**3. Discussion**

Intestinal barrier integrity was commonly assessed by gu<sup>t</sup> morphology, serum DAO activity, and D-LA activity. The gu<sup>t</sup> morphology was a useful biomarker of the stress response of the intestinal tract [9,23,24]. The DAO activity and D-LA content were biomarkers of intestinal permeability [3,11,25]. In this study, oxidative stress decreased the VH of the small intestine, and enhanced DAO content and D-LA activity, demonstrating that oxidative stress resulted in intestinal injury by increasing intestinal permeability and decreasing VH [3,9]. As expected, COS decreased DAO content and D-LA activity, and increased the VH of the small intestine; hence, COS had positive effects on intestinal permeability. These results are also in line with former studies by Li et al. [21] and Zhao et al. [26], who indicated COS decreased serum DAO activity. The structure of the intestinal morphology reflected gu<sup>t</sup> health status. Li et al. [21] illustrated COS increased the VH of the duodenum and ileum in broilers. Liu et al. [27] and Liu et al. [28] reported dietary COS supplementation increased the VH of the jejunum and ileum in weaning pigs. The positive effects of COS on intestinal permeability and morphology could explain the improving intestinal function with COS supplementation.

The tight junction proteins maintained and regulated the intestinal barrier function. The tight junction proteins mainly consisted of the transmembrane proteins claudin and occludin, and peripheral membrane protein ZO-1. Therefore, the decreased mRNA expression of ZO-1, claudin, and occludin reflected intestinal barrier dysfunction [4]. In this study, oxidative stress downregulated the protein expression of ZO-1, occludin, and claudin in the jejunum; and the mRNA expression of claudin in the duodenum and ZO-1 in the jejunum and ileum, which were all consistent with the results reported by Song et al. [29] and Cao et al. [1]. COS upregulated the protein expression of ZO-1 and claudin in the jejunum, and the mRNA expression of ZO-1 in the jejunum and ileum, and claudin in the duodenum—similarly to other studies on mice fed high-fat diets [30], dexamethasonechallenged broilers [31], and weaning pigs [32,33]. These results demonstrated that COS could alleviate oxidative-induced intestinal barrier function partly by maintaining the intestinal structure, intestinal permeability, and tight junction functionality.

Accumulating evidence indicates that oxidative status was an important factor in intestinal barrier function. An imbalance between oxidation and the antioxidant defense system leads to oxidative stress and inflammation, and finally, induces intestinal barrier dysfunction [12]. SOD, GSH-Px, and CAT were regarded as the main antioxidant enzymes for scavenging free radicals. GSH was regarded as the most important non-enzymatic antioxidant which scavenges single oxygen molecules and hydroxyl radicals. The T-AOC can reflect the total antioxidant capacity. MDA is an indicator of oxidative stress [34]. H2O2 can stimulate ROS over-production, disrupt the activity of antioxidant enzymes, and induce lipid peroxidation [35,36]. Consistently, in this study, oxidative stress induced higher small intestine MDA content, along with lower duodenum and ileum mucosal GSH-Px activity, jejunum mucosal SOD activity, and jejunum and ileum mucosal T-AOC activity. COS reduced duodenum, jejunum, and ileum mucosal MDA content; and increased duodenum and ileum mucosal GSH-Px activity, and jejunum and ileum mucosal T-AOC activity, suggesting COS could alleviate intestinal mucosal oxidative stress by improving the antioxidative enzyme activity and decreasing MDA content. These results are consistent with Lan et al. [20], who indicated COS could increase SOD, CAT, GSH-Px, and T-AOC activity, and decrease the MDA level with an H2O2 challenge. Li et al. [21] also indicated COS increased SOD activity in the duodenum's mucosa and decrease the MDA level in the jejunum and ileum's mucosa in broilers. Similarly, Li et al. [22] indicated that dietary COS supplementation increased the inhibition of hydroxy radical capacity, and GSH, T-AOC, GSH-Px, and SOD activity, whereas decreased MDA content in the ileum mucosa of broilers. Nrf2 is a nuclear transcription factor and plays a vital role in antagonizing oxidative stress [37]. Our results show decreased mRNA expression of Nrf2 in the jejunum and ileum, and HO-1 in the duodenum and ileum by H2O2 challenge. Other studies illustrated the increased Nrf2 mRNA expression level could increase the mRNA expression of SOD and GSH-Px [3]; the decreased Nrf2 and HO-1 mRNA expression levels may be related to a response to oxidative stress. As expected, COS enhanced the mRNA expression of Nrf2 in the jejunum and HO-1 in the duodenum and ileum—similarly to other studies on doxorubicin-challenged rats [38] and mice fed a high-fat diet [39]. Collectively, the combined results illustrate that the increased antioxidant enzyme activity may be mediated by Nrf2/HO-1 signaling pathway. Additionally, the efficiency of the free-radical scavenging capacities reflected the neutralization of free radical capacities, or hydrogen donor capacity [40]. In this study, COS improved the radical scavenging capacity of the jejunum mucosa; that may relate to antioxidant capacity and hydrogen donation ability [17].

