title: Gill structural integrity changes in fish deficient or excessive in dietary isoleucine: Towards the modulation of tight junction protein, inflammation, apoptosis and antioxidant defense via NF-κB, TOR and Nrf2 signaling pathways.
abstract: This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp (Ctenopharyngodon idella) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1β (IL-1β), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill.
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.
authors.forename: Lu
authors.initials: L
authors.lastname: Gan
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: 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: 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: Jun
authors.initials: J
authors.lastname: Jiang
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: Sheng-Yao
authors.initials: SY
authors.lastname: Kuang
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; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis; D002347:Carps / Q000033:anatomy & histology*; D004032:Diet / Q000662:veterinary*; D019587:Dietary Supplements; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D005880:Gills / Q000166:cytology*; D007532:Isoleucine / Q000172:deficiency* / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000975:Antioxidants; D029941:Fish Proteins; D007532:Isoleucine
keywords: Antioxidant defense; Gill; Grass carp (Ctenopharyngodon idella); Inflammation; Isoleucine; Structural integrity; Tight junction