Short-Wavelength Blue Light Contributes to Pyroptosis of Human Lens Epithelial Cells (hLECs) by Caspase-1-GSDMD Signalling Axis Activation

Backgroud: To examine the effects of short-wavelength blue light (SWBL) on cultured human lens epithelial cells (hLECs). The nosogenesis of cataracts after SWBL exposure was discussed. Methods: HLE-B3 hLECs were divided into 3 groups randomly: A: normal control group, which consisted of hLECs cultured in the dark; B: the caspase-1 inhibitor group; and C: the SWBL exposure group. After the SWBL (2500 lux) irradiation (for 8, 16, 24, and 32 h), the caspase-1 and gasdermin D (GSDMD) expression levels in HLE-B3 hLECs were examined using ELISA, immunouorescence, and Western blotting analyses. Double-positive staining of HLE-B3 hLECs for activated and inhibited caspase-1 was used to conrm pyroptosis in hLECs by ow cytometry. Results: SWBL can cause cell death in HLE-B3 hLECs, but a caspase-1 inhibitor suppressed cell death. The ow cytometry results also conrmed the does-dependent of short-wavelength blue light irradiation on pyroptotic death of hLECs. Caspase-1 and GSDMD expression levels of all hLECs groups changed with short-wavelength blue light exposure times (8, 16, 24, and 32 h) and were higher in groups B and C than group A. The immunouorescence results demonstrated that the expression of GSDMD-N was higher in the cell membrane in both the B and C groups than in the A group. Conclusion: The data indicate that SWBL induces pyroptotic programmed cell death by activation of the GSDMD signalling axis in HLE-B3 hLECs. These results provide new insights into the exploitation of new candidates for the prevention of cataracts. Our study showed that short-wavelength blue light induces pyroptotic programmed cell death by activation of the caspase-1/GSDMD signalling axis in HLE-B3 hLECs. These ndings provide evidence and novel insights for new drug candidates for the prevention and treatment of cataracts.


Background
Cataract remains the leading cause of treatable blindness in the world, are characterized by opaci cation of the lens, and account for nearly half of all blindness cases [1]. Currently, cataract extraction and intraocular lens (IOL) implantation are the main treatment methods for cataracts. However, surgery of cataract might bring many serious postoperative complications, such as infection, corneal oedema, and high intraocular pressure. Poor postoperative outcomes, cost, and fear of cataract surgery are the most common barriers to cataract surgery [2,3]. Therefore, it is necessary to elucidate pathogenic mechanisms of cataracts from a new point of view and further provide new targets for effective prevention strategies.
To date, pathogenesis of cataract is not clearly elucidated. A previous study showed that endogenous photosensitive substances in the lens, such as tryptophan and ribo avin, are extremely sensitive to shortwavelength light, which includes UVA and short-wavelength blue light [4].
It is estimated that arti cial light sources, including uorescent light tubes and LEDs, consumed approximately 19% of the electricity produced worldwide in 2012 [5]. SWBL mainly comes from arti cial light sources. With the widespread use of blue backlight electronic display devices, such as computers and mobile, the human eyes are increasingly exposed to more SWBL [6].
SWBL (400-500 nm), induced oxidative stress and cellular injury, has attracted increasing attention because of the probably injury to the retina [7][8][9]. The transmission of visible light, especially in the blue spectrum region, is signi cantly reduced with age. Majority of them is absorbed in lens [10,11]. It has been suggested a link between exposure to SWBL and the development of cataracts, but additional studies are needed to con rm it.
According to the classi cation established by the NCCD, cell death is roughly classi ed as apoptosis, autophagic cell death, necrosis, pyroptosis, etc [12]. In the pathogenesis of cataracts, at least three types of programmed cell death pathways have been studied in deed, including apoptosis [13], autophagy [14], and pyroptosis [15]. The formation of cataracts is not entirely dependent on any single programmed cell death pathway. Of all the types of programmed cell death, apoptosis is the most well understood forms of death. Apoptosis of LECs is a common cellular basis for the formation of cataracts [15]. However, the speci c pyroptosis pathway implicated in cataract formation remains to be elucidated.
Our previous animal study indicated that pyroptosis plays a vital role in cataracts formation caused by SWBL exposure. In the present study, SWBL exposure systems were established to discuss the photobiological effects on cultured human LECs, including morphological changes, cell viability and protein levels, as well as the localization of caspase-1 and GSDMD. The results indicated that the caspase-1/GSDMD signalling axis may be involved in the pathological process under SWBL exposure.

