Role of Ref-1 and NF-κB in Modulation Mechanism of Oxidative Damage in the Cochlea Induced by Aminoglycoside Antibiotics

PartⅠRef-1 expression in the guinea pig cochlea of gentamicin-induced ototoxic damage and prevention with antioxidant treatmentBackgrounds Increasing evidences indicated that ototoxicity induced by aminoglycoside antibiotics may be related to free radical damage. Imbanlance between production and clearance of free radicals may result in alteration of redox state of the inner ear and cause cell death or apoptosis. Many complicated cell death pathways and modulatory mechanisms are involved in aminoglycoside ototoxicity, but the exact mechanisms remain unclear. Alteration of redox state could trigger intracellular expression of the signal-transduction factors involved in the regulation of redox state and initiate a series of gene expression and transcription. Among these factors, redox factor 1 (Ref-1) plays important roles in oxidation/reduction regulation.Objective To investigate the expression of Ref-1 in the guinea pig cochlea of gentamicin-induced ototoxic damage and prevention with antioxidant sodium salicylate, and to explore the roles of Ref-1 in the ototoxic mechanism of aminoglycoside antibiotics.Methods Fifty healthy male guinea pigs with normal Preyer’s reflex weighed from 250 to 350 g were involved in this study. All the animals were randomly divided into 5 groups and received intraperitoneal injections according to their arranged group. GroupⅠ(control), treated with normal saline. GroupⅡ(GM7d), treated with gentamicin alone for 7 days. GroupⅢ(GMSA7d), treated with gentamicin in combination with sodium salicylate for 7 days. GoupⅣ(GM14d), treated with gentamicin alone for 14 days. GroupⅤ(GMSA14d), treated with gentamicin in combination with sodium salicylate for 14 days. The auditory function was evaluated by evoked auditory brainstem responses (ABR) and the surface preparation of basilar membrane stained by silver nitrate was performed for cochlea histopathologic observation in each animal. Paraffin-embedded immunohistochemistry was used for evaluation of Ref-1 expression in the cochlea. The protein was extracted from the cochlea tissues, and Ref-1 protein levels in the cochlea were detected by Western blot assay. The RNA was extracted from the cochlea tissues and Ref-1 mRNA levels in the cochlea were measured by reverse transcription-polymase chain reaction (RT-PCR).Results①Auditory function. ABR thresholds of the experimental groups significantly increased compared with the control, and the order of the ABR thresholds from high to low was GM14d＞GMSA14d＞GM7d＞GMSA7d＞the control group. There were significant differences between each two groups.②Morphologic changes. There was no obvious outer hair cells loss in the control group. The order of the outer hair cell loss from high to low was GM14d＞GMSA14d＞GM7d＞GMSA7d＞the control group.③Immunohistochemistry. A slightly positive reaction for Ref-1 staining was found in the cochlea of the control group, mainly shown in the cytoplasm. The expression of Ref-1 immunoreactivity prominently increased in the cytoplasm and/or nucleus in GM7d group. A highly positive expression was shown in GMSA7d group, and the amounts of stained cells in nucleus increased obviously. The expression decreased in GM14 and GMSA14 groups compared with GM7d and GMSA7d groups, respectively. Image analysis showed that the differences of Ref-1 expression between each two groups were statistically significant (P＜0.01).④Western blot assay. Semi-quantitative data showed prominently upregulative expression of Ref-1 in GM7d group. It reached the peak expression in GMSA7d group and decreased in GM14d group. The positive Ref-1 expression was stronger in GMSA14d group in comparison with GM14d. Statistics analysis showed that the expression between each two groups had a significant difference (P＜0.01). ⑤RT-PCR assay. PCR products were detected and the semi-quantitative data were analyzed by electrophoresis on 1% agarose gel. A positive expression ofβ-actin mRNA (internal control) was detected in every group. Semi-quantitative analysis showed upregulation of Ref-1 mRNA expression in GM7d group. The highest expression of Ref-1 mRNA was shown in GMSA7d group. The differences of expression were statistically significant compared in each two groups (P＜0.01).Conclusions A slightly positive reaction for Ref-1 staining was found in the cochlea of normal guinea pigs, mainly observed in the cytoplasm of the spiral ganglion and organ of Corti. At the early period of gentamicin administration, upregulation of Ref-1 protein and mRNA expression was shown in the cochlea, and the immunostaining transferred from cytoplasm to nuclei was observed in partial cells. Simutaneous administration of antioxidant sodium salicylate could further promote the transposition of Ref-1 from cytoplasm to nuclei and significantly increase the expression of Ref-1 protein and mRNA. The expression levels of Ref-1 protein and mRNA in each experimental group were consistant with the reservation of outer hair cell amounts and auditory function. These results indicated that Ref-1 may play roles that prevent the cochlea from gentamicin-induced oxidative damage. PartⅡRole of NF-κB in gentamicin-induced ototoxicity and antioxidant protection in guinea pig cochleaBackgrouds Nuclear factor-κB (NF-κB) is a signal transcription factor that has emerged as an important modulator of altered gene programs in cochlea redox pathology. But the mechanism about how redox state activating NF-κB remains unclear. Recently, a few researches implicated that Ref-1 regulates the transcription activity of NF-κB through reduction of p50 subunit.Objective To investigate the effects of NF-κB on gentamicin-induced ototoxicity and antioxidant protection of sodium salicylate, and to explore the role of NF-κB in ototoxic mechanism of aminoglycoside and its relationship to Ref-1.Methods Fourty healthy male guinea pigs were randomly divided into 5 groups according to grouping methods in PartⅠof this study. Each group received the corresponding treatment. Cochlear paraffin-embedded immunohistochemistry for NF-κB p50 was performed and electrophoretic mobility shift assay (EMSA) was used to detect the nuclear protein DNA binding activity of NF-κB.Results①A slightly positive reaction for NF-κB p50 staining was found in the cochlea of normal control group, mainly expressed in cytoplasm. Positive expression of NF-κB p50 immunoreactivity was prominent in the cytoplasm and/or partly in the nuclei of GM7d group. The strongest positive expression was shown in GMSA7d group and most of the positive staining was observed in the nuclei. Compared with GM7d and GMSA7d, NF-κB p50 expression decreased in GM14d and GMSA14d, respectively. Image analysis showed that the expression of NF-κB p50 had statistically significant differences between each two groups(P＜0.01).②EMSA showed that radiolabelled probe differently bound to nuclear protein extracted from each group. Prominent upregulation activation of NF-κB was obtained in GM7d group. It reached the peak activation in GMSA7d group and decreased in GM14d group. The levels of NF-κB activation were higher in GMSA14d group than that in GM14d groups.③The distribution of NF-κB expression and its alteration of DNA binding activity were consistant with the expression of Ref-1 protein and mRNA.Conclusions Gentamicin administration induced NF-κB transposition from cytoplasm to nuclei in partial cells of cochlea and resulted in enhancement of its DNA binding activity. Sodium salicylate could further promote NF-κB transposition and enhance the DNA binding activity. The expression and DNA binding activity of NF-κB were consistant with Ref-1 expression. These results implicated that gentamicin might initiate the Ref-1/NF-κB pathway, and activation of both factors could probably provide protection from ototoxicity in some extent. The prevention effect of antioxidant sodium salicylate might be related to upregulation of Ref-1 and activation of NF-κB.