A method for constructing a model of type 2 diabetes mellitus and ulcerative colitis

Abstract

The present application relates to the technical field of disease animal model construction, and particularly relates to a method for constructing a type 2 diabetes and ulcerative colitis co-morbidity model. The method comprises the following steps: S1, experimental animal and grouping, S2, type 2 diabetes model induction; S3, superimposed induction of ulcerative colitis model. The method constructs a type 2 diabetes mouse model by using a high-fat diet and a low-dose streptozotocin solution to induce insulin resistance and hyperglycemia. On this basis, a 3.0% dextran sulfate sodium solution is used to chemically stimulate and induce acute ulcerative colitis, and the ulcerative colitis model is superimposed and induced. The present application solves the technical problems of mutual interference of the two disease model phenotypes, low model formation rate and poor model consistency, and has the technical advantages of good repeatability, high model formation rate and no mutual interference of the two model phenotypes.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology for diseases, specifically to a method for constructing a comorbid model of type 2 diabetes and ulcerative colitis. Background Technology

[0002] Type 2 diabetes is a chronic disease caused by insufficient insulin use or reduced insulin efficiency. It is common in adults and is also called adult-onset diabetes. This disease is caused by a combination of genetic and environmental factors, such as lifestyle, overnutrition, and insufficient physical activity. Often, the initial symptoms are mild, and many people are only diagnosed when complications develop or during routine checkups. Ulcerative colitis is a long-term inflammatory bowel disease that primarily affects the rectum and large intestine. Its cause is not yet clear, but it may be related to environmental, genetic, gut microbiota, and immune factors. It can occur at any age, but is most common in young adults aged 20 to 49, with no significant gender difference. The mortality rate is low, but long-term illness may increase the risk of intestinal cancer. Typical symptoms of ulcerative colitis include recurrent diarrhea, abdominal pain, and stools often containing blood, mucus, or pus.

[0003] Type 2 diabetes and ulcerative colitis are both common chronic and refractory diseases in clinical practice. Recent epidemiological evidence and basic research indicate a close interaction between type 2 diabetes and ulcerative colitis in their pathogenesis. A prospective cohort study showed that patients with type 2 diabetes had a significantly increased risk of developing ulcerative colitis (Li L, et al. Type II diabetes mellitus increases the risk of inflammatory bowel disease in a prospective cohort study. Clin Nutr ESPEN. 2024;61:212-218. PMID: 38777435.). Further research on immunological mechanisms indicates that ulcerative colitis is the subtype of inflammatory bowel disease most strongly associated with type 2 diabetes (Frumento D, et al. Immunological Linkages Between Inflammatory Bowel Diseases and Type 2 Diabetes. Biomedicines. 2025;13(9):2224. PMID: 41007787. The Chinese translation of the title is "Immunological Linkages Between Inflammatory Bowel Diseases and Type 2 Diabetes"). In terms of shared pathological conditions, abnormal activation of the NLRP3 inflammasome and gut microbiota dysbiosis are considered key links connecting the two diseases. Changes in microbial composition affect intestinal permeability and exacerbate inflammation and metabolic disorders (Malicevic U, et al. NLRP3 Inflammasome and Gut Dysbiosis Linking Diabetes Mellitus and Inflammatory Bowel Disease. Arch Intern Med Res. 2024. PMID: 39328924. The Chinese translation of the title is NLRP3 Inflammasome and Gut Microbiota Dysbiosis: The Association Between Diabetes Mellitus and Inflammatory Bowel Disease).Furthermore, research on the therapeutic mechanisms of gut microbiota based on multi-target interactions with the intestinal barrier has confirmed that gut microbiota dysbiosis and intestinal mucosal barrier damage are common pathogenic bases for a variety of gut-related diseases, including ulcerative colitis and type 2 diabetes (Common mechanisms of Gut microbe-based strategies for the treatment of intestine-related diseases: based on multi-target interactions with the intestinal barrier. Cell Communication and Signaling. 2025;23:288.). Both diseases involve chronic low-grade inflammation and alterations in immune signaling pathways, including cytokine dysregulation, T cell imbalance, and abnormal activation of innate immunity (ibid., Frumento D, et al., 2025). Patients suffering from both diseases have significantly higher hospitalization rates and healthcare resource consumption, and face greater difficulties in treatment options (ibid., Li L, et al., 2024).

