慢性氟中毒大鼠脑组织P-NMDAR及CaMKⅡ的表达

Expressions of P-NMDAR and CaMKⅡin brain tissue of rats with chronic fluorosis

目的观察慢性氟中毒大鼠脑组织中磷酸化-N-甲基-D-天冬氨酸受体(phosphorylated N-methyl-D-aspartat receptor,P-NMDAR)亚基及钙调蛋白依赖性蛋白激酶Ⅱ(calmodulin-dependent kinaseⅡ,CaMKⅡ)蛋白表达变化,探讨慢性氟中毒神经损伤的分子发病机制。方法选择1月龄SD大鼠18只(雌雄各半),适应性喂养1周,按体质量[(100±20)g]采用随机数字表法分为对照组(饮用自来水,含氟量<0.5 mg/L)、低氟组(饮水含氟量为10.0 mg/L)、高氟组(饮水含氟量为100.0 mg/L),每组6只(雌雄各半);饲养180 d后处死取大鼠脑组织。HE染色观察大鼠脑组织形态学改变;尼氏染色观察神经细胞尼氏体变化;免疫组织化学染色(免疫组化)观察大鼠脑组织中P-NMDAR亚基(P-NMDAR1、P-NMDAR2A)及CaMKⅡ蛋白表达水平。结果光镜下,HE染色可见高氟组大鼠脑组织海马区神经元排列紊乱,细胞核浓染、嗜碱性增强。尼氏染色结果显示,对照组、低氟组、高氟组大鼠脑组织海马区神经细胞尼氏体平均光密度值分别为0.0244±0.0097、0.0240±0.0031、0.0239±0.0138,各组间比较差异无统计学意义(F=0.010,P>0.05)。免疫组化结果显示,高氟组大鼠脑组织P-NMDAR1、P-NMDAR2A、CaMKⅡ蛋白表达水平(0.0263±0.0057、0.0863±0.0090、0.2109±0.0487)与对照组(0.0118±0.0065、0.0656±0.0111、0.1438±0.0299)和低氟组(0.0172±0.0068、0.0626±0.0178、0.1356±0.0296)比较显著升高(P均<0.05),且低氟组P-NMDAR1蛋白表达水平明显高于对照组(P<0.05)。结论长期过量氟摄入可导致大鼠神经细胞损伤,其损伤机制可能与NMDAR亚基过度激活所致的钙离子(Ca2+)超载诱导的兴奋性神经毒性有关。

Objective To observe the changes of phosphorylated N-methyl-D-aspartate receptor(P-NMDAR)subunit and calmodulin-dependent protein kinaseⅡ(CaMKⅡ)protein expression in the brain tissue of rats with chronic fluorosis,and to explore the molecular pathogenesis of chronic fluorosis nerve injury.Methods Eighteen one-month-old SD rats(half male and half female),weighing(100±20)g,were randomly divided into three groups after adaptive feeding for 1 week:control group(drinking tap water,fluoride content<0.5 mg/L),low fluoride group(drinking water fluoride content was 10.0 mg/L)and high fluoride group(drinking water fluoride content was 100.0 mg/L),with 6 rats in each group(half male and half female).Brain tissue was harvested after 180 days of feeding.HE staining was used to observe the morphological changes of rat brain tissue,Nissl staining was used to observe the changes of Nissl bodies in nerve cells,and immunohistochemical staining was used to observe the expressions of P-NMDAR subunits(P-NMDAR1,P-NMDAR2A)and CaMKⅡprotein in rat brain tissue.Results Under light microscope,HE staining showed disordered arrangement of hippocampal neurons,nuclear hyperstaining and basophilic enhancement in the high fluoride group.The results of Nissl staining showed that the average optical density of Nissl bodies in nerve cells in the hippocampus of rats in the control group,low fluoride group,and high fluoride group were 0.0244±0.0097,0.0240±0.0031,and 0.0239±0.0138,respectively.There was no statistically significant difference between the groups(F=0.010,P>0.05).Compared with the control group(0.0118±0.0065,0.0656±0.0111,0.1438±0.0299)and low fluoride group(0.0172±0.0068,0.0626±0.0178,0.1356±0.0296),the protein expressions of P-NMDAR1,P-NMDAR2A and CaMKⅡin high fluoride group were significantly increased(0.0263±0.0057,0.0863±0.0090,0.2109±0.0487,P<0.05);and the protein expression of P-NMDAR1 in low fluoride group was higher than that in the control group(P<0.05).Conclusion Long-term excessive fluoride intake can lead to nerve cell injury in rats,and the mechanism of injury maybe related to the excitotoxicity induced by calcium ion(Ca2+)overload caused by overactivation of NMDAR subunits.