Putative free radical-scavenging activity of an extract of Cineraria maritima in preventing selenite-induced cataractogenesis in Wistar rat pups

Abstract

Purpose: To investigate the possible free radical-scavenging activity of an extract of Cineraria maritima on selenite-induced cataractous lenses in Wistar rat pups.

Methods: In the present study, Wistar rat pups were divided into three experimental groups. On P10, Group I (control) rat pups received an intraperitoneal injection of 0.89% saline. Rats in groups II (selenite-challenged, untreated) and III (selenite-challenged, C. maritima treated) received a subcutaneous injection of sodium selenite (19 μmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of the extract of C. maritima (350 mg/kg bodyweight) once daily P9-14. Both eyes of each pup were examined from P16 until P30. Cytochemical localization of nitroblue tetrazolium salts and generation of superoxide, hydroxyl, and nitric oxide levels were measured. The expression of the inducible nitric oxide synthase gene was evaluated with reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of the inducible nitric oxide synthase protein.

Results: Subcutaneous injection of sodium selenite led to severe oxidative damage in the lenticular tissues, shown by increased formation of formazan crystals, elevated generation of superoxide, hydroxyl, and nitric oxide radicals, and elevated inducible nitric oxide synthase gene and protein expression that possibly contributed to the opacification of the lens and thus cataract formation. When rat pups were treated with intraperitoneal administration of the extract of C. maritima, the generation of free radicals as well as the messenger ribonucleic acid and protein expression of inducible nitric oxide synthase were maintained at near normal levels.

Conclusions: The data generated by this study suggest that an ethanolic extract of C. maritima possibly prevents cataractogenesis in a rat model by minimizing free radical generation.

Figure 1

Results of nitroblue tetrazolium (NBT) salt reduction assay in the ocular lens of Wistar rat pups. A: No blue color formation was observed upon incubation with NBT for 45 min in Group I rat lenses. B: Formation of intensely blue-colored formazan deposits, suggestive of superoxide (O₂ −) generation was observed in Group II rat lenses. C: Negligible blue formazan deposition was observed in Group III rat lenses.

Figure 2

Superoxide anion radical generation in lenticular tissues of Wistar rat pups. Group I=Control rat lenses; Group II=Selenite-challenged, untreated rat lenses; Group III=Selenite-challenged, C. maritima extract-treated rat lenses. ^aGroup II versus Group I (p<0.05); ^bGroup III versus Group I (p<0.05).

Figure 3

Hydroxyl radical generation in lenticular tissues of Wistar rat pups. Group I=Control rat lenses; Group II=Selenite-challenged, untreated rat lenses; Group III=Selenite-challenged, C. maritima extract-treated rat lenses. ^aGroup II versus Group I (p<0.05); ^bGroup III versus Group I (p<0.05).

Figure 4

Nitric oxide generation in lenticular tissues of Wistar rat pups. Group I=Control rat lenses; Group II=Selenite-challenged, untreated rat lenses; Group III=Selenite-challenged, C. maritima extract-treated rat lenses. ^aGroup II versus Group I (p<0.05); ^bGroup III versus Group I (p<0.05).

Figure 5

Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of inducible nitric oxide synthase messenger RNA (mRNA) in rat lenses visualized on an ethidium bromide-stained agarose gel alongside messenger ribonucleic acid of glyceraldehydes-3-phosphate dehydrogenase. A: M=100 bp DNA ladder; N=Group I (control); S=Group II (selenite-challenged, cataract-untreated); T=Group III (selenite-challenged, C. maritima extract-treated). B: The results depicted are normalized to levels of glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Data are mean value (experiments run in triplicate) of ratios of intensity for gene of interest divided by that for GAPDH. ^aGroup I versus Group II and III values (p<0.05); ^bGroup II versus Group III values (p<0.05).

Figure 6

Immunoblot analysis of inducible nitric oxide synthase (iNOS) in lysate of whole lens. A: Representative western blot for iNOS (with β-actin as loading control) in lenses of 16-day-old Wistar rat pups. N-Group I (control); S-Group II (selenitechallenged,cataract untreated); T-Group III (selenite-challenged, C.maritima extract-treated). B: Bar graphs of mean normalized densitometry readings. N-Group I (control); S- Group II (selenite-challenged, cataract-untreated); T-Group III (selenite-challenged, C.maritima extract-treated). ^aGroup I vs Group II & III values (p<0.05); ^bGroup II vs Group III values (p<0.05).