title: Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella).
abstract: This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp (Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4 (C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2 mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively.
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China.
authors.forename: Hui-Jun
authors.initials: HJ
authors.lastname: Xu
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China; Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China.
authors.forename: Wei-Dan
authors.initials: WD
authors.lastname: Jiang
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China; Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Feng
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China; Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China; Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Wu
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China; Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China.
authors.forename: Sheng-Yao
authors.initials: SY
authors.lastname: Kuang
authors.affiliation: Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Tang
authors.affiliation: Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China.
authors.forename: Wu-Neng
authors.initials: WN
authors.lastname: Tang
authors.affiliation: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Yong-An
authors.initials: YA
authors.lastname: Zhang
authors.affiliation: Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China; Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China; Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China. Electronic address: xqzhouqq@tom.com.
authors.forename: Xiao-Qiu
authors.initials: XQ
authors.lastname: Zhou
mesh_terms: D016980:Aeromonas hydrophila / Q000502:physiology; D000821:Animal Feed / Q000032:analysis; D000818:Animals; D017209:Apoptosis; D001205:Ascorbic Acid / Q000378:metabolism; D001206:Ascorbic Acid Deficiency / Q000503:physiopathology*; D002347:Carps / Q000235:genetics / Q000254:growth & development / Q000276:immunology*; D004032:Diet / Q000662:veterinary; D019587:Dietary Supplements / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D005393:Fish Diseases / Q000276:immunology*; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D016905:Gram-Negative Bacterial Infections / Q000276:immunology / Q000662:veterinary*; D060226:Head Kidney / Q000187:drug effects / Q000276:immunology; D007113:Immunity, Innate; D011897:Random Allocation; D015398:Signal Transduction; D013154:Spleen / Q000276:immunology
chemical_list: D029941:Fish Proteins; D001205:Ascorbic Acid
keywords: Head kidney; Immunity; Spleen; Structural integrity; Vitamin C; Young grass carp (Ctenopharyngodon idella)