Chronic atrophic gastritis precancerous lesion animal model and construction method and application thereof

Abstract

The invention discloses a construction method of a chronic atrophic gastritis precancerous lesion animal model, and the construction method comprises the following specific steps: selecting experimental mice, randomly and averagely dividing the experimental mice into a model group and a blank control group, the experimental mice of the model group drink an MNU solution I by themselves and use an MNU solution II for intragastric administration once a week, and the blank control group normally diets; after modeling, dissecting and taking stomach tissues for later use; the MNU solution I is an N-nitroso-N-methylurea solution with the concentration of 290 mg / L to 310 mg / L; the MNU solution II is an N-nitroso-N-methylurea solution with the concentration of 1.6 g / L to 1.7 g / L; according to the method, 14-week modeling can be achieved, intestinal metaplasia, heterodysplasia and even a small number of cancer cells can occur in 14 weeks, and compared with a traditional MNNG combined multi-factor modeling method, the modeling method has the advantages that the modeling method is simplified, experimental errors and individual differences are reduced, the modeling period is shortened, and the modeling success rate is increased.

Description

Technical Field

[0001] This invention belongs to the field of experimental animal science, and relates to animal models of precancerous lesions of gastritis, and particularly to an animal model of precancerous lesions of chronic atrophic gastritis, its construction method, and its application. Background Technology

[0002] Precancerous lesions of gastric cancer (PLGC) are a key pathological stage in the transformation of chronic gastritis into gastric cancer. According to the classic Correa model, gastric cancer develops from normal gastric mucosa to chronic non-atrophic gastritis, then to chronic atrophic gastritis, intestinal metaplasia, dysplasia, and finally gastric cancer. Intervening in this pathological evolution to block or reverse the inflammatory-cancer transformation process is crucial for gastric cancer prevention. Therefore, establishing reliable and stable animal models is particularly important in drug and basic medical research on anti-PLGC drugs.

[0003] In recent years, although significant progress has been made in the research of chronic atrophic gastritis (PLGC) models, problems such as model instability and long modeling cycles often arise. Therefore, establishing an efficient and stable animal model of PLGC and using multi-index analysis to evaluate the reliability and stability of the model is essential for basic clinical research and new drug development in PLGC. Summary of the Invention

[0004] The purpose of this invention is to establish a reliable and stable animal model. This invention uses a combination of free drinking of MNU and oral administration to replicate the chronic atrophic gastritis PLGC mouse model. The model is comprehensively evaluated using multiple indicators such as gastric tissue pathology, gastric mucosal scanning electron microscopy analysis, and gastric function, providing a practical and reliable animal model of chronic atrophic gastritis PLGC for clinical basic and drug research.

[0005] The technical solution adopted in this invention provides a method for constructing an animal model of precancerous lesions of chronic atrophic gastritis. The key point is that the specific steps of the above-mentioned construction method are as follows:

[0006] Experimental mice were randomly divided into a model group and a blank control group. Mice in the model group were given MNU solution I to drink and MNU solution II to be administered by gavage once a week. Mice in the blank control group had a normal diet.

[0007] After modeling is completed, stomach tissue is dissected and used for later use.

[0008] The MNU solution I mentioned above is an N-nitroso-N-methylurea solution with a concentration of 290 mg / L to 310 mg / L;

[0009] The MNU solution II mentioned above is an N-nitroso-N-methylurea solution with a concentration of 1.6 g / L to 1.7 g / L.

[0010] Preferably, the experimental mice mentioned above are SPF-grade BALB / c males aged 6 to 8 weeks and weighing 20g to 22g. The experimental mice need to undergo acclimatization feeding for 1 week.

[0011] Specifically, MNU solution II was administered via gavage weekly at a dose of 0.1 mL / 10 g.

[0012] Furthermore, the modeling period was 14 weeks, and successful modeling was determined by the appearance of gastric mucosal damage in experimental mice, followed by intestinal metaplasia and dysplasia.

[0013] Furthermore, after the model is established, blood needs to be drawn from the orbital cavity of the experimental mice in both the model group and the blank control group, and they should be euthanized by dislocation of the neck.

[0014] A precancerous animal model of chronic atrophic gastritis is provided, the key point being that the animal model is obtained by the above-mentioned method for constructing a precancerous animal model of chronic atrophic gastritis.

