Therapeutic Effects And Mechanism Of Grape Seed Proanthocyanidins Extract Against Aflatoxin B₁ Toxicity In Broiler Chickens

Aflatoxin B₁（AFB₁）is one of the most poisonous mycotoxins that are hazardous to human and animal’s health worldwide because of their frequent occurrence in food and/or feedstuffs.AFB₁ is well known for its hepatotoxic,carcinogenic,immunosuppressive,teratogenic,and other devastating effects in mammals and poultry.To further extend,AFB₁ has been classified by the International Agency for Research on Cancer（IARC）as a group-1 carcinogen to humans with no safe dose.Thus,in order to prevent economic losses and safeguard human and animal’s health,there is a need for developing cost-effective methods for reducing the AFB₁ poisoning in poultry and other livestock species.One of the feasible solutions is to look at the possibility of minimizing AFB₁ poisoning through the application of plants herb extracts for better performance and reducing the risk of diseases in poultry birds.Grape seed proanthocyanidins extract（GSPE）is a powerful natural antioxidant as it has the ability to scavenge free oxygen radicals.Meanwhile,GSPE demonstrates a broad range of biological and therapeutic effects,including anti-carcinogenic,anti-inflammatory,anti-apoptotic,anti-mutagenic,and neuroprotective properties.By virtue of these properties,this research was designed to explore the possibility of ameliorating the toxic effects of AFB₁ in broiler chickens through the supplementation of grape seed proanthocyanidins extract.1.Ameliorative effects of GSPE on growth performance,immune function,antioxidant capacity,liver histopathology and aflatoxin residues in broilers exposed to AFB₁The purpose of this research was to investigate the effectiveness of GSPE in the detoxification of AFB₁ in broilers.A total of 300 one-day-old Cobb chicks were randomly allocated into five treatments of six replicates（10 birds per replicate）,fed ad libitum for four weeks with the following dietary treatments:1)basal diet（control）;2)basal diet+1 mg/kg AFB₁ contaminated corn（AFB₁）;3)basal diet+GSPE 250 mg/kg（GSPE 250 mg/kg）;4)basal diet+AFB₁（1 mg/kg）+GSPE 250 mg/kg（AFB₁+GSPE250 mg/kg）;5)basal diet+AFB₁（1mg/kg）+GSPE 500 mg/kg（AFB₁+GSPE 500mg/kg）.Compared with the control group,feeding broilers with AFB₁ alone significantly reduced growth performance,serum immunoglobulin（IgA,IgG,and IgM）contents,negatively altered serum biochemical profiles alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,gamma-glutamyltransferase（GGT）,alkaline phosphatase（ALP）,albumin（ALB）,globulin（GLU）and total protein.Additionally,AFB₁ aggravated histopathological lesions in the liver,liver tissue from AFB₁ fed group had periportal fibrosis,hydropic degeneration/fatty changes,and bile duct hyperplasia when compared with the tissue of birds fed with uncontaminated diet.Moreover,AFB₁ significantly increased malondialdehyde（MDA）content and decreased total superoxide dismutase（T-SOD）,catalase（CAT）,glutathione peroxide（GSH-Px）,glutathione-S transferase（GST）,glutathione reductase（GR）activities,and glutathione（GSH）concentration within the liver and serum.On the other hand,GSPE treatment significantly improved growth performance,serum immunoglobulins contents,biochemical profile,and ameliorated histopathological lesions in the liver induced by AFB₁.Furthermore,supplementation of GSPE（250 and 500 mg/kg）to AFB₁ contaminated diet（P<0.05）decreased MDA content and enhanced the activities of antioxidant enzymes（T-SOD,CAT,GSH-Px,GST,GR,and GSH）.In addition,the inclusion of GSPE（250 and 500 mg/kg）to AFB₁ diet resulted in a significant decrease in AFB₁ residues in the liver by 51%and 42%,respectively,when compared to the AFB₁ fed group.From these results,it can be concluded that dietary supplementation of GSPE has protective effects against aflatoxicosis caused by AFB₁ in broiler chickens.2.GSPE alleviates AFB₁-induced immunotoxicity and oxidative stress via modulation of NF-κB and Nrf2 signaling pathways in broilersBased on the first study,this study was designed to understand the molecular mechanism of GSPE against AFB₁-induced immunotoxicity and oxidative stress via NF-κB and Nrf2 signaling pathways in broiler chickens.All the samples were taken from the first experiment.The result showed that AFB₁ fed group significantly increased serum inflammatory cytokines tumor necrosis factor alpha（TNF-α）,interferon gamma（IFN-γ）,interleukin-1 beta（IL-1β）,interleukin 10（IL-10）,and interleukin 6（IL-6）as compared to the control and experimental groups.Similarly,AFB₁ treated group revealed a significant increase in mRNA expressions of pro-inflammatory cytokines（TNF-α,IFN-γ,IL-1β,and IL-6）in the splenic tissue compared to the control group.Moreover,western blotting results manifested that AFB₁ treatment markedly enhanced the phosphorylation of nuclear factor kappa B（p65）and degradation of IκBαprotein.Furthermore,the mRNA and protein expression level of nuclear factor-erythroid-2-related factor（Nrf2）and it’s down streaming associated genes heme oxygenase-1（HO-1）,glutathione peroxidase 1（GPx1）,quinone oxidoreductase 1（NQO1）,and glutamate-cysteine ligase catalytic（GCLC）was markedly down-regulated in AFB₁-fed group（P<0.05）.Contrastingly,GSPE treatment significantly decreased serum inflammatory cytokines（TNF-α,IFN-γ,IL-1β,IL-10,and IL-6）induced by AFB₁.Likewise,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 fed group.Moreover,GSPE treatment normalized the phosphorylation of nuclear factor kappa B（p65）and the degradation of IκBαprotein induced by AFB₁.Additionally,GSPE supplementation ameliorated AFB₁-induced oxidative damage via enhancing the mRNA and protein expression level of Nrf2,and it’s down streaming associated genes（HO-1,GPx1,and NQO1）.Nevertheless,the numerical difference was found in GCLC protein expression,as compared to the AFB₁group.