Wang Limin, Cai Shanjun, Wu Zhipeng, Gong Xin, Lyu Jianping, Su Gang, Wang Lili
Department of Ophthalmology, the Hospital Affiliated to Zunyi Medical School, Zunyi 563003, China.
Department of Ophthalmology, the Hospital Affiliated to Zunyi Medical School, Zunyi 563003, China; Email:
Zhonghua Yan Ke Za Zhi. 2015 Nov;51(11):839-43.
To investigate the concentrations of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), inositol triphosphate (IP3) and diacylglycerol (DAG) in human retinal pigment epithelium (RPE) cells after exposuring to blue light, and to explore the relationship with Ca2+-PKC signaling pathways, to evaluate the role of Ca2+-PKC signaling pathways of blue-light irradiation induced apoptosis in RPE cells.
The fourth generation human RPE cells in vitro were exposured to blue light (2000±500 lux) for 6 hours, 24 hours prolongation of post-exposure culture. The concentrations of VEGF, PEDF, IP3 and DAG were assayed by enzyme linked immunosorbent assay (ELISA). Cells were randomly divided into 6 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+PMA), group D (exposure to blue light+Calphostin C), group E (exposure to blue light+Nifedipine), group F (exposure to blue light+Calphostin C+Nifedipine). Flow cytometry was used to detect the apoptosis rate of human RPE cells in A, B and F group.
Comparing with group A (584.38±10.66), the concentration of VEGF in group B (700.70±5.88), group C (698.21±6.66) and group E (648.30±4.91) was higher, the difference was statistically significant (P=0.002, 0.002, 0.016). Comparing with group B (700.70±5.88), the concentration of VEGF in Group D (623.87±3.12) and E (648.30±4.91) was lower (P=0.001, 0.002). Comparing with group A (75.96±1.70), the concentration of PEDF in Group B (71.82±1.67) and C (72.43±0.58) was lower (P=0.004, 0.011), but the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group B (71.82±1.67), the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group A (7.70±0.29), the ratio of VEGF to PEDF in Group B (9.85±0.34) and Croup C (9.64±0.02) was higher (P=0.008, 0.027) Comparing with group B, The ratio of VEGF to PEDF in Group D (7.23±0.08) and E (6.92±0.06) was lower (P=0.016, 0.015). Comparing with group A (108.42±0.75, 995.47± 13.61), the concentration of IP3 and DAG in Group B (117.24±1.06, 1070.10±10.07), C (137.12±2.71, 1046.40±7.90), D (139.17±1.40, 1041.13±9.76) and E (149.61±0.77, 1273.14±10.89) was higher, the difference was statistically significant (P=0.003, 0.007, 0.000, 0.000, 0.000, 0.000, 0.000, 0.000). Comparing with group B, the concentration of IP3 in Group C, D and E was higher (P=0.011, 0.000, 0.000). Comparing with group B, the concentration of DAG in Group C and D was lower (P=0.021, 0.007). Comparing with group B, the concentration of DAG in Group E was higher (P=0.000). Comparing with group A (10.27±1.88), the apoptosis rate of RPE cells in Group B(25.07±2.66) and F(19.37±3.23) was higher, the difference was statistically significant (P=0.001, 0.009). Comparing with group B (25.07±2.66), the apoptosis rate of RPE cells in Group F (19.37±3.23) was lower (P=0.038).
(1) After exposuring to blue light, the concentrations of VEGF, IP3 and DAG are increased and the ratio of VEGF to PEDF is also increased and the concentration of PEDF is decreased in human RPE cells. (2) L-Type Calcium Channels and Ca2+-PKC signaling pathways may be regulate the concentrations of VEGF, PEDF, IP3 and DAG in RPE cells after exposuring to blue light by feedback regulation. (3) The application of Calphostin C combined with Nifedipine may be restrain the apoptosis of RPE cells after exposuring to blue light.
研究蓝光照射后人视网膜色素上皮（RPE）细胞中血管内皮生长因子（VEGF）、色素上皮衍生因子（PEDF）、三磷酸肌醇（IP3）和二酰甘油（DAG）的浓度，并探讨其与Ca2+-蛋白激酶C（PKC）信号通路的关系，以评估Ca2+-PKC信号通路在蓝光照射诱导RPE细胞凋亡中的作用。
将体外培养的第四代人RPE细胞暴露于蓝光（2000±500勒克斯）下6小时，照射后延长培养24小时。采用酶联免疫吸附测定（ELISA）法检测VEGF、PEDF、IP3和DAG的浓度。细胞随机分为6组，A组（对照组）、B组（蓝光照射组）、C组（蓝光照射+佛波酯（PMA）组）、D组（蓝光照射+钙泊三醇（Calphostin C）组）、E组（蓝光照射+硝苯地平组）、F组（蓝光照射+钙泊三醇+硝苯地平组）。采用流式细胞术检测A、B和F组人RPE细胞的凋亡率。
与A组（584.38±10.66）相比，B组（700.70±5.88）、C组（698.21±6.66）和E组（648.30±4.91）中VEGF的浓度较高，差异有统计学意义（P=0.002、0.002、0.016）。与B组（700.70±5.88）相比，D组（623.87±3.12）和E组（648.30±4.91）中VEGF的浓度较低（P=0.001、0.002）。与A组（75.96±1.70）相比，B组（71.82±1.67）和C组（72.43±0.58）中PEDF的浓度较低（P=0.004、0.0??1），但D组（86.31±1.35）和E组（93.72±1.24）中PEDF的浓度较高（P=0.000、0.000）。与B组（71.82±1.67）相比，D组（86.31±1.35）和E组（93.72±1.24）中PEDF 的浓度较高（P=0.000、0.000）。与A组（7.70±0.29）相比，B组（9.85±0.34）和C组（9.64±0.02）中VEGF与PEDF的比值较高（P=0.008、0.027）。与B组相比D组（7.23±0.08）和E组（6.92±0.06）中VEGF与PEDF的比值较低（P=0.016、0.015）。与A组（108.42±0.75，995.47±13.61）相比，B组（117.24±1.06，1070.10±10.07）、C组（137.12±2.71，1046.40±7.90）、D组（139.17±1.40，1041.1??3±9.76）和E组（149.61±0.77，1273.14±10.89）中IP3和DAG的浓度较高，差异有统计学意义（P=0.003、0.007、0.000、0.000、0.000、0.000、0.000、0.000）。与B组相比，C组、D组和E组中IP3的浓度较高（P=0.011、0.000、0.000）。与B组相比，C组和D组中DAG的浓度较低（P=0.021、0.007）。与B组相比，E组中DAG的浓度较高（P=0.000）。与A组（10.27±1.8??）相比，B组（25.07±2.66）和F组（19.37±3.23）中RPE细胞的凋亡率较高，差异有统计学意义（P=0.001、0.009）。与B组（25.07±2.66）相比，F组（19.37±3.23）中RPE细胞的凋亡率较低（P=0.038）。
（1）蓝光照射后人RPE细胞中VEGF、IP3和DAG的浓度升高，VEGF与PEDF的比值升高，PEDF的浓度降低。（2）L型钙通道和Ca2+-PKC信号通路可能通过反馈调节来调控蓝光照射后RPE细胞中VEGF、PEDF、IP3和DAG的浓度。（3）钙泊三醇联合硝苯地平的应用可能抑制蓝光照射后RPE细胞的凋亡。
