Application of Mucor racemosus and its metabolites in preparing drugs for preventing and / or treating ulcerative colitis

Abstract

The present invention provides the application of Mucor racemosus and its metabolites in the preparation of a drug for preventing and / or treating ulcerative colitis, and belongs to the field of biomedicine technology. Mucor racemosus CGMCC NO: 40320 and its metabolite 5mc (5-methylcytosine) can significantly improve the problem of reduced disease index, body weight, and immune-related organ (such as spleen) index in mice with ulcerative colitis, and can alleviate the inflammation of colon tissue, improve the integrity of the intestinal wall of intestinal tissue, and can be used for the preparation of a drug for preventing and / or treating ulcerative colitis. The present invention expands the application field of Mucor racemosus and provides guidance for its clinical application in the treatment of colitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of Mucor racemosus and its metabolites in the preparation of drugs for preventing and / or treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a nonspecific, chronic, and intractable intestinal inflammatory disease. Long-term treatment carries a risk of cancer, and the incidence of cancer increases with duration. Clinical manifestations of UC are primarily diarrhea, bloody and mucus-purulent stools, and abdominal pain. The disease is characterized by recurrent exacerbations and remissions, severely impacting patients' quality of life. Severe cases or complications can be life-threatening. Currently, the treatment of UC primarily relies on pharmacological interventions, with drugs primarily focusing on anti-inflammatory, immunomodulatory, and biologic agents, such as 5-aminosalicylic acid, glucocorticoids, and monoclonal antibodies. However, these drugs have limitations, including unstable efficacy, high relapse rates after discontinuation, side effects such as liver and kidney damage and even bone marrow suppression, increased potential infection risks, and high treatment costs. Consequently, there is a lack of safe, stable, effective, and universally available treatments for UC.

[0003] The exact cause of ulcerative colitis remains unclear, but it may be related to multiple factors, including genetics, immunity, environment, and infection. However, numerous studies have shown that it is associated with the pathological interaction between the immune system and intestinal commensal bacteria. Gut microbial imbalance often leads to a high incidence of ulcerative colitis. Gut microbes play a beneficial role in regulating immune responses and improving ulcerative colitis and other infectious diseases. This beneficial effect is not limited to intestinal tissue but can also extend to extraintestinal organs, enhancing the response of immune cells and thus helping to treat extraintestinal diseases. For example, Bifidobacterium adolescentis can alleviate colitis by inducing protective Treg and Th2 cells (Fan, Lina et al. "B.adolescentisameliorates chronic colitis by regulating Treg / Th2 response and gutmicrobiotaremodeling." Gut microbes vol.13, 1 (2021): 1-17.doi: 10.1080 / 19490976.2020.1826746); Chinese patent CN118497080A discloses that a fermented mucus lactobacillus is used to regulate intestinal oxidative stress levels and improve the structure of intestinal microbiota to alleviate ulcerative colitis in mice. In addition to using microorganisms themselves, there are also studies on the use of microbial metabolites to regulate immune responses. For example, tryptophan indole derivatives produced by Lactobacillus reuteri can stimulate intestinal differentiation to produce CD4 + CD8αα + Double-positive intraepithelial lymphocytes, thereby improving inflammatory bowel disease (Cervantes-Barragan, Luisa et al. "Lactobacillus reuteriinduces gutintraepithelial CD4 + CD8αα + T cells." Science (New York, NY) vol. 357, 6353 (2017): 806-810. doi: 10.1126 / science.aah5825), etc. Although the above-mentioned microorganisms and microbial metabolites have shown unlimited potential in alleviating ulcerative colitis, their safety in clinical application has not been effectively confirmed.

[0004] In addition to the probiotics mentioned above, fungi also live in the intestines. Although fungi only account for 0.01% to 0.1% of the intestinal microbiome, they play an indispensable role in maintaining the dynamic balance of the intestinal microbiome. However, there are currently no studies on the use of fungi and their metabolites in the treatment of enteritis. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a use of Mucor racemosus and its metabolites in the preparation of drugs for preventing and / or treating ulcerative colitis, expand the application field of Mucor racemosus, and provide safe, reasonable and effective drugs for the clinical treatment of ulcerative colitis.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides use of Mucor racemosus or a preparation containing Mucor racemosus in preparing a medicament for preventing and / or treating ulcerative colitis. The deposit number of the Mucor racemosus is CGMCCNO: 40320.

