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目的观察兴奋性谷氨酸受体(NMDA 受体)拮抗剂地佐环平(MK801)对边缘叶癫痫大鼠发作后 P-糖蛋白(P-gp)表达的影响,探讨 NMDA 受体是否能对癫痫发作后 P-gp 表达进行调控。方法制备氯化锂-匹罗卡品癫痫发作大鼠模型,60只大鼠,随机分为6组:对照组、癫痫发作终止后0、3、6、24和72 h 组,用实时荧光定量 RT-PCR 检测癫痫大鼠发作终止后不同时程海马组织 P-gpmRNA 的表达。另取60只大鼠随机分为对照组、癫痫持续状态发作模型(SE)组、MK801干预组,各组均分为发作终止后6 h 和24 h 时间点。用实时荧光定量 RT-PCR 检测癫痫发作终止后6 h 大鼠海马 P-gp mRNA 的表达,用免疫组化检测大鼠癫痫发作终止后24 h 海马组织 P-gp 蛋白的表达。结果癫痫发作终止后72 h 内的所有动物海马组织多药耐药基因1a(mdr1a)的表达均显著高于对照组,SE终止即刻其表达显著升高[5.6(2.9)×10~5 mRNA 拷贝数/40 ng 总 RNA,P<0.05],SE 终止后3 h 时其表达进一步升高[7.6(6.3)×10~5,P<0.01],此后至 SE 终止后72 h,mdr1a mRNA 的表达一直维持在该水平;mdr1b 的表达在边缘叶发作动物呈一过性增高,发作终止后3、6 h[分别为(3.3±0.4)×10~4和(3.6±1.0)×10~4,为对照组的2.2、2.4倍]均明显高于对照组(P<0.01)。另外,在发作终止后6 h 时,MK801组 mdr1a[(4.3±0.8)×10~5]和 mdr1b[(2.0±0.7)×10~4]基因表达明显低于 SE组(P<0.01);发作终止24 h 时,MK801组 P-gp[(26.6±5.0)个微血管/400倍视野]蛋白表达也明显低于 SE 组[(39.0±4.1)个微血管/400倍视野,P<0.01]。结论 NMDA 受体拮抗剂 MK801下调癫痫大鼠发作后 P-gp 的过度表达,提示 NMDA 受体可能参与癫痫发作过程中 P-gp 表达的调控,有效抑制 NMDA 受体的过度活化可能有助于防止癫痫发作后 P-gp 的过度表达。
Objective To investigate the effect of MK801, an antagonist of excitatory glutamate receptor (NMDA receptor), on the expression of P-glycoprotein (P-gp) after the onset of limbic epilepsy in rats. P-gp expression after seizure regulation. Methods A rat model of lithium-pilocarpine seizure was prepared. Sixty rats were randomly divided into 6 groups: control group, 0, 3, 6, 24 and 72 h groups after termination of seizure. Real-time fluorescence quantitative RT-PCR was used to detect the expression of P-gpmRNA in hippocampus at different time points after the onset of epilepsy. Another 60 rats were randomly divided into control group, epileptic seizure model group (SE) and MK801 intervention group, and each group was divided into 6 h and 24 h after the termination of seizure. The expression of P-gp mRNA in hippocampus of hippocampus was detected by real-time fluorescence quantitative RT-PCR 6 h after the onset of seizure. The expression of P-gp protein in hippocampus 24 h after the termination of seizure was detected by immunohistochemistry. Results The expression of multidrug resistance gene 1a (mdr1a) in hippocampus of all animals within 72 h after the termination of seizure was significantly higher than that of the control group (5.6 (2.9) × 10 -5 mRNA copies immediately after SE termination) / 40 ng of total RNA, P <0.05]. The expression of mdr1a mRNA was further increased 3 h after SE termination [7.6 (6.3) × 10 -5, P <0.01] (3.3 ± 0.4) × 10 ~ 4 and (3.6 ± 1.0) × 10 ~ 4, respectively. The expression of mdr1b was transiently increased in the marginal lobe at 3 and 6 h after the onset of seizures 2.2, 2.4 times than the control group] were significantly higher than the control group (P <0.01). In addition, the expression of mdr1a [(4.3 ± 0.8) × 10 ~ 5] and mdr1b [(2.0 ± 0.7) × 10 ~ 4] in MK801 group was significantly lower than that in SE group at 6 h after the onset of seizures (P <0.01) The expression of P-gp [(26.6 ± 5.0) microvessels per 400 field of hyaluronic acid) in MK801 group was also significantly lower than that in SE group [(39.0 ± 4.1) microvessels / 400 times field, P <0.01] Conclusion NMDA receptor antagonist MK801 down-regulates the overexpression of P-gp in epileptic rats, suggesting that NMDA receptor may be involved in the regulation of P-gp expression during epileptic seizure and effectively inhibiting the over-activation of NMDA receptor may help prevent P-gp overexpression after seizures.