Application of NMN in the preparation of gastric mucosal intestinal metaplasia drugs

Abstract

The present invention discloses the use of nicotinamide mononucleotide (NMN) in the preparation of drugs for treating intestinal metaplasia of gastric mucosa, and belongs to the field of biomedicine technology. The invention specifically includes: constructing a mouse gastric mucosal intestinal metaplasia model, collecting gastric tissue after NMN intervention, and performing H&E staining, AB-PAS staining, HID-AB staining, immunohistochemistry, immunofluorescence, etc. to evaluate changes in intestinal metaplasia-related indicators. The results showed that NMN has a therapeutic effect on gastric mucosal intestinal metaplasia, especially after long-term intervention in vivo, it significantly reversed the intestinal metaplasia lesions of gastric mucosa. This provides a theoretical basis for the clinical treatment of atrophic gastritis and intestinal metaplasia.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedicine technology, and specifically to the application of NMN in the preparation of gastric mucosal intestinal metaplasia drugs. Background Art

[0002] Gastric cancer is the leading cause of cancer death worldwide. Its incidence varies greatly by region, and this heterogeneity is attributed to multiple factors, including infection, environment, and genetic traits. Most cases of gastric cancer are associated with Helicobacter pylori infection. According to the Correa cascade model, the development of most gastric cancers progresses through a multi-step progression: normal gastric mucosa → atrophic gastritis → intestinal metaplasia → intraepithelial neoplasia → gastric cancer. Gastric intestinal metaplasia (GIM) is a critical precancerous stage in the progression of gastric cancer and is closely associated with an increased risk of gastric cancer. GIM can present in two histological types: complete GIM expresses only intestinal mucin but not gastric mucin. Incomplete GIM exhibits a mixed gastrointestinal glandular pattern, expressing both gastric mucin and intestinal mucin markers. Incomplete GIM or extensive GIM has a higher risk of progression to gastric cancer, and some researchers even consider it a mild form of dysplasia. Therefore, effectively reversing intestinal metaplasia and reducing its transformation to gastric cancer is crucial for the prevention and treatment of gastric cancer.

[0003] Recent studies have highlighted the role of nicotinamide adenine dinucleotide (NAD + ) metabolism regulates stem cell homeostasis. + It is a redox cofactor and substrate of key metabolic enzymes, participating in metabolic pathways such as glycolysis, fatty acid oxidation, tricarboxylic acid (TCA) cycle and electron transport chain (ETC), and receiving hydrogen ions from metabolites of these pathways to form NADH. By regulating these key physiological metabolic processes, cells can adapt to environmental changes to regulate stress responses to genotoxic factors, infection, inflammation and exogenous substances. These effects are mainly through NAD + This is achieved by acting as an enzyme cofactor to drive the metabolic pathway of transferring hydrogen in redox reactions. In addition to its important role as a coenzyme in energy metabolism, NAD + It is also a co-substrate of multiple key enzymes, including sirtuins (SIRTs), PARPs (poly-(ADP-ribose) polymerases), CD38 and SARM1, etc., by regulating these NAD + NAD-dependent enzyme activity affects multiple physiological processes such as energy metabolism, DNA repair, epigenetic modification, inflammation, and circadian rhythm. +Long-term metabolic disorders can lead to diseases such as metabolic diseases, cancer, aging, and neurodegenerative diseases.

[0004] Targeting NAD + Metabolic interventions have shown some efficacy in the treatment of gastric cancer. + Precursors of nicotinic acid (NA), nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) have shown good efficacy in the treatment of diabetes, Alzheimer's disease, endothelial dysfunction and inflammation. Some studies have shown that in addition to NAD and NADP, CD38 can also directly degrade NMN into NAM and ribose monophosphate (RMP). In addition, in the presence of NAD + In the salvage synthesis pathway, NMN is NAD + The direct precursor of NMN is rapidly absorbed in the body and is an ideal supplement. Therefore, the present invention selected NMN for intervention to explore the potential effect of NMN on reversing intestinal metaplasia.

[0005] The present invention constructed multiple mouse intestinal metaplasia models and used NMN to intervene in mice to explore the role of NMN in reversing intestinal metaplasia, providing new clues for the diagnosis, prevention and treatment of gastric precancerous lesions. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of NMN in the preparation of gastric mucosal intestinal metaplasia drugs. The present invention constructs an intestinal metaplasia model, uses NMN for intervention, and explores the therapeutic effect of NMN on intestinal metaplasia. The results of the present invention found that long-term intervention with NMN promoted the recovery of parietal cells, inhibited gastric cancer stem cell markers and DNA damage, and significantly reversed the intestinal metaplasia lesions of the gastric mucosa of mice, which has potential application prospects for the clinical treatment of intestinal metaplasia of gastric mucosa.