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.3.Protective effect of GSPE on AFB₁-induced histopathological lesions,oxidative stress,and apoptosis through mitochondrial pathway in spleen and bursa of Fabricius of broilersThe aim of this study was to investigate the ameliorative effects of GSPE against AFB₁-induced histopathology,oxidative stress,and apoptosis via the mitochondrial pathway in the spleen and bursa of Fabricius of broilers.For this experiment,all the samples were taken from the first experiment.The present study results showed that consumption of AFB₁ diet-induced oxidative stress in the spleen and bursa of Fabricius as evidenced by reduced total superoxide dismutase（T-SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）and glutathione S-transferase（GST）activities and increased lipid peroxidation（LPO）contents.Similarly,AFB₁ fed group markedly decreased mRNA expression of antioxidant genes（SOD,GST,CAT,and GPx1）in comparison with the control group（P<0.05）.Furthermore,histological results showed that AFB₁ aggravated lymphocytic depletion in splenic tissue of broilers.The bursa of Fabricius tissue from AFB₁ fed group birds manifested apoptotic cells as compared to control and experimental groups.Moreover,the relative mRNA expression results manifested that mitochondrial-apoptosis associated genes Bcl-2-associated X protein（Bax）,caspase-3,caspase-9,and tumor protein p53（p53）showed up-regulation,while B-cell lymphoma 2（Bcl-2）showed down-regulation in both organs of AFB₁ fed group.Strikingly,the same trends were noted in Bax,Bcl-2,p53,and caspase-3 protein expressions as indicated in the mRNA expression levels.AFB₁ fed group exhibited significant up-regulation in the Bax,p53,and caspase-3,while down-regulation in the Bcl-2 protein expression in both organs.Interestingly,dietary GSPE treatment ameliorated AFB₁-induced oxidative stress in the spleen and bursa of Fabricius tissue via inhibiting the accumulation of LPO content and enhancing the antioxidant enzymes activities（T-SOD,CAT,GSH-Px,and GST）.Likewise,GSPE markedly enhanced mRNA expression of antioxidant genes（SOD,GST,CAT,and GPx1）in comparison with AFB₁ fed group（P<0.05）.Furthermore,GSPE alleviated AFB₁-induced histopathological alterations in spleen and bursa of Fabricius tissue of broilers.Histological findings supported that GSPE has potential protective effects against AFB₁-induced toxicity.Additionally,the supplementation of GSPE to AFB₁ diet significantly reversed AFB₁-induced mRNA expression of mitochondrial apoptosis-associated genes（Bax,Bcl-2,caspase-3,caspase-9,and p53）as compared to the AFB₁ fed group.Further confirmation was achieved by similar results of protein expression of mitochondrial apoptosis-associated genes（Bax,Bcl-2,p53,and caspase-3）through western blotting analysis.Conclusively,our results demonstrated that GSPE ameliorated AFB₁-induced oxidative stress by reducing the LPO accumulation and enhancing the antioxidant defense system.In addition,GSPE attenuates AFB₁-induced excessive apoptosis in spleen and bursa of Fabricius of broilers via the mitochondrial pathway.In summary,our studies come to the following conclusions:1)Exposure to AFB₁ suppressed the growth performance,antioxidant capacity and immune function of broilers.Moreover,AFB₁ aggravated histopathological lesions in the liver.Dietary supplementation of GSPE exhibited a good protection against AFB₁-induced aflatoxicosis in broilers.GSPE could improve growth performance,biochemical profile,and immune function and alleviate AFB₁-induced hepatic injury in broilers through enhancing the antioxidant defense system.2)Consumption of AFB₁ could provoke an inflammatory response,immunotoxicity,and oxidative damage in broilers.However,GSPE inhibited the inflammatory response via modulating the secretion of inflammatory and pro-inflammatory cytokines.Meanwhile,GSPE mitigates AFB₁-induced immunotoxicity by inhibiting the NF-κB expression.Additionally,GSPE ameliorates AFB₁-induced oxidative damage through activating the Nrf2 signaling pathway in broilers.3)AFB₁ showed toxic effects in spleen and bursa of Fabricius of broilers.The consumption of AFB₁ exhibited histopathological lesions in both organs.Moreover,AFB₁triggered oxidative stress and excessive apoptosis in broilers.Meanwhile,dietary supplementation of GSPE alleviates AFB₁-induced histopathological lesions and oxidative damage via reducing lipid peroxidation accumulation and enhancing the activity of antioxidant-related enzymes and mRNA expression.Furthermore,GSPE ameliorated excessive apoptosis induced by AFB₁ through regulating the mitochondrial-mediated apoptosis pathway in spleen and bursa of Fabricius of broilers.