[0004] As the current situation demonstrates, establishing a standardized animal model capable of simulating the combined pathological state of type 2 diabetes mellitus and ulcerative colitis in humans is crucial for a deeper understanding of the comorbidity mechanisms, the discovery of new therapeutic targets, and the evaluation of candidate drugs. Existing technologies have mature methods for inducing either type 2 diabetes or ulcerative colitis models. However, simply overlapping or arbitrarily applying these two induction methods at different times generally suffers from three major bottlenecks: first, extremely high animal mortality rates, as diarrhea and bloody stools during the ulcerative colitis induction period can rapidly worsen metabolic disorders and body condition in diabetic mice; second, the two model phenotypes interfere with each other, for example, severe ulcerative colitis may lead to reduced food intake, resulting in a passive decrease in blood glucose levels and making it impossible to maintain a stable diabetic phenotype; and third, low model success rate and poor model consistency, making it difficult to obtain a standardized comorbid animal population for experimental research. Therefore, a scientific and reproducible modeling method is urgently needed to overcome these shortcomings. Summary of the Invention

[0005] To overcome the technical problems of mutual interference between the two phenotypes and low model success rate and poor model consistency in the above-mentioned comorbidity models of type 2 diabetes and ulcerative colitis, the present invention provides a method for constructing a comorbidity model of diabetes and ulcerative colitis. This method has good repeatability, high model success rate, and the two phenotypes do not interfere with each other.

[0006] The present invention provides a method for constructing a comorbid model of diabetes and ulcerative colitis as follows: A method for constructing a comorbid model of type 2 diabetes and ulcerative colitis includes the following steps: S1. Experimental animals and grouping: SPF-grade mice were selected and housed in cages in a standard SPF-grade barrier environment animal room. They were allowed free access to food and water. After acclimatizing to the environment for one week, they were randomly divided into a normal control group and a model group. Induction of S2 and Type 2 Diabetes Models: Mice in the model group were initially fed a high-fat diet continuously. Before the end of feeding, they were injected intraperitoneally with streptozotocin solution. Mice in the normal control group were fed a standard maintenance diet throughout the process and injected with an equal volume of citrate buffer at the same time intervals. Mouse weight was monitored weekly, and fasting blood glucose was measured on day 7 after the streptozotocin injection. A stable and consistently high fasting blood glucose level ≥11.1 mmol / L was used as the standard for successful type 2 diabetes modeling, thus obtaining the type 2 diabetes mouse model. S3. Induction of the comorbid ulcerative colitis model: After confirming the successful establishment of the type 2 diabetes model in step S2, the type 2 diabetes model mice were allowed to enter an intermittent recovery period, during which they were fed a normal maintenance diet and ordinary purified water to stabilize their condition. After the intermittent recovery period, the type 2 diabetes model mice were fed a high-fat diet, and the ordinary purified water was replaced with dextran sulfate sodium solution to induce ulcerative colitis. The normal control mice drank ordinary purified water throughout the entire process, and each mouse had free access to drinking water for 7 consecutive days. Thus, a comorbid type 2 diabetes and ulcerative colitis model mouse was obtained. Furthermore, in step S1, the mice are selected from the C57BL / 6J strain, are male, and are 6 to 8 weeks old.

[0007] Furthermore, in step S2, the mice were fed a high-fat diet for 6-8 weeks; the model group mice were injected with streptozotocin solution daily for 7 consecutive days.

[0008] Furthermore, the mice were fed a high-fat diet for 8 weeks. At the end of the 7th week, the model group mice were injected with streptozotocin solution.

[0009] Further, in step S2, streptozotocin solid powder is dissolved in freshly prepared citrate buffer solution and stirred evenly to form a streptozotocin solution with a concentration of 10 mg / mL.

[0010] Furthermore, the dosage of streptozotocin solution for injection is 50 mg / kg body weight.

[0011] Furthermore, in step S3, the duration of the intermittent recovery period is 1-2 weeks.