[0007] Based on the above objectives, the present invention adopts the following technical solutions:

[0008] The use of NMN in the preparation of drugs for preventing and / or treating intestinal metaplasia of gastric mucosa.

[0009] Furthermore, the intestinal metaplasia is induced by tamoxifen or Hp infection or Atp4a gene knockout.

[0010] Use of NMN in the preparation of a drug for preventing and / or treating spasmolytic polypeptide-expressing metaplasia.

[0011] Furthermore, the spasmolytic polypeptide expression metaplasia is induced by tamoxifen or Hp infection or Atp4a gene knockout.

[0012] Preferably, the NMN is administered by injection or oral administration.

[0013] Preferably, the NMN is the only active ingredient.

[0014] Preferably, NMN is mixed with other active ingredients.

[0015] A pharmaceutical composition comprising NMN for the above-mentioned use, wherein the pharmaceutical composition has at least one of the following functions 1) to 3):

[0016] 1) Inhibit intestinal metaplasia in gastric mucosa;

[0017] 2) inhibiting the expression of stem cell markers in intestinal metaplastic mucosa;

[0018] 3) Inhibit DNA damage and repair markers in intestinal metaplasia mucosa.

[0019] A pharmaceutical kit comprising the NMN for the above-mentioned use.

[0020] The present invention found that:

[0021] (1) After NMN intervention in the mouse metaplasia model, NAD + The content increased significantly;

[0022] (2) Compared with the model group, the expression of MUC5AC in the gastric mucosa of mice in the NMN intervention group was upregulated, while the expression of MUC2 and CDX2 was downregulated. In particular, the expression and proportion of ATP4a-positive cells, a parietal cell marker, were significantly increased;

[0023] (3) Compared with the gastric mucosa of the control group, the expression of gastric cancer stemness markers LGR5 and CD44 was significantly downregulated in the NMN intervention group, and the DNA damage marker γ-H2AX was downregulated, while the expression of CD38 did not change;

[0024] Overall, NMN significantly reversed intestinal metaplasia in mice by promoting parietal cell recovery, inhibiting gastric cancer stem cell markers, and promoting DNA damage repair to maintain gastric stem cell characteristics. NMN provides new clinical insights and theoretical guidance for the treatment of intestinal metaplasia in clinical work. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 3 shows the NMN intervention pattern and H&E staining results of gastric mucosa in three intestinal metaplasia mouse models: A. Gastric mucosa of Tam-treated mice, B. Gastric mucosa of Hp-infected mice, C. Atp4a - / - Mouse gastric mucosa;

[0026] Figure 2RT-qPCR was used to detect the expression of inflammatory factor Il8 in the gastric mucosa of mice after H. pylori infection (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0027] Figure 3 Staining for AB-PAS and HID-AB in the GIM mouse model: A. Atp4a - / - Mucin staining of mouse gastric mucosa. B. Scoring statistics for Figure A. (**P < 0.01, ***P < 0.001, ****P < 0.0001);

[0028] Figure 4 Immunohistochemical staining of ATP4a in three mouse models of intestinal metaplasia: A. Gastric mucosa of Tam-treated mice. B. Scoring of panel A. C. Gastric mucosa of H. pylori-infected mice. D. Scoring of panel C. E. Gastric mucosa of Atp4a- / - mice. F. Scoring of panel E. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001);

[0029] Figure 5 To detect MUC5AC expression in GIM mice: A. Atp4a - / - Immunohistochemical staining of MUC5AC in mouse gastric mucosa. B. Scoring of Figure A. (*P < 0.05, **P < 0.01, ****P < 0.0001);

[0030] Figure 6 To detect MUC2 expression in GIM mice: A. Atp4a - / - Immunohistochemical staining of MUC2 in mouse gastric mucosa. B. Scoring of Figure A. (**P < 0.01, ****P < 0.0001);

[0031] Figure 7 Immunohistochemical staining of CDX2 in mouse gastric mucosa: A. CDX2 expression in gastric mucosa of Hp-infected mice before and after NMN intervention. BA score. C. Atp4a - / - CDX2 immunohistochemical staining of mouse gastric mucosa. D. Scoring of Figure C. (***P < 0.001, ****P < 0.0001);

[0032] Figure 8 Detection of MIST1 expression in the gastric mucosa of NMN-treated Tam-treated mice: A. Immunofluorescence identification of the expression of MIST1 and TFF2 in the gastric mucosa of NMN-treated Tam-treated mice. B. Statistics of mean fluorescence intensity.

