谷氨酸对视神经脑膜上皮细胞的兴奋性毒性作用及其氧化应激机制

Excitatory toxicity of glutamate on meningothelial cells of optic nerve sheaths and its oxidative stress mechanism

背景作为脑脊液－视神经屏障的主要细胞成分，视神经脑膜上皮细胞（MECs）直接与脑脊液相接触，脑脊液中的组分改变可影响MECs的功能，进而对视神经的功能产生不利影响。 目的探讨脑脊液中兴奋性神经递质谷氨酸对MECs功能的影响，为视神经疾病发病机制的研究提供新的线索。方法将培养的人MECs株制备成悬液并接种于96孔培养板中，密度为1×104/孔，分别在培养液中用添加100、200、400、600、800和1 000 μmol/L谷氨酸孵育12、24、36、48和72 h，未添加谷氨酸者作为正常对照组，采用MTS法测定各组MECs的增生率（吸光度A490）值。采用600 μmol/L谷氨酸分别孵育MECs 24 h和48 h，未添加谷氨酸者作为正常对照组，采用实时定量PCR法检测细胞中超氧化物歧化酶（SOD）mRNA和热休克蛋白90（HSP90）mRNA的相对表达量；采用ELISA法检测细胞的总抗氧化能力（T-AOC），并通过荧光探针DCFH-DA观察细胞活性氧（ROS）的荧光强度。结果培养MECs生长良好，培养后72 h约80%细胞达到融合。不同浓度谷氨酸培养组随着谷氨酸浓度的增加及作用时间的延长细胞增生率逐渐下降，总体比较差异均有统计学意义（F浓度＝52.501，P〈0.001；F时间＝8.505，P〈0.001）。实验后24 h和48 h谷氨酸处理组细胞中SOD mRNA相对表达量均明显低于同时间点正常对照组，差异均有统计学意义（t＝20.278、t＝16.724，均P〈0.001）；谷氨酸作用MECs后24 h细胞中HSP90 mRNA相对表达量明显高于正常对照组，差异有统计学意义（t＝5.065，P＝0.002）。实验后24 h谷氨酸处理组细胞中T-AOC活性为（30.835±2.094）nmol/（min·L），低于正常对照组的（32.873±2.317）nmol/（min·L），但差异无统计学意义（t＝1.599，P＝1.414）；实验后48 h谷氨酸处理组细胞中T-AOC活性为（29.561±1.831）nmol/（min·L），低于正常对照组的（33.680±2.039）nmol/（min·L），差异有统计学意义（t＝3.682，P＝0.004）。谷氨酸处理组细胞中ROS荧光强于正常对照组，谷氨酸处理组处理后24 h及48 h细胞中ROS含量分别为48.110±1.712和40.982±1.853，均明显高于正常对照组的36.608±1.009和37.153±1.424，差异均有统计学意义（t＝14.178，P〈0.001；t＝4.012，P＝0.002）。结论谷氨酸可抑制体外培养MECs的增生，其对MECs的兴奋毒性作用与氧化应激刺激作用有关。

Background Meningothelial cells （MECs） occupy the predominant cell component of barrier between optic nerve and the cerebral spinal fluid, and any change of cerebral fluid components probably affects the MECs function and further impairs the optic nerve.ObjectiveThis study was designed to investigate the influence of glutamate, a potentially excitotoxic amino acid, to the functional changes of MECs and provide a theoretical evidence for clarifying the mechanism of optic nerve disorders.MethodsHuman MECs strains were cultured in vitro and prepared into cell suspension.The cells were inoculated to 96-well plates with the densities of 1×104/well.The glutamate of 100, 200, 400, 600, 800 and 1 000 μmol/L was added into medium for 12, 24, 36, 48 and 72 hours, respectively, and the cultured cells without glutamate were used as normal control group.MTS assay was employed to measure the proliferative rate （absorbency） of the cells.The regularly cultured MECs were divided into 600 μmol/L glutamate-treated group and normal control group and the cells were treated for 12 and 24 hours respectively, and the expression of superoxide dismutase （SOD） mRNA and heat shock protein 90 （HSP90） mRNA in the cells was detected by real-time PCR; the level of total anti-oxidative capacity （T-AOC） of the cells was processed by enzyme linked immunosorbent assay （ELISA）, and the reactive oxygen species （ROS） production was determined by DCFH-DA probe.ResultsCultured MECs grew well and formed 80% confluence after 72 hours culture.The proliferative rate of the cells were gradually decreased with the increase of glutamate dose and the lapse of affected time, with significant differences among different concentrations of glutamate and various time points （Fconcentration=52.501, P〈0.001; Ftime=8.505, P〈0.001）. The relative expression level of SOD mRNA was significantly reduced in the glutamate-treated group compared with the normal control group in both 24 hours and 48 hours after culture （t=20.278, t=16.724, both at P〈0.001）, and the expression of HSP90 mRNA in the cells was significantly lower in the glutamate-treated group than that in the normal control group in 24 hours after culture （t=5.065, P=0.002）. No significant difference was found in T-AOC activity between glutamate-treated group and normal control group in 24 hours after culture （[30.835±2.094] nmol/（min·L） vs. [32.873±2.317] nmol/（min·L）） （t=1.599, P=1.414）. In 48 hours after culture, T-AOC activity was （29.561±1.831） nmol/（min·L） in the glutamate-treated group, which was significantly lower in comparison with normal control group（33.680±2.039） nmol/（min·L） （t=3.682, P=0.004）. Fluorescence staining showed that the intensity of green fluorescence of ROS in MECs in the normal control group was weaker than that in the glutamate-treated group under the immunofluorescense microscope.The ROS level was 48.110±1.712 and 40.982±1.853 at 24 hours and 48 hours in the glutamate-treated cells, and which was significantly elevated in comparison with 36.608±1.009 and 37.153±1.424 in the normal control group （t=14.178, P〈0.001; t=4.012, P=0.002）.ConclusionsGlutamate inhibits the proliferation of MECs in vitro, and excitatory toxicity of glutamate on MECs probably is associated with oxidative stress response.