| Objective: Excited toxicity of glutamte (Glu) is an improtant molecular mechanism of traumatic brain injury (TBI). To further interprete the pathologic process of TBI, It is essential to invastigate the structure of Glu recepter subunit, their switch of combination and regulatory process of expression and polymeriztion. Recent researches have shown that the high expression level of phosphatase and tensin homology deleted on chromosome ten (PTEN) could deteriorate TBI through involving in metabolism of Glu receptor and regulating the excited toxicity of Glu receptor. PTEN can boost excitability of NMDA receptor by regulating phosphorylation process of NMDA receptor to induce Ca2+ overload and neural apoptosis. But, the regulation of PTEN on AMPA receptor, another crucial receptor of Glu, is unclear and need further research.AMPA receptor contains four kinds of subunit coded by different genes, which are GluR1,GluR2,GluR3 and GluR4. Because of the low permeability of GluR2 subunit to Ca2+ induced by mRNAQ/R site editing, the relative content of GluR2 determines the functional characteristics of AMPA receptor. Kinds of neuron have a high content of GluR2 which can restrict Ca2+ influx. So, GluR2 may be a crucial target in the process that PTEN regulate the metabolism of AMPA receptor.Methods: we cutured primary hippocanlpal neuron from newborn SD rat and establish stretch damage model of neuron. Western-blot and Tunel staining were done to detect the expression level of PTEN and positioning of different subunits of AMPA receptor at different phases. ShRNA lentivirus was ultilized to block the expression of PTEN in neuron. Meanwhile, we assessed the regulation of PTEN on expression and positioning of AMPA-GluR2 in rat primary hippocampal neuron after stretch damage to provide experimental basis to the prevention and treatment strategy of TBI target PTEN.Results:1. The relationship between the expression level of PTEN and AMPA receptor positioning of hippocanlpal neurons after stretch damage.(1) After stretch damage, the apoptosis ratio of rat hippocanlpal neurons increased with the passage of time.(2) The expression level of PTEN increased after stretch damage, the neuons at 2h after damage contained higer level expression of PTEN than neurons without damage (P < 0. 05). The expression level of PTEN peaked in neuons at 24h after damage.(3) The results of Western-blot showed the expression level of AMPA-GluR1,GluR2 and GluR3 in total protein of neuron had no statistically difference at 0h, 2h, 6h, 24h, 48h after stretch damage.(4) The results of Western-blot showed the expression level of AMPA-GluR1 and GluR3 in membrane protein of neuron had no statistically difference at 0h, 2h, 6h, 24h, 48h after stretch damage. But, the expression level of AMPA-GluR2 in membrane protein of neuron decreased evidently at 6h, 24h, 48h after stretch damage.2.The regulation of PTEN siRNA induced by lentivirus on expression and positioning of AMPA-GluR2 in rat primary hippocampal neuron after stretch damage.(1) PTEN siRNA induced by lentivirus can block the expression of PTEN in rat primary hippocampal neuron effectively.(2) The neurons swelled at 2h after damage. The neuron shrinked, some connection of cells ruptured and the celluar network thinned out at 6h after damage. The neurons shrinked more evidently, most of cells ruptured, many dead cells suspended and the celluar network disappeared at 24h after damage. The number of neuron decreased more evidently, the cell debris and degenerative neuron could be seen anywhere. The morphological changes between Neuron-PTEN-ND and Neuron was rare at 0h, 2h and 6h after damage, the injuries of Neuron-PTEN-ND were obviously lighter than that of Neuron at 24h and 48h after damage.(3) The results of Western-bolt showed the content of AMPA-GluR2 had no statistical difference between the total protein of Neuron-PTEN-ND and Neuron at each phase. The content of AMPA-GluR2 in membrane protein of Neuron decreased evidently at 6h, 24h, 48h after damage. The content of AMPA-GluR2 in membrane protein of Neuron-PTEN-ND showed no evident decrease at 6h after damage and a light decrease at 24h and 48h after damage. The content of AMPA-GluR2 in membrane protein of Neuron-PTEN-ND were higher than that of Neuron at 6h, 24h and 48h after damage.Conclusion: The expression level of PTEN and the content of AMPA-GluR2 shows a negative corrlation in hippocampal neuron after stretch damage. It hints PTEN may regulate the transport and polymerise process of GluR2. Downregulation of PTEN can inhibit the decrease of GluR2 content in membrane protein of neuron effectively and relieve apoptosis of neuron. The regulation of PTEN on AMPA receptor may provide a new passway to prevention and treatmen of TBI. |
