Chunjie Tian

Korean red ginseng prevents gentamicin-induced hearing loss in rats†‡§

To evaluate the preventive effects of Korean red ginseng (KRG) on gentamicin (GM)‐induced ototoxicity and to identify the effective components of KRG.

A combination of cilostazol and Ginkgo biloba extract protects against cisplatin-induced Cochleo-vestibular dysfunction by inhibiting the mitochondrial apoptotic and ERK pathways.

Cisplatin (cis-diammine-dichloroplatinum; CDDP) is an anticancer drug that induces significant hearing loss and balance dysfunction as side effects. Cilostazol (CS, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3, 4-dihydro-2-(1H)-quinolinone) has neuroprotective and antioxidant effects, whereas Ginkgo biloba extract (GbE) has preventive effects on CDDP-induced hearing loss in rats, and GbE enhances the antiatherogenic effect of CS by inhibiting the generation of reactive oxygen species (ROS). The purpose of this study was to investigate the effects of renexin (RXN), which contains GbE and CS, against CDDP-induced cochleo-vestibular dysfunction in rats and to elucidate the mechanism underlying the protective effects of RXN on auditory cells. Rats intraperitoneally injected with CDDP exhibited an increase in hearing threshold and vestibular dysfunction, which agreed with hair cell damage in the Organ of Corti and otoliths. However, these impairments were significantly prevented in a dose-dependent manner by pre- and co-treatment with RXN, and these preventive effects in RXN-treated rats were more prominent than those in GbE-treated rats. In a CDDP pharmacokinetic study, platinum concentration was very similar between CDDP-only treated and RXN+CDDP cotreated rats. RXN markedly attenuated CDDP-induced intracellular ROS and significantly reduced CDDP-activated expression of p-extracellular regulated kinase (ERK), BAX, cytochrome c, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, but increased BCL-XL expression. These results show that RXN may have a synergistic effect by strongly protecting hearing and vestibular dysfunction induced by CDDP by inhibiting ROS production, mitochondrial pathways and the ERK pathway, without interfering with CDDP pharmacokinetics. Therefore, RXN could potentially be used to reduce CDDP-related hearing loss and dizziness.

Gingko biloba extracts protect auditory hair cells from cisplatin-induced ototoxicity by inhibiting perturbation of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) may play an important role in the hearing process. Cisplatin is an anticancer drug that causes hearing loss and Gingko biloba extracts (EGb 761) have been used as an antioxidant and enhancer for GJIC. The purpose of this study was to examine the efficiency of EGb 761 in protecting against cisplatin-induced apoptosis and disturbance of GJIC. House Ear Institute-Organ of Corti 1 auditory cells were cultured and treated with cisplatin (50 μM) and EGb (300 μg/ml) for 24h, and then analyzed by immunocytochemistry (Annexin V/propidium iodide) and Western blots. GJIC was evaluated by scrape-loading dye transfer (SLDT). Basal turn organ of Corti (oC) explants from neonatal (p3) rats were exposed to cisplatin (1-10 μM) and EGb (50-400 μg/ml). The number of intact hair cells was counted by co-labeling with phalloidin and MyoVIIa. EGb prevented cisplatin-induced apoptosis in immunostaining and decreased caspase 3 and poly-ADP-ribose polymerase bands, which were increased in cisplatin-treated cells in Western blots. EGb prevented abnormal intracellular locations of connexin (Cx) 26, 30, 31, and 43 in cells treated with cisplatin and increased quantities of Cx bands. EGb also prevented cisplatin-induced disturbance of GJIC in SLDT. In oC explants, EGb significantly prevented hair cell damage induced by cisplatin. In animal studies, EGb significantly prevented cisplatin-induced hearing loss across 16 and 32 kHz. These results show that cisplatin induces ototoxicity including hearing loss as well as down-regulation of GJIC and inhibition of Cxs in auditory cells. EGb prevents hearing loss in cisplatin-treated rats by inhibiting down-regulation of Cx expression and GJIC. The disturbance of GJIC or Cx expression may be one of the important mechanisms of cisplatin-induced ototoxicity.

Autophagic flux, a possible mechanism for delayed gentamicin-induced ototoxicity.