[0033] (**P<0.01, ****P<0.0001);

[0034] Figure 9 CD44 immunohistochemical staining of mouse gastric mucosa: A. CD44 expression in gastric mucosa of Hp-infected mice before and after NMN intervention. BA score. C. Atp4a - / - CD44 immunohistochemical staining of mouse gastric mucosa. D. Scoring of Figure C. (**P < 0.01, ***P < 0.001, ****P < 0.0001);

[0035] Figure 10 Immunohistochemical staining of LGR5 in mouse gastric mucosa: A. LGR5 expression in the gastric mucosa of H. pylori-infected mice before and after NMN treatment. BA score. C. LGR5 immunohistochemical staining in the gastric mucosa of Atp4a- / - mice. D. Score for panel C. (*P < 0.05, ***P < 0.001, ****P < 0.0001);

[0036] Figure 11 Immunohistochemical staining of γ-H2AX in mouse gastric mucosa: A. γ-H2AX expression in gastric mucosa of Tam-treated mice after NMN treatment. B. Histochemical score for panel A. C. γ-H2AX expression in gastric mucosa of H. pylori-infected mice before and after NMN treatment. D. Histochemical score for panel C. E. γ-H2AX immunohistochemical staining in gastric mucosa of Atp4a- / - mice. F. Histochemical score for panel E (**P < 0.01, ***P < 0.001, ****P < 0.0001). DETAILED DESCRIPTION

[0037] The present application will be further explained below in conjunction with the embodiments. Before introducing the specific embodiments, a brief description of the basic conditions of some biological materials and experimental equipment involved in the following embodiments is given as follows.

[0038] Biomaterials:

[0039] Atp4a knockout mice (Atp4a - / - ) was constructed and characterized by Nanjing Model Organisms Co., Ltd. Wild-type C57BL / 6cnc mice were purchased from Beijing Weitonglihua Co., Ltd. Mouse-adapted H. pylori strain SS1 was purchased from the Guangdong Microbial Culture Bank and cultured on Columbia agar plates in a humidified, 37°C anaerobic incubator, passaged every 3 days.

[0040] Experimental reagents:

[0041]

[0042]

[0043] Example 1

[0044] This example utilizes various mouse models of intestinal metaplasia to explore the potential effects of NMN on intestinal metaplasia. The relevant experimental procedures are briefly described below:

[0045] 1. Construction of a high-dose tamoxifen (Tam)-induced SPEM mouse model and NMN intervention;

[0046] (1) Tamoxifen preparation: Weigh 250 mg of dried Tamoxifen into a 15 mL centrifuge tube; add 9 mL of sterile sunflower oil, cap, and vortex the solution to ensure thorough mixing. Aliquot and store at -20°C until use.

[0047] (2) Ten male C57 mice aged 4-5 weeks were divided into vehicle group, Tam group, vehicle + NMN group, and Tam + NMN group.

[0048] In the Tam group, mice were weighed and the injection site was disinfected by wiping the abdomen of the mouse with alcohol at a ratio of 200 μL / 20 g body weight. The Tam solution prepared in step (1) was injected intraperitoneally once a day for 3 consecutive days.

[0049] The mice in the vehicle group were intraperitoneally injected with an equal amount of solvent (sterile sunflower oil).

[0050] Tam+NMN group: NMN was intervened at a dose of 300 mg / kg / day and prepared into a 0.6 g / L aqueous solution. It was placed in drinking water for free drinking, and fresh drinking water was replaced twice a week. After 7 weeks, Tam treatment was given. The mouse abdomen was wiped with alcohol to disinfect the injection site at a ratio of 200 μL / 20 g body weight. The Tam solution prepared in step (1) was injected intraperitoneally once a day for 3 consecutive days (NMN intervention was also given during this period). The mice were euthanized on the morning of the 4th day after the last Tam intervention, and the gastric mucosa was collected.