[0015] This invention discloses the application of an animal model of precancerous lesions of chronic atrophic gastritis in drug screening for the treatment of precancerous lesions of chronic atrophic gastritis.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] *Helicobacter pylori* (Hp) infection is a significant contributing factor to chronic atrophic gastritis. Hp infection can induce widespread dysplasia along the lesser curvature of the gastric body at the squamocolumnar junction and polypoid lesions in the antrum, eventually leading to invasive tumors in the gastric body. However, these invasive lesions are submucosal cystic lesions with low malignant potential; therefore, this model cannot fully replicate intestinal-type adenocarcinoma with invasive or distant metastatic potential. High-salt and pickled diets are high-risk factors for gastric cancer. High-concentration NaCl intake reduces gastric mucoprotein viscosity and damages the gastric mucosal barrier, but high-salt intake alone does not induce gastric cancer; it often synergistically causes cancer with other factors, increasing the risk of gastric cancer.

[0018] Existing chemical carcinogen-induced models often utilize free ingestion of carcinogens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or MNU, which suffer from limitations such as uncertainty in the timing, location, number, and size of tumor appearance, as well as significant genetic heterogeneity of tumors. Some researchers have attempted to combine MNU and MNNG with multi-factor induction models incorporating Helicobacter spp. infection and diet; however, multi-factor modeling methods often suffer from low model establishment rates and low survival rates due to the inability to control experimental variables and ensure the accuracy and reliability of experimental results. With the development of gene editing technology, gene-edited mouse models are widely used both domestically and internationally. These models simulate the occurrence of gastric cancer by abnormally expressing proteins in specific inflammatory factors or pathways, and by mutating oncogenes or tumor suppressor genes. However, systemic gene editing in mice often leads to premature death due to extragastric lesions, hindering the full observation of gastric pathological progression. This invention addresses the shortcomings of current PLGC animal models, such as poor stability and long model establishment time, by establishing a highly efficient and stable PLGC animal model.

[0019] The model established in this invention employs a chemical carcinogen-induced model, using MNU, a more sensitive inducer for mouse gastric glands. After entering cells, MNU can undergo alkylation reactions with intracellular biomolecules such as DNA via nitrosyl groups, thereby altering the normal structure and function of DNA, causing errors in DNA replication and leading to gene mutations or cell carcinogenesis. MNU's mutagenic effect is stronger than MNNG, effectively shortening the modeling time. Furthermore, because MNU is readily soluble in water and has no obvious odor, using an aqueous solution of MNU effectively reduces avoidance and refusal to drink in mice during free access to the water, compared to MNNG. Simultaneously, since low doses of MNU can effectively induce gastric adenocarcinoma, this invention reduces the concentration of the MNU solution, effectively alleviating not only refusal to eat but also reducing mouse mortality. This invention also addresses the issue that free access alone cannot accurately control the amount of water consumed by mice in the same group, leading to unstable model establishment rates and atypical PLGC performance. Therefore, regular gavage administration is added to effectively control the amount and timing of MNU intake in mice, improving the stability and success rate of model establishment.

[0020] In this invention, BALB / c mice were selected as experimental animals because BALB / c mice have a relatively shorter lifespan compared to the commonly used SD rats. When conducting experiments that require observing changes throughout the entire life cycle or are related to long-term chronic diseases, complete experimental data can be obtained in a relatively short time. In addition, BALB / c mice have a clear and stable genetic background and a high degree of gene homozygosity, making them more stable in experiments.

[0021] The experimental results of this invention show that the MNU free drinking combined with gavage method can achieve model establishment in 14 weeks. Furthermore, the model group mice exhibit intestinal metaplasia, dysplasia, and even a small number of cancer cells at 14 weeks. Combined with scanning electron microscopy results and gastrointestinal hormone secretion indicators, this indicates that the indicators of the chronic atrophic gastritis PLGC mouse model are clear and stable. In summary, compared with the traditional MNNG combined with multifactorial modeling method, the modeling method of this invention simplifies the modeling process, reduces experimental errors and individual differences, shortens the modeling cycle, and improves the modeling success rate. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the construction of the animal model of precancerous lesions of chronic atrophic gastritis according to the present invention.

[0023] Figure 2 This is a graph showing the trend of body weight change in mice during the modeling process according to an embodiment of the present invention.

[0024] Figure 3 This is a mouse gastric mucosal tissue pathological morphology-HE staining during the modeling process of the present invention.

[0025] Figure 4 The image shows the pathological morphology of gastric mucosal tissue in mice during the modeling process according to this invention – AB-PAS staining.

[0026] Figure 5 These are the results of HE staining observation and pathological comparison of the embodiments and comparative examples of the present invention.

[0027] Figure 6 These are the survival rate results of the embodiments and comparative examples of the present invention.

[0028] Figure 7 The results of transmission electron microscopy observation of the ultrastructure of mouse gastric mucosa tissue in this embodiment of the invention are shown.

