*2.3. Antioxidant Capacity*

Figure 3 presents the antioxidant indicators in the small intestinal mucosa. In duodenum, the malondialdehyde (MDA) content in the AS group was higher (*p* < 0.05) than the contents in the CON and ASC groups, but the glutathione peroxidase (GSH-Px) activity was lower (*p* < 0.05). In the jejunum, the MDA content in the AS group was higher (*p* < 0.05) than in the CON and ASC groups; the superoxide dismutase (SOD) activity in the CON group was higher (*p* < 0.05) than in the AS and ASC groups; the total antioxidant capacity (T-AOC) activity in the AS group was lower (*p* < 0.05) than in the CON and ASC groups. In the ileum, the MDA contents in the CON and ASC groups were lower (*p* < 0.05) than that in the AS group, but the GSH-Px and T-AOC activities were higher.

**Figure 3.** Effects of chitosan oligosaccharides on small intestine antioxidant indicators when challenged with hydrogen peroxide. CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. (**A**) MDA content; (**B**) SOD activity; (**C**) GSH-Px activity; (**D**) CAT activity; (**E**) GSH content; (**F**) T-AOC activity. Values indicate means ± standard errors. a,b Columns that have different letter statistically differ (*p* < 0.05).

#### *2.4. Free Radical Scavenging Activity*

Figure 4 presents the free-radical scavenging capacities in jejunum mucosa. The 1,1- diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), O2− (superoxide radical) and hydroxyl radical (OH−) scavenging activities in the CON and ASC groups were higher (*p* < 0.05) than in the AS group.

#### *2.5. Intestinal Mucosal Immunity*

In the duodenum, the IL-6 and TNF-α contents in the AS group were enhanced (*p* < 0.05) compared to CON and ASC groups (Figure 5), but the IL-10 content was decreased (*p* < 0.05). In the ileum, the IL-6 content in the AS group was increased (*p* < 0.05) over CON and ASC groups, but the IL-10 content was decreased (*p* < 0.05).

**Figure 4.** Effects of chitosan oligosaccharides on the free-radical scavenging activity of the jejunum when challenged with hydrogen peroxide. CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC group, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. The free-radical scavenging activity was calculated on the basis of the protein content (mg/mL) of the jejunum mucosa. Values indicate means ± standard errors. a,b,c Columns that have different letter statistically differ (*p* < 0.05).

**Figure 5.** Effects of chitosan oligosaccharides on inflammatory cytokines in the small intestine when challenged with hydrogen peroxide. CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC group, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. (**A**) IL-1β content; (**B**) IL-6 content; (**C**) IL-10 content; (**D**) TNF-α content. Values are indicated as means ± standard errors. a,b Columns that have different letter statistically differ (*p* < 0.05).

Furthermore, we also examined the related cytokines' gene expression in the small intestine (Figure 6). In the duodenum, the relative gene expression levels of IL-1β and TNF-α in the AS group were higher (*p* < 0.05) than in the CON group; IL-6 in the AS group was enhanced (*p* < 0.05) over CON and ASC groups; IL-10 in the AS and ASC groups was less present (*p* < 0.05) compared to the CON group. In the jejunum, the relative gene expression levels of IL-6 and TNF-α in the AS group were increased (*p* < 0.05) compared to CON and ASC groups; IL-10 in the AS and ASC groups was decreased (*p* < 0.05) compared to the CON group. In the ileum, the relative gene expression of IL-1β in the AS group was enhanced (*p* < 0.05) compared to the CON group; TNF-α in the AS group was higher (*p* < 0.05) than in CON and ASC groups.

**Figure 6.** Effects of chitosan oligosaccharides on inflammatory cytokine-related gene mRNA expression in the small intestine when challenged with hydrogen peroxide. CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC group, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. (**A**) relative IL-1β mRNA level; (**B**) relative IL-6 mRNA level; (**C**) relative IL-10 mRNA level; (**D**) relative TNF-α mRNA level. Values indicate means ± standard errors. a,b Columns that have different letter statistically differ (*p* < 0.05).

#### *2.6. Intestinal Barrier Function-Related Gene Expression*

In the duodenum, the relative gene expression of claudin in the AS group was decreased (*p* < 0.05) compared to CON and ASC groups (Figure 7). In the jejunum and ileum, the relative gene expression of zonula occludens (ZO)-1 in the AS group was decreased (*p* < 0.05) compared to CON and ASC groups. In addition, we found that the expression levels of ZO-1 and claudin protein in the AS group were decreased (*p* < 0.05) compared to CON and ASC groups (Figure 8); and the expression of occludin protein in the AS and ASC groups was decreased (*p* < 0.05) compared to the CON group in jejunum mucosa.

We also examined the critical gene expression in the antioxidant signaling pathway. As shown in Figure 9, in the duodenum, the relative gene expression of heme oxygenase (HO)-1 in the AS group was decreased (*p* < 0.05) compared to CON and ASC groups. In the jejunum, the relative gene expression of nuclear factor-erythroid 2-like 2 (Nrf2) in the AS group was lower (*p* < 0.05) than that in the CON group. In the ileum, the relative gene expression of Nrf2 in the AS group was decreased (*p* < 0.05) compared to the CON group; and the relative gene expression of HO-1 in the AS group was decreased (*p* < 0.05) compared to CON and ASC groups.

**Figure 7.** Effects of chitosan oligosaccharides on intestinal barrier-related gene mRNA expression of the small intestine when challenged with hydrogen peroxide. CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC group, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. (**A**) relative ZO-1 mRNA expression level; (**B**) relative occludin mRNA expression level; (**C**) relative claudin mRNA expression level. Values indicated as means ± standard errors. a,b Columns that have different letter statistically differ (*p* < 0.05).

**Figure 8.** Effects of chitosan oligosaccharides on tight junction protein expression in the jejunum when challenged with hydrogen peroxide. CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC group, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. a,b,c Columns that have different letter statistically differ (*p* < 0.05).

**Figure 9.** Effects of chitosan oligosaccharides on Nrf2 and HO-1 gene mRNA expression in the small intestine when challenged with hydrogen peroxide. CON group, basal diet; AS group, basal diet + 0.1% H2O2 in drinking water; ASC group, basal diet + 200 mg/kg COS + 0.1% H2O2 in drinking water. (**A**) relative Nrf2 mRNA expression level; (**B**) relative HO-1 mRNA expression level. Values indicated as means ± standard errors. a,b Columns that have different letter statistically differ (*p* < 0.05).