[0051] Vehicle+NMN group: NMN was intervened at a dose of 300 mg / kg / day, prepared into a 0.6 g / L aqueous solution, and placed in the drinking water for free drinking. Fresh drinking water was replaced twice a week. After 7 weeks, the mice were intraperitoneally injected with an equal amount of solvent (sterile sunflower oil) (NMN intervention was also given during this period). The mice were euthanized on the morning of the 4th day after the last solvent injection, and the gastric mucosa was collected.

[0052] 2. Construction of a chronic SPEM mouse model induced by H. pylori infection and intervention with NMN;

[0053] (1) Methylenenitrosourea (MNU) pretreatment: A total of 20 5-6 week-old C57BL / 6cnc mice were randomly divided into a control group (Uni) and an Hp infection group, with 10 mice in each group.

[0054] The control group (Uni) received normal drinking water. The control group (Uni) was randomly divided into two groups, with five mice in each group. One group did not require special treatment and had free access to water. The other group (Uni-NMN) was given 300 mg / kg / day NMN by gavage at week 20. The mice were euthanized after 8 consecutive weeks, and the sample collection procedure was the same as before.

[0055] The H. pylori-infected group was first treated with MNU. 0.15g of MNU was dissolved in 1L of clean water. The water bottles were wrapped in tin foil to protect from light and allowed to drink freely. The MNU treatment concentration was 150ppm each time for five consecutive weeks. After five weeks, the water was replaced with normal drinking water, and the subjects were allowed to recover for two weeks.

[0056] (2) Hp culture: Take the frozen mouse-adapted Hp SS1 strain and thaw it on ice. Use a cotton swab to gently scoop up a small amount of bacterial solution and evenly spread it on a culture plate. Place the culture plate upside down in an anaerobic incubator at 37°C and culture for 4 days.

[0057] (3) Subculture every 3 days. Take bacteria from the 4th generation to infect mice. Collect H. pylori with a cotton swab into a centrifuge tube containing 5 mL of BHI liquid medium.

[0058] (4) Measure the absorbance at OD440 nm using a spectrophotometer, and then adjust the OD value to approximately 0.1 using BHI liquid medium (supplemented with 10% FBS and 1% antibiotics);

[0059] (5) Hp was transferred to a 100 mL conical culture flask, placed on a horizontal shaker, and cultured in an anaerobic incubator at 37°C for 1 day;

[0060] (6) Measure the OD440 value, then centrifuge at 4500 rpm for 5 min, measure the OD value of the supernatant, and then use the standard curve to estimate the bacterial count. OD440 = 0.2 means the concentration of Hp is 4 × 10 7 CFU / mL (colony forming unit);

[0061] (7) Hp was then resuspended in BHI medium without FBS and antibiotics to adjust the concentration to 3×10 9 CFU / mL;

[0062] (8) After fasting for 24 hours, each mouse was gavaged with 50 μL of 1% sodium bicarbonate solution to neutralize gastric acid. Ten minutes later, 0.1 mL of H. pylori suspension was gavaged. This was repeated seven times every other day to establish an H. pylori-infected mouse model. The mice were then randomly divided into two groups, with five mice in each group.

[0063] (9) NMN intervention: For one group of Hp-infected mouse models, 300 mg / kg / day NMN was administered orally at week 10 after Hp gavage. The mice were euthanized after 8 consecutive weeks, and the sample collection procedure was the same as before. The other group served as the Hp control group and did not require special treatment and was allowed to drink water freely.

[0064] 3. Atp4a - / - Mouse spontaneous intestinal metaplasia model and NMN intervention;

[0065] (1) Atp4a knockout mice (Atp4a - / - ) was constructed and genotyped by Nanjing Model Organisms Company. Atp4a is a parietal cell-specific gene that encodes H + K + -ATPase, responsible for gastric acid secretion. After its gene knockout, the parietal cells of mice gradually atrophy and spontaneous intestinal metaplasia occurs within 4-5 months;

[0066] (2) NMN intervention: For Atp4a - / - Mice, 6 male mice in each group were given 300 mg / kg / day NMN in drinking water at 16 weeks of age and euthanized after 8 weeks of free drinking. WT mice of the same age were used as controls, and the sample collection procedure was the same as before.