[0029] Figure 8 The results show the changes in the levels of gastrin GAS17 and inflammatory factors IL-1β, IL-2, and IL-6 in embodiments of the present invention.

[0030] Figure 9 This is a comparison of the contents of gastrin GAS17 and inflammatory factor IL-6 in the embodiments and comparative examples of the present invention.

[0031] Figure 10 This is the immunofluorescence staining result 1 of an embodiment of the present invention.

[0032] Figure 11 This is the immunofluorescence staining result 2 of the embodiment of the present invention.

[0033] Figure 12 These are the Western blot detection results of an embodiment of the present invention. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Unless otherwise specified in the examples, the procedures can be followed according to conventional conditions; unless the manufacturers of the reagents or instruments used are specified, they are all conventional products that can be purchased commercially.

[0040] The MNU used in this invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: K2320260.

[0041] The experimental mice used in this invention are SPF-grade male BALB / c mice, 6 to 8 weeks old, weighing 20g to 22g, purchased from Liaoning Changsheng Biotechnology Co., Ltd., Animal Production License No. SCXK(Liaoning)2020-0001, Animal Qualification Certificate No. 210726251100181456.

[0042] The experimental mice were housed at the Experimental Animal Center of Hebei University of Traditional Chinese Medicine, with free access to food and water. They were kept under alternating light and dark conditions every 12 hours, with a temperature of 20℃~22℃ and a humidity of 50%~60%. The bedding was changed every other day.

[0043] This invention was reviewed and approved by the Animal Experiment Ethics Committee of Hebei University of Traditional Chinese Medicine during its research, with the ethics review approval number being DWLL202305004.

[0044] Example

[0045] This embodiment is based on the appendix. Figure 1 The flowchart is used to construct an animal model of precancerous lesions of chronic atrophic gastritis.

[0046] Forty experimental mice were selected and acclimatized for one week, then randomly divided into two groups: a blank control group and a model group, also known as the free access to drinking water + medium-dose gavage group, with 20 mice in each group. Except for the blank control group, the experimental mice could freely drink 300 mg / L MNU solution I, and were gavaged once a week with 1.67 g / L MNU solution II at a dose of 0.1 ml / 10 g. MNU solution I was dispensed into drinking bottles for the mice to drink freely, and the drinking bottles were wrapped with aluminum foil.

[0047] The blank control group had a normal diet.

[0048] Blood was collected from the orbital cavity 14 weeks after modeling, the mice were euthanized by dislocation of the neck, and the stomach tissue was dissected for later use.

[0049] Comparative Example 1

[0050] Twenty experimental mice were selected and acclimatized for one week, and then placed in the free drinking group. Mice in the free drinking group could freely drink 300 mg / L MNU solution I, but were not given gavage once a week. MNU solution I was dispensed into water bottles for the mice to drink freely, and the water bottles were wrapped with aluminum foil.

[0051] The blank control group had a normal diet.

[0052] Comparative Example 2

[0053] Twenty experimental mice were selected and acclimatized for one week, and then used as the free drinking + low-dose gavage group. Mice in the free drinking + low-dose gavage group could drink 300 mg / L MNU solution I freely, and were gavaged once a week with 0.1 ml / 10 g of 0.84 g / L MNU solution II. MNU solution I was dispensed into drinking bottles for mice to drink freely, and the drinking bottles were wrapped with aluminum foil.

[0054] Comparative Example 3

[0055] Twenty experimental mice were selected and acclimatized for one week, and then placed in the free drinking + high-dose gavage group. Mice in the free drinking + high-dose gavage group could freely drink 300 mg / L MNU solution I, and were gavaged once a week with 3.34 g / L MNU solution II at a dose of 0.1 ml / 10 g. MNU solution I was dispensed into water bottles for mice to drink freely, and the water bottles were wrapped with aluminum foil.

[0056] Analysis and Testing

[0057] I. General condition and changes in body weight of mice

[0058] During the experiment, the general condition and body weight changes of the mice were observed. The results showed that after the modeling process began, compared with the blank control group, the mice in the model group gradually exhibited reduced free movement, decreased food intake, and yellowing and shedding of their fur. The body weight of the blank control group showed a rapid increase in the early stage of modeling, which gradually leveled off over time. Compared with the blank control group, the body weight of the model group showed a gradual decrease during the modeling process, indicating that the modeling method of this invention has a significant impact on the diet of mice.

[0059] The changes in body weight of mice in the model group and the blank control group are shown in the appendix. Figure 2 ,from Figure 2 The trend shows that the modeling method in the model group can significantly affect the gastrointestinal function of mice.

