Benzoylhypaconitine br Materials and methods br Results br

Materials and methods
Results
Discussion An HMC is a blinding type of cataract frequently observed in Asian countries due to the appallingly high incidence of high myopia in those areas [4], [5] and typically features earlier onset and more rapid progression of nuclear cataract [1], [2]. The Blue Mountains Eye Study, a large population-based prospective study conducted in Australia, confirmed the association between high myopia and increased incidence of nuclear cataract [26]. In normal eyes, an intact vitreous body is responsible for maintaining a low-oxygen environment surrounding the lens. Previous studies have shown that vitrectomy might lead to nuclear sclerotic cataracts due to alterations in lens metabolism [27], [28]. Beebe et al. found that these problems may have resulted from the increased exposure of the lens to the oxygen from the fundus due to the destruction of vitreous gel [7]. Yan et al. removed the vitreous gel of rabbits with vitrectomy and exposed the lens to increased oxygen and found aggravated lens oxidation [29]. The lens may also lose transparency in other conditions related to vitreous destruction, such as high myopia [30], [31]. In highly myopic eyes, with much longer axial lengths and larger eyeballs, vitreous liquefaction occurs much earlier than in normal eyes [32], generating a high-oxygen environment surrounding the lens, thus resulting in earlier onset and higher severity of cataracts than those of ARC. Therefore, the antioxidant capacity within the lens plays an essential role in preventing the development of cataracts. In this study, we measured two classic markers related to oxidative stress, T-AOC and MDA, and found significantly lower T-AOC and higher levels of MDA in the HMC group, suggesting that the antioxidant defense of HMC is much weaker than that of ARC. Thus, we propose that the earlier onset of nuclear cataracts in highly myopic eyes is probably related to the higher oxidative stress and the lower antioxidant capacity in HMC than in ARC. DNA methylation is a classic epigenetic mechanism of gene silencing that participates in various ocular diseases, such as pterygium [33], age-related macular degeneration and retinitis pigmentosa [34]. A high-oxygen environment may induce epigenetic alterations, especially to Benzoylhypaconitine of antioxidant enzymes. In our previous study, we identified that hypermethylation of the CRYAA promoter played an important role in the development of high myopia-induced dark nuclear cataracts [3]. In the present study, we examined the methylation status of six important antioxidant genes as described in the results (Fig. 2), and GSTP1 and TXNRD2 were selected for further study because their promoters were hypermethylated. GSTP1 is widely expressed in the epithelial tissue of the eyes and belongs to a large supergene family with a powerful capacity in detoxification of xenobiotics and in antioxidant defense, especially during exposure to increased oxygen [35], [36]. Glutathione S-transferase (GST) distribution and activity were significantly decreased in cataractous human lens compared with healthy lens [37]. Fan et al. previously reported hypermethylation of the GSTP1 promoter in response to systemic oxidative stress in peripheral mononuclear cells of patients with chronic hepatitis B [38]. Interestingly, Chen et al. reported epigenetic alterations of GSTP1 in age-related nuclear cataracts (ARNCs) and suggested that the methylation levels of two regions of the GSTP1 promoter (− 819 bp to − 533 bp and from − 201 bp to + 86 bp) might be correlated with the severity of ARNCs [15]. In our study, we showed that the methylation level of the GSTP1 promoter was even higher in HMC than in ARC (we can also use ARNCs here, considering that the included ARC samples were all of the nuclear type). Thioredoxin reductase could also catalyze the reduction of numerous oxidized cell constituents, including protein-thiol mixed disulfide. It is crucial to restore thioredoxin, a core antioxidant molecule in the lens, to its reduced state for defense against oxidants. Downregulated TXNRD2, the mitochondrial form of thioredoxin reductase, may weaken the mitochondrial thioredoxin system, resulting in mitochondrial injury to LECs. Our preliminary data showed that in the lens-induced myopia mouse model, the expression of TXNRD2 was also significantly reduced in the lens of the myopic eye, while the GSTP1 level was relatively stable (Supplementary Fig. 9). In addition, the importance of hypermethylated CpG units of GSTP1 and TXNRD2 in transcriptional activity was validated by luciferase reporter assay, as displayed in Fig. 6. However, to confirm the repressive effect of the hypermethylated spots on transcriptional activity, specific methylation of the CpG units 21–25 of GSTP1 and CpG unit 34 of TXNRD2 followed by expression assessment is ideal. Although it is possible to perform such site-specific epigenetic modification using a recently developed DNA editing tool [39], this procedure is beyond our current technical capacity.