活性氧在贝伐单抗诱导视网膜色素上皮细胞上皮间质转化中的作用

Reactive oxygen species participate in the mesenchymal transition of retinal pigment epithelial cells induced by bevacizumab

目的观察贝伐单抗(BEV)对人视网膜色素上皮(RPE)细胞氧化应激损伤和上皮间质转化(EMT)的影响,进一步探讨抑制活性氧(ROS)在EMT中的作用。方法选取人ARPE-19细胞株,根据实验需要将ARPE-19细胞分为4组空白对照组、BEV组、BEV+NAC组和BEV+DPI组,其中空白对照组细胞不进行任何干预,BEV组用0.25 g·L-1 BEV处理细胞72 h,另外两组在BEV处理细胞24 h后分别再用ROS抑制剂NAC和NADPH氧化酶抑制剂DPI处理细胞48 h。采用2,7-二氯二氢荧光素二乙酸酯(DCFH-DA)染色观察各组ARPE-19细胞内ROS和H2O2的生成堆积情况;细胞免疫荧光观察各组ARPE-19细胞中EMT标志物紧密连接相关蛋白闭锁带蛋白-1(ZO-1)、α-平滑肌肌动蛋白(α-SMA)和纤维连接蛋白(FN)的表达情况;再通过RT-PCR和Western blot检测各组细胞中EMT标志物的mRNA和蛋白的表达水平。结果DCFH-DA染色观察发现,ARPE-19细胞加入BEV继续培养后,ROS和H2O2表达均明显上调(均为P<0.05)。与空白对照组相比,BEV组EMT指标变化显著上皮标志物ZO-1的表达降低,间质标志物α-SMA和FN的表达升高,两组间各指标相比差异均有统计学意义(均为P<0.05);与BEV组相比,BEV+NAC组和BEV+DPI组中各项指标mRNA表达变化显著,ZO-1上升至0.955±0.048、1.056±0.017,而α-SMA下降至0.982±0.165、1.058±0.165,此外FN表达(0.666±0.063、0.983±0.125)也显著降低,差异均有统计学意义(均为P<0.05)。各种因子的蛋白表达变化趋势与其相应mRNA表达相一致,与单纯BEV组相比,ROS抑制剂NAC和NADPH氧化酶抑制剂DPI都显著改变了EMT标志物的表达,ZO-1蛋白相对表达量升高了0.195±0.010、0.770±0.175,而α-SMA下降了0.353±0.098、0.482±0.037,FN下降了0.528±0.161、0.612±0.134,差异均有统计学意义(均为P<0.05)。结论ROS参与了BEV诱导的RPE细胞EMT,抑制ROS可减轻BEV诱导的人RPE细胞的EMT程度。

Objective To observe the effects of bevacizumab(BEV)on oxidative stress damage and epithelial-mesenchymal transition(EMT)of human retinal pigment epithelium(RPE)cells,and to further explore the effect of inhibiting reactive oxygen species in EMT.Methods Human retinal pigment epithelial cell line(ARPE-19)was selected,and ARPE-19 cells were divided into 4 groups according to experimental needscontrol group,BEV group,BEV+NAC group,and BEV+DPI group.The cells in the control group did not undergo any intervention.The BEV group was the drug control group,that is,the cells were treated with 0.25 g·L-1 BEV for 72 hours.The other two groups were treated with the reactive oxygen species inhibitor NAC and the NADPH oxidase inhibitor DPI for 48 hours after the BEV treatment for 24 hours.We observed the accumulation of reactive oxygen species and H2O2 in ARPE-19 cells by 2,7-dichlorodihydrofluorescein diacetate(DCFH-DA)staining,and the EMT markers in each group by cell immunofluorescence,including zona occludens-1(ZO-1),α-smooth muscle actin(α-SMA)and fibronectin(FN)in ARPE-19 cells.qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of EMT markers in each group.Results DCFH-DA staining showed that the expression of reactive oxygen species and H2O2 were significantly up-regulated after adding BEV to ARPE-19 cells(both P<0.05).Compared with the control group,the EMT indicators of the BEV group changed significantlythe expression level of the epithelial marker ZO-1 decreased,and the levels of the interstitial markersα-SMA and FN increased,and the differences in each index were statistically significant(all P<0.05).Compared with the BEV group,the mRNA expression of various indicators in the BEV+NAC group and BEV+DPI group changed significantly,ZO-1 increased to 0.955±0.048,1.056±0.017,andα-SMA decreased to 0.982±0.165,1.058±0.165,and FN(0.666±0.063,0.983±0.125)also significantly decreased,and the differences were statistically significant(all P<0.05).The protein change trend was consistent with mRNA.Compared with the pure BEV group,both the reactive oxygen species inhibitor NAC and the NADPH oxidase inhibitor DPI significantly changed the expression of EMT markers,ZO-1 increased by 0.195±0.010 and 0.770±0.175,whileα-SMA and FN decreased by 0.353±0.098,0.482±0.037 and 0.528±0.161,0.612±0.134,respectively,and the differences were statistically significant(all P<0.05).Conclusion Reactive oxygen species are involved in the BEV-induced interstitial transformation of RPE cells.Inhibition of reactive oxygen species can reduce the degree of EMT of human RPE cells induced by BEV.