Application of N-acetylglutamine in product for preventing or treating ulcerative colitis and product

Abstract

The invention provides application of N-acetylglutamine in preparation of a product for preventing or treating ulcerative colitis and the product, and belongs to the technical field of medicines. The N-acetylglutamine can relieve the phenomena of intestinal barrier injury, intestinal inflammation and the like of an ulcerative colitis mouse model, and can be used for treating ulcerative colitis. N-acetylglutamine is used as an intestinal flora metabolite, and when N-acetylglutamine is applied to a product for preventing or treating ulcerative colitis, the expression of proinflammatory factors can be inhibited, the expression of anti-inflammatory factors can be improved, the expression of mechanical barrier Zo1, Occludin and Claudin1 proteins can be improved, the intestinal barrier injury, intestinal inflammation and the like of acute ulcerative colitis model mice can be relieved, and the application of N-acetylglutamine to the product for preventing or treating ulcerative colitis can be promoted. A novel medicine choice is provided for the treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to N-acetylglutamine, and particularly relates to the application of N-acetylglutamine in preparing products for preventing or treating ulcerative colitis and the products. Background Art

[0002] Ulcerative colitis is a type of inflammatory bowel disease (IBD). It is a chronic nonspecific inflammatory disease of the colon and rectum with an unclear etiology. IBD is associated with immune and microbial imbalance, but its effects and mechanisms on inflammatory bowel disease through intestinal microbial and metabolic changes are still unclear. The lesions of ulcerative colitis are limited to the mucosa and submucosa of the large intestine, mostly located in the sigmoid colon and rectum, but can also extend to the descending colon or even the entire colon. The most common symptoms of ulcerative colitis are diarrhea and bloody stools. Depending on the severity and location of the disease, patients experience varying degrees of abdominal pain, nocturnal diarrhea, and mucus discharge. Damage to the barrier function of the colonic mucosa is one of its pathogenesis. The disease has a long course, recurrent attacks, and a high risk of cancer.

[0003] The primary goal of clinical drug treatment for ulcerative colitis is to alleviate clinical symptoms and improve the inflammatory response of the intestinal mucosa. Currently, a variety of drugs are used to treat ulcerative colitis, primarily aminosalicylic acid preparations, glucocorticoids, and immunosuppressants. Despite this diverse range of therapeutic agents, currently developed drugs still suffer from poor prognosis and inappropriate use. The aminosalicylic acid drug sulfasalazine can cause leukopenia, hepatitis, and discomfort symptoms such as nausea, vomiting, abdominal pain, and bloating. Glucocorticoids are prone to dependence, metabolic disorders, and the risk of infection. Immunomodulators have adverse reactions such as pancreatitis and hepatotoxicity, and their high cost limit their widespread use. Therefore, it is of great significance to discover novel targets for the treatment of ulcerative colitis and develop new drugs that can alleviate the disease, can be used long-term, are inexpensive, and have no side effects.

[0004] N-acetylglutamine has the effects of improving nerve cell metabolism, maintaining nerve stress capacity and lowering blood ammonia. It can pass through the blood-brain barrier and is used for coma caused by brain trauma, hepatic coma, hemiplegia, sequelae of polio, sequelae of brain trauma, mental retardation, neuralgia and low back pain.

[0005] Patent publication number CN1047162C discloses an acetylglutamine zinc compound, its preparation, and application. Acetylglutamine zinc is prepared from N-acetyl-L-glutamine and basic zinc carbonate or zinc oxide. Acetylglutamine zinc has certain physiological activity and is absorbed by the body after oral administration. It can be used to prevent and treat zinc deficiency, significantly improving anhydrous ethanol-induced gastric ulcers in rats, and also has a protective effect on gastric cells.