[0067] (3) Evaluation methods of intestinal metaplasia-related indicators;

[0068] Using NMN to intervene in the mouse model of intestinal metaplasia in vivo, gastric tissue was collected for H&E staining and inflammatory factors

[0069] IL-8

[0070] Detection, AB-PAS staining, HID-AB staining, immunofluorescence, immunohistochemistry, etc. were used to evaluate the changes in intestinal metaplasia-related indicators.

[0071] 1. H&E staining;

[0072] (1) The embedded paraffin block was cut into 4 μm sections on a paraffin microtome and then baked in a drying oven at 65°C for 1 h;

[0073] (2) The tissue sections were then dewaxed and immersed in xylene for 15 min twice; then immersed in anhydrous ethanol (twice), 95%, 85%, and 75% alcohol for 10 min each to fully hydrate the tissue sections;

[0074] (3) Rinse in running tap water for 5 min, then wash three times with PBS for 5 min each;

[0075] (4) Place the sections in hematoxylin staining solution for 5 minutes. Rinse with running water for 5 minutes.

[0076] (5) Add 1% concentrated hydrochloric acid to 75% alcohol, and then immerse the slices in it for 1 second;

[0077] (6) Rinse with running water for 5 minutes and observe the staining under a microscope. If the staining is light, restain.

[0078] (7) Add 1% concentrated ammonia solution to distilled water, immerse the sections in anti-blue water for 3 seconds, and rinse with running water;

[0079] (8) Place the sections in eosin staining solution for 3 minutes and rinse with running water;

[0080] (9) Observe the staining under a microscope. Then, hydrate and transparentize the sections: soak in 75%, 85%, and 95% alcohol for 1 minute, in anhydrous ethanol for 5 minutes (twice), and in xylene for 10 minutes (twice);

[0081] (10) Place a small drop of neutral gum on the tissue area and seal with a coverslip. Allow to dry thoroughly in a fume hood and then photograph under a microscope.

[0082] 2. Detection of inflammatory factor IL-8

[0083] RNA extraction

[0084] (1) Place the tissue sample in a 1.5 mL EP tube. Use surgical scissors to mince the tissue, and then add 1 mL of Trizol reagent to each EP tube. Grind the tissue until it becomes a homogenate to ensure that the cells in the tissue are fully lysed. This process should be performed on ice to keep the sample cold and avoid RNA degradation;

[0085] (2) Next, add 400 μL of chloroform to each EP tube containing the lysate, mix thoroughly by inverting, and then let stand at room temperature for 5 min.

[0086] (3) Centrifuge at 12,000 rpm for 15 min at 4°C. After centrifugation, the sample will separate into three layers, with RNA in the supernatant.

[0087] (4) Use a 200 μL pipette to transfer the upper aqueous phase to a new enzyme-free EP tube. Add an equal amount of isopropanol, gently shake and invert to mix, and then centrifuge at 12,000 rpm for 10 minutes.

[0088] (5) After centrifugation, a white precipitate is visible. Prepare 70% alcohol with enzyme-free water and anhydrous ethanol. Then add 1 mL of 70% alcohol and gently resuspend the precipitate. Centrifuge as before and repeat twice.

[0089] (6) Aspirate the supernatant as much as possible, open the tube, and let it dry for 5 minutes. Then, depending on the amount of precipitation, add about 5-10 μL of enzyme-free water to resuspend the RNA, and use a micro-nucleic acid analyzer to detect the concentration and purity.

[0090] Reverse transcription

[0091] (1) Denature the RNA extracted in the previous step at 65°C for 5 minutes. Then cool on ice for 2 minutes. Denature the RNA in a 65°C water bath for 5 minutes. Prepare the reaction system according to the following table to remove genomic DNA:

[0092]

[0093] (2) Place the centrifuge tube in a microcentrifuge for instant centrifugation, and then place it in a PCR instrument at 42°C for 2 minutes.

[0094] (3) Then, 2 μL of each of 10× RT Mix and HiScript II Enzyme Mix were added sequentially. The mixture was centrifuged briefly and then placed in a PCR instrument for reverse transcription at 50°C for 15 min and 85°C for 2 min. The resulting product can be diluted 5-10 times with water for RT-qPCR analysis.

