Dietary Supplementation with Chitosan Oligosaccharides Alleviates Oxidative Stress in Rats Challenged with Hydrogen Peroxide

Simple Summary Oxidative stress adversely affects animal health and performance. Feed additives with antioxidant abilities supplementation can alleviate oxidative stress. The aim of this study was to evaluate the hypothesis that dietary supplementation with COS alleviates the damage caused by oxidative stress in Sprague Dawley rats challenged with hydrogen peroxide (H2O2). The results shown that COS exhibited better radical scavenging capacity of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion (O2−), H2O2, and ferric ion reducing antioxidant power (FRAP) than butylated hydroxy anisole (BHA), increasing activity of SOD, CAT, GSH-Px, GSH, and T-AOC, as well as decreasing MDA level in serum, liver, spleen, and kidney. Our results indicated that COS can protect Sprague Dawley rats from H2O2 challenge by reducing lipid peroxidation and restoring antioxidant capacity. Abstract Oxidative stress is induced by excessive oxidative radicals, which directly react with biomolecules, and damage lipids, proteins and DNA, leading to cell or organ injury. Supplementation of antioxidants to animals can be an effective way to modulate the antioxidant system. Chitosan oligosaccharides (COS) are the degraded products of chitosan or chitin, which has strong antioxidant, anti-inflammatory, and immune-enhancing competency. Therefore, the current study was conducted to evaluate the hypothesis that dietary supplementation with COS alleviates the damage caused by oxidative stress in Sprague Dawley rats challenged with hydrogen peroxide (H2O2). The rats were randomly divided into three groups: CON, control group, in which rats were fed a basal diet with normal drinking water; AS, H2O2 group, in which rats were fed the basal diet and 0.1% H2O2 in the drinking water; ASC, AS + COS group, in which rats were fed the basal diet with 200 mg/kg COS, and with 0.1% H2O2 in the drinking water. In vitro, COS exhibited better radical scavenging capacity of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion (O2−), H2O2, and ferric ion reducing antioxidant power (FRAP) than butylated hydroxy anisole (BHA). In vivo, dietary supplementation with COS alleviated the H2O2-induced oxidative damage, evidenced by comparatively increasing activity of SOD, CAT, GSH-Px, GSH, and T-AOC, and comparatively decreasing level of MDA in serum, liver, spleen, and kidney. COS also comparatively alleviated the H2O2-induced inflammation. In conclusion, COS supplementation reduced lipid peroxidation and restored antioxidant capacity in Sprague Dawley rats, which were challenged with H2O2.


Introduction
Oxidative stress is induced by excessive oxidative radicals, which directly react with biomolecules, damaging lipids, proteins, and DNA, and leading to cell or organ injury [1][2][3][4]. Moreover, oxidative the FRAP reagent consisted of acetate buffer (0.3 M, pH = 3.6), TPTZ (10 mM) in hydrochloric acid (40 mM), and ferric chloride (20 mM), at a ratio of 10:1:1. The FRAP reagent was prepared just before the reaction, then 1.5 mL of FRAP reagent was mixed with 150 µL distilled water and 50 µL sample solutions at 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 g/L. It was then incubated for 30 min at room temperature in the darkness and the absorbance was determined at 593 nm. The reducing power is presented as micromoles Fe/gram fresh weight.

Animals, Experiment Design, and Diets
The male Sprague Dawley rats (8-10 weeks, 178.39 ± 5.12 g) used in this study were obtained from the Beijing Administration Office of Laboratory Animals (Beijing, China). The rats were individually housed in polycarbonate cages with soft wood granulate floors, and kept at 24 • C, with a 12 h light-dark cycle. After a week of acclimatization, 30 rats were randomly divided into one of 3 groups with 10 rats in each group for this 10 day experiment: CON, control group, rats were fed basal diet and normal drinking water; AS, H 2 O 2 group, rats were fed basal diet with 0.1% H 2 O 2 in drinking water [29]; ASC, AS + COS group, rats were fed basal diet with 200 mg/kg COS, and with 0.1% H 2 O 2 in drinking water. The supplementation level of COS was based on our previous preliminary research. All rats had free access to water and diet. The composition of the basal diet is listed in Table 1, and was made according to the nutritional requirement recommendations by the American Institute of Nutrition-93 diet [30].

