The Effect Of Propofol Post-conditioning On ADAR2-AMPAR Receptor GluR2 Pathway In Oxygen-glucose Deprivation Induced Primary Hippocampus Neuron Injury

In China, the number of stroke patients is 7 million, and the annual stroke mortality rate is approximately 1.6 million, which approximately 60% patients are with different degree of loss labor. Because of the high disabling and deadly, the patient and the society always are under heavy burdens. Meanwhile, in clinical works, perioperative surgical operations, such as intracranial aneurysm, inevitably caused ischemia-reperfusion injury. As anesthesiologists, choosing the appropriate anesthetics which could preserve the compromised brain function for the patients who have suffered cerebral injury or with high risk of perioperative stroke is a major research endeavor. Our team previous research demonstrated that propofol at dose of 20mg·kg-1·h-1 infused at the onset of reperfusion could provide neuroprotection to rats which had undergone 2h of middle cerebral artery occlusion(MCAO) by contributing to stabilization of GluR2 on α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid(AMPA) receptors which play a critical role in central nervous systerm ischemic insults. Howerver, the upstream mechanism is not completely clear. Therefore, the study aimed to investigate the role of adenosine deaminase acting on RNA2(ADAR2)-AMPA receptor GluR2 subunit pathway in the neuroprotection induced by propofol postconditioning in ischemia model in vitro. The study chose primary hippocampus neuros to make oxygen-glucose deprivation(OGD)-reperfusion model to stimulate the ischemia-reperfusion injury in rats. The experiments divided into two parts. The first part, the culture of primary hippocampus neurons and the building of OGD model in vitro. The second part, investigate the effect of the ADAR2-AMPA receptor GluR2 pathway in neuroprotection induced by propofol postconditioning. The primary cultured neurons were randomly divided into 4 groups: normal control group(group C); oxygen-glucose deprivation(OGD) group(group O: OGD 1h followed by cultured routinely); propofol post-conditioning group(group Pro-post: after OGD 1h, the cells were exposed to medium with 1.2μg/ml propofol then cultured routinely) and siADAR2 group(group siADAR2: in cultured 3th day, neurons were infected by lentivirus with GFP for 24 h, and in 7th day, after OGD 1h, the cells were exposed to medium with 1.2μg/ml propofol then cultured routinely). After 24 h, the neuron morphology was evaluated by observing under light microscope. The cellular viability and cytotoxicity were respectively tested by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) and lactate dehydrogenase(LDH) release. The expression of ADAR2 protein at 24 h after collection was analyzed by Western Bolt. The ratio of GluR2 mRNA Q/R edited was analyzed by Nest reverse transcriptionpolymerase chain reaction(RT-PCR) and BbV1. The intracellular Ca2+ concentration were evaluated by fluo-3 AM and flow cytomety. The first part, our outcome had demonstrated that the majority of cultured hippocampus cells were neurons. Microtubule associated protein(MAP2) and 4’6-diamidino-2-phenylindole(DAPI) were used for identifying the purity of cultured primary hippcampal neurons. The percentage of cultured neurons was above 95%. Morevoer, compared with control group, the cell viability of 1h, 2h and 4h had decreased signicantly, which provided an important signal for susscess of OGD model. As the result, we chose the 1h as the time of OGD model. The second part, compared with group C, there are statistically decreased in neurons viability, the ratios of ADAR2 plasmosin/total protein and the ratio of GluR2 mRNA Q/R edited/unedited in group O, and decreased in intracellular Ca2+ concentration(p<0.05). However compared with group O, propofol post-conditioning importantly increased neurons viability, the ratios of ADAR2 plasmosin/total protein and the ratio of GluR2 mRNA Q/R edited/unedited, increased in intracellular Ca2+ concentration(p<0.05). ADAR2 knockdown by small interfering RNA weakened neuroprotection effect induced by propofol post-conditioning. As the conclusion, we presently shown that propofol post-conditioning provided acute neuroprotection against the OGD injury through promoting nuclear expression of ADAR2 protein, further increased the ratio of AMPAR GluR2 subunit Q/R site edited, stabilized the structure of postsynaptic AMPA receptor GluR2 subunit and inhibited the internalization of AMPAR GluR2 subunit, ultimate avoided damage cascade reaction induced by intracellular Ca2+ overload in vitro.