Application of glucopyranose sulfate octasaccharide in preparation of medicine for preventing or treating inflammatory bowel disease

Abstract

The invention discloses an application of glucose-6-galactooligosaccharide sulfate in preparation of a medicine for preventing or treating inflammatory bowel diseases. The sulphated galacto-octasaccharide can effectively relieve TNBS-induced mouse colitis, obviously inhibit continuous decrease of mouse weight, promote colon length recovery, reduce expression level of inflammatory factors in intestinal tissues, repair pathological damage of the colon tissues and regulate composition of intestinal microorganisms, so that intestinal inflammation is relieved, structural integrity of the colon tissues is recovered, and the curative effect is improved. Wide application prospects are shown in prevention and treatment of the inflammatory bowel disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of a sulfated galactofuranose in preparation of a medicine for preventing or treating inflammatory bowel disease. BACKGROUND

[0002] Inflammatory bowel disease (IBD) is a kind of chronic, autoimmune-related intestinal disease, mainly including ulcerative colitis (UC) and Crohn's disease (CD). UC mainly manifests as inflammation and ulcer of colonic mucosa, and is often accompanied by symptoms such as diarrhea, abdominal pain, and hematochezia. CD is a kind of intestinal disease mainly characterized by chronic transmural inflammation, and can occur at any site of the digestive tract, most commonly in the terminal ileum and colon. Typical clinical symptoms include abdominal pain, chronic diarrhea, weight loss, and fatigue. Some CD patients only manifest intestinal inflammation in the early stage, and may develop a large number of complications such as intestinal stenosis, fistula, and perforation as the disease progresses, and finally need surgical intervention, but cannot be cured and have a high recurrence rate. Epidemiological studies have shown that CD has no gender difference, is common in people aged 20 to 40 years, and its incidence is continuously increasing worldwide, which seriously affects the quality of life of patients and increases the social medical burden. The exact cause of CD has not been completely clear, and may be caused by multiple factors, including genetic susceptibility, environmental influence, and intestinal flora imbalance, etc. These factors jointly cause abnormal activation of the mucosal immune system, damage the intestinal barrier function, and thus promote the persistence of inflammatory response.

[0003] At present, there is no cure for CD, and the existing treatment strategies mainly focus on inhibiting inflammation, delaying disease progression, maintaining remission, and preventing recurrence. In clinical practice, commonly used drugs include traditional immunosuppressants (such as mesalazine, dexamethasone, and methotrexate, etc.) and biological agents (such as infliximab, adalimumab, and certolizumab), which are used to alleviate and control the inflammatory response of CD. However, long-term use of these drugs may cause side effects such as immunogenicity, immunosuppression, drug resistance, and allergic reactions. Therefore, it is urgent to develop a lower-cost, safer, and more effective medicine for UC patients to overcome the limitations of the current therapeutic drugs.

[0004] The inventors reported in previous studies that octaparin (OP), a new heparin analogue synthesized by one-pot sulfation reaction with ricolinoctaose as the starting material, has good anticoagulant function, and compared with traditional heparin, octaparin shows significantly reduced bleeding risk and less platelet-related adverse reactions, and has higher safety (Yu N, Fang R, Ding Z, et al. Preparation and structural characterization of a sulfated octasaccharide with heparin-like anticoagulant activity [J]. Carbohydrate Polymers, 2025, 347: 122782.). However, whether octaparin has a therapeutic effect on inflammatory bowel disease has not been reported. SUMMARY

[0005] The purpose of the present application is to provide an application of octaparin in the preparation of a drug for preventing or treating inflammatory bowel disease.

[0006] The structural formula of the octaparin according to the present application is:

[0007]

[0008] The dosage form of the drug according to the present application is an oral dosage or an injection dosage known to those skilled in the art, and the oral dosage type is, for example, a tablet, a capsule, a pill, a powder, a granule, a suspension, etc., and the injection dosage type is, for example, an injection solution, a powder injection, a transfusion agent, etc.

[0009] The inflammatory bowel disease according to the present application includes, but is not limited to, ulcerative colitis, Crohn's disease, infectious enteritis, radiation enteritis, drug-induced enteritis, allergic enteritis, autoimmune enteritis, etc.

