Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light

Abstract

Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm(2)). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light.

Figure 1. The effects of blue, white, and green LED lights on the cell viability.

(A) The exposure of blue, white, and green LED light to cells cultured in a 96-well plate. (B) The observation of cell morphology using bright field microscopy, showing blue LED light caused the morphological changes compared with the control. Green LED light did not change the cells. (C–E) The quantitative evaluation of cell viability by the CCK-8 assay. This result is consistent with the observed change in cell morphology. Cell viability was reduced by blue and white LED light exposure, but not green LED light. The scale bar represents 50 μm. Data are expressed as mean ± SEM (n = 6). ^## indicates p < 0.01 vs. control (ANOVA).

Figure 2. ROS production by blue, white, and green LED light exposure.

(A–C) Blue LED light and white LED light exposure increased each 1.4-fold and 1.2-fold ROS production, and green LED light did not increase ROS level. Data are expressed as mean ± SEM (n = 6). ^## indicates p < 0.01 vs. control (ANOVA). (D) Representative images show JC-1 stained cells. The healthy cells with mainly JC-1 J-aggregates (red) and apoptotic or unhealthy cells with mainly JC-1 monomers (green). Merged cells (yellow) were considered to be pre-apoptotic (early or middle state of transition to cell death) cells. Scale bar represents 50 μm. (E) The number of cells with red or yellow color were counted. The ratio of merged cells to red color cells was increased by blue LED light exposure for 12 h or 24 h. Data are expressed as mean ± SEM (n = 6). ^## indicates p < 0.01 vs. control (ANOVA).

Figure 3. Changes in protein levels induced by blue LED light exposure.

(A) Western blotting showed changes in the levels of phosphorylated NF-κB, p38, and ERK (p-NF-κB, p-p38, and p-ERK). The bands indicate protein expression levels at each 3 h (NF-κB), 6 h (p38), and 6 h (ERK) after LED light exposure. (B–D) Quantitative analysis of protein levels. Quantitative data of the groups of white LED and green LED exposure indicates 3 h (NF-κB), 6 h (p38), and 6 h (ERK) results. The phosphorylated NF-κB level is increased 3 h after exposure to blue LED light. The phosphorylated p38 level is increased 6 h after blue and white LED light exposure. The phosphorylated ERK level is decreased 6 h after blue LED light exposure. These changes were not observed after green LED light exposure. Data are expressed as mean ± SEM (n = 3 to 6). ^# indicates p < 0.05, ^## indicates p < 0.01 vs. control (B, E, H; one-way ANOVA followed by Dunnett's test, F; ANOVA). The cropped blots are used in this Figure and the full-length blots are presented in Supplementary Figure S5–7.

Figure 4. The aggregation of S-opsin induced by blue LED light exposure.

(A) Representative immunostaining images of S-opsin after LED light exposure for 24 h. Blue and white LED light-induced the perinuclear aggregation of S-opsin compared to control and green LED light. n = 4. (B) Representative immunostaining images of S-opsin shows that the S-opsin aggregated cells after blue LED light exposure for 3 or 6 h (arrowhead). (C) Quantitative analysis of immunostaining images. The ratio of the S-opsin aggregated cells was increased by blue LED light exposure for 3 or 6 h. Data are expressed as mean ± SEM (n = 3 or 4). ^# indicates p < 0.05 vs. control (one-way ANOVA followed by Dunnett's test). The scale bars represent 5 μm (A), 50 μm and 10 μm (B).

Figure 5. Blue LED light caused the primary retinal cell damage.

(A) Primary retinal cells were exposed to blue LED light for 24 h. The cell viability was evaluated by the CCK-8 assay. Blue LED light decreased the primary retinal cell viability. (B) Blue LED light increased the ROS level in primary retinal cells. (C, D) Immunostaining of cleaved caspase-3. Blue LED light increased the cleaved caspase-3 positive cells compared to control. (E, F) Double immunostaining for S-opsin and cleaved caspase-3. Blue LED light increased the S-opsin and cleaved caspase-3 double positive cells (arrowhead). Data are expressed as mean ± SEM (n = 3 or 4). ^# indicates p < 0.05, ^## indicates p < 0.01 vs. control (ANOVA). The scale bar represents 50 μm.

Figure 6. NAC suppressed the blue LED light-induced damage and inhibited NF-κB activation.

(A–C) Evaluation of the cell viability by the CCK-8assay and the rate of cell death by Hoechst and PI staining. The rate of cell death indicates by the ratio of PI (red) stained cells per Hoechst (blue) stained cells. NAC at 1 mM significantly improved the cell viability reduced by blue LED light. (D) NAC reduced the ROS level elevated by blue LED light. (E–H) The effect of NAC against blue LED light-induced changes in protein expression was assessed by Western blots. NAC suppressed the blue LED light-induced increase in activated NF-κB levels, but did not suppress activated p38. NAC did not alter the reduced ERK level. Data are expressed as mean ± SEM (n = 5 or 6). ** indicates p < 0.01 vs. vehicle; ^## indicates p < 0.01 vs. control (one-way ANOVA followed by Tukey's test). The scale bar represents 50 μm. The cropped blots are used in this Figure and the full-length blots are presented in Supplementary Figure S8.

Figure 7. NAC suppressed blue LED light-induced caspase-3/7 activation and autophagy activation.

(A) Measurement of caspase-3/7 activity by Caspase-Glo® 3/7 Assay kit. Activation of caspase-3/7 was observed after blue LED light exposure. NAC treatment significantly inhibited the activation. Data are expressed as mean ± SEM (n = 3 or 4). ** indicates p < 0.01 vs. vehicle; ^## indicates p < 0.01 vs. control (one-way ANOVA followed by Tukey's test). (B) Western blots of LC3-II/LC3-I indicated an increase in the expression level after blue LED light exposure. NAC treatment significantly reduced the expression. Data are expressed as mean ± SEM (n = 6). * indicates p < 0.05 vs. vehicle; ^## indicates p < 0.01 vs. control (one-way ANOVA followed by Tukey's test). The cropped blots are used in this Figure and the full-length blots are presented in Supplementary Figure S9.

Figure 8. The putative pathway of blue LED light-induced retinal photoreceptor-derived cell damage.

In 661 W cells, blue LED light induces ROS production and S-opsin aggregation. The rapid ROS increase leads to mitochondrial damage and the MAPK activation or the nuclear translocation of NF-κB. Activated MAPK and NF-κB induces the activation of caspase and leads to apoptotic cell death. Active NF-κB also activates autophagy, and excessive autophagy leads to cell death. While, S-opsin aggregation causes endoplasmic reticulum (ER) stress. Blue LED light-induced retinal photoreceptor-derived cell death may be associated with both oxidative stress and ER stress.