Nanoeugenol Prevents Apoptotic Damage in Selenite-induced Cataracts in Cultured Lenses From Wistar Rats and in Human Lenticular Epithelial Cell Lines: Molecular Validation

Senile cataract is the most common cause of severe visual impairment and blindness. Lenticular epithelial cells apoptosis induced by oxidative stress is a major factor in senile cataract pathogenesis, but there are still many blind nodes in this progress. The aim of the present experiment was to investigate the possibility that nanoeugenol prevents selenite-induced cataractogenesis by regulating the reactive oxygen species (ROS) generation, expressions of apoptotic genes and corresponding proteins. Nanoeugenol found to be inhibited oxidative stress-induced downregulation cytochrome c oxidase subunit I (COX-1), B-cell lymphoma 2 (Bcl-2) genes and upregulation of early growth response protein- 1 (EGR-1), Bcl-2-associated X (Bax), caspase-3, caspase-8 and caspase-9 genes in cultured lenses from Wistar rats. Nanoeugenol signicantly reduced oxidative stress-induced cell apoptosis and generation of ROS in human lenticular epithelial cells (HLE-B3 cells). These ndings suggested that, nanoeugenol can regulate cataract progression by inuencing cell vitality and apoptosis, which could provide new ideas for the clinical treatment of senile cataract.


Introduction
Free radical-induced oxidative stress is postulated to be, perhaps, the key factor leading to senile cataract formation [1]. Oxidative stress-induced apoptosis in lenticular epithelial cells plays a key role in cataract formation, and its prevention is of therapeutic [2]. Apoptosis, or programmed cell death, is the most common form of physiological cell death; it is characterized by the presence of DNA condensation in the nuclei, DNA fragmentation at the nucleosome linkage regions, and cell shrinkage, and ultimately results in the formation of apoptotic bodies [3]. Apoptosis is triggered by a number of physiological and pathological stimuli, including external signals such as hormonal stimulation [4], withdrawal of growth factors [5], viral infection [6], and oxidative stress [7].
In oxidative stress-mediated apoptosis, there is disruption of mitochondrial membranes, release of cytochrome C (Cyt-c) from the mitochondria and subsequent activation of the caspase-mediated apoptotic pathway [8]. Two representative proteins of the B-cell lymphoma (Bcl) family that restrain or promote apoptosis, namely B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax), are thought to play key roles in regulating the effect of mitochondrial membrane permeability, mitochondrial function and Cyt-c release [9]. Normal and selenite cataract lenses were found to vary in the expression of 91 different [10], with the most noticeable variations being noted in the cytochrome c oxidase subunit I (COX-I) (expression of this gene was found to be decreased in selenite cataractous lenses) and in the early growth response protein-1 (EGR-1) gene (expression of this gene was found to be increased in selenite cataractous lenses) [11]. Interestingly, EGR-1 and COX-I are involved in lenticular apoptosis [11]. Caspases are crucial mediators of apoptosis. Among them, caspase-3 is a frequently activated death protease, which catalyzes the speci c cleavage of many key cellular proteins [12]. Caspase-3 is activated by caspase-9 and both are involved in the mitochondria-dependent pathway. Caspase-8 lies at the apex of an apoptotic cascade and initiates proteolytic activation of downstream caspase family members, resulting in apoptosis [13,14]. Lenticular epithelial cell apoptosis has been documented to play a signi cant role in age-related (senile) cataracts and chemical-induced cataracts under experimental conditions [15]. Previous studies have also shown that lenticular epithelial cell apoptosis can disrupt normal lenticular homeostasis and transparency of lenticular ber cells, thereby triggering cataractogenesis [2,16,17].
In recent years, nanoparticles have emerged as potentially useful modalities for administration of important medicinal compounds to the eye. This is because nanoparticles, compared to conventional drugs, possess several key advantages, including a large surface area, good tissue penetration and improved bioavailability, with enhanced aqueous solubility and targeted drug-delivery to a speci c location in the eye [18]. Our earlier study suggest that nanoeugenol is more effective than plain eugenol in preventing oxidative stress-induced cataractogenesis in an in-vitro experimental model (communicated). In the present study, a description is provided of a study to assess the potential of nanoeugenol to modulate changes in the expression of apoptotic-cascade components, and to regulate lenticular apoptosis in an in-vitro model, and to modulate apoptosis in human lenticular epithelial-B3 (HLE-B3) cells.

In vitro phase of the study
The animal experiments in the present study were conducted to comply with guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) and protocols approved by the Association for Research in Vision and Ophthalmology (ARVO); the study protocols were approved by the Institutional Animal Ethical Committee (IAEC; Approval No. BDU/IAEC/P23/2018/24/07.08.2018). Wistar rats (Rattus norvegicus; 75-90 g in weight) were anesthetized with diethyl ether and then sacri ced by cervical dislocation. The lens of each eye was dissected out carefully and then immersed in 3 ml of Dulbecco's modi ed Eagle's medium (DMEM) (supplemented with fetal calf serum [10%] and sodium bicarbonate [0.9 g/L]) in a 12-well Falcon plastic culture plate. Streptomycin (60 µg/ml) and penicillin (60 µg/ml) were also added to avoid microbial contamination. After incubation for 2 h, opaque lenses were removed from the set-up; only lenses manifesting complete transparency were chosen for subsequent experimental studies. The selected lenses were placed in DMEM and were assigned to four groups: 1. Group I (n = 8) lenses were cultured in DMEM alone (normal control); 2. Group II (n = 8) lenses were cultured in DMEM with sodium selenite only (100 µM selenite/ml DMEM) (selenite only); 3. Group III (n = 8) lenses were cultured in DMEM with sodium selenite (100 µM selenite/ml DMEM) and plain eugenol (250 µM/ml DMEM) (eugenol-treated); 4. Group IV (n = 8) lenses were cultured in DMEM with sodium selenite (100 µM selenite/ml DMEM) and eugenol-loaded chitosan nanoparticles (nanoeugenol) (150 µM/ml DMEM) (nanoeugenol-treated).
These lenses were cultured for 24 h at 37°C in cell culture test plates placed in an incubator with 5% CO 2 .
At the end of 24 h, all lenses were subjected to gross morphological examination, and then processed for DNA fragmentation assay and other molecular investigations.

DNA fragmentation assay
DNA was extracted from entire lenses in each group. The fragmentation assay was performed as described by [19], with minor modi cations. Brie y, the lenses were homogenized in 500 µl extraction RT-PCR was performed using a one-step RT-PCR kit (Qiagen, Venlo, Netherlands), as per the manufacturer's instructions. Brie y, 1 µg of template RNA and 0.6 µM of each of the forward and reverse primers speci c to: the apoptotic cascade genes, namely Bcl-2, Bax, caspase-3, caspase-9 and caspase-8, to related genes such as EGR-1 and COX-1, and also to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, the housekeeping gene; After the completion of the PCR reaction, a 10 µl portion of the PCR product was electrophoresed in a 2% agarose gel. The ethidium bromide-stained gel was photographed with a DS-34 type Polaroid camera and the band was scanned with an imaging densitometer (Bio-Rad, Hercules, CA, USA). The GAPDH gene was used as an internal standard for the RT-PCR reaction. To quantitate the transcript level, the ratio of the study gene product to the GAPDH gene product was calculated. Experiments were performed in replicate.

Immunoblot analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 10% gels using the Tris-glycine buffer system, essentially as described by Laemmli [20]. Immunoblotting for Bcl-2, Bax, caspase-3, caspase-8, caspase-9, EGR-1, COX-I, and β-actin proteins was performed by electro transferring proteins from the SDS-PAGE gel onto a nitrocellulose membrane (0.45 mm pore size, Bio-Rad, Hercules, CA, USA) using a semidry blotting apparatus (Bio-Rad, Hercules, CA, USA). Blotting was done at Images of the cells were captured using 40x objectives of the uorescence microscope.

Assay for detection of apoptotic cell death by acridine orange/ethidium bromide (AO/EB) staining
For measuring apoptotic cell death in the HLE-B3 cells, 5x10 5 cells were seeded on a cover slip in a 6-well plate and allowed to attach overnight. The next day, the cell culture medium (DMEM) was replaced as described earlier for the experimental groups. The cells were then incubated for 24 h, following which the cover slip was removed from the culture plate and stained with 50 µl/ml of AO/EB, and incubated at 37°C with 5% CO 2 for 30 min. The stained cover slip was washed with 1 X PBS to remove extra dye, and then xed on a glass slide. Images of the cells were captured using 40x objectives of the uorescence microscope (Floid Cell Imaging uorescence microscope, Thermo Fisher Scienti c, Waltham, MA, USA).

Statistical analysis
The values are documented as mean ± the standard deviation of multiple readings. The statistical signi cance of differences between groups was calculated by One-Way Analysis of Variance (ANOVA) using Statistical Package for Social Sciences (SPSS) software package for Windows, version 21, IBM Corporation, Armonk, NY, USA. Where one-way ANOVA yielded signi cant results, post-hoc testing was performed for inter-group comparisons using the least signi cant difference test. Values of P < 0.05 were deemed statistically signi cant (highlighted by distinct symbols in gures). Table 1 In-vitro study on Wistar rat lenses cultured for 24 h in Dulbecco's modi ed Eagle's medium: primer sequences and expected product sizes for the genes ampli ed.
S. No. Name of the gene Primer sequence Size of the amplicon

Analysis of DNA fragmentation
To validate the occurrence of apoptosis in Wistar rat lenses, a DNA fragmentation assay was performed by subjecting the DNA sample to agarose gel electrophoresis. DNA fragmentation in lenticular tissue samples of selenite only (Group II) lenses was inferred by the appearance of a number of low-molecular weight bands with a speci c laddering pattern, which is recognized as a typical feature of apoptosis (Fig. 1). However, such a "ladder" pattern was not observed in the DNA samples of lenticular tissue of eugenol-treated (Group III) lenses as well as of nanoeugenol-treated (Group IV) lenses. Interestingly, the intact DNA was more prominent in nanoeugenol-treated lenses than in eugenol treated lenses (Fig. 1).

Molecular investigations
3.2.1. Inferred effects of nanoeugenol on mRNA transcript levels of the apoptotic-cascade component genes and related genes The mean levels of mRNA transcripts of the EGR-1, Bax, caspase-3, caspase-8 and caspase-9 genes in selenite only (Group II) lenses were signi cantly (P < 0.05) higher than those noted in normal control (Group I) lenses, in plain eugenol (Group III)-treated lenses and in nanoeugenol (Group IV)-treated lenses (Figs. 2 and 3). Interestingly, the mean mRNA transcript levels of these genes in the nanoeugenol-treated lenses approached the mean values noted in normal control lenses (Figs. 2 and 3). The mean mRNA transcript levels of the COX-1 and Bcl-2 genes were signi cantly (p < 0.05) lower in Group II lenses than those observed in Group I, Group III and Group IV lenses (Fig. 2). Treatment with nanoeugenol appeared to be more effective than treatment with plain eugenol in maintaining the mean mRNA transcript levels of the COX-1 and Bcl-2 genes at near control (normal) levels (Fig. 2).

Effects of nanoeugenol on the levels of apoptoticcascade proteins
To corroborate the data (provided above) obtained from RT-PCR analysis of the apoptotic-cascade component genes and related genes, immunoblotting was performed to detect and quantitate the level of expression of the corresponding proteins. Immunoblot analysis revealed signi cantly (P < 0.05) higher mean band intensities of the EGR-I and Bax (Fig. 4) proteins in selenite only (Group II) lenses than those in normal control (Group I) lenses, eugenol-treated (Group III) and nanoeugenol-treated (Group IV) lenses (Fig. 4). Conversely, lower mean band intensities of the COX-1 and Bcl-2 proteins were observed in Group II lenses than those in Group I, Group III and Group IV lenses (Fig. 4). The mean levels of these proteins in Group III and in Group IV lenses approached the mean levels noted in the control (Group I) lenses (Fig. 4).
Possible proteolytic processing of procaspase-3 was evaluated by Western blotting using a monoclonal antibody to caspase-3 (Fig. 5). This revealed a band at 32 kDa, which was interpreted as being uncleaved caspase-3; this was most intense in samples from control (Group I) lenses (Fig. 5). In selenite only (Group II) lenses, an additional band was present at 17 kDa, which was possibly a fragment of cleaved caspase-3. However, in protein samples from eugenol-treated (Group III) lenses, such fragments were drastically reduced, while in samples from nanoeugenol-treated (Group IV) lenses, such fragments were not observed at all (Fig. 5). Interestingly, the band at 32 kDa was more intense in Group IV lenses than that noted in Group III lenses.
Putative changes in the basal pro le of effector caspase proteins were sought in the cultured lenses by Western blotting analysis. In selenite only (Group II) lenses, there appeared to be activation of caspase-8, since there was a reduction in the intensity of what was inferred to be a 55 kDa band, with the simultaneous appearance of a 42 kDa fraction, which was inferred to have been cleaved from the 55 kDa product (Fig. 5). However, in protein samples from eugenol-treated (Group III) and from nanoeugenol-treated (Group IV) lenses, such activation appeared to have been prevented, which was inferred from the absence of the 42 kDa fragment. Interestingly, the basal pro le of effector caspase proteins in Group IV lenses approximated that seen in cultured normal (Group I) lenses (Fig. 5).
To understand the possible involvement of the mitochondrial apoptotic pathway, the mean level of caspase-9 protein was also determined, since it has been reported that procaspase-9 (45 kDa), upon activation, cleaves into a fragment of approximately 35 kDa. Such cleavage appeared to occur in samples from the selenite only (Group II) lenses (Fig. 5). However, in eugenol-treated (Group III) lenses and in nanoeugenol-treated (Group IV) lenses, there did not appear to be any such activation of procaspase-9 since there was no cleaved fragment of 35 kDa (Fig. 5).

Effect of nanoeugenol on ROS generation in HLE-B3 cells
The intensity of ROS generation induced by sodium selenite was morphologically examined using DCFH-DA staining. Intense green uorescence, a sign of ROS generation, was noted in Group II (selenite only) cells; only minimal green uorescence was noted in Group I (normal control) cells (Fig. 6). Interestingly, in the cells that had been challenged with selenite and treated with nanoeugenol (Group III), minimal intensity of green uorescence was observed (Fig. 6), suggesting that ROS generation was prevented in these cells.

Effect of nanoeugenol on prevention of apoptotic cell death in HLE-B3 cells stained with AO/EB
To determine the apoptosis-associated changes of cell membranes in HLE-B3 cells, the AO/EB uorescence staining method was adopted. Selenite only (Group II) cells stained by AO/EB exhibited morphological characteristics of non-viable cells, with cell shrinkage, impaction of nuclei, chromatin condensation and nuclear fragmentation all being noted; normal control (Group I) and nanoeugenoltreated (Group III) cells did not exhibit such characteristics (Fig. 7). In Group II cells, nuclei which had undergone condensation of chromatin uoresced uniformly as bright-red or orange. Interestingly, the selenite-challenged cells that had been treated with nanoeugenol did not exhibit such uorescence; this suggests that treatment with nanoeugenol prevented the condensation of chromatin noted in Group II cells and permitted maintenance of morphology of the cells at near-normal levels (similar to control cells that uoresced green [ Fig. 7]).

Discussion
Apoptotic cell death, an essential and natural process that occurs in all tissues under both physiological and pathological conditions, mainly acts to eliminate cellular wastes [22]. However, under certain conditions, apoptosis may actually kill cells, resulting in various pathological conditions, including cataract. The results of a previous study [23] suggested that normal lenticular epithelial cells exhibited less apoptosis than did cataractous cells. Thus, lenticular epithelial cell apoptosis may be a common cellular basis for the initiation of non-congenital cataract formation. Apoptosis, the end-result of cataractogenesis, is also believed to arise due to stimuli such as calcium in ux, oxidative stress, hypoxia, heat, and ionizing radiation [24]. Therefore, prevention of cataractogenesis by medical means seeks to inhibit oxidative stress and apoptosis in lenticular epithelial cells.
In the present study, a DNA fragmentation assay of genomic DNA from selenite only (Group II) lenses revealed the characteristic "laddering" pattern that has been reported to occur in apoptotic cell death. A similar "laddering" pattern has also been noted in DNA samples of lenticular tissue from patients with anterior polar cataract [25]. This "laddering" is believed to be due to internucleosomal Ca 2+ -Mg 2+ dependent endonuclease-mediated cleavage [26]. It has previously been reported that calcimycin initiates epithelial cell apoptosis in lens organ culture [23]. In the current study, such "laddering" was drastically reduced in eugenol-treated lenses and not seen at all in nanoeugenol-treated lenses. Moreover, genomic DNA appeared to be intact in the nanoeugenol-treated lenses, as noted in control lenses (Fig. 1). This result is consistent with earlier observations wherein doxorubicin-loaded methyl ether-poly ethylene glycol nanoparticles prevented DNA damage more e ciently than did the free drug in preventing posterior capsular opaci cation in New Zealand white rabbits [27].
Elevated intracellular calcium results in activation of calpain in selenite cataractous lenses. It has been documented that chrysin modulates these effects and therein retards experimental selenite cataractogenesis [28]. Hence, in the present study, an attempt was made to investigate whether this effect of calcium extends up to the activation of the apoptotic pathway, and whether nanoeugenol modulates or blocks the caspase cascade, therein retarding selenite-induced cataractogenesis. This hypothesis was tested by assessing the expression of ve essential genes involved in the apoptotic pathway, namely the EGR-1, COX-1, caspase-3, caspase-8 and caspase-9 genes. Freyssenet et al. [29] demonstrated a direct correlation between increased intracellular calcium and upregulation of the gene encoding EGR-1. A similar association appears to have occurred in the present investigation in that selenite only lenses exhibited elevated mean calcium levels (data not shown, communicated) as well as increased expression of the EGR-1 gene (Fig. 2) and the EGR-1 protein (Fig. 4). Nakajima et al. [30] were of the opinion that the loss of epithelial barrier function contributes to an increase in the intracellular calcium pool, therein leading to increased expression of EGR-1. However, in the present investigation, treatment of selenitechallenged lenses with eugenol (Group III) or nanoeugenol (Group IV) appeared to maintain the lenticular mRNA transcript level of EGR-1, and the lenticular concentration of the EGR-1 protein itself, at near normal levels (Figs. 2 and 4). A similar pattern of EGR-1 expression has also been reported following treatment with other antioxidants, such as curcumin in endothelial cells and broblasts [31] and acetyl-L-carnitine in selenite-induced cataractous lenses of Wistar rats [32].
Dysfunction of the COX-1 enzyme leads to compromised mitochondrial membrane potential and a decreased ATP level [33]. In the current investigation, selenite only (Group II) lenses have shown signi cantly lower mean levels of COX-I mRNA transcripts (Fig. 2) and COX-I protein than those in normal control lenses (Fig. 4). Decreased expression of COX-I has also been noted in selenite-cataractous lenses [11,32] and in UPL rats [34]. Yang et al. [35] have suggested that down-regulation of COX-1 might result in decreased synthesis of ATP. However, in the present study, such a decline in COX-1 expression, both at the transcriptional and translational levels, appears to have been prevented in eugenol-treated and nanoeugenol-treated lenses, suggesting a protective role for these compounds. Similarly, chrysin was found to maintain mRNA transcript levels of COX-1 at near normal levels in lenses challenged with selenite [28].
In apoptosis, there is direct damage to the mitochondria by ROS or indirect mitochondrial depolarization by proapoptotic Bcl-2 family proteins [2]. Bax and Bcl-2 are two important pro-and anti-apoptotic, respectively, proteins [36]. Over-expressed Bax counters the death repressor activity of Bcl-2 and accelerates apoptotic death induced by cytokine deprivation [37]. Bcl-2 is a membrane-bound protein which strongly inhibits apoptosis [38]. Bcl-2 functions as an antiapoptotic protein by forming homo-and heterodimerization with other members of the Bcl-2 family of proteins [39]. In the present study, a signi cantly (P < 0.05) lower mean mRNA transcript level of Bcl-2 and a higher mean mRNA transcript level of Bax was observed in selenite only (Group II) lenses than the corresponding values in normal control (Group I) lenses (Fig. 2). These expression patterns of Bcl-2 and Bax genes in samples from cataractous lenses are similar to those noted in samples from human anterior polar cataract; it was suggested that the lower Bcl-2 and higher Bax mRNA transcript levels represent an "active" means of cell death in lenticular epithelial cells of anterior polar cataract [25]. In the present study, treatment of selenitechallenged lenses with either plain eugenol or with nanoeugenol appeared to have prevented such alterations in mean mRNA transcript levels of Bax and Bcl-2; however, this effect was apparent to a greater extent in the nanoeugenol-treated lenses (Fig. 2), suggesting that the regulatory in uence of eugenol as a nanoform is superior to that of plain eugenol. The immunoblot results of Bcl-2 and Bax protein levels in the cultured lenses (Fig. 4) appeared to mirror the RT-PCR results (Fig. 2). Similarly, Pinus densi ora bark extract was found to maintain mRNA transcript levels of Bcl-2 and Bax at near normal levels in lenses challenged with selenite [40].
Caspases are crucial mediators of apoptosis; they transduce the apoptotic signal cascade and engage cellular targets, leading to programmed cell death [41]. Following activation, both caspase-9 and caspase-8 activate procaspase-3, therein forming active caspase-3, an "executioner caspase", which is reported to play a vital role in regulating and executing apoptosis in mammalian cells [42]. The activation of caspase-3 has been shown to be an essential step in multiple apoptotic signaling pathways triggered by different apoptotic signals [43]. In the present study, the mean mRNA transcript levels of the caspase-3, caspase-8, and caspase-9 genes in selenite only (Group II) lenses were found to be signi cantly higher than those in the normal control (Group I), plain eugenol-treated (Group III) and nanoeugenol-treated (Group IV) lenses (Fig. 3). In order to con rm the above nding at the translational level by detection of the proteins, speci c antibodies were used. In samples of selenite only lenses, the band intensity of caspase-3 was higher than that seen in samples of cultured normal control lenses; a proteolytic fragment of active caspase-3 (17 kDa) was also noted. However, in samples of Group III and Group IV lenses, the band intensity of caspase-3 protein was similar to that noted in normal control lenses; moreover, the proteolytic fragment (active form) was present in traces (Group III) or not at all (Group IV) (Fig. 5). This suggests that activation of caspase-3 was prevented in Group III and Group IV lenses, possibly due to the antiapoptotic potential of plain eugenol and nanoeugenol (Fig. 5). The results of the present study are similar to those obtained in studies on mRNA transcript and protein levels of caspase 3 in selenitechallenged rat pups that had been treated with a Pinus densi ora bark extract, to prevent selenite-induced oxidative stress and apoptosis [40].
Caspase-8, one of the initiator caspases responsible for the activation of the effector caspases (caspase-3, -6 and − 7), plays a pivotal role in the extrinsic apoptotic signaling pathway via death receptors [44]. In the present study, caspase-8 was found to be activated in selenite only lenses, as suggested by the presence of a cleaved fragment of 42 kDa (Fig. 5). However, in plain eugenol-treated (Group III) and in nanoeugenol-treated (Group IV) lenses, the activation of caspase-8 appeared to have been prevented, as there was absence of these 42 kDa fragments (Fig. 5). Procaspase-9 (approximately 45 kDa), upon activation, is reported to cleave into fragments of approximately 35 kDa [45,46]. In the present set of experiments, a similar activation of procaspase-9 was also noted in samples from selenite only lenses which showed cleaved fragments of approximately 35 kDa (Fig. 5). However, nanoeugenol treatment (in Group IV lenses) appeared to prevent such activation, and hence it appeared as intact procaspase-9 ( Fig. 5). The results obtained in the current investigation are similar to those of an earlier study [47].
