Polyphenol-enriched fraction of Vaccinium uliginosum L. protects selenite-induced cataract formation in the lens of Sprague-Dawley rat pups

Abstract

Purpose: As the aging population is increasing, the incidence of age-related cataract is expected to increase globally. The surgical intervention, a treatment for cataract, still has complications and is limited to developed countries. In this study, we investigated whether the polyphenol-enriched fraction of Vaccinium uliginosum L. (FH) prevents cataract formation in Sprague-Dawley (SD) rat pups.

Methods: Sixty rat pups were randomly divided into six groups: CTL, Se, FH40, FH80, FH120, and Cur80. The cataract was induced with subcutaneous injection of sodium selenite (18 μmol/kg bodyweight) on postnatal (P) day 10. All groups, except CTL, were injected with sodium selenite, and the FH40, FH80, and FH120 groups were given gastric intubation with FH40 mg/kg, 80 mg/kg, and 120 mg/kg on P9, P10, and P11. The Cur80 group was also given gastric intubation with curcumin 80 mg/kg on P9, P10, and P11. All rat pups were euthanized on P30.

Results: Lens morphological analysis showed that FH dose-dependently inhibited cataract formation. In the Se group, soluble proteins were insolubilized, and the gene expression of the α-, β-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, β-, and γ-crystallins. In the Se group, the gene and protein levels of m-calpain were downregulated, which were attenuated with FH treatment. In addition, sodium selenite injection caused reduced antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)), glutathione (GSH) depletion, and malondialdehyde (MDA) production in the lens. The administration of FH inhibited sodium selenite-induced oxidative stress in a dose-dependent manner. The mechanism of protection against oxidative stress by FH involves NF-E2-related factor (Nrf-2) and hemoxygenase-1 (HO-1). FH treatment inhibited decrease of Nrf-2 in the nucleus fraction and HO-1 in the cytosol fraction. Finally, the FH treatment protected poly (ADP)-ribose polymerase (PARP) from cleavage, determined with western blotting.

Conclusions: FH showed a preventive effect against cataract formation by inhibiting m-calpain-mediated proteolysis and oxidative stress in the lens. These results suggest that FH could be a potential anticataract agent in age-related cataract.

Figure 1

Effects of FH on cataract formation. Representative anterior images of all groups were taken (A) without or (B) with a white-light lamp on P16. The CTL group shows a clear lens (stage 0); however, the Se group shows severe cataract formation (stage 5). FH treatment dose-dependently inhibited cataract formation. CTL: normal control, Se: selenite-treated, FH40/80/120: selenite/FH-treated (40, 80, and 120 mg/kg, respectively), Cur80: selenite/curcumin-treated (80 mg/kg).

Figure 2

Effects of FH on soluble and insoluble proteins and α-, β-, and γ-crystallins in the lens. (A) Soluble and (B) insoluble proteins of lenses were analyzed in all groups. The results were represented as the mean ± standard deviation (SD) of three independent experiments (n=6). C: The gene expression of the α-, β-, and γ-crystallins were analyzed using real-time polymerase chain reaction, and then normalized to glyceraldehyde 3-phosphated dehydrogenase (GAPDH). The transcript levels were expressed as the mean ± standard deviation (SD) of three independent experiments (n=4). #p<0.05, ##p<0.01, ###p<0.001 statistically significant different from CTL. *p<0.05, **p<0.01, ***p<0.001 statistically significant different from Se. CTL: normal control, Se: selenite-treated, FH40/80/120: selenite/FH-treated (40, 80, 120 mg/kg, respectively), Cur80: selenite/curcumin-treated (80 mg/kg).

Figure 3

Effects of FH on m-calpain in the lens. A: Gene expression of m-calpain was analyzed using real-time polymerase chain reaction, and then normalized to glyceraldehyde 3-phosphated dehydrogenase (GAPDH). The transcript levels were expressed as mean ± standard deviation (SD) of three independent experiments (n=4). B: Protein expression of m-calpain was analyzed using western blotting. The protein levels were quantified with band density. The protein levels were presented as mean ± standard deviation (SD) of three independent experiments (n=4). ###p<0.001 statistically significant different from CTL. *p<0.05, **p<0.01, ***p<0.001 statistically significant different from Se. CTL: normal control, Se: selenite-treated, FH80: selenite/FH-treated (80 mg/kg), Cur80: selenite/curcumin-treated (80 mg/kg).

Figure 4

Effects of FH on GSH, MDA, SOD, and GPx in the lens. We measured the (A) glutathione (GSH), (B) malondialdehyde (MDA), (C) superoxide dismutase (SOD), and (D) glutathione peroxidase (GPx) levels to find antioxidant effects of FH in the lens. Each value represents the mean ± standard deviation (SD) of three independent experiments (n=6). ###p<0.001 statistically significant different from CTL. *p<0.05, **p<0.01, ***p<0.001 statistically significant different from Se. CTL: normal control, Se: selenite-treated, FH40/80/120: selenite/FH-treated (40, 80, 120 mg/kg, respectively), Cur80: selenite/curcumin-treated (80 mg/kg).

Figure 5

FH inhibits the selenite-induced decrease in Nrf-2 and HO-1 in the lens. A: Gene expression levels of Nrf-2 and HO-1 were analyzed with real-time polymerase chain reaction and normalized to glyceraldehyde 3-phosphated dehydrogenase (GAPDH) in the lens. The transcript level represents the mean ± standard deviation (SD) of three independent experiments (n=4). B: The protein level of Nrf-2 was measured in the nuclear fraction of the lens, and the protein level of HO-1 was measured in the cytosol fraction of the lens using western blotting. The protein level represents the mean ± standard deviation (SD) of three independent experiments (n=4). ##p<0.01, ###p<0.001 statistically significant different from CTL. *p<0.05, **p<0.01 statistically significant different from Se. CTL: normal control, Se: selenite-treated, FH80: selenite/FH-treated (80 mg/kg), Cur80: selenite/curcumin-treated (80 mg/kg).

Figure 6

FH inhibits selenite-induced apoptosis in the lens. Poly (ADP)-ribose polymerase (PARP) and a cleaved form of PARP were analyzed using western blotting in the lens. The protein level represents the mean ± standard deviation (SD) of three independent experiments (n=4). ##p<0.01 statistically significant different from CTL. *p<0.05 statistically significant different from Se. CTL: normal control, Se: selenite-treated, FH80: selenite/FH-treated (80 mg/kg), Cur80: selenite/curcumin-treated (80 mg/kg).