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Effects of Korean Red Ginseng against Ototoxic Drugs and Age-related Cochleo-vestibular Dysfunction

  • Chunjie Tian (Department of Otolaryngology Ajou University School of Medicine)

초록/요약

Gentamicin (GM) is one of the most widely used aminoglycoside antibiotics. However, it can damage inner ear hair cells and cause both hearing loss and vestibular impairment. 3-Nitropropionic acid (3-NP), a mitochondrial toxin, has been reported to induce an acute cochlear damage. There are no therapeutic interventions have been developed for age related inner ear disorder which is one of the most common sensory deficits in the elderly. Korean red ginseng (KRG) with its ginsenosides has neuron beneficial effects and anti-ageing effects. In the present study, the authors evaluated (1) the protective effects of KRG against gentamicin (GM)-induced unilateral vestibular and hearing dysfunction and investigated its effective mechanism using in vitro cell cultures; (2) the protective effects of KRG in an ototoxic animal model BALB/c mice using 3-NP; (3) the effects of KRG on age-related hearing loss (AHL) and balance disturbance in C57BL/6 mouse. In the first study, Sprague-Dawley rats were classified into GM group (n = 12) and KRG+GM group (n = 10). Head tilt, tail hanging, and swimming tests for balance function and auditory brainstem response test (ABR) for hearing function were performed for hearing and vestibular function evaluation. Cochleae and utricles/saccules were harvested for scanning electron microscope (SEM). The ventral otocyst-epithelial clone 36 (VOT-E36) cell was used to explore protective mechanism of Ginsenoside Rb1. In the second study, Dose-dependent toxic effects of 3-NP were investigated for an appropriate toxicity level of 3-NP, and then 23 mice were grouped into 3-NP (n = 12) and KRG + 3-NP (n = 11) groups for observation of the protective effects of KRG. ABR and cochlear morphological evaluations were performed before and after drug administration. In the third study, 22 mice were randomly distributed into control (n = 8), KRG (150 mg/kg, n = 7), and KRG (500 mg/kg, n = 7) groups. Hearing/vestibular function and related morphology were evaluation during ageing process. In the first study, Vestibular function was comprehensively evaluated by a scoring system that ranged from 0 (normal) to 3 (worst) points, using head tilt, tail hanging, and swimming tests. The GM group showed significantly more deteriorated vestibular function (0 point – 5 rats, 1 point – 1 rat, 2 points – 3 rats, and 3 points – 3 rats) than the KRG + GM group (0 point – 9 rats and 1 point – 1 rat) (p < 0.01). The hearing thresholds were better in the KRG + GM group than in the GM group (p < 0.05). Quantitative analysis of hair cell damage in the scanning electron microscopy was closely related with vestibular and hearing functional results. In vitro study showed that ginsenoside Rb1 (gRb1) attenuated reactive oxygen species production, suppressed JNK activation, up-regulated Bcl-xL and down-regulated Bax, cytochrome c, caspase 3, and cleaved poly (ADP-ribose) polymerase in GM-treated VOT-E36 cells. In the second study, The ABR thresholds in the 800–5000 mM groups exceeded the maximum recording limit at 16 and 32 kHz 1 day after 3-NP administration. The ABR threshold in the 500 mM 3-NP + KRG group was significantly lower than that in the 500 mM 3-NP group from post 1 week to 1 month. The mean type II fibrocyte counts significantly differed between the control and 3-NP groups and between the 3-NP and 3-NP + KRG groups. Spiral ganglion cell degeneration in the 3-NP group was more severe than that in the 3-NP + KRG group. In the third study, No mice in the control and KRG1 groups died at the age of 12 month, however, 4 in 7 mice died in the KRG (500 mg/kg) group. ABR recording demonstrated high frequency hearing loss at 32 kHz (38.8 ± 5.6 dB) at 6 month of age, and 16 kHz (41.9 ± 6.0 dB) at 9 month of age, elevated to 52.2 ± 8.6 dB at 16 kHz and 55.6 ± 8.3 dB at 32 kHz at 12 month in the control group. However, the thresholds shift was delayed significantly (P < 0.05) in the KRG (150 mg/kg) group (from the age of 6 month (32.1 ± 5.7 dB) to 12 month (50.0 ± 16.6 dB) at 32 kHz, and 9 month (34.6 ± 8.2 dB) to 12 month (47.9 ± 18.1 dB) at 16 kHz). The hearing thresholds increased to 61.7 ± 20.7 dB at 16 kHz and 64.2 ± 16.9 dB at 32 kHz at 12 month in the 500 mg/kg fed mice. The age-associated vestibular dysfunction was observed with tail hanging and swimming tests, the severity score in the tail hanging test and swimming time in swimming test were significantly different between the KRG (150 mg/kg) group and the control group at 12 month (P < 0.05). Histological observation supported the hearing and vestibular function findings. The number of the Bcl-xL immunopositive SGCs and type II fibrocytes was markedly more in KRG (150 mg/kg) group than that in the control group (P < 0.05). These findings suggest that KRG including gRb1 component protects against vestibular/hearing dysfunction by inhibiting apoptotic pathways when ototoxicity is induced by unilateral intratympanic injection with GM in rats. Mice model of 3-NP-induced hearing loss exhibited a dose-dependent hearing loss with histological changes. KRG administration ameliorated the deterioration of hearing by 3-NP. C57BL/6J mouse showed early onset of hearing and vestibular dysfunction with ageing, which were delayed by KRG treatment in dose of 150 mg/kg by inhibiting mitochondrial apoptotic pathway; however, long-term treatment of 500 mg/kg KRG may induce aggressive behavior and aggravate age-related hearing and balance dysfunction. In conclusion, Korean red ginseng might be an effective treatment for protecting hearing loss caused by various etiologies.

