莱菔硫烷对过氧化氢诱导牛眼小梁细胞氧化应激损伤的保护作用

摘要

背景 研究表明小梁网组织的氧化应激损伤是开角型青光眼的基本病理过程之一,莱菔硫烷(SFN)可通过激活Nrf2/ARE通路发挥其抗氧化应激作用,但其是否可对氧化应激损伤的小梁细胞发挥作用目前尚不清楚. 目的 探讨SFN对H2O2诱导的牛眼小梁细胞氧化应激损伤的防护作用. 方法 采用组织块培养法对新鲜摘取的黑牛眼球小梁组织进行小梁细胞原代培养,将第3代牛小梁细胞以1x 103/孔的细胞密度接种于96孔板中培养24 h后将细胞分为4个组,空白对照组加入100μl无血清培养基,H2O2组细胞在空白对照组的基础上加入100 μl终浓度为100 μmol/L H2O2建立细胞氧化应激损伤模型,SFN组在培养液中加入100μl终浓度为10μmol/L SFN,而SFN+H2O2组在SFN组的基础上加入100 μmol/L H2O2,细胞培养后6h采用CCK-8法检测各组牛眼小梁细胞活力,采用Annexin V-FITC/PI双染流式细胞仪检测各组细胞的凋亡率.结果 传至第3代的细胞呈梭形,形状和大小较均一,细胞质丰富,可见色素颗粒,核仁大.H2O2组和SFN+H2O2组相对细胞活力分别为空白对照组的(67.00±1.27)％和(80.00±6.25)％,其中SFN+H2O2组相对细胞活力低于空白对照组和SFN组,但明显高于H2O2组,差异均有统计学意义(均P＜0.01).空白对照组、H2O2组、SFN组和SFN+H2O2组细胞凋亡率分别为(11.33±0.77)％、(32.31±1.03)％、(10.44±0.68)％和(17.68±0.21)％,各组间总体比较差异均有统计学意义(F-539.96,P＜0.01),其中SFN+H2O2组细胞凋亡率明显低于H2O2组但高于空白对照组和SFN组,差异均有统计学意义(均P＜0.01).结论 SFN可以增强H2O2诱导的牛眼小梁细胞的抗氧化应激能力,减轻氧化应激对牛眼小梁细胞的损伤.%Background Evidences indicated that oxidative stress damage is an essential pathological process in primary open angle glaucoma.Sulforaphane (SFN) can play an antioxidative stress role to many tissues and cells by activating Nrf2/ARE single pathway.However,whether SFN has a protective role to oxidative stress induced damage of trabecular meshwork cells is still unclear.Objective This study was to investigate the antioxidant effect of SFN against H2O2-induced oxidative damage in bovine trabecular meshwork cells.Methods Trabecular cells were isolated from fresh black bovine eyeballs and primarily cultured and passaged.The third generation of cells were incubated to 96-well dish at a density of 1 ×103/well for 24 hours and divided into 4 groups.The cells were incubated using 100 μl serum-free medium in the blank control group.Oxidative damage models were established by adding 100 μmol/L H2O2(100 μl) in medium in the H2O2 group.The cells were cultured with the medium containing 10 μmol/L SFN (100 μl) in the SFN group,and 100 μl H2O2 at the final concentration of 100 μmol/L was added in the SFN-treated cell medium in the SFN +H2O2 group.The cell vitality in various groups was assayed by using cell counting kit-8 (CCK-8).The apoptosis rate of the cells was detected by Annexin V-FITC/PI double-staining with flow cytometry.Results Cultured cells showed a spindle shape with uniform size,abundant cytoplasm,numberous pigmented particles and big nucleolus.The relative cell vitality reduced to (67.00± 1.27)％ and (80.00±6.25)％ in the H2O2 group and SFN+H2O2 group in comparison with 100％ in the blank control group,and the cell vitality in the SFN+ H2O2 group was lower than that in the SFN group but higher than that in the H2 O2 group (both at P＜0.01).The mean apoptosis rate was (11.33 ±0.77) ％,(32.31 ± 1.03) ％,(10.44 ±0.68) ％ and (17.68 ±0.21) ％ in the blank control group,H2 O2 group,SFN group and SFN+H2O2 group,respectively,showing a significant difference among the groups (F=539.96,P＜0.01),and the apoptosis rate in the SFN+H2O2 group was significantly lower than that in the H2O2 group but higher than that in the blank control group and SFN group (all at P＜0.01).Conclusions SFN can improve the antioxidative stress ability of trabecular meshwork cells and alleviate the damage induced by oxidative stress.