Protective effect of mussel polysaccharide on cyclophosphamide‐induced intestinal oxidative stress injury via Nrf2‐Keap1 signaling pathway

Abstract The hard‐shelled mussel (Mytilus coruscus) has been used as a traditional Chinese medicine and health food in China for centuries. Polysaccharides from mussel has been reported to have multiple biological functions, however, it remains unclear whether mussel polysaccharide (MP) exerts protective effects in intestinal functions, and the underlying mechanisms of action remain unclear. The aim of this study was to investigate the protective effects and mechanism of MP on intestinal oxidative injury in mice. In this study, 40 male BALB/C mice were used, with 30 utilized to produce an animal model of intestinal oxidative injury with intraperitoneal injection of cyclophosphamide (Cy) for four consecutive days. The protective effects of two different doses of MP (300 and 600 mg/kg) were assessed by investigating the change in body weight, visceral index, and observing colon histomorphology. Moreover, the underlying molecular mechanisms were investigated by measuring the antioxidant enzymes and related signaling molecules through ELISA, real‐time PCR, and western blot methods. The results showed that MP pretreatment effectively protected the intestinal from Cy‐induced injury: improved the colon tissue morphology and villus structure, increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH‐Px) activities, and reduced malondialdehyde (MDA) content in serum and colon tissues. Meanwhile, MP also significantly increased the expression levels of SOD, GSH‐Px, heme oxygenase‐1 (HO‐1), and nuclear factor E2‐related factor 2 (Nrf2) mRNA in colon tissues. Further, western blot results showed that the expression of Nrf2 protein was significantly upregulated while kelch‐like ECH‐associated protein 1 (Keap1) was significantly downregulated by MP in the colonic tissues. This study indicates that MP can ameliorate Cy‐induced oxidative stress injury in mice, and Nrf2‐Keap1 signaling pathway may mediate these protective effects.


| INTRODUC TI ON
Reactive oxygen species (ROS) are chemically reactive oxygencontaining species, and hydrogen peroxide and superoxide anion are two examples of ROS (Sies & Jones, 2020). A high level of ROS can damage DNA, proteins, and lipids if it is not quenched immediately, and this damage is termed oxidative stress (Martin et al., 2020).
Excessive oxidative stress provokes a wide variety of disorders, including cell dysfunction and apoptosis, organism aging, and the occurrence of multiple chronic diseases (Ortega et al., 2021;Pizzino et al., 2017;Shen et al., 2017). The intestine is an essential organ for digestion, absorption, and secretion (Gong et al., 2021). However, as the gateway to the outside world, the intestinal is easily impacted by the surrounding environment, especially the oxidative stress conditions. Oxidative stress plays an important role in intestinal barrier disruption, which will lead to increased infiltration of toxins. It is possible that excessive oxygen free radical accumulation will destroy the mucosal barrier, increase intestinal permeability, and disrupt the intestinal flora, thereby destroying the body's steady state (Turan et al., 2017;Xue et al., 2020). Several studies showed that intestinal barrier dysfunction is associated with diverse chronic disorders, such as inflammatory bowel disease, obesity, diabetes, and rheumatologic diseases (Ramos & Papadakis, 2019). Therefore, many studies have focused their attention on finding substances that could counter intestinal oxidative stress injury.
Natural polysaccharides, an important bioactive substance, are widely existed in plants, animals, microorganisms, and marine organisms. Polysaccharides have drawn greater attention for their health benefits and exhibit great structural variability and the ability to hold biological information (Gan et al., 2021). Polysaccharides have various biochemical functions, including antioxidant , antiviral , anti-inflammatory (Xiang et al., 2021), immune regulation (Ruijun et al., 2015), and antitumor effects (Ye et al., 2021). The hard-shelled mussel (Mytilus coruscus) is a kind of marine shellfish with homology of food and medicine, which mainly grows in the shallow sea between the rocks . It is widely distributed in the Bohai Sea and the Yellow Sea of China. Owing to its high nutritional value and good medicinal potential, mussels have been widely artificially farmed throughout the world and have a high yield (Li et al., 2020). According to previous studies, mussel polysaccharide (MP) is the main active ingredient for its nutritional and medicinal values (Liu et al., 2017).
Particularly, MP has been reported to have significant antioxidant, free radical scavenger, and immunomodulatory effects. Xiang et al. found that MP had a certain scavenging ability, reducing ability, and strong anti-lipid peroxidation ability on hydroxyl free radical and superoxide anion free radical in vitro (Umasuthan et al., 2012).
In 2021, Xiang et al. further examined the anti-inflammatory and antioxidant activities of MP on RAW264.7 cells and DSS-induced colitis mouse model, their results found that the MP was able to reduce inflammation and relieve some clinical symptoms of colitis (Xiang et al., 2021). A review article also summarized the current understanding of the potential health benefits of MP, discussed several studies that have demonstrated the antioxidant and antiinflammatory properties of the polysaccharide, as well as its potential applications in the prevention and treatment of various diseases related to oxidative stress and inflammation . In addition, MP was also found can boost immune activity in mice (Xiang, Wang, Chen, Chen, Zheng, et al., 2022). It is well-known that the biological activities of polysaccharides as complex biomolecules are closely related to their composition, molecular size, and conformational shapes . A recent study has revealed the structure of MP , which is mainly composed of glucose, with a small amount of galactosamine hydrochloride, glucosamine hydrochloride, and galactose. The main chain connection mode of MP was determined to Glcp-(1→ was bonded to the main chain via O-6. However, there are few studies investigating its protective action for the intestinal in vivo, and the exact mechanism of action is also needed to identify. Cyclophosphamide (Cy) is a widely used agent in the treatment of systemic autoimmune diseases and cancer (Emadi et al., 2009;Pilz et al., 2008), including germ cell tumors, sarcomas, lymphoma, and lung cancer. However, Cy has a variety of side effects according to clinical studies, such as impairing ovarian and fertility function, decreasing immune function, and causing excessive production of oxygen free radicals which in turn leads to gastrointestinal mucosal barrier damage (Shi et al., 2017;Xue et al., 2020). Therefore, the present study established the intestinal oxidative damage mouse model by intraperitoneal injection of Cy and sought to investigate the protective effect of MP on Cy-induced intestinal oxidative injury and elucidate the underlying molecular mechanisms of its protective actions. This study provided evidence that MP can be used in food and pharmaceuticals as natural intestinal antioxidant protection.