[0008] The present invention also provides the use of a metabolite of Mucor racemosus or a preparation containing the metabolite of Mucor racemosus in preparing a drug for preventing and / or treating ulcerative colitis. The metabolite of Mucor racemosus includes 5-methylcytosine.

[0009] Furthermore, the drug is any one or more of the following drugs:

[0010] 1) The drug is a drug for alleviating weight loss in patients with ulcerative colitis;

[0011] 2) The drug is a drug for increasing the length of the colon in patients with ulcerative colitis;

[0012] 3) The drug is a drug that has the ability to regulate immunity;

[0013] 4) The drug is an anti-inflammatory drug;

[0014] 5) The drug is a drug with antioxidant capacity;

[0015] 6) The drug is a drug that increases the integrity of the intestinal wall of colon tissue.

[0016] Among them, 3) the drug with immunomodulatory ability can reduce the spleen index of patients with ulcerative colitis.

[0017] Among them, 4) the drug with anti-inflammatory effect can reduce the content of any one or more of IL-6, IL-1β and TNF-α in ulcerative colitis tissue.

[0018] Among them, 5) the drug with antioxidant ability can reduce the content of ROS in ulcerative colitis tissue.

[0019] Among them, 6) the drug for increasing the integrity of the intestinal wall of colon tissue can increase the expression level of Claudin-2 in ulcerative colitis tissue.

[0020] The present invention also provides a medicine for preventing and / or treating ulcerative colitis, which comprises one or both of Mucor racemosus and 5-methylcytosine.

[0021] In the present invention, the ulcerative colitis includes remission, mild, moderate, severe or acute severe ulcerative colitis.

[0022] Preferably, the drug for preventing and / or treating ulcerative colitis comprises an oral preparation.

[0023] The present invention provides the use of Mucor racemosus and its metabolites in the preparation of a drug for preventing and / or treating ulcerative colitis. The deposit number of the Mucor racemosus is CGMCC NO: 40320. The present invention proves that Mucor racemosus and its metabolite 5mc (5-methylcytosine) have a good effect on ulcerative colitis through experiments on an ulcerative colitis animal model. Experiments have confirmed that Mucor racemosus and 5mc can alleviate the problem of weight loss in mice with ulcerative colitis and increase the colon length of mice with ulcerative colitis. At the same time, Mucor racemosus and 5mc have the ability to regulate immunity, which can reduce the spleen index and disease index of mice with colitis, thereby alleviating the symptoms and severity of colitis. In addition, Mucor racemosus and its metabolite 5mc have certain anti-inflammatory effects and can significantly reduce the levels of ROS, IL-6, IL-1β and TNF-α in the colon tissue of ulcerative colitis. In addition, Mucor racemosus and 5mc can significantly increase the expression of Claudin-2 in colitis tissue, thereby increasing the integrity of the intestinal wall of the colon tissue. The present invention reveals the action pathways of Mucor racemosus and its metabolites in preventing and / or treating ulcerative colitis, providing important clues for exploring the impact of fungi and their metabolites on human health, and also offering new ideas for their clinical application in the treatment of colitis.

[0024] The present invention also provides a drug for preventing and / or treating ulcerative colitis, which expands the application field of Mucor racemosus and also provides a new, safe and effective drug for treating ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Effect of Mucor racemosus on body weight of mice with colitis, *** represents p < 0.001.

[0026] Figure 2 Effect of Mucor racemosus on colon length in mice with colitis, *** represents p < 0.001.

[0027] Figure 3 Effects of Mucor racemosus on the spleen of colitis mice, *** represents p < 0.001.

[0028] Figure 4Effect of Mucor racemosus on the disease index of colitis mice: the disease index of the blank group was 0. Compared with the blank group, the model group had a disease index of ***, which indicated p<0.001 and ns indicated no significant difference. Compared with the blank group, the Mucor racemosus-treated group had a disease index of #, which indicated p<0.05 and ## indicated p<0.01 and ns indicated no significant difference.

