The use of amikacin (AMK) in present treatment strategies results in severe ototoxicity; however, the underlying molecular mechanisms of this toxicity remain unclear. In this study, we investigated the effectiveness of orally administered pomegranate peel extract (PPE), a strong antioxidant, as a protective agent against AMK-induced ototoxicity. To this end, PPE was orally administered to adult BALB/c mice for 5 days, and the mice were then concurrently treated with AMK (500 mg/kg/day for 15 consecutive days). Auditory threshold shifts induced by AMK were significantly attenuated. The results of immunohistochemical staining and western blot analysis revealed that PPE exerted its protective effects by by downregulating the phosphorylation of Forkhead box O3a (FoxO3a), an important transcription factor which is involved in the responses to oxidative stress. The results also showed that PPE treatment inhibited mitogen-activated protein kinase phosphorylation, prevented the activation of pro-apoptotic protein caspase-3, decreased the levels of apoptosis-inducing Bax protein, and increased the levels of the anti-apoptotic mediator, Bcl-2, induced by AMK in the mouse cochlea. Taken together, our experimental findings suggest that phosphorylated FoxO3a mediates AMK-induced apoptosis in BALB/c mice cochlea. PPE effectively attenuated oxidative stress and ototoxicity by regulating FoxO3a, and may thus prove to be beneficial in protecting auditory cells from ototoxic drugs.
Amikacin (AMK) is one of the aminoglycoside antibiotics most often used for the treatment of severe, hospital-acquired infections due to multidrug resistant Gram-negative bacteria (
Forkhead box proteins O (FoxO) are a subclass of the forkhead family of transcription factors, and have been linked to the regulation of oxidative stress and cellular apoptosis (
In this study, we evaluated the protective effects of PPE on AMK-induced ototoxicity and the potential underlying mechanisms
PPE was purchased from Xi'an Acetar Bio-Tech Inc. (Shaanxi, China). PPE, a brown powder that contains ≥98% ellagic acid (phenolic compound with antioxidant and anti-inflammatory effects), was dissolved in saline for gavage. AMK sulfate injections (0.1 g/ml) were purchased from Qilu Pharmaceutical Co., Ltd. (Shandong, China). Rabbit polyclonal anti-FoxO3a (ab109629), anti-phospho-FoxO3a (ab47285) and anti-4-hydroxynonenal (4-HNE; ab46545) primary antibodies were from Abcam (Cambridge, UK); rabbit polyclonal anti-p38 (8690), anti-phospho-p38 (9215), anti-ERK 1/2 (4695), anti-phospho-ERK (4370), anti-JNK (9252), anti-p-JNK (9251), anti-Bcl-2 (2876), anti-Bax (5023) and anti-cleaved caspase-3 (9579) were from Cell Signaling Technology, Inc. (Danvers, MA, USA).
All animal manipulations were conducted in accordance with the regulations for the Management of Laboratory Animals published by the Ministry of Science and Technology of the People's Republic of China, and was approved by the Institutional Animal Care and Use Committee of Jinzhou Medical University. BALB/c mice (18–22 g), which were 6–8 weeks old and had normal auropalpebral reflexes and otomicroscopic examination results, were purchased from the Animal Experimental Center of Dalian Medical University [Liaoning, China; license no. SCXK (Liao) 2008-0002]. All mice were fed a standard commercial diet, and housed at an ambient temperature of 22°C with a relative humidity of 50±5% under 12 h/12 h light-dark cycle in a specific pathogen-free facility.
