P2X7受体抑制剂通过神经连接蛋白负性调控癫痫发生过程

目的分析癫痫发作后24 h内P2X7受体(P2X7R)和神经连接蛋白(NLGN)的表达特点,并探讨P2X7R抑制剂负性调控癫痫发生过程的可能机制。方法将健康雄性SD大鼠按随机数字表法分为6组,分别为1 h、3 h、6 h、12 h、24 h组及空白对照组(每组3只);除空白对照组外,其余组均制备为氯化锂-匹鲁卡品癫痫模型,Racine评分量表评级达4或5级为造模成功。依次于癫痫发作后1 h、3 h、6 h、12 h及24 h将各组大鼠处死,空白对照组与1 h组一同处理,采用蛋白免疫印迹法检测P2X7R、NLGN1及NLGN2在各组大鼠海马和内嗅皮质中的表达情况。进一步按随机数字表法将健康雄性SD大鼠分为干预组(造模前腹腔注射P2X7R抑制剂+载体)和对照组(造模前腹腔注射等剂量生理盐水+载体),每组3只。记录两组大鼠药物诱发癫痫的潜伏时间,以判断癫痫持续状态的严重程度;之后采用蛋白免疫印迹法检测两组大鼠海马和内嗅皮质中NLGN1、NLGN2的表达情况,以判断P2X7R抑制剂对其表达的影响。结果1 h、3 h、6 h、12 h及24 h组大鼠均造模成功。蛋白免疫印迹法结果显示,癫痫发作后1 h或3 h,P2X7R、NLGN1及NLGN2在癫痫模型海马和内嗅皮质中的相对表达量(海马:分别为1.194±0.053、2.367±0.336、2.319±0.234,内嗅皮质:分别为1.185±0.104、2.094±0.178、1.512±0.332)均高于空白对照组(海马:分别为1.014±0.046、1.108±0.101、1.134±0.177,内嗅皮质:分别为0.917±0.081、1.046±0.093、1.017±0.028;均P<0.05);且随发作时间延长,表达均有降低的趋势(均P<0.001)。干预组药物诱发癫痫的潜伏时间较对照组长[分别为(34.5±2.6)min、(20.0±3.1)min,P=0.029]。蛋白免疫印迹法检测结果显示,干预组海马中NLGN1和NLGN2的相对表达量(NLGN1:0.678±0.011,NLGN2:0.904±0.060)均低于对照组(NLGN1:1.031±0.039,NLGN2:1.135±0.079;均P<0.05),而内嗅皮质中NLGN1和NLGN2的相对表达量,两组间差异均无统计学意义(均P>0.05)。结论癫痫发作后1 h或3 h内,P2X7R和NLGN蛋白在海马和内嗅皮质中的表达量均明显增加,且在发作后24 h内,随发作时间延长呈降低的趋势;P2X7R抑制剂可通过抑制海马区NLGN的表达进而负性调控癫痫发生过程。

Negative modulation of epielptogenesis by P2X7 receptor inhibitors via neuroligins

Objective To analyze the expression patterns of P2X7 receptor(P2X7R)and neuroligin(NLGN)within 24 hours after seizures and to explore the possible mechanism of P2X7R inhibitors in negative regulation of epileptogenesis.Methods Healthy male Sprague Dawley(SD)rats were divided into 6 groups using the random number table,which were 1 h,3 h,6 h,12 h,and 24 h groups and a control group with each group containing 3 rats.Except the control group,the other groups of rats were administered with lithium chloride-pilocarpine to establish the epilepsy model,which was considered as successful in case of grade 4 or 5 based on Racine scale.Those rats were sacrificed at 1 h,3 h,6 h,12 h,and 24 h post seizures.Rats in control and 1 h groups were sacrificed at the same time.Protein immunoprinting was used to detect expression of P2X7R,NLGN1 and NLGN2 in the hippocampus and entorhinal cortex in each group of rats.Using the random number table,healthy male SD rats were further divided into an intervention group(intraperitoneal injection of P2X7R inhibitor+vehicle)and a control group(intraperitoneal injection of equivalent dose of saline+vehicle)with 3 rats in each group.The latency of drug-induced epilepsy in two groups of rats was documented to determine the effect on epileptogenesis,and then the expression of NLGN1 and NLGN2 in the hippocampus and entorhinal cortex was detected by protein immunoprinting to determine the effect of P2X7R inhibitors on their expression.Results Epileptic rat models were successfully made in the 1 h,3 h,6 h,12 h,and 24 h groups.Protein immunoprinting results showed that the relative expression levels of P2X7R,NLGN1 and NLGN2 after the seizure 1 hour or 3 hours(hippocampus:1.194±0.053,2.367±0.336,2.319±0.234;entorhinal cortex:1.185±0.104,2.094±0.178,1.512±0.332)were significantly higher than those in the control group(hippocampus:1.014±0.046,1.108±0.101,1.134±0.177;entorhinal cortex:0.917±0.081,1.046±0.093,1.017±0.028;all P<0.05).With the increase of seizure duration,the protein expression showed a tendency of decrease(P<0.001).The latency of drug-induced epilepsy in the intervention group was significantly longer than that of control group(34.5±2.6 min vs.20.0±3.1 min,P=0.029).Protein immunoprinting showed that the relative expression levels of NLGN1 and NLGN2 in the hippocampus of the intervention group(0.678±0.011 and 0.904±0.060 respectively)were lower than those of the control group(1.031±0.039 and 1.135±0.079 respectively,both P<0.05),while the relative expression levels of NLGN1 and NLGN2 in the entorhinal cortex of the intervention group were not statistically different from those of the control group(both P>0.05).Conclusions After the seizure 1 hour or 3 hours,the expression of P2X7R and NLGN proteins in both the hippocampus and entorhinal cortex increases significantly,and decreases with the duration of seizures within 24 h,and P2X7R inhibitors could suppress epileptogenesis by inhibiting the expression of NLGN in the hippocampus.