[0060] II. Pathological and Morphological Observation of Gastric Tissue

[0061] Gastric tissue was routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE) and AB-PAS, and observed under a microscope for gastric histopathological examination. The histopathological morphology of the gastric mucosa during the modeling process—HE staining results—are shown in the appendix. Figure 3 See the appendix for AB-PAS staining results. Figure 4 .

[0062] Figure 3 The results showed that in the blank control group, the gastric mucosal glands of mice were neatly arranged, uniform in size and shape, and single-column in shape. The boundaries between parietal cells and chief cells were clear. The gastric glands in the lamina propria were tightly and neatly arranged, with few or no inflammatory cells and no edema in the submucosa. In the model group, the gastric fundic glands of mice gradually thinned from 4 to 8 weeks, and the glands atrophied from 8 to 10 weeks. Intestinal metaplasia appeared in the antrum of the stomach, with increased nuclear division and decreased B cells. After 12 weeks, intestinal metaplasia increased, lymphocyte infiltration was observed in the lamina propria, and the submucosa was significantly edematous. After 14 weeks, the glands were disordered, the muscularis mucosae were disordered, B cells were decreased, and gastric mucosal intestinal metaplasia and dysplasia were obvious.

[0063] Figure 4The results showed that no obvious purplish-red and blue staining was observed in the gastric mucosa of mice in the blank control group, indicating that normal gastric mucosal epithelial cells generally contain a small amount of glycogen. In the model group, the degree of blue staining and purplish-red staining in the gastric mucosa gradually increased and became denser from 4 weeks onwards, suggesting the presence of intestinal metaplasia lesions and less mucus secretion on the mucosal surface.

[0064] In addition, the examples were compared with Comparative Examples 1 to 3, and the HE staining results are shown in the appendix. Figure 5 Among them, the free drinking + medium-dose gavage group is the model group of the embodiment.

[0065] Depend on Figure 5 The results showed that in Comparative Example 3, the combination of free drinking water and high-dose gavage induced gastric mucosal damage in mice within a shorter time, resulting in stable pathological manifestations of precancerous lesions of chronic atrophic gastritis, such as intestinal metaplasia. However, the survival rate in Comparative Example 3 was low. Figure 6 As shown, the survival rate was lowest in the free drinking + high-dose gavage group. The survival rates of the free drinking, free drinking + low-dose, and free drinking + medium-dose gavage groups were in the same range. However, pathological staining revealed that the free drinking model showed unstable intestinal metaplasia and mucosal atrophy. Therefore, to ensure successful model establishment, the group with the higher survival rate, namely the free drinking + medium-dose gavage group, was selected as the final model group.

[0066] III. Observation of the ultrastructure of gastric mucosal epithelial cells

[0067] Transmission electron microscopy was used to observe the ultrastructural changes of gastric mucosal epithelial cells. See the appendix for details on the ultrastructural changes of mouse gastric mucosal tissue. Figure 7 .

[0068] Observation of the ultrastructure of chief cells and parietal cells in mouse gastric tissue using transmission electron microscopy. Figure 7 The results showed that in the blank control group, the cell nuclei were normal in morphology and oval in shape, and the cytoplasm contained a large number of mitochondria; most mitochondria had normal structure, regular shape, neat arrangement, and dense cristae, and the rough endoplasmic reticulum was not swollen. In the model group, the cell nuclei were shrunken, the number of mitochondria in the cytoplasm was reduced, the mitochondrial structure was abnormal, the cristae were sparse and broken, and the rough endoplasmic reticulum was swollen.

[0069] IV. ELISA detection of serum gastric function indicators and inflammatory markers

[0070] The levels of pepsinogen (PGⅠ, PGⅡ), gastrin (GAS17), and inflammatory factors IL-1β, IL-2, and IL-6 in serum were detected using ELISA. The specific procedures were performed according to the kit instructions. Results are shown in the appendix. Figure 8 Meanwhile, comparative models one through three used gastric function index GAS and inflammatory marker IL-6 as evaluation criteria; the results are shown in the appendix. Figure 9 .

[0071] in, Figure 8 In the middle horizontal axis, Con represents the results of the blank control group, and Model represents the results of the model group. Figure 8 The results showed that, compared with the blank control group, the serum levels of gastrin GAS17, pepsinogen PGⅠ and PGⅡ in the model group were significantly reduced (P < 0.01), while the levels of inflammatory factors IL-1β, IL-2 and IL-6 were significantly increased (P < 0.01). This indicates that the model constructed in this invention can induce gastric dysfunction and inflammatory factor infiltration.

