Application of mucor racemosus and metabolite thereof in preparation of medicine for preventing and / or treating ulcerative colitis

Abstract

The invention provides application of Mucor racemosus and a metabolite of the Mucor racemosus in preparation of a medicine for preventing and / or treating ulcerative colitis, and belongs to the technical field of biological medicine. The mucor racemosus CGMCC NO: 40320 and the metabolite 5mc (5-methylcytosine) of the mucor racemosus CGMCC NO: 40320 can remarkably improve the problem that the disease index, the weight and the index of immune related organs (such as the spleen) of a mouse with the ulcerative colitis disease are reduced, can relieve inflammation of colon tissue, improves the integrity of the intestinal wall of the intestinal tissue, and improves the curative effect of the mouse with the ulcerative colitis disease. The compound can be used for preparing medicines for preventing and / or treating ulcerative colitis. The application field of the mucor racemosus is expanded, and guidance is provided for clinical application of the mucor racemosus in colitis treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application 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 non-specific, chronic, and difficult-to-heal intestinal inflammation. If it is not cured for a long time, there is a risk of cancer, and its canceration rate gradually increases with the duration of illness. The clinical manifestations of ulcerative colitis are mainly diarrhea, mucus, pus, and blood in the stool, and abdominal pain. The disease is characterized by repeated attacks and remissions, which seriously affects the quality of life of patients. When the disease is severe or complications occur, it may be life-threatening. At present, the treatment of ulcerative colitis is mainly based on drug intervention, and the drugs for the treatment of ulcerative colitis are mainly concentrated in anti-inflammatory, immunomodulatory, and biological agents, such as 5-aminosalicylic acid, glucocorticoids, and monoclonal antibodies. However, these drugs have certain limitations, such as unstable efficacy, easy recurrence after discontinuation of medication, side effects such as liver and kidney damage and even bone marrow suppression, increased potential infection risks, and high treatment costs. Therefore, there is a lack of safe, stable, effective, and universal treatment drugs for ulcerative colitis in clinical practice.

[0003] The exact cause of ulcerative colitis is still unclear. It may be related to multiple factors such as genetics, immunity, environment, and infection. However, a large number of studies have shown that it is related to the pathological interaction between the immune system and intestinal symbiotic bacteria. Intestinal microbial imbalance usually leads to a high incidence of ulcerative colitis. Intestinal microorganisms play a beneficial role in regulating immune responses, improving ulcerative colitis and other infectious diseases. This beneficial effect is not limited to intestinal tissues, but can also extend to extra-intestinal organs, enhancing the response ability of immune cells, thereby helping to treat extra-intestinal diseases. For example, Bifidobacterium adolescentis can relieve 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 relieve 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, there are also fungi in the intestine. Although fungi only account for 0.01% to 0.1% of the intestinal microbiota, they play an indispensable role in maintaining the dynamic balance of the intestinal microbiota. However, there is no research on 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 an application 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 invention provides application of Mucor racemosus or a preparation containing Mucor racemosus in preparing a medicine 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 the metabolites of Mucor racemosus or the preparation containing the metabolites of Mucor racemosus in the preparation of drugs for preventing and / or treating ulcerative colitis, wherein the metabolites of Mucor racemosus include 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 a drug with anti-inflammatory effect;

[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 an application of Mucor racemosus and its metabolites in the preparation of a drug for preventing and / or treating ulcerative colitis, and 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 an ulcerative colitis animal model experiment. Experiments have confirmed that Mucor racemosus and 5mc can alleviate the problem of weight loss in mice with ulcerative colitis, and can also increase the colon length of mice with ulcerative colitis. At the same time, Mucor racemosus and 5mc have the ability to regulate immunity, can reduce the spleen index and disease index of colitis mice, and thus reduce the symptoms and severity of colitis. In addition, Mucor racemosus and its metabolite 5mc have a certain anti-inflammatory effect, which can significantly reduce the content 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 colon tissue. The present invention reveals the action pathway of Mucor racemosus and its metabolites in preventing and / or treating ulcerative colitis, provides important clues for exploring the effects of fungi and their metabolites on human health, and also provides 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, *** represents p<0.001 and ns represents no significant difference in the model group; compared with the blank group, # represents p<0.05 and ## represents p<0.01 and ns represents no significant difference in the Mucor racemosus drug-treated group.

[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 colitis mice. *** represents p<0.001.

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

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

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

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

[0036] Fig.12 Effect 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 in the model group; compared with the blank group, # represents p<0.05 and ## represents p<0.01 and ns represents no significant difference in the 5mc oral administration group.

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

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

[0039] Fig.15 Effect of 5mc, a metabolite of Mucor racemosus, on the expression of Claudin-2, a marker of colon wall integrity in colitis mice. *** 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 Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit unit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit time is September 19, 2022, and the deposit number is CGMCCNO.40320 (disclosed in patent CN116286390A).

