Protective effect of chitosan oligosaccharides on dextran sulfate induced colitis mice
Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn’s disease (CD). Ulcerative colitis was discovered by Wilk and Moxon in 1875, defined by Boas and Wilk in 1903, and officially designated as a special clinical condition by WHO in 1905. Ulcerative colitis is a non-specific colitis mainly located in the sigmoid colon and rectum of the large intestine. The lesions are continuously distributed in the mucosa and submucosa, and recur slowly. There are also a few cases of acute onset, mainly manifested as abdominal pain, diarrhea, and sticky stool with pus and blood, accompanied by various extraintestinal symptoms. The exact etiology and pathogenesis of ulcerative colitis are still unclear, and it is generally believed to be related to various factors such as infection, immune system disorders, genetic sensitivity, environment, mental state, and dysbiosis. It is very common in western countries, with 10-20 people suffering from it for every 105 people, and the incidence rate rate in North America and Western Europe is higher. In recent years, the incidence of UC in China has also been on the rise. At present, there are symptomatic drugs such as 5-aminosalicylic acid (sulfasalazine) and immunosuppressants available for the treatment of colitis, but they have certain side effects, making the search for safe drugs a research hotspot.
Chitooligosaccharides (COS) are produced by hydrolysis of chitosan (CS), with a polymerization degree of 2-20 and an average molecular weight of less than 3900 Da. Its molecular weight is small, easily soluble in water, and can be directly absorbed by biological organisms. It has biological activities such as reducing fat, lowering blood sugar, inhibiting bacteria, fighting cancer, and anti-inflammatory, and has no toxic side effects. It can be widely used in various fields such as medicine, cosmetics, food, and agriculture. Chitosan can be obtained by deacetylation of chitin, the second most abundant polymer in the world after cellulose (commonly found in crustacean shells, insect epidermis, and fungal cell walls). At present, there have been some studies on the effect of chitosan oligosaccharides on colitis, such as Yang et al.’s discovery that chitosan oligosaccharides can achieve protective effects on colitis through AMPK and NF – κ B. However, the application concentration is significantly higher (generally 500 mg/kg, equivalent to a human dose of about 3.6 g), far exceeding the national recommended intake dose of 0.5 g/day (see Announcement on the Approval of Six New Food Ingredients including Chitosan Oligosaccharides (2014 No. 6), equivalent to a mouse dose of 70 mg/kg). Yousef found that chitosan oligosaccharides at a dose of 20 mg/kg can also have a protective effect on colitis using acetic acid infusion colitis model and intestinal epithelial cell experiments. Therefore, this article mainly uses a 3% DSS drinking water induced colitis mouse model to verify the intervention of chitosan oligosaccharides on colitis at different doses (70 and 140 mg/kg), explore the role of chitosan oligosaccharides in regulating colitis symptoms and physiological status in mice, and provide data support for further research on the protective effect of chitosan oligosaccharides on DSS induced colitis mice. In this experiment, 50 male C57BL/6 mice were randomly divided into 5 groups: blank group, model group, positive group (SASP, 50 mg/kg), and low – and high-dose chitosan groups (70 and 140 mg/kg) by drinking water containing 3% sodium dextran sulfate (DSS) for 7 days to establish a model. Evaluate the effect of chitooligosaccharides through weight changes, disease activity index, pathological score, and myeloperoxidase activity. Detect the levels of inflammatory factors in colitis by ELISA.

The DSS colitis model is an internationally recognized and widely used experimental UC animal model established in 1990. Its pathological changes include ulcer formation, crypt abscess, small vessel inflammation, decreased goblet cells, and infiltration of various inflammatory cells, which are similar to the clinical symptoms of human colitis. The recommended human dose of chitosan by the country is 0.5 g/person, which is converted to a mouse dose of 70 mg/kg. Therefore, using this model, we selected two doses of chitosan, 70 mg/kg and 140 mg/kg, to investigate their intervention effects on DSS induced colitis. A Korean literature conducted acute toxicity experiments on oligosaccharides and found that the LD50 of oligosaccharides in mice exceeded 5.0 g/kg. Zhao et al.’s safety evaluation of chitosan functional foods showed that no acute toxic reactions were observed within 10 days after administration to mice (1.25 g/kg/day) and rats (1.47 g/kg/day) (approximately 312-368 times the clinically recommended dose). This experiment established an ulcerative colitis model by allowing mice to freely drink 3% DSS for 7 days. On the 6th day of the experiment, typical physiological symptoms of colitis were observed in the model group mice, such as weight loss, loss of appetite, uneven hair, diarrhea, sticky feces with blood, and even rectal bleeding. On the eighth day of dissection, it was found that the colon of the model group mice was shortened, and the colon tissue was congested and edematous. HE staining revealed edema of the colon mucosa, incomplete epithelial cells, disappearance of crypt glandular structure, and severe inflammatory infiltration. The above symptoms were improved in mice treated with low-dose (70 mg/kg) and high-dose (140 mg/kg) chitosan intervention. Consistent with the results of Yousef et al., he pointed out that chitosan oligosaccharides can reduce apoptosis of intestinal epithelial cells, inhibit inflammation, and protect the structural and functional integrity of the intestinal epithelial barrier. Some studies suggest that inflammatory bowel disease is caused by defects in the intestinal epithelial barrier and dysregulation of mucosal immune response. Chitosan oligosaccharides can protect intestinal epithelial cells, indicating their important protective role in enteritis. In this experiment, the chitosan 70 mg/kg dose group showed significant differences compared to the model group in improving colon shortening, alleviating colonic mucosal edema, and protecting goblet cells and crypt structures. However, the 140 mg/kg dose group showed no significant difference in improving colon shortening compared to the model group, with only a significant difference in improving pathological tissue scores. This indicates that the low-dose chitosan group has a better protective effect on DSS induced colitis mice. The results showed that a dose of 70 mg/kg chitosan had a certain effect on alleviating colon shortening and protecting colon structure in mice, which is helpful for the development and application of chitosan at the nationally recommended dose.