Plasma Collection and Tissue Preparation
On the last day of the experiment, after 12 h fasting, all rats were euthanized with diethyl ether. Blood was collected from the posterior vena orbitalis, then centrifuged at 3000× g × 10 min to collect the serum, and stored at −20 • C until analysis. The liver, kidney, and spleen were collected and a 10% homogenate was prepared in PBS and centrifuged at 3000× g × 10 min at 4 • C, the supernatant was used for further biochemical assays.

Antioxidant and Inflammatory Cytokines Assays
The content or the activity of MDA, SOD, CAT, GSH-Px, GSH, T-AOC, IL-1β, IL-6, IL-10, and TNF-α in serum, liver, kidney, and spleen were measured according to the manufacturer's instruction.

Statistical Analysis
Data were analyzed by ANOVA using the GLM procedures of SAS (V9.1, SAS Inst., Inc., Cary, NC, USA). Duncan's multiple range tests were done to check the differences among treatments, and p < 0.05 was considered significant.

Antioxidant and Inflammatory Cytokines Assays
The content or the activity of MDA, SOD, CAT, GSH-Px, GSH, T-AOC, IL-1β, IL-6, IL-10, and TNF-α in serum, liver, kidney, and spleen were measured according to the manufacturer's instruction.

Statistical Analysis
Data were analyzed by ANOVA using the GLM procedures of SAS (V9.1, SAS Inst., Inc., Cary, NC, USA). Duncan's multiple range tests were done to check the differences among treatments, and p < 0.05 was considered significant.

DPPH, O2 -, H2O2, and FRAP Scavenging Capacity
The DPPH, O2 -, H2O2, and FRAP scavenging capacity of COS and BHA is shown in Figure 1. The concentration ranges from 0.01 to 0.06 g/L, the scavenging activity of COS ranges from 50.19% to 61.77%, and the scavenging activity of BHA ranges from 24.52% to 36.10%. For O2 − scavenging capacity, the scavenging activity of COS ranges from 32.94% to 40.31%, and the scavenging activity of BHA ranges from 23.45% to 30.02%. For the H2O2 scavenging capacity, the scavenging activity of COS ranges from 61.65% to 86.21%, and the scavenging activity of BHA ranges from 38.07% to 49.10%. For ferric reducing antioxidant power (FRAP) scavenging capacity, the scavenging activity of COS ranges from 44.24% to 42.62%, and the scavenging activity of BHA ranges from 23.73% to 24.93%.

Effects of COS on Antioxidant Status in Serum
Administration of H2O2 in drinking water increased (p < 0.05) the content of MDA in serum (Figure 2A), and decreased (p < 0.05) the activity of SOD ( Figure 2B) and CAT ( Figure 2C) in the AS

Effects of COS on Antioxidant Status in Serum
Administration of H 2 O 2 in drinking water increased (p < 0.05) the content of MDA in serum (Figure 2A), and decreased (p < 0.05) the activity of SOD ( Figure 2B) and CAT ( Figure 2C) in the AS group compared with the CON group. No significant differences were observed on the activity of MDA, SOD, CAT, GSH-Px, GSH, or T-AOC.
Animals 2020, 10, x FOR PEER REVIEW 5 of 13 group compared with the CON group. No significant differences were observed on the activity of MDA, SOD, CAT, GSH-Px, GSH, or T-AOC.

Effects of COS on Antioxidant Status in the Liver
The activity of SOD and GSH in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figures 3B,E) in the liver. The activity of CAT ( Figure 3C), GSH-Px ( Figure 3D), and T-AOC ( Figure 3F), and the content of MDA ( Figure 3A) were not significantly affected by exposure to H2O2 or supplementation with COS.

Effects of COS on Antioxidant Status in the Liver
The activity of SOD and GSH in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure  group compared with the CON group. No significant differences were observed on the activity of MDA, SOD, CAT, GSH-Px, GSH, or T-AOC.

Effects of COS on Antioxidant Status in the Liver
The activity of SOD and GSH in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figures

Effects of COS on Antioxidant Status in the Spleen
The activity of CAT and GSH-Px in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure

Effects of COS on Antioxidant Status in the Spleen
The activity of CAT and GSH-Px in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure

Effects of COS on Antioxidant Status in the Kidney
The activity of SOD, GSH-Px, GSH, and T-AOC in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure 5B-F, respectively), the activity of GSH and T-AOC in the AS group was also lower (p < 0.05) than that in the ASC group. The activity of CAT ( Figure 5C) and the content of MDA ( Figure 4A) in the kidney were not significantly affected by exposure to H2O2 or supplementation with COS.

