| Objective: To observe the changes of oxidative stress,specific excitatory neurotransmitter NMDA receptor's subtypes(NR1,NR2 A,NR2B)and their phosphorylated proteins in the hippocampus of offspring rats and the antagonistic effect of NMDA receptor antagonist MK-801.Methods: The model of offspring rats with water-drinking fluorosis was established in vitro.The offspring rats was divided into 6 groups:(1)control group(tap water,fluorine content less than 0.5 mg/L);(2)low fluorine group(fluorine content: 10 mg/L);(3)high fluorine group(fluorine content: 100 mg/L);(4)antagonistic group(MK-801: 1.0 mg/kg);(5)low fluorine antagonistic group(fluorine content: 10 mg/L,MK-801: 1.0 mg/kg);(6)high fluorine antagonistic group(fluorine content: 100 mg/L,MK-801: 1.0 mg/kg).The growth and neurobehavioral development of the offspring rats was observed after birth;PND28,Morris water maze was used to detect the learning and study abilities of the offspring rats;HE staining and Nissl staining was used to observe the brain histomorphology changes of the offspring rats;The oxidative stress in serum of offspring rats was detected by NO and NOS synthetase kits;Western blot was used to detect the changes of NMDA receptor subunits(NR1,NR2 A,NR2B)and their phosphorylated proteins in hippocampus of the offspring rats.Results: Compared with control group,the body weight of PND1,PND7 and PND14 in low fluorine group and high fluorine group had no significant changes(P >0.05);PND21,compared with control group,the body weight of the offspring rats in high fluorine group was decreased(P < 0.05);PND28,compared with control group and low fluorine group,the body weight of the offspring rats in high fluorine group was increased slowly(P < 0.05);Compared with control group,the brain weight of high fluorine group and low fluorine group was decreased significantly;However,there was no significant difference of organ index of brain between each group.Compared with control group,the time of eyes opening in high fluorine group was prolonged(P < 0.05);However,there was no significant difference in auricle separation,teeth eruption and hair growth between each group(P > 0.05).Compared with control group and low fluorine group,the time of completing cliff avoidance reflex in high fluorine group was delayed(P < 0.05);In addition,compared with control group,the time of completing surface righting reflex in high fluorine group was prolonged(P < 0.05);However,there was no significant difference in the time of completing auditory startle and vibrissa positioning reflex in each group(P > 0.05).In Morris water maze,compared with control group,the time of escape latency of high fluorine group and low fluorine group was delayed,the number of crossing platform and the time of dwelling target in high fluorine group and low fluorine group was decreased(P < 0.05);In addition,compared with low fluorine group,the time of escape latency of high fluorine group was delayed,the number of crossing platform and the time of dwelling target high fluorine group was decreased(P < 0.05);After treated with MK-801,compared with high fluorine group,the time of escape latency of high fluorine antagonistic group was delayed,the number of crossing platform and the time of dwelling target in high fluorine antagonistic group was decreased(P<0.05).HE staining showed that in high fluorine group,the basophilic of cytoplasm enhanced slightly in CA1 area;In CA3 area,the neurons was arranged sightly disorder,the basophilic of cytoplasm enhanced,some nuclein became pyknotic;After the treatment of MK-801,the neurons in the antagonistic group showed pathological changes similar to those in low fluorine and high fluorine groups,and the morphology of neurons in low fluorine and high fluorine antagonistic groups improved.Nissl staining showed that in CA1 area,compared with control group,the number of nissl bodies in low fluorine group and high fluorine group was decreased(P < 0.05);Additionally,compared with low fluorine group,thenumber of nissl bodies in high fluorine group was decreased too(P < 0.05);After treated with MK-801,compared with high fluorine group,the number of nissl bodies in high fluorine antagonistic group was increased(P < 0.05).In CA3 area,compared with control group,the number of nisslbodies in low fluorine group and high fluorine group was decreased(P < 0.05);Besides,after treated with MK-801,compared with high fluorine group,the number of nissl bodies in high fluorine antagonistic group was increased(P < 0.05).Compared with control group and low fluorine group,in oxidative stress,the NO content of in high fluorine group was significantly increased(P < 0.05);After treated with MK-801,the content of NO in high fluorine antagonistic group was decreased(P< 0.05).Compared with control group,the activity of NOS and i NOS in high fluorine group and low fluorine group was significantly increased(P < 0.05).Besides,compared with low fluorine group,the activity of NOS and i NOS was increased too(P < 0.05);After treated with MK-801,the activity of NOS and i NOS in high fluorine antagonistic group was decreased(P < 0.05);Besides,compred with antagonist group,the activity of i NOS in high fluorine group was significantly increased.Western blot showed that compared with control group the expression level of NR2 B in hippocampus of high fluorine group and low fluorine group was significantly increased(P < 0.05);After treated with MK-801,compared with high fluorine group,the expression of NR2 B protein in high fluorine antagonistic group decreased(P <0.05).Similarly,compared with control group,the expression of p-NR2 A in hippocampus of low fluorine group and high fluorine group was significantly increased(P < 0.05);After the treatment of MK-801,the expression of p-NR2 A protein in each antagonist group decreased compared with that in the non-antagonist group.Compared with control group,the expression of p-NR2 B protein in low fluorine group and high fluorine group decreased(P < 0.05);After the treatment of MK-801,the expression of p-NR2 B in high fluorine antagonistic group decreased(P< 0.05).Conclusion: Excessive fluoride can activate the activity of free radicals and NMDA receptors in brain,and cause the damage of brain morphology and function. |
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