丁基苯酞对过氧化氢诱导下视网膜Müller细胞凋亡的影响

摘要

Objective To observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).Methods Human retinal Müller cells cultured in vitro were divided into normal control group,model group (H2O2 group) and experimental group (H2O2+NBP group).The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μ mol/L H2O2 for 2 h.Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 tmol/L NBP.The normal control group was a conventional cultured cells.Müller cells were identified by immunofluorescence staining.Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes.MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention.Hoechst33258 staining was used to observe the apoptosis.LIVE/DEAD (R)cell activity/cytotoxicity kit was used to detect cell viability.Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells.One-way ANOVA combined with Dunnett statistical method were used for data analysis.Results HE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group.MTT assay showed that after 24 h and 48 h of NBP intervention,the differences in cell viability between the normal control group and the H2O2 group,the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96,3.658,47.58,20.33;P＜0.001,0.022).The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced,and the blue fluorescence of the remaining cells was uniform.The LIVE/DEAD ~ cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly,and the number of viable cells with green fluorescence decreased significantly.In the H2O2+NBP group,the number of viable cells with green fluorescence increased,and the number of dead cells with red fluorescence decreased.The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced;the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.Conclusion NBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.%目的 观察丁基苯酞(NBP)对过氧化氢(H2O2)诱导下视网膜Müller细胞凋亡的保护作用.方法 体外培养的人视网膜Müller细胞分为正常对照组、模型组(H2O2组)、实验组(H2O2+NBP组).H2O2组、H2O2+NBP组细胞培养液中加入200 μmol/LH2O2刺激2h后,H2O2组更换为完全培养基,H2O2+NBP组更换为含l μmol/L NBP的完全培养基.正常对照组为常规培养细胞.苏木精-伊红(HE)染色观察各组细胞形态改变;噻唑蓝(MTT)比色法检测NBP作用24、48 h后各组细胞活性;Hoechst33258染色观察各组细胞凋亡情况;LIVE/DEAD(R)细胞活性/细胞毒性试剂盒检测各组细胞生存力;二氯荧光素二乙酸酯(DCFH-DA)+内质网(ER)红色荧光探针(ER-Tracker Red)双染色观察各组细胞ER中活性氧(ROS)的表达水平.单因素方差分析联合Dunnett统计学方法进行数据分析.结果 HE染色结果显示,H2O2+NBP组细胞数量较H2O2组增加.MTT比色法检测结果发现,NBP作用24、48 h后,正常对照组与H2O2组、H2O2组与H2O2+NBP组细胞生存力比较,差异均有统计学意义(t=28.96、3.658、47.58、20.33,P＜0.001、0.022).Hoechst33258结果显示,H2O2+NBP组细胞少数细胞核边集呈新月状且细胞核碎裂减少,其余细胞蓝色荧光均匀.LIVE/DEAD@细胞活性/细胞毒性试剂盒检测结果显示,H2O2组中呈红色荧光的死亡细胞明显增多,呈绿色荧光的活细胞数量显著减少;H2O2+NBP组呈绿色荧光的活细胞数量增多,呈红色荧光的死亡细胞减少.DCFH-DA+ER-TrackerRed双染色结果显示,H2O2组细胞中绿色荧光强度明显增强;H2O2+NBP组细胞中绿色荧光强度较H2O2组明显降低.结论 NBP通过抑制ROS的产生缓解H2O2诱导的人视网膜Müller细胞凋亡.