1,6-anhydro-beta-d-glucose for use in the preparation of a medicament for maintaining the integrity of the colonic epithelial barrier and crypts and for use in the treatment of colitis

Abstract

The present invention belongs to the field of medicine, and in particular relates to the use of 1,6-anhydro-β-D-glucose in the preparation of drugs for maintaining the integrity of the colon epithelial barrier and crypts and for anti-colitis. The present invention detects the colitis-related metabolite 1,6-anhydro-β-D-glucose by detecting mouse serum, and applies it to mice. It is found that 1,6-anhydro-β-D-glucose can alleviate the weight loss and shortening of colon length in mice, reduce the disease score index (DAI), maintain the integrity of the colon epithelial barrier and crypts, and exert anti-inflammatory effects, thereby alleviating DSS-induced colitis. Therefore, 1,6-anhydro-β-D-glucose is expected to be used in the research and development of related drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, in particular to the use of 1,6-anhydro-beta-D-glucose for the preparation of a medicament for maintaining the integrity of the colonic epithelial barrier and crypts and for anti-colitis. BACKGROUND

[0002] Metabolomics is a research approach that quantitatively analyzes all metabolites in the body and finds the relative relationship between metabolites and physiological and pathological changes, which is similar to the research ideas of genomics and proteomics. Metabolomics is widely used in various research fields. Non-targeted metabolomics is a common metabolomics research method. By comparing and analyzing the metabolic levels of the experimental group and the control group, the differential metabolites are found out, and the screening of biomarkers and the research of disease pathogenesis and drug treatment mechanism are completed.

[0003] The present application is based on the finding in the research that mannuronic acid oligosaccharide (MAOS) can alleviate DSS-induced colitis by affecting intestinal flora and exerting anti-inflammatory effect, but the metabolism and probiotic mechanism of MAOS in the intestine are not clear. Therefore, based on the two aspects of intestinal flora and MAOS metabolic pathway, it is of important guiding significance to further understand and seek effective drugs for the treatment of inflammatory bowel disease (IBD). Further, it is found that 1,6-anhydro-beta-D-glucose has the effects of maintaining the integrity of the colonic epithelial barrier and crypts and anti-inflammation, and can be applied to the preparation of related drugs. SUMMARY

[0004] The present application uses a mouse cohabitation model to find that the intestinal flora after intervention of mannuronic acid oligosaccharide (MAOS) can be successfully transplanted to the recipient mice, which changes the intestinal microecology and metabolic level to a certain extent. Through non-targeted metabolomics method, the serum of mice is detected, and the differential metabolite 1,6-anhydro-beta-D-glucose (CAS number 498-07-7) of the experimental group and the control group is found in the negative ion mode. Subsequently, 1,6-anhydro-beta-D-glucose is used to further verify that it can alleviate the weight loss, shortening of colon length and increase of colitis score caused by DSS induction in mice. At the same time, H&E staining of colon tissue and expression of inflammatory factors also confirm that 1,6-anhydro-beta-D-glucose has the probiotic function of maintaining the integrity of the colonic epithelial barrier and crypts and anti-inflammation.

[0005] The present application aims to provide a method for screening anti-colitis substances based on metabolomics, the screened substance is 1,6-anhydro-beta-D-glucose and can be applied to the preparation of anti-colitis drugs and drugs for maintaining the integrity of the colon epithelial barrier and crypt, comprising the following steps:

[0006] Step 1. Understand the relationship between mannuronic acid oligosaccharide and its mediated intestinal microbiota and metabolites and DSS-induced bacterial population metabolic differences, set up mouse experiment: 24 mice in total, after 3 days of adaptation, evenly divided into two groups according to the weight of the mice: DSS group and mannuronic acid oligosaccharide pretreatment group, 12 in each group, among which the mice in the DSS group drink PBS during pretreatment, and the mice in the mannuronic acid oligosaccharide pretreatment group drink mannuronic acid oligosaccharide water prepared by PBS with a concentration of 100mg / kg; After 21 days of pretreatment, 6 mice were taken from each of the above two groups respectively, and cohabitation was carried out, that is, two treatment groups were included, namely Cohousing-PBS+DSS group and Cohousing-MAOS+DSS group, and the remaining mice were DSS group and MAOS+DSS group; Then, 2% DSS challenge was carried out for 7 days, and finally the mice eyeball blood was taken for subsequent metabolomics analysis experiment;

[0007] Step 2. Collection of animal serum samples and principal component analysis of each group of samples and screening of differential metabolites: after the mice were sacrificed, 500ul of eyeball blood of each mouse was taken into a 1.5ml EP tube, centrifuged at 2500rpm for 15min at 4℃, and the supernatant was collected as serum;

[0008] Step 3. Use non-targeted metabolomics technology to study the changes of metabolites in different treatment groups of mice; detect the metabolic principal components and differences between groups by PLS-DA model, and analyze the differential metabolites with the largest difference in the cohabitation mouse metabolome in the negative ion mode, which is 1,6-anhydro-beta-D-glucose.