[0029] Figure 5 Effect of Mucor racemosus on ROS content in the colon of mice with colitis, *** represents p < 0.001.

[0030] Figure 6 Effects of Mucor racemosus on the levels of IL-6, IL-1β, and TNF-α in the colon of mice with colitis. *** represents p < 0.001.

[0031] Figure 7 Effect of Mucor racemosus on the expression of Claudin-2, a marker of colon wall integrity in mice with colitis. *** represents p < 0.001.

[0032] Figure 8 Statistical results of the metabolite 5mc content in the culture medium of Mucor racemosus in vitro. *** represents p < 0.001.

[0033] Figure 9 Statistical results of 5mc content in feces of colitis mice after oral administration of Mucor racemosus. *** represents p < 0.001.

[0034] Figure 10 Effect of 5mc, a metabolite of Mucor racemosus, on body weight in mice with colitis. *** represents p < 0.001.

[0035] Figure 11 Effect of 5mc, a metabolite of Mucor racemosus, on colon length in mice with colitis. *** represents p < 0.001.

[0036] Figure 12 Effects of 5mc, a metabolite of Mucor racemosus, on the disease index of colitis mice: compared with the blank group, *** represents p<0.001, and ns represents no significant difference; compared with the blank group, # represents p<0.05, ## represents p<0.01, and ns represents no significant difference.

[0037] Figure 13 Effect of 5mc, a metabolite of Mucor racemosus, on ROS content in the colon of mice with colitis. *** represents p < 0.001.

[0038] Figure 14 Effects of 5mc, a metabolite of Mucor racemosus, on the levels of IL-6, IL1-β, and TNF-α in the colon of mice with colitis. *** indicates p < 0.001.

[0039] Figure 15 Effect of 5mc, a metabolite of Mucor racemosus, on the expression of Claudin-2, a marker of colon wall integrity, in mice with colitis. *** represents p < 0.001. DETAILED DESCRIPTION

[0040] The present invention provides the use of Mucor racemosus or a preparation containing Mucor racemosus in the preparation of a drug for preventing and / or treating ulcerative colitis. In the present invention, the Mucor racemosus used is classified and named Mucor racemosus, and is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with the deposit address at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on September 19, 2022, with a deposit number of CGMCCNO.40320 (disclosed in patent CN116286390A).

[0041] In the present invention, the preparation containing Mucor racemosus is a preparation containing the strain CGMCC NO. 40320, and the preparation can be an oral preparation such as a suspension, lyophilized powder, tablet, capsule, pill, oral liquid, etc. containing Mucor racemosus CGMCC NO. 40320. In the present invention, the preparation containing Mucor racemosus further includes pharmaceutically acceptable excipients, including one or more of a diluent, a colorant, a sweetener, a coating agent, a binder, an absorbent, a disintegrant, a dispersant, a wetting agent, a solubilizer, a buffer, and a surfactant.

[0042] The present invention also provides the use of a metabolite of Mucor racemosus or a preparation containing a metabolite of Mucor racemosus in preparing a medicament for preventing and / or treating ulcerative colitis. The metabolite of Mucor racemosus is preferably 5-methylcytosine, a metabolite produced by Mucor racemosus with a deposit number of CGMCC No. 40320. The preparation containing the metabolite of Mucor racemosus contains 5-methylcytosine, which is produced by the metabolism of Mucor racemosus with a deposit number of CGMCC No. 40320 and can also be prepared by chemical methods.

