This study attempted to evaluate the possible impact and mechanism of leucine (Leu) on fish intestinal barrier function. One hundred and five hybrid Pelteobagrus vachelli ♀ × Leiocassis longirostris ♂ catfish were fed with six diets in graded levels of Leu 10.0 (control group), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g/kg diet for 56 days. Results showed that the intestinal activities of LZM, ACP, and AKP and contents of C3, C4, and IgM had positive linear and/or quadratic responses to dietary Leu levels. The mRNA expressions of itnl1, itnl2, c-LZM, g-LZM, and β-defensin increased linearly and/or quadratically (p < 0.05). The ROS, PC, and MDA contents had a negative linear and/or quadratic response, but GSH content and ASA, AHR, T-SOD, and GR activities had positive quadratic responses to dietary Leu levels (p < 0.05). No significant differences on the CAT and GPX activities were detected among treatments (p > 0.05). Increasing dietary Leu level linearly and/or quadratically increased the mRNA expressions of CuZnSOD, CAT, and GPX1α. The GST mRNA expression decreased linearly while the GCLC and Nrf2 mRNA expressions were not significantly affected by different dietary Leu levels. The Nrf2 protein level quadratically increased, whereas the Keap1 mRNA expression and protein level decreased quadratically (p < 0.05). The translational levels of ZO-1 and occludin increased linearly. No significant differences were indicated in Claudin-2 mRNA expression and protein level. The transcriptional levels of Beclin1, ULK1b, ATG5, ATG7, ATG9a, ATG4b, LC3b, and P62 and translational levels of ULK1, LC3Ⅱ/Ⅰ, and P62 linearly and quadratically decreased. The Beclin1 protein level was quadratically decreased with increasing dietary Leu levels. These results suggested that dietary Leu could improve fish intestinal barrier function by increasing humoral immunity, antioxidative capacities, and tight junction protein levels.

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