核酸内切酶G在慢性氟中毒大鼠脑组织中的表达及其意义

Expression of endonuclease G in the brain tissue of rats with chronic fluorosis

目的 了解核酸内切酶G（endonuclease G，Endo G）介导的非Caspase依赖性细胞凋亡通路参与慢性氟中毒大鼠大脑神经细胞凋亡的可能机制。方法 清洁级SD大鼠60只，体重100 ~ 120 g，按体重采用随机数字表法分成对照组和染氟组，每组30只，雌雄各半，均饲以标准营养动物饲料（含氟量 〈 0.5 mg／kg）。复制慢性氟中毒大鼠模型，对照组自由饮用自来水（含氟量 〈 0.5 mg／L）；染氟组自由饮用含氟化钠蒸馏水（氟含量为50.0 mg／L）。饲养10个月，处死大鼠，取脑组织备用。采用流式细胞术检测脑组织细胞凋亡率；免疫组织化学法检测EndoG在大鼠脑组织皮质及海马区的细胞分布；蛋白免疫印迹（Western blot）法检测Endo G在大鼠脑组织线粒体和细胞核中的蛋白表达。结果 细胞凋亡结果：染氟组大鼠海马、皮质神经细胞凋亡率［（2.6 ± 0.6）%、（3.1 ± 0.7）%］与对照组［（1.3 ± 0.6）%、（1.9 ± 0.3）%］比较明显升高，差异均有统计学意义（t = 3.1、3.4，P均 〈 0.05）。免疫组织化学结果：海马CA1、CA2、CA3、CA4区染氟组Endo G评分［（11.1 ± 2.2）、（10.2 ± 1.9）、（9.8 ± 3.1）、（9.9 ± 1.6）分］与对照组［（5.8 ± 1.8）、（6.7 ± 2.6）、（5.2 ± 2.4）、（7.2 ± 2.1）分］比较明显升高，差异有统计学意义（t = 2.9、2.5、2.4、2.3，P 〈 0.01或 〈 0.05）；额叶上层、额叶下层、顶叶上层染氟组Endo G评分［（10.6 ± 2.9）、（8.2 ± 2.4）、（11.1 ± 2.8）分］与对照组［（7.7 ± 2.6）、（6.1 ± 1.9）、（8.1 ± 2.6）分］比较明显升高，差异有统计学意义（t = 2.2、2.5、2.3，P均〈0.05）。Western blot结果：在海马以及皮质的线粒体中，染氟组EndoG 的蛋白表达［（86.4 ±7.2）%、（83.9 ± 6.8）%］与对照组［（100.0 ± 6.1）%、（100.0 ± 5.5）%］比较明显降低，差异有统计学意义（t = 2.6、2.3，P均 〈 0.05）；在海马和皮质的细胞核中，染氟组Endo G的蛋白表达［（117.5 ± 6.4）%、（115.2 ± 6.2）%］与对照组［（100.0 ± 5.2）%、（100.0 ± 5.5）%］比较明显升高，差异有统计学意义（t = 2.5、2.2， P均 〈 0.05）。结论 Endo G的高表达以及核转移与慢性氟中毒大鼠脑组织神经细胞凋亡有关，是慢性氟中毒引起脑损伤的发生机制之一。

Objective To investigate the possible mechanism of endonuclease G （Endo G）-mediated non-Caspase-dependent apoptotic pathway in brain neuronal apoptosis in chronic fluorosis rats. Methods Sixty Sprague Dawley （SD） rats （half male and half female） were randomly divided into two groups： control group fed with tap water with fluoride content 〈 0.5 mg/L and fluorine group in which sodium fluoride was added into drinking water with fluoride content of 50.0 mg/L. Both groups were fed with standard food with fluorine content 〈 0.5 mg/kg. The experiment period was 10 months. At the end of the experiment, all the animals were sacrificed, and brain tissue was taken. Flow cytometry was used to examine apoptosis rate, immune-histochemistry was employed to detect the distribution of Endo G in brain tissue; Western blotting was used to test theprotein expression of Endo G. Results Compared to the apoptosis rate of control group [（1.3 ± 0.6）%, （1.9 ± 0.3）%], the apoptosis rate in hippocampus and cortex of rats with chronic fluorosis [（2.6 ± 0.6）%, （3.1 ± 0.7）%] was significantly increased （t = 3.1, 3.4, all P 〈 0.05）. The Endo G positive neurons and their degree of staining in CA1, CA2, CA3 and CA4 of hippocampus, frontal cortex as well as the upper layer of parietal cortex [（11.1 ± 2.2）, （10.2 ± 1.9）, （9.8 ± 3.1）, （9.9± 1.6）, （10.6 ± 2.9）, （8.2 ± 2.4）, （11.1 ± 2.8） scores] in rats with chronic fluorosis were significantly higher than those in the control group [（5.8 ± 1.8）, （6.7 ± 2.6）, （5.2 ± 2.4）, （7.2 ± 2.1）, （7.7 ± 2.6）, （6.1 ± 1.9）, （8.1 ± 2.6）scores, t = 2.9, 2.5, 2.4, 2.3, 2.2, 2.5, 2.3, P 〈 0.01 or 〈 0.05]. The protein level of Endo G in the mitochondria of rat brains with chronic fluorosis [（86.4 ± 7.2）%, （83.9 ± 6.8）%] was significantly lower than that of control group [（100.0 ± 6.1）%, （100.0 ± 5.5）%, t = 2.6, 2.3, all P 〈 0.05 ]. Meanwhile, the protein level of Endo G in the nucleus of neurons from chronic fluorosis rats [（117.5 ± 6.4）%, （115.2 ± 6.2）%] was significantly higher than that of the control [（100.0 ± 5.2）%,（100.0 ± 5.5）%, t = 2.5, 2.2, all P 〈 0.05]. Conclusion The high expression of Endo G and nuclear transfer are related to the neuron apoptosis in chronic fluorosis rat, which may be one of the mechanisms of brain injury of the disease.