Effect Of Glutathione On Gentamicin-induced Cochlear Hair Cell Apoptosis And Phosphorylated JNK In Rat

IntroductionApoptosis is a predominant mode of aminoglycoside antibiotics (AmAn) -induced death of inner ear hair cells (HCs). However, the molecular mechanisms are poorly defined. The generation of reactive oxygen species (ROS) has been demonstrated in ototoxic drug - induced HCs damage. Some authors have previously shown that in apoptotic HCs, the c-jun N-terminal kinase (JNK) pathway—a stress -activated protein kinase that can promote apoptotic cell death in a variety of systems—is activated in HCs. In other studies, ROS has been shown to activate JNK. Thus, we choose GM to construct a model of AmAn -induced cochlear damage and examine the effect of glutathione on apoptosis of HCs and activity of JNK. The conclusion show that the ototoxic action of AmAn leading to the loss of cochlear HCs is mainly through apoptosis and the damage mode may be: the generation of ROS→the activity of JNK→the opoptosis of HCs.Materials and methodsAnimals: 40 healthy Wistar rats of either sex with body mass of 250g-300g, with sensitive preyer's reflexes and normal amplitudes of distortion products otoacoustic emission (DPOAE) were used.Main drugs and equipments: gentamicin; glutathione; TUNEL detection kit; rabbit anti-p-JNK; scanning electron-microscopy; upright microscopyMethods: The 40 Wistar rats were divided into 4 groups randomly, 10 in each group: control group, GM group, GSH group and GM+GSH group. Control group was injected with 0.9% Natrium chloride; GM group was injected with gentamicin 120mg/kg·d^-1 intramuscularly; GSH group was injected with glutathione 400mg/kg·d^-1 intramuscularly; GM+GSH group was first injected with glutathione 400mg/kg·d^-1 intramuscularly, after 4 hours it was gentamicin 120mg/kg·d^-1 intramuscularly. All groups were injected drugs for successive 10 days.DPOAE was determined from each ear 1 day before the injections started and 1 day after the last injection. After the last DPOAE measurement, animals were decapitated immediately, and the temporal bones were quickly removed. Left ear was perfused with 2.5% glutaraldehyde in PBS in order to observe the modality of HCs by scanning electron-microscopy. Right ear was perfused with 4% paraformaldehyde in PBS in order to make 10μm sections. Odd sections were detected the apoptosis of HCs by TUNEL methods and even sections were detected the labeling of phosphorylated JNK by immunohistochemistry. An upright microscope was used for observing stain-result. The data of DPOAE were statistical analyzed by t-test and the result of TUNEL and immunostaining were analyzed by Fishers exact probabilities in 2×2 table.ResultAfter 10 days of drug injection, GSH group had no change in either function or morphology of cochlear; in GM group, the amplitudes of DPOAE at 0.75-8kHz frequency declined remarkably (P<0.01), and the staining of TUNEL and p-JNK were the most obviously; in GM+GSH group, the amplitudes of DPOAE were descend only at high frequency region (4,6,8kHz), and they were lower than those in GM group, and the staining of either TUNEL or p-JNK were less than GM group (p<0.05). The function and morphology of HCs are protected perfectly by GSH.DiscussionThe ototoxic action of AmAn leading to the apoptotic death of HC of the inner ear is well documented. Apoptosis is also named programmed cell death (PCD). Based on morphological and biochemical criteria, apoptosis and necrosis are two forms of cell death. Apoptosis is a gene-directed self-destruction program, which is involved in intracellular signaling cascades. Many studies have demonstrated the JNK pathway is activated in apoptotic cells, and the inhibitor of JNK can obviously protect HCs from the ototoxic action of AmAn. In other studies, ROS has been shown to activated JNK. In ototoxic AmAn-induced HCs damage, the generation of ROS has been demonstrated. So, ROS may be upstream regulators of JNK activation. Thus, we think the mode of AmAn-induced inner ear damage may be: the generation of ROS→the activity of JNK→the apoptosis of HCs.Conclusion1. Cellular apoptosis is a predominant method of HCs in gentamicin ototoxicity.2. JNK is participated in gentamicin-induced HCs apoptosis.3. Through the effect of glutathione on the activity of JNK, we indirect demonstrate that ROS can activate JNK pathway, and the mode of AmAn-induced inner ear damage may be: the generation of ROS results the activity of JNK, and then results the apoptosis of HCs ultimately.