Astaxanthin Inhibits Expression of Retinal Oxidative Stress and Inflammatory Mediators in Streptozotocin-Induced Diabetic Rats

Abstract

Purpose: We evaluated whether orally administered astaxanthin (AST) protects against oxidative damage in the ocular tissues of streptozotocin (STZ)-induced diabetic rats.

Methods and results: Fifty 6-week-old female Wistar rats were randomly assigned to receive an injection of STZ to induce diabetes (n = 40) or to remain uninduced (n = 10). The diabetic rats were randomly selected into four groups and they were separately administered normal saline, 0.6 mg/kg AST, 3 mg/kg AST, or 0.5 mg/kg lutein daily for eight weeks. Retinal functions of each group were evaluated by electroretinography. The expression of oxidative stress and inflammatory mediators in the ocular tissues was then assessed by immunohistochemistry, western blot analysis, ELISA, RT-PCR, and electrophoretic mobility shift assay (EMSA). Retinal functions were preserved by AST and lutein in different levels. Ocular tissues from AST- and lutein-treated rats had significantly reduced levels of oxidative stress mediators (8-hydroxy-2'-deoxyguanosine, nitrotyrosine, and acrolein) and inflammatory mediators (intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and fractalkine), increased levels of antioxidant enzymes (heme oxygenase-1 and peroxiredoxin), and reduced activity of the transcription factor nuclear factor-kappaB (NF-κB).

Conclusion: The xanthophyll carotenoids AST and lutein have neuroprotective effects and reduce ocular oxidative stress, and inflammation in the STZ diabetic rat model, which may be mediated by downregulation of NF-κB activity.

Fig 1. Evaluation of functional changes of the retina by electroretinography (ERG).

The ERG were performed on untreated rats (Control) or STZ-induced diabetic rats treated for 8 weeks with normal saline (Diabetes), 3 mg/kg AST (AST High), 0.6 mg/kg AST (AST Low), or 0.5 mg/kg lutein (Lutein). The relative b-wave ratio was significantly decreased in the Diabetes, AST Low and Lutein groups compared with the Control group. The relative b-wave ratio in AST High group had no significant difference from Control group. The ratio in AST High group was significantly higher than that in the AST Low and the Lutein groups (p = 0.046 and p < 0.001). The data are expressed as the mean ± SD in 4 rats for each group (bar graph). *, p < 0.05 compared with the control group. #, p < 0.05 compared with the Diabetes group. Differences among groups were analyzed by one-way analysis of variance followed by Bonferroni’s test for multiple comparisons.

Fig 2. Immunofluorescence staining of oxidative stress mediators in retinas.

Retinal sections were prepared from Control, Diabetes, AST High, AST Low, or Lutein groups. Retinal oxidative damages were evaluated by immunofluorescence staining of (A) 8-hydroxy-2'-deoxyguanosine (8-OHdG) (B) nitrotysine, and (C) acrolein (original magnification 200×). (D) The relative density of immunostaining was defined as immunostaining index of control group. For quantitation of immunostaining, we first determined the immunostaining index, which could be measured and calculated from the following formula: Σ [(immunostaining density-threshold) × area (pixels)] / total cell number. Treatment with AST and lutein decreased the staining for nitrotysine, acrolein and 8-OHdG in the retinas compared with Diabetes group but the staining density of nitrotyrosine and acrolein in lutein group were significantly higher than control group. Data are presented as the mean ± SD. *, p < 0.05 versus the Control group; #, p < 0.05 versus the Diabetes group.

Fig 3. mRNA levels of inflammatory mediators in retinas.

(A) RNA was subjected to RT-PCR using gene-specific primers for ICAM-1, MCP-1, FKN, and β-actin. (B) mRNA expression of ICAM-1, MCP-1, and FKN, normalized to the expression of β-actin. Data are presented as the mean ± SD. *, p < 0.05 versus the Control group; #, p < 0.05 versus the Diabetes group.

Fig 4. Western blot analysis of ICAM-1, MCP-1, and FKN protein expression in retinas.

Retinal cell extracts were prepared from animals treated as described in Fig 1. (A) Blots were probed with antibodies specific for ICAM-1, MCP-1, and FKN. β-actin was probed as a loading control. (B) The relative intensities of the bands in (A) were determined by ImageJ software and normalized to the expression of β-actin. Data are presented as the mean ± SD. *, p < 0.05 versus the Control group; #, p < 0.05 versus the Diabetes group.

Fig 5. IHC staining of inflammatory mediators in retinas.

Retinal sections were prepared from animals by each group. (A) Photomicrographs show immunocytochemical localization of ICAM-1, MCP-1, and FKN (original magnification 400×). (B) Images in (A) were quantified with Image-Pro software. Data are presented as the mean ± SD. *, p < 0.01 versus the Control group; #, p < 0.01 versus the Diabetes group.

Fig 6. Effect of AST and lutein on ICAM-1, MCP-1, and FKN expression in aqueous humors.

Aqueous humor was isolated and pooled from the eyes of rats by each group. ICAM-1, MCP-1, and FKN levels were quantified by 3 repeat ELISA experiments. Data are presented as the mean ± SD. *, p < 0.05 versus the Control group; #, p < 0.05 versus the Diabetes group.

Fig 7. mRNA levels of antioxidant defense enzymes in retinas.

RNA was isolated from the retinas of rats by each group. (A) RNA was subjected to RT-PCR using gene-specific primers for HO-1, PRDX, Trx, and β-actin. (B) mRNA expression of HO-1, PRDX, and Trx, normalized to the expression of β-actin. Data are presented as the mean ± SD. *, p < 0.05 versus the Control group; #, p < 0.05 versus the Diabetes group.

Fig 8. Effect of AST and lutein on NF-κB activity in retinas.

(A) Photomicrographs of IHC staining for NF-κB p65 in retinal sections from animals treated as described in Fig 1. (B) The images in (A) were analyzed with Image-Pro software and staining intensity was quantified. Data are presented as the mean ± SD. *, p < 0.05 versus the Control group; #, p < 0.05 versus the Diabetes group. (C) Nuclear proteins were prepared from the retinas of rats treated as described in Fig 1. EMSA was performed by incubation of extracts with a biotinylated oligonucleotide containing an NF-κB consensus sequence. Lane 1: p50 subunit of NF-κB; lane 2: free probe (FP); lane 3: control rats; lane 4: diabetic rats; lane 5: diabetic rats treated with high dose astaxanthin (3.0mg/kg/day); lane 6: diabetic rats treated with low dose astaxanthin (0.6mg/kg/day); lane 7: diabetic rats treated with lutein (0.5mg/kg/day); lane 8: 100–fold molar excess of unlabeled NF-κB probe, and lane 9: p65, biotinylated probe with anti-p65 antibody.