An Antioxidant Supplement Function Exploration: Rescue of Intestinal Structure Injury by Mannan Oligosaccharides after Aeromonas hydrophila Infection in Grass Carp (Ctenopharyngodon idella)

Abstract

Mannan oligosaccharides (MOS) are a type of functional oligosaccharide which have received increased attention because of their beneficial effects on fish intestinal health. However, intestinal structural integrity is a necessary prerequisite for intestinal health. This study focused on exploring the protective effects of dietary MOS supplementation on the grass carp’s (Ctenopharyngodon idella) intestinal structural integrity (including tight junction (TJ) and adherent junction (AJ)) and its related signalling molecule mechanism. A total of 540 grass carp (215.85 ± 0.30 g) were fed six diets containing graded levels of dietary MOS supplementation (0, 200, 400, 600, 800 and 1000 mg/kg) for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila for 14 days. We used ELISA, spectrophotometry, transmission electron microscope, immunohistochemistry, qRT-PCR and Western blotting to determine the effect of dietary MOS supplementation on intestinal structural integrity and antioxidant capacity. The results revealed that dietary MOS supplementation protected the microvillus of the intestine; reduced serum diamine oxidase and d-lactate levels (p < 0.05); enhanced intestinal total antioxidant capacity (p < 0.01); up-regulated most intestinal TJ and AJ mRNA levels; and decreased GTP-RhoA protein levels (p < 0.01). In addition, we also found several interesting results suggesting that MOS supplementation has no effects on ZO-2 and Claudin-15b. Overall, these findings suggested that dietary MOS supplementation could protect intestinal ultrastructure, reduce intestinal mucosal permeability and maintain intestinal structural integrity via inhibiting MLCK and RhoA/ROCK signalling pathways.

Keywords: adherent junction; antioxidant capacity; intestine; permeability; tight junction.

Figure 1

The effect of MOS on phenotypic indicators of intestinal structural integrity after infection with Aeromonas hydrophila. (A,B) Ultrastructural observation of intestine; (C,D) intestinal permeability parameters; (E–G) intestinal total antioxidant capacity in the PI, MI and DI. DAO: diamine oxidase (U/L); d-lactate: malondialdehyde (μmol/L); T-AOC: total antioxidant capacity; PI: proximal intestine; MI: middle intestine; DI: distal intestine. N = 6 for each MOS level, p-values indicate a significant quadratic dose–response relationship (p < 0.05).

Figure 2

The effect of MOS on Occludin expression with the immunohistochemistry method in three intestinal segments after infection with Aeromonas hydrophila. (A) Occludin protein expression in the intestine in the 0 mg/kg MOS, 400 mg/kg MOS and 1000 mg/kg MOS groups; (B) Quantification of the positive area as revealed by Image Pro Plus 6.0. N = 6 for each MOS level. Differences among the variables were assessed using Student’s t-tests. Statistical significance: * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001; ns: not significant.

Figure 3

The effect of MOS on ZO-1 expression by the immunohistochemistry method in three intestinal segments after infection with Aeromonas hydrophila. (A) ZO-1 protein expression of intestine in the 0 mg/kg MOS, 400 mg/kg MOS and 1000 mg/kg MOS groups; (B) Quantification of the positive area as revealed by Image Pro Plus 6.0. N = 6 for each MOS level. Differences among the variables were assessed using Student’s t-tests. Statistical significance: * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001; ns: not significant.

Figure 4

Heat map showing MOS (mg/kg diet) changed expression of AJCs and related signalling molecules genes in three intestinal segments of grass carp after infection with Aeromonas hydrophila. The signal values of up-regulation (red) and down-regulation (blue) were expressed and ranged from 0.5 to 2 fold. Data represent means of six fish in each group (N = 6).

Figure 5

Correlation analysis of parameters in the three intestinal segments of grass carp after infection of Aeromonas hydrophila. (A) proximal intestine; (B) middle intestine; (C) distal intestine. R > 0.7, strong correlation; 0.5 < R < 0.7, moderate correlation; R < 0.5, weak correlation.

Figure 6

Western blot analysis of GTP-RhoA levels in the intestine of grass carp after infection with Aeromonas hydrophila. (A) Proximal intestine; (B) middle intestine; (C) distal intestine. Data represent means of three fish in each group, error bars indicate S.D. Values with different letters are significantly different (p < 0.05). Quantification and analysis were performed through NIH Image J software (version 1.42 q, National Institutes of Health, Bethesda, MD, USA).

Figure 7

Potential action pathways of MOS on intestinal structural integrity and its related mechanisms after infection with Aeromonas hydrophila. (A) A. hydrophila group; (B) optimal MOS group. This picture is drawn by Figdraw (www.figdraw.com, accessed on 24 March 2022).