title: Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.
abstract: This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, β-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264-1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264-1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 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: D000821:Animal Feed / Q000032:analysis; D000818:Animals; D001205:Ascorbic Acid; D001206:Ascorbic Acid Deficiency / Q000276:immunology / Q000662:veterinary*; D002347:Carps / Q000276:immunology*; D004032:Diet / Q000662:veterinary*; D019587:Dietary Supplements; D005393:Fish Diseases / Q000178:diet therapy / Q000276:immunology / Q000382:microbiology; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D045826:Flavobacteriaceae Infections / Q000178:diet therapy / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D005417:Flavobacterium / Q000502:physiology; D005880:Gills / Q000276:immunology / Q000502:physiology; D007113:Immunity, Innate; D011897:Random Allocation; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D029941:Fish Proteins; D001205:Ascorbic Acid
keywords: Gill; Grass carp (Ctenopharyngodon idella); Immune barrier; Physical barrier; Vitamin C