绿茶提取物EGCG对视网膜神经节细胞氧化应激损伤的防护作用

Neuroprotective effect of epigallocatechin gallate on oxidative-stress-injured retinal ganglion cells

目的研究绿茶提取物表没食子儿茶素没食子酸酯（EGCG）对视网膜神经节细胞（RGC-5）氧化应激损伤的防护作用。方法利用外源性活性氧（H2O2）造成RGC-5细胞的氧化应激损伤，同时应用EGCG进行保护。通过末端脱氧核苷酸转移酶介导的脱氧尿嘧啶核苷三磷酸缺口末端标记（TUNEL）验证RGC-5细胞的死亡方式，2’，7＇-二氢乙啡啶（DHE）染色实验检测细胞内活性氧簇（ROS）生成情况，蛋白质印迹法定量分析细胞核酶（PARP-1）的激活，噻唑蓝（MTT）法分析比较EGCG、维生素E水溶性衍生物（Trolox）和PARP-1抑制剂NU1025对RGC-5细胞的保护作用。结果H2O2以浓度和时间依赖的方式降低RGC-5细胞活性，500μmol／L H2O2在24h可以导致RGC-5细胞活性下降50％。H2O2可以导致RGC-5细胞凋亡，并显著增加细胞内ROS的生成，上调细胞核酶PARP-1的表达；而预先给予EGCG可以明显减少RGC-5细胞凋亡和细胞内ROS生成。MTT实验显示EGCG可以使氧化应激损伤中RGC-5细胞活性恢复到87％，而Trolox和NU1025可以分别提高细胞活性到62％和71％。结论EGCG能够有效地防护氧化应激对视网膜神经节细胞的损伤，是一种极具开发潜力的神经保护性药物。

Objective To investigate the neuroprotective effect of epigallocatechin gallate（EGCG）, the main extract from green tea, on the oxidative-stress-injured retinal ganglion cells. Methods Rat retinal ganglion cells （RGC-5） were cultured into 3 groups （normal control; H2O2; H2O2 ＋ EGCG or Trolox or NU1025）. In-situ TUNEL was used to detect the apoptosis of the RGC-5 cells. Dihydroethidium （DHE） assay was used to observe the intracellular ROS generation. The activation of nuclear enzyme, PARP-1 was quantitatively detected by Western blot and the cell viability was measured by MTT method. Results Hydrogen peroxide reduced RGC-5 cell viability in a time-concentration-dependent manner. The treatment of 500 μmol/L H2O2 for 24 hours reduced RGC-5 cell viability by about 50% of control. Hydrogen peroxide caused apoptosis of the RGC-5 cell, obviously increased intracellular ROS generation and up-regulated the PARP-1 expression. The pretreatment with EGCG was able to markedly reduce the number of apoptotic cells, attenuate intracellular ROS generation. Furthermore, MTT assay showed that the pretreatment with EGCG （50 μmol/L） increased the most cell viability to 87% of control, but pretreatment with Trolox （100 μmol/L） and NU1025（ 100 μmol/L, a PARP-1 inhibitor） recovered the most cell viability to 62% and 71% of control respectively. Conclusion EGCG is able to effectively protect retinal ganglion cell against oxidative-stressed injury and can be used as a very potential neuroprotective drug.