Cell culture
The HLE-B3 hLEC line (ATCC, Manassas, VA, USA) was cultured in DMEM containing 10% FBS at 37 °C. Under these conditions, the doubling time of the cells was about 24 hours and the cells passage rate were generally 1:2. The cells were inoculated onto 6-well plates (5 × 10 5 -1 × 10 6 cells) for the experiments.
We used AC-YVAD-CMK as the caspase-1 inhibitor and evaluated its protective effects against shortwavelength blue light exposure. The concentration of AC-YVAD-CMK was decided by the cell counting kit-8 assay (SAB, USA) [25]. Detailed methods are provided in the supplementary materials.
HLE-B3 LECs were divided into 3 groups randomly: A: normal control group, which consisted of HLE-B3 cells cultured in the dark; B: the caspase-1 inhibitor group; and C: the short-wavelength blue light exposure. Groups A, B, and C were subdivided into four groups according to blue light exposure times (8,16,24, and 32 h). In the caspase-1 inhibitor group, hLECs were treated with ACYVADCMK (20 µmol/L).

Short-wavelength blue light exposure
An illuminance intensity of 2500 lux was used to study the potential in uence of SWBL on HLE-B3 hLECs in vitro [26]. Detailed methods are provided in the supplementary materials.

Cell morphology analysis
The number and dynamic morphological changes of HLE-B3 hLECs were observed under an inverted microscope (× 200). HLE-B3 hLECs morphological changes in both groups were photographed and compared after 8, 16, 24 and 32 h.

Enzyme-linked immunosorbent assay
The protein expressions of caspase-1, -4, and GSDMD in the three groups were decided by using individual ELISA kits. Detailed methods are provided in the supplementary materials.

Flow cytometry
To distinguish pyroptosis from apoptosis in HLE-B3 hLECs, a cell viability assay was performed by ow cytometry [28] The assay was repeated 3 times. Detailed methods are provided in the supplementary materials.

Immuno uorescence localization
Immunohistochemistry was performed to measure the expression levels of caspase-1 and GSDMD. The uorescence images were further processed using Image-Pro plus 6.0. Six random sections per smear at a magni cation of × 400 over a microscopic eld were observed within 1 h [29]. Detailed methods are provided in the supplementary materials.

Western blot analysis
Western blot was applied to determine protein expression levels that were previously described by Mei, et al [30]. Band intensities were analysed with ImageJ software (NIH, Bethesda, MD, USA). Detailed methods are provided in the supplementary materials.
Statistical analyses SPSS 23.0 statistical software (SPSS Inc., USA) was used to perform statistical analysis. The results are presented as the mean ± SD. Data were analysed by two-way ANOVA. A value of p < 0.05 was considered statistically signi cant.

Results
Short-wavelength blue light can cause cell death, but ACYVADCMK suppresses HLE-B3 hLECs death The number and dynamic morphological changes in HLE-B3 hLECs were observed. After 8 h of SWBL exposure, the number of cells in group B was signi cantly decreased, and there were no prominent morphological changes. However, the number of HLE-B3 hLECs in group C decreased by a moderate amount, and the cells were slightly swollen. After 24 h of exposure, the number of hLECs decreased signi cantly, and the cells were obviously swollen in group C. After 32 h of exposure, the number of hLECs in groups B and C was signi cantly decreased. Furthermore, the HLE-B3 hLECs in group B were swollen, and the LECs in group C were severely swollen and morphologically deformed (Fig. 1).
Double-positive staining for activated and inhibited caspase-1 using ow cytometry was selected to assess pyroptotic programmed cell death. As shown in Fig. 2, in the B group, the proportions of doublepositive hLECs were increased at 8, 16, 24, and 32 h compared with those in the control group. Although there was no signi cant difference at 16 h compared to 8 h, the proportion of double-positive hLECs was increased at a does-dependent manner. In the short-wavelength blue light group, the proportion of doublepositive hLECs was also signi cantly increased at 8, 16, 24, and 32 h in a does-dependent manner (p < 0.05). Moreover, the proportions of double-positive hLECs were increased at 8, 16, 24, and 32 h in the short-wavelength blue light exposure subgroups compared with the same treatment times in the ACYVADCMK subgroups (p < 0.05). Our ow cytometry results also con rmed that SWBL exposure motivated pyroptotic death in HLE-B3 hLECs at a does-dependent manner and that ACYVADCMK suppressed cell death.

