To Investigate The Protective Effect Of Simvastatin On Ischemic Stroke Based On Up - Regulation Of NR3A And PP2A Expression

BackgroundStroke is the commonly encountered and frequently-occuring disease in the aged and is ranked as three major lethal diseases with heart disease and malignant tumour for its high mortality and disability, of which60%-70%is ischemic stroke, its attack rates increases with years and a trend of getting younger appearing. In the pathogenesis of ischemic stroke, brain ischemia and reperfusion is the leading cause of neuronal death, and neuronal excitotoxicity is always the core issue in ischemia and reperfusion, so it is urgent to make great efforts in researching and searching the best remedy to prevent and treat this disease. Statins could decrease the synthesis of cholesterol by inhibiting hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductases, and the protective effects of statins on cardiovascular and cerebrovascular diseases are called as "pleiotropic effects", which are independent of reducing cholesterol. By copmparing many parameters among statins, simvastatin showed the best effect on cerebral ischemic injury for its higher liposolubility and blood-brain barrier permeability.N-methyl-D-aspartate receptor (NMDAR) is constituted by NR1, NR2and NR3subunits, and NR2including four subtypes, NR2A, NR2B, NR2C and NR2D, while NR3A and NR3B belonging to NR3. Traditional NMDAR is composed of NR1and different NR2subtypes, and as it activated by glutamate, the cations, such as Na+、K+, Ca2+(mainly), would inflow to activate the downstream signaling pathways and regulate the functional activities of neural system. Under pathological conditions of brain ischemia and reperfusion, the overload of Ca2+by the overactivition of NMDAR along with large amount of glutamate leads to the downstream signaling pathways amplifying and then the excitotoxicity producing. In1995, NR3A was firstly reported, and the expression of which showed temporal and spatial specificity. The NR3A-contained receptors in hippocampus neuron of transgene mice, and the noval NMDAR with NR3A generated by gene fusion in Xenopus oocytes, HEK293all showed insensitive to glutamate and the influx of Ca2+decreasing while it activated.Serine/threonine protein kinase and protein phosphotase play a role in regulating functional activities of many kinds of proteins by phosphorylation and dephosphorylation. Two serine phosphorylation sites (Ser896and Ser897) of NR1could be phosphorylated, and the phosphorylation would increase NMDAR activities and Ca2+influxes. However protein phosphatase2A could make these phosphorylation sites dephosphorylated and thus inhibiting the activities of NMDAR.AimsThe present research would uncover that whether NR3A and PP2A participate in the protective effect of simvastatin on ishchemic stroke and the phosphorylation levels of serine sites on NR1. As the downstream of NMDAR, Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) may reflect the overactivation of NMDAR, so we would also detect that how the autophosphorylation of CaMK II is changed and if PP2A have a regulative and positive effect on NR3A.MethodsPretreatment of simvastatin for7days, and then adopted middle cerebral artery occlusion (MCAO) model following1hour’occlusion and24or12hour’reperfusion.The animals were divided into four groups:sham group, MCAO group, vehicle+MCAO group and sim+MCAO group.1. Neurological deficit scoring, infarct area and brain water content measuring with2,3,5-triphenyltetrazolium chloride (TTC) staining, dry/wet weighting and magnetic resonance imaging (MRI) scanning;2. Observing the morphosis of neurons with hematoxylin eosin (HE), Nissl and Hoechst33258staining;3. Detecting enzymatic activities of PP2A in hippocampus CA1region with serine/threonine phosphotase assay system;4. Detecting the mRNA expression of NR1and NR3A in hippocampus CA1region with real-time qPCR;5. Detecting the phosphorylation of NR1(Ser896, Ser897) and CaMKⅡ (Thr286), and the protein levels of NR3A, cytalytic subunit(C subunit) of PP2A, and AQP4with western blot;6. Single administration of simvastatin for7days to normal rats, and then detecting the enzymatic activities of PP2A with sering/threonine phosphotase assay system, and the protein levels of PP2A C subunit and NR3A with western blot;7. Observing the immunocomplex between NR3A and NR1, AQP4, CaMKⅡ, PP2A in normal adult rats with co-immunoprecipitation;8. After pretreatment of simvastatin for7days, okadaic acid (OA) was microinjected into hippocampus CA1and then adopted MCAO to detect the protein levels of PP2A and NR3A;9. Microinjection of sphingosine or OA into hippocampus CA1of normal rats, and then detected the protein levels of PP2A, NR3A and NR1.Results1. Simvastatin decreased neurological deficit signs, infarct area and brain water contentMCAO group presented a poor neurological function and brain infarction when compared with those of sham group, which, however, showed no neurological deficit and infarction. Simvastatin pretreatment decreased the behavior scores (n=8, P<0.01) and brain infarction (n=6, P<0.01) significantly in sim+MCAO when compared with MCAO group.The brain water content