喹啉酸对大鼠螺旋神经节细胞的神经兴奋毒性作用及其机制探讨

Analysis of quinolinic acid neurotoxicity to excitability of spiral ganglion cells and its mechanism in rat

目的 探讨喹啉酸（quinolinic acid,QA）对大鼠螺旋神经节细胞（spiral ganglion cell,SGC）的神经兴奋毒性作用,观察N-甲基-D-天冬氨酸（N-methyl-D-aspartate,NMDA）受体拮抗剂5-甲基二氢丙环庚烯业胺马来酸（MK-801）对QA神经兴奋毒性的拮抗作用,同时观察镁离子对SGC的保护作用.方法 新生SD大鼠SGC原代培养72 h后,更换培养基,分为空白对照组、QA损伤组（1 mmol/L QA）、MK-801拮抗组（1 mmol/L QA＋20 μmol/L MK-801）、MgCl2保护组（1 mmoL/L QA＋1 mmol/L MgCl2）,继续培养24 h后,通过磷脂酰丝氨酸外翻分析法在荧光显微镜下测定SGC凋亡率.SGC原代培养72 h后,分为四组：低浓度QA组（100 μmol/L QA）,高浓度QA组（1 mmnol/LQA）,MK-801拮抗组（20 μmol/L MK-801＋l mmol/QA）,MgCl2保护组（1 mmol/L MgCl2＋l mmol/L QA）;激光共聚焦显微镜动态检测各组SGC胞内瞬时钙离子浓度的变化.结果 与空白对照组（凋亡率9.2%±0.9%,（x）±s,下同）相比,QA损伤组可见大量SGC凋亡（凋亡率59.1%±7.5%）,差异具有统计学意义（q=11.9,P〈0.05）;MgCl2保护组凋亡细胞比QA组明显减少,凋亡率为27.5%±8.3%,二者筹异具有统计学意义（q=7.5,P〈0.05）;MK-801组细胞凋亡率（12.8%±5.7%）与空白对照组差异无统计学意义（q=0.9,P〉0.05）.在高浓度QA（1 mmoL/L）的作用下,SGC内钙离子浓度明显升高,190 s时达高峰,随后逐渐下降,450 s时基本恢复加药前水平;低浓度QA（100 μmol/L）对SGC胞内钙离子浓度没有明赤影响;Mgcl2组SGC胞内钙离子浓度曲线峰值降低,时程缩短;MK-801组未见明显SGC胞内钙离子浓度变化.结论 QA可过度激活细胞膜上的NMDA受体而造成大鼠离体培养SGC的损伤,镁离子可以降低QA对SGC的神经兴奋毒性作用.

Objective To investigate the neurotoxicity and its mechanism of quinolinic acid （QA） to spiral ganglion cells（SGC） and observe the protectable potential of MgCl2 on SGC. Methods SGC were cultured in vitro for 72 h, and then were divided into 4 groups; control group, QA group（ 1 mmol/L QA） , MK-801 group （1 mmol/L QA ＋ 20 μmol/L MK-801 ） and MgCl2 protected group （ 1 mmol/L QA ＋ 1 mmol/L MgCl2）. SGC apoptosis rate was analyzed by Annexin V staining and PI staining measurements after 24 h exposure to different medium. SGC cultured as methods above were divided into 4 groups as following： 100 （μmol/L QA, 1 mmol/L QA, 20 μmol/L MK-801 ＋ 1 mmol/L QA and 1 mmol/L MgCl2 ＋ 1 mmol/L QA. The intracellular calcium concentration was measured by laser scanning confocal microscope finally. Results Apoptosis rate in QA group was higher than that in both of control group （59. 1% ±7.5% vs 9.2% ±0. 9%, x±s, q = 11.9,P〈0. 05） and MgCl2 group（59. 1% ±7.5% vs 27.5% ±8.3%, q =7.5 ,P 〈0.05）. There was no significant difference between apoptosis rate of control and MK-801 group （12.8% ±5.7% vs 9.2% ±0.9% ,q = 0.9,P〉0.05）. It was shown that there was a significant increase of Ca2＋ in SGC in the presence of QA by laser scanning confocal microscope. MK-801 may completely block the increase of Ca2＋ , and the increase of Ca2＋ can be reduce by the application of MgCl2. Conclusions QA might injure SGC by excessive activating NMDA receptors on the cell membrane. Mg2＋ may have the function to reduce the neurotoxicity of QA.