氟诱导大鼠脊髓神经细胞凋亡及氧化应激的研究

Fluoride induced apoptosis and oxidative stress in rat spinal cord nerve cells

目的探讨氟对大鼠脊髓神经细胞凋亡及氧化应激水平的影响。方法取54只6周龄Sprague-Dawley雌性大鼠,体重为150~200 g,适应性喂养1周后,采用随机数字表法分为对照组[给予含0 mg/L氟化钠(NaF)去离子水]、低氟组(给予含50 mg/L NaF去离子水)、高氟组(给予含100 mg/L NaF去离子水),每组18只,各组均食用标准饲料。染氟4、8、12周,每组选择6只大鼠观察氟斑牙发生情况,并采用Basso-Beattie-Bresnahan(BBB)评分对大鼠后肢运动功能进行评估;随后腹腔注射5%水合氯醛进行麻醉,经心脏穿刺处死大鼠,采集脊髓组织,检测氧化应激因子超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)、过氧化氢酶(CAT)含量。染氟12周,利用尼氏(Nissl)染色观察大鼠脊髓神经元形态学变化,原位缺口末端标记法(TUNEL)细胞凋亡检测试剂盒检测脊髓神经细胞凋亡情况,蛋白质印迹法检测大鼠脊髓组织B淋巴细胞瘤-2(Bcl-2)基因相关X蛋白(Bax)、Bcl-2基因相关启动子(Bad)、Bcl-2蛋白表达情况,免疫荧光染色观察脊髓神经元中Bax、Bcl-2蛋白表达情况。结果染氟12周,低、高氟组大鼠均发生不同程度氟斑牙;对照、低氟、高氟组大鼠BBB评分比较,差异有统计学意义(F=14.09,P<0.001)。染氟12周,对照、低氟、高氟组SOD[(124.04±4.87)、(96.66±15.01)、(91.12±15.87)U/mg prot]、GSH-Px活性[(561.92±59.65)、(456.83±29.51)、(385.07±74.87)U/mg prot]及MDA[(9.96±1.50)、(16.64±2.05)、(20.80±3.37)nmol/mg prot]、CAT含量[(8.97±1.05)、(6.39±0.97)、(6.42±0.83)nmol/mg prot]比较,差异均有统计学意义(F=11.17、14.19、30.12、14.52,均P<0.05);其中,低、高氟组SOD、GSH-Px活性及CAT含量均低于对照组,MDA含量均高于对照组(均P<0.05);且高氟组GSH-Px活性低于低氟组,MDA含量高于低氟组(均P<0.05)。对照组大鼠脊髓神经元结构完整、细胞核清晰可见,Nissl小体染色均匀、数量较多,未见凋亡细胞;低、高氟组大鼠脊髓神经元Nissl小体染色不均匀、数量较少,凋亡细胞较多。对照、低氟、高氟组大鼠脊髓神经细胞凋亡率,脊髓组织Bax、Bad、Bcl-2蛋白表达水平比较,差异均有统计学意义(F=272.81、35.53、17.57、92.50,均P<0.05)。免疫荧光染色显示,对照、低氟、高氟组大鼠脊髓神经元中Bax、Bcl-2蛋白荧光强度比较,差异均有统计学意义(F=12.67、22.14,均P<0.05)。结论慢性氟中毒可诱导大鼠脊髓神经抗氧化酶活性降低、脂质过氧化水平升高,神经细胞凋亡增加。

Objective To study the effects of fluoride on apoptosis and oxidative stress levels of spinal cord nerve cells in rats.Methods A total of 546-week-old Sprague-Dawley female rats,weighing 150-200 g,were selected and fed for 1 week.They were divided into a control group[given deionized water containing 0 mg/L sodium fluoride(NaF)],a low fluoride group(given deionized water containing 50 mg/L NaF),and a high fluoride group(given deionized water containing 100 mg/L NaF)using a random number table method,with 18 rats in each group.All groups received standard feed.After 4,8,and 12 weeks of fluoride exposure,six rats were selected from each group to observe the occurrence of dental fluorosis,and the motor function of hind limbs in rats was evaluated based on the Basso-Beattie-Bresnahan(BBB)score.Then the rats were anesthetized with 5%chloral hydrate via intraperitoneal injection and euthanized by cardiac puncture.Spinal cord tissue of the rats was collected to detect the activities of oxidative stress factors such as superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),as well as the contents of malondialdehyde(MDA)and catalase(CAT).After 12 weeks of fluoride exposure,morphologic changes in rat spinal cord neurons were observed using Nissl staining,and apoptosis of spinal cord nerve cells was detected using the TdT mediated dUTP nick end labeling(TUNEL)cell apoptosis detection kit.The Western blotting was used to detect the expression of B-lymphoblastoma-2(Bcl-2)gene related X protein(Bax),Bcl-2 promoter(Bad),and Bcl-2 protein in rat spinal cord tissue;immunofluorescence staining was used to observe the expression of Bax and Bcl-2 protein in spinal cord neurons.Results After 12 weeks of fluoride exposure,rats in both the low fluoride and high fluoride groups developed varying degrees of dental fluorosis;the differences of BBB scores of rats in the control,low fluoride,and high fluoride groups were statistically significant(F=14.09,P<0.001).The differences of SOD[(124.04±4.87),(96.66±15.01),(91.12±15.87)U/mg prot]and GSH-Px activitives[(561.92±59.65),(456.83±29.51),(385.07±74.87)U/mg prot],MDA[(9.96±1.50),(16.64±2.05),(20.80±3.37)nmol/mg prot]and CAT contents[(8.97±1.05),(6.39±0.97),(6.42±0.83)nmol/mg prot]among the control,low fluoride,and high fluoride groups were statistically significant(F=11.17,14.19,30.12,14.52,P<0.05).Among them,the SOD,GSH-Px activities,and CAT content in the low fluoride and high fluoride groups were lower than those in the control group,while the MDA content was higher than that in the control group(P<0.05).The GSH-Px activity in the high fluoride group was lower than that in the low fluoride group,and MDA content was higher than that in the low fluoride group(P<0.05).The intact neuronal structures and clear visible nuclei were seen,and Nissl bodies were uniformly stained in the spinal cord neurons of the control group rats,with more numbers,and no apoptotic cells were observed;the staining of Nissl bodies in the spinal cord neurons of rats was uneven in the low fluoride and high fluoride groups,with fewer numbers,and more apoptotic cells.There were statistically significant differences in the apoptosis rate of spinal cord nerve cells and the expression levels of Bax,Bad,and Bcl-2 protein in the spinal cord tissues of rats in the control,low fluoride,and high fluoride groups(F=272.81,35.53,17.57,92.50,P<0.05).The results of immunofluorescence staining showed that there were statistically significant differences in the fluorescent intensity of Bax and Bcl-2 proteins in the spinal cord neurons of rats in the control,low fluoride,and high fluoride groups(F=12.67,22.14,P<0.05).Conclusion Chronic fluorosis induces a decrease in antioxidant enzyme activity,an increase in lipid peroxidation levels,and an increase in neuronal apoptosis in the spinal cord of rats.