NADPH氧化酶在D-半乳糖诱导的老化大鼠听觉系统氧化损伤过程中的作用

目的研究NADPH氧化酶（NADPH oxidase,NOX）在D-半乳糖诱导的老化大鼠外周听觉系统耳蜗和中枢听觉系统听皮层组织中的表达,探讨老年性耳聋氧化性损伤的发生机制。方法 48只1个月龄雄性Spragua-Dawley大鼠按数字随机法分成两组,每组各24只。D-半乳糖组：每日颈背部皮下注射D-半乳糖（500 mg/kg）,连续8周;对照组：每日颈背部皮下注射同体积的生理盐水,连续8周。造模完成后,取两组大鼠外周听觉系统耳蜗和中枢听觉系统听皮层组织,利用免疫组织化学法检测DNA氧化损伤生物标记物8-羟基-2-脱氧鸟苷（8-hydroxy-2-deoxyguanosine,8-OHdG）;利用实时定量PCR检测线粒体DNA（mitochondrial DNA,mtDNA）普遍缺失（common deletion,CD）和NOX基因的表达;利用Western blot检测NOX蛋白表达;应用Taqman PCR检测mt DNA CD。所有实验数据采用两样本的t检验进行分析。结果 D-半乳糖组和对照组大鼠相比较,D-半乳糖诱导的老化大鼠8-OHdG的表达在耳蜗和听皮层组织中明显增多,两组比较差异具有统计学意义（t分别为6.341、10.589,P均〈0.05）;NOX3基因和蛋白的表达在耳蜗组织中明显增强,两组比较差异具有统计学意义（t分别为8.491、18.983,P均〈0.05）;NOX2基因和蛋白的表达在听皮层组织中明显增强,两组比较差异具有统计学意义（t分别为3.548、8.219,P均〈0.05）;mtDNA CD的累积在耳蜗和听皮层组织中明显增多,两组比较差异具有统计学意义（t分别为9.796、7.901,P均〈0.05）。结论在听觉系统老化过程中,NOX是活性氧的重要来源,可能是导致老年性耳聋氧化性损伤发生的重要原因。

Effect of NADPH oxidase on oxidative damage process auditory system of D-galactose-induced aging rats

Objective To study the effect of NADPH oxidase （ NOX ） on the cochlea of peripheral auditory system and auditory cortex of central auditory system in D-g a lactose-induced aging rats and to investigate the mechanism of oxidative damage in the auditory system of age-related hearing loss. Methods 48 male Sprague Dawley rats of one-month-old were randomly divided into two groups （24 rats in each group ） , i. e. , D-galactose group and control group. Rats of D-galaetose group were injected subcutaneously with D-gal of 500 mg/kg once a day for 8 weeks, and those of the control group received injection of normal saline as the same volume, frequency and duration as those of the D-galactose group. At the end of the experiment, tissues were harvested from the cochlea and auditory cortex in both groups. The expression of 8-hydroxy-2-deoxyguanosine （8-OHdG） , a biomarker of DNA oxidative damage, was investigated by immunohistochemical analysis. Accumulation of mitochondrial DNA （ mtDNA ） common deletion （ CD ） and mRNA level of NOX were detected by real-time PCR. The protein level of NOX was evaluated by Western blot. All experimental data were analyzed using two-sample t-test. Results Compared with the results of the control group, in the D-galactose group, the expression of 8-OHdG in the cochlea and auditory cortex was significantly increased both P 〈 0.05 ） , the expression of NOX3 mRNA and protein in the cochlea was significantly increased （ both P 〈 0.05 ） , and the expression of NOX2 mRNA and protein in the auditory cortex was significantly increased （ both P 〈 0. 05 ） , and the accumulation of mtDNA CD in the cochlea and auditory cortex was also significantly increased （ both P 〈 0. 05 ）. Conclusion In the aging process of auditory system, NOX may be an important source of reactive oxygen species and may induce oxidative damage of age-related hearing loss.