in MCAO group (n=6, P<0.01) significantly increased as compared with that in sham group, while comparing with MCAO group, simvastatin decreased water content in sim+MCAO group (n=6, P<0.05). The brain water content of contraletaral hemisphere among the four groups showed no significant difference.2. Simvastatin improved the morphological structure of neurons in hippocampus CA1regionAs compared with sham group, the hippocampus CA1regions of vehicle+MCAO group showed degenerated neurons with pyknosis and cytoplasm vacuoles, and increased apoptosis, but in sim+MCAO group, the degenerated neurons significantly decreased.3. Simvastatin decreased phosphorylation of NR1(ser896, ser897)At I1R24, it was found that the phosphorylation of NR1(ser896, ser897) in MCAO group (n=5, P<0.01) increased markedly in comparison with those of sham group, but the two indexes in sim+MCAO group (n=5, P<0.01and P<0.05) decreased when compared with those of MCAO group. The protein levels of total NR1in MCAO group and sim+MCAO group were all decreased as compared with that of sham group (n=5, P<0.05).At I1R12, the mRNA of NR1in MCAO group and sim+MCAO group decreased when compared with that of sham group (n=5, P<0.01).4. Simvastatin increased NR3A levelsWith real-time qPCR analysis, it was found that the mRNA expression of NR3A in MCAO group (n=5, P<0.01) were all lowered at I1R12and I1R24when compared with that of sham group. At I1R12, the mRNA expression of NR3A in sim+MCAO group (n=5, P<0.05) increased when compared with that of vehicle+MCAO group. At I1R24, the mRNA expression of NR3A in sim+MCAO group (n=5, P<0.01) significantly increased as compared with that of MCAO group and vehicle+MCAO group.With western blot analysis, the protein levels of NR3A among the four groups showed no significant difference at I1R12. At I1R24, the protein levels of NR3A in MCAO group (n=5, P<0.01) significantly decreased as compared with that of sham group, however, the protein levels of NR3A in sim+MCAO group (n=5, P<0.01) significantly increased when compared with that of MCAO group.5. Simvastatin decreased phosphorylation of CaMK Ⅱ (Thr286)At I1R24, the phosphorylation of CaMK Ⅱ(Thr286) in MCAO group (n=6, P<0.01) increased markedly in comparison with that of sham group, but the index in sim+MCAO group (n=6, P<0.01) decreased as compared with that of MCAO group.6. Simvastatin increased PP2A levels At I1R24, the enzymatic activities of PP2A in MCAO group (n=6,P<0.01) decreased when compared with that of sham group, but the enzymatic activities in sim+MCAO group (n=6, P<0.01) increased as compared with that in MCAO group.At I1R12, the protein levels of PP2A C subunit showed no significant difference. At I1R24, the protein levels of PP2A C subunit in MCAO group (n=6, P<0.05) decreased as compared with that of sham group, while the protein levels of PP2A C subunit in sim+MCAO group (n=6,P<0.05) increased as compared with that of MCAO group.7. Simvastatin decreased AQP4levelsAt I1R24, the protein levels of AQP4in MCAO group (n=6, P<0.01) increased markedly in comparison with that of sham group, but the index decreased in sim+MCAO group (n=6, P<0.05) when compared with that of MCAO group.8. Single pretreatment of simvastatin had no effect on PP2A and NR3ASingle pretreatment of simvastatin on normal rats for7days, the protein levels of PP2A C subunit and NR3A in sim group showed no significant difference while compared with those of vehicle group.9. Immunocomplex formed was detected in hippocampus of normal rats with co-immunoprecipitation In normal rats, immunocomplex existed between NR3A and NR1, AQP4, and CaMK II, but the complex was not detected between NR3A and PP2A.10. The regulative effect of PP2A on NR3AAs compared with sim+DMSO+MCAO group, the protein levels of PP2A C subunit in sim+OA+MCAO group showed no significant difference, but the protein levels of NR3Ain sim+OA+MCAO group (n=6, P<0.01) markedly decreased.Microinjection of sphingosine or OA into hippocampus CA1of normal rats had no effect on the protein levels of PP2A C subunit. The protein levels of NR3A in normal+SP group (n=5, P<0.01) increased as compared with that of normal+DMSO group, but the index in normal+OA group (n=5, P<0.05) decreased as compared with that of normal+DMSO group. The protein levels of NR1in normal+SP group (n=5, P<0.01) decreased as compared with that of normal+DMSO group, however the index in normal+OA group (n=5, P<0.05) increased as compared with that of normal+DMSO group.Conclusions1. The protective effect of simvastatin on ischemic stroke could be elucidated by enhancing the expression levels of NR3A and PP2A, and then decreasing the phosphorylation of NR1(ser896, ser897) and the autophosphorylation of CaMK Ⅱ.2. Simvastatin could reduce brain edema by decreasing the protein levelsvof AQP4in ischemic stroke.3. NR3A interacted with AQP4, CaMK Ⅱ, and NR3A may be the intermediate to promote the signal transduction between AQP and CaMK.4. No complex was detected between NR3A and PP2A, but PP2A may exert a regulative and positive effect on the expression of NR3A, and the upregulation of NR3A competitively inhibited the expression of NR1, which may benefit the formation of noval NMDAR.