[0010] The administration subject of the drug according to the present application is any animal that may have or has had inflammatory bowel disease. These animals include humans and non-human animals, such as pets or livestock, etc.

[0011] The administration dosage of the drug according to the present application depends on the age, health and weight of the recipient, the treatment frequency, the administration route, etc.

[0012] In the specific embodiment of the present application, the administration dosage of the drug is 20 mg / kg to 500 mg / kg per day, based on the mass of octaparin.

[0013] The present application also provides a medicine for preventing or treating inflammatory bowel disease, which contains the above sialyl-lactose sulfate.

[0014] Further, the medicine for preventing or treating inflammatory bowel disease also contains a medically acceptable adjuvant.

[0015] Compared with the prior art, the present application has the following advantages:

[0016] (1) The inventors first discovered that sialyl-lactose sulfate can be used as a medicine for treating inflammatory bowel disease, which plays a therapeutic role by inhibiting intestinal inflammatory response. Sialyl-lactose sulfate is safe and non-toxic, has strong efficacy, and has good prospects for medical use.

[0017] (2) The sialyl-lactose sulfate of the present application has the effects of alleviating weight loss and restoring colon length in mice caused by 2,4,6-trinitrobenzenesulfonic acid (TNBS), and has a positive effect on improving systemic inflammation.

[0018] (3) The sialyl-lactose sulfate of the present application has the effect of improving pathological damage of the colon in TNBS-induced mice, and can increase the number of goblet cells and promote mucosal recovery and regeneration.

[0019] (4) The sialyl-lactose sulfate of the present application can significantly reduce the gene expression level of inflammatory factors in the colon tissue of TNBS-induced mice, and effectively alleviate local inflammatory response.

[0020] (5) The sialyl-lactose sulfate of the present application can significantly inhibit the increase of myeloperoxidase (MPO) activity in the colon tissue of TNBS-induced mice, inhibit neutrophil infiltration, and alleviate intestinal inflammation.

[0021] (6) The sialyl-lactose sulfate of the present application can regulate the composition of intestinal microorganisms in TNBS-induced colitis mice, increase the relative abundance of beneficial bacteria, reduce the level of harmful bacteria closely related to inflammation, and improve the intestinal microenvironment.

[0022] (7) The medicine prepared from the sialyl-lactose sulfate of the present application has the effects of inhibiting inflammatory response of RAW264.7 macrophages induced by lipopolysaccharide (LPS) and improving the inflammatory pathological state, and has good in vitro anti-inflammatory effect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is a graph of the changes in the body weight of mice in each group in Example 1.

[0024] Figure 2 Figure 2 is a photograph of the colon of mice in each group in Example 1 and a graph of the length of the colon of mice in each group, wherein A is a representative photograph of the colon of mice in each group, and B is the length of the colon of mice in each group.

[0025] Figure 3Representative hematoxylin / eosin (H&E) staining images of the colon of each group of mice in Example 1, wherein A is a representative H&E staining photograph (40x) of the colon of each group of colitis mice after lesions, in which the solid arrows indicate goblet cells and the dotted arrows indicate inflammatory cell infiltration, and B is the colitis pathology score of each group of mice.

[0026] Figure 4 Representative periodic acid-Schiff (PAS) staining images of the colon of each group of mice in Example 1 (40x).

[0027] Figure 5 Figure 1 is the gene expression diagram of inflammatory factors in the colon tissue of each group of mice in Example 1, where AD is the relative expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and interferon-γ (IFN-γ) mRNA, respectively.

[0028] Figure 6 Statistical graph of myeloperoxidase (MPO) activity in colon tissue of each group of mice in Example 1.

[0029] Figure 7 This is the distribution diagram of the genus level in the intestinal microorganisms of each group of mice in Example 1.

[0030] Figure 8 These are the inflammatory factor gene expression diagrams of LPS-induced RAW264.7 cells in each group in Example 2, where A represents the TNF-α mRNA expression level and B represents the IL-6 mRNA expression level. DETAILED DESCRIPTION

[0031] Unless otherwise specified, the methods used in the following examples are all commonly used methods in the art. All raw materials used in the following examples are commercially available products unless otherwise specified. Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs.