[0095] Real-time fluorescence quantitative qPCR

[0096] (1) The primers used in this invention were synthesized by Shanghai Sangon Biotechnology. Remove the 2×ChamQ Universal SYBR qPCR Master Mix, primers, and enzyme-free water from the -20°C refrigerator in advance and place them on ice to fully thaw. In a clean row of eight centrifuge tubes, prepare the reaction system according to the following table:

[0097]

[0098] (2) After centrifugation in a microcentrifuge, ensure that no bubbles are generated and place the eight tubes in a Roche real-time fluorescence quantitative qPCR instrument for qPCR reaction detection:

[0099]

[0100] (3) After obtaining the Ct value of gene expression, GAPDH was used as a housekeeping gene for expression correction, and the relative gene expression value was analyzed according to the 2-ΔΔct method.

[0101] 3. Mucin staining;

[0102] (1) Alcian Blue Stain (AB): Tissue sections were dewaxed and hydrated as described above, then immersed in Alcian Blue acidified solution for 3 min, and then stained with Alcian Blue staining solution for 30 min. After rinsing with tap water, the sections were counterstained with Nuclear Fast Red reagent for 5 min. After rinsing, the sections were hydrated and mounted as described above.

[0103] (2) Alcian Blue-Periodic Acid Schiff (AB-PAS) staining: Dewax and hydrate the sections as described above, then immerse in AB staining solution for 20 minutes, rinse with distilled water for 3 minutes, three times; add oxidant for 6 minutes, rinse with water three times; add Schiff stain for 20 minutes, rinse with distilled water for 5 minutes, three times. After completion, hydrate and seal the sections.

[0104] (3) High Iron Diamine / Alcian Blue (HID-AB) staining: The sections were dewaxed and hydrated as before. The sections were placed in a humidified chamber and HID staining solution A and B were mixed in a ratio of 50:3 to prepare a working solution. After adding HID staining solution, the sections were left at room temperature overnight. The next day, the sections were washed with running water for 5 minutes, then immersed in Alcian Blue staining solution for 20 minutes, washed with water, and then stained with nuclear fast red reagent for 5 minutes. After the sections were hydrated and sealed, the sections were scored and statistically analyzed according to the previously published method. The specific criteria were: 5 points for the percentage of positive cells in the visual field of 75-100%, 4 points for 50%-75%, 3 points for 25%-50%, 2 points for 5%-25%, 1 point for less than 5%, and 0 points for negative.

[0105] 4. Immunofluorescence;

[0106] (1) Sectioning and dewaxing procedures were performed as described above;

[0107] (2) Rinse thoroughly with distilled water and then with PBS solution for 3 min, 3 times;

[0108] (3) Heat the prepared sodium citrate antigen retrieval solution in a microwave oven at medium-high heat for 5 minutes, place the slide on the microscope slide, and retrieval at medium-high heat for 20 minutes. Remove the slide and allow it to cool naturally at room temperature (for more than 2 hours).

[0109] (4) Remove the sections and wash them thoroughly in PBS-T solution (PBS solution with 0.1% Tween 20 added) for 5 min, three times;

[0110] (5) After gently shaking off the water stains, use an immunohistochemistry pen to circle along the edge of the tissue to mark the tissue position. Place the slice flat in a staining wet box, then add tissue autofluorescence quencher A solution, incubate at room temperature for 30 minutes, and wash with distilled water for 5 minutes;

[0111] (6) Add 50-100 μL of blocking solution (3% BSA dissolved in PBS-T, 1% volume of Triton X100 is required for nuclear and cytoplasmic protein staining, and no Triton X100 is required for membrane protein staining; use directly after filtering through a 0.22 μm filter) to each area of ​​the tissue until it fully covers the tissue. Block at room temperature for 1 hour.

[0112] (7) Dilute the corresponding primary antibody directly with blocking solution in an appropriate ratio. After blocking, carefully absorb the blocking solution with absorbent paper, then directly add 50-100 μL of diluted primary antibody to fully cover the tissue and incubate at room temperature for 1 hour;

[0113] (8) After the primary antibody incubation is completed, the sections are washed three times with PBS-T for 5 minutes each, and the fluorescent secondary antibody is diluted in blocking solution at an appropriate ratio; 50-100 μL of the diluted secondary antibody is added to the tissue area and incubated in the dark at room temperature for 1 hour;

[0114] (9) Discard the secondary antibody and wash the cells three times with PBS-T for 5 minutes. Then, stain the cell nuclei with DAPI solution for 10 minutes.

[0115] (10) Add tissue autofluorescence quencher B solution, incubate at room temperature in the dark for 5 minutes, and rinse with running water for 5 minutes;

[0116] (11) Wash three times with PBS (note that it does not contain Tween 20, otherwise it will reduce the efficacy of autofluorescence quencher B). Seal the slides with anti-fluorescence quenching mounting medium and dry them at room temperature. Observe and photograph them using a confocal microscope.