[0041] In the present invention, the preparation containing Mucor racemosus is a preparation containing the strain of Mucor racemosus 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 also includes pharmaceutically acceptable excipients, including one or more of diluents, colorants, sweeteners, coating agents, adhesives, absorbents, disintegrants, dispersants, wetting agents, cosolvents, buffers, and surfactants.

[0042] The present invention also provides the use of a metabolite of Mucor racemosus or a preparation containing the metabolite of Mucor racemosus in the preparation of a drug 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 a chemical method.

[0043] In the present invention, the C57BL / 6J mouse model is used as an 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 construction method of the C57BL / 6J mouse model, and the ulcerative colitis model can be constructed using sodium dextran sulfate well known in the art. The body weight, colon length, spleen index and disease index of the ulcerative colitis mouse model are calculated, and the contents of reactive oxygen species ROS, inflammatory factors IL-6, IL-1β and TNF-α in colon tissue and the expression of Claudin-2, a marker of colon wall integrity, are detected. The results showed that after 5 days of intragastric treatment with Mucor racemosus or 5-methylcytosine, the weight of mice with ulcerative colitis increased significantly, the length of the colon increased significantly, the disease progression index decreased significantly with the extension of modeling time, the ROS content in colon tissue and the content of inflammatory related factors IL-6, IL1-β and TNF-α decreased, and the expression 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, relieve 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 drug for preventing and / or treating ulcerative colitis, the drug including one or both of Mucor racemosus and 5-methylcytosine. According to the "Guidelines for the Diagnosis and Treatment of Ulcerative Colitis in China (2023·Xi'an)", according to the severity assessment of ulcerative colitis, ulcerative colitis is divided into active phase and remission phase, wherein the active phase is further divided into mild, moderate, severe and acute severe. The drug 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 purpose, technical scheme and advantages of the present invention more clearly understood, the application of 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 embodiments, but they should not be construed as limiting the scope of protection of the present invention.

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

[0047] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[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, with a deposit number of CGMCC3.4941, purchased from China General Microbiological Culture Collection Center) is inoculated onto a PDA medium covered with cellophane under aseptic conditions, and is statically cultured at 30°C under aerobic conditions for 48 to 96 hours until the medium is covered with Mucor racemosus, and then used. The PDA medium is a potato dextrose agar medium, and the components are 6 g / L potato extract powder, 20 g / L glucose, and 20 g / L agar.

[0051] Take fresh feces from healthy 6-8 week old C57BL / 6J mice, add sterile saline at a ratio of 0.1g feces: 1mL saline and mix thoroughly, take 300μL of the prepared fecal liquid and evenly smear it on the PDA+BHI medium, transfer the cellophane covered with Mucor racemosus to the PDA+BHI medium containing fecal liquid, and culture it statically at 37°C under strict anaerobic conditions for 5 days for acclimation culture, and replace the PDA+BHI medium smeared with fresh fecal liquid on the third day of acclimation culture. Wherein, the BHI medium is a brain heart infusion broth medium, and the components are peptone 10g / L, dehydrated calf extract powder 12.5g / L, dehydrated bull heart extract powder 5g / L, sodium chloride 5g / L, glucose 2g / L, disodium hydrogen phosphate 2.5g / L. 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, 39 g PDA and 52 g BHI medium are weighed and mixed evenly, 1 L of distilled water is added, and sterilized at 121° C. and high temperature and high pressure for 15 min.

[0052] On the fifth day, the domesticated Mucor racemosus was obtained and scraped off the glass paper for preservation, with the preservation number being CGMCC NO: 40320. Sterile physiological saline and grinding beads were added to the above Mucor racemosus for grinding, and the mixture was quantitatively prepared into 10 8 CFU / mL of Mucor racemosus suspension.

[0053] Example 2 Effect of Mucor racemosus on ulcerative colitis mice

[0054] 1. Materials and Methods

[0055] 1.1 Experimental Materials

[0056] Male C57BL / 6J mice, 6-8 weeks old, weighing 19-21 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. All mice were housed in the SPF laboratory of the Animal Center of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (preliminary: 22±2°C, humidity: 50%-60%, 12:12 hours of day and night cycle). They were raised for one week before the experiment and had free access to food and water during this period. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, and all operations in the experiment strictly followed the Manual for the Care and Use of Laboratory Animals of Nankai University.

[0057] 1.2 Drug administration protocol

[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 mouse colitis model was established using a 3% dextran sulfate sodium salt (DSS) solution. Preparation method: Weigh 3 grams of dextran sulfate sodium salt powder, dissolve it in 100 mL of sterile water, shake it evenly, and filter it with a 0.22 μm filter membrane to sterilize it, which is a 3% dextran sulfate sodium salt solution.