[0032] The sulfated glucogalactooctaose of the present invention is prepared according to the document [Yu N, Fang R, Ding Z, et al. Preparation and structural characterization of a sulfated octasaccharide with heparin-like anticoagulant activity [J]. Carbohydrate Polymers, 2025, 347: 122782.].

[0033] Example 1

[0034] Preventive and therapeutic effects of sialyl-lactose on inflammatory bowel disease

[0035] 1. Establishment of animal model of inflammatory bowel disease

[0036] In this experiment, TNBS-induced Crohn's colitis mice were used as the animal model of inflammatory bowel disease. TNBS-induced Crohn's colitis is a model of colitis characterized by persistent inflammation, easy recurrence, destruction of mucosal barrier and imbalance of intestinal immunity. Male BALB / c mice, 6-8 weeks old, weighing 22-26 g, were used in the experiment. All mice were raised under standard feeding conditions, with a 12-hour light-12-hour dark cycle, and free access to food and water. In order to establish the animal model of inflammatory bowel disease, the mice were fasted but not watered 24 hours before the experiment. On the first day of the experiment, a 3.5F polyethylene hose was connected to an insulin syringe and slowly inserted into the colon 4 cm from the anus. The experimental group mice were given 2.5% TNBS ethanol solution (5% TNBS mixed with absolute ethanol at a volume ratio of 1:1) at a dose of 160 mg / kg body weight (calculated by the mass of TNBS), and the control group mice were given the same volume of 50% ethanol solution. After injection, the mice were inverted for 4 min to ensure that all the modeling agents were dropped into the intestinal cavity.

[0037] 2. Experimental grouping

[0038] After the establishment of the animal model of inflammatory bowel disease for 24 hours, the experimental group mice were randomly divided into 4 groups (6 mice in each group): (1) model group (MC), given physiological saline by gavage every day; (2) low-dose group (OP-L), given sialyl-lactose solution by gavage every day at a dose of 50 mg / kg body weight (calculated by the mass of sialyl-lactose, the same below); (3) high-dose group (OP-H), given sialyl-lactose solution by gavage every day at a dose of 100 mg / kg body weight; (4) cyclosporine (CsA) positive control group (PC), given cyclosporine solution by gavage every day at a dose of 25 mg / kg body weight. At the same time, a control group (CK) was set up, in which the mice were given the same volume of physiological saline by gavage every day. All gavage drugs were prepared with physiological saline to the required concentration, and the gavage volume was calculated according to the body weight of the mice in each group, gavaged once a day for a total of 6 days.

[0039] 3. Body weight recording: the body weight of the mice was recorded every day.

[0040] 4. Measurement of colon length

[0041] The mice were sacrificed 24 hours after the last gavage, and the colon was isolated from the end of the cecum to the anus, with gentle stripping of the mesentery and adipose tissue. The total length of the mouse from the cecum to the anus was measured using a ruler.

[0042] 5. Histological analysis of colon

[0043] The excised colon was immediately fixed with 4% paraformaldehyde and paraffin-embedded. Sections were stained with H&E and PAS for histological analysis. The colon pathology was observed by microscope.

[0044] 6. MPO activity assay

[0045] MPO activity was determined by enzymatic chromogenic quantitative analysis method, according to the operation steps in the kit instructions, and finally the activity was determined by colorimetric method at 460 nm wavelength.

[0046] 7. Gene expression analysis

[0047] After all the mice were sacrificed, the colon tissue was taken to extract total RNA, and the RNA was reverse transcribed into cDNA by using reverse transcription kit. Then real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression level of inflammation-related factors (TNF-a, IL-6, IL-1b, IFN-g). The relative gene expression was calculated by 2 ΔΔCt method, and b-Actin was used as the internal reference.

[0048] 8. Intestinal microbiota sequencing

[0049] The mouse fecal DNA was extracted, and 16S rDNA high-throughput sequencing was used to detect the intestinal flora. The samples were sequenced according to the standardization program Illumina NovaSeq 6000 platform, and the sequencing library was combined according to the effective concentration and target data volume.