[0006] Patent publication number CN103070295B discloses a feed additive for enhancing the intestinal barrier function of livestock and poultry, and its use. The feed additive is a mixture of N-acetyl-L-glutamine and N-acetylcysteine, formulated in a suitable ratio. It effectively promotes intestinal mucosal development, enhances intestinal antioxidant capacity and immune function, thereby improving intestinal barrier function, reducing the incidence of intestinal diseases, promoting livestock health, and enhancing production performance. Compared to antibiotic additives, it has the advantages of being non-toxic, non-residual, and pollution-free, and is beneficial to both animal and human health.

[0007] Patent publication number CN101707877A discloses materials and methods for diagnosing and treating oxidative stress disorders, including methods and compositions for treating inflammatory bowel diseases, including inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, where symptoms are associated with oxidative stress or conditions that weaken the immune system. In one embodiment, the oral formulation comprises alpha-lipoic acid and / or N-acetyl-L-cysteine ​​(NAC) and / or L-glutamine.

[0008] None of the above technologies disclose the use of N-acetylglutamine in preventing or treating ulcerative colitis. Given the widespread problems of current ulcerative colitis medications, such as poor specificity, severe side effects, and difficulty in administering, there is an urgent need to develop new drugs. Therefore, the present invention provides the use of N-acetylglutamine in preventing or treating ulcerative colitis. Summary of the Invention

[0009] The present invention addresses the problems existing in the prior art and provides the use of N-acetylglutamine in the preparation of products for preventing or treating ulcerative colitis and the products thereof. Ulcerative colitis is a type of inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease. Different diseases have different pathogenesis and different treatment directions. Ulcerative colitis occurs in the colon, only affecting the mucosa of the innermost layer of the colon, and the inflamed area is continuous; Crohn's disease occurs in any part of the gastrointestinal tract, but is most common in the tail end of the small intestine, affecting all layers of the intestinal wall, and the inflamed area may appear in patches next to healthy parts of the intestine. The present invention uses N-acetylglutamine, a metabolite of intestinal flora, to prevent or treat ulcerative colitis, alleviate intestinal barrier damage, intestinal inflammation, etc., and provides a new drug option for the treatment of ulcerative colitis.

[0010] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides use of N-acetylglutamine or a stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing or treating ulcerative colitis.

[0012] The ulcerative colitis includes one or more of intestinal barrier damage and intestinal inflammation.

[0013] In a second aspect, the present invention provides a product for preventing or treating ulcerative colitis, comprising N-acetylglutamine or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0014] Preferably, the product further comprises auxiliary materials, and the auxiliary materials include excipients. The excipients are commonly used excipients in the art and are not limited here.

[0015] Preferably, the product is a tablet, capsule, powder, granule, paste, suppository, rectal enema or foam.

[0016] Preferably, the product is administered orally or rectally, preferably orally.

[0017] Preferably, the dosage of N-acetylglutamine or its stereoisomers, or pharmaceutically acceptable salts in the product is 0.1-1.5 g / d, preferably 0.2-0.5 g / d.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides the use of N-acetylglutamine in the preparation of a product for preventing or treating ulcerative colitis. The N-acetylglutamine is a metabolite of intestinal flora, which can inhibit the expression of pro-inflammatory factors, increase the expression of anti-inflammatory factors, increase the expression of mechanical barrier Zo1, Occludin, and Claudin1 proteins, alleviate intestinal barrier damage and intestinal inflammation in acute ulcerative colitis model mice, etc., can be used for the treatment of ulcerative colitis, and provides a new drug option for the treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the effect of N-acetylglutamine on the body weight, enteritis disease activity index (DAI) score, survival rate, and colon length of mice, wherein A is the effect of N-acetylglutamine on the body weight of mice, B is the effect of N-acetylglutamine on the enteritis disease activity index (DAI) score of mice, C is the effect of N-acetylglutamine on the survival rate of mice, and D is the effect of N-acetylglutamine on the colon length of mice;

[0020] Figure 2 The effect of N-acetylglutamine on the colon histology of mice;