Effects of COS on Antioxidant Status in the Kidney
The activity of SOD, GSH-Px, GSH, and T-AOC in the AS group was significantly (p < 0.05) lower than that in the CON group ( Figure 5B-F, respectively), the activity of GSH and T-AOC in the AS group was also lower (p < 0.05) than that in the ASC group. The activity of CAT ( Figure 5C) and the content of MDA ( Figure 4A) in the kidney were not significantly affected by exposure to H 2 O 2 or supplementation with COS.

Effects of COS on Inflammatory Cytokines in the Serum, Liver, Spleen, and Kidney
The content of IL-1β, IL-6, TNF-α, and IL-10 in the serum, liver, spleen, and kidney are presented in Figures 6-9, respectively. The content of IL-1β ( Figures 6A-9A), IL-6 ( Figures 6B-9B), IL-10 ( Figures 6C, 7C and 9C), and TNF-α ( Figures 6D-9D) in the serum, liver, spleen, and kidney did not differ among treatments, except the content of IL-10 ( Figure 8C) in the spleen. The amount of IL-10 was significantly lower (p < 0.05) in the AS group compared with the CON group.

Discussion
Increasing attention has been paid to the field of antioxidants in recent years. Food scientists have paid attention to antioxidants because of their ability to prevent fat from oxidative rancidity. Doctors are interested because of their ability to protect from oxidative injury. COS possess stronger antioxidant competency as evidenced by their reaction with unstable free radicals to form stable radicals [31]. In the present study, the effects of COS on antioxidant property were analyzed both in vitro and in vivo.
DPPH is a stable nitrogen radical, and is widely used to evaluate radical quenching capacities. The DPPH scavenging ability of COS was higher than BHA. H2O2, and O2 − act as signaling intermediates, and the over-production of H2O2 and O2 − is an indicator of oxidative stress [32]. DPPH reflects limited oxidation situations because it only exists in vitro; therefore, we further evaluated the H2O2 and O2 − scavenging capacity of COS. H2O2 can be synthesized and destroyed in response to external stimuli [33], and O2 − acts as a potential precursor to generate reactive radical species. Thus, evaluating the scavenging capacity of H2O2 and O2 − [34] is important for clarifying the antioxidant capacity. The results showed that the scavenging capacity of COS against H2O2 and O2 − was higher than BHA. There is an association between antioxidant capacity of antioxidants and their reducing power. We investigated the reducing power of COS using the FRAP assay. The results showed the scavenging capacity of COS against FRAP was higher than BHA. The antioxidant capacity of COS is related to its characteristic structure, including its deacetylation ratio, molecular weight, and the source of the material. The molecular weight and deacetylation ratio of COS also exerts some synergistic effects on the biological capacities, where, generally, the low molecular weight gives the stronger scavenging activity of DPPH, H2O2 and O2 − [35].
Many reports indicated that oxidative stress was related to animal health [36]. H2O2 is generally used as an oxidative stress stimulus, and studies have indicated that oral administration or intraperitoneal administration of H2O2 induces oxidative stress [37,38]. The detrimental effects of H2O2 depends on its conversation to hydroxy ions and other subsequent redox products. In this study, H2O2 challenge increased the content of MDA in the serum, liver, and kidney. Increasing content of MDA is an indicator of lipid peroxidation. COS supplementation resulted in a comparative