[0050] 9. Data analysis

[0051] The comparison between multiple groups was statistically analyzed by One-way / Two-way ANOVA statistical method. P<0.05 was considered to have significant effect.

[0052] 10. Result analysis

[0053] As shown in Figure 1 , the stigmasterol could alleviate the body weight loss of TNBS-induced colitis mice. The body weight of TNBS-induced mice decreased significantly from the first day, and the body weight of the model group decreased most obviously, with a decrease of about 18% on the third day, indicating that the inflammatory reaction in vivo was severe. After treatment with different concentrations of stigmasterol and cyclosporine, the body weight of mice gradually recovered, especially in the OP-H group, which recovered most obviously, and the body weight gradually recovered after the third day, and approached the control group level on the seventh day.

[0054] As shown in Figure 2As shown, the colon length of normal mice was about 11 cm, while the colon of MC group mice was obviously congested, edematous, and shortened to about 7 cm. After treatment with different doses of ST, the colon length of mice was significantly longer than that of the MC group, and the congestion and bleeding were significantly reduced, indicating that ST could effectively alleviate the shortening of the colon caused by intestinal inflammation and improve the overall pathological damage of the intestinal tract.

[0055] As shown in FIG. 1A, Figure 3 As shown in FIG. 2A, H&E staining results showed that the colon epithelial cells and mucosal structure of the control group mice were complete, the crypts were arranged regularly, and no obvious inflammatory cell infiltration was observed in the lamina propria. The colon tissue of the MC group mice had a large number of inflammatory cell infiltration, leading to disordered mucosal structure, significant reduction of goblet cells, crypt abscess, and severe ulcer. In contrast, only a small amount of inflammatory cell infiltration was observed in the OP group, and the intestinal mucosal structure was relatively complete, and the number of goblet cells was significantly increased. Further statistical analysis showed that the pathological score of the OP group was significantly lower than that of the MC group Figure 3 B), indicating that ST could effectively alleviate the pathological damage of the colon induced by TNBS.

[0056] As shown in FIG. 2B, Figure 4 As shown in FIG. 3A, PAS staining results showed that the mucin secreted by the goblet cells in the colon of the control group mice was purple (black arrow), and the number was more, and regularly distributed in the glands, and the mucosal structure was complete. The mucosal and mucosal layer structure of the colon of the MC group mice was severely damaged, part of the gland was missing, the number of goblet cells was significantly reduced, and the secretion of mucin was significantly reduced. After treatment with ST, the number of mucin was significantly increased, the number of goblet cells was increased, and the gland structure was also recovered. The results showed that ST could effectively alleviate the colon tissue damage induced by TNBS, protect the function of goblet cells, and maintain the integrity of the intestinal mucus barrier.

[0057] As shown in FIG. 3B, Figure 5 As shown in FIG. 4, ST could significantly reduce the mRNA levels of TNF-a, IL-6, IL-1b and IFN-g in colon tissue. Compared with the CK group, the mRNA levels of TNF-a, IL-6, IL-1b and IFN-g in the colon of mice induced by TNBS were significantly increased, indicating that the inflammatory response was active; while in the mice treated with ST, the expression of these inflammatory factors was significantly reduced and showed a dose-dependent manner. The results showed that ST could effectively inhibit the overexpression of inflammatory factors induced by TNBS, and reduce the local inflammatory response of mice with colitis.

[0058] As shown in FIG. 5A, Figure 6As shown, compared with the CK group, the MPO enzyme activity in the colon tissue of the MC group of mice was significantly increased, indicating that neutrophils infiltrated in large quantities and the intestinal inflammatory response was enhanced; after treatment with galactose sulfate, the MPO activity was significantly reduced and almost returned to the level of the control group of mice. The results showed that galactose sulfate can reduce the inflammatory response mediated by neutrophils and play an anti-inflammatory role.