[0021] Figure 3Figure 3 is the effect of N-acetylglutamine on the expression of inflammatory factor-related genes in the colon tissue of mice, where AI are the effects of N-acetylglutamine on Colon AhR, Colon IL-22, Colon INF-γ, Colon Muc2, Colon Occludin, Colon Claudin1, Colon TNF-α, Colon S100A8, and Colon IL-10, respectively (*P < 0.1, **P < 0.01, ns: no significant difference);

[0022] Figure 4 The effect of N-acetylglutamine on the mucus barrier of mice, where the red arrow indicates the mucus barrier;

[0023] Figure 5 Immunofluorescence was used to detect the effect of N-acetylglutamine on the expression of mechanical barriers (Zo1, Occludin, Claudin1 proteins) in mice. DETAILED DESCRIPTION

[0024] It is worth noting that, unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0025] The following raw material sources are illustrative:

[0026] N-Acetylglutamine: N-acetyl-L-glutamine, Shanghai MacLean Biochemical Technology Co., Ltd., product number: G6203-25g;

[0027] Dextran sulfate sodium: MP sodium sulfate dextran (DSS), MP Biomedicals, USA, product number: 216011090.

[0028] Example 1

[0029] Twenty-four male mice (C57BL / 6 strain), 6-7 weeks old, weighing 20-23 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. (License No.: SCXK (Beijing) 2019-0010). The mice were fed with standard rodent chow and purified water in a specific pathogen-free (SPF) animal room with a 12-h light-dark cycle. The relative humidity of the environment was maintained at 40%-60%, and the growth environment temperature was 25°C ± 2°C.

[0030] After one week of adaptive feeding, the mice were randomly divided into three groups: blank control group, DSS model group, and 200 mg / d N-acetylglutamine (N-Gln) administration group.

[0031] The specific treatment method for mice in the 200 mg / d N-acetylglutamine (N-Gln) administration group is as follows: mice that have undergone adaptive feeding were gavaged with 200 mg / d N-acetylglutamine for a total of 10 days. Among them, on days 1 to 7 of gavage, the mice were given a 2.5% weight percent DSS purified water solution to drink freely, and on days 8 to 10 of gavage, the mice were given a purified water solution to drink freely.

[0032] The specific treatment method for mice in the DSS model group is as follows: mice that have undergone adaptive feeding are gavaged with an equal volume of normal saline containing N-acetylglutamine for a total of 10 days. Among them, on days 1 to 7 of gavage, the mice are given a 2.5% weight percentage DSS pure water solution to drink freely, and on days 8 to 10 of gavage, the mice are given a pure water solution to drink freely.

[0033] The specific treatment method for the blank control group mice is as follows: the adaptively fed mice were gavaged with an equal volume of normal saline containing N-acetylglutamine for a total of 10 days, and the mice were given pure aqueous solution to drink freely on the 1st to 10th day of gavage.

[0034] The survival rate, body weight and feces of each group of mice were measured every day. After 10 days, the mice were anesthetized and colon tissue was collected to measure the colon length for later use.

[0035] Effects of N-acetylglutamine on body weight, disease activity index (DAI) score, survival rate, and colon length of mice Figure 1 As shown, Figure 1 The figures show the effects of N-acetylglutamine on the body weight, enteritis disease activity index (DAI) score, survival rate and colon length of mice; A shows the effect of N-acetylglutamine on the body weight of mice; B shows the effect of N-acetylglutamine on the enteritis disease activity index (DAI) score of mice; C shows the effect of N-acetylglutamine on the survival rate of mice, with the black line overlapping the blue line; and D shows the effect of N-acetylglutamine on the colon length of mice.

[0036] DAI scoring criteria:

[0037] DAI = stool consistency score + stool bleeding score + weight loss score

[0038] DAI total score 0-10 points;

[0039] Stool consistency score: 0, normal stool; 1, slightly soft stool; 2, very soft stool; 3, watery stool;

[0040] Fecal bleeding score: 0, normal stool; 1, brown stool; 2, red stool; 3, bloody stool;

[0041] Weight loss score: 0, weight loss <2%; 1, weight loss 2-5%; 2, weight loss 5-10%; 3, weight loss 10-15%; 4, weight loss ≥15%.