[0043] In the present invention, the C57BL / 6J mouse model was used as the experimental material to verify the preventive and therapeutic effects of CGMCC NO.40320 Mucor racemosus and its metabolite 5-methylcytosine on ulcerative colitis. The present invention has no special restrictions on the method for constructing the C57BL / 6J mouse model, and the ulcerative colitis model can be constructed using dextran sulfate sodium salt well known in the art. The body weight, colon length, spleen index and disease index of the ulcerative colitis mouse model were calculated, and the contents of reactive oxygen species (ROS), inflammatory factors (IL-6, IL-1β and TNF-α) in the colon tissue and the expression of the colon wall integrity marker Claudin-2 were detected. The results showed that after 5 days of oral administration of Mucor racemosus or 5-methylcytosine, the weight of mice with ulcerative colitis increased significantly, the colon length increased significantly, the disease progression index decreased significantly with the extension of modeling time, the ROS content and the inflammatory-related factors IL-6, IL1-β and TNF-α in the colon tissue decreased, and the expression level of the colon wall integrity marker gene Claudin-2 increased significantly. Therefore, the Mucor racemosus or the preparation containing Mucor racemosus, the metabolite 5-methylcytosine of Mucor racemosus or the preparation containing 5-methylcytosine can effectively treat ulcerative colitis, alleviate weight loss in patients with ulcerative colitis, increase the length of the colon in patients with ulcerative colitis, have the ability to regulate immunity, anti-inflammatory and antioxidant, and can also increase the integrity of the colon tissue wall.

[0044] The present invention also provides a kind of medicine for preventing and / or treating ulcerative colitis, and the medicine includes one or both of Mucor racemosus and 5-methylcytosine.According to " China's Guidelines for the Diagnosis and Treatment of Ulcerative Colitis (2023 Xi'an) " record, according to the severity assessment of ulcerative colitis, ulcerative colitis is divided into active phase and remission phase, wherein the active phase is divided into mild, moderate, severe and acute severe.The medicine for preventing and / or treating ulcerative colitis provided by the present invention can be directed to various types of ulcerative colitis, including remission phase, mild, moderate, severe or acute severe ulcerative colitis.In addition, the pharmaceutical dosage form for preventing and / or treating ulcerative colitis includes oral preparations, for example, lyophilized powder, tablets, capsules, pills, oral liquids etc.

[0045] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the application of the Mucor racemosus and its metabolites provided by the present invention in the preparation of drugs for preventing and / or treating ulcerative colitis is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0046] In the examples of the present invention, all data were statistically analyzed using GraphPad Prism 8.0 software, and the measurement data were expressed as mean ± standard deviation. Inter-group comparisons were performed using one-way ANOVA or two-way ANOVA.

[0047] In the following examples, unless otherwise specified, all methods are conventional.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] Example 1 Preparation of Mucor racemosus suspension

[0050] Mucor racemosus (Mucor racemosus f. racemosus, deposit number CGMCC3.4941, purchased from the China General Microbiological Culture Collection Center) was aseptically inoculated onto a PDA medium covered with cellophane and incubated aerobically at 30°C for 48 to 96 hours until the medium was fully grown with Mucor racemosus. The PDA medium was a potato dextrose agar medium composed of 6 g / L potato extract powder, 20 g / L glucose, and 20 g / L agar.

[0051] Fresh feces from healthy 6-8 week old C57BL / 6J mice were collected and sterile saline was added at a ratio of 0.1 g feces to 1 mL of saline, mixed thoroughly, and 300 μL of the prepared fecal solution was evenly spread on a PDA+BHI medium. A piece of cellophane covered with Mucor racemosus was transferred to the PDA+BHI medium containing the fecal solution. The culture was incubated at 37°C under strict anaerobic conditions for 5 days for acclimation. On the third day of acclimation, the PDA+BHI medium with fresh fecal solution was replaced. The BHI medium was brain heart infusion broth, composed of 10 g / L peptone, 12.5 g / L dehydrated calf extract powder, 5 g / L dehydrated buffalo heart extract powder, 5 g / L sodium chloride, 2 g / L glucose, and 2.5 g / L disodium hydrogen phosphate. The PDA+BHI medium is prepared by mixing and sterilizing PDA medium powder and BHI medium powder in a ratio of 13:4, that is, 39g PDA and 52g BHI medium are weighed and mixed evenly, 1L distilled water is added, and the mixture is sterilized at 121°C and high temperature and high pressure for 15min.

[0052] On the 5th day, the domesticated Mucor racemosus was obtained and scraped off the cellophane for preservation. The preservation number is CGMCC NO: 40320. Sterile physiological saline and grinding beads were added to the above Mucor racemosus for grinding. After quantitative preparation, 10 8 CFU / mL of Mucor racemosus suspension.

