| Background and Objective: It is generally accepted that AmAn(aminoglycoside antibiotics) has severe ototoxicity which inhibits its clinical usage. There are many studies on how to antagonize this side effect, but most of them focused on the free radical and other molecule caused hair cell damage. With the rapid development of molecular biochemistry , people have more knowledge about the mechanism of AmAn's ototoxicity. In recent years, studies show that apoptosis maybe involved in the inner ear vestibular sensation hair cells loss caused by AmAn. It is unclear whether cochlear cells is the target tissue of this apoptosis pathway. Caspases are cysteine proteases. They act on each other to form a cascade, which can take part in many types of cell's apoptosis procedure. Among those analogs, Caspase-3 is the "executor" and act in the center of apoptosis signal pathways. The Bcl-2 family is one of the most important adjusting genes, it may adjust the release of cyto-c, and activation of Caspase furthermore. Both the Caspase and Bcl-2 family play the important role in the damage of cell. But it is unclear whether AmAn can through the Caspases pathway to induce cochlear cells's apoptosis and how it works. The present study investigated Caspase-3's effect on cochlear cells induced by kanamycin. Calcium antagonists(nimodipine) are used to block Ca2+ overloading induced by kanamycin. And we studied whether the DNA damage of cochlear cells in guinea pig is related with Caspase-3 and Bcl-2 and Bax and Ca2+ overloading. Thus, we hope to offer a new idea to prevent and cure the inner ear lesion of AmAn.Method: We choose 126 healthy albino guinea pig which weight ranges from 250g to 300g, divide into nine groups:(1)Kanamycin group: Guinea pig are given intramuscular injections of kanamycin(200mg/kg/d) for 14 consecutive days; (2)Nimodipine group: At the same time of intramuscular injections of kanamycin(200mg/kg/d), irrigate stomach to administrate nimodipine 20mg/kg/d for 14 consecutive days;(3)Control group: injected normal saline for 14 Consecutive days. We observe ABR threshold and kill those animals after drug withdrawal 1d , 3d ,7d and 14d. After that, We detecte HE, TUNEL, Caspase-3, Bcl-2 and Bax and detect fluorescence staining, electron microscope, laser confocal scanning respectively to observe hair cell apoptosis and ultrastructure and Ca2+ changes.Results: l.Electrophysiological change: In kanamycin groups, guinea pigs'ABR threshold raise gradually companying with the increase time of the kanamycin withdrawal. The lesion of hearing decrease after treatment of nimodipine, but still have a little rising of the ABR threshold. 2.TUNEL: Kanamycin 3d group's cochlear cells area have positive cells , the absolute gray value compare with control group which have obvious difference(P<0.05), and with the rising time of kanamycin withdrawal, there were significantly more positive cells, and the color are more deepened, their absolute gray value compare with control group which have significant difference(P<0.01). In nimodipine treatment groups, positive cells decrease. Nimodipine 3d group's staining positive cells decrease significantly compare with kanamycin 3d group (P<0.05), 7 and 14d group decrease very significantly(P<0.01). Kanamycin 14d group have more TUNEL positive cells in basal turn than apical turn and have more positive cells than nimodipine 14d group. 3.Expression of Caspase-3 protein: The staining of Caspase-3 shows a lot of buffy yellow particles distributing in the cytoplasm. Guinea pig's cochlear cells show positive reaction in the kanamycin withdrawal 1d group, their absolute gray value compare with control group which increase significantly (P<0.05), and3,7,14d kanamycin group show strong positive reaction which increase very significantly compare with control group (P<0.01). In nimodipine treatment groups, positive cells decrease. Nimodipine 3d group's staining positive cells decrease significantly compare with kanamycin 3d group (P<0.05), 7 and 14d group which decreas... |
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