贝伐珠单抗对H2O2诱导的视网膜色素上皮细胞功能的影响

Effect of bevacizumab on the function of retinal pigment epithelial cells induced by HO

目的研究贝伐珠单抗对H_(2)O_(2)诱导的视网膜色素上皮细胞凋亡及PTEN/AKT信号通路的影响。方法ARPE-19细胞分为空白组(正常DMEM培养液培养)、模型组(200μmol·L^(-1)H_(2)O_(2))、低剂量实验组(200μmol·L^(-1)H_(2)O_(2)+0.125 mg·mL^(-1)贝伐珠单抗)、高剂量实验组(200μmol·L^(-1)H_(2)O_(2)+1.25 mg·mL^(-1)贝伐珠单抗)、SF1670组(200μmol·L^(-1)H_(2)O_(2)+2μmol·L^(-1)SF1670)、SF1670+高剂量实验组(200μmol·L^(-1)H_(2)O_(2)+2μmol·L^(-1)SF1670+1.25 mg·mL^(-1)贝伐珠单抗)。以CCK-8法检测细胞ARPE-19存活率,以流式细胞术检测ARPE-19细胞凋亡率,以DCFH-DA荧光探针法检测细胞中活性氧(ROS)水平,检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,以蛋白质印迹法检测ARPE-19细胞凋亡相关及PTEN/AKT信号通路相关蛋白的表达情况。结果给药24 h,空白组、模型组和低、高剂量实验组ARPE-19细胞存活率分别为(98.79±3.57)%,(52.36±2.14)%,(76.74±3.72)%,(91.23±2.19)%;凋亡率分别为(3.64±0.52)%,(15.47±3.15)%,(9.14±1.77)%,(6.82±0.69)%;给药24 h,模型组、SF1670组、SF1670+高剂量实验组ARPE-19细胞存活率分别为(53.95±4.12)%,(80.14±2.95)%,(96.12±1.92)%;凋亡率分别为(16.31±2.66)%,(10.96±1.94)%,(5.58±2.38)%,差异均有统计学意义(均P<0.05)。各组ROS荧光强度、SOD活性、MDA含量、Bax和Bcl-2蛋白相对表达量,差异均有统计学意义(均P<0.05)。结论贝伐珠单抗可明显减少H_(2)O_(2)诱导的视网膜色素上皮细胞活力的损伤和细胞凋亡,其作用机制可能与促进PTEN/AKT信号通路相关蛋白转导有关。

Objective To study the effect of bevacizumab on the apoptosis of retinal pigment epithelial cells induced by H_(2)O_(2)and the PTEN/AKT signaling pathway.Methods ARPE-19 cells were divided into blank group(DMEM),model group(200μmol·L^(-1)H_(2)O_(2)),Exp-L group(200μmol·L^(-1)H_(2)O_(2)+0.125 mg·mL^(-1)bevacizumab),Exp-H group(200μmol·L^(-1)H_(2)O_(2)+1.25 mg·mL^(-1)bevacizumab),SF1670 group(200μmol·L^(-1)H_(2)O_(2)+2μmol·L^(-1)SF1670),SF1670+Exp-H group(200μmol·L^(-1)H_(2)O_(2)+2μmol·L^(-1)SF1670+1.25 mg·mL^(-1)bevacizumab).CCK-8 method was used to detect cell survival rate of ARPE-19,flow cytometry was used to detect ARPE-19 cell apoptosis rate,DCFH-DA fluorescent probe method was used to detect cell reactive oxygen species(ROS)level,detect the Superoxide dismutase(SOD)activity and Malondialdehyde(MDA)content.Western blotting was used to detect the expression of ARPE-19 cell apoptosis-related and PTEN/AKT signaling pathway related proteins.Results After 24 hours of administration,the survival rates of ARPE-19 cells in blank group,model group,Exp-L group and Exp-H group were(98.79±3.57)%,(52.36±2.14)%,(76.74±3.72)%,(91.23±2.19)%;apoptosis rates were(3.64±0.52)%,(15.47±3.15)%,(9.14±1.77)%,(6.82±0.69)%,respectively.After 24 hours of administration,the survival rates of ARPE-19 cells in model group,SF1670 group,and SF1670+Exp-H group were(53.95±4.12)%,(80.14±2.95)%,(96.12±1.92)%;apoptasis rates were(16.31±2.66)%,(10.96±1.94)%,(5.58±2.38)%,all with significant difference(all P<0.05).The ROS,SOD activity,MDA content,relative expression of Bax protein and Bcl-2 protein in each group were all with significant differences(all P<0.05).Conclusion Bevacizumab can significantly reduce the damage and apoptosis of retinal pigment epithelial cells induced by H_(2)O_(2),and its mechanism of action may be related to the promotion of protein transduction in the PTEN/AKT signaling pathway.