[0009] Compared with the prior art, the beneficial effects of the technical scheme are: I. By using a mouse cohabitation model, it is found that the intestinal flora after intervention of mannuronic acid oligosaccharide (MAOS) can be successfully transplanted to the recipient mice, which changes the intestinal microecology and metabolic level to a certain extent, and through non-targeted metabolomics method, 1, 6-anhydro-beta-D-glucose related to colitis is detected in the serum of mice, which is applied to mice, and it is found that 1, 6-anhydro-beta-D-glucose can relieve the decrease of mouse body weight and the shortening of colon length, reduce disease activity index (DAI), maintain the integrity of colon epithelial barrier and crypt, and play an anti-inflammatory role, thereby relieving DSS-induced colitis. Therefore, 1, 6-anhydro-beta-D-glucose is expected to be applied to the research and development of related drugs, and the application can also provide a metabolomics method for the research of intestinal anti-inflammatory.

[0010] II. A method for screening substances against colitis based on metabolomics is provided, and the biological information of endogenous metabolites in the body can be obtained by the method of non-targeted metabolomics. The changes in the content of these metabolites in mice to a certain extent reflect the changes in the metabolic level of DSS-induced colitis mice, which provides a certain basis for detecting the changes of metabolites in the body under pathological and physiological conditions and the prevention and improvement of colitis. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 PLS-DA analysis of the metabolome of the MAOS cohabitation mouse in negative ion mode;

[0012] Figure 2 Relative abundance of 1, 6-anhydro-beta-D-glucose in the MAOS cohabitation mouse in negative ion mode;

[0013] Figure 3 Body weight change graph of the mouse treated with 1, 6-anhydro-beta-D-glucose;

[0014] Figure 4 Colonic lesions and disease activity index of the mouse treated with 1, 6-anhydro-beta-D-glucose;

[0015] Figure 5 H&E staining and expression of inflammatory factors of the colon tissue of the mouse treated with 1, 6-anhydro-beta-D-glucose. DETAILED DESCRIPTION

[0016] The application will be described in detail below in combination with the drawings and examples, and the content of the application is not limited to the following examples.

[0017] Example 1

[0018] Relationship between MAOS-induced gut microbiota and metabolic differences in DSS-induced colitis

[0019] The experimental mice were C57BL / 6 mice (Three Gorges University, Yichang, China), 5-week-old male, a total of 24, maintained under pathogen-free conditions for 12 h of circadian cycle (21 ± 2°C). During the entire study period, all animals had free access to drinking water and food. All animal experiments were approved by the Scientific Ethics and Safety Committee of South China University of Nationalities (No. 2020-SCUEC-006; Wuhan, China). After 3 days of adaptation, the mice were divided into PBS group and MAOS intervention group (n = 12 in each group), and MAOS in the MAOS intervention group was dissolved in 100 mL PBS to prepare 100 mg / kg MAOS (Qingdao Bozhihuilibio Technology Co., Ltd., China), which lasted for 21 d. After the intervention was completed, 6 mice from each group were cohabited, and all mice were subjected to DSS challenge for 7 d, i.e. four experimental groups were obtained, namely DSS group, Cohousing+PBS+DSS group, Cohousing+MAOS+DSS group and MAOS+DSS group. After the challenge, about 500 μL of blood was taken from each mouse, centrifuged at 4°C 2500 rpm for 15 min, and about 200 μL of supernatant was collected as serum, which could be stored at -80°C. The animal experiment grouping and drug administration are shown in Figure 1 .

[0020] Partial least squares discriminant analysis (PLS-DA) is a multivariate statistical method for discriminant analysis, and we can use PLS-DA to establish a model between metabolite expression and sample category, so as to compare the differences in component metabolite expression. Our PLS-DA analysis of the metabolome of MAOS cohabited mice in negative ion mode showed that there were significant changes in the expression of metabolites between the DSS control group and the MAOS+DSS group, and the metabolite components of the Cohousing+PBS+DSS group and the Cohousing+MAOS+DSS group were partially the same, and the metabolite expressions of the two groups were different from those of the DSS control group and the MAOS+DSS group, respectively Figure 1 ), indicating that the intervention of MAOS can change the metabolic level of DSS-induced colitis mice, and the change in the metabolic level of cohabited mice also indicates the effect of cohabitation-mediated gut microbial transfer on the metabolic level of mice.