Short-wavelength blue light initiates caspase-1-induced pyroptosis of hLECs in culture
Enzyme-linked immunosorbent assay (ELISA) was performed and revealed that the expression levels of caspase-1 and GSDMD in all LEC groups changed with the short-wavelength blue light exposure time (8,16,24, and 32 h); caspase-1 and GSDMD expression levels were signi cantly increased in ACYVADCMK groups and SWBL group compared with control group. (Fig. 3a, b). Interestingly, we found that the expression of caspase-4, a member of a cysteine protease family in humans, was also signi cantly suppressed when the caspase-1 inhibitor was used (Fig. 3c).
Next, we performed immunohistochemical assays to examine the expression levels of caspase-1 and GSDMD-N. The data showed that SWBL exposure activated the expression of both proteins. The protein expressions of caspase-1 p20 and GSDMD in ACYVADCMK and SWBL group were increased. However, ACYVADCMK signi cantly reversed the increase in expression compared to that of the SWBL group (Fig. 4).
Western blot was performed to examine the in uence of ACYVADCMK on caspase-1 and GSDMD-N protein expression (Fig. 5). The protein expression levels (Fig. 5) of caspase-1 and GSDMD-N in the SWBL exposure group were increased compared to those in the control group (p < 0.05). The inhibitor-treated group exhibited reversal of the increased expression levels of caspase-1 compared to that of the SWBL exposure group. The results suggest that SWBL exposure could activate caspase-1 and GSDMD in cultured HLE-B3 hLECs. However, AC-YVAD-CMK can reduce caspase-1-induced pyroptosis of hLECs in culture and suppressing the progression of cataracts.

Short-wavelength blue light activates GSDMD-N in the HLE-B3 cell membrane via caspase-1
The immuno uorescence results showed that in the control group, both caspase-1 and GSDMD-N proteins were expressed in the cytoplasm rather than in other organelles. Compared to that of the group A, the protein expression levels of caspase-1 in the cytoplasm increased with increasing exposure to SWBL, and the distribution was not uniform, but the caspase-1 protein was not expressed on the cell membrane or in the nucleus. However, the protein expression of GSDMD-N was increased in the cell membrane in both the short-wavelength blue light and caspase-1 inhibitor groups (Fig. 6).

