatory response, oxidative stress and endoplasmic reticulum stress in HEI-OC1 cells. Moreover, melatonin treatment dose-dependently alleviated the nicotine-induced cytotoxicity in HEI-OC1 cells (0, 10 25, 50 and 100 μM). Further investigation showed that melatonin (100 μM) effectively attenuated the nicotine-induced apoptosis, inflammation, oxidative stress and endoplasmic reticulum stress in HEI-OC1 cells. Collectively, we demonstrated that nicotine induced apoptosis, inflammation, oxidative stress and endoplasmic reticulum stress of cochlear hair cells in an in vitro cell model. Melatonin showed protective effect on these aspects, suggesting that melatonin may be a potential agent for treating smoking-induced hearing loss.

Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells

Several studies have shown how different cell lines can influence the differentiation of stem cells through co-culture systems. The House Ear Institute-Organ of Corti 1 (HEI-OC1) is considered an important cell line for in vitro auditory research. However, it is unknown if HEI-OC1 cells can promote the differentiation of embryonic stem cells (ESCs). In this study, we investigated whether co-culture of ESCs with HEI-OC1 cells promotes differentiation. To this end, we developed a co-culture system of mouse ESCs with HEI-OC1 cells. Dissociated or embryonic bodies (EBs) of ESCs were introduced to a conditioned and inactivated confluent layer of HEI-OC1 cells for 14 days. The dissociated ESCs coalesced into an EB-like form that was smaller than the co-cultured EBs. Contact co-culture generated cells expressing several otic progenitor markers as well as hair cell specific markers. ESCs and EBs were also cultured in non-contact setup but using conditioned medium from HEI-OC1 cells, indicating that soluble factors alone could have a similar effect. The ESCs did not form into aggregates but were still Myo7a-positive, while the EBs degenerated. However, in the fully differentiated EBs, evidence to prove mature differentiation of inner ear hair cell was still rudimentary. Nevertheless, these results suggest that cellular interactions between ESCs and HEI-OC1 cells may both stimulate ESC differentiation.

Resveratrol upregulates miR-455-5p to antagonize cisplatin ototoxicity via modulating the PTEN-PI3K-AKT axis.

Resveratrol is a non-flavonoid polyphenol compound that exists in many plants, and is considered an antitoxin. This study explores the effects from the regulation of miR-455-5p by resveratrol on cisplatin-induced ototoxicity via the PTEN-PI3K-AKT signaling pathway. For this, House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were transfected with miR-455-5p inhibitor and treated with cisplatin and resveratrol, then cell proliferation, apoptosis, and oxidative stress were evaluated. A mouse model of hearing loss was established, and these mice were treated with cisplatin, resveratrol, or cisplatin combined with resveratrol, by intraperitoneal injection. The auditory brainstem response (ABR) threshold was measured, and hair cells were examined using immunofluorescence staining. The expression levels of miR-455-5p, PTEN, and PI3K/Akt proteins were examined. The results from our in-vitro experiments indicate that resveratrol promoted viability and reduced apoptosis and oxidative stress in cisplatin-induced HEI-OC1 cells. Resveratrol upregulated miR-455-5p, downregulated PTEN, and activated the PI3K-Akt axis. These effects of resveratrol were reversed by knock-down of miR-455-5p. The results from our in-vivo experiments indicate that resveratrol protected hearing and inhibited the hair-cell injury caused by cisplatin ototoxicity. Resveratrol also upregulated miR-455-5p, downregulated PTEN, and activated the PTEN-PI3K-Akt axis in cochlear tissues from cisplatin-treated mice. These results indicate that resveratrol upregulates miR-455-5p to target PTEN and activate the PI3K-Akt signaling pathway to counteract cisplatin ototoxicity.

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.

U0126 pretreatment inhibits cisplatin-induced apoptosis and autophagy in HEI-OC1 cells and cochlear hair cells

Deafness is the most common sensory disorder in the world. Ototoxic drugs are common inducing factors of sensorineural hearing loss, and cochlear hair cell (HC) damage is the main concern of the present studies. Cisplatin is a widely used, highly effective antitumor drug, but some patients have experienced irreversible hearing loss as a result of its application. This hearing loss is closely related to HC apoptosis and autophagy. U0126 is a specific inhibitor of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathway and has neuroprotective effects. For example, the neuroprotective effect of U0126 on ischemic stroke has been widely recognized. In neural cells, U0126 can prevent death due to excess glutamate, dopamine, or zinc ions. However, no studies of U0126 and ototoxic drug-induced injury have been reported to date. In the present study, we found that U0126 pretreatment significantly reduced the apoptosis and autophagy of HCs in auditory House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear HCs. In addition, U0126 reduced the cisplatin-induced production of reactive oxygen species as well as the cisplatin-induced decrease in the mitochondrial membrane potential. These findings suggest that U0126 may be a potential therapeutic candidate for the prevention of cisplatin-induced ototoxicity.