The experimental mice were divided into 4 groups (n=20 ears in each group): i) the control group received physiological saline (100
For the analysis of the auditory threshold, the auditory brainstem response (ABR) was recorded 1 day before and 15 days after AMK treatment with tone bursts of 8, 12, 24 and 32 kHz (1-msec rise/fall time, 2-msec plateau) using the Smart EP and OAE auditory evoked potential recording system (Intelligent Hearing Systems Co., Miami, FL, USA). The mice were anesthetized using pentobarbital sodium (40 mg/kg) and kept warm with a heating pad during ABR recording. A subdermal (active) needle electrode was inserted at the vertex, while ground and reference electrodes were inserted subdermally in the loose skin beneath the pinnae of opposite ears. The technique used to record ABRs has been previously described in detail (
After the ABR test, the temporal bones were harvested. Each bulla was opened using rongeurs to expose the cochlea. The oval and round windows were then opened. Following the creation of a hole in the cochlea apex, 4% paraformaldehyde was perfused through the cochlea for at least 24 h. The cochlea was decalcified in 4% EDTA for 7 days at 4°C. Subsequently, the basilar membrane was dissected under a dissecting microscope, and the stria vascularis and tectorial membrane were removed. To identify F-actin in the organ of Corti, tetramethyl rhodamine isothiocyanate (TRITC) (Sigma-Aldrich) was applied for 20 min at room temperature and protected from light. The specimens were then rinsed 3 times with 0.01 M phosphate-buffered saline (PBS) (pH 7.4). Fluorescence signals from the hair cells were counted under a BX41 microscope with epifluorescence (Olympus, Tokyo, Japan), and the images were obtained with TCS-SP5II laser-scanning confocal microscope (Leica Biosystems, Wetzlar, Germany). Three rows of the outer hair cells (OHCs) were counted from the apex through the basilar turn of the cochlea under ×200 magnification in 20 consecutive fields.
Tissue sections were incubated with rabbit anti-4-HNE antibody (1:200 dilution; Abcam), followed by appropriate fluorescent-secondary antibody for 1 h at room temperature. After the samples were counterstained with DAPI, immunohistofluorescence images were obtained via confocal microscopy (TCS-SP5II; Leica Biosystems). Immunofluorescence analysis was performed as previously described (
Cochlea tissue was homogenized in 0.01 M cold phosphate buffer (pH 7.4) using a homogenizer. The homogenate was centrifuged at 1,500 × g for 10 min. Tissue supernatant was collected and measured for oxidative stress using the MDA assay kit [2-thio-barbituric acid (TBA) method]. SOD and CAT activities were assessed using the SOD and CAT assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's instructions.
Total cochlea tissue was homogenized in RIPA buffer (Aidlab Biotechnologies Co., Ltd, Beijing, China) containing protease inhibitor cocktail tablets and phosphostop cocktails (cOmplete; Roche Diagnostics GmbH, Mannheim, Germany). The tissue homogenate was sonicated for 30 sec and centrifuged at 12,000 rpm at 4°C for 30 min to extract the supernatant. Protein concentrations were determined using the BCA kit (Thermo Fisher Scientific, Inc., San Francisco, CA, USA). The protein samples (30
Data were analyzed using Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA), and presented as mean ± SD. Comparisons of parameters between different groups were performed by one-way analysis of variance (ANOVA), followed by a Newman-Keuls test. A value of P<0.05 was considered to indicate a statistically significant difference.
To verify whether PPE prevents hearing loss induced by AMK, the ABR of the experimental groups (n=20 ears for each group, two from each animal) was recorded. The ABR test showed that mice in the control group maintained stable thresholds throughout the experiment. By contrast, ABR threshold shifts in the AMK group were significantly elevated at 16, 24 and 32 kHz (25.50±3.40, 45.75±9.07 and 53.55±8.97 dB, respectively) compared with the control group (6.30±2.08, 7.15±2.94 and 8.00±2.73 dB, respectively) after continuous injections for 15 days, and the hearing functional deficit was greater at the higher frequency. However, following concurrent treatment with PPE and AMK for 15 days, the ABR threshold shifts were markedly reduced compared to the AMK group at 16, 24 and 32 kHz (13.95±2.98, 30.15±3.85 and 33.50±5.40 dB). PPE alone had no effect on the ABR threshold shifts (
