FOXO4对高糖环境下视网膜血管内皮细胞氧化应激和凋亡的影响

Effects of FOXO4 on oxidative stress and apoptosis of retinal vascular endothelial cells under high glucose environmental conditions

摘要:

目的：研究叉头框转录因子O4(FOXO4)对高糖环境下视网膜血管内皮细胞氧化应激和凋亡的影响。

方法：以高糖培养人视网膜血管内皮细胞，Real-Time PCR和Western blot检测细胞中FOXO4表达变化。以FOXO4 RNAi慢病毒、对照载体慢病毒感染视网膜血管内皮细胞，以高糖培养液培养，Real-Time PCR和Western blot检测干扰效率。收集培养液上清和细胞，用试剂盒检测培养液上清中超氧化物歧化酶(superoxide orgotein dismutase，SOD)活性、丙二醛(malonaldehyde，MDA)含量和细胞中活性氧(reactive oxygen species，ROS)水平。流式细胞术测定细胞凋亡变化，Western blot检测细胞中凋亡蛋白Cleaved Caspase-3、Cleaved Caspase-9的表达变化。

结果：高糖处理后的视网膜血管内皮细胞中FOXO4表达上调，FOXO4 RNAi慢病毒感染下调高糖环境下视网膜血管内皮细胞中FOXO4表达水平，对照载体慢病毒对细胞中FOXO4表达水平没有影响。高糖诱导视网膜血管内皮细胞中ROS水平升高，降低细胞培养液中SOD活性，提高MDA含量，增加细胞凋亡率，促进细胞中Cleaved Caspase-3、Cleaved Caspase-9蛋白表达。下调FOXO4表达的视网膜血管内皮细胞经高糖诱导后，细胞培养液中的SOD活性升高，MDA水平降低，细胞中ROS水平降低，细胞凋亡减少，细胞中Cleaved Caspase-3、Cleaved Caspase-9蛋白水平降低，与未干扰FOXO4表达的视网膜血管内皮细胞比较，差异有统计学意义(P<0.05)。

结论：高糖诱导视网膜血管内皮细胞表达FOXO4，敲低其表达降低高糖诱导的细胞氧化应激和凋亡水平。

Abstract:

AIM: To study the effects of FOXO4 on oxidative stress and apoptosis of retinal vascular endothelial cells under high glucose environmental conditions.

METHODS: Human retinal vascular endothelial cells were cultured with high glucose medium. Real-time PCR and Western blot were used to detect the expressions of FOXO4 within cells; meanwhile, both FOXO4 RNAi lentivirus and control vector lentivirus were infected the retinal vascular endothelial cells cultured in high sugar culture medium. Real-time PCR and Western blot techniques were used to detect the interference efficiency. After collection of supernatant and cells treated with various interferences, the SOD activity, MDA content in the supernatants and ROS level within cells were detected. Flow cytometry was used to determine the changes of cell apoptosis. Western blot was used to detect the expressions of apoptotic protein cleaved Caspase-3 and cleaved Caspase-9 within cells.

RESULTS: The expression of FOXO4 was increased in retinal vascular endothelial cells after treatment with high glucose medium. FOXO4 RNAi lentivirus infection reduced the expression level of FOXO4 in retinal vascular endothelial cells under high glucose environmental conditions. By contrast, control vector lentivirus had no effect on FOXO4 expression in cells. High glucose induced elevated levels of ROS in retinal vascular endothelial cells, reduced the activity of SOD in cell culture medium, increased the content of MDA, elevated the rate of apoptosis, and promoted the expressions of cleaved Caspase-3 and cleaved Caspase-9 proteins in cells. After down-regulation of FOXO4 expression, retinal endothelial cells were induced by high glucose, the activity of SOD in the cell culture medium increased, the levels of MDA, ROS, apoptosis, and the levels of cleaved Caspase-3 and cleaved Caspase-9 proteins decreased in cells as compared with those of retinal vascular endothelial cells. Moreover, compared with those did not interfere with FOXO4 expression, there was statistically significant differences(P<0.05).

CONCLUSION: High glucose induces the expression of FOXO4 in retinal vascular endothelial cells. Knocking-down of FOXO4 expression reduces oxidative stress and apoptosis induced by high glucose medium.