CXCR4拮抗剂AMD3100通过增强抑制性神经传递减弱大鼠痫性活动

CXCR4 antagonist AMD3100 attenuates epileptic activity by enhancing inhibitory neurotransmission in rats

目的探讨CXCR4特异性拮抗剂AMD3100影响癫痫发作的分子机制。方法(1)动物实验:36只成年雄性SD大鼠按随机数字表法分为对照组、癫痫组和AMD3100处理组,每组12只。癫痫组大鼠腹腔注射戊四氮(PTZ)构建癫痫模型,剂量为40 mg/kg;AMD3100处理组大鼠侧脑室注射5μL(5 mg/mL)AMD310020 min后腹腔注射同等剂量PTZ;对照组大鼠腹腔注射等量生理盐水。采用Racine分级评估各组大鼠癫痫发作等级并记录其癫痫发作潜伏期,采用脑电图(EEG)记录各组大鼠脑神经元异常放电情况,采用ELISA试剂盒检测各组大鼠海马组织γ-氨基丁酸(GABA)含量。应用实时荧光定量PCR(qRT-PCR)技术检测各组大鼠海马组织神经元γ-氨基丁酸A受体α1亚单位(GABA_(A)Rα1)mRNA水平。(2)细胞实验:另取SD大鼠鼠婴(1日龄)培养原代海马神经元,培养7 d后将细胞分为对照组、癫痫组和AMD3100处理组。癫痫组神经元采用无镁外液诱导的方法构建癫痫细胞模型;AMD3100处理组神经元在含有10 nmol/L AMD3100的无镁外液中培养3 h,随后换为Neurobasal继续培养;对照组采用Neurobasal培养基常规培养。采用全细胞膜片钳技术检测各组神经元灌流AMD3100(10 nmol/L)后自发性抑制性突触后电流(sIPSC)。结果(1)动物实验:AMD3100处理组大鼠发作潜伏期较癫痫组大鼠明显缩短[分别为(663.30±74.84)s、(164.40±17.20)s],差异有统计学意义(t=6.490,P<0.001);AMD3100处理组大鼠4级以上发作次数较癫痫组大鼠明显减少[分别为(3.75±0.39)次、(9.00±0.73)次],差异有统计学意义(t=4.680,P<0.001)。ELISA实验结果显示,3组大鼠GABA含量差异有统计学意义(F=17.850,P<0.001),其中癫痫组明显低于对照组,AMD3100处理组大鼠明显高于癫痫组,差异均有统计学意义(P<0.05)。qRT-PCR实验结果显示,3组大鼠GABA_(A)Rα1 mRNA含量差异有统计学意义(F=14.400,P<0.001),其中癫痫组明显低于对照组,AMD3100处理组大鼠明显高于癫痫组,差异均有统计学意义(P<0.05)。EEG结果显示,与癫痫组大鼠比较,AMD3100处理组大鼠放电频率有所降低。3组大鼠EEG功率差异无统计学意义(F=3.220,P=0.062),但与癫痫组、对照组大鼠比较,AMD3100处理组大鼠EEG功率有所降低。(2)细胞实验:膜片钳技术检测结果显示,3组神经元sIPSCs的频率和波幅差异均有统计学意义(F=13.670,P<0.001;F=10.920,P<0.001)。与对照组、癫痫组比较,AMD3100处理组神经元sIPSCs的频率和波幅均显著增加,差异均有统计学意义(P<0.05)。结论CXCR4拮抗剂AMD3100可通过增强抑制性神经传递降低癫痫发作频率。

Objective To investigative the molecular mechanism of C-X-C chemokine receptor type 4(CXCR4)antagonist AMD3100 in epileptic seizure.Methods(1)Animal experiment:36 adult male SD rats were randomly divided into control group(Con,n=12),epilepsy group(Epi,n=12),Epi+AMD3100 group(n=12).Experimental epilepsy rat models in the Epi group were induced by intraperitoneal injection of pentrazole(PTZ,40 mg/kg);rats in the Epi+AMD3100 group were given intraperitoneal injection of PTZ(40 mg/kg)20 min after lateral intracerebroventricular injection of 5μL(5 mg/mL)AMD3100;rats in the Con group were given intraperitoneal injection of normal saline.Racine grading was used to evaluate the levels of epileptic seizure and the latency of epileptic seizure was recorded in rats from each group.EEG was used to record the abnormal discharges of brain neurons in rats from each group.The content ofγ-aminobutyric acid(GABA)in the hippocampus was detected by ELISA kit;γ-aminobutyric acid A receptorα1 subunit(GABA_(A)Rα1)mRNA levels of hippocampal neurons in each group were detected by real-time fluorescent quantitative PCR(qRT-PCR).(2)Cell experiment:the hippocampal neurons from 1-d-old SD rats were primarily cultured;7 d after cultivation,these cells were divided into control group,epilepsy group and AMD3100 group;the cellular epileptic models in the epileptic group were induced by magnesium-free external fluid;neurons in the AMD3100 group were cultured in magnesium-free external solution containing 10 nmol/L AMD3100 for 3 h,and then changed to Neurobasal medium for further culture;cells in the control group were cultured with Neurobasal medium.Whole cell patch clamp technique was used to detect the spontaneous inhibitory postsynaptic currents(sIPSCs)after AMD3100(10 nmol/L)perfusion.Results(1)Animal experiment:the seizure latency in Epi+AMD3100 group was significantly shorter than that in Epi group([663.30±74.84]s vs.[164.40±17.20]s,t=6.490,P<0.001).The frequency of seizures>grading 4 in the Epi+AMD3100 group was significantly decreased as compared with that in the Epi group(3.75±0.39 vs.9.00±0.73,t=4.680,P<0.001).ELISA results showed that GABA content in the 3 groups was significantly different(F=17.850,P<0.001):that in the Epi group was significantly lower than that in the Con group,and that in the Epi+AMD3100 group was significantly higher than that in Epi group(P<0.05).The qRT-PCR results showed that GABA_(A)Rα1 mRNA content among the 3 groups was significantly different(F=14.400,P<0.001):that in the Epi group was significantly lower than that in the Con group,and that in the Epi+AMD3100 group was significantly higher than that in the Epi group(P<0.05).EEG results showed that the discharge frequency of rats in the Epi+AMD3100 group was lower than that in Epi group;there was no significant difference in EEG power among the 3 groups(F=3.220,P<0.001),but the EEG power in the Epi+AMD3100 group was lower than that in Epi group and control group.(2)Cell experiment:patch clamp technique showed that the average frequency and amplitude of sIPSCs in the 3 groups were statistically significant(F=13.670,P<0.001;F=10.920,P<0.001).As compared with those in the control group and epilepsy group,the average frequency and amplitude of sIPSCs in AMD3100 group were significantly increased(P<0.05).Conclusion CXCR4 antagonist AMD3100 can reduce seizure frequency by enhancing inhibitory neurotransmission.