丁基苯酞对H2O2诱导下视网膜色素上皮细胞凋亡的保护作用

Effects of butylphthalide on hydrogen peroxide induced retinal pigment epithelial cells injury

目的观察丁基苯酞(NBP)对H2O2诱导下RPE细胞凋亡的保护作用。方法以人ARPE-19细胞系为实验细胞,并将其分为对照组、模型组及NBP组。对照组单纯采用完全培养基培养细胞。模型组采用200 μmol/L的H2O2刺激细胞2 h,换液后给予完全培养基培养细胞。NBP组采用200 μmol/L的H2O2及1 μmol/L的NBP共同培养细胞2 h,换液后给予完全培养基联合1 μmol/L的NBP继续培养细胞。采用MTT法检测细胞受到氧化应激刺激后的细胞活力;HE染色对RPE细胞形态进行观察;Hoechst33258染色观察RPE细胞凋亡形态;线粒体膜电位检测试剂盒(JC-1)观察细胞膜电位改变及细胞早期凋亡变化;2,7-二氯二氢荧光素二乙酸酯(DCFH-DA)染色观察细胞内ROS生成堆积情况;细胞免疫荧光和Western blot分析NBP对血红素氧合酶1 (HO-1)表达的影响。结果MTT法检测结果显示,细胞培养24、48 h,对照组(t=17.710、13.760,P<0.000 1、<0.000 1)、NBP组(t=4.857、9.225,P=0.000 7、P<0.000 1)细胞生存力均较模型组更强,差异均有统计学意义。HE染色及Hoechst33258染色观察发现,与对照组比较,模型组细胞数量明显变少,细胞形态不完整;与模型组比较,NBP组细胞数量明显增多,细胞形态更好。JC-1法检测结果显示,模型组凋亡细胞数较对照组明显增多,NBP组凋亡细胞数较模型组明显减少。DCFH-DA染色观察发现,模型组细胞内ROS堆积较对照组更多,NBP组细胞内ROS堆积较模型组更少。免疫染色观察发现,NBP组细胞的HO-1荧光强度较对照组明显增高,差异有统计学意义(t=10.270,P=0.000 5)。Western blot检测结果显示,NBP以时间依赖性的方式上调HO-1的表达水平;NBP作用4、8、12 h时HO-1相对表达量较0 h时呈明确升高趋势,差异有统计学意义(F=164.91,P<0.05)。结论氧化应激损伤可下调RPE细胞的生存力并诱导其出现凋亡,NBP通过上调HO-1的表达,有效提高RPE细胞抗氧化能力,减轻细胞损伤并抑制细胞凋亡。

Objective To investigate the protective effect of butylphenyphthalein (NBP) on RPE apoptosis induced by H2O2. Methods The human RPE cell line (human ARPE-19 cell line) were used as the experimental cells and were divided as control group, model group, NBP group. Complete medium was used in control group. The model group was stimulated with 200 μmol/L H2O2 for 2 h, and the cells were cultured in complete medium. The NBP group was cultured with 200 μmol/L H2O2 and 1 μmol/L NBP for 2 h. After changing the medium, complete medium was combined with 1 μmol/L NBP to continue the culture of the cells. Cell viability were detected by MTT assay while the morphology of RPE were observed by HE staining. Moreover, Hoechst 33258 was used to detect RPE cell apoptosis. Mitochondrial membrane potential (JC-1) staining were performed to monitor changes in cell membrane potential and the characteristic change of apoptosis in RPE cells. Furthermore, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining were used to analyze the effect of NBP treatment on the expression of ROS. The effect of NBP on the expression of Heme oxygenase-1(HO-1) was analyzed by cellular immunofluorescence and western blotting. Results The results of MTT assay showed that the cells were cultured for 24 and 48 hours, cell viability of control group (t=17.710, 13.760;P<0.000 1,<0.000 1) and treatment group (t=4.857, 9.225;P=0.000 7,<0.000 1) were stronger than that of model group, and the difference was statistically significant. HE staining and Hoechst33258 staining showed that compared with the control group, the number of cells in the model group was significantly less, and the cell morphology was incomplete. Compared with the model group, the number of cells in the treatment group was significantly increased, and the cell morphology was better. The results of JC-1 assay showed that the number of apoptotic cells in the model group was significantly higher than that in the control group, and the number of apoptotic cells in the treatment group was significantly lower than that in the model group. DCFH-DA staining showed that the ROS accumulation in the model group was more than that in the control group, and the ROS accumulation in the treatment group was less than that in the model group. Immunostaining observation showed that the HO-1 fluorescence intensity of the cells in the treatment group was significantly higher than that of the control group, and the difference was statistically significant (t=10.270, P=0.000 5). Western blot analysis showed that NBP up-regulated the expression level of HO-1 in a time-dependent manner. The relative expression of HO-1 at 4, 8, and 12 h of NBP showed a clear increase trend compared with 0 h, and the difference was statistically significant (F=164.91, P<0.05). Conclusions Oxidative stress injury can down-regulate the viability of RPE cells and induce apoptosis. NBP can increase the antioxidant capacity of RPE cells, reduce cell damage and inhibit cell apoptosis by up-regulating HO-1 expression.