AMK signifincantly increased the staining for 4-HNE in the organ of Corti, spiral ganglion and stria vascularis of the mouse cochleae. However, the production of 4-HNE was inhibited in the mice treated with AMK plus PPE. However, PPE alone had no effect on the expression of 4-HNE in the mouse cochleae, as indicated by immunohistochemical analysis (
Immunofluorescence staining revealed a significantly increased phosphorylation of FoxO3a in the organ of Corti, spiral ganglion and stria vascularis of the cochleae of mice treated with AMK, which was prevented to a significant degree in mice treated with AMK plus PPE (
The results of western blot analysis revealed the activated phosphorylation of ERK 1/2 (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185) and phospho-p38 MAPK (Thr180/Tyr182), and the upregulated protein expression of JNK in the cochleae of mice treated with AMK. Of note, all these effects were inhibited in the mice treated with AMK plus PPE (
The results of western blot analysis revealed that the protein expression of Bcl-2 was degraded, while the expression of Bax and cleaved caspase-3 was upregulated in the cochleae of mice in the AMK group. However, PPE inhibited the degradation of Bcl-2 and prevented the activation of caspase-3 in the cochleae of mice treated with AMK plus PPE (
Hair cell loss and the activation of oxidative stress may play crucial roles in the development and progression of aminoglycoside-induced ototoxicity (
AMK has been shown to significantly enhance the ABR threshold shifts in guinea pigs (
Oxidative stress plays an important role in the pathogenesis of drug-induced ototoxicity. 4-HNE, an aldehydic product of lipid peroxidation, has been implicated in the etiology of pathological changes under oxidative stress as a key mediator of oxidative stress induced cell death (
FoxO3a has recently been widely investigated as a transcription factor that is involved in regulation of the stress response, apoptosis and autophagy (
Recent studies have shown that the overexpression of Bcl-xL prevented gentamicin-induced hair cell apoptosis on the cochlea in mice (
In conclusion, our
This study was funded by the Jinzhou Medical University Youth Science and Technology Staring Foundation Program (Y2012Z018), Liaoning Science and Technology Program (2014022029), Liaoning Education Program (L2015316), Undergraduate of Liaoning Province Innovation and Entrepreneurship Training Program (201410160023).
Pomegranate peel extract (PPE) prevents amikacin (AMK)-induced hearing loss in mouse. Auditory brainstem response (ABR) threshold shifts. Data are presented as the means ± SD (*P<0.05, the AMK group vs. the control group; #P<0.05, the AMK plus PPE group vs. the AMK group, n=20 ears for each group).
Pomegranate peel extract (PPE) prevents amikacin (AMK)-induced hair cell loss in mice. (A) Hair cells outlined by tetramethyl rhodamine isothiocyanate (TRITC) staining in the lower basal turn of the cochlea. Scale bar, 20
Pomegranate peel extract (PPE) alleviates oxidative stress in mice with amikacin (AMK)-induced ototoxicity. (A) Effect of PPE on AMK-induced expression of 4-hydroxynonenal (4-HNE) in mouse cochlea. Images of paraffin sections of the organ of Corti, spiral ganglion, and stria vascularis from BALB/c mice. The sections were labeled for 4-HNE (red) and nuclear-stained with DAPI (blue). The arrows indicate the location of 4-HNE-positive expression. White scale bar, 25
Pomegranate peel extract (PPE) inhibits amikacin (AMK)-induced phosphorylation of forkhead box O3a (FoxO3a) in mouse cochlea. (A) Representative immunohistochemical images of staining for phosphorylation of FoxO3a in cochlea of mice treated with AMK. Scale bar, 25
Pomegranate peel extract (PPE) inhibits the activation of the mitogen-activated protein kinase (MAPK) signaling pathway in cochlea of mice treated with amikacin (AMK). (A) Western blot analysis was used to quantify total and phosphorylated protein expression of ERK 1/2, JNK and p38 in cochlea, with β-actin as a loading control. Statistical analysis of the expression levels of (B) p-ERK/ERK; (C) p-JNK/JNK; and (D) p-p38/p38 (*P<0.05; n=6 ears for each group, and mean values were obtained in n=3 independent experiments).
Pomegranate peel extract (PPE) attenuates the expression of apoptotic family proteins in cochleae of mice treated with amikacin (AMK). (A) Western blot analysis of the protein expression levels of Bcl-2 and Bax in cochlea, with β-actin as a loading control; (C) Western blot analysis of the protein expression levels of cleaved caspase-3, with β-actin as loading control; statistical analysis of the expression levels of (B) Bcl-2/Bax, and (D) caspase-3/β-actin (*P<0.05; n=6 ears for each group, and mean values were obtained in n=3 independent experiments).