Inflammation cytokines play vital roles in the inflammatory and immune responses. The accumulating literature illustrates inflammation is an important marker in intestinal disfunction [3,12]. Chen et al. [12] indicated that over-production of cytokines could change the intestinal permeability and tight junction structure by modulating tight junction-related genes expression in weaning piglets. The over-production of IL-1β, IL-6, and TNFα directly resulted in intestinal mucosal injury [41,42]. Therefore, suppressing the overproduction of intestinal mucosal IL-1β, IL-6, and TNFα was a useful way to maintain the intestinal function. Previous studies indicated that stressors could disturb the balance between anti- and pro-inflammatory responses by increasing pro-inflammatory cytokines' production [12,43,44]. In this study, the duodenum and ileum mucosal IL-6 content, and jejunum mucosal TNFα level were higher, whereas the duodenum and ileum mucosal IL-10 levels were decreased, in the AS group compared to the CON group, indicating that oxidative stress resulted in inflammation in the intestine. Furthermore, the levels of mRNA expression of IL-1β in the duodenum and ileum, IL-6 in the duodenum and jejunum, and TNFα in the small intestine in the AS group were increased compared to the CON group, but the expression levels of IL-10 in the duodenum and jejunum were decreased. Dietary COS supplementation decreased the duodenum and ileum mucosal IL-6 level and jejunum mucosal TNFα level; inhibited the expression of IL-6 in the jejunum and ileum, and TNFα in the jejunum and ileum; and increased the duodenum and ileum mucosal IL-10 levels, all of which was in consistent with the results of Hu et al. [33], who reported COS reduced IL-1β and TNFα mRNA expression levels in jejunum mucosa in weaning pigs. Besides, COS decreased the IL-6 and TNFα mRNA expression levels in the liver of mice fed a high-fat diet [39]. These results sugges<sup>t</sup> that COS may alleviate intestinal inflammation by suppressing the levels of IL-1β, IL-6, and TNFα [45,46]. However, in this study, we

ignored the immune cells in the mucosal immune system, especially the mast cells, which play a vital role in the regulation of intestinal mucosal immune function and intestinal barrier function. Further study would focus on this point.

In conclusion, COS had beneficial effects on intestinal integrity by improving the antioxidant capacity and suppressing the release of inflammatory cytokines. Dietary COS supplementation may be an effective nutritional strategy to alleviate the detrimental effects of oxidative stress.

#### **4. Materials and Methods**

#### *4.1. Animals, Diets, and Experimental Design*

In total, 30 male Sprague–Dawley rats (8–10 weeks old, 178.39 ± 5.12 g) were purchased from Beijing Administration Office of Laboratory Animals and acclimatized for 7 days before the experiment. The rats were provided with a pelleted diet, had free access to diet and water, and were housed at constant temperature (24 ± 2 ◦C) and relative humidity (60% ± 5%) on a 12-h light–dark cycle. The basal diet composition is shown in Table 2. The experimental protocol and use of rats were approved by the Animal Care and Use Committee of Guangdong Ocean University, Zhanjiang, China (SYXK-2018-0147, 2018).

**Table 2.** The composition of basal diet.


1 Mineral premix (mg/kg of premix): CaCO3, 3.70 × 105; KH2PO4, 1.96 × 105; K3C6H5O7·H2O, 7.08 × 104; NaCl, 7.4 × 104; K2SO4, 4.66 × 104; MgO, 2.4 × 104; FeC6H5O7H2O, 6.06 × 103; ZnCO3, 1.65 × 103; MnCO3, 630; CuCO3, 324; NaSiO3·9H2O, 1.45 × 103; CrK(SO4)·12H2O, 275; LiCl, 17.4; H3BO3, 81.5; NaF, 63.5; NiCO3·2Ni(OH)2·4H2O, 30.6; NH4VO3, 6.6; sucrose was added to make a total of 1 kg; 2 vitamin premix (mg/kg of premix): nicotinic, 3.0 × 103; calcium pantothenate, 1.6 × 103; pyridoxine hydrochloride, 700; thiamine hydrochloride, 600; riboflavin, 600; folic acid, 200; D-biotin, 20; cyanocobalamin, 2.5 × 103; a-tocopherol, 1.5 × 104; cholecalciferol, 250; phylloquinone, 75; sucrose was added to make a total of 1 kg.

H2O2 induced oxidative stress by generation of potent ROS [23–25]. ROS caused lipid peroxidation, membrane disintegration, and endothelial cell damage [26]. The 30 rats were randomly divided into three groups: CON, 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.
