A method for constructing an animal model of ankylosing spondylitis and chronic enteritis and its application

Abstract

The present invention discloses a method for constructing an animal model of ankylosing spondylitis and chronic enteritis. By using proteoglycan PG immune induction and sodium dextran sulfate chemical induction methods, an animal model is established that simulates the coexistence of ankylosing spondylitis and chronic enteritis in terms of symptoms, imaging changes, and pathological characteristics. The model mice exhibit imaging changes similar to those of human ankylosing spondylitis, such as sacroiliac joint bone erosion and sclerosis, narrowing and roughening of the joint space, and worm-eaten changes. Furthermore, they exhibit pathological changes similar to those of human chronic enteritis, such as intestinal epithelial cell destruction, glandular disorder, crypt structure destruction, goblet cell reduction, and inflammatory cell infiltration. This model can be used to study the interaction mechanism between the two diseases, explore new therapeutic targets, and help evaluate the potential application of new drugs in the treatment of ankylosing spondylitis and chronic enteritis, providing an important tool for mechanism research and drug development of such co-morbidities.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical research, in particular to the field of animal model construction for inflammatory diseases and immune diseases, and specifically relates to an animal model for studying the comorbidity of human ankylosing spondylitis combined with chronic enteritis. Background Art

[0002] Ankylosing spondylitis (AS) is a common, immune-mediated, chronic inflammatory disease that primarily affects the spine and sacroiliac joints. Chronic enteritis (or colitis) is a long-term (chronic) inflammatory disease affecting the digestive tract. Its pathological features include massive infiltration of lymphocytes and plasma cells in the intestinal mucosal lamina propria, along with structural abnormalities including altered crypt architecture, metaplastic changes, increased basal plasma cells, and inflammatory polyps. Chronic enteritis is the histological basis of inflammatory bowel disease (IBD). Recent studies have shown that 70% of AS patients experience intestinal inflammation, and approximately 5-10% of these patients will develop IBD. Bradtzaeg first proposed the concept of the "gut-joint axis" in 1997, positing that immune cells home from the intestine to sites of synovial inflammation, establishing a communication channel between intestinal immunity and joint disease. Recent studies have further elucidated the underlying mechanisms, including increased intestinal barrier permeability, dysbiotic intestinal flora and their metabolites that induce autoimmunity, and the migration of gut-derived immune cells and molecules to the joints. Mendelian randomization also found a positive causal association between IBD and AS, indicating an increased risk of developing AS. This genetic evidence further confirms the close association between intestinal inflammation and AS. However, the current lack of an animal model that simulates the coexistence of ankylosing spondylitis and chronic enteritis in terms of symptoms, imaging changes, and pathological features has limited research on the pathogenesis of this comorbidity and the development of therapeutic drugs.

[0003] Because obtaining human tissue relevant to AS pathology is difficult, the development of experimental animal models is crucial for AS research. Experimental animal models for AS can be broadly categorized into four categories: HLA-B27 transgenic animal models, inflammation-related animal models, ankylosing enthesitis animal models, and other animal models. The HLA-B27 / human β2-microglobulin (hβ2m) double transgenic rat model is primarily used for genetic research on AS, but it suffers from drawbacks such as long modeling time, technical difficulties, complex operation, and high cost. Ankylosing enthesitis animal models and other animal models also suffer from significant environmental influences and demanding housing conditions. Inflammation-related animal models, such as the proteoglycan PG-immunized BALB / c mouse model, focus on immune-inflammation studies. These models closely mimic the clinical course of AS and are valuable for studying autoimmunity in spondylitis and mapping genetic loci of spondyloarthritis. There are various animal models for chronic enteritis, including chemical, immunological, genetically modified, and combined approaches. The latter three approaches, however, have limited widespread adoption both domestically and internationally due to their low success rates and high cost. The disadvantage of acetic acid and oxazolone modeling in the chemical method is the lack of a chronic relapse process similar to that of humans after a short-term acute attack. Trinitrobenzenesulfonic acid and sodium dextran sulfate can be divided into acute and chronic colitis models according to the time and cycle of administration. Although both have the disadvantage of being easy to heal on their own, both can well simulate the symptoms and pathological changes of human chronic enteritis, and sodium dextran sulfate modeling is safer for animals. Animal models of simple AS or simple chronic enteritis often cannot fully reflect the comorbidity of ankylosing spondylitis and chronic enteritis. Therefore, the development of an animal model that can simulate the coexistence of ankylosing spondylitis and chronic enteritis in terms of symptoms, imaging changes and pathological characteristics can be used to study the pathogenesis of the comorbidity of ankylosing spondylitis and chronic enteritis, explore the correlation between the two, and have positive significance for drug screening and development of AS. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and provide an animal model of co-morbidity of ankylosing spondylitis and chronic enteritis. An animal model simulating the coexistence of ankylosing spondylitis and chronic enteritis is established through proteoglycan PG immune induction and dextran sodium sulfate chemical induction methods.

