槲皮素对N-甲基-D-天冬氨酸（NMDA）诱导损伤的视网膜神经节细胞的保护作用及其机制

目的　探讨槲皮素对N-甲基-D-天冬氨酸（N-methyl-D-aspartic acid，NMDA）诱导的视网膜神经节细胞（retinal ganglion cell，RGC）损伤的保护作用及其机制。方法　将小鼠RGC随机分为对照组，NMDA组，NMDA+1 μmol·L^-1、10 μmol·L^-1、100 μmol·L^-1槲皮素处理组，槲皮素处理组，NMDA+槲皮素+茴香霉素（anisomycin，ANISO）处理组，NMDA+槲皮素+P79350处理组。NMDA组细胞加入100 μmol·L^-1的NMDA作用24 h，NMDA+1 μmol·L^-1、10 μmol·L^-1、100 μmol·L^-1槲皮素处理组则在NMDA作用前分别加入相应浓度槲皮素预处理2 h，槲皮素处理组只加入10 μmol·L^-1槲皮素处理，NMDA+槲皮素+ANISO处理组和NMDA+槲皮素+P79350处理组在NMDA和槲皮素处理后分别加入25 μg·L^-1的ANISO和50 μmol·L^-1的P79350处理。随后利用噻唑蓝（methyl thiazolyl tetrazolium，MTT）试剂盒检测细胞活性，流式细胞术检测细胞凋亡率，酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）检测细胞培养上清液中活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）、乳酸脱氢酶（lactate dehydrogenase，LDH）、超氧化物歧化酶（superoxide dismutase，SOD）和一氧化氮（nitric oxide，NO）水平，RT-PCR检测细胞中神经型一氧化氮合酶（neuronal nitric oxide synthase，nNOS）和诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）mRNA表达，Western blot检测细胞中Bcl-2、Bax、p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase，p38 MAPK）、磷酸化p38 MAPK（phosphorylated p38 MAPK，p-p38 MAPK）、c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）和p-JNK蛋白表达水平，Caspase-3活性试剂盒检测Caspase-3活性。结果　与对照组比较，NMDA组细胞活性、SOD、Bcl-2 mRNA表达水平降低，LDH，ROS，MDA，NO，iNOS mRNA、nNOS mRNA、Bax mRNA和蛋白表达水平，Caspase-3活性，p-JNK蛋白表达水平，p-p38 MAPK蛋白表达水平，细胞凋亡率均升高（均为P<0.05）。相比于NMDA组，NMDA+槲皮素处理组则提高细胞活性和SOD水平，上调Bcl-2 mRNA和蛋白表达，降低LDH、ROS、MDA和NO，下调iNOS mRNA、nNOS mRNA、Bax mRNA和蛋白，p-JNK蛋白以及p-p38 MAPK蛋白表达，并降低Caspase-3活性和细胞凋亡率（均为P<0.05）。ANISO和P79350抵消槲皮素的作用。结论　槲皮素通过JNK/p38 MAPK信号通路防止NMDA诱导的RGC损伤。

Protective effects of quercetin on N-methyl-D-aspartic acid induced retinal ganglion cells injury and its mechanisms

Objective　To explore the protective effects and its underlying mechanism of quercetin onN-methyl-D-aspartic acid (NMDA) induced retinal ganglion cell (RGC) injury.Methods　Mouse RGC were randomly allocated into control group，NMDA group，NMDA+1 μmol·L^-1，10 μmol·L^-1，and 100 μmol·L^-1 quercetin group，quercetin group，NMDA+quercetin+anisomycin (ANISO) group，NMDA+quercetin+P79350 group.Cells in NMDA group were treated with 100 μmol·L^-1 NMDA for 24 h，cells in NMDA+1 μmol·L^-1，10 μmol·L^-1，and 100 μmol·L^-1 quercetin group were pre-treated with the indicated concentration of quercetin for 2 h，following with NMDA，quercetin group were treated with 10 μmol·L^-1 quercetin only.NMDA+quercetin+ANISO group and NMDA+quercetin+P79350 group were treated with 25 μg·L^-1 ANISO and 50 μmol·L^-1 P79350 after exposing to NMDA and quercetin，respectively.Cell viability was examined by methyl thiazolyl tetrazolium (MTT) kit，cell apoptosis rate was detected by flow cytometry，the levels of reactive oxygen species (ROS)，malondialdehyde (MDA)，lactate dehydrogenase (LDH)，superoxide dismutase (SOD) and nitric oxide (NO) were determined by enzyme-linked immunosorbent assay (ELISA)，the mRNA expression of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) expression were analyzed by RT-PCR，the protein expression levels of Bcl-2，Bax，p38 mitogen-activated protein kinase (p38 MAPK)，phosphorylated p38 MAPK (p-p38 MAPK)，c-Jun N-terminal kinase (JNK) and p-JNK was analyzed by Western blot，Caspase-3 activity was measured using Caspase-3 activity assay kit.Results　Compared with control group，cell viability，SOD levels and Bcl-2 mRNA expression were decreased，while LDH levels，ROS levels，MDA levels，NO levels，iNOS mRNA expression，nNOS mRNA expression，Bax mRNA and protein expression，Caspase-3 activity，p-JNK protein，p-p38 MAPK protein，and apoptotic rates were enhanced in NMDA group (all P<0.05).Compared with NMDA group，treatment of NMDA+quercetin promoted cell viability and SOD levels，up-regulated Bcl-2 mRNA and protein expression，decreased LDH，ROS，MDA and NO levels，down-regulated the expression of iNOS mRNA，nNOS mRNA，Bax mRNA and protein，p-JNK and p-p38 MAPK protein，and reduced Caspase-3 activity and apoptotic rates (all P<0.05).ANISO and P79350 could significantly compensate the effects caused by quercetin.Conclusion　Quercetin protects against NMDA-induce RGC injury via the JNK/p38 MAPK pathway.