| Animals and study design
In this study, the SPF BALB/C mice (8-week-year old, weighted 18- The mice were randomly divided into four groups (n = 10) after 1 week of adaptive feeding, the detailed grouping information is shown in Table 1. Each group received 21 consecutive days of intragastric administration of MP or vehicle. From day 18 to 21, Cy was given through intraperitoneal injection for 4 days. Each week, food intake and body weight were recorded during the experiments.
Finally, the blood and tissues were collected after the mice were fully anesthetized and sacrificed. Tissues were snap-frozen in liquid nitrogen and stored at −80°C until assay.

| Chemicals and reagents
The standard feed for mice was purchased from Beijing Keaoki

| Preparation of mussel polysaccharide
Fresh mussels were purchased from the Hangzhou Seafood Market, Zhejiang Province. Mussel polysaccharide (MP) was extracted and prepared by Dr. Xiang's laboratory in Zhejiang University of Technology, the method was described previously . Briefly, the fresh mussel flesh and its soft tissues were collected and homogenized immediately. Then the homogenized solution was degreased by the Soxhlet extraction method and then dissolved in distilled water, and 1% papain and 1% alkaline protease were added, respectively. After extraction, the enzyme was removed at 99°C for 20 min, cooled to room temperature, and adjusted the pH value to neutral. Centrifuged at 3000× g for 15 min, the supernatant was taken and the protein was removed by the Sevage method  (the volume ratio of chloroform to n-butanol was 4:1) to obtain the extract. The supernatant was concentrated to 1/3 of the original volume by rotary evaporator and was sunk with 95% ethanol, four times the volume, and placed in a refrigerator at 4°C overnight. The next day, the precipitation was taken from the centrifugal solution and freeze-dried. The dried sample was mussel polysaccharide.

| Measurement of visceral index
In this study, all mice were sacrificed after 21 days' treatment.
Viscera index of the main organs, including the liver, spleen, and thymus were isolated from the treated and control mice to evaluate the toxicity of Cy and the protective effects of MP. The liver, spleen, and thymus were taken and weighted, the organ index was calculated as follows:

| Histomorphological observation of colon tissues
Mice were sacrificed with posterior neck dislocation and then dissected immediately. The whole section of the node tissue from the cecal node to the anus was removed, and the colon was quickly cut open along the mesentery direction, and the intestinal contents and residual blood were rinsed with normal saline, and then dried with clean filter paper. Fixed colon tissues were dehydrated in graded ethanol for 45 min, followed by paraffin-embedded sections and HE staining. The dried HE sections were placed into an optical microscope for observation.

| Detection of antioxidant factors in serum of mice
One milliliter of blood was taken from the abdominal aorta and placed in a clean tube. The serum was centrifuged at 4°C and 3000× g for 10 min. SOD, GSH-Px, CAT, and MDA activities in serum were determined according to kit instructions.

| Detection of antioxidant factors in colon tissue
One hundred milligrams of colon tissues was taken and homogenized in a tissue grinder with 1 mL 0.9% saline on ice. The supernatant was centrifuged at 3000× g for 10 min at 4°C, and the contents of antioxidant enzymes SOD, GSH-Px, CAT, and MDA in colon homogenate were determined according to the production instructions of commercial kits.