[0060] Blank group: The mice were given normal drinking water. The drugs were administered starting from the 6th day. The mice were gavaged 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 freely for 7 days. The drug was administered on the 6th day of modeling by gavage with normal saline for 5 consecutive days, with a gavage dose of 100 μL / mouse / time.

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

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

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

[0065] Mouse weight is one of the indicators for judging the severity of ulcerative colitis and is related to the health of mice. Ulcerative colitis causes mice to lose weight severely. Therefore, before the establishment of the mouse model, that is, on day 0, the initial weight of each mouse was recorded, and the weight of each mouse was recorded every day after the model was established.

[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 mice in the model group decreased significantly 10 days after the modeling began. Compared with the mice in the model group, the weight of the mice in the group treated with Mucor racemosus increased significantly 10 days after the modeling began, and there was a trend of recovering to the weight of the mice in the blank group. Conclusion: Mucor racemosus 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 the length of the mouse colon to be severely reduced, the mice were euthanized on the 10th day, the mice were dissected and the mouse colon tissue was taken for length measurement and comparison, and the colon length was measured and photographed for record.

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

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

[0071] Since colitis causes the spleen of mice to swell, the spleen index = spleen weight / mouse body weight, which is one of the indicators for judging the severity of ulcerative colitis, that is, ulcerative colitis increases the spleen index of mice. Therefore, the mice were weighed on the 10th day, and after the mice were euthanized, the mice were dissected and the spleen tissues were weighed to calculate the spleen index.

[0072] The results are as follows Figure 3 As shown in the figure, compared with the normal mice in the blank group, the spleen index of the mice in the model group was significantly increased, and compared with the mice in the model group, the spleen index of the mice in the group treated with Mucor racemosus was significantly reduced, and the spleen index of the mice in the group treated with Mucor racemosus was close to that of the mice in the blank group. Conclusion: Mucor racemosus 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 ulcerative colitis mice

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

[0075] Table 1 Disease index scoring parameters

[0076]

[0077] The results are as follows Figure 4 , the disease index of the blank group, i.e., wild-type mice, was always 0; compared with the blank group, the disease index of the model group increased significantly with the increase of modeling time; compared with the model group, the disease index of the mice in the Mucor racemosus administration group decreased significantly with the increase of modeling time. Conclusion: Mucor racemosus can significantly reduce the disease index of colitis mice 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] Since 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 causing 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 determined 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 saline, that is, 1 mL of saline was added to 0.1 g of tissue, the tissue was crushed, and centrifuged at 3000 g / min for 20 minutes to take the supernatant. The 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 figure, compared with the normal mice in the blank group, the ROS content in the colon of the model group mice was significantly increased, and compared with the model group mice, the ROS content in the colon of the mice treated with Mucor racemosus was significantly reduced, close to 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 the levels of IL-6, IL-1β and TNF-α in the colon of mice with ulcerative colitis

[0082] Since IL-6, IL-1β and TNF-α are indicators of colitis and reflect the degree of inflammation in the colon tissue of mice, the contents of IL-6, IL-1β and TNF-α in the colon tissue were detected to detect the effect of Mucor racemosus on colon tissue inflammation. The contents of the three factors in colon tissue were detected using IL-6 detection kit, IL-1β detection kit and TNF-α detection kit (ELISA, China).

[0083] The results are as follows Figure 6 As shown in the figure, compared with the blank group mice, the contents of IL-6, IL-1β and TNF-α in the colon of the model group mice were significantly increased; compared with the model group mice, the contents of IL-6, IL-1β and TNF-α in the colon of the mice treated with Mucor racemosus were significantly reduced, close to the contents of IL-6, IL-1β and TNF-α in the colon of the blank group mice. Conclusion: Mucor racemosus can significantly reduce the contents of IL-6, IL-1β and TNF-α in the colon tissue of colitis mice, play an anti-inflammatory role, and confirm 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 in the figure, compared with the normal mice in the blank group, the Claudin-2 content in the colon of the model group mice was significantly reduced; compared with the model group mice, the Claudin-2 content in the colon of the mice treated with Mucor racemosus was significantly increased, close to the Claudin-2 content in the colon of the mice in the blank group. Conclusion: Mucor racemosus can significantly increase the integrity of the intestinal wall of the colon tissue of colitis mice and effectively delay the progression of ulcerative colitis.

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

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

[0089] The PDB and BHI medium powders were mixed in a ratio of 6:13 and sterilized to prepare PDB+BHI liquid medium, i.e., 24 g PDB and 52 g BHI medium were weighed and mixed evenly, 1 L distilled water was added, and the mixture was 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 inoculation amounts, i.e., the inoculation units were 5×10 6 / 100mL, 10 7 / 100mL, 2×10 7 / 100mL. Cultivated under strict anaerobic conditions at 37°C for 3 days, three groups of different suspensions of Mucor racemosus with low inoculation amount, medium inoculation amount and high inoculation amount were obtained. The PDB medium is a potato glucose medium, and the components are 6g / L potato powder and 20g / L glucose.