[0053] Example 2 Effects of Mucor racemosus on Mice with Ulcerative Colitis

[0054] 1. Materials and Methods

[0055] 1.1 Experimental Materials

[0056] Male C57BL / 6J mice, 6–8 weeks old and weighing 19–21 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. All mice were housed in a SPF-grade laboratory at the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (preliminary: 22 ± 2°C, humidity: 50%–60%, 12:12 hr light-dark cycle). They were housed for one week before the experiment and had free access to food and water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures were in strict accordance with the Manual for the Care and Use of Laboratory Animals of Nankai University.

[0057] 1.2 Dosage experimental plan

[0058] Healthy C57BL / 6J mice were randomly divided into three groups according to their body weight, with 6 mice in each group.

[0059] Model Establishment: A 3% dextran sulfate sodium salt (DSS) solution was used to establish a colitis model in mice. Preparation: Weigh 3 grams of dextran sulfate sodium salt powder and dissolve it in 100 mL of sterile water. Shake thoroughly and filter through a 0.22 μm filter to sterilize the solution. This yields a 3% dextran sulfate sodium salt solution.

[0060] Blank group: The mice were given normal drinking water. On the 6th day, the drugs were administered by gavage with normal saline for 5 consecutive days. The gavage dose was 100 μL / mouse / time.

[0061] Model group: mice were given 3% DSS solution and allowed to drink DSS solution freely for 7 days. The drug was started on the 6th day of modeling, and normal saline was gavaged for 5 consecutive days. The gavage dose was 100 μL / mouse / time.

[0062] Mucor racemosus administration group: 3% DSS solution was administered to mice, and the mice were allowed to drink the DSS solution freely for 7 days. The administration began on the 6th day of modeling, and 10% of the prepared in Example 1 were administered orally every day. 8 CFU / mL of Mucor racemosus suspension was administered orally for 5 consecutive days with a dose of 100 μL per animal per time.

[0063] After feeding, samples are taken separately, and the samples are processed and stored according to different samples and subsequent experimental requirements to meet the needs of subsequent testing and analysis.

[0064] 2. Effect of Mucor racemosus on the body weight of mice with ulcerative colitis

[0065] Mouse weight is one of the indicators for determining the severity of ulcerative colitis and is related to the health of the mice. Ulcerative colitis causes severe weight loss in mice. Therefore, before establishing the mouse model (i.e., on day 0), the initial weight of each mouse was recorded. The weight of each mouse was then recorded daily thereafter.

[0066] The results are as follows Figure 1 The results showed that compared with the normal mice in the blank group, the weight of the model group mice decreased significantly 10 days after the start of modeling. Compared with the model group mice, the weight of the mice in the Mucor racemosa treatment group increased significantly 10 days after the start of modeling, and showed a trend of recovering to the weight of the blank group mice. Conclusion: Mucor racemosa can significantly alleviate the weight loss of colitis mice and has a certain therapeutic effect on ulcerative colitis.

[0067] 3. Effect of Mucor racemosus on colon length in mice with ulcerative colitis

[0068] Since the length of the mouse colon is one of the indicators for judging the severity of ulcerative colitis, that is, ulcerative colitis causes a severe decrease in the length of the mouse colon, the mice were euthanized on day 10, and the mouse colon tissue was dissected and measured for length comparison. The colon length was also measured and photographed.

[0069] The results are as follows Figure 2 As shown, compared with the normal mice in the blank group, the colon length of the model group mice was significantly reduced. Compared with the model group mice, the colon length of the mice in the Mucor racemosa treatment group was significantly increased, approaching the colon length of the blank group mice. Conclusion: Mucor racemosa can significantly increase the colon length of mice with colitis and alleviate the colon shortening caused by ulcerative colitis.

[0070] 4. Effects of Mucor racemosus on the spleen of mice with ulcerative colitis

[0071] Because colitis causes spleen enlargement in mice, the spleen index (SLI) = spleen weight / mouse body weight. This is one indicator of the severity of ulcerative colitis, indicating that ulcerative colitis increases the SLI in mice. Therefore, mice were weighed on day 10 and euthanized. Spleen tissue was then dissected and weighed to calculate the SLI.

