Racl在正常及庆大霉素耳毒性损伤豚鼠耳蜗中的表达

Expression of Racl in normal and gentamicin-induced ototoxic damaged cochlea of guinea pigs

目的研究庆大霉素所致耳毒性损伤及应用抗氧化剂水杨酸钠保护时Racl在豚鼠耳蜗中的表达，探讨RacI在庆大霉素耳毒性机制中的作用。方法健康雄性白色红目豚鼠30只，按随机数字表法分成3组。对照组：腹腔内注射生理盐水，连续7d；庆大霉素组：腹腔内注射硫酸庆大霉素，连续7d；庆大霉素＋水杨酸钠组：腹腔内注射硫酸庆大霉素和水杨酸钠，连续7d。7d后处死大鼠，采用石蜡切片免疫组织化学染色技术检测各组豚鼠耳蜗Racl蛋白的表达与分布情况；提取耳蜗蛋白，采用Westernblot检测各组Racl蛋白表达水平。结果对照组耳蜗螺旋器和螺旋神经节细胞Racl呈弱阳性表达，主要表达于细胞浆和细胞膜；庆大霉素组耳蜗Racl表达增强；庆大霉素＋水杨酸钠组呈阳性表达，其表达程度介于对照组和庆大霉素组之间；各组灰度值比较差异有统计学意义（P〈0．05）。对照组耳蜗Racl蛋白有低强度表达；庆大霉素组耳蜗Racl蛋白表达最强；庆大霉素＋水杨酸钠组耳蜗Racl蛋白表达程度介于对照组和庆大霉素组之间；各组间蛋白电泳积分光密度值比较差异有统计学意义（P〈0．05）。结论Racl在正常豚鼠耳蜗螺旋器和螺旋神经节细胞的胞浆及胞膜弱表达，庆大霉素给药后耳蜗中Racl表达增强，同时给予抗氧化剂水杨酸钠后可使RacI表达较庆大霉素组减弱。提示Racl可能在庆大霉素导致耳蜗损伤过程中起作用。

Objective To investigate the expression of Racl in the guinea pig cochlea of gentamicin-induced ototoxic damage and prevention with antioxidant sodium salicylate, and to explore the roles of Racl in the ototoxic mechanism of aminoglycoside antibi- otics. Methods Thirty healthy male guinea pigs were involved in this study. All the guinea pigs were randomly divided into 3 groups and received intraperitoneal injections according to their arranged group. Group I （control） was treated with normal saline for 7 days. Group II[ Gentamicin （GM） ] was treated with gentamicin alone for 7 days. Group III[ GM ＋ sodium salicylate （SA） ] was treated with gentamicin in combination with sodium salicylate for 7 days. Paraffin-embedded cochlear section with immunohistochemical stai- ning was used for evaluation of Racl expression in the cochlea. The protein was extracted from the cochlea tissues, and Racl protein levels in the cochlea were detected by Western blot assay. Results Immunohistochemistry showed a slightly positive reaction for Racl staining found in the cochlea of the control group was mainly shown in the cytoplasm and cytomembrane of ganglion and organ of Corti ; a highly positive expression was shown in GM group; and the extent of Racl expression of GM ＋ SA group was between the control and GM groups. Image analysis showed that the differences in Racl expression between each two groups were statistically significant （ P 〈 0. 05）. Western blot assay showed that expression of Racl protein was highest in GM group and decreased in GM ＋ SA group, while the intensity of Racl protein expression in GM ＋ SA group was between the control and GM group. Statistics analysis showed that the expression between each two groups had significant difference （ P 〈 0.05）. Conclusions A slightly positive reaction for Racl stai- ning was found in the cochlea of guinea pigs, and mainly observed in the cytoplasm and cytomembrane of spiral ganglion and organ of Corti. With the administration of gentamicin, Racl protein expression was upiegulated in the cochlea. Simultaneous administration of antioxidant sodium salicylate could significantly decrease the expression of Racl protein. These results indicated that Racl might play an important role in the processes of gentamicin-induced oxidative damage of cochlea.