Aminoglycoside antibiotics including gentamicin (GM) induce delayed ototoxic effects such as hearing loss after long-term use, unlike the early-onset ototoxicity caused by cisplatin. The purpose of the study was to identify the mechanism of the delayed GM-induced ototoxicity by exploring the role of autophagy in vitro and in vivo. Treating HEI-OC1 auditory cells with GM led to a time-dependent increase of the autophagosome marker LC3-II, which was accompanied by cell death. In contrast, cisplatin and penicillin caused a rapid increase and had no effect on LC3-II levels, respectively. LC3-II-expressing autophagosomes co-localized with the labeled GM. GM-treated autophagosomes expressed reduced levels of Rab7, which is necessary for the fusion of autophagosomes with lysosomes. When the autophagic flux enhancer rapamycin was applied to GM-treated cells, Rab7 and the lysosomal enzyme cathepsin D were upregulated, and increased cell survival was observed. In animal studies, the intraperitoneal injection of GM worsened hearing thresholds and induced the accumulation of LC3 in the organ of Corti. This hearing impairment was attenuated by rapamycin. These findings suggest that the delayed onset-ototoxicity of GM may be closely related to the accumulation of autophagosomes via impaired autophagy. This GM-induced auditory cell death could be inhibited by enhancing autophagic flux.

Latent progenitor cells as potential regulators for tympanic membrane regeneration

Tympanic membrane (TM) perforation, in particular chronic otitis media, is one of the most common clinical problems in the world and can present with sensorineural healing loss. Here, we explored an approach for TM regeneration where the latent progenitor or stem cells within TM epithelial layers may play an important regulatory role. We showed that potential TM stem cells present highly positive staining for epithelial stem cell markers in all areas of normal TM tissue. Additionally, they are present at high levels in perforated TMs, especially in proximity to the holes, regardless of acute or chronic status, suggesting that TM stem cells may be a potential factor for TM regeneration. Our study suggests that latent TM stem cells could be potential regulators of regeneration, which provides a new insight into this clinically important process and a potential target for new therapies for chronic otitis media and other eardrum injuries.

Prevention of cisplatin-induced ototoxicity by the inhibition of gap junctional intercellular communication in auditory cells

Cis-diamminedichloroplatinum (cisplatin) is an effective chemotherapeutic drug for cancer therapy. However, most patients treated with cisplatin are at a high risk of ototoxicity, which causes severe hearing loss. Inspired by the “Good Samaritan effect” or “bystander effect” from gap junction coupling, we investigated the role of gap junctions in cisplatin-induced ototoxicity as a potential therapeutic method. We showed that connexin 43 (Cx43) was highly expressed in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, mediating cell–cell communication. The viability of HEI-OC1 cells was greatly decreased by cisplatin treatment, and cisplatin-treated HEI-OC1 cells showed lower Cx43 expression compared to that of untreated HEI-OC1 cells. In particular, high accumulation of Cx43 was observed around the nucleus of cisplatin-treated cells, whereas scattered punctuate expression of Cx43 was observed in the cytoplasm and membrane in normal cells, suggesting that cisplatin may interrupt the normal gap junction communication by inhibiting the trafficking of Cx43 to cell membranes in HEI-OC1 cells. Interestingly, we found that the inhibition of gap junction activity reduced cisplatin-induced apoptosis of auditory hair cells. Cx43 siRNA- or 18α-GA-treated HEI-OC1 cells showed higher cell viability compared to control HEI-OC1 cells during cisplatin treatment; this was also supported by fluorescence recovery after photobleaching studies. Inhibition of gap junction activity reduced recovery of calcein acetoxymethyl ester fluorescence compared to control cells. Additionally, analysis of the mechanisms involved demonstrated that highly activate extracellular signal-regulated kinase and protein kinase B, combined with inhibition of gap junctions may promote cell viability during cisplatin treatment.

