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

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。

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

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 (H₂O₂) group, and AS+H₂O₂ group. Cells in the AS+H₂O₂ group were pretreated with 1 μmol·L^-1, 10 μmol·L^-1, and 30 μmol·L^-1 AS for 2 h, respectively. The cell viability was evaluated by MTT assay. In subsequent experiments, the AS+H₂O₂ group selected 30 μmol·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 200 μmol·L^-1 H₂O₂ for 24 h after being treated with 30 μmol·L^-1 AS alone. The cell viability was determined by MTT assay. Results　Compared with the control group, the cell viability in the H₂O₂ group decreased significantly (P<0.01). Compared with the H₂O₂ group, the viability of cells pretreated with 10 μmol·L^-1 and 30 μmol·L^-1 AS in the AS+H₂O₂ group increased significantly (P<0.05, P<0.01). Compared with the control group, H₂O₂ induced an increase in ROS and a decrease in mitochondrial membrane potential in the H₂O₂ group; In addition, the Bcl-2/Bax ratio decreased, and the relative expression of Caspase-3 increased (all P<0.05). Compared with the H₂O₂ group, ROS decreased, mitochondrial membrane potential increased, and the Bcl-2/Bax ratio also increased, inhibiting the activation of Caspase-3 in the AS+H₂O₂ 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 H₂O₂-induced injury. Conclusion　AS can inhibit H₂O₂-induced oxidative stress injury by up-regulating HO-1 expression in RGC-5 cells.