凋亡诱导因子在慢性氟中毒大鼠脑组织神经细胞凋亡中的作用

Role of apoptosis-inducing factor in neuron apoptosis in brain tissue of rats with chronic fluorosis

目的了解凋亡诱导因子（apoptosis—inducing factor，AIF）介导的非凋亡经典途径在慢性氟中毒大鼠大脑神经细胞凋亡过程中的作用。方法清洁级SD大鼠60只，体质量100～120g，按体质量采用随机数字表法分为对照组与染氟组，每组30只，雌雄各半，均饲以标准营养动物饲料（含氟量〈0．5mg／kg）。对照组自由饮用自来水（含氟量〈0．5mg／L）；染氟组自由饮用含氟水，氟含量[用氟化钠（NaF）配制]为50．0mg／L，复制慢性氟中毒大鼠模型。10个月后，采用磷酸盐缓冲液经心脏灌流处死动物，断头并取脑组织备用。采用免疫组织化学方法检测大脑皮质及海马AIF分布；蛋白免疫印迹方法检测脑组织中AIF、具有活性的已剪切的（cl）-caspase-3及cl—caspase-9的蛋白表达水平；流式细胞术检测大鼠脑组织细胞凋亡率。结果免疫组织化学结果：染氟组大鼠海马（CAI．CA2、CA3）及皮质顶叶神经细胞AIF阳性表达（7．50±2．17、9．00±1．63、8．00±0．82、10．24±1．80）明显高于对照组（5．18±1．66、6．27±1．42、6．36±1．96、6．96±2．62），差异有统计学意义（t=2．76、4．09、2．45和5．77，P均〈0．05）。蛋白免疫印迹结果：染氟组大鼠脑组织神经细胞线粒体内AIF的蛋白表达水平[（89．46±8．47）％]明显低于对照组[（100．00±7．12）％，t=3．16，P〈0．01]，而细胞核中AIF的蛋白表达水平[（112．80±7．10）％]明显高于对照组[（100．00±8．20）％，t=3．75，P〈0．01]；染氟组大鼠脑组织神经细胞cl—caspase-3、cl-caspase-9蛋白表达[海马与皮质分别为（132．14±18．66）％、（107．31±2．58）％。（121．33±14．86）％、（112．97±7．97）％]明显高于对照组[（100．00±11．99）％、（100．00±3．74）％，（100．00±16．87）％、（100．00±8．04）％，t=3．55、3．94、2．32、2．81，P〈0．01或〈0．05]。细胞凋亡结果：染氟组大鼠海马与皮质神经细胞凋亡率[（2．55±0．58）％、（3．05±0．65）％]与对照组[（1．28±0．59）％、（1．88±0．25）％]比较显著增高，差异有统计学意义（t=3．08、3．40，P均〈0．05）。结论以AIF为主的非easpase依赖性凋亡途径与通过激活caspase介导的经典途径共同参与慢性氟中毒大鼠脑组织神经细胞凋亡，是慢性氟中毒引起的脑损伤发生机制之一。

Objective To explore the role of apoptosis-inducing factors （AIF） mediated non-apoptosis classic pathway involved in neuron apoptosis of rats with chronic fluorosis. Methods Sixty Sprague Dawley （SD） rats （body weight 100 - 120 g） were divided into two groups （30 rats in each group, half male and half female） by random number table according to body weight. Control group was fed with tap water with fluoride content 〈 0.5 mg/L and fluorine group was fed with water with fluoride content of 50.0 mg/L. Both groups were fed with standard food with fluorine content 〈 0.5 mg/kg. After 10 months, all the animals were sacrificed though heart perfusion using phosphate buffer, and brain tissue was taken. Immunohistochemical method was employed to detect the distribution of AIF in brain tissue. Western blotting was used to test the protein expression of AIF, cl-caspase-3 and cl-caspase-9. Flow cytometry was used to examine apoptosis rate. Results The AIF positive distribution and degree of staining in the neurons of hippocampal CA1, CA2 and CA3, as well as the parietal cortex （7.50±2A7, 9.00±1.63, 8.00±0.82, 10.24±1.80） in rats with chronic fluorosis were significantly higher than those of the control group （5.18±1.66, 6.27±1.42, 6.36±1.96, 6.96± 2.62, t = 2.76, 4.09, 2.45, 5.77, all P 〈 0.05）. The AIF protein expression of neuronal mitochondria in the cerebral tissue of rats with chronic fluorosis [（89.46±8.47）%] was significantly lower than that of the control group [（100.00±7.12）%, t = 3.16, P 〈 0.01], while the AIF protein expression of the neuronal nucleus [（112.80±7.10）%] was significantly higher than that of the control group [（100.00±8.20）%, t = 3.75, P 〈 0.01]; cl-caspase-3 and cl-caspase-9 protein expression in the neurons of hippocampus and cortex from the rats with chronic fluorosis [（132.14±18.66）%, （107.31±2.58）%, （121.33±14.86）%, （112.97±7.97）%] were significantly higher than those of the control group [（100.00±11.99）%, （100.00±3.74）%, （100.00±16.87）%, （100.00±8.04）%, t = 3.55, 3.94, 2.32, 2.81, P 〈 0.01 or 〈 0.05]. As compared with those of the control group [（1.28±0.59）%, （1.88±0.25）%], the apoptosis rates in hippocampus and cortex of the rats with chronic fluorosis [（2.55±0.58）%, （3.05±0.65）%] were significanily increased （t = 3.08, 3.40, all P 〈 0.05）. Conclusion Both of AIF-mediated caspase-independent apoptosis and the classic caspase-dependent apoptosis pathways have participated in neuron apoptosis in rat induced by chronic fluorosis, which may be one of the mechanisms of brain damage of the disease.