P2X受体在大鼠海马氧糖缺失时IL-1β合成和释放中的作用

Role of P2X receptors in synthesis and release of IL-1β during oxygen-glucose deprivation in rat hippocampus

目的 探讨P2X受体在大鼠海马氧糖缺失时IL-1β合成和释放中的作用.方法 成年雄性SD大鼠,体重150～200 g,制备海马脑片,取160张,随机分为4组（n=40）,C组：用95%O2-5%CO2饱和aCSF孵育;OGD组：脑片移至经95%N2-5%CO2饱和的无糖aCSF中,并置于持续通入95%N2-5%CO2的容器内进行氧糖缺失处理;BBG组：脑片移至含P2X7受体特异性拮抗剂亮蓝G（BBG,终浓度1μmol/L）的经95%O2-5%CO2饱和的aCSF中,孵育20 min,随后进行氧糖缺失处理,无糖aCSF中加入终浓度1 μmol/L的BBG;OP组：脑片移至加入P2X4受体单克隆抗体（终浓度1.5μg/ml）的经95%O2-5%CO2饱和的aCSF中孵育60 min,随后进行氧糖缺失处理,无糖aCSF中加入终浓度1.5 μg/ml的P2X4受体单克隆抗体.于氧糖缺失前及氧糖缺失20、40和60 min时测定LDH和IL-1β的释放量,并观察病理学结果.于氧糖缺失60 min时测定细胞内IL-1β前体蛋白（pro-IL-1β）的表达水平.结果 与C组比较,其余各组LDH和IL-1β释放量升高,细胞内pro-IL-1β表达上调（P＜0.05）;与OGD组比较,BBG组LDH和IL-1β释放量降低,细胞内pro-IL-1β表达上调（P＜0.05）,OP组上述指标差异无统计学意义（P＞0.05）.结论 P2X7受体介导了大鼠海马氧糖缺失时IL-1β的合成和释放,而P2X4受体没有介导IL-1β的合成和释放.

Objective To evaluate the role of P2X receptors in the synthesis and release of IL-1β during oxygen-glucose deprivation （OGD） in rat hippocampus. Methods Male SD rats weighing 150-200 g were anesthetized with ether and decapitated. The hippocampi were removed and sagittally sliced （400 μm thick） and placed in artificial cerebrospinal fluid （aCSF） aerated with 95% O2-5% CO2. One hundred and sixty hippocampal slices were randomly divided into 4 groups （ n = 40 each）： Ⅰ control group （group C）; Ⅱ OGD group; Ⅲ OGD ＋BBG group; Ⅳ OGD ＋ anti-P2X4 group （group OP）. In group C, the hippocampal slices were continously incubated with aCSF aerated with 95% O2-5% CO2 . In group OGD, the hippocampal slices were incubated with glucose-free aCSF and aerated with 95% N2-5%CO2 . In group OGD ＋ BBG, the hippocampal slices were incubated with aCSF containning P2X7 receptor-specific antagonist G （BBG, final concentration 1 μmol/L） and aerated with 95% O2-5% CO2 for 20 min, then exposed to OGD, and BBG （final concentration 1 μ mol/L） was added in glucose-free aCSF. In group OP, the hippocampal slices were incubated with aCSF containning P2X4 receptor antibody （final concentration 1.5 μg/ml） and aerated with 95% O2-5% CO2 for 60 min, then exposed to OGD, and P2X4 receptor antibody （final concentration 1.5 μg/ml） was added in glucose-free aCSF. LDH and IL-1β release was detected before OGD and at 20, 40 and 60 min of OGD. Histological changes were observed using HE staining.Intracellular pro-IL-1β precursor protein expression was detected by Western blot at 60 min of OGD. Results LDH and IL-1β release and expression of intracellular pro-IL-1β precursor protein were signifcantly higher in the other groups than in group C （ P ＜ 0.05 ）. Compared with group OGD, LDH and IL- 1 β release was signifcantly decreased, while the expression of intracellular pro-IL-1β precursor protein un-regulated in group OGD ＋ BBG （ P ＜0.05）, but no signifcant difference was found in the prarameters mentioned above in group OP （ P ＞ 0.05）. Conclusion P2X7 receptor mediates the synthesis and release of IL-1β during OGD in rat hippocampus, but P2X4 receptor does not.