[0012] Furthermore, in step S3, the duration of the intermittent recovery period is 2 weeks.

[0013] Furthermore, in step S3, the mass fraction of the solute in the dextran sulfate solution is 2.5% to 3.5%.

[0014] Furthermore, the mass fraction of the solute in the dextran sulfate solution is 3.0%.

[0015] Based on the above technical solution, the beneficial effects of the present invention compared to the prior art are as follows: 1. The comorbidity model phenotypes of this invention do not interfere with each other. In this study, insulin resistance and hyperglycemia were induced by a high-fat diet plus a low-dose streptozotocin solution, successfully establishing a type 2 diabetic mouse model. Based on this, acute ulcerative colitis was successfully induced by chemical stimulation with 3.0% sodium dextran sulfate solution. This comorbidity model mouse simultaneously exhibited a stable hyperglycemic state and typical clinical and pathological features of colitis, simulating the comorbidity state of type 2 diabetes complicated by ulcerative colitis.

[0016] 2. The comorbidity model construction method of this invention has good reproducibility and a high model success rate. Through experimental screening, this invention has determined that in diabetic model mice, allowing free drinking of a 3.0% sodium dextran sulfate solution for 7 days can reliably induce a moderate to severe colitis phenotype. Furthermore, the method avoids either failure to develop the disease due to excessively low concentrations or death of the animals before the observation endpoint due to excessively high concentrations. This comorbidity model construction method exhibits a high model success rate and good reproducibility.

[0017] 3. This invention integrates a comorbidity assessment system. Breaking through the limitations of previous methods that evaluated single disease indicators separately, this invention is the first to confirm at the histopathological level that, and simultaneously observes more significant pathological changes in the colonic mucosa of mice in the model group under the background of diabetes, the comorbidity status is identified morphologically, providing an identification standard for the construction of a comorbidity model of diabetes and ulcerative colitis. Attached Figure Description

[0018] Figure 1 This is a graph showing the change in mouse body weight. Figure 2 This is a comparison chart of fasting blood glucose levels in mice; Figure 3 This is a graph of the Disease Activity Index (DAI) score in mice; Figure 4 This is a comparison chart of mouse colon length; Figure 5 This is a comparison of the results of hematoxylin-eosin staining of mouse colon tissue. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0020] It should be noted that the mice used in this invention are all selected from the C57BL / 6J strain, are male, and are 6 to 8 weeks old.

[0021] It should be noted that the preparation and use method of the streptozotocin solution injected into the model group mice in this invention is as follows: Weigh an appropriate amount of streptozotocin (abbreviated as STZ) solid powder, and dissolve it in freshly prepared 0.1 mol / L sodium citrate buffer solution with pH 4.2~4.5 under ice bath and light protection conditions. Stir well to prepare an STZ solution with a concentration of 10 mg / mL. Prepare and use immediately, and complete the injection within 30 minutes. The injection dose of streptozotocin solution is 50 mg / kg body weight.

[0022] Example 1 This invention discloses a method for constructing a comorbid model of type 2 diabetes and ulcerative colitis, comprising the following steps: S1. Experimental animals and grouping: SPF-grade mice were selected and housed in cages in a standard SPF-grade barrier environment animal room. They were allowed free access to food and water. After acclimatizing to the environment for one week, they were randomly divided into a normal control group and a model group. Induction of S2 and Type 2 Diabetes Models: Mice in the model group were initially fed a high-fat diet continuously. Before the end of feeding, they were injected intraperitoneally with streptozotocin solution. Mice in the normal control group were fed a standard maintenance diet throughout the process and injected with an equal volume of citrate buffer at the same time intervals. Mouse weight was monitored weekly, and fasting blood glucose was measured on day 7 after the streptozotocin injection. A stable and consistently high fasting blood glucose level ≥11.1 mmol / L was used as the standard for successful type 2 diabetes modeling, thus obtaining the type 2 diabetes mouse model. S3. Induction of the comorbid ulcerative colitis model: After confirming the successful establishment of the type 2 diabetes model in step S2, the type 2 diabetes model mice were allowed to enter an intermittent recovery period, during which they were fed a normal maintenance diet and ordinary purified water to stabilize their condition. After the intermittent recovery period, the type 2 diabetes model mice were fed a high-fat diet, and the ordinary purified water was replaced with dextran sulfate sodium solution to induce ulcerative colitis. The normal control mice drank ordinary purified water throughout the entire process, and each mouse had free access to drinking water for 7 consecutive days. Thus, a comorbid type 2 diabetes and ulcerative colitis model mouse was obtained. In step S2 above, the mice were fed a high-fat diet for 8 weeks. At the end of the 7th week, the model group mice were injected with streptozotocin solution daily for 7 consecutive days.