[0072] The results are as follows Figure 3 As shown, compared with the normal mice in the blank group, the spleen index of the model group mice was significantly increased. Compared with the model group mice, the spleen index of the mice treated with Mucor racemosa was significantly reduced. At the same time, the spleen index of the mice treated with Mucor racemosa was close to that of the mice in the blank group. Conclusion: Mucor racemosa can significantly reduce the spleen index of mice with colitis and play a regulatory role in the immune system.

[0073] 5. Effect of Mucor racemosus on the disease index of mice with ulcerative colitis

[0074] The colitis disease index can intuitively show the degree of colitis development in mice. The disease index is a comprehensive score of three indicators: weight loss, stool viscosity, and blood in stool. Starting from the sixth day of the initial administration of Mucor racemosus, the above three indicators are tested daily and scored according to Table 1. A higher score indicates a more severe disease.

[0075] Table 1 Disease index scoring parameters

[0076]

[0077] The results are as follows Figure 4 The disease index of the blank group (wild-type mice) remained at 0. Compared with the blank group, the disease index of the model group increased significantly with increasing modeling time. Compared with the model group, the disease index of the mice treated with Mucor racemosa significantly decreased with increasing modeling time. Conclusion: Mucor racemosa can significantly reduce the disease index of mice with colitis and alleviate the symptoms and severity of colitis.

[0078] 6. Effect of Mucor racemosus on ROS content in the colon of mice with ulcerative colitis

[0079] Because colitis causes a large amount of ROS to be produced in colon tissue cells, excessive ROS can cause DNA mutations and mitochondrial dysfunction, thereby damaging biological molecules and cells and leading to cell death. At the same time, ROS also dominates the oxidative stress in the body. When the production of ROS exceeds the antioxidant capacity of the cells, it will lead to an oxidative stress state, causing damage to cellular macromolecules (such as lipids, proteins and DNA). Therefore, the ROS content in colon tissue was measured to detect the effect of Mucor racemosus on the ROS content in colon tissue. After the mice were euthanized on the 10th day, the mice were dissected and the mouse colon tissue was taken. The tissue was added with an appropriate amount of normal saline, that is, 1 mL of normal saline was added to 0.1 g of tissue, the tissue was crushed, centrifuged at 3000g / min for 20 minutes, and the supernatant was taken. A ROS detection kit (ELISA, China) was used to detect and obtain the ROS content in the supernatant.

[0080] The results are as follows Figure 5 As shown in the results, compared with the normal mice in the blank group, the ROS content in the colon of the model group mice was significantly increased. Compared with the model group mice, the ROS content in the colon of the mice treated with Mucor racemosus was significantly reduced, approaching the ROS content in the colon of the mice in the blank group. Conclusion: Mucor racemosus can significantly reduce the ROS content in the colon tissue of mice with colitis, reflecting that Mucor racemosus has a certain antioxidant capacity.

[0081] 7. Effect of Mucor racemosus on IL-6, IL-1β, and TNF-α levels in the colon of mice with ulcerative colitis

[0082] Because IL-6, IL-1β, and TNF-α are indicators of colitis and reflect the extent of inflammation in the mouse colon, the levels of IL-6, IL-1β, and TNF-α in colonic tissue were measured to examine the effects of Mucor racemosus on colonic inflammation. IL-6, IL-1β, and TNF-α assays (ELISA, China) were used to measure the levels of these three factors in colonic tissue.

[0083] The results are as follows Figure 6 As shown, compared with the blank group, the levels of IL-6, IL-1β, and TNF-α in the colon of the model group mice were significantly increased; compared with the model group, the levels of IL-6, IL-1β, and TNF-α in the colon of the mice treated with Mucor racemosus were significantly decreased, approaching the levels of IL-6, IL-1β, and TNF-α in the colon of the blank group mice. Conclusion: Mucor racemosus can significantly reduce the levels of IL-6, IL-1β, and TNF-α in the colon tissue of mice with colitis, exerting an anti-inflammatory effect, confirming that Mucor racemosus can treat ulcerative colitis from an anti-inflammatory perspective.