Discussion
To reduce the occurrence of sightlessness and delay the onset of cataracts, it is vital to determine cataract pathogenesis. In recent decades, researches have focused on the pyroptosis signalling pathways in a varity of diseases, which is implicated in this process. Recently, an in vitro study of LECs indicated that the caspase-1/IL-1β signalling pathways may be involved in the pathological process of cataract formation [15]. Data presented in a previous report demonstrated that pyroptosis plays a vital role in cataracts formation under SWBL exposure in vivo. In this study, we further investigated the in uences of caspase-1/GSDMD on HLE-B3 hLECs in culture and the underlying pathogenesis of cataract formation under SWBL exposure. The results of this study showed that SWBL could initiate caspase-1-induced pyroptosis of hLECs. LEC dysfunction may lead to oedema in super cial cortical lens bres, which in turn may lead to mature cataracts [31].
Caspase-1, a representative marker in the pyroptosis signalling pathway, is an in ammatory caspase that is activated in not only immune cells but also mesenchymal and epithelial cells [32]. The most important function of cleaved caspase-1 is to facilitate GSDMD cleavage into an N-terminal effector domain and a C-terminal inhibitory domain. The N-terminal domain can bind to PIPs in the cell membrane and promote the release of intracellular in ammatory substances, such as IL-1β, through pore opening in cell membrane, ultimately leading to pyroptosis [33]. In agreement with the previous reports, this research results con rmed that caspase-1 and GSDMD protein expression levels, determined by ELISA, immuno uorescence and western blot, were increased in HLE-B3 hLECs at a does-dependent manner, as. As expected, AC-YVAD-CMK effectively reversed the increased expression trend of GSDMD compared to that of the SWBL exposure group. Our study identi ed for the rst time that SWBL could induce caspase-1-mediated pyroptosis in HLE-B3 LECs in vitro. This work further suggests that AC-YVAD-CMK may be an effective reagent to suppress the formation of cataracts and defend against LEC damage by suppressing the caspase-1/GSDMD pathways in response to SWBL exposure. Our results demonstrated that the higher expression of human caspase-4, which has the highest homology to murine caspase-11 [22], was also signi cantly suppressed when a caspase-1 inhibitor was used. Theoretically, murine caspase-11 and its human orthologue caspase-4 are activated in noncanonical pyroptosis. Caspase-1 knockout mice lack both caspase-11 and caspase-1 [34,35]. In this study, the caspase-1 inhibitor may also have suppressed the expression of caspase-4 due to the close genomic location of caspase-1 and caspase-4. Further studies are needed to investigate their relative effects in response to SWBL exposure.
Although the increased expression of caspase-1 and GSDMD and the inhibitory activity of caspase-1 indicate that pyroptosis may be involved in the cataract formation under SWBL exposure, pyroptosis and apoptosis are still di cult to distinguish. It has been reported that apoptosis is involved in cataract formation 18 . Multiple studies show that the characteristic morphological changes of various types of cell death are cell rounding and shrinkage in apoptosis, intact cell membrane and vacuolated cytoplasm in autophagy, and the appearance of membrane bubbles or swelling in pyroptosis [36,37].
The number and types of morphological changes in HLE-B3 hLECs were observed under an inverted microscope in our study. During the 32-hour observation period, there were no signi cant changes in the number and status of cells in the control group. However, after 32 h of exposure, the numbers of LECs in the AC-YVAD-CMK group and SWBL exposure group were signi cantly decreased, and the LECs were swollen in the AC-YVAD-CMK group and severely swollen and deformed morphologically in the blue light exposure group. Flow cytometry, a strength of this work, was used to assess pyroptotic programme cell death based on double-positive staining for activated and inhibited caspase-1. Flow cytometry were selected to analyse the ratio of viable cells and the degree of pyroptosis of HLE-B3 cells after exposure to SWBL for different times. Our results suggested that SWBL exposure causes pyroptotic death in HLE-B3 hLECs in a does-dependent manner.
Some potential limitations of the present study need to be addressed, however. First, the conclusion has some limitations because of the relatively lower knockout e ciency of caspase-1 by AC-YVAD-CMK and further studies are required. Second, although the morphological changes, double-positive staining to indicate pyroptotic cell death, protein levels, and localization of caspase-1 and GSDMD were elucidated in our study, these results, which do not include mRNA data, seem to be insu cient to reveal the exact molecular mechanisms. Third, direct evidence of plasma membrane rupture when pyroptosis occurs was not examined. Additional studies, such as transmission electron microscopy observations of the cell membrane, are needed.

Conclusion
Our study showed that short-wavelength blue light induces pyroptotic programmed cell death by activation of the caspase-1/GSDMD signalling axis in HLE-B3 hLECs. These ndings provide evidence and novel insights for new drug candidates for the prevention and treatment of cataracts.

Availability of data and materials
The datasets used during this current study are available from the corresponding author on reasonable request.    The protein expression of Caspase-1 and GSDMD in HLE-B3 hLECs using Western blotting (a) The in uence of the caspase-1 inhibitor on caspase-1 expression was detected by Western blot. (b) The in uence of the caspase-1 inhibitor on GSDMD expression was detected by Western blot. The protein expression levels of caspase-1 and GSDMD in response to short-wavelength blue light exposure were upregulated at 8 h and 24 h compared to those in the control and AC-YVAD-CMK groups (p<0.05), as determined by Western blot analysis. However, the expression of caspase-1 and GSDMD in the caspase-1 inhibitor groups decreased in the presence of AC-YVAD-CMK. *p < 0.05 versus the control group. #p<0.05 versus the caspase-1 inhibitor group. The differences were statistically signi cant.

Figure 6
Cellular immuno uorescence results of Caspase-1 p20 and GSDMD-N expression in HLE-B3 hLECs at different time points DAPI-stained nuclei are blue, FITC-labelled Caspase-1 p20 is green, and TRITClabelled GSDMD-N is red. Confocal uorescence images of the control, caspase-1 inhibitor, and shortwavelength blue light groups at different time points. The uorescence intensity in the control group did not change with time. Both Caspase-1 p20 and GSDMD-N were localized in the cytoplasm. With longer blue light exposure, the expression of Caspase-1 p20 in the cytoplasm gradually increased and localized in a nonspeci c position in the cytoplasm. However, the expression of GSDMD-N in the cytoplasm gradually increased, and the localization gradually transferred from the cytoplasm to the cell membrane, but neither protein was expressed in the nucleus.