摘要
目的 研究石斛碱对过氧化氢(H2O2)诱导的视神经节细胞氧化损伤的保护作用.方法 将培养的RGC-5细胞分为正常对照组、H2O2模型组、石斛碱1 nmol/L组、石斛碱10 nmol/L组及石斛碱100 nmol/L组.采用细胞技术试剂盒-8(CCK-8)检测RGC-5细胞活力;试剂盒法检测细胞中氧化和抗氧化物质的含量、细胞中ROS水平;蛋白免疫印迹法(West-ern Blot)检测细胞中核因子E2相关因子2(Nrf2)以及下游靶蛋白表达.结果 石斛碱在1、10、100 nmol/L浓度对RGC-5细胞活力无毒性作用.H2O2从200 µmol/L浓度开始显著降低RGC-5细胞活力.100 nmol/L石斛碱可显著降低H2O2诱导的RGC-5细胞中LDH外漏率、活性氧和丙二醛含量,显著升高超氧化物歧化酶和谷胱甘肽含量.石斛碱可诱导RGC-5细胞中Nrf2从胞浆向胞核转移,同时正向调控其下游抗氧化蛋白GPx1、GCLC、NQO 1和HO-1的蛋白表达.结论 石斛碱对H2O2诱导的RGC-5细胞氧化损伤有明显的保护作用,其机制可能是石斛碱促进Nrf2核转位,正向调控Nrf2下游抗氧化蛋白表达发挥抗氧化作用.
Abstract
Objective To study the protective effect of dendrobine on H2O2-induced oxidative damage of optic ganglion cells.Methods The cultured RGC-5 cells were divided into normal control group,H2O2 model group,dendrobine 1 nmol/L group,dendrobine 10 nmol/L group and dendrobine 100 nmol/L group,and the cell via-bility of RGC-5 cells was detected by CCK-8.The content of oxidation and antioxidant substances and the level of ROS in cells were detected by kit method.Western Blot was used to detect the expression of nuclear factor E2-related factor 2(Nrf2)and the downstream target protein.Results Dendrobine at the concentrations of 1,10 and 100 nmol/L has no toxic effect on RGC-5 cell viability.H2O2 significantly reduced the activity of RGC-5 cells from the concentration of 200 μmol/L.Dendrobine at the concentrations of 100 nmol/L can significantly re-duce the leakage rate of LDH,the contents of reactive oxygen species and malondialdehyde in RGC-5 cells in-duced by H2O2,and significantly increase the contents of superoxide dismutase and glutathione.Dendrobine can induce the transfer of Nrf2 from cytoplasm to nucleus in RGC-5 cells,and at the same time positively regulate the protein expression of its downstream antioxidant proteins GPx1,GCLC,NQO 1 and HO-1.Conclusion Dendrob-ine has obvious protective effect on H2O2-induced oxidative damage of RGC-5 cells.Its mechanism may be that Dendrobine promotes nuclear translocation of Nrf2,positively regulates the expression of downstream antioxidant protein on Nrf2 pathway,and thus plays an antioxidant role.
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