[0084] 8. Effect of Mucor racemosus on the expression of Claudin-2, a marker of colon wall integrity in colitis mice

[0085] Since colitis causes severe damage to the colon tissue of mice, that is, the integrity of the colon wall is destroyed, the expression level of Claudin-2 (tight junction protein-2), a marker gene for colon wall integrity, can be detected to determine the integrity of the colon wall.

[0086] The results are as follows Figure 7 As shown, compared with the normal mice in the blank group, the colonic Claudin-2 content of the model group mice was significantly reduced; compared with the model group mice, the colonic Claudin-2 content of the mice treated with Mucor racemosus was significantly increased, approaching the colonic Claudin-2 content of the mice in the blank group. Conclusion: Mucor racemosus can significantly improve the integrity of the colonic tissue wall of colitis mice and effectively delay the progression of ulcerative colitis.

[0087] Example 3 Detection of 5-methylcytosine, a metabolite produced in vitro and in vivo by Mucor racemosus

[0088] 1. Propagation of Mucor racemosus with different inoculum sizes

[0089] PDB and BHI medium powders were mixed in a ratio of 6:13 and sterilized to prepare PDB+BHI liquid medium. 24 g of PDB and 52 g of BHI medium were weighed and mixed evenly. 1 L of distilled water was added and sterilized at 121°C for 15 min. The suspension of Mucor racemosus prepared in Example 1 was inoculated into the PDB+BHI liquid medium at different inoculum sizes, i.e., the inoculum units were 5×10 6 / 100mL, 10 7 / 100mL, 2×10 7 / 100mL. Three different groups of Mucor racemosus suspensions were obtained under strict anaerobic conditions at 37°C for 3 days: low, medium, and high inoculum sizes. The PDB medium is potato dextrose medium, consisting of 6g / L potato extract powder and 20g / L glucose.

[0090] 2. Detection

[0091] The content of 5-methylcytosine produced by Mucor racemosus in the host and in vitro was determined using a 5-methylcytosine detection kit (ELISA, China).

[0092] The three groups of suspensions of Mucor racemosus with different inoculation amounts were collected, centrifuged at 3000 g / min for 20 minutes, and the supernatant was taken. In addition, the feces of the three groups of mice in the blank group, model group, and Mucor racemosus administration group in Example 1 on the 10th day were taken, and the mouse feces were added with an appropriate amount of normal saline, that is, 1 mL of normal saline was added to 0.1 g of feces, and the feces were crushed and centrifuged at 3000 g / min for 20 minutes, and the supernatant was taken.

[0093] Dilute the six sets of supernatants 1:1 with sample diluent and add 50 μL to each reaction well. Add 50 μL of the diluted standard to each well, and then add 50 μL of the supernatant sample to each well. Immediately add 50 μL of the biotinylated antibody. Cover the plate, gently vortex to mix, and incubate at 37°C for 1 hour. Discard all liquid from the wells, fill each well with wash buffer, vortex for 30 seconds, discard the wash buffer, and pat dry with absorbent paper. Repeat this process three times. If using a plate washer, add one additional wash cycle. Add 80 μL of streptavidin-HRP to each well, gently vortex to mix, and incubate at 37°C for 30 minutes. Discard all liquid from the wells, fill each well with wash buffer, vortex for 30 seconds, discard the wash buffer, and pat dry with absorbent paper. Repeat this process three times. If using a plate washer, add one additional wash cycle. Add 50 μL each of substrates A and B to each well, gently shake to mix, and incubate at 37°C for 10 minutes. Protect from light. Remove the plate and quickly add 50 μL of stop solution. Measure the results immediately after adding the stop solution. Measure the OD value of each well at a wavelength of 450 nm. Create a curve with the absorbance OD value as the vertical axis (Y) and the corresponding concentration of the standard substance to be tested as the horizontal axis (X). The content of the test substance in the sample can be calculated from the standard curve based on its OD value.