[0021] Example 2

[0022] Screening of differential metabolite 1,6-anhydro-β-D-glucose with potential anti-inflammatory effect

[0023] To observe the change rule of the relative content of metabolites, we standardized the original relative content of the differential metabolites identified by applying the screening criteria by using Unit Variance Scaling (UV) standardization, and observed the differential metabolite information by drawing a heat map through the R software Complex-Heatmap package. According to the heat map of the metabolites of the MAOS cohabiting mice in the negative ion mode, it can be seen that compared with the DSS control group, the expression level of the 1,6-anhydro-β-D-glucose metabolite in the MAOS+DSS group was significantly up-regulated, and the expression was stable within the group. The relative abundance of 1,6-anhydro-β-D-glucose in the negative ion mode is shown in FIG. 6. Figure 2

[0024] Example 3

[0025] Verification of the anti-inflammatory effect of the differential metabolite 1,6-anhydro-β-D-glucose.

[0026] To verify whether the differential metabolite screened has the anti-colitis probiotic effect, we verified it at the animal level. The experimental mice used in the experiment were C57BL / 6 mice (Sanxia University, Yichang, China), 5-week-old male, a total of 12, kept under the condition of 12h of day-night cycle (21±2℃) without pathogens. During the entire study period, all animals had free access to drinking water and food. All animal experiments were approved by the Scientific Ethics and Safety Committee of South China University of Nationalities (No. 2020-SCUEC-006; Wuhan, China). After the mice were adapted for 3d, they were divided into a DSS control group and a 1,6-anhydro-β-D-glucose+DSS experimental group (n=6 in each group). The pretreatment lasted for 21d, during which the control group was treated with PBS drinking water, while the experimental group was given 1,6-anhydro-β-D-glucose with a reference concentration of 250mg / kg / d, and the concentration was configured to be 1mg / ml, prepared with PBS. Finally, DSS was used for attack for 7d, during which the body weight and the condition of loose stool and blood stool of the mice were recorded, and the DAI score was completed. After the attack, the mice were sacrificed, the colon tissue was taken for length measurement, and the colon tissue was subjected to H&E staining and determination of the expression of the inflammatory factor IL-6 in the serum.

[0027] Through the following indicators, we can see that 1,6-anhydro-β-D-glucose can alleviate DSS-induced colitis:

[0028] 1. Change in body weight of mice: FIG. 7 shows the change in body weight of mice. Figure 3 ​)showed: after the end of pretreatment, the body weight of both groups of mice increased to a certain extent; in the first 3 days of DSS treatment, the body weight of mice in the control group and the experimental group fluctuated up and down; while in the 4-7 days of DSS treatment, the body weight of mice in the DSS group began to decrease, compared with the DSS group, the 1,6-anhydro-β-D-glucose pretreatment group could alleviate the decrease of the body weight of mice.

[0029] 2. Changes in colon length and DAI score: after DSS challenge, the colon length of mice was shortened, while 1,6-anhydro-β-D-glucose pretreatment could alleviate the shortening of colon length induced by DSS Figure 4 A). By recording the loose stool and bloody stool of mice during DSS challenge, we scored the DAI of two groups of mice Figure 4 B), the results showed that after DSS challenge, the DAI score increased, while 1,6-anhydro-β-D-glucose pretreatment could reduce the loose stool and bloody stool of mice, thereby reducing the DAI score.

[0030] 3. H&E staining of mouse colon tissue: colon tissue was fixed with 4% paraformaldehyde and paraffin-embedded. After cutting the tissue section, place it on a glass slide, and observe it under a microscope after a series of H&E staining. Figure 5 A is the colon tissue section of the DSS group and the 1,6-anhydro-β-D-glucose pretreatment group, in which the colon tissue of the DSS group is severely damaged, while the colon tissue of the 1,6-anhydro-β-D-glucose pretreatment group is more complete, i.e. 1,6-anhydro-β-D-glucose has a beneficial function on the intestinal barrier of mice, and can alleviate the destruction of colon tissue induced by DSS.

[0031] 4. Expression of inflammatory factor IL-6 in mouse serum: serum treatment is the same as in Example 1, and the expression of inflammatory factor IL-6 in serum is detected by ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., China). Refer to the standard curve of inflammatory factor IL-6 in the instruction manual of the kit. Finally, use the enzyme marker (BioTek Synergy Htx) to determine at OD450 nm, and calculate the expression of IL-6. The results showed that the expression of serum inflammatory factor IL-6 in the 1,6-anhydro-β-D-glucose pretreatment group was significantly lower than that in the DSS group, which had a statistically significant difference, i.e. 1,6-anhydro-β-D-glucose could play an anti-inflammatory role Figure 5 B).

Claims

1. Use of 1,6-anhydro-β-D-glucose as the sole active ingredient in the preparation of anti-colitis medicines.

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