The Neuroprotective Effects Of Phosphorylation Of Tyrosine 1472 NR2B On Hypoxia Tolerance In Hypoxia Preconditioning Mice Hippocampus

Objective The aim of this study was to explore the neuroprotective effects of tyrosine phosphorylation site 1472 of NMDA receptor(N-methyl-D-aspartate receptor)2B subunit(NR2B)on the hypoxia tolerance using hypoxic preconditioning animal and cells model.At the same time,the role of DNA methylation in the change of Y1472-NR2 B was detected.Methods there are two parts in this study: in vivo experiment and in vitro experiment.(1)In vivo experimental part: mice were divided into two groups.The control group mice were injected with 0.1% BSA and experiment group mice were was injected with 5 μl of 10 μM DNA methyltransferase inhibitor 5-aza-cd R group.Then the hippocampus tissue was isolated form the hypoxic preconditioning model for following experiment.TRIzol method was used to extract the RNA from the tissue and it was reverse-transcribed into c DNA.Real-time PCR detect the NR2 B m RNA level.Total protein,membrane protein and TXP were isolated from mouse hippocampus for detection of p-NR2B-1472 changes by Western-blot,immunofluorescence staining was adopted to analyze the expression of p-NR2B-1472 in the whole brain.(2)In vitro experimental part: HT22 cells(mouse hippocampal neurons)were induced to differentiate and establish a hypoxic preconditioning model of HT22 cells.Cell proliferation was detected by MTS,cell apoptosis and cell cycle were analyzed by flow cytometry,cell extract membrane protein was collected to detect p-NR2B-1472 protein expression by Western-blot,immunofluorescence staining was performed.The p-NR2B-1472 level in hypoxic preconditioning HT22 cells which treated with 5-aza-cd R were detected.Results(1)The hypoxic tolerance time of mice was increased with repeated hypoxia times increased.(2)The expression of NR2 B m RNA in the 5-aza-cd R group was lower than the BSA group,and the NR2 B m RNA of the mice injected with5-aza-cd R in the hypoxic group was significantly reduced.(3)Compared with the BSA group,whole protein p-NR2B-1472 in experimental group was significantly increased and whole protein NR2 B was not significantly changed in experimental group,Only p-NR2B-1472 decreased significantly,however,there was no significant change in membrane proteins.(4)The content of whole protein p-NR2B-1472 in after hypoxia preconditioning was gradually increased in BSA group,and decreased after reaching the peak,the p-NR2B-1472 in cell membrane protein has increasing trend,and the p-NR2B-1472 protein content on the postsynaptic membrane has a tendency to decrease.In 5-aza-cd R group,the whole protein p-NR2B-1472 after hypoxia and hypoxia preconditioning treatment showed decline and then them gradually increased,while the membrane protein was more stable,and the content of p-NR2B-1472 in the postsynaptic membrane showed gradual decline.(5)Fluorescence intensity of p-NR2B-1472 in HT22 cells were simair to that in hippocampus.Conclusion The changes in NR2 B and p-NR2B-1472 after hypoxia and hypoxia preconditioning treatment indicate that NR2 B and its major phosphorylation site p-NR2B-1472 are involved in hypoxic preconditioning both in vivo and in vitro.The protective effect of neurological excitatory damage may be related to the endocytosis and metastasis of p-NR2B-1472 on the cell membrane,and DNA methylation was involved in the change of p-NR2B-1472.