[0094] The results are as follows Figure 8 and Figure 9 As shown in the results, when Mucor racemosus was cultured in liquid culture medium in vitro, the amount of 5-methylcytosine produced by it also increased significantly with the increase in the inoculum size of Mucor racemosus, indicating that 5-methylcytosine is a metabolite of Mucor racemosus. In addition, in vivo animal experiments found that the content of 5-methylcytosine in the feces of mice in the model group was significantly reduced compared with the blank group. Compared with the model group, the content of 5-methylcytosine in the feces of mice in the Mucor racemosus administration group was significantly increased, close to the content of 5-methylcytosine in the feces of mice in the blank group. Therefore, Mucor racemosus can metabolize and produce 5-methylcytosine both in the host body and in vitro culture, which also confirms that 5-methylcytosine is a metabolite of Mucor racemosus.

[0095] Example 4 Effects of 5mc, a metabolite of Mucor racemosus, on mice with ulcerative colitis

[0096] 1. Materials and Methods

[0097] 1.1 Experimental Materials

[0098] Same as Example 2, "1.1 Experimental Materials".

[0099] 1.2 Experimental methods

[0100] Healthy C57BL / 6J mice were randomly divided into three groups according to their body weight, namely blank group, model group and 5mc gavage group, with 6 mice in each group.

[0101] The model establishment and administration method were the same as those in Example 2, "1.2 Administration Experimental Plan", except that the 5mc gavage group was gavaged with 2 mg / mL 5mc starting on the 6th day of modeling and continued for 5 consecutive days at a gavage dose of 100 μL / animal / time.

[0102] After 10 days of feeding, samples were taken and processed and stored according to different samples and subsequent experimental requirements to meet the needs of subsequent testing and analysis.

[0103] 2. Detection

[0104] The detection indicators and operating methods are the same as those in items 2 to 8 under Example 2, specifically including mouse weight, colon length, disease index, ROS and inflammatory factor (IL-6, IL1-β and TNF-α) content, and Claudin-2 expression detection items.

[0105] The results are as follows Figures 10-15 As shown, compared with the normal mice in the blank group, the model group had a significant decrease in body weight and a significant decrease in colon length. Furthermore, the disease progression index of the mice increased significantly with the prolongation of modeling time. The levels of ROS and the inflammatory factors IL-6, IL1-β, and TNF-α in colon tissue increased significantly, while the expression of Claudin-2, a gene that indicates colon wall integrity, decreased significantly. However, compared with the model group, the body weight and colon length of the mice in the 5-methylcytosine gavage group increased significantly. The disease progression index decreased significantly with the prolongation of modeling time. The levels of ROS and the inflammatory factors IL-6, IL1-β, and TNF-α in colon tissue decreased, while the expression of Claudin-2, a gene that indicates colon wall integrity, increased significantly. Conclusion: 5-methylcytosine, a metabolite of Mucor racemosus, is an effective treatment for ulcerative colitis.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of Mucor racemosus or a preparation containing Mucor racemosus in the preparation of a medicament for preventing and / or treating ulcerative colitis, characterized in that: The deposit number of the Mucor racemosus is CGMCC NO: 40320.

2. The use according to claim 1, characterized in that The drug is any one or more of the following drugs: 1) The drug is a drug for alleviating weight loss in patients with ulcerative colitis; 2) The drug is a drug for increasing the length of the colon in patients with ulcerative colitis; 3) The drug is a drug that has immune regulation effects; 4) The drug is an anti-inflammatory drug; 5) The drug is a drug with antioxidant capacity; 6) The drug is a drug that increases the integrity of the intestinal wall of colon tissue.

3. The use according to claim 2, characterized in that 3) The drug having the ability to regulate immunity can reduce the spleen index of patients with ulcerative colitis.

4. The use according to claim 2, characterized in that 4) The drug with anti-inflammatory effect can reduce the content of any one or more of IL-6, IL-1β and TNF-α in ulcerative colitis tissue.

5. The use according to claim 2, characterized in that 5) The drug with antioxidant capacity can reduce the content of ROS in ulcerative colitis tissue.

6. The use according to claim 2, characterized in that 6) The drug for increasing the integrity of the intestinal wall of colon tissue can increase the expression of Claudin-2 in ulcerative colitis tissue.

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