[0042] Figure 1 The results showed that 200 mg / d of N-acetylglutamine in DSS model mice increased their body weight, reduced the enteritis disease activity index, improved their survival rate, and lengthened their colon, significantly improving their clinical symptoms.

[0043] Colonic tissue staining: Sections of the same mouse colon were fixed with 4% paraformaldehyde (PFA), dehydrated, and embedded in paraffin. Sections were then sectioned at 3 μm thickness, deparaffinized, and rehydrated for subsequent experiments. Hematoxylin and eosin staining was performed according to the manufacturer's instructions. The procedure was as follows: staining with hematoxylin solution (Servicebio, Cat#G1004-500ML) for 3-5 minutes, followed by rinsing with tap water; then staining with hematoxylin differentiation solution for 2-5 seconds, followed by rinsing with tap water; then staining with hematoxylin bluing solution for 2-5 seconds, followed by rinsing with tap water. Microscopic observation revealed blue nuclei and a nearly colorless tissue background. Sections were then dehydrated in 70%, 80%, and 95% ethanol, each for 1-3 minutes. Sections were then stained with eosin solution (ethanol-soluble, Servicebio, Cat#G1001-500ML) for 4-5 minutes, followed by dehydration in anhydrous ethanol three times, each for 4-5 minutes. Sections were then cleared in xylene for 5 minutes, followed by fresh xylene for another 5 minutes. Finally, seal the slides with neutral gum.

[0044] Colon histological changes such as Figure 2 As shown, Figure 2 The effect of N-acetylglutamine on the colon histology of mice, Figure 2 The results showed that the colon mucosal layer of DSS model mice was severely infiltrated with inflammatory cells and the crypts were significantly damaged. N-acetylglutamine could significantly alleviate the cellular inflammation and restore the cell arrangement to normal.

[0045] qPCR detection of the expression of inflammatory factor-related genes in colon tissue:

[0046] Total RNA was extracted from mouse colon using Trizol reagent (Accor Biotech, Cat#AG21102). The purified RNA was quantified and reverse transcribed into cDNA using the ReverTraAce qPCR RT Kit (Accor Biotech, Cat#AG11706). To quantify gene expression, gene-specific primers and SYBR Green PCR Master Mix (Vazyme, Cat#Q311) were mixed and real-time PCR was performed using a real-time quantitative PCR detection system. GAPDH gene expression was used as a reference, and the relative expression of specific genes was analyzed using the 2-ΔΔCT technique.

[0047] qPCR was used to detect the expression of inflammatory factor-related genes in colon tissue. Figure 3 As shown, Figure 3 The figures are the effects of N-acetylglutamine on the expression of inflammatory factor-related genes in the colon tissue of mice, where AIs are the effects of N-acetylglutamine on Colon AhR, Colon IL-22, Colon IFN-γ, Colon Muc2, Colon Occludin, Colon Claudin1, Colon TNF-α, Colon S100A8 and Colon IL-10, respectively.

[0048] The results showed that N-acetylglutamine could significantly alleviate the decrease in the level of Colon AhR (inhibiting the expression of proinflammatory factors) caused by DSS mice, increase the expression of Colon IL-22 (barrier-related repair factor), reduce the expression of proinflammatory factors (Colon IFN-γ), significantly alleviate the increase in the levels of proinflammatory factors (Colon TNF-α and Colon S100A8), significantly alleviate the decrease in the level of anti-inflammatory factors (Colon IL-10), and increase the expression of intestinal barrier-related indicators (Colon Muc2, Colon Occludin and Colon Claudin1).