The oncoprotein, gankyrin, is up-regulated in middle ear cholesteatoma

Abstract Conclusion: Gankyrin seems to be a better biomarker for cholesteatoma compared with Ki-67. Objective: Gankyrin is an oncoprotein, and occurs in cancers but not in benign diseases. The goal of this study was to compare expression of gankyrin, p53, and a proliferation marker (Ki-67) in cholesteatoma and retroauricular skin (RAS), and to evaluate their significance as clinical parameters. Methods: The levels of expression of gankyrin, Ki-67, and p53 in 10 cholesteatoma and 10 paired samples of normal RAS were evaluated by immunohistochemical staining and Western blot. The results were compared with clinical profiles to investigate a correlation. Results: The expression of gankyrin, Ki-67, and p53 proteins was observed in both basal and suprabasal layers of cholesteatoma. The intensity of gankyrin expression was ‘positive’ in two cases (20%) and ‘strongly positive’ in eight cases (80%); p53 expression in the suprabasal layer was ‘positive’ in 70% of cases; and the Ki-67 staining was ‘focal’ in 80% of cases. In RAS, these proteins were expressed dominantly in the basal layer. Western blot analysis showed that the gankyrin band was more intense in cholesteatoma than in RAS for three of four cases (p < 0.05). However, there was no significant difference in the expression of gankyrin, Ki-67, and p53 according to clinical variables.

Korean red ginseng ameliorates acute 3-nitropropionic acid-induced cochlear damage in mice.

3-Nitropropionic acid (3-NP), a mitochondrial toxin, has been reported to induce an acute cochlear damage. Korean red ginseng (KRG) is known to have protective effects from some types of hearing loss. This study aimed to observe the protective effect of KRG in an ototoxic animal model using 3-NP intratympanic injection. BALB/c mice were classified into 5 groups (n=15) and dose-dependent toxic effects after intratympanic injection with 3-NP (300-5000 mM) on the left ear were investigated to determine the appropriate toxicity level of 3-NP. For observation of the protective effects of KRG, 23 mice were grouped into 3-NP (500 mM, n=12) and KRG+3-NP groups (300 mg/kg KRG for 7 days before 500 mM 3-NP administration, n=11). Auditory brain response (ABR) and cochlear morphological evaluations were performed before and after drug administration. 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. This animal model exhibited a dose-dependent hearing loss with histological changes. KRG administration ameliorated the deterioration of hearing by 3-NP.

Red ginseng delays age-related hearing and vestibular dysfunction in C57BL/6 mice.

Since Korean red ginseng (KRG) has been proven to protect against gentamicin-induced vestibular and hearing dysfunction, the effects of KRG on age-related inner ear disorder in C57BL/6 mice were investigated. While age-related hearing loss was detected at the age of 6months (32kHz) and 9months (16kHz) in the control group, it was significantly delayed (p<0.05) in the 150mg/kg KRG-treated group. Vestibular dysfunction was observed in the tail-hanging and swimming tests, with significantly different severity scores and swimming times detected between the control and 150mg/kg KRG-treated group at the age of 12months (p<0.05). Mice treated with 500mg/kg KRG exhibited irritability and aggravated inner ear dysfunction. Histological observation supported the findings of hearing and vestibular function defects. In conclusion, C57BL/6 mice showed early-onset hearing loss and progressive vestibular dysfunction with aging, which were delayed by treatment with 150mg/kg KRG. However, 500mg/kg KRG treatment may induce aggressive behavior.

Isolation and identification of mesenchymal stem cells from human mastoid bone marrow

A new type of mesenchymal stem cells (MSCs) isolated from mastoid bone marrow obtained during mastoidectomies were identified. The isolated cells were analyzed by fluorescence-activated cell sorting (FACS) using CD45, CD90, CD105, and CD29 for investigation of the characteristics of the isolated cells. Stem cell phenotypes of human mastoid bone marrow-derived cells (hMBMCs) were maintained during passages 0–4. In the proliferation profiles of stem cells, FACS showed positive results for CD90, CD105, CD29, and negative for CD45. We found that the cells differentiated into adipocytes, chondrocytes, osteocytes, neuronal, and epithelial cells. The morphological features of the differentiated cells were also observed. Our results demonstrated that hMBMCs showed general features of MSCs that can differentiate into multiple cell types under specific stimulation, which could be used as a valuable source of MSCs for various applications in tissue engineering and regenerative medicine, especially for otolaryngology such as one stage reconstruction of functional ears.