[0023] In step S3 above, the duration of the intermittent recovery period is 2 weeks, and the mass fraction of the solute in the dextran sulfate sodium (DSS) solution is 3.0%.

[0024] Example 2 This invention discloses a method for constructing a comorbid model of type 2 diabetes and ulcerative colitis, comprising the following steps: S1. Experimental animals and grouping: SPF-grade mice were selected and housed in cages in a standard SPF-grade barrier environment animal room. They were allowed free access to food and water. After acclimatizing to the environment for one week, they were randomly divided into a normal control group and a model group. Induction of S2 and Type 2 Diabetes Models: Mice in the model group were initially fed a high-fat diet continuously. Before the end of feeding, they were injected intraperitoneally with streptozotocin solution. Mice in the normal control group were fed a standard maintenance diet throughout the process and injected with an equal volume of citrate buffer at the same time intervals. Mouse weight was monitored weekly, and fasting blood glucose was measured on day 7 after the streptozotocin injection. A stable and consistently high fasting blood glucose level ≥11.1 mmol / L was used as the standard for successful type 2 diabetes modeling, thus obtaining the type 2 diabetes mouse model. S3. Induction of the comorbid ulcerative colitis model: After confirming the successful establishment of the type 2 diabetes model in step S2, the type 2 diabetes model mice were allowed to enter an intermittent recovery period, during which they were fed a normal maintenance diet and ordinary purified water to stabilize their condition. After the intermittent recovery period, the type 2 diabetes model mice were fed a high-fat diet, and the ordinary purified water was replaced with dextran sulfate sodium solution to induce ulcerative colitis. The normal control mice drank ordinary purified water throughout the entire process, and each mouse had free access to drinking water for 7 consecutive days. Thus, a comorbid type 2 diabetes and ulcerative colitis model mouse was obtained. In step S2 above, the mice were fed a high-fat diet for 6 weeks. At the end of the 5th week, the model group mice were injected with streptozotocin solution daily for 7 consecutive days.

[0025] In step S3 above, the duration of the intermittent recovery period is 1 week, and the mass fraction of the solute in the dextran sulfate sodium solution is 2.5%.

[0026] Example 3 This invention discloses a method for constructing a comorbid model of type 2 diabetes and ulcerative colitis, comprising the following steps: S1. Experimental animals and grouping: SPF-grade mice were selected and housed in cages in a standard SPF-grade barrier environment animal room. They were allowed free access to food and water. After acclimatizing to the environment for one week, they were randomly divided into a normal control group and a model group. Induction of S2 and Type 2 Diabetes Models: Mice in the model group were initially fed a high-fat diet continuously. Before the end of feeding, they were injected intraperitoneally with streptozotocin solution. Mice in the normal control group were fed a standard maintenance diet throughout the process and injected with an equal volume of citrate buffer at the same time intervals. Mouse weight was monitored weekly, and fasting blood glucose was measured on day 7 after the streptozotocin injection. A stable and consistently high fasting blood glucose level ≥11.1 mmol / L was used as the standard for successful type 2 diabetes modeling, thus obtaining the type 2 diabetes mouse model. S3. Induction of the comorbid ulcerative colitis model: After confirming the successful establishment of the type 2 diabetes model in step S2, the type 2 diabetes model mice were allowed to enter an intermittent recovery period, during which they were fed a normal maintenance diet and ordinary purified water to stabilize their condition. After the intermittent recovery period, the type 2 diabetes model mice were fed a high-fat diet, and the ordinary purified water was replaced with dextran sulfate sodium solution to induce ulcerative colitis. The normal control mice drank ordinary purified water throughout the entire process, and each mouse had free access to drinking water for 7 consecutive days. Thus, a comorbid type 2 diabetes and ulcerative colitis model mouse was obtained. In step S2 above, the mice were fed a high-fat diet for 6 weeks. At the end of the 5th week, the model group mice were injected with streptozotocin solution daily for 7 consecutive days.

