银杏叶提取物抗 N-甲基-D-天冬氨酸受体介导兴奋毒性作用及机制研究

Effects of Ginkgo biloba extract against excitotoxicity induced by NMDA receptors and mechanism thereof

目的观察银杏叶提取物(GBE)对 N-甲基-D-天冬氨酸(NMDA)受体过度激活导致海马神经元和脑损伤作用的影响并探讨其可能机制。方法用锥虫蓝染色和乳酸脱氢酶(LDH)测定等方法评估 GBE 对海马神经元兴奋毒性损伤的影响,并运用全细胞膜片钳记录观察 GBE 对大鼠海马急性分离神经元 NMDA 受体激活电流的调制作用;以 Longa 评分、红四氮唑、神经元核蛋白和微管相关蛋白-2免疫组化染色等方法评估 GBE 对大鼠局灶性脑缺血损伤的保护作用。以上 GBE 干预作用均与 NMDA 受体特异性拮抗剂 MK-801作比较。结果 150μg/ml GBE 预处理可有效地保护暴露于 NMDA+甘氨酸的海马神经元,使细胞生存率提高到85.2%±5.2%,LDH 漏出减少到87 U/L±8U/L,但此效应弱于 MK-801(P<0.05);预加0.1 mg/ml GBE 可明显抑制由 NMDA 和甘氨酸共同作用能引起的内向电流(I_(NMDA)),抑制率为40%±17%,50 μmol/L MK-801的抑制率为78%±18%,两组差异有统计学意义(P<0.01);用标准细胞外液进行冲洗后,GBE 组 I_(NMDA)可恢复至91%±8%,而MK-801组不能(P<0.05)。GBE 预处理可有效缓解大鼠局灶性脑缺血损伤,其保护作用弱于 MK-801(P<0.05),但没有 MK-801所引起大鼠严重的行为学毒性反应。结论 GBE 预处理能对抗NMDA 受体过度激活所致海马神经元毒性损伤和大鼠局灶性脑缺血损伤,可能与通过拮抗 NMDA 受体有关,此保护作用及其与 NMDA 受体结合力均弱于 MK-801,无行为学毒性反应,使抗兴奋毒性临床干预成为可能.

Objective To observe the effects of Ginkgo biloba extract （GBE） on N-methyl-Dasparate （NMDA） excitotoxicity and focal cerebral ischemia, and further explore the neuroprotective mechanisms of GBE. Methods Neonatal SD rat hippocampus was taken out to make into cell suspension. immunohistochemistry with neuron neucleoprotein monoclonal antibody （NeuN） was used to calculate the percentage of NeuN positive cells. Twelve days after incubation the suspension of neurons were randomly divided into 4 groups ： normal control group （ exposed to normal saline for 15 min and then to DMEM without NMDA and glycine for 24 h ）, NMDA group （exposed to culture fluid with NMDA of the terminal concentration of 100 μmol/L and glycine of the terminal concentration of 10 μmol/L for 15 min and then to DMEM without NMDA and glycine for 24 h）, MK-801 group （exposed to MK-801, an NMDAR antagonist, for 2 min, to culture fluid with NMDA for 15 min, and then to DMEM without NMDA and glycine for 24 h）, and GBE pretreatment group （ exposed to GBE of the terminal concentration of 150 μg/ml for 3 d, culture fluid with NMDS for 15 min, and then to DMEM without NMDA and glycine foe 24 h）. Trypan blue staining was used to calculate the survival rate of the neurons. The lactic dehydrogenase （ LDH ） level in the supernatant of cultured cell suspension was detected. Whole-cell patch clamp recording was carried out to evaluate the modulatory effects of GBE on NMDA-activated currents in the rat hippocampal neurons. 108 SD rats were randomly divided into 5 groups ： sham operation group （n = 12）, standard middle cerebral artery occlusion （MCAO） group （n = 24, undergoing MCAO and then reperfusion）, MK-801 acute administration group （ n = 24, undergoing MCAO and immediate peritoneal administration of MK-801 1 mg/kg ）, GBE acute administration group （ n = 24, undergoing peritoneal injection of GBE 100 mg/kg immediately after the MCAO）, and GBE pretreatment group （ n = 24, undergoing peritoneal administration of GBE every day for 7 days before the MCAO）. The 4 groups were re-divided into 4 subgroups with 3 -4 rats： 0.5 h ischemia, and 3 h, 1 d, and 7 d ischemia-reperfusion （IR） subgroups. The neurological symptoms were evaluated by Longa＇s scoring after the rats became conscious. The rats were killed at different time-points, their brains were taken out to undergo 2,3,5-triphenyl-tetrazolium chloride staining, the areas of cerebral infarction were calculated, and immunohistochemiistry was used to evaluate the contents of NeuN and microtubule-associated protein （MAP-2）. Results The cell viability of the GBE group was 85% ± 5%, significantly higher than that of the NMDA group （39.8% ±2. 1% , P 〈0.01 ）, and significantly lower than that of the MK-801 group （93.8% ±2.7%, P〈0. 05）. The LDH efflux of the GBE group was 87 U/L±8 U/L, significantly lower than that of the NMDA group （ 138 U/L ± 12 U/L, P 〈 0. 01 ） and significantly higher than that of the MK-801 group （ 47 U/L ± 7 U/L, P 〈 0. 05 ）. The inward current （ INMDA ） of the NMDA group was significantly activated, The inhibitory rate of the NMDA-activated INMDA of the GBE group was 40% ± 17%, significantly lower than that of the MK-801 group （ 78% ± 18% , P 〈 0. 05 ） ; After washing out with standard extracellular solution, the INMDA could recover to 91% ± 8% in the GBE group, but not in the MK-801 group （P 〈 0. 05 ）, which indicated that GBE had lower affinity to NMDA receptor than MK-801. The Longa＇s scores of the 3 h and 24 h IR subgroups of the GBS pretreatment group were all significantly lower than those of the corresponding subgroups of the standard MCAO and GBE acute administration groups. The symptoms of the MK-801 were the most severe. Cerebral infarction began to appear in the 1-day subgroups. The cerebral infarction areas of the 1 d subgroups of the GBF pretreatment and MK-801 groups were 11.5% ± 1.3% and 6. 5% ±0. 9% respectively, both significantly smaller than those of the MCAO and GBE acute administration groups （ 24.5% ± % and 22. 9% ± 1.3% respectively, both P 〈0. 01 ）, however, there was no significant difference in the cerebral infarction area between the GBE acute administration and MCAO group. It was true too for the cerebral infarction areas of the 7 d subgroups. Except in the control group, loss of NeuN positive neuron was seen in all groups, especially the MCAO and GBE acute administration groups. Except in the control group, the MAP-2 positive neurons were decreased in all groups, especially the MCAO and GBE acute administration groups, and 1 day and 7 days after the IR MAP-2 positive neurons were almost unseen in the MCAO and GBE acute administration groups, however, could be seen in small amounts in the GBE and MK-801 groups （ all P 〈 0.01 ）. Conclusion GBE pretreatment protects the neurons from excitotoxicity induced by over-activated NMDA receptor and focal cerebral ischemia, which can be explained by the mild blocking effect of GBE on NMDA receptor with low affinity, comparing with MK-801, and GBE is expected to interfere in excitotoxicity in clinic without neurotoxic behaviors.