[0005] In order to solve the above technical problems, the present invention discloses a method for constructing an animal model of ankylosing spondylitis and chronic enteritis co-morbidity, wherein mice are continuously fed with drinking water containing sodium dextran sulfate for 6 to 8 days, and then given normal drinking water and fed for 13 to 15 days as a treatment cycle, and this feeding cycle is repeated for 4 cycles. At the same time, proteoglycan PG immune inducer is injected into the mice at 0 to 1 week, 3 to 4 week, and 6 to 7 week of modeling, respectively, and an animal model of ankylosing spondylitis and chronic enteritis co-morbidity is obtained after 12 to 24 weeks of modeling.

[0006] In a specific embodiment, mice are continuously fed with drinking water containing sodium dextran sulfate for 7 days, followed by normal drinking water and feeding for 14 days as a treatment cycle, and this feeding cycle is repeated for 4 cycles. At the same time, proteoglycan PG immune inducer is injected into the mice on the 1st, 21st and 42nd day of modeling, and an animal model of ankylosing spondylitis and chronic enteritis co-morbidity is obtained after the 140th day of modeling.

[0007] Preferably, the animal is a female Balb / c mouse, 6 to 8 weeks old.

[0008] Preferably, the molecular weight of the sodium dextran sulfate is 36,000 to 50,000, with a concentration of 2 wt% for the first three cycles and 4 wt% for the final cycle. Drinking water should be replaced each time the concentration is changed, and freshly prepared solutions should be used immediately to ensure a fresh solution. Studies have shown that sodium dextran sulfate has poor stability, so daily changes in drinking water are necessary.

[0009] Preferably, the proteoglycan immune inducer is prepared by dissolving 100 μg of proteoglycan PG in 100 μL of phosphate buffered saline (PBS) solution, vortexing to form a proteoglycan solution, mixing the proteoglycan solution with complete Freund's immune adjuvant at a volume ratio of 0.8:1 to 1.2:1, and then ultrasonically emulsifying to obtain a white emulsified suspension, which is the proteoglycan PG immune inducer. Preferably, the proteoglycan solution and complete Freund's immune adjuvant are mixed at a volume ratio of 1:1.

[0010] Preferably, the proteoglycan is bovine cartilage proteoglycan, and the prepared proteoglycan immune inducer has a dosage of 0.8-1.2 μg / μL and a dosage of 0.1-0.3 mL / 20 g. The administration route is intraperitoneal injection. Preferably, the dosage is 1.0 μg / μL and the dosage is 0.2 mL / 20 g.

[0011] Preferably, in order to verify the phenotype of the animal model of co-morbidity of ankylosing spondylitis and chronic enteritis, the sample includes sacroiliac joints and colon, and the coexistence of ankylosing spondylitis and chronic enteritis is confirmed by imaging, pathology or other evaluation criteria.

[0012] Preferably, the imaging manifestations of ankylosing spondylitis include sacroiliac joint bone erosion and sclerosis, narrowing and roughening of the joint space, and worm-eaten changes. Pathological manifestations of chronic enteritis include destruction of intestinal epithelial cells, disordered glandular arrangement, crypt structure disruption, goblet cell reduction, and inflammatory cell infiltration. Other evaluation criteria include the general condition of the mice, body weight, intestinal disease activity index (DAI) score, colon length, and morphological observations.

[0013] Beneficial effects: The invention provides an optimized animal model that simulates the co-morbidity of ankylosing spondylitis and chