Research ArticleExperimental studies
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Bisdemethoxycurcumin-mediated Attenuation of Apoptosis Prevents Gentamicin-induced Ototoxicity in Mouse Cochlear UB/OC-2 Cells

TING-YA KANG, CHUAN-JEN HSU, JIA-NI LIN, CHEN-CHI WU, JEN-SHU WANG, HUI-YI LIN, SZU-HUI YU, RONG-SHUAN WU, YU-HSUAN WEN, GUO-FANG TSENG and HUNG-PIN WU
In Vivo May 2022, 36 (3) 1095-1105; DOI: https://doi.org/10.21873/invivo.12807

Abstract

Background/Aim: Gentamicin has been widely prescribed since the last two decades despite its ototoxicity and nephrotoxicity. Bisdemethoxycurcumin (BDMC) is an affordable and safe curcuminoid with medicinal properties. We aimed to understand the effects of BDMC on the gentamicin-induced hair cell damage in mouse cochlear UB/OC-2 cells, in order to elucidate the therapeutic potential of BDMC against gentamicin-induced ototoxicity. Materials and Methods: We quantified the cell membrane potential and examined the regulators and cascade proteins in the intrinsic pathway of hair cell apoptosis. Mouse cochlear UB/OC-2 cells were treated with BDMC before exposure to gentamicin. The effects of BDMC on hair cell viability, mitochondrial function, and apoptosis-related proteins were examined by flow cytometry, western blot, and fluorescent staining. Results: Our results revealed that BDMC reversed gentamicin-mediated cycle arrest at the G2/M phase, stabilizing the mitochondrial membrane potential, decreasing cleaved caspase proteins, and successfully reversing hair cell apoptosis. Conclusion: BDMC is a potential agent for reducing gentamicin-induced ototoxicity.

Gentamicin is a broad-spectrum aminoglycoside (AG) antibiotic effective against aerobic gram-negative and gram-positive bacteria. It is known for its efficacy against neonatal sepsis and tuberculosis (1-3). Owing to their affordable cost, AGs have been widely used in the last two decades, especially in developing countries. However, gentamicin has serious adverse effects such as ototoxicity and nephrotoxicity (4-6).

For several years, researchers have attempted to find novel therapies against gentamicin-induced ototoxicity (6-8). Curcuminoids, including curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC), reportedly exert antioxidant effects (9-11). Curcumin, the major component in turmeric, has been extensively studied (12-15). It protects rat cochlear fibroblasts and might prevent apoptotic pathway in gentamicin-induced ototoxicity (16, 17). To the best of our knowledge, there are no reports on BDMC, one of the rarest compounds in nature, in relation to AG-induced ototoxicity. As BDMC is safe with a highly stable structure and good bioavailability among curcuminoids, it might be a potential candidate worth of in-depth research (18).

The aim of our study was to clarify the effects of BDMC on hair cell death pathway during gentamicin-induced damage in mouse cochlear UB/OC-2 cells. We also quantified the cell membrane potential, regulators, and cascade in the hair cells.

Materials and Methods

Drug preparation. Powdered rhizomes of Curcuma longa (2.5 kg) were extracted with ethanol (570 g) at room temperature. The crude extract was suspended in water and partitioned with n-hexane, CHCl3, and ethyl acetate to obtain four fractions. The ethyl acetate-soluble fraction (20 g) was dissolved in methanol, passed through a Sephadex LH-20 column (Sigma-Aldrich, St. Louis, MO, USA), and monitored by a silica gel thin-layer chromatography. The subfraction (2 g) was chromatographed on a silica gel column using gradient elution (70-230 mesh, 0-10% methanol in CHCl3) to obtain a pigment. The pigment was identified on a 400 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) and liquid chromatography-ESI-mass spectrometer (liquid chromatography from Dionex Ultimate 3000, Thermo Scientific, Waltham, MA, USA; HCT ultra PTM discovery system from Bruker Daltonics, Bremen, Germany). The purified component was at least 99% pure, which was determined using an HPLC gradient system and a UV-visible detector at 425 nm.

Cell culture and drug treatment. Mouse cochlear UB/OC-2 cells were obtained from Ximbo (London, UK) and cultured in MEM GlutaMAXTM medium (Gibco, NY, USA) containing 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA) and 50 U/ml interferon-γ (R&D system, Minneapolis, MN, USA) under a humidified atmosphere of 5% CO2 at 33°C. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin (Standard Chem & Pharm Co., Ltd, Tainan, Taiwan, ROC) for 24 h. UB/OC-2 cells were divided into four groups: the control group (no treatment), the gentamicin group (treated with 1.25 mM gentamicin), the BDMC pretreatment group (pretreated with 5 μM BDMC for 2 h and then co-incubated with 1.25 mM gentamicin for the indicated times), and the BDMC group (treated with 5 μM BDMC).

Cell viability assay. Cells were seeded into 24-well plates at a density of 4×104 cells/well and incubated overnight. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT solution; VWR International, Radnor, PA, USA) was added to each well to achieve a final concentration of 0.5 mg/ml for the last 4 h. Subsequently, the medium was removed, and dimethyl sulfoxide was added to dissolve crystals. The absorbance was determined at 570 nm using a microplate reader (Infinite 200 PRO Series Multimode Reader; TECAN, Zürich, Switzerland), and the control absorbance was normalized to 100% cell viability.

Cell cytotoxicity assay. Cells were seeded into 24-well plates at a density of 4×104 cells/well and incubated overnight. After cells were subjected to different treatments for 24 h, lactate dehydrogenase (LDH) activity was measured in the cell supernatant according to the manufacturer’s instructions using the LDH Cytotoxicity Assay Kit (Enzo Life Sciences, Farmingdale, NY, USA). The absorbance was measured using a microplate reader at 490 nm (Infinite 200 PRO Series Multimode Reader).

Cell cycle. Cells were seeded into 6-well plates at a density of 4×105 cells/well and incubated for 24 h. Subsequently, cells under different treatments were collected, fixed with 70% ethanol, and stored at -20°C overnight. Cells were then washed with phosphate-buffered saline (PBS) and stained with 50 μg/ml propidium iodide (PI; Sigma-Aldrich) at 33°C for 30 min. The cell population was detected using a BD Accuri™ C6 flow cytometry system (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm.

Analysis of intracellular reactive oxygen species (ROS) production. Cells were seeded into 6-well plates at a density of 4×105 cells/well and incubated for 24 h. Then, those under different treatments were incubated with 10 μM 2’,7’-dichlorofluorescein diacetate (DCFDA; Enzo Life Sciences) for 30 min at 33°C and re-suspended in PBS to detect intracellular ROS levels. Approximately one million cells per group were acquired and the fluorescence intensity was measured using a BD Accuri™ C6 flow cytometry system (BD Biosciences) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Mitochondrial membrane potential analysis. Cells were seeded into 6-well plates at a density of 4×105 cells/well and incubated for 24 h. Cells under different treatments were incubated with 5 μM JC-1 dye (Enzo Life Sciences) for 10 min at 33°C and re-suspended in PBS to examine changes in mitochondrial membrane potential. Approximately one million cells per group were acquired and cell fluorescence was measured using a BD Accuri™ C6 flow cytometry system (BD Biosciences) at an excitation wavelength of 488 nm and an emission wavelength of 520-570 nm.

Cell fractionation. Cells were seeded into 10-cm dishes at a density of 3×106 cells and incubated overnight. After cells were subjected to different treatments for 4 or 24 h, cytosolic and mitochondrial fractions were isolated using the Cell Fractionation Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. Each fraction (30 μg of protein) was resolved on a 15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA), and immunoblotted with anti-cytochrome c antibody (Cell Signaling Technology, Beverly, MA, USA).

Western blot analysis. Total cellular protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitors (Sigma-Aldrich). Proteins (30 μg) were resolved on 8-15% SDS-PAGE and transferred to polyvinylidene fluoride membranes, blocked with 3% (w/v) bovine serum albumin (Sigma-Aldrich) at 37°C for 1 h, and then incubated with specific primary antibodies at 1:2000 dilutions in blocking buffer at 4°C for 24 h. The primary antibodies used were against: Caspase-9, cleaved Caspase-9, Caspase-3, cleaved Caspase-3, PARP, cleaved PARP, Bcl-2, Bax, cytochrome c, COX IV, and β-actin (Cell Signaling Technology). Protein bands were detected after incubating with a horseradish peroxidase-conjugated secondary antibody (PerkinElmer Life Sciences, Boston, MA, USA), visualized by enhanced chemiluminescence (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and detected using the KETA C Chemi imaging system (Wealtec Corporation, Sparks, NV, USA).

Cell apoptosis analysis. Cells were seeded into 6-well plates at a density of 4×105 cells/well and incubated overnight. After cells were subjected to different treatments for 24 h, the level of apoptotic cells was measured using the Annexin V-FITC Apoptosis Detection Kit (Abcam) following the manufacturer’s instructions. Approximately one million cells per group were acquired and the cell population was detected using a BD Accuri™ C6 flow cytometry system (BD Biosciences).

Nucleic acid detection. Cells were seeded into 6-well plates at a density of 4×105 cells/well and incubated for 24 h. Thereafter, cells under different treatments were fixed with methanol for 10 min at room temperature and incubated with 20 μg/ml Hoechst 33258 dye (Enzo Life Sciences) for 20 min at 33°C to detect chromatin changes in apoptotic cells. Fluorescence images were obtained using an Olympus BX41 microscope (Tokyo, Japan) at an excitation wavelength of 352 nm.

Statistical analysis. Data are presented as the mean±standard deviation (SD) of at least three independent experiments. Comparisons between two groups were performed using the Student’s t-test. Statistical significance was set at p<0.05.

Results

Gentamicin-induced cytotoxicity was reversed by treatment with BDMC. To investigate the cytotoxicity of gentamicin in mouse cochlear UB/OC-2 cells, we tested the cell viability for 24 h using the MTT assay. Gentamicin significantly inhibited cell viability in a dose-dependent manner (Figure 1A). The viability of UB/OC-2 cells decreased to approximately 50% at 1.25 mM gentamicin, and thus 1.25 mM gentamicin was used for subsequent experiments. Next, we examined the cytotoxicity of BDMC on mouse cochlear UB/OC-2 cells. Concentrations up to 5 μM BDMC had little to no effect on cell viability. However, 7.5 and 10 μM BDMC demonstrated cytotoxic effects on cells (Figure 1B). Therefore, 5 μM BDMC was selected for subsequent experiments. To examine the protective effect of BDMC against toxicity induced by gentamicin, we performed the LDH release analysis. Compared to the control, the cell cytotoxicity significantly increased in the gentamicin group and decreased in the BDMC pretreatment group (Figure 1C). These results suggested that BDMC protected UB/OC-2 cells from gentamicin-induced cell injury.

Figure 1.
Figure 1.

Effect of gentamicin and bisdemethoxycurcumin (BDMC) on cell viability and cytotoxicity in gentamicin-treated UB/OC-2 cells. Cell viability after treatment with (A) gentamicin (0-1.5 mM) and (B) BDMC (0-10 μM), for 24 h. (C) Cells were pretreated with or without 5 μM BDMC for 2 h and then incubated with or without 1.25 mM gentamicin for 24 h. The cytotoxicity was measured by the lactate dehydrogenase (LDH) release assay. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01; ***p<0.001.

Treatment with BDMC reversed gentamicin-induced cell cycle arrest at the G2/M phase. To determine the effects of BDMC on gentamicin-induced changes in the cell population, we analyzed the cell cycle by flow cytometry, which revealed that the gentamicin caused cell cycle arrest in the G2/M phase in the gentamicin group, while BDMC effectively reversed gentamicin-mediate G2/M cell cycle arrest in the BDMC pretreatment group (Figure 2A and B). Thus, pretreatment with BDMC inhibited gentamicin-induced cell damage at least partially by attenuating the G2/M-arrest.

Figure 2.
Figure 2.

Effect of bisdemethoxycurcumin (BDMC) on cell cycle progression in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. (A) Cell cycle distribution was measured by flow cytometry. (B) Quantitative analysis of the proportion of cells in each cycle phase. Data are presented as the mean±SD of three independent experiments. **p<0.01; ***p<0.001.

Treatment with BDMC inhibited gentamicin-induced ROS production and mitochondrial dysfunction. Gentamicin-induced apoptosis relies on ROS production and mitochondrial dysfunction (19). Hence, to explore the protective effects of BDMC on cells against gentamicin-induced apoptosis, we evaluated intracellular ROS levels using the fluorescent DCFDA dye. Intracellular ROS generation significantly increased in UB/OC-2 cells in the gentamicin group, but was remarkably reduced upon pretreatment with BDMC (Figure 3A). Excessive generation of ROS after gentamicin exposure leads to toxicity due to mitochondrial membrane permeability (20). To unravel the influence of BDMC on gentamicin-induced oxidative damage, mitochondrial membrane potential was analyzed by the ratio of JC-1 green/red fluorescence intensity after different treatments. The ratio of JC-1 green/red fluorescence intensity increased in the gentamicin group than that in the control group, and the ratio decreased in the BDMC pretreatment group, compared to the gentamicin group (Figure 3B).

Figure 3.
Figure 3.

Effect of bisdemethoxycurcumin (BDMC) on intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential in gentamicin-treated UB/OC-2 cells. (A) Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. (A) ROS production [dichlorofluorescein diacetate (DCFDA) assay] and (B) mitochondrial permeability (JC-1 staining), respectively, and analyzed by flow cytometry. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01.

Cytochrome c is released from the mitochondria to the cytosol via the disruption of membrane integrity in damaged mitochondria and is implicated in the mitochondrial apoptotic pathway (21). To elucidate whether BDMC exerts protective effects against gentamicin-induced apoptotic cell death by regulating cytochrome c, we examined the cytochrome c distribution between the cytosol and mitochondria. Compared to the control group, gentamicin exposure led to a cytosolic accumulation of cytochrome c, whereas pretreatment with BDMC significantly reduced cytochrome c release from mitochondria (Figure 4A and B). Hence, pretreatment with BDMC may have inhibited gentamicin-induced oxidative toxicity by suppressing ROS production and ameliorating mitochondrial dysfunction.

Next, we analyzed the expression of Bcl-2 family proteins, including pro-apoptotic (Bax) and anti-apoptotic proteins (Bcl-2), following different treatments. The expression of the pro-apoptotic protein Bax increased in the gentamicin group compared to the control group, while it was decreased in the BDMC pretreatment group compared to the gentamicin group. Furthermore, the level of the anti-apoptotic protein Bcl-2 was down-regulated in the gentamicin group compared to the control group, whereas Bcl-2 expression was significantly increased in the BDMC pretreatment group compared to the gentamicin group (Figure 4C).

Figure 4.
Figure 4.

Effect of bisdemethoxycurcumin (BDMC) on mitochondrial-related proteins in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. Expression levels of cytochrome c in cytosolic (A) and mitochondrial fractions (B) were measured by western blot analysis. (C) Expression levels of Bcl-2 and Bax were evaluated by western blot analysis. β-actin and COX IV were used as loading controls. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01.

Treatment with BDMC reduced gentamicin-induced apoptosis. To examine the intrinsic apoptotic pathway involved in gentamicin-induced cell death, western blot analysis was used to detect apoptosis-related proteins, such as the active cleaved form of Caspase-3, Caspase-9, and PARP. As shown in Figure 5A-C, the protein expression of cleaved Caspase-3, cleaved Caspase-9, and cleaved PARP significantly increased in the gentamicin group; while pretreatment with BDMC reduced the expression of the aforementioned proteins induced by gentamicin. These data support the inhibitory effect of BDMC on gentamicin-induced apoptosis in UB/OC-2 cells.

Figure 5.
Figure 5.

Effect of bisdemethoxycurcumin (BDMC) on apoptosis-related proteins in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. Expression levels of Caspase-9 and cleaved Caspase-9 (A), Caspase-3 and cleaved Caspase-3 (B), and PARP and cleaved PARP (C) were measured by western blot analysis. β-actin was used as the loading control. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01; ***p<0.001.

We next investigated the effects of BDMC on gentamicin-mediated cell death by flow cytometry using Annexin V/PI double staining to determine cell damage. Compared to the control group, the percentage of apoptotic cells significantly increased in the gentamicin group, whereas the number of apoptotic cells in the BDMC pretreatment group substantially decreased compared to that of the gentamicin group (Figure 6A). Furthermore, the bright blue fluorescence was significantly lower in the BDMC pretreatment group than in the gentamicin group (Figure 6B), showing that BDMC pretreatment prevented the apoptotic nuclear condensation caused by gentamicin.

Figure 6.
Figure 6.

Effect of bisdemethoxycurcumin (BDMC) on cell death in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. (A) The percentage of apoptotic cells was assessed by Annexin V/propidium iodide (PI) assays using flow cytometry. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01. (B) Condensed or fragmented nuclei of the apoptotic cells were observed by Hoechst 33258 staining using a fluorescence microscope (100×). Scale bar=200 μm.

Discussion

In our study, BDMC, isolated from turmeric, was evaluated for its effects on cell cycle progression and apoptosis pathway. Our results revealed that BDMC down-regulated cell cycle arrest in the G2/M phase, stabilized mitochondrial membrane potential, decreased cleaved caspase proteins, and successfully reversed hair cell apoptosis. Therefore, BDMC may serve as a potential agent to reduce gentamicin-induced ototoxicity.

Comparison of BDMC with other curcuminoids. Ramsewak et al. reported that all extracts of Curcuma longa (curcumin, DMC, and BDMC) showed antioxidative capacity (9, 11). The antioxidant effects are associated with the hydrogen on the phenolic or central methylene groups (22). Although curcumin is extensively studied owing to its abundance in turmeric (curcumin:DMC:BDMC=71:26:3), BDMC is highly stable in vivo and presents good bioavailability (15, 22). Kharat et al. reported that curcumin easily degrades in alkaline environments (pH≥7) (23). The enol form of curcumin, present at alkaline pH, becomes water insoluble (22). These disadvantages are overcome by BDMC, which differs from DMC and curcumin by lack of o-methoxy groups on its phenolic rings. Sudeep et al. examined the gastrointestinal digestion of BDMC and curcumin in male rats and reported significantly higher bioavailability of BDMC, with a 4.4-fold higher plasma concentration than that of curcumin (18). Moreover, the antioxidant capacity of BDMC was superior to that of curcumin at the same concentration (18).

Administration routes of BDMC into the cochlea. The routes of medicine delivery into the cochlear include the following: locally administered intratympanically, systemic administration by either orally or intravenously, and intracochlear or intrasemicircular canal administrations, which are more invasive and are avoided (24-26). In the intratympanic route, the major burden is the round window membrane (26). After entering the cochlea, medicines typically form an axial gradient, with the concentration index from base to apex. However, no definite model can precisely predict the permeability of membranes in the ear (25). Generally, lipid-soluble, small, and non-polar molecules tend to pass through the round window membrane more readily (25). BDMC is the most stable in non-polar solvents of the curcuminoids (18).

Further research on BDMC intratympanic delivery is needed. Systemic administration of BDMC is more commonly used, as BDMC has good bioavailability. The exact effects of BDMC passing through the tight junction within the blood–labyrinth barrier is still not clear.

BDMC as a potent agent against gentamicin ototoxicity. In our study, the cochlear cell line UB/OC-2 was treated with gentamicin at different doses to determine the experimental concentration, which was 1.25 mM, to reach 50% cell viability. The results elucidated the cytotoxicity of gentamicin; they also showed that UB/OC-2 cell death was dose dependent. We found that BDMC reversed gentamicin-induced cell cycle distribution in the G2/M phase (Figure 2). In the following experiments, BDMC successfully reversed cochlear cell death induced by gentamicin, with decreased LDH formation (Figure 1C). Serial examinations revealed that BDMC decreased intracellular ROS level, stabilized mitochondrial membrane potential (Figure 3), decreases the release of cytochrome c from the mitochondria to cytosol (Figure 4), leading to the down-regulation of mitochondrial-dependent apoptotic proteins (Figure 5), and eventually inhibiting cochlear cell apoptosis caused by gentamicin (Figure 6).

Limitations of the study. Our results revealed that BDMC has potential protective effect against gentamicin-induced ototoxicity. However, this effect was only proved in the UB/OC-2 cell line. Owing to the lack of information on the half-life of BDMC in the inner ear in vivo, the exact timing of treatment with different concentrations is worth further testing. Routes of medicine delivery into the inner ear, such as intra-tympanic and systemic administrations, should be further analyzed in animal models. These studies will help determine the appropriate route and optimal treatment concentration in the inner ear, without cytotoxicity to other organ systems. Furthermore, it is not clear whether BDMC influences the antimicrobial efficacy of gentamicin or not.

Future perspectives of BDMC. The study suggests BDMC as a safe agent for cochlear cells and with protective effects against gentamicin ototoxicity. Since gentamicin may activate cochlear cell apoptotic intrinsic pathway mainly through generation of ROS (27), antioxidant effects of BDMC and related signaling pathways may be warranted for further investigation.

Conclusion

BDMC attenuates gentamicin-induced ototoxicity in mouse cochlear UB/OC-2 cells, including cytotoxicity, cell cycle arrest, ROS production, mitochondrial membrane potential reduction, and apoptosis-related protein expression. Thus, our study provides evidence that BDMC might be a potent agent for alleviating gentamicin ototoxicity.

Acknowledgements

This research was supported by grants from the Buddhist Tzu Chi Medical Foundation (TCMF-CP 111-09, TCMF-CM2-111-02 and TCMF-A 108-03) and the Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (TTCRD 110-03).

Footnotes

  • Authors’ Contributions

    Kang TY, Hsu CJ and Lin JN conceived and designed the experiments. Kang TY, Lin JN, Lin HY, Yu SH and Wu RS performed the experiments. Kang TY, Lin JN, Yu SH and Wu RS analyzed the data. Wu CC, Wang JS, Wen YH and Tseng GF contributed reagents/materials/analysis tools. Kang TY and Lin JN wrote the article. Hsu CJ and Wu HP revised the article. All authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest with the contents of this article.

  • Received February 11, 2022.
  • Revision received March 18, 2022.
  • Accepted April 1, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Bisdemethoxycurcumin-mediated Attenuation of Apoptosis Prevents Gentamicin-induced Ototoxicity in Mouse Cochlear UB/OC-2 Cells
TING-YA KANG, CHUAN-JEN HSU, JIA-NI LIN, CHEN-CHI WU, JEN-SHU WANG, HUI-YI LIN, SZU-HUI YU, RONG-SHUAN WU, YU-HSUAN WEN, GUO-FANG TSENG, HUNG-PIN WU
In Vivo May 2022, 36 (3) 1095-1105; DOI: 10.21873/invivo.12807
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