Use of nnmt in the diagnosis and treatment of inflammatory bowel disease

Abstract

The application belongs to the field of biological medicine, and specifically discloses a use of NNMT in diagnosis and treatment of inflammatory bowel disease. The application of NNMTi in preparation of a medicine for preventing and treating inflammatory bowel disease, including Crohn's disease and ulcerative colitis. The application of a reagent for detecting the expression level of NNMT in preparation of a product for diagnosing or detecting inflammatory bowel disease. The application provides a new treatment scheme for inflammatory bowel disease, and further studies the treatability of NNMTi for Crohn's disease and ulcerative colitis through a DSS mouse model. Meanwhile, the application also provides a new scheme for detection of inflammatory bowel disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the use of NNMT in the diagnosis and treatment of inflammatory bowel disease. BACKGROUND

[0002] Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC), which is a complex systemic inflammatory immune response caused by multiple pathogenic factors, and is manifested as epithelial barrier dysfunction and mucosal immune response disorder. The pathogenesis of IBD is the result of changes in intestinal immune response, which is highly affected by host microbiota in addition to genetic predisposition. Long-term self-sustaining recurrent chronic intestinal inflammation may lead to the occurrence of colorectal cancer (CRC). At present, the incidence of IBD is high worldwide, which causes a great burden on medical resources, and is considered as a major pathogenic risk factor for the progression of colorectal cancer. A better understanding of the pathophysiology and molecular biochemical processes of IBD pathogenesis and progression can provide us with a new perspective for early diagnosis and treatment of IBD disease.

[0003] Nicotinamide N-methyltransferase (NNMT) is a cytoplasmic metabolic enzyme that catalyzes the n-methylation of nicotinamide (NAM) with s-adenosyl-1-methionine as the methyl donor, producing n1-methyl nicotinamide (MNA) and releasing s-adenosyl-1-homocysteine (SAH). NNMT plays a key role in the metabolic pathway of nicotinic acid and nicotinamide, and NNMT methylates NAM and promotes its excretion from the body, thereby reducing the precursor material for nicotinamide adenine dinucleotide (NAD+) synthesis. Most NAD+ decomposition enzymes are inhibited by the concentration of their product NAM, but NNMT can remove excess NAM, which helps to sustain the conduction of NAD+-dependent pro-inflammatory signals. Inhibition of NNMT can increase the content of NAD+. There is sufficient evidence that NAD+ has a regulatory effect on IBD. The role of NNMT inhibitor NNMTi in IBD has not been reported. SUMMARY

[0004] In view of the above problems, the present application provides the use of NNMT in the diagnosis and treatment of inflammatory bowel disease, which mainly fills the gap of the unclear pathogenicity of NNMT in inflammatory bowel disease, fills the research blank of NNMTi in the treatment of inflammatory bowel disease, and further studies how to diagnose or detect inflammatory bowel disease and the like.

[0005] In order to solve the above problems, the present application adopts the following technical scheme:

[0006] The first aspect of the application relates to The application of NNMTi in the preparation of a drug for preventing and treating inflammatory bowel disease. Among them, prevention and treatment means prevention and treatment.

[0007] In some aspects, the inflammatory bowel disease comprises Crohn's disease and ulcerative colitis.

[0008] In some aspects, the effect of the NNMTi is manifested as improving the permeability of the intestinal tract, repairing the barrier dysfunction of the intestinal tract, and improving the oxidative stress and inflammatory damage of the intestinal tissue.

[0009] In some aspects, the NNMTi is This part focuses on a common NNMTi, but other mature NNMTis should also be considered feasible for the same reason. The present application verifies the effect of adjusting the expression level of NNMT on relieving inflammatory bowel disease, and the expression of NNMT can be effectively relieved by inhibiting the expression of NNMT.

[0010] The second aspect of the application relates to The use of a reagent for detecting the expression level of NNMT in the preparation of a product for diagnosing or detecting inflammatory bowel disease. Among them, because the expression level of NNMT is positively correlated with the occurrence of inflammatory bowel disease, the reagent for detecting the expression level of NNMT can more targetedly assist in the diagnosis of inflammatory bowel disease, and provide a reference for the diagnosis of the disease. Of course, the expression level of NNMT can also be used as an index in the diagnosis of inflammatory bowel disease. The reagent for detecting the expression level of NNMT should also include some high-throughput detection products.

