## 4 Discussion
The protective role against the toxicity of blue-violet light on human corneal epithelial cells of increased expression of HO-1, Prx-1, CAT and SOD-2 and the consequent attenuation of ROS production by some plant extracts has been shown in a recent paper by [PERSON] and collaborators [62]. Moreover, it has been shown that both the oral and topical administration of astaxanthin may effectively protect corneal cells from apoptosis and significantly reduce oxidative stress following UV-induced photokeraitis damage [63; 64]. In this study, we have focused our attention on the Nrf-2 pathway, which is known to be involved in the regulation of endogenous antioxidant defense also after blue light irradiation. In fact, the long-term exposure (24 to 48 h) of murine photoreceptor cells (661W) in vitro to low-intensity blue light was reported to increase ROS production and induce Nrf-2 expression [29; 65]. In the same model system, astaxanthin was shown to induce the Nrf-2 protection system and decrease blue light cell damage [61]. Under our experimental conditions of acute photooxidative damage (50 J/cm\({}^{2}\) for 30 min), we observed a dramatic increase of ROS, a relevant decrease in cell viability and a parallel decrease of Nrf-2 expression (Figures 1 and 5). The presence of lutein and astaxanthin blunted ROS increase, thus improving cell viability and rescuing Nrf-2 expression (Figures 2, 3 and 5). The likely explanation of these data on Nrf-2--opposite to what shown in the previously cited studies--could rely on the observation that when an acute oxidative stress is given in the absence of an appropriate antioxidant defense (such as in our case), there is a sudden loss of cell viability due to mitochondrial toxicity as detected by the MTT assay [66], thus leading to a decrease of Nrf-2 expression. However, if a milder oxidative stress is given over a sufficiently wide time frame (as in the previous referenced studies on 661W cells), the Nrf-2 pathway is induced, and an increase of Nrf-2 expression can be observed. In either case, the shielding effect of lutein and astaxanthin, combined with their intrinsic antioxidant properties, may result in the recovery of the original situation: in our case, in a recovery of cell viability and Nrf-2 expression. Similar in vitro results were also reported for a human corneal epithelial cell line treated by hyperosmolar stress and for a rabbit corneal cell line treated by hyperglycemic stress, each treatment resulting in a decrease of cell viability and a concomitant reduction of Nrf-2 expression. In these cases, the decrease of both cell viability and Nrf-2 expression could be prevented by pretreatment with the antioxidant edaravone, in the case of hyperosmotic stress [67], or by dimethyl-fumarate, in the case of hyperglycemic stress [68]. An additional consideration is that Nrf-2 response to oxidative stress could be differently modulated in epithelial (a decrease is observed) and neuronal (an increase is reported) cells. A similar situation has been reported for scleral or choroidal fibroblasts, in which atropine, respectively, induced an increase or a decrease of collagen production [69]. Most important, in our opinion, is the behavior of Keap-1. This protein binds Nrf-2 and induces its degradation, thus preventing its functioning. Oxidants disrupt this interaction so that more Nrf-2 is free to activate the DNA transcription of antioxidant genes [(38; 70)]. In our study, we found a relevant decrease of Kaep-1 expression (Figure 5B), unrelated to the presence of xanthophylls and cell survival. Such low levels of Kaep-1, significantly lower in the presence of both xanthophylls, implies that more Nrf-2 can be available to induce endogenous antioxidant protection, thus contributing to enhanced cell viability. In particular, the ratio Nrf-2/Keap-1 is the highest when both astaxanthin and lutein were present together to shield HCEF cells from blue-violet light (Figure 5E).