P-糖蛋白对H2O2诱导的视网膜色素上皮细胞氧化损伤的抑制作用

Inhibition effect of P-glycoprotein functional expression on oxidative damage of retinal pigment epithelial cells

目的探讨P-糖蛋白对H 2O 2诱导的视网膜色素上皮(retinal pigment epithelium,RPE)细胞氧化损伤的抑制作用。方法取人RPE细胞株D407细胞,放入体积分数5%CO 2培养箱中用含体积分数10%胎牛血清和双抗的DMEM培养基于37℃传代培养。先将细胞按H 2O 2浓度梯度处理,使用免疫印迹实验和P-糖蛋白荧光性底物罗丹明123蓄积实验确定H 2O 2对P-糖蛋白表达的最佳诱导浓度。预先使用1μmol·L-1 P-糖蛋白抑制剂Tariquidar处理细胞,通过测量细胞内罗丹明123蓄积量检测P-糖蛋白功能活性。将细胞分成3组:对照组、H 2O 2模型组(400μmol·L-1)、H 2O 2+P-糖蛋白抑制剂组(H 2O 2+Tariquidar),通过检测细胞内ATP水平以及采用Annexin V-FITC凋亡检测试剂盒检测细胞凋亡率来验证氧化应激下P-糖蛋白功能性表达对细胞氧化损伤的抑制作用。结果免疫印迹实验结果显示,随着H 2O 2浓度的增加,P-糖蛋白相对表达量呈剂量依赖性增加并在400μmol·L-1时达到最高值。H 2O 2在浓度400μmol·L-1时对P-糖蛋白表达和功能均有最明显的诱导作用,该浓度为最佳处理浓度。H 2O 2+P-糖蛋白抑制剂组中细胞内罗丹明123水平较H 2O 2模型组增加,差异有统计学意义(P<0.01),提示Tariquidar能有效抑制P-糖蛋白功能。H 2O 2模型组ATP水平较对照组显著下降,差异有统计学意义(P<0.001);而H 2O 2+P-糖蛋白抑制剂组ATP水平则较H 2O 2模型组更低,差异亦有统计学意义(P<0.05),提示抑制P-糖蛋白功能可进一步加剧氧化应激导致的ATP水平下降。与对照组相比,H 2O 2模型组细胞凋亡率显著增加,而H 2O 2+P-糖蛋白抑制剂组细胞凋亡率则较H 2O 2模型组增加,提示抑制P-糖蛋白功能可加重氧化应激下的细胞凋亡。结论P-糖蛋白对H 2O 2诱导的RPE细胞氧化损伤具有一定的抑制作用。

Objective To investigate the inhibition role of P-glycoprotein on oxidative damage of retinal pigment epithelium(RPE)cells induced by hydrogen peroxide(H 2O 2).Methods The human RPE cell line D407 was cultured in 5%CO 2 incubator with DMEM containing 10%fetal bovine serum and double antibody,and subcultured at 37℃.Cells were treated with different concentration of H 2O 2,and the optimal concentration of H 2O 2 on p-glycoprotein expression was determined by western blot and rhodamine 123 accumulation tests.P-glycoprotein inhibitor tariquidar(1μmol·L-1)was pretreated prior to H 2O 2 exposure and the inhibition efficiency was detected by measuring the intracellular accumulation of rhodamine 123,a fluorescent substrate of P-glycoprotein.Cells were divided into three groups:control group,H 2O 2 model group(400μmol·L-1)and H 2O 2+P-glycoprotein inhibitor group(H 2O 2+tariquidar group).The inhibition effect of P-glycoprotein on oxidative damage was verified by detecting intracellular ATP level,and the apoptosis rate was detected by Annexin V-FITC apoptosis detection kit.Results The relative expression level of P-glycoprotein increased in a dose-dependent manner with the increase of H 2O 2 concentration,and it reached the highest value at 400μmol·L-1.H 2O 2 could induce the expression and function of P-glycoprotein at the concentration of 400μmol·L-1.The level of rhodamine 123 in the H 2O 2+tariquidar group was higher than that in H 2O 2 model group(P<0.01),which suggested that tariqudar can effectively inhibit the function of P-glycoprotein.The ATP level of the H 2O 2 model group was significantly lower than that of the control group,and the difference was statistically significant(P<0.001);while the ATP level of the H 2O 2 model group was lower than that of the H 2O 2 model group,and the difference was also statistically significant(P<0.05),suggesting that inhibiting the function of P-glycoprotein can further aggravate the decline in ATP levels caused by oxidative stress.Compared with the control group,the apoptosis rate of H 2O 2 model group was significantly increased,while that of H 2O 2+tariquidar group was higher than that of H 2O 2 model group,suggesting that inhibition of H 2O 2 model group aggravate apoptosis under oxidative stress.Conclusion P-glycoprotein can inhibit RPE cells from oxidative damage induced by H 2O 2.