At present, the main treatment directions for IBD are oxidative stress and inflammation. It is believed that oxidative stress is the pathogenic basis of IBD, and excessive oxygen free radicals are a key factor in the formation of ulcerative inflammatory tissue damage and colitis. With the production of MDA, excessive NO generates oxygen free radicals, causing oxidative damage and leading to the infiltration of a large number of inflammatory cells in the tissue. Neutrophils are the fastest immune cells recruited to the site of inflammation in the early stages of UC. It has a dual function. On the one hand, it aggregates in epithelial crypts and intestinal cavities, and secretes IL-1, IL-6, MPO, etc. by releasing reactive oxygen species (ROS), inducing further tissue damage. On the other hand, it actively maintains tissue homeostasis by secreting anti-inflammatory factors and can limit microbial invasion. Qiao et al. found that chitosan oligosaccharides can reduce the infiltration of neutrophils and lipid peroxidation in damaged organs in LPS induced sepsis mouse models. Dou et al. found that chitosan oligosaccharides can inhibit the production of reactive oxygen species, degranulation, and adhesion of activated neutrophils in rabbits, thereby exerting anti-inflammatory effects. The myeloperoxidase value (MPO) is a marker of tissue damage and neutrophil (PMN) infiltration, which can lead to mucosal rupture and ulceration, and is closely related to IBD.

In the experiment, the MPO activity in the colitis model group mice was significantly increased compared to the blank group, indicating a significant increase in neutrophils in intestinal tissue and severe infiltration. After chitosan oligosaccharide intervention, the MPO activity in colon tissue decreased, although not significantly different from the model group, there was a trend of slowing down neutrophil aggregation in the intestine and reducing further tissue damage. This is consistent with Dou et al.’s discovery that chitosan oligosaccharides can promote neutrophil apoptosis and reduce MPO release in mice with glycogen peritonitis. It is speculated that this may be related to the activation of PLD and PI3K, inducing neutrophil production of superoxide and other substances.

UC is the result of multiple factors working together, among which cytokines also play an important role. IL-6 can stimulate T cell proliferation at low concentrations, activate B cell proliferation, secrete antibodies, and participate in the body’s inflammatory response at high concentrations. TNF-α， Also known as tumor necrosis factor alpha, it is produced by activated macrophages II monocytes and promotes apoptosis. IL-6 and TNF – α, as two important inflammatory cytokines, are the most rapid and significant inflammatory factors that respond to pathogenic stimuli in the body. They are commonly found in various pathological processes such as immune diseases, inflammatory responses, and tumor immunity. In the colonic mucosal tissue of UC patients, the expression of IL-6 and TNF – α is much higher in the active phase than in the remission phase, and they also play important roles in inflammation and immune response of intestinal mucosa, and are considered as key cytokines mediating IBD. Nuclear factor kappa B (NF – κ B) is an important transcription factor related to the expression of inflammatory cytokines. When the body is stimulated, I κ B can be activated and degraded, activating NF – κ B in the cytoplasm and entering the nucleus, inducing gene transcription, and generating various inflammatory or chemokines, such as IL-1 β, IL-6, IL-12, and TNF – α, which are further exacerbated through feedback amplification. Youself et al. believe that chitooligosaccharides alleviate inflammatory reactions in the body by inhibiting the NF – κ B pathway. In this experiment, the expression of IL-6 and TNF – α in the colon tissue of ulcerative colitis mice was significantly increased, while intervention with low and high doses of chitosan oligosaccharides could significantly reduce their expression levels, consistent with the results of Yang and Youself et al. Indicating that oligosaccharides can effectively improve colitis symptoms by alleviating inflammatory reactions.

By comparing the experimental results of two doses of chitosan oligosaccharides, it was found that both doses of chitosan oligosaccharides slowed down DSS induced weight loss, colon shortening, and inhibited disease activity index, as well as increased MPO activity. They significantly improved histopathological changes and increased tissue TNF – α and IL-6 levels. Among them, the low-dose group and high-dose group had better effects in alleviating colon length and histopathological changes. This indicates that the low-dose group of COS has a better effect in alleviating colitis symptoms. Yousef et al. treated colitis mice with COS at doses of 1, 5, 10, 20, 50, and 100 mg/kg and found that 10 and 20 mg/kg COS had the best effect. Similar bell shaped relationships between COS dose and effect were also observed in T84 cells induced by LPS and TNF – α, suggesting that COS may stimulate two different signaling pathways, produce opposite effects, and cause functional antagonism. The relationship between dose effect was also found in previous studies in our laboratory. Ding applied COS at doses of 35, 70, 140, and 210 mg/kg to alcoholic fatty liver mice, and found that the 70 and 140 mg/kg groups had better protective effects. The 70 and 140 mg/kg COS selected in this article may be located in the middle and back of the bell shape, and the experimental results show that the low-dose group has slightly better effects than the high-dose group.

In summary, chitosan oligosaccharides can effectively alleviate DSS induced colitis symptoms. Among them, 70 mg/kg chitosan oligosaccharides are more effective in relieving colon length and histopathological changes compared to 140 mg/kg, and are more suitable as the recommended dosage for human use. Meanwhile, chitosan oligosaccharides may play a role in reducing inflammation and alleviating IBD symptoms by alleviating neutrophil infiltration and inhibiting the release of pro-inflammatory cytokines. However, the specific mechanism of action of chitooligosaccharides in inhibiting the release of inflammatory cytokines still needs further verification.