[0059] As shown, compared with the CK group, the MPO enzyme activity in the colon tissue of the MC group of mice was significantly increased, indicating that neutrophils infiltrated in large quantities and the intestinal inflammatory response was enhanced; after treatment with galactose sulfate, the MPO activity was significantly reduced and almost returned to the level of the control group of mice. The results showed that galactose sulfate can reduce the inflammatory response mediated by neutrophils and play an anti-inflammatory role. Figure 7 As shown, compared with the CK group, the MPO enzyme activity in the colon tissue of the MC group of mice was significantly increased, indicating that neutrophils infiltrated in large quantities and the intestinal inflammatory response was enhanced; after treatment with galactose sulfate, the MPO activity was significantly reduced and almost returned to the level of the control group of mice. The results showed that galactose sulfate can reduce the inflammatory response mediated by neutrophils and play an anti-inflammatory role.

[0060] Example 2

[0061] Effect of galactose sulfate on the expression of inflammatory factors in mouse monocyte macrophage leukemia cells (RAW264.7):

[0062] 1. Cell culture

[0063] The cells used in the experiment were RAW264.7 cells, and the culture process was carried out in DMEM high-sugar medium containing 10% fetal bovine serum, 1% streptomycin and 1% erythromycin. The cells were cultured in a carbon dioxide incubator at a temperature of 37°C, and when subcultured, the cells were gently blown off the bottom of the culture dish to fall off, divided into two, and continued to be cultured.

[0064] 2. Model establishment

[0065] (1) RAW264.7 cells were inoculated in 6-well plates at a cell density of 5×10 5 cells per well, divided into four groups, namely the CK group, the LPS induction group (MC), the LPS+200 μg / mL galactose sulfate (OP-200) group and the LPS+400 μg / mL galactose sulfate (OP-400) group.

[0066] (2) After the cells adhered, LPS was added to the MC, OP-200 and OP-400 groups at a final concentration of 1 μg / mL, and the CK group was added with the same volume of PBS (pH 7.2) as a control. Subsequently, dextran sulfate was added to the OP-200 and OP-400 groups at a final concentration of 200 μg / mL and 400 μg / mL, respectively; the CK and MC groups were added with the same volume of PBS. After mixing, the cells were incubated in a 37°C, 5% CO2 incubator.

[0067] 3. Gene expression analysis

[0068] RAW264.7 cells were collected after 12 h of culture, total RNA was extracted, and RNA was reverse transcribed into cDNA using a reverse transcription kit. Subsequently, real-time fluorescence quantitative PCR (qRT-PCR) was performed to detect the expression levels of inflammatory-related factors (TNF-α, IL-6). The relative gene expression was calculated using the 2 ΔΔCt method, and β-Actin was used as an internal reference.

[0069] 4. Data analysis

[0070] The comparison between multiple groups was statistically analyzed using One-way / Two-way ANOVA statistical methods. P < 0.05 was considered to have a significant effect.

[0071] 5. Results analysis

[0072] As shown in Figure 8 , in the LPS-induced RAW264.7 cell inflammation model, dextran sulfate can significantly inhibit the expression of inflammatory factors TNF-α and IL-6 mRNA induced by LPS, indicating that it has good anti-inflammatory activity in the in vitro model.

Claims

1. Use of glucogalactoctaose sulfate in the preparation of a medicament for preventing or treating inflammatory bowel disease, characterized in that: The structural formula of galactoctaose sulfate is: 。 2. The use according to claim 1, characterized in that The drug is available as an oral dose or an injection.

3. The use according to claim 2, characterized in that Oral dosage forms include tablets, capsules, pills, powders, granules or suspensions; injection forms include injection solutions, powder injections or infusions.

4. The use according to claim 1, characterized in that Inflammatory bowel disease includes ulcerative colitis, Crohn's disease, infectious enteritis, radiation enteritis, drug-induced enteritis, allergic enteritis or autoimmune enteritis.

5. The use according to claim 1, characterized in that The drug is administered to animals that may develop or have developed inflammatory bowel disease.

6. The use according to claim 5, characterized in that Animals are human or non-human animals.

7. The use according to claim 1, characterized in that The dosage of glucopyranoside sulfate is 20 mg / kg to 500 mg / kg / day.

8. A drug for preventing or treating inflammatory bowel disease, characterized in that: Contains the glucogalactoctaose sulfate according to claim 1.

9. The drug according to claim 8, characterized in that Also contains medically acceptable excipients.

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