Grape Seed Proanthocyanidin Extract Alleviates AflatoxinB₁-Induced Immunotoxicity and Oxidative Stress via Modulation of NF-κB and Nrf2 Signaling Pathways in Broilers

Abstract

Aflatoxin B₁ (AFB₁) is a widely spread mycotoxin contaminates food and feed, causing severe oxidative stress damages and immunotoxicity. Grape seed proanthocyanidin (GSPE), a natural antioxidant with wide range of pharmacological and medicinal properties. The goal of the present study was to investigate the protective effects of GSPE against AFB₁-induced immunotoxicity and oxidative stress via NF-κB and Nrf2 signaling pathways in broiler chickens. For the experiment, 240 one-day old Cobb chicks were allocated into four dietary treatment groups of six replicates (10 birds per replicate): 1. Basal diet (control); 2. Basal diet + AFB₁ 1mg/kg contaminated corn (AFB₁); 3. Basal diet + GSPE 250 mg/kg (GSPE); 4. Basal diet + AFB₁ 1 mg/kg + GSPE 250 mg/kg (AFB₁ + GSPE). The results showed that GSPE significantly decreased serum inflammatory cytokines TNF-α, IFN-γ, IL-1β, IL-10, and IL-6 induced by AFB₁. Similarly, GSPE + AFB₁ treated group revealed a significant decrease in mRNA expressions of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) in the splenic tissue compared to the AFB₁ treatment group. In addition, western blotting results manifested that GSPE treatment normalized the phosphorylation of nuclear factor kappa B (p65) and the degradation of IκBα protein induced by AFB₁. Furthermore, GSPE enhanced the antioxidant defense system through activating the nuclear factor-erythroid-2-related factor (Nrf2) signaling pathway. The mRNA and protein expression level of Nrf2 and its down streaming associated genes were noted up-regulated by the addition of GSPE, and down-regulated in the AFB₁ group. Taken together, GSPE alleviates AFB₁-induced immunotoxicity and oxidative damage by inhibiting the NF-κB and activating the Nrf2 signaling pathways in broiler chickens. Conclusively, our results suggest that GSPE could be considered as a potential natural agent for the prevention of AFB₁-induced immunotoxicity and oxidative damage.

Keywords: Aflatoxin B1; Broilers; Grape Seed Proanthocyanidin Extract; Immunotoxicity; NF-κB; Nrf2; oxidative stress.

Figure 1

Protective role of grape seed proanthocyanidin extract (GSPE) treatment on Aflatoxin B₁ (AFB₁)-induced levels of the inflammatory cytokines in the serum of broilers. All data were expressed as mean ± SD (n = 6). Different symbols among groups indicate significant difference by least significant difference test (LSD) test (p < 0.05). * (p < 0.05) significant differences compared to the control group and ^ (p < 0.05) significant differences compared to the AFB₁ group. (A) Tumor necrosis factor alpha (TNF-α); (B) interferon gamma (IFN-γ); (C) interleukin-1 beta (IL-1β); (D) interleukin 10 (IL-10); (E) interleukin 6 (IL-6).

Figure 2

Protective role of GSPE treatment on AFB₁-induced mRNA expression levels of the pro-inflammatory genes in the spleen of broilers. The mRNA expression of TNF-α, IFN-γ, IL-1β, and IL-6 were detected by quantitative real-time PCR. All data were expressed as mean ± SD (n = 6). Different symbols among groups indicate significant difference by LSD test (p < 0.05). * (p < 0.05) significant differences compared to the control group and ^ (p < 0.05) significant differences compared to the AFB₁ group. (A) Tumor necrosis factor alpha (TNF-α); (B) interferon gamma (IFN-γ); (C) interleukin-1 beta (IL-1β); (D) interleukin 6 (IL-6).

Figure 3

Protective role of GSPE treatment on the AFB₁-induced degradation IκBα (A) and phosphorylation of NF-κB (p65) (B). The protein expression of β-actin, p65, pp65, and IκBα protein were detected by western blotting in the spleen of broilers. All data were expressed as mean ± SD. Different symbols among groups indicate significant difference by LSD test (p < 0.05). * (p < 0.05) significant differences compared to the control group and ^ (p < 0.05) significant differences compared to the AFB₁ group.

Figure 4

Protective role of GSPE treatment on AFB₁-induced mRNA expression levels of the Nrf2 signaling pathway in the liver of broilers. The mRNA expression of Nrf2, HO-1, GPx1, NQO1, and GCLC were detected by quantitative real-time PCR. All data were expressed as mean ± SD (n = 6). Different symbols among groups indicate significant difference by LSD test (p < 0.05). * (p < 0.05) significant differences compared to the control group and ^ (p < 0.05) significant differences compared to the AFB₁ group. (A) Nuclear erythroid-2-related factor (Nrf2); (B) heme oxygenase-1 (HO-1); (C) glutathione peroxidase (GPx1); (D) quinone oxidoreductase 1 (NQO1); (E) glutamate-cysteine ligase catalytic subunit (GCLC).

Figure 5

Protective role of GSPE treatment on AFB₁-induced protein expression levels of the Nrf2 signaling pathway in the liver of broilers. The protein expression of β-actin, Nrf2, HO-1, GPx1, NQO1, and GCLC were detected by western blotting. All data were expressed as mean ± SD. Different symbols among groups indicate significant difference by LSD test (p < 0.05). * (p < 0.05) significant differences compared to the control group and ^ (p < 0.05) significant differences compared to the AFB₁ group. (A) Nuclear erythroid-2-related factor (Nrf2); (B) heme oxygenase-1 (HO-1); (C) glutathione peroxidase 1 (GPx1); (D) quinone oxidoreductase 1 (NQO1); (E) glutamate-cysteine ligase catalytic subunit (GCLC).