Authors: Li Dandan, Zou Xiulan, Chen Jingxia, Xu Zhe, Yu Yongzhen, Zhou Wenjie, Wang Guanfeng, Rao Benqiang, Zou Yuping
Light-induced retinal damage results in the damage of retinl pigment epithelial (RPE) cells and therefore affects the pathogenesis and development of age-related macular degeneration (AMD). Studies showed that tissue factor (TF) is overexpressed in oxidative damaged RPE cells and the choroidal neovascularization (CNV) of AMD, speculating that the suppression of TF can prevent the damage of RPE cells and inhibit CNV.
This study was conducted to observe the protective effects of TF targeting peptide (TF-TP), a new drug of autologous synthesis, on human RPE-cells induced by blue light.
Human RPE cells were isolated from donor eye and cultured.Cultured cells were divided into blank control group, model group and TF-TP treated group.Light-induced RPE cell damage model was established by exposuring the cells in the blue light of (4.0±0.5)mW/cm2 for 12 hours in the model group, and different concentrations (10, 100, 150, 200, 300 μmol/L) of TF-TP were added into the medium to pretreat the cells for 24 hours and then exposed the cells to the blue light for 12 hours in the TF-TP groups.The cell viability was determined by CCK-8 assay.The morphology and ultrastructure in the cells were observed under the inverted microscope and transmission electron microscope.The apoptosis of the cells was assayed by Hoechst staining.The expressions of TF and apoptosis-related protein bax, bcl-2 in the cells were determined by Western blot.
CCK-8 assay showed that there was no significant difference in the cell viability among blank control group and different concentrations TF-TP groups (F=2.15, P=0.11). The cell survival rate of blank control group, model group and 150 μmol/L TF-TP group was (100.0±0.00)%, (43.79±6.55)% and (63.45±3.57)%, and the survial rate was increased in the 150 μmol/L TF-TP group compared with the model group (P=0.00), and 150 μmol/L was detemined as a optimal concentration of TF-TP.A lot of shrinkage, deformation, suspension cells were exhibited under the optical microscope, and decrease of microvilli structure, rupture of mitochondrial cristae and vacuolar degeneration of the cells were found in the model group, and the damage of the cells were evidently lightened in the 150 μmol/L TF-TP group.The apoptosis rate of the cells were (0.98±0.19)%, (9.98±0.82)% and (5.73±0.88)% in the blank group, model group and 150 μmol/L TF-TP group, respectively, with a significant difference among the groups (F=206.18, P=0.00), and the apoptosis rate of the cells in the 150 μmol/L TF-TP group was significantly lower than that in the model group (P<0.05). Compared with the blank control group, the relative expression of bax and TF was obviously increased and that of bcl-2 was decreased in the model group; while the expression of bax and TF was lower, and that of bcl-2 was higher in the 150 μmol/L TF-TP group compared with the model group (all at P<0.05).
Pretreation of TF-TP can lessen cell apoptosis and increase cell survival rate and therefore plays a protective role to blue light-induced human RPE cells possibly by inhibiting bax/bcl-2 apoptotic pathways mediated by TF.