[0049] Mucus barrier Alcian blue staining:

[0050] The specific steps are: use methanol-Carnoy's fixative (60%, v / v) dry ethanol, 30% (v / v) chloroform, 10% (v / v) glacial acetic acid) to fix the colon containing feces and preserve intestinal mucus, wash and fix the tissue with methanol, anhydrous ethanol and xylene, dehydrate and embed in paraffin. Finally, the colon tissue sections are 3 μm thick, dewaxed, hydrated, and stained with Alcian Blue staining kit (Cat#G1560, Solarbio) according to the instructions. The specific steps are: 1. Dewax the sections to water; 2. Soak in Alcian acidification solution for 3 minutes; 3. Stain in Alcian staining solution for 30 minutes, rinse with running water; 4. Restain in nuclear fast red staining solution for 5 minutes, rinse with running water for 1 minute; 5. Dehydrate with gradient ethanol, make transparent with xylene, and seal with neutral resin. Observe the mucus barrier by Alcian blue staining. Figure 4 As shown, Figure 4 The effect of N-acetylglutamine on the mucus barrier of mice, where the red arrow indicates the thickness of the mucus layer in the mucus barrier. Figure 4 The results showed that N-acetylglutamine could significantly improve the destruction of the intestinal mucus barrier in DSS mice and treat ulcerative colitis.

[0051] Immunofluorescence was used to detect the expression of mechanical barrier-related proteins: Zo1 (zonal occludens), Occludin (occludens), and Claudin1 (tight junction protein-1):

[0052] Colon tissue sections were sliced ​​at 3 μm thickness and antigen retrieval was performed with Tris-EDTA. Tissue sections were then blocked with 5% BSA at room temperature for 60 minutes and incubated with primary antibodies against ZO1 (Cat#21773-1-AP, Proteintech), anti-occludin (Cat#E6B4R, Cell Signaling), and anti-claudin1 (Cat#13050-1-AP, Proteintech) overnight at 4°C. The next day, sections were washed twice for 10 minutes in PBST on a shaker and once for 10 minutes in PBS. Sections were then incubated with Alexa Fluor 488 / Cy3-conjugated goat anti-rabbit IgG (Cat#A0428 / A0516, Beytime) for 1 hour at room temperature. Tissue sections were then washed twice for 10 minutes in PBST on a shaker and once for 10 minutes in PBS to remove any residual secondary antibody. DAPI was then added for 20 minutes, followed by mounting and imaging under a fluorescence microscope.

[0053] Immunofluorescence detection of mechanical barrier-related Zo1, Occludin, and Claudin1 protein expressions Figure 5 As shown, Figure 5Immunofluorescence was used to detect the effect of N-acetylglutamine on the expression of mechanical barriers (Zo1, Occludin, Claudin1 proteins) in mice.

[0054] The results showed that N-acetylglutamine can increase the expression of key proteins claudin1, ZO1, and occludin, repair colon mucosal damage, and treat ulcerative colitis.

[0055] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of N-acetylglutamine or its stereoisomers and pharmaceutically acceptable salts in the preparation of products for preventing or treating ulcerative colitis.

2. The application according to claim 1, characterized in that: The ulcerative colitis includes one or more of intestinal barrier damage and intestinal inflammation.

3. A product for preventing or treating ulcerative colitis, characterized in that: It includes N-acetylglutamine or its stereoisomers and pharmaceutically acceptable salts.

4. The product according to claim 3, characterized in that: The product also includes auxiliary materials.

5. The product according to claim 4, characterized in that: The auxiliary materials include excipients.

6. The product according to claim 3, characterized in that: The product is in the form of tablets, capsules, powders, granules, pastes, suppositories, rectal enemas or foams.

7. The product according to claim 3, characterized in that: The product can be administered orally or rectally.

8. The product according to claim 7, characterized in that: The product is administered orally.

9. The product according to claim 3, characterized in that: The dosage of N-acetylglutamine or its stereoisomers and pharmaceutically acceptable salts in the product is 0.1-1.5 g / d.

10. The product according to claim 9, characterized in that: The dosage of N-acetylglutamine or its stereoisomers and pharmaceutically acceptable salts in the product is 0.2-0.5 g / d.

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