Objective To observe the expression of vascular endothelial growth factor A (VEGFA) and its receptors sFlt-1, kinase insert domain receptor (KDR) in lightinjured human retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells (8th - 12th generations) were divided into normal control group and light damage group. The cells of two groups were exposed to the 18 W cold white light (2200±300) Lux for 12 hours to induce light damage responses, but the cells of normal control group were packed by tinfoil with doubledeck high pressure disinfection. The VEGF-A, sFlt-1 and KDR mRNA and protein expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot at 0, 6, 12, 24 hours after light damage. Results The VEGF-A mRNA and protein expressions in light damage group were significantly increased at 6 hours, and reached its peak at 12 hours after light damage which obviously higher than that in normal group (t=2.74, 2.93; P＜0.05), and then went down gradually. The sFlt-1 mRNA and protein expressions in light damage group reached its peak at 12 hours after light damage which obviously higher than that in normal group (t=4.32, P＜0.01), but obviously lower than that in normal group at 24 hours after light damage (t=2.41, P＜0.05). The KDR mRNA and protein expressions in light damage group were obviously higher than that in normal group at 24 hours after light damage （t=2.89, P＜0.05）,but there was no changes at 6, 12 hours after light damage (t=1.84, P＞0.05). Conclusions At 6, 12 hours after light damage, the expressions of VEGF-A and sFlt-1 increases significantly and KDR expression is stable in lightinjured RPE cells. At 24 hours after light damage, the expression of VEGF-A and sFlt-1 decreases, but KDR expression increases in light-injured RPE cells.
Objective To assess the effects of 670nm LED (lightemitting diode) to protect the photoreceptor from the lightinduced damage in a rat model. Methods 32 SD rats were randomly assigned to one of eight groups: untreated control group, the LEDtreated control group, three groups of lightinduced damage，and three groups of lightinduced damage treated with LED. Lightinduced damage result from exposing to constant light for 3 hours of different illuminations of 900，1800 and 2700 lx, respectively. The LED treatment (50 mW) was delivered for 30 minutes at 3 hours before the light damage and 0，24 and 48 hours after the light damage. Retinal function and morphology were measured by electroretinogram (ERG) and histopathology assay. Results The illumination of 900 lx for 3 hours did not damage the rat retina. The illumination of 1800 lx for 3 hours resulted in thinner ONL and no OS and IS. The ratio of damaged area/total retinal area was 048plusmn;012， the damaged thickness of ONL/normal ONL (L5 ) was 039plusmn;007，and the amplitude of ERG b wave was (431plusmn;120) mu;V. With the LED treatment the ratio of damaged area decreased (M6=017plusmn;0.12, P5/6=0.002), and the ratio of the damaged thickness of ONL also decreased (L6=0.22plusmn;0.09, P5/6lt;0.01), and the amplitude of ERG b wave increased to (1011plusmn;83) mu;V（P5/6lt;0.001）. The illumination of 2700 lx for 3 hours caused severed damage to the rat retina and the LED could not protect them significantly. Conclusions 670 nm LED treatment has an evident protective effect on retinal cells against light-induced damage, which may be a simple and effective therapy to prevent or to delay agerelated macular degeneration.
Objective To explore the protective mechanism and effect of coenzyme Q10 on the retinal photic injury in experimental rats. Methods Thirty Sprague-Dawley rats were divided randomly into 3 groups: normal control group, positive control group, and coenzyme Q10 group. The experimental model of photic injury in rats was established by being exposed to intense green fluorescent light with an illuminance level of (2000plusmn;120) Lux for 24 hours. The physiological saline and coenzyme Q10 were given through tail intravenous injection at 24 hours and 30 min before light exposure in positive control group and coenzyme Q10 group, respectively. Ophthalmectomy was performed 1 day after the illumination. Changes of retinal histopathology and ultrastructure were observed by light and electron microscope. The apoptosis rate of retinal cells was detected by flow cytometry. Results The result of histopathological examination showed that in coenzyme Q10 group, the outer segments arranged trimly with only few cell apoptosis; the inner and outer segments slightly swelled, and compared with the positive group, the histopathological changes alleviated obviously. The result of flow cytometry revealed that the apoptosis rate of retinal cells was (1.65plusmn;1.48)% in normal control group, (25.83plusmn;2.92)% in positive control group, and (12.43plusmn;2.25)% in coenzyme Q10 group, respectively. The apoptosis rate of retinal cells was higher in positive control group than that in the normal control group (t=18.28, Plt;0.01), and lower in coenzyme Q10 group than that in the positive control group (t=9.07, Plt;0.01). Conclusion Coenzyme Q10 plays an important role in preventing the photic injury of retina and optic cell apoptosis. (Chin J Ocul Fundus Dis, 2007, 23: 122-125)
Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage. Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted. Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05). Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
ObjectiveTo observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells. MethodsCultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000±500) Lux for 6 hours to establish the light injured model. Light injured cells were divided into HtrA1 siRNA group, negative control group and blank control group. HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively. The proliferation of cells was assayed by CCK-8 method. Transwell test was used to detect the invasion ability of these three groups. Flow cytometry was used to detect the cell cycle and apoptosis. The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively. ResultsThe mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62, 15.09; P<0.05). Compared with negative control group and blank control group, the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37, 4.52), migration (t=9.56, 12.13), apoptosis (t=23.37, 29.08) and decreased mRNA (t=17.36, 11.32, 7.29, 4.05) and protein levels (t=12.02, 15.28, 4.98, 6.24) of HtrA1 and VEGF-A. Cells of HtrA1 siRNA group mainly remained in G0/G1 phase, the difference was statistically significant (t=6.24, 4.93; P<0.05). ConclusionKnockdown of HtrA1 gene may reduce the proliferation, migration capability and apoptosis of light-injured RPE cells, and decrease the expression of VEGF-A.
ObjectiveTo observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3).MethodsCultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR).ResultsBlue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like double-concave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04), IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P＜0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P＜0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P＜0.001).ConclusionThe expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells.