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 influx, 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 Ca2+-Mg2+ 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 efficiently than did the free drug in preventing posterior capsular opacification 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 five 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 selenite-challenged 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 fibroblasts [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 significantly 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 significantly (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 selenite-challenged 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 influence 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 densiflora 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 significantly 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 confirm the above finding at the translational level by detection of the proteins, specific 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 selenite-challenged rat pups that had been treated with a Pinus densiflora 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 significantly 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 significantly 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 H2O2 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 fluorescence 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 fluorescence 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 fluorescence 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.