[0072] V. Immunofluorescence staining detection

[0073] The expression and localization of inflammatory markers interleukin-6 and tumor necrosis factor-α in gastric mucosal tissue were detected using immunofluorescence staining, denoted as IL-6 and TNF-α, respectively. The expression and localization of glycolytic markers hexokinase 2 and pyruvate kinase M2 were also detected, denoted as HK2 and PKM2, respectively. Results of immunofluorescence staining are attached. Figure 10 and 11 , Figure 10 and 11 The Con on the left represents the results of the blank control group, and the Model represents the results of the model group.

[0074] IL-6 and TNF-α play crucial roles in the body's immune response, inflammatory response, and various physiological and pathological processes. Figure 10 The results showed that the expression of IL-6 and TNF-α in the gastric mucosa of the model group was significantly enhanced, and IL-6 and TNF-α were positively correlated. They were mainly located in gastric mucosal epithelial cells and inflammatory cells in the lamina propria, indicating that the model constructed in this invention successfully induced a gastrointestinal inflammatory environment.

[0075] HK2 and PKM2 are key enzymes in the glycolysis pathway. Figure 11 The results showed that the expression of HK2 and PKM2 in the gastric mucosa of the model group was enhanced, indicating that the mice enhanced their glucose uptake and utilization after modeling. Since glycolysis provides energy and material basis for the rapid proliferation of tumor cells, the changes in HK2 and PKM2 may be closely related to the emergence of gastric cancer cells.

[0076] In summary, this demonstrates that the model constructed by this invention was successfully replicated and reached the PLGC stage.

[0077] VI. Western blot analysis of the expression of key indicator proteins

[0078] Proteins were extracted from gastric tissue and analyzed using Western blot to determine the expression of key proteins IL-1β and TNF-α. The Western blot results are attached. Figure 12 , Figure 12 In this context, Con represents the blank control group, and Model represents the model group.

[0079] Figure 12 The results showed that the protein expression levels of key inflammatory proteins IL-1β and TNF-α in the gastric mucosa of the model group were significantly higher than those in the blank control group.

[0080] VII. Statistical Analysis

[0081] This invention uses SPSS 29.0 software for statistical analysis. Quantitative data are expressed as mean ± standard deviation (x ± s). The independent samples t-test (for normally distributed data with homogeneous variances) or Mann-Whitney U test (for non-normally distributed data or data with unequal variances) is used for comparison between two groups. Categorical data are expressed as rates. The chi-square test is used for comparison between two groups. P < 0.05 indicates that the difference is statistically significant.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing an animal model of precancerous lesions in chronic atrophic gastritis, characterized in that, The specific steps of the construction method are as follows: Experimental mice were randomly divided into a model group and a blank control group. Mice in the model group were given MNU solution I to drink and MNU solution II to be administered by gavage once a week. Mice in the blank control group had a normal diet. After modeling is completed, stomach tissue is dissected and used for later use. The MNU solution I is an N-nitroso-N-methylurea solution with a concentration of 290 mg / L to 310 mg / L; The MNU solution II is an N-nitroso-N-methylurea solution with a concentration of 1.6 g / L to 1.7 g / L.

2. The method for constructing an animal model of precancerous lesions of chronic atrophic gastritis according to claim 1, characterized in that, The experimental mice were SPF-grade BALB / c males aged 6 to 8 weeks and weighing 20g to 22g. The experimental mice needed to undergo an acclimatization period of 1 week.

3. The method for constructing an animal model of precancerous lesions of chronic atrophic gastritis according to claim 1, characterized in that, The medication was administered weekly via gavage at a dose of 0.1 mL / 10 g.

4. The method for constructing an animal model of precancerous lesions of chronic atrophic gastritis according to claim 1, characterized in that, The modeling period was 14 weeks. Successful modeling was determined by the appearance of gastric mucosal damage in experimental mice, followed by intestinal metaplasia and dysplasia.

5. The method for constructing an animal model of precancerous lesions of chronic atrophic gastritis according to claim 1, characterized in that, After the model is established, blood needs to be drawn from the orbital cavity of the experimental mice in the model group and the blank control group, and they should be euthanized by dislocation of the neck.

6. An animal model of precancerous lesions in chronic atrophic gastritis, characterized in that, The animal model is obtained by the method for constructing an animal model of precancerous lesions of chronic atrophic gastritis as described in any one of claims 1-5.

7. The application of the animal model of chronic atrophic gastritis precancerous lesions as described in claim 6 in the screening of drugs for the treatment of chronic atrophic gastritis precancerous lesions.

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