[0117] (12) Zeiss ZEN software was used to obtain the mean fluorescence intensity for semi-quantitative statistical analysis of protein expression. Protein colocalization analysis was performed using Image J software.

[0118] 5. Immunohistochemistry;

[0119] (1) Dewaxing, dehydration, and antigen retrieval procedures are the same as above;

[0120] (2) Wash with PBS solution three times for 5 min each; seal the edge of the tissue with an immunohistochemical pen;

[0121] (3) Place the sections in a humidified chamber, add 50–100 μL of 3% hydrogen peroxide in deionized water, and incubate at room temperature for 15 min to block endogenous peroxidase activity.

[0122] (4) After completion, wash in PBS three times for 5 min, then add 50 μL of goat serum blocking solution to each section and let it stand at room temperature for 15 min;

[0123] (5) Preparation of primary antibody diluent: Add 1% goat serum and 0.5% Triton X100 (not required for membrane protein staining) to PBS. Dilute the primary antibody in an appropriate ratio. After blocking, remove the goat serum blocking solution with absorbent paper, then carefully add 50-100 μL of the prepared primary antibody and incubate overnight in a 4°C refrigerator.

[0124] (6) The next day, the cells were equilibrated at room temperature for 1 h; washed with PBS solution; 50-100 μL of reaction enhancement solution was added dropwise, and the cells were incubated in a 37°C oven for 20 min; and washed with PBS solution.

[0125] (7) Add 50-100 μL of enhanced enzyme-labeled goat anti-rabbit IgG polymer and incubate in a 37°C oven for 20 min; wash with PBS solution;

[0126] (8) Mix the DAB colorimetric solution A and solution B in a ratio of 1:20, add 50-100 μL of the prepared DAB colorimetric reagent, and colorimetric for an appropriate time. When the target area appears distinctly yellow under a microscope, stop colorimetric development and rinse with running water for 15 minutes.

[0127] (9) Counterstain the cell nuclei with hematoxylin for 2 min and then rinse in running water;

[0128] (10) Differentiate in hydrochloric acid and alcohol for 2 seconds, then rinse with running water for 10 minutes;

[0129] (11) The steps of hydration, transparency, and sealing are the same as above;

[0130] (12) Protein expression was statistically analyzed according to the German semi-quantitative scoring standard. The scores were scored by two pathologists in a double-blind manner and the average value was taken. Staining intensity score: negative: 0 points, light yellow: 1 point, brownish yellow: 2 points, brownish brown: 3 points. Positive cell ratio score: 0 points if the proportion of stained cells to the total number of cells is less than 5%; 1 point if 5% ≤ proportion ≤ 25%; 2 points if 25% ≤ proportion ≤ 50%; 3 points if 25% ≤ proportion ≤ 50%; 4 points if proportion ≥ 75%. The final score was the product of the staining intensity and the proportion of positive cells, and statistical analysis was performed.

[0131] (1) NMN inhibits intestinal metaplasia lesions in gastric mucosa of mice: The present invention uses an intestinal metaplasia mouse model to identify whether NMN has a therapeutic effect on intestinal metaplasia of gastric mucosa. Acute SPEM induced by high-dose Tam usually recovers on its own within a week. Therefore, the present invention intervenes in mice with NMN for 7 weeks in advance before administering Tam, and then treats them with Tam, and continuously gavages them for 3 days and 3 times, and then kills them after 4 days (NMN intervention is also given during this period), and harvests gastric mucosa for testing, that is, the total NMN intervention time is 8 weeks. Compared with the Tam group, H&E staining of the gastric mucosa in the NMN intervention group showed a certain degree of recovery, the degree of mucosal disorder was reduced, and the proportion of eosinophilic parietal cells increased ( Figure 1 A); In the chronic SPEM model induced by Hp, the NMN intervention group showed reduced inflammatory cell infiltration and glandular atrophy, especially the number of parietal cells was significantly higher than that in the infection group ( Figure 1 B); In addition, the mRNA expression of inflammatory factor IL-8 in the NMN intervention group was significantly lower than that in the infection group ( Figure 2 ).

