虾青素激活Nrf2/HO-1通路对蓝光损伤视锥细胞的保护作用

Protective Effects of Astaxanthin on Blue Light Damaged Cones Through Activating Nrf2/HO-1 Pathway

关键词:  虾青素  视锥细胞  蓝光  氧化应激  核因子E2相关因子

Key words:  astaxanthin  cones  blue light  oxidative stress  nuclear factor E2-related factor

摘要:

目的 探究虾青素对蓝光诱导的小鼠视锥细胞(661W)氧化损伤的保护作用。方法 将661W细胞分成3组：正常组、蓝光组、虾青素干预组。虾青素干预组细胞予以15 μmol·L^-1虾青素溶液预处理24 h，其余2组不干预；24 h后，将虾青素干预组与蓝光组的细胞暴露于波长为450 nm的短波蓝光中6 h，正常组细胞常规培养；12 h后收集细胞，用CCK-8检测试剂盒筛选虾青素最佳给药浓度，活性氧(reactive oxygen species，ROS)检测试剂盒检测细胞内ROS的生成，通过实时荧光PCR和细胞免疫荧光法测定各组大鼠视网膜组织中核因子E2相关因子2(nuclear factor E2-related factor 2，Nrf2)和血红素氧合酶1(heme oxygenase-1，HO-1)的相对表达量。结果 CCK-8法检测发现虾青素最佳给药浓度为15 μmol·L^-1；与正常组相比，蓝光照射后视锥细胞内ROS水平升高(P < 0.01)，氧化应激的增强导致细胞增殖活性明显减弱，Nrf2和HO-1 mRNA表达水平显著升高，Nrf2蛋白表达水平降低，HO-1蛋白表达水平升高，差异均具有统计学意义(P < 0.01)；与蓝光组相比，虾青素的预处理使得虾青素干预组细胞内ROS含量明显减少(P < 0.01，细胞增殖活性提高，Nrf2和HO-1 mRNA表达水平降低(P < 0.01)，Nrf2和HO-1蛋白表达水平升高(P < 0.05)。结论 虾青素可抑制短波蓝光所诱导的细胞氧化应激损伤以及应激性的Nrf2/HO-1通路激活，维持Nrf2/HO-1通路稳态，有望为蓝光导致的视网膜损伤提供新的保护策略。

Abstract:

OBJECTIVE To explore the protective effect of astaxanthin on blue light induced oxidative damage in mouse cone cells(661W).METHODS 661W cells were divided into three groups: normal group, blue light group and astaxanthin intervention group. Cells in astaxanthin intervention group were pretreated with 15 μmol·L^-1 astaxanthin solution for 24 h, and the other two groups did not give the drug intervention. After 24 h, cells in astaxanthin intervention group and blue light group were exposed to 450 nm short-wave blue light for 6 h, while cells in normal group were cultured routinely. After 12 h, cells were collected. The optimal astaxanthian concentration were screened by CCK-8 detection kit. Reactive oxygen species(ROS) detection kit was used to detect the intracellular ROS generation. The relative expressions of nuclear factor E2-related factor 2(Nrf2) and heme oxygenase-1(HO-1) in cone cells were detected by Real-time PCR and immunofluorescence assay.RESULTS CCK-8 assay showed that the optimal concentration of astaxanthin was 15 μmol·L^-1. Compared with control group, the generation of ROS was increased after blue light irradiation(P < 0.01). The enhancement of oxidative stress led to a significant decrease in cell proliferation activity. The mRNA relative expressions level of Nrf2 and HO-1 were increased. Immunofluorescence assay showed that the level of Nrf2 was decreased, while the level of HO-1 was increased. All the differences were statistically significant(P < 0.01). Compared with blue light group, astaxanthin pretreatment significantly reduced ROS content while increased cell proliferation activity(P < 0.01). The mRNA relative expressions levels of Nrf2 and HO-1 were decreased(P < 0.01), and the protein expression level of Nrf2 and HO-1 were increased(P < 0.05).CONCLUSION Astaxanthin can inhibit oxidative stress injury and the over-activation of the Nrf2/HO-1 pathway induced by short-wave blue light, and maintain the Nrf2/HO-1 pathway homeostasis, which is expected to provide a new protective strategy for blue light-induced retinal injury.