Discussion
Increasing attention has been paid to the field of antioxidants in recent years. Food scientists have paid attention to antioxidants because of their ability to prevent fat from oxidative rancidity. Doctors are interested because of their ability to protect from oxidative injury. COS possess stronger antioxidant competency as evidenced by their reaction with unstable free radicals to form stable radicals [31]. In the present study, the effects of COS on antioxidant property were analyzed both in vitro and in vivo.
DPPH is a stable nitrogen radical, and is widely used to evaluate radical quenching capacities. There is an association between antioxidant capacity of antioxidants and their reducing power. We investigated the reducing power of COS using the FRAP assay. The results showed the scavenging capacity of COS against FRAP was higher than BHA. The antioxidant capacity of COS is related to its characteristic structure, including its deacetylation ratio, molecular weight, and the source of the material. The molecular weight and deacetylation ratio of COS also exerts some synergistic effects on the biological capacities, where, generally, the low molecular weight gives the stronger scavenging activity of DPPH, H 2 O 2 and O 2 − [35].
Many reports indicated that oxidative stress was related to animal health [36]. H 2 O 2 is generally used as an oxidative stress stimulus, and studies have indicated that oral administration or intraperitoneal administration of H 2 O 2 induces oxidative stress [37,38]. The detrimental effects of H 2 O 2 depends on its conversation to hydroxy ions and other subsequent redox products. In this study, H 2 O 2 challenge increased the content of MDA in the serum, liver, and kidney. Increasing content of MDA is an indicator of lipid peroxidation. COS supplementation resulted in a comparative decrease in MDA content in the serum, liver, and kidney, indicating that COS had protective effects due to its antioxidant capacity [39].
Both SOD and CAT can scavenge superoxide ions and hydroxyl ions. The present study showed decreasing activity of SOD in the serum, liver and kidney, and decreasing activity of CAT in the serum and spleen of H 2 O 2 -exposed rats. H 2 O 2 induced a dramatic decrease in the activity of SOD and CAT, which may be related to the formation of reactive oxygen species (ROS) [29]. Dietary COS increased the activity of SOD and CAT, which can save the depletion of the two enzymes. GSH-Px is a glutathione-related enzyme, and there was a significant decrease in the activity of GSH-Px in the spleen and kidney of H 2 O 2 -exposed rats in this study. H 2 O 2 can efficiently be scavenged by GSH-Px; therefore, the decreasing activity of GSH-Px reflects perturbations in the normal oxidative balance by H 2 O 2 exposure. GSH is a typical non-enzyme antioxidant, which defends against reactive free radicals and other oxidant species in cellular defense systems. In this study, there was a significant decrease in the activity of GSH in the liver and kidney of H 2 O 2 -exposed rats, indicating that the depletion of GSH led to enhanced formation of ROS [40]. Meanwhile, COS supplementation increased the activity of GSH in the kidney, which suggests that COS maintained the redox balance of ROS through both the enzymatic and non-enzymatic antioxidant defense system. The value of T-AOC reflects the total antioxidant capacity [41]. In this study, the T-AOC value of the kidney decreased by H 2 O 2 exposure, but increased with COS supplementation, which suggests that COS supplementation can alleviate oxidative stress induced by H 2 O 2 through the non-enzymatic antioxidant system in the kidney. Both in vitro and in vivo experiments showed that COS has strong radical scavenging activity and antioxidant capacity. The radical scavenging activity of COS is associated with their proton donation ability [42]. Dietary supplementation with COS prevented H 2 O 2 -induced lipid peroxidation and reserved depletion of SOD, CAT, GSH-Px, and GSH activity, which was consistent with former reports indicating antioxidant and protective properties of COS [43][44][45].
Oxidative stress and inflammation are highly related [46]. Oxidative stress and excessive production of ROS is associated with inflammation, leading to the synthesis and release of pro-inflammatory cytokines. No significant differences were detected in the concentration of TNF-α, IL-1β, IL-6, and IL-10 in the serum, liver, spleen and kidney, except a significant decreasing concentration of IL-10 in the AS group compared to the CON group in the spleen. With COS supplementation, the level of TNF-α, IL-1β, and IL-6 in the ASC group was comparatively lower than that in the AS group in the serum, liver, spleen, and kidney, while the level of IL-10 in the ASC group was comparatively higher than that in the AS group, which may be due to the anti-inflammatory activity of COS [24].

Conclusions
In conclusion, COS had higher antioxidant activities than BHA when checked by DPPH, O 2 − , H 2 O 2 , and FRAP scavenging capacity in vitro. COS significantly increased the content of GSH and T-AOC in the kidney, and comparatively decreased the content of MDA in the serum, liver and kidney, which suggested COS had protective effects against H 2 O 2 -induced oxidative damage and can be used as a potential antioxidant in feed.
Author Contributions: R.L., L.A. and Z.Z. participated in the experiment design and manuscript writing. R.L. and Q.C. involved in feeding trail, sample and data collection, and analysis. All authors have read and agreed to the published version of the manuscript.