[0132] Atp4a - / - In mice, H&E staining results showed that the NMN intervention group almost completely eliminated intestinal metaplasia lesions, improved gastric mucosal morphology, and significantly improved lesions such as hyperplasia of glands and inflammatory cells ( Figure 1 C); NMN did not significantly change the gastric mucosa in WT C57 mice. AB-PAS staining and HID-AB staining were further used to identify intestinal metaplastic glands in the mucosa. AB-PAS staining showed a significant decrease in the number of blue intestinal metaplastic glands and a significantly lower positive score. HID-AB staining also showed a significant decrease in the number of brown intestinal metaplastic glands ( Figure 3 A, B). (all P < 0.05).

[0133] The immunohistochemical results of ATP4a, a marker of parietal cells, showed that in Tam-intervention mice, Hp-infected mice and Atp4a - / - In the gastric mucosa of mice, the mechanism of intestinal metaplasia caused by these three models largely depends on the loss of parietal cells. The number and expression level of ATP4a positive cells in the NMN intervention group were significantly higher than those in the untreated group (all P < 0.05) ( Figure 4 ). In summary, NMN intervention promoted the recovery of gastric mucosal parietal cells and improved intestinal metaplasia. Immunohistochemistry results showed that NMN intervention of Atp4a - / - The expression of MUC5AC in the gastric mucosa of mice was significantly higher than that in the non-intervention group ( Figure 5 ), while the expression level of MUC2 was lower than that in the non-intervention group ( Figure 6 ), and the expression of intestinal marker CDX2 was also downregulated ( Figure 7 , all P < 0.05). In particular, despite upregulation of CDX2 expression in H. pylori-infected mice, goblet cells were not produced. These results suggest that long-term NMN intervention significantly reduces intestinal metaplasia markers in multiple models and promotes the restoration of gastric phenotype.

[0134] (II) NMN inhibits the expression of stem cell markers in intestinal metaplastic mucosa: The enhancement of gastric cancer stem cell characteristics and the increase in number are important mechanisms for the generation of GIM in response to injury-induced. In order to further explore the mechanism by which NMN inhibits intestinal metaplasia in vivo, the present invention further explored the effect of NMN on the expression of stemness markers. Based on immunofluorescence, compared with the gastric mucosa of mice treated with high-dose Tam, the gastric mucosa of mice pretreated with NMN showed a significant decrease in TFF2 expression, while the expression of the stem cell marker MIST1 was significantly increased ( Figure 8 The expression of stem cell markers CD44 and LGR5 in the gastric mucosa of other model mice was detected by immunohistochemistry. In Hp-infected mice, the expression of chronic SPEM marker CD44 was significantly decreased after NMN intervention ( Figure 9 A, B). NMN treatment did not change the expression of cancer stem cell markers LGR5 and CD44, Atp4a in WT mice. - / - The expression of both in the gastric mucosa of mice was significantly higher than that in WT mice. - / - The expression of CD44 in the gastric mucosa of mice was significantly lower than that in the unintervention group ( Figure 9 C, D). The expression of another cancer stemness marker LGR5 also showed the same trend. NMN intervention had a significant effect on the expression of Hp chronic infected mice and Atp4a. - / - In the gastric mucosa of mice, LGR5 expression was also significantly lower than that in the non-intervention group ( Figure 10 ).

[0135] (III) NMN inhibits DNA damage repair markers in intestinal metaplasia mucosa: DNA damage is also an important mechanism leading to intestinal metaplasia. The present invention also analyzed the DNA damage in the gastric mucosa of mice treated with NMN. By immunohistochemical staining, the expression of DNA damage marker γ-H2AX in the NMN pretreatment group was significantly reduced compared with the Tam intervention group alone ( Figure 11 A, B; In the gastric mucosa of Hp-infected mice treated with NMN, the expression of γ-H2AX was significantly lower than that in the single infection group ( Figure 11 C, D); Consistently, in Atp4a - / - In mice, NMN treatment also reduced γ-H2AX expression ( Figure 11 E, F), the differences were statistically significant.

[0136] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of NMN in the preparation of drugs for preventing and / or treating intestinal metaplasia of gastric mucosa.

2. The use according to claim 1, characterized in that The intestinal metaplasia is induced by tamoxifen or Hp infection or Atp4a gene knockout.

3. The use according to claim 1, characterized in that The NMN administration method is injection or oral administration.

4. The use according to claim 1, characterized in that The NMN is the only active ingredient.

5. The use according to claim 1, characterized in that In the medicine, NMN is mixed with other active ingredients.

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