[0027] In step S3 above, the duration of the intermittent recovery period is 1 week, and the mass fraction of the solute in the dextran sulfate sodium solution is 3.5%.

[0028] All of the above embodiments can achieve the technical effects of the present invention.

[0029] To confirm the success of the comorbidity model construction in this invention, the following observation and detection index experiments were conducted and evaluated: 1. Observation and detection indicators (1) General condition observation: The mental state, spontaneous activity, hair color and fecal characteristics of each group of mice were observed and recorded at fixed times every day. The weight of the mice was weighed and recorded at fixed times every week, and the weight change curve was plotted.

[0030] (2) Disease Activity Index (DAI) score: Starting from the day of administration of DSS solution, the DAI score of mice in the model group and normal control group was performed daily. The DAI score is composed of three components: percentage of body weight loss, fecal characteristics score, and degree of fecal hemorrhage score. The specific scoring criteria are shown in Table 1. Among them, the percentage of body weight loss was calculated based on the body weight on the day DSS solution induction began; fecal characteristics and degree of fecal hemorrhage were determined by observation and fecal occult blood test strip detection.

[0031] The DAI score ranges from 0 to 12. A DAI score of ≥2 indicates successful ulcerative colitis phenotype superposition. See the table below for specific scoring criteria: (3) Sample collection and processing: On the day following the end of the DSS solution drinking cycle, mice in each group were euthanized by an overdose of the anesthetic sodium pentobarbital. The mice were fixed in a supine position, and the abdominal cavity was cut open along the midline. The entire colon from the end of the cecum to the anus was bluntly dissected and completely removed. The natural length of the colon was measured with a ruler and photographed for recording.

[0032] (4) Histopathological evaluation of colon tissue: Approximately 0.5 cm of distal colon tissue was excised from about 1 cm from the anus and fixed in 4% paraformaldehyde solution for at least 24 hours. After dehydration with graded ethanol, clearing with xylene, and embedding in paraffin, continuous sections with a thickness of 4-5 μm were cut and stained with hematoxylin and eosin (HE). The pathological morphological changes of the colonic mucosa were observed under an optical microscope, with a focus on evaluating the following features: the integrity of the mucosal epithelium, the depth and density of inflammatory cell infiltration, the degree of damage to the crypt structure, and microvascular pathological changes associated with diabetes.

[0033] 2. Evaluation (1) Type 2 diabetes model successfully established: By monitoring fasting blood glucose in comorbidity model mice, it was confirmed that the diabetes phenotype of comorbidity model mice was maintained throughout the entire process of ulcerative colitis induction, such as stable fasting blood glucose and a sustained ≥11.1 mmol / L. like Figure 1 and Figure 2 As shown, compared with the normal control group, the mice in the model group fed a high-fat diet combined with STZ solution injection showed a significant increase in body weight at the end of the modeling period, and exhibited polydipsia and polyphagia. Blood glucose testing showed that the fasting blood glucose level of the model group mice was stable and significantly higher than that of the normal control group, meeting the diagnostic criteria for diabetes, and a type 2 diabetes mouse model was successfully established.

[0034] (2) Successful superposition of ulcerative colitis phenotype: The presence of typical clinical manifestations of ulcerative colitis was confirmed by daily Disease Activity Index (DAI) scoring.

[0035] like Figure 1 and Figure 3 As shown, during the period of drinking 3.0% DSS solution, the mice's body weight decreased significantly in the following days, while their DAI scores increased significantly over time. Figure 4 Dissection of the colon tissue revealed that the colon length of the model group mice was significantly shorter than that of the normal control group, and the colon wall was congested and edematous, with some showing punctate hemorrhages or ulcers.

