阿克替苷通过激活血红素加氧酶-1抑制H_(2)O_(2)诱导的视网膜神经节细胞氧化损伤

Inhibitory effect of acteoside on hydrogen peroxide-induced oxidative damage in retinal ganglion cells by activating heme oxygenase-1

目的研究阿克替苷对视网膜神经节细胞(RGC-5)的保护作用及其机制。方法将RGC-5细胞分为对照组、H_(2)O_(2)组、阿克替苷+H_(2)O_(2)组(1μmol·L^(-1)、10μmol·L^(-1)、30μmol·L^(-1)阿克替苷分别预处理细胞2 h),MTT法检测各组细胞活力;后续实验阿克替苷+H_(2)O_(2)组选择阿克替苷30μmol·L^(-1)作为治疗浓度,荧光探针H2DCF-DA检测活性氧(ROS)产生和罗丹明123检测线粒体膜电位,Western blot检测各组细胞中Bcl-2、Bcl-2/Bax、半胱氨酸蛋白酶-3(Caspase-3)蛋白表达。机制研究中首先用不同浓度阿克替苷单独处理RGC-5细胞24 h,Western blot检测血红素加氧酶-1(HO-1)蛋白表达水平,进一步锌原卟啉(ZnPP)预处理RGC-5细胞1 h,而后30μmol·L^(-1)阿克替苷单独预处理后与200μmol·L^(-1)H_(2)O_(2)共孵育24 h,通过MTT法测定细胞活力。结果与对照组相比,H_(2)O_(2)组中细胞活力明显降低(P<0.01)。与H_(2)O_(2)组相比,阿克替苷+H_(2)O_(2)组中10μmol·L^(-1)、30μmol·L^(-1)阿克替苷预处理可显著提高细胞活力(P<0.05、P<0.01)。与对照组相比,H_(2)O_(2)组RGC-5细胞ROS生成明显增加,线料体膜电位显著降低,同时Bcl-2/Bax比值降低和Caspase-3蛋白相对表达量增加(均为P<0.05)。与H_(2)O_(2)组相比,阿克替苷+H_(2)O_(2)组RGC-5细胞ROS生成减少,线粒体膜电位增加,Bcl-2/Bax比值提高,Caspase-3蛋白相对表达量减少(均为P<0.05)。与阿克替苷未处理组相比,阿克替苷剂量依赖性诱导HO-1蛋白的表达,与阿克替苷+H_(2)O_(2)组相比,加入ZnPP后细胞活力降低(P<0.01),说明HO-1抑制剂ZnPP降低了阿克替苷对H_(2)O_(2)损伤的抑制作用。结论阿克替苷能够通过上调HO-1表达抑制H_(2)O_(2)诱导的RGC-5氧化应激损伤。

Objective To study the protective effect of acteoside(AS)on retinal ganglion cells(RGC-5)and its possible mechanism.Methods RGC-5 cells were divided into the control group,hydrogen peroxide(H_(2)O_(2))group,and AS+H_(2)O_(2)group.Cells in the AS+H_(2)O_(2)group were pretreated with 1μmol·L^(-1),10μmol·L^(-1),and 30μmol·L^(-1)AS for 2 h,respectively.The cell viability was evaluated by MTT assay.In subsequent experiments,the AS+H_(2)O_(2)group selected 30μmol·L^(-1)as the therapeutic concentration.The reactive oxygen species(ROS)was detected by H2DCF-DA and the mitochondrial membrane potential by Rhodamine123.The expression levels of Bcl-2,Bcl-2/Bax and cysteine protease-3(Caspase-3)proteins were measured by Western blot.To study the possible mechanism,RGC-5 cells were treated separately with different concentrations of AS for 24 h,and the expression level of heme oxygenase-1(HO-1)protein was measured by Western blot.Further,RGC-5 cells were pretreated with ZnPP for 1 h and then cultured with 200μmol·L^(-1)H_(2)O_(2)for 24 h after being treated with 30μmol·L^(-1)AS alone.The cell viability was determined by MTT assay.Results Compared with the control group,the cell viability in the H_(2)O_(2)group decreased significantly(P<0.01).Compared with the H_(2)O_(2)group,the viability of cells pretreated with 10μmol·L^(-1)and 30μmol·L^(-1)AS in the AS+H_(2)O_(2)group increased significantly(P<0.05,P<0.01).Compared with the control group,H_(2)O_(2)induced an increase in ROS and a decrease in mitochondrial membrane potential in the H_(2)O_(2)group;In addition,the Bcl-2/Bax ratio decreased,and the relative expression of Caspase-3 increased(all P<0.05).Compared with the H_(2)O_(2)group,ROS decreased,mitochondrial membrane potential increased,and the Bcl-2/Bax ratio also increased,inhibiting the activation of Caspase-3 in the AS+H_(2)O_(2)group(all P<0.05).AS induced the expression of HO-1 protein in a dose-dependent manner,while the HO-1 inhibitor ZnPP decreased the cell viability(P<0.01)and the inhibitory effect of AS on H_(2)O_(2)-induced injury.Conclusion AS can inhibit H_(2)O_(2)-induced oxidative stress injury by up-regulating HO-1 expression in RGC-5 cells.