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목차

TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES ix

PART 1 Korean red ginseng protects against gentamicin-induced balance dysfunction and hearing loss in rats through antiapoptotic functions of ginsenoside Rb1 １
I. INTRODUCTION １
II. MATERIALS AND METHODS ３
A. Preparation of animal study groups ３
B. Evaluation of vestibular function ５
C. Evaluation of hearing function ５
E. Cell culture and viability assays ６
F. Intracellular levels of reactive oxygen species (ROS) ６
G. Western blot assay ７
H. Statistical analysis ８
III. RESULTS ９
A. Evaluation of vestibular/balance function ９
B. Otolith organ (saccule and utricle) morphology １３
C. Evaluation of hearing function １５
D. Cochlear morphology １５
E. gRb1 exerts a protective effect in vitro １７
IV. DISCUSSION ２３
V. CONCLUSION ２６

PART 2 Korean red ginseng ameliorates acute 3-nitropropionic acid-induced cochlear damage in mice ２７
I. INTRODUCTION ２７
II. MATERIALS AND METHODS ３０
A. Animals ３０
B. Korean red ginseng ３０
C. Study design ３０
D. Intratympanic administration of 3-NP ３１
E. Auditory brainstem response (ABR) measurement ３１
F. Morphological evaluation ３２
G. Statistical analysis ３３
Ш. RESULTS ３４
A. 3-NP dose-dependent hearing threshold shift ３４
B. Histological analysis after 3-NP administration ３６
C. Protective effects of KRG on 3-NP-induced hearing threshold shift ４０
D. Histological analysis after KRG treatment ４２
IV. DISCUSSION ４８
V. CONCLUSION ５４

PART 3 Korean red ginseng delays age-related hearing loss and vestibular dysfunction in C57BL/6 mouse ５５
I. INTRODUCTION ５５
II. MATERIALS AND METHODS ５８
A.Study design ５８
B. Auditory and vestibular function assessment. ５８
C. Morphological evaluation of hair cells. ５９
D. Immunohistochemistry ５９
E. Statistical analysis ５９
III. RESULTS ６１
A. Mortality ６１
B. KRG’s effect on age related hearing loss in C57BL/6J mouse ６３
C. KRG’s effect on age related balance dysfunction ６７
D. Detection of Bcl-xL and cytochrome c expression ７１
IV. DISCUSSION ７３
V. CONCLUSION ７７

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