银杏叶提取物对体外培养的人Müller细胞氧化损伤的保护作用

摘要

背景 氧化应激是视网膜损伤中重要的病理生理过程,探讨保护视网膜免受氧化应激损伤的方法具有重要的临床意义.研究证实银杏叶提取物具有抗氧化、抗凋亡、抗血栓、抗炎等多种功能,但其对人Müller细胞的抗氧化作用研究较少. 目的 研究银杏叶提取物(EGb)对氧化剂三氧化二砷(As2O3)诱导的人Müller细胞氧化应激损伤的保护作用及其机制. 方法 人Müller细胞株用含体积分数10％胎牛血清(FBS)、2μmol/L谷氨酰胺和质量分数1％青链霉素双抗的DMEM/F12培养液进行培养和传代,取对数生长期的Müller细胞分为5个组,阴性对照组细胞采用常规培养法进行培养,在培养基中加入终质量浓度为5 mg/L的As2 O3处理细胞24 h以建立细胞氧化损伤模型,然后将5、10和20 mg/L EGb分别加入模型细胞培养液中作用24 h,采用MTT法检测各组细胞的活力;采用CM-H2DCFDA荧光探针法测定各组细胞活性氧簇(ROS)含量;采用逆转录PCR(RT-PCR)法检测细胞中easpase-3 mRNA的相对表达量;采用Western blot法分别检测细胞质和细胞核中Nrf2蛋白的表达. 结果 Müller细胞接种至培养瓶后24 h,倒置显微镜下可见接种细胞贴壁良好,培养6～7d时Müller细胞的细胞体积较大,细胞质丰富,呈镶嵌样排列.As2O3处理组部分细胞凋亡,呈卵圆形悬浮在培养液中.MTT法测定结果表明,阴性对照组细胞活力(A值)最高,As2 O3处理组细胞活力最低,而As2O3 +5 mg/L EGb组、As2O3+10 mg/L EGb组和As2O3+20 mg/L EGb组A值随着EGb质量浓度的增加而逐渐增加,各组间总体比较差异有统计学意义(F=163.57,P=0.00).阴性对照组细胞中ROS荧光强度最弱,As2O3处理组最强,As2O3 +5 mg/L EGb组、As2O3+10 mg/L EGb组和As2 O3 +20 mg/L EGb组细胞中ROS荧光强度值随着EGb质量浓度的升高而逐渐减弱,各组间细胞中ROS荧光强度值的总体比较差异有统计学意义(F=4 013.61,P=0.00).细胞内caspase-3 mRNA的相对表达量随EGb质量浓度的升高逐渐下降,各组间总体比较差异有统计学意义(F=2 199.72,P=0.00).各组细胞质中Nrf2蛋白的表达比较,差异无统计学意义(F=15.42,P=0.40);阴性对照组、As2O3处理组及As2O3+5 mg/L EGb组、As2O3+ 10 mg/L EGb组和As2 O3 +20 mg/L EGb组细胞核Nrf2蛋白表达量(灰度值)分别为100.01±0.04、46.59±0.63、54.51 ±0.62、59.93±0.17和67.60±0.24,总体比较差异有统计学意义(F=7271.72,P=0.00),其中阴性对照组细胞核Nrf2蛋白表达量最低,而As2O3处理组最高,差异均有统计学意义(均P＜0.05). 结论 EGb以剂量依赖的方式减轻体外培养的人Müller细胞的氧化应激损伤,其机制与抗氧化和抗凋亡作用有关,其减轻Müller细胞氧化应激损伤的调控主要发生在细胞核.%Background Oxidative stress is a pathophysiological process of retina,so it is very important to explore a protective way against retinal oxidative stress.Studies determined that extract of ginkgo biloba (EGb) has antioxidant,anti-apoptosis,anti-thrombosis and anti-inflammatory effects,however,the effect of EGb on human Müller cells in oxidative stress is still below understood.Objective This study was to investigate the protection of EGb against oxidative stress of human retinal Müller cells induced by As2O3 in vitro.Methods Human retinal Müllercell line was cultured in DMEM/F12 with 10％ fetal bovine serum (FBS),2 μmol/L glutamine and 1％ antibiotics.As2O3 solution at the final concentration 5 mg/L was added in the medium for 24 hours to establish oxidative models,and then the EGb with the final concentrations of 5,10 and 20 mg/L was used to cell models for 24 hours,respectively.Cell viability was detected by MTT assay,and reactive oxygen species (ROS) levels in the cells were detected with CM-H2DCFDA fluorescent probe.The relative expression levels of caspase-3 mRNA in cytoplasm and cell nuclei were assayed by reverse-transcription PCR (RT-PCR),and the expressions of Nrf2 protein were quantitatively detected by Western blot.Results Müller cells adhered well 24 hours after cultured.At 6-7 days after culture,Müller cell body was large with abundant cytoplasm and mosaic-like arrangement.However,floating cells were seen after As2 O3 treatment.Cell viability (absorbance) was significantly different among the normal culture group,As2 O3-treated group,As2 O3 + 5 mg/L EGb group,As2 O3 + 10 mg/L EGb group and As2 O3 + 20 mg/L EGb group,with the strongest viability in the normal culture group and the weakest viability in the As2 O3-treated groups (F=163.57,P =0.00).The fluorescence intensity of ROS was the weakest in the normal culture group and the strongest in the As2 O3-treated group and was gradually weakened with the increase of EGb doses,showing a remarkable difference among the groups (F =4 013.61,P =0.00).The relative expression level of caspase-3 mRNA in the cells was gradually reduced with the increase of EGb doses,with a statistically significant difference among the groups (F =2 199.72,P =0.00).In addition,no considerable difference was seen in the expression level of Nrf2 protein (grey scale) in cytoplasm among the groups (F=15.42,P=0.40);while in the nuclei,the expression levels of Nrf2 protein were 100.01 ±0.04,46.59±0.63,54.51 ±0.62,59.93 ±0.17 and 67.60±0.24 in the normal culture group,As2 O3-treated group and As2O3+5 mg/L EGb group,As2O3+10 mg/L EGb group,As2O3+20 mg/L EGb group respectively,with a significant difference among them (F=7 271.72,P=0.00).Conclusions EGb can protect human retinal Müller cells against As2O3-induced damage in a dose-dependant manner by antioxidant and antiapoptotic effects in vitro,and the activities occur primarily in cell nucleus.