[0090] 2. Detection

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

[0092] The above three groups of Mucor racemosus suspensions 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 blank group, model group, and Mucor racemosus administration group on the 10th day in Example 1 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 crushed, centrifuged at 3000 g / min for 20 minutes, and the supernatant was taken.

[0093] Dilute the above 6 groups of supernatants with sample diluent 1:1 and add 50uL to the reaction wells, add 50uL of the diluted standard to the reaction wells, and add 50uL of the above supernatant samples to the reaction wells. Immediately add 50uL of biotin-labeled antibody. Cover the membrane plate, gently shake and mix, and incubate at 37℃ for 1 hour. Shake off the liquid in the wells, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, increase the number of washes once. Add 80uL of affinity streptavidin-HRP to each well, shake and mix gently, and incubate at 37℃ for 30 minutes. Shake off the liquid in the wells, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, increase the number of washes once. Add 50uL of substrate A and B to each well, shake gently to mix, and incubate at 37℃ for 10 minutes. Avoid light. Take out the ELISA plate and quickly add 50ul of stop solution. The result should be measured immediately after adding the stop solution. Measure the OD value of each well at a wavelength of 450nm. With the absorbance OD value as the ordinate (Y) and the corresponding concentration of the standard substance to be tested as the abscissa (X), make a corresponding curve. The content of the substance to be tested in the sample can be converted to the corresponding concentration from the standard curve according to its OD value.

[0094] The results are as follows Figure 8 and Fig. 9 As shown in the figure, when Mucor racemosus is cultured in an in vitro liquid culture medium, the amount of 5-methylcytosine produced by it also increases significantly with the increase in the inoculation amount of Mucor racemosus, which indicates that 5-methylcytosine is a metabolite of Mucor racemosus. In addition, in vivo animal experiments found that compared with the blank group mice, the content of 5-methylcytosine in the feces of the model group mice was significantly reduced. Compared with the model group mice, the content of 5-methylcytosine in the feces of the mice in the Mucor racemosus administration group was significantly increased, close to the content of 5-methylcytosine in the feces of the blank group mice. 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 Effect 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 in 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 Scheme", except that the 5mc gavage group was gavaged with 2 mg / mL 5mc starting on the 6th day of modeling for 5 consecutive days with a gavage dose of 100 μL / mouse / 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 to 15 As shown in the results, compared with the normal mice in the blank group, the body weight of the mice in the model group decreased significantly, the colon length was significantly shortened, and the disease development index of the mice increased significantly with the extension of the modeling time. The ROS content and the inflammatory factors IL-6, IL1-β and TNF-α in the colon tissue increased significantly, and the expression of the colon wall integrity marker gene Claudin-2 decreased significantly. However, compared with the mice in the model group, the body weight of the mice in the 5-methylcytosine gavage group increased significantly, the colon length increased significantly, the disease development index decreased significantly with the extension of the modeling time, the ROS content and the inflammatory factors IL-6, IL1-β and TNF-α in the colon tissue decreased, and the expression of the colon wall integrity marker gene Claudin-2 increased significantly. Conclusion: 5-methylcytosine, a metabolite of Mucor racemosus, can effectively treat 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 principle of the present invention. These improvements and modifications should also be regarded as 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. Use of a metabolite of Mucor racemosus or a preparation containing a metabolite of Mucor racemosus in the preparation of a drug for preventing and / or treating ulcerative colitis, characterized in that: The metabolites of Mucor racemosus include 5-methylcytosine.

3. The use according to claim 1 or 2, characterized in that: The drug is any one or more of the following: 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 the ability to regulate immunity; 4) The drug is a drug with anti-inflammatory effect; 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.

4. The use according to claim 3, characterized in that: Among them, 3) the drug with immunomodulatory ability can reduce the spleen index of patients with ulcerative colitis.

5. The use according to claim 3, characterized in that: 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.

6. The use according to claim 3, characterized in that: Among them, 5) the drug with antioxidant ability can reduce the content of ROS in ulcerative colitis tissue.

7. The use according to claim 3, characterized in that: 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.

8. A drug for preventing and / or treating ulcerative colitis, characterized in that: Includes one or both of Mucor racemosus and 5-methylcytosine.

9. The drug for preventing and / or treating ulcerative colitis according to claim 8, characterized in that: The ulcerative colitis includes remission, mild, moderate, severe or acute severe ulcerative colitis.

10. The drug for preventing and / or treating ulcerative colitis according to claim 8, characterized in that: The medicine includes oral preparations.

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