| Quantitative real-time reverse transcription PCR
A reverse transcription kit (TIANGEN Biotech Co. Ltd., Beijing, China) was used to reverse transcribe total RNA from colon tissues into cDNA and qRT-PCR was used to determine the levels of mRNA.
The primers were designed with Primer Premier 6.0 software and synthesized by Shenggong Bioengineering Technology Limited (Shanghai, China). The sequences of primers used can be found in Table 2. A Roche LightCycler 480 II RT-PCR Detection System was used for all quantitative real-time PCR reactions. Gene expression was quantified using the 2 −ΔΔCt method (Livak & Schmittgen, 2001).
The housekeeping gene β-actin was used as a control.

| Western blot analysis
RIPA lysis buffer with protease and phosphatase inhibitor cocktail was used to homogenize colon tissues. After centrifugation at 12,000× g for 15 min at 4°C, tissue debris was removed. Then, the total protein was quantified by BCA quantitative kit (Solaibao, Beijing, China) and diluted to 2-4 mg/mL. Equal amounts of total proteins were electrophoresed. Afterward, the gel was transferred to a 0.45 μm PVDF membrane (Millipore). Then, the membrane was blocked with TBST (Tris-buffered saline with 0.05% Tween-20) containing 5% skim milk powder for 2 h. The membranes were then incubated at 4°C with the primary antibody overnight. Subsequently, the membranes were washed 5 min with TBST for three times, and then, HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. Finally, after rewashing the membranes with TBST for three times, images of the protein bands were obtained using electrochemiluminescence (ECL).

| Statistical analysis
The figure legends for each experiment provide all the statistical information, including the statistical tests used, number of mice (represented as n) in the animal experiments. Date were expressed as mean ± standard deviation (SD), and the Student's t-test was adopted to analyze the significant differences between the two groups using GraphPad Prism 6.0 (GraphPad Software, Inc.), and significant differences were designated as p < .05.

| Mussel polysaccharides enhanced antioxidant capacity in colon tissues
The effect of MP on the antioxidant levels in colon tissues was evaluated, results can be found in Figure 4. Compared with the normal

| Effect of mussel polysaccharides on Nrf2 and Nrf2-modulated phase II detoxifying gene expression levels in colon tissues
As an important antioxidant protein, the mRNA expression levels of Nrf2 and its driving detoxifying genes HO-1 and NQO1 were evaluated, results are indicated in Figure 6. Compared with the normal group, the Nrf2 expression levels were significantly de-

| Effect of mussel polysaccharides on Nrf2-Keap1 signaling pathway
It is well known that activation of the Nrf2-Keap1 signaling pathway plays a critical role in protecting cells from oxidative stress damage. Western blot analysis was used to evaluate the effect of MP on Nrf2-Keap1 signaling pathway, Nrf2 and Keap1 protein expression levels present in Figure 7. As compared to the normal group, Cy

| DISCUSS ION
Mussels are considered a high-quality food choice for consumers due to their nutritional value. According to previous reports, the nutritional value of mussels could be attributed to the presence of high levels of polysaccharides . Our recent work characterized MP's structure and evaluated its antioxidant capacity on RAW264.7 cells . In view of the obvious anti-inflammatory activities of MP in vitro, we further carry out animal experiments to investigate its beneficial effect in vivo. Cy as a common clinical chemotherapy drug, has antitumor effect, which is also used to treat autoimmune diseases. However, the active alkylating compounds of Cy in vivo (4-hydroxy-cyclophosphamide and aldophosphamide) will combine with macromolecules in the cells and change the import-   (Li et al., 2018). These results further confirmed that MP can effectively alleviate oxidative stress injury by activating the Nrf2-Keap1 antioxidant signaling pathway, and this mechanism is schematically represented in Figure 8.

| CON CLUS ION
The present study showed that SOD, CAT, and GSH-Px levels in serum and colon tissues were significantly improved with MP, while MDA levels were significantly reduced. Further, MP pretreatment greatly enhanced SOD, GSH-Px, Nrf2, HO-1, and NQO1 mRNA expression in colon tissues, increased Nrf2 protein expression, and decreased Keap1 protein expression. Taken together, the above findings indicated that MP could restore and relieve the intestinal oxidative stress induced by Cy in mice, this is likely to be due to the regulation of antioxidative enzyme secretion and activation of the Nrf2-Keap1 pathway that leads to antioxidant effects. Based on these findings, we concluded that MP has a great therapeutic potential in the treatment of several diseases related to intestinal oxidative damage.

ACK N OWLED G M ENTS
We thank all participants, including Prof. Xing-Wei Xiang and Dr. Yu-Feng Chen from Zhejiang University of Technology, for their enthusiastic participation and cooperation in this study.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original contributions presented in this study are included in the article, further inquiries can be directed to the corresponding authors.