吗啡预处理对小鼠海马脑片氧-糖缺失／恢复时CaMKⅡ和NMDA受体的影响

Effect of morphine preconditioning on the changes of CaMKⅡand NMDA receptors in mice hippocampal slices induced by oxygen-glucose deprivation and recovery

目的评价吗啡预处理对小鼠海马脑片氧-糖缺失／恢复时钙／钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)膜转位及N-甲基-D-天冬氨酸(NMDA)受体磷酸化的影响。方法成年BALB／C小鼠,体重18-22 g,雌雄不拘。每次将5只小鼠同批断头取脑,制备海马脑片,随机分为5组:正常对照组(Ⅰ组)、氧-糖缺失／恢复组(Ⅱ组)、吗啡预处理组(Ⅲ组)、纳络酮+吗啡预处理组(Ⅳ组)及纳络酮预处理组(Ⅴ组)。Ⅰ组脑片正常体外培养,假操作换液。Ⅲ、Ⅳ、Ⅴ组小鼠海马脑片分别作相应预处理30 min,间隔30min。Ⅱ、Ⅲ、Ⅳ、Ⅴ组建立小鼠海马脑片体外缺血再灌注损伤模型,分别氧-糖缺失20 min,氧-糖恢复2 h。各组于氧-糖缺失前即刻(T0)、氧-糖缺失20 min(T1)、氧-糖恢复1 h(T2)和2 h(T3)取若干脑片匀浆、超声粉碎、离心,分离胞浆蛋白和膜相关蛋白;部分脑片分离总蛋白,Western blot检测CaMKⅡα蛋白表达量以及NMDA受体NR1亚单位的磷酸化。结果海马脑片在T1、T2、T3时, CaMKⅡα膜相关蛋白含量增多,同时胞浆蛋白含量减少(P<0．05);T2、T3时,NMDA受体NR1亚单位磷酸化水平增高(P<0．05);而吗啡预处理明显地抑制上述CaMKⅡα膜转位及NMDA受体NR1亚单位磷酸化(P<0．05)。纳络酮完全阻断吗啡预处理对CaMKⅡα膜转位及NR1磷酸化的抑制作用(P <0．05)。CaMKⅡα总蛋白表达水平未见明显变化。结论CaMKⅡ膜转位的抑制及NMDA受体磷酸化的抑制在吗啡预处理对小鼠海马脑片缺血再灌注的脑保护作用机制中起重要作用。

Objective To investigate the effects of morphine preconditioning on the changes of membrane translocation of calcium/calmodulin-dependent kinase Ⅱ （CaMK Ⅱ ） and phosphorylation of NMDA receptors in hippocampal slices induced by oxygen-glucose deprivation and recovery （ OGDR）. Methods Hippocampal slices （400 tun thick） were prepared from adult BALB/C mice of both sexes and incubated in normal artificial cerebrospinal fluid （nACSF） aerated with 95% O2 and 5% CO2 at 37℃ for 30 min. The slices were then divided into 5 groups： group Ⅰ control （C） ; group Ⅱ OGDR; group Ⅲ morphine preconditioning ＋ OGDR （M） ; group Ⅳ naloxone ＋ morphine preconditioning ＋ OGDR （N ＋ M） and group Ⅴ naloxone preconditioning ＋ OGDR （N）. In group Ⅱ , Ⅲ, Ⅳ and Ⅴ the hippocampal slices were deprived of O2 and glucose for 20 min followed by 2 h reoxygenation and glucose supply. The slices were treated with morphine 3 μmol· L^-1 × 30 min （ group m ） or naloxone 50 μmol· L^- 1 × 30 min followed by naloxone 50 μmol· L^- 1 ＋ morphine 3 μmol· L^-1× 30 min （ group Ⅳ ） or naloxone 50 μmol· L^- 1 × 1 h （ group Ⅴ ） before OGDR. The slices were homogenized, supersonicated and centrifuged to separate particulate, cytosolic and total protein immediately before OGDR （ T0 ）, at the end of 20 min OGDR （%） and at 1 h （T2） and 2 h （T3） of reoxygenation and glucose supply for determination of the changes of membrane translocation and total protein of CaMK Ⅱ a and the level of phosphorylation of NRI subtype of NMDA receptors in hippocampal slices （ Western blot）. Results The levels of CaMK Ⅱ a were significantly increased in the particulate fraction but decreased in the cytosolic fraction at T1, T2, and T3 as compared with the basaeline values at To, while the levels of phosphorylation of NR1 subtype of NMDA receptors were significantly increased at T2 and T3 as compared with the baseline values at To in OGDR group. Morphine preconditioning significantly inhibited the changes of the membrane translocation of CaMK Ⅱα and phosphorylation of NR1 subtype of NMDA receptors induced by OGDR. The inhibitory effects of morphine preconditioning were completely blocked by naloxone. There was no significant change in protein expression of CaMK Ⅱα. Conclusion Inhibition of membrane translocation of CaMK Ⅱα and phosphorylation of NMDA receptors by morphine preconditioning plays an important role in the neuroprotection against ischemia-reperfusion injury.