[0036] (3) Histopathological confirmation of comorbidity model characteristics: After induction, mice in each group were over-anesthetized and euthanized. Then, colon tissue was dissected and hematoxylin-eosin stained to observe and confirm the pathological damage of the colonic mucosa.

[0037] like Figure 5 HE staining of colon tissue revealed that the colonic mucosa of normal control mice was structurally intact with neatly arranged glands. In contrast, the colonic mucosa of model mice exhibited typical pathological changes characteristic of ulcerative colitis, including: disruption of mucosal epithelial integrity, extensive inflammatory cell infiltration, distorted or absent crypt structures, reduced goblet cells, and varying degrees of ulceration. These pathological changes were more pronounced in the context of diabetes.

Claims

1. A method for constructing a comorbid model of type 2 diabetes and ulcerative colitis, comprising the following steps: S1. Experimental animals and grouping: SPF-grade mice were selected and housed in cages in a standard SPF-grade barrier environment animal room. They were allowed free access to food and water. After acclimatizing to the environment for one week, they were randomly divided into a normal control group and a model group. Induction of S2 and Type 2 Diabetes Models: Mice in the model group were initially fed a high-fat diet continuously. Before the end of feeding, they were injected intraperitoneally with streptozotocin solution. Mice in the normal control group were fed a standard maintenance diet throughout the process and injected with an equal volume of citrate buffer at the same time intervals. Mouse weight was monitored weekly, and fasting blood glucose was measured on day 7 after the streptozotocin injection. A stable fasting blood glucose level ≥11.1 mmol / L for one week was used as the standard for successful type 2 diabetes modeling, thus obtaining the type 2 diabetes mouse model. S3. Induction of ulcerative colitis model: After confirming the successful construction of the type 2 diabetes model in step S2, the type 2 diabetes model mice were allowed to enter an intermittent recovery period, during which they were fed a normal maintenance diet and ordinary purified water to stabilize their condition. After the intermittent recovery period, the type 2 diabetes model mice were fed a high-fat diet, and the ordinary purified water was replaced with dextran sulfate sodium solution to induce ulcerative colitis. The normal control group mice drank ordinary purified water throughout the process, and each group had free access to drinking water for 7 consecutive days. This resulted in a comorbid model of type 2 diabetes and ulcerative colitis in mice.

2. The method for constructing a comorbid model of type 2 diabetes and ulcerative colitis according to claim 1, wherein in step S1, the mice are selected from the C57BL / 6J strain, are male, and are 6 to 8 weeks old.

3. In the method for constructing a comorbidity model of type 2 diabetes and ulcerative colitis according to claim 1, in step S2, the mice are fed a high-fat diet for 6-8 weeks; the mice in the model group are injected with streptozotocin solution daily for 7 consecutive days.

4. According to claim 3, a method for constructing a comorbidity model of type 2 diabetes and ulcerative colitis is to feed mice a high-fat diet for 8 weeks, and at the end of the 7th week, inject streptozotocin solution into the model group mice.

5. The method for constructing a comorbidity model of type 2 diabetes and ulcerative colitis according to claim 1, wherein in step S2, streptozotocin solid powder is dissolved in freshly prepared citrate buffer solution and stirred evenly to form a streptozotocin solution with a concentration of 10 mg / mL.

6. In the method for constructing a comorbidity model of type 2 diabetes and ulcerative colitis according to claim 5, the streptozotocin solution injection dose is 50 mg / kg body weight.

7. In the method for constructing a comorbid model of type 2 diabetes and ulcerative colitis according to claim 1, in step S3, the duration of the intermittent recovery period is 1-2 weeks.

8. In the method for constructing a comorbid model of type 2 diabetes and ulcerative colitis according to claim 7, in step S3, the duration of the intermittent recovery period is 2 weeks.

9. In the method for constructing a comorbidity model of type 2 diabetes and ulcerative colitis according to claim 1, in step S3, the mass fraction of the solute in the dextran sulfate sodium solution is 2.5%~3.5%.

10. The method for constructing a comorbidity model of type 2 diabetes and ulcerative colitis according to claim 9, wherein the mass fraction of the solute in the dextran sulfate sodium solution is 3.0%.

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