Thus, the results of molecular investigation of apoptotic-related genes in the current study suggest that nanoeugenol protects against apoptotic cell death in lenticular cells of selenite-challenged lenses by reducing or blocking the activation of the apoptotic cascade, thereby preventing caspase-mediated cell death. Numerous antioxidants have been reported to possess anti-apoptotic properties in various animal models; these include quercetin [48], beta-carotene [49], acetyl-L-carnitine [32], melatonin [50] and extract of leaves of Nerium oleander [51].
Death of lenticular epithelial cells interrupts the lifelong growth of the human lens, therein contributing to the thinness of cataractous lenses [10,23] and to the lower density of epithelial cells in cataractous lenses [52,53]. Excessive ROS production in oxidative stress is signi cantly implicated in mitochondrial damage and cell death [54,55]. Substantial evidence suggests that exposure to selenite in experimental animal models of cataract leads to increased generation of ROS [40,56]. So also, in the present investigation, ROS generation appeared to be signi cantly higher in selenite only (Group II) cells than that in control (Group I) and that in nanoeugenol-treated (Group III) cells, as assessed by DCFH-DA staining (Fig. 6). These results suggest that due to the elevation of ROS levels, oxidative stress occurs in HLE-B3 cells exposed to selenite. Interestingly, simultaneous nanoeugenol treatment of such selenite-challenged cells (Group III) appeared to effectively prevent excessive ROS production, and ROS levels were maintained at near-normal (Fig. 6). So also, Zhou et al. [57] reported, that excessive ROS generation induced by H 2 O 2 in HLE-B3 cells was prevented by rutin, an antioxidant.
Apoptosis of lenticular epithelial cells appears to be a common cellular basis for the initiation and progression of non-congenital cataracts in humans and animals [58]. There appears to be a close relationship between apoptosis of lenticular epithelial cells and cataract formation since death of lenticular epithelial cells due to stress leads to oxidation, hydration and, ultimately, cataract formation [59]. Apoptotic cells are characterized by a series of morphological events, including shrinkage in the size of the cells and the nucleus, loss of adhesion to adjacent cells, membrane blebbing, chromatin condensation, and DNA fragmentation [60]. It has been reported that following AO/EB uorescence staining, the cells that stain green represent viable cells with a highly-organized structure whereas the cells that stain orange/red represent cells in late apoptosis, with condensed or fragmented chromatin [61]. In the present investigation, among the experimental groups of cells that underwent AO/EB staining, the normal (Group I) cells stained green, suggesting viable cells with a highly organized cellular structure (Fig. 7). However, selenite only (Group II) cells revealed intense orange/red uorescence staining, suggesting that the cells were in the late apoptotic stage with condensed or fragmented chromatin (Fig. 7). Interestingly, the cells that were selenite-challenged and simultaneously treated with nanoeugenol (Group III) exhibited green uorescence staining with only very few orange/red spots (Fig. 7). This observation clearly suggests that treatment of selenite-challenged cells with nanoeugenol prevented, or greatly minimized, apoptosis of the cells. Thus, nanoeugenol possibly had a protective effect on the selenite-challenged HLE-B3 cells by inhibiting selenite-induced cell apoptosis.

Conclusion
In conclusion, the results of the present investigation suggest that nanoeugenol, and, to a lesser extent, plain eugenol, confer protection against apoptotic cell death in cultured selenite-challenged, Wistar rat lenses by regulating expression of EGR-1, COX-1, Bcl-2 and Bax, both at the transcriptional and translational levels, and by blocking the activation of the caspase cascade in both the 'extrinsic' and the 'intrinsic' pathways, thereby preventing caspase-mediated DNA damage and cell death. The results also suggest that nanoeugenol confers protection against selenite-induced oxidative damage and apoptosis that would otherwise lead to cataractogenesis in HLE-B3 cells. The observations of the present study strongly suggest that nanoeugenol possesses greater antiapoptotic potential than that of plain eugenol by virtue of its action on the apoptotic-cascade components involved in selenite-induced cataractogenesis.

Declarations
Compliance with ethical standards

Con ict of interest
The authors report no potential con ict of interest relevant to this article.

Funding
No nancial support was received for this submission.