P2X7R拮抗剂及p38MAPK抑制剂对氧糖剥夺诱导的原代神经元凋亡的影响  

P2X7R antagonist inhibits primary neuronal apoptosis induced by oxygen and glucose deprivation through p38MAPK

作　　者：张涛 胡金霞 张聪慧 张成 郝琦 阴菡菡 昝坤 祖洁 张作慧 时宏娟 杨新新 叶新春 崔桂云 ZHANG Tao;HU Jinxia;ZHANG Conghui;ZHANG Cheng;HAO Qi;YIN Hanhan;ZAN Kun;ZU Jie;ZHANG Zuohui;SHI Hongjuan;YANG Xinxin;YE Xinchun;CUI Guiyun(Department of Neurology,the Affiliated Hospital of Xuzhou Medical University,Xuzhou,Jiangsu 221002,China)

机构地区：[1]徐州医科大学附属医院神经内科,江苏徐州221002

出　　处：《徐州医科大学学报》2020年第4期235-239,共5页Journal of Xuzhou Medical University

基　　金：国家自然科学基金(81771282);江苏省高校自然科学基金(17KJB320017);徐州市科技项目(KC18055)。

摘　　要：目的探讨嘌呤能P2X7受体(P2X7R)拮抗剂通过p38丝裂原活化蛋白激酶(p38MAPK)在原代神经元氧糖剥夺(OGD)模型中发挥的抗凋亡作用。方法培养C57BL/6J胎鼠大脑皮质原代神经元,构建OGD模型。将原代神经元分为对照组和OGD0.5、1、2、3、6h组,Westernblot检测P2X7R的表达变化。再将原代神经元分为对照组、OGD组、OGD+亮蓝G(BBG)、OGD+SB203580组,Westernblot检测P2X7R、磷酸化的p38MAPK(p-p38MAPK)、p38MAPK和活化的半胱天冬酶-3(cleavedcaspase-3)的蛋白表达,免疫荧光染色检测神经元凋亡。结果Westernblot结果显示:与对照组相比,P2X7R在原代神经元OGD1、2、3、6h表达均增加(P<0.05),3h表达最高。与对照组相比,OGD组P2X7R、p-p38MAPK和cleavedcaspase-3的表达均明显升高(P<0.05);而与OGD组相比,OGD+BBG组P2X7R、p-p38MAPK和cleavedcaspase-3的表达均下降(P<0.05);与OGD组比较,OGD+SB203580组p-p38MAPK和cleavedcaspase-3表达下调(P<0.05)。免疫荧光染色显示:与对照组相比,OGD组神经元凋亡明显增加(P<0.05);而与OGD组相比,OGD+BBG组神经元凋亡明显减少(P<0.05)。荧光强度比值分析显示:与对照组相比,OGD组p38MAPK磷酸化水平明显升高(P<0.05),BBG和SB203580预处理后p38MAPK磷酸化水平降低(P<0.05)。结论P2X7R拮抗剂通过抑制p38MAPK磷酸化在原代神经元OGD模型中发挥抗凋亡作用。Objective To investigate the anti-apoptotic effect of purinergic P2X7 receptor(P2X7R)antagonist through p38 mitogen-activated protein kinase(p38MAPK)in primary neuron of oxygen-glucose deprivation(OGD)model.Methods The primary neurons in the cerebral cortex of C57BL/6J fetal mice were cultured and the OGD model was established.The primary neurons were divided into control group and OGD 0.5,1,2,3,and 6 h groups.The expression of P2X7R was detected by Western blot.Then the primary neurons were divided into control group,OGD group,OGD+inhibitor(Brilliant Blue G,BBG)or SB203580)group.The protein expression of P2X7R,phosphorylated p38MAPK,p38MAPK,and activated caspase-3(cleaved caspase-3)were detected by Western blot,and apoptosis was detected by immunofluorescence staining.Results The results of Western blot showed that P2X7R expression in OGD 1,2,3 and 6 h of primary neurons increased(P<0.05),and the highest expression was at 3 h.Compared with the control group,the expression of P2X7R,P-P38MAPK and cleaved caspase-3 in OGD group increased significantly(P<0.05).Compared with OGD group,the expression of P2X7R,P-P38MAPK and cleaved caspase-3 in OGD+BBG group was decreased(P<0.05).Compared with OGD group,the expression of P-P38MAPK and cleaved caspase-3 in OGD+SB203580 group decreased(P<0.05).Immunofluorescence staining showed that compared with the control group,the apoptosis of neurons in OGD group increased significantly(P<0.05),while compared with OGD group,the apoptosis of neurons in OGD+BBG group decreased significantly(P<0.05).Compared with the control group,the phosphorylation level of p38MAPK in OGD group was significantly higher(P<0.05),and the phosphorylation level of p38MAPK in BBG and SB203580 pretreatment group was lower(P<0.05).Conclusions P2X7R antagonists play an anti-apoptotic role in primary neuronal OGD model by inhibiting p38MAPK phosphorylation.

关 键 词：嘌呤能受体 拮抗剂 氧糖剥夺 神经元 凋亡 

分 类 号：R743.33[医药卫生—神经病学与精神病学]