Radioprotective Effect of Walnut Oligopeptides Against Gamma Radiation-Induced Splenocyte Apoptosis and Intestinal Injury in Mice

Abstract

Walnut oligopeptides (WOPs) intake is associated with the augment of the antioxidant defense system and immune system. The chief object of this study is to evaluate the radioprotective effect of walnut oligopeptides extracted from walnut seed protein against ⁶⁰Coγ-irradiation induced damage in mice. Female BALB/c mice were administered WOPs through drinking water for 14 days until a single dose of whole-body ⁶⁰Coγ-irradiation. The 30-day survival test was carried out in the first group (8 Gy), and the other two groups (3.5 Gy) were sacrificed at 3 days and 14 days post-irradiation. Blood and organ samples of mice in the three groups were collected, the histopathological analysis and immunohistochemistry were conducted. The number of peripheral blood leukocytes, bone marrow DNA content, inflammatory cytokines, antioxidant capacity, and intestinal permeability were measured. We found that the administration of WOPs augmented antioxidant defense system, accelerated hematopoietic recovery and showed the significant trend toward higher survival rate and less weight loss compared with non-administrated control mice. In addition, WOPs administration appeared to be important to limit IR-induced splenocyte apoptosis and inflammatory cascade as well as reduce intestine epithelial barrier dysfunction and promote epithelial integrity. These results suggest that pre and post-treatment of WOPs may help to ameliorate acute damage, which is induced by ionizing radiation in mice and accelerate its recovery.

Keywords: antioxidant; epithelial barrier; immunosuppression; splenocyte apoptosis; walnut oligopeptides.

Figure 1

Effect of WOPs on survival time in mice after whole-body irradiation. Survival functions. 1: vehicle control group; 2: IR control group; 3: IR+whey protein group; 4: IR + WOPs 0.22 g/kg BW group; 5: IR + WOPs 0.44 g/kg BW group. 6: IR + WOPs 0.88 g/kg BW group.

Figure 2

Effect of WOPs on body weight and immune organ indices in mice after whole-body irradiation. (A) Body weight 3d (g); (B) Body weight 3d (g); (C) Liver index (mg/g) = liver weight (mg)/ body weight (g); (D) Thymus index (mg/g) = thymus weight (mg)/ body weight (g); (E) Spleen index (mg/g) = spleen weight (mg)/ body weight (g). Values represented the mean ± S.D. (n = 10 per group), which were analyzed by ANOVA test followed by least significant difference for post hoc test between multiple groups. * p < 0.05 versus vehicle control group, # p < 0.05 versus IR control group, and Δp < 0.05 versus IR + whey protein group.

Figure 3

Effect of WOPs on white blood cell count and bone marrow hematopoietic system damage. (A) Number of WBC (10⁹/mL); (B) dsDNA (ng/μl). Values represented the mean ± S.D. (n = 10 per group), which were analyzed by ANOVA test followed by least significant difference for post hoc test between multiple groups. * p < 0.05 versus vehicle control group, # p < 0.05 versus IR control group, and Δp < 0.05 versus IR + whey protein group.

Figure 4

Effect of WOPs on irradiation-induced depletion of endogenous antioxidant defense systems. (A) Liver SOD (U/L); (B) Liver GSH-Px (U/L); (C) Liver MDA (mmol/L); (D) Serum SOD (U/L); (E) Serum GSH-Px (U/L); and (F) Serum MDA (mmol/L). Values represented the mean ± S.D. (n = 10 per group), which were analyzed by ANOVA test followed by least significant difference for post hoc test between multiple groups. * p < 0.05 versus vehicle control group, # p < 0.05 versus IR control group, and Δp < 0.05 versus IR + whey protein group.

Figure 5

Effect of WOPs on pro-inflammatory cytokine levels in serum. (A) Serum IL-6 level; (B) Serum TNF-αlevel. Values represented the mean ± S.D. (n = 10 per group), which were analyzed by ANOVA test followed by least significant difference for post hoc test between multiple groups. * p < 0.05 versus vehicle control group, # p < 0.05 versus IR control group, and Δp < 0.05 versus IR + whey protein group.

Figure 6

Effect of WOPs on intestinal morphology in irradiated mice (200×). Duodenum at 3 days, Jejunum at 3 days and Ileum at 3 days were intestinal morphology in mice at 3 days postirradiation; Duodenum at 14 days, Jejunum at 14 days and Ileum at 14 days were intestinal morphology in mice at 14 days postirradiation.

Figure 7

Effect of WOPs on villus height and crypt depth in irradiated mice. (A) Villus height at 3 days postirradiation; (B) Crypt depth at 3 days postirradiation; (C) Villus height at 3 days postirradiation; (D) Crypt depth at14 days postirradiation. Values represented the mean ± SD (n = 12 per group). * p < 0.05 versus vehicle control group, # p < 0.05 versus IR control, and Δp < 0.05 versus IR+whey protein control group.

Figure 8

Effect of WOPs on intestinal permeability in irradiated mice. (A) Serum D-lactate content; (B) Serum DAO content; (C) Serum LPS content. Values represented the mean ± SD (n = 12 per group). * p < 0.05 versus vehicle control group, # p < 0.05 versus IR control, and Δp < 0.05 versus IR+whey protein control group.

Figure 9

Effect of WOPs on the expression of spleen apoptosis related proteins. (A) The expression of BAX, Bcl-2, IB NF-B and Caspase-3; (B) mean optical density of BAX, Bcl-2, IB NF-B, and Caspase-3. Values represented the mean ± S.D. (n = 5 per group), which were analyzed by ANOVA test followed by least significant difference for post hoc test between multiple groups. * p < 0.05 versus vehicle control group, # p < 0.05 versus IR control, and Δp < 0.05 versus IR+whey protein control group.