[0011] In some aspects, the inflammatory bowel disease comprises Crohn's disease and ulcerative colitis.

[0012] The reagent for detecting the expression level of NNMT includes primers, antibodies and other detection products. For example, the primer for detecting the expression level of NNMT is

[0013] Upstream primer: 5'-CCATCTGTTCTAAAAGAAGGGC-3',

[0014] Downstream primer: 5'-GGAGGTGAAGCCTGATTCCA-3'.

[0015] The beneficial effects of the present application are:

[0016] A new treatment scheme for inflammatory bowel disease is provided, and the treatability of NNMTi for Crohn's disease and ulcerative colitis is further studied through a DSS mouse model. At the same time, a new scheme for detecting inflammatory bowel disease is also provided. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The expression of NNMT in the intestinal tract and serum of IBD patients;

[0018] Figure 2 The graph of the relief of intestinal inflammation of mice by NNMTi;

[0019] Figure 3 Graphs showing that NNMTi increased the expression of intestinal barrier tight junction proteins in mice;

[0020] Figure 4 Graphs showing that NNMTi reduced oxidative stress and inflammatory damage in the intestines of mice. DETAILED DESCRIPTION

[0021] The present application is further described below:

[0022] I. Materials

[0023] 1. Mice: C57BL / 6J mice (purchased from Beijing Vantoll Life Co., Ltd.) were raised in a SPF environment to 20-22 g at about 8 weeks of age.

[0024] 2. NNMT inhibitor NNMTi (purchased from Med Chem Express Co., Ltd. in the United States), molecular formula C 10 H 11 IN2, molecular weight 286.11 Da; purity 99.64%.

[0025] Compound 1:

[0026] II. Methods

[0027] 1. Construct a dextran sulfate sodium (DSS)-induced colitis mouse model and use NNMTi for intervention.

[0028] ① Grouping of experimental mice: 24 male C57BL / 6 mice were randomly divided into a control group, a DSS group, a DSS+1 mg / kg NNMTi group, and a DSS+10 mg / kg NNMTi group, 6 mice per group. The mice were adaptively fed for 7 days, and their diet, activity, and stool were observed and their body weight was measured.

[0029] ② Intervention of mice:

[0030] Prepare the solvent: 10% DMSO+40% PEG300+5% Tween-80+45% normal saline;

[0031] Control group: freely drink normal water for 7 days, and subcutaneously inject 200ul of the above prepared solvent on days 0, 1, 2, 3, 4, 5, 6, and 7;

[0032] DSS group: mice freely drink 2.5% DSS solution for 7 days, and subcutaneously inject 200ul of the above prepared solvent on days 0, 1, 2, 3, 4, 5, 6, and 7;

[0033] DSS+1mg / kg NNMTi group and DSS+10mg / kg NNMTi group: mice were allowed to drink 2.5% DSS solution for 7 days, and 1 mg / kg and 10 mg / kg of NNMTi were dissolved in the above-mentioned solvent, respectively. 200 ul of the solution containing 1 mg / kg and 10 mg / kg of NNMTi was subcutaneously injected on day 0, 1, 2, 3, 4, 5, 6, and 7.

[0034] ③General condition of mice: After the modeling began, the body weight, stool characteristics (normal, paste or watery stool), blood condition (none, occult or obvious visible blood stool), and activity (active or lethargic) of the mice were recorded every day.

[0035] ④Mouse sacrifice and specimen collection: the mice were sacrificed on day 7, and the mice were anesthetized with sodium pentobarbital intraperitoneally, and then the eyeball blood was taken, the abdominal cavity was opened along the midline, the entire colon from above the rectum to the ileocecal junction was taken, the colon length was measured and recorded, and the spleen was also taken and weighed. The colon feces were washed, and the distal colon tissue was stored at -80°C for RT-qPCR, western-blot and oxidative stress detection.

[0036] ⑤Intestinal mucosal permeability detection of mice: each mouse was given 200 ul of FITC-dextran 4kd (25 mg / ml) by gavage, and after 4 hours, the mice in each group were sacrificed, and about 500 ul of whole blood samples were taken, which were placed at room temperature for 2 hours, then centrifuged at 3000 rpm at 4°C for 15 minutes, and the supernatant serum was taken, and the fluorescence level in the serum was detected at 490 nm and 520 nm using a fluorescence spectrophotometer.

[0037] 2. Primer sequences for RT-qPCR

[0038] Gene Upstream primer Downstream primer Human NNMT 5'-CCATCTGTTCTAAAAGAAGGGC-3' 5'-GGAGGTGAAGCCTGATTCCA-3' Mouse β-actin 5'-GTTGGAGCAAACATCCCCCA-3' 5'-CGCGACCATCCTCCTCTTAG-3' Mouse IL-1 β 5'-CCTCGTGCTGTCGGACCCATA-3' 5'-CAGGCTTGTGCTCTGCTTGTGA-3' Mouse TNF-α 5'-CCTGTAGCCCACGTCGTAG-3' 5'-GGGAGTAGACAAGGTACAACCC-3' Mouse IL-6 5'-TAGTCCTTCCTACCCCAATTTCC-3' 5'-TTGGTCCTTAGCCACTCCTTC-3' Mouse IFN-γ 5'-CCAGCGCCAAGCATTCAATGAG-3' 5'-GACAATCTCTTCCCCACCCCG-3'

[0039] III. Results

[0040] As Figure 1 In the present application, RT-qPCR was used to detect that the expression of NNMT in the lesion tissue of IBD patients was increased, while the expression in the adjacent tissue of the colon lesion was not significantly changed; ELISA was used to detect that the expression of NNMT in the serum of IBD patients in the active stage was increased, and was positively correlated with CRP, ESR, and NE%.

[0041] As Figure 2The specific grouping of mice is shown in the method section. The ratio of the daily body weight of each mouse to the body weight of the mouse on day 0 was measured and calculated, and the average and standard deviation of each group were calculated; the results showed that the average body weight of the mice in the NNMTi group was heavier than that in the DSS group, and there was statistical significance, and there was no statistical difference in the body weight of the mice treated with different doses of NNMTi. The daily disease activity index (DAI) of each mouse was evaluated, and the average and standard deviation of each group were calculated; the results showed that the DAI value of the mice in the NNMTi group was smaller than that in the DSS group, and there was statistical significance, and there was no statistical difference in the DAI index of the mice treated with different doses of NNMTi. RT-qPCR detection found that the treatment of the NNMTi group successfully reduced the expression of NNMT in the intestinal tissue of the mice. HE staining was performed on the colon tissue of each group of mice, and the tissue activity index (HAI) was evaluated, and it was found that the HAI of the colon tissue of the mice in the NNMTi group was significantly lower than that in the DSS group. The average spleen weight of the mice in the 10 mg / kg NNMTi group was lighter than that in the DSS group, and the average colon length of the mice in the 10 mg / kg group was longer than that in the DSS group, and both had statistical significance.

[0042] As Figure 3 In the experiment, the tight junction proteins ZO-1 and occludin in the terminal colon tissue of the four groups of mice were detected by western-blot method, and the expression of GAPDH was standardized, the column height represented the average value of the ratio of protein expression level to internal reference gene GAPDH, and the distance between the horizontal line above the column and the column represented the standard deviation. The results showed that the protein expression of ZO-1 and occludin in the NNMTi group and the DSS group mice increased to different degrees. FD4 reflects the permeability of the intestinal tract of mice, and the intestinal permeability of the mice in the NNMTi group is higher than that in the DSS group, which proves that NNMTi can repair the intestinal barrier of mice.

[0043] As Figure 4 In the experiment, the total oxidative capacity (T-AOC), glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) of each group of mice were detected, and the results showed that NNMTi intervention alleviated the oxidative stress damage of the colon tissue of DSS mice.

[0044] Those skilled in the art can clearly make various modifications to the above embodiments without departing from the overall spirit and concept of the present application. All fall within the scope of the present application. The protection scheme of the present application is subject to the claims attached to the present application.

Claims

1. The application of NNMTi in the preparation of drugs for the prevention and treatment of inflammatory bowel disease, characterized in that, The NNMTi is .

2. The application according to claim 1, characterized in that, The inflammatory bowel disease referred to is Crohn's disease or ulcerative colitis.

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