氟对大鼠血浆中蛋白质氧化损伤作用的研究

Effects of fluoride on oxidative damage of protein in rat plasma

目的观察大鼠血浆中氧化应激水平、糖基化终产物(AGEs)和晚期氧化蛋白产物(AOPP)含量,探讨氟对大鼠血浆中蛋白质氧化损伤的作用。方法80只SPF级雄性3周龄Wistar大鼠,体重为(82.34±10.60)g,按体重采用随机数字表法分为4组,每组20只,对照组饮用蒸馏水,染氟组分别饮用氟含量为25、50和100mg/L的蒸馏水。各组大鼠自由饮食,分别于饲养1个月和3个月时处死大鼠,取尿液、股骨、外周血等用于实验。尿氟和骨氟含量检测采用离子选择电极法,超氧化物歧化酶(SOD)活性检测采用羟胺法,丙二醛(MDA)含量检测采用硫代巴比妥酸(TBA)法,AGEs和AOPP含量检测采用酶联免疫吸附测定法(ELISA)。结果饲养1个月和3个月时,对照组和25、50、100mg/L染氟组大鼠尿氟含量(mg/L:2.088±0.638、9.170±2.865、20.094±8.186、54.866±2.866,2.202±1.282、9.112±2.364、21.854±8.325、52.513±16.211)、骨氟含量(mg/kg:324.985±127.094、846.148±331.861、1886.601±250.140、2420.971±135.883,417.591±88.324、1582.243±347.975、2163.519±614.932、2755.434±265.370)组间比较差异有统计学意义(F=88.379、29.225,87.440、33.998,P均<0.05)。饲养1个月和3个月时,对照组和25、50、100mg/L染氟组大鼠血浆SOD活性(U/ml:32.469±5.674、35.931±2.262、36.746±3.994、38.042±4.632,31.027±4.147、30.777±4.791、34.148±1.755、36.585±2.860)、AGEs含量(μg/L:26.977±5.285、33.303±6.226、28.021±5.946、34.117±6.706,35.681±3.802、33.651±7.214、28.114±4.660、24.330±3.581)组间比较差异有统计学意义(F=2.896、5.780,3.565、10.195,P均<0.05)。经析因设计方差分析,氟暴露剂量和暴露时间与AGEs含量存在交互作用(F=8.957,P<0.01)。结论过量氟暴露可影响大鼠尿氟、骨氟含量,SOD活性和AGEs含量,提示过量氟有可能通过蛋白质直接和间接氧化损伤途径而调节蛋白质的表达,从而导致氟中毒的发生。

Objective To investigate the effects of fluoride on protein oxidative damage in rat plasma by measuring oxidative stress levels, advanced glycation end products (AGEs) and advanced oxidation protein products (AOPP). Methods Eighty SPF male 3-week-old Wistar rats weighing (82.34 ± 10.60) g were randomly divided into 4 groups, 20 rats in each group. The control group drank distilled water, and the fluoride groups drank distilled water with fluoride concentrations of 25, 50 and 100 mg/L, respectively. Rats were allowed to eat and drink freely, and they were sacrificed at 1 month and 3 month, respectively, and samples such as urine, femur and peripheral blood were collected for experiments. Fluoride contents in urine and bone were detected by ion selective electrode method, the superoxide dismutase (SOD) activity was detected by hydroxylamine method, malondialdehyde (MDA) content was detected by thiobarbituric acid (TBA) method, and AGEs and AOPP contents were detected by enzyme linked immunosorbent assay (ELISA). Results For 1 month and 3 months, compared urinary fluoride contents (mg/L: 2.088 ± 0.638, 9.170 ± 2.865, 20.094 ± 8.186, 54.866 ± 2.866;2.202 ± 1.282, 9.112 ± 2.364, 21.854 ± 8.325, 52.513 ± 16.211), and bone fluoride contents (mg/kg: 324.985 ± 127.094, 846.148 ± 331.861, 1 886.601 ± 250.140, 2 420.971 ± 135.883;417.591 ± 88.324, 1 582.243 ± 347.975, 2 163.519 ± 614.932, 2 755.434 ± 265.370) in control group and fluoride concentrations of 25, 50 and 100 mg/L groups, the differences were statistically significant (F = 88.379, 29.225;87.440, 33.998, P < 0.05). For 1 month and 3 months, compared SOD activity (U/ml: 32.469 ± 5.674, 35.931 ± 2.262, 36.746 ± 3.994, 38.042 ± 4.632;31.027 ± 4.147, 30.777 ± 4.791, 34.148 ± 1.755, 36.585 ± 2.860) and AGEs contents (μg/L: 26.977 ± 5.285, 33.303 ± 6.226, 28.021 ± 5.946, 34.117 ± 6.706;35.681 ± 3.802, 33.651 ± 7.214, 28.114 ± 4.660, 24.330 ± 3.581) in control group and fluoride concentrations of 25, 50 and 100 mg/L groups, the differences were statistically significant (F = 2.896, 5.780;3.565, 10.195, P < 0.05). By factorial design anova, there was an interaction between the exposure concentration and exposure time of fluorine and the content of AGEs (F = 8.957, P < 0.01). Conclusion Excessive fluoride can affect urinary, bone fluoride contents, SOD activity, AGEs content, suggesting that excessive fluoride may regulate protein expression through direct and indirect oxidative damage pathways, which leading to fluorosis.