Exploratory Research On The Association Of NMDAR Single Nucleotide Polymorphism With The Injury Of Brain Induced By Microwave Radiation

Objective: With the wide application of electronic equipment, the biological effects of microwave become more serious. And the central nervous system is a sensitive target organ of microwave radiation. The epidemiological survey showed that microwave radiation could cause fatigue, headache, excitement, dreaminess memory decline and other symptoms. Therefore, it is extremely important to search the sensitive prediction and monitoring indexes of prevetion, diagnosis and treatment for population exposed to microwave radiation. In view of the complexity of microwave parameters and exposed environment, and the difference of individuals sensitivity, it need to be concerned that the relation between the risk of brain structure and function abnormality induced by microwave exposure and the genetic factors. This study focused on the brain injury induced by microwave radiation and finding the prediction indexes by single nucleotide polymorphisms. NMDA receptors is the most important excitatory amino acid receptor in the central nervous system. And microwave radiation could change the activity, numbers, gene and protein expression of NR1 and NR2B of, thereby which might further affecting the important brain functions, such as learning and memory. Therefore, it is significant to study the gene polymorphisms of NR1 and NR2B, and its correlation with the change of brain structure and function induced by microwave radiation for defending and finding the sensitive prediction and monitoring indexes.Materials and Methods: Bioinformatics were used to analyze SNP of NR1 and NR2B gene promoter region. Rapid blood sampling- automatic DNA extraction - multiple PCR amplification - three sequencing primers were used to explore of 10 candidate areas in 15 Wistar rats from animal center of Academy of Military Medical Sciences. The whole blood DNA of 96 male rats from three animal centers and in four strains were extracted. NR1 and NR2B gene core promoter sequences and the SNP located sequences reported in NCBI-SNP database were amplified by PCR. DNA sequencing and RFLP were used for screening, validating and genotyping of the SNP. Further, 48 male rats from four animal centers and in two strains were exposed to 30mW/cm2 microwave radiation , hippocampal DNA extraction method, DNA sequencing, RFLP, HE stainin, HPLC, Western Blot and image analysis were used at 7d after radiation, to study the change of DNA sequences in SNP location and the hippocampal function, structural and NR2B protein expression in different strain and genotypes rats after microwave radiation.Results:(1) A SNP site was found in NR2B core promoter region(705bp)of Wistar rats and SD rats from different sources, -217C/T polymorphism.(2) -217C/T different alleles in sequences had different binding sites: C allele had 3 Hb, 1 HSF and 1 C-ETs-transcriptional binding elements; T allele had 4 Hb 1 HSF transcription binding elements.(3) The DNA sequence in SNP location had no change at 7d after 30mW/cm2 microwave radiation.(4)At 7d after 30mW/cm2 microwave radiation,compared with sham-treated group, Asp, GABA were increased (p<0.05) in CC type of radiation group, Asp was increased significantly (p<0.01), in TT type of radiation group; Compared with CC type, Asp was increased(p<0.05) in TT type in Wistar rats.Gly was increased(p <0.05) ,Asp and GABA were increased significantly(p<0.01) in CC type of radiation group, Glu was increased (p <0.05), Gly and GABA were increased significantly (p<0.01) in CT type of radiation group, Glu and GABA were increased (p<0.05), Asp and Gly were increased significantly (p <0.01) in TT type of radiation group in SD rats compared with sham-treated group; and GABA was increased (p <0.05) in TT type compared to TT type.(5)At 7d after 30mW/cm2 microwave radiation,hippocampal neurons in rat of different strains and genotypes showed different degrees of degeneration and necrosis, mainly as nuclear condensation and stained to irregular dark blue mass, cell body shrinkage of the pyramidal cells and granule cells, gaps of capillary broaden and engorged and perivascular space widened, especially in the dentate gyrus and CA3 area. Compared with sham group, there were significant differences (P<0.05) in the junjury of each genotype in Wistar and SD rats, but there was no significant difference in the different strains and different genotypes.(6)At 7d after 30mW/cm2 microwave radiation,compared to sham group, NR2B protein expression was increased significantly (p<0.01) in CC type of radiation group in Wistar rats, that of TT type and CT type were decreased (p <0.05); NR2B protein expression in CC type of radiation group in SD rats was increased significantly (p<0.01) , that of CT type was decreased significantly (p <0.01).(7)There is no SNP site in the promoter region of NR1, the upstream promoter region of NR2B and 4 SNP sites reported in NCBI-SNP database in rats from different animal centers of four different strains ,which shows the regions may not exist SNP. Conclusions:(1) The region-217C/T polymorphism is found for the first time in the core promoter of NR2B in rats from different animal centers of four different strains:1) The different alleles has different binding sites in the sites, which suggests the SNP may be closely related to NR2B gene transcription, translation, expression, and changes in regulation and signaling pathways.2) Microwave radiation doesn't not cause the change of DNA sequences in SNP location and the site might be stable after radiation.(2) 30mW/cm2 microwave radiation can cause1) hippocampal dysfunction, such as metabolism disorder of amino acid neurotransmitters in three genotypes of Wistar and SD rats. And the changes in different genotypes of the same strain (Wistar rat) and, different strains (Wistar and SD rats) are different.2) the damage of hippocampal structure in three genotypes of Wistar and SD rats. And its injury characteristics are similar, all neurons shows nuclear condensation, cell body shrinkage, gaps of capillary broaden and engorged and perivascular space widened.3) NR2B protein expression increased in CC type and decreased in CT type .(3) The changes of CC type protein expression and amino acid neurotransmitters after radiation might be involved in the injury and repair process, mainly in the repair process. And those of CT and TT type could mainly involved in the process of injury, which shows genetic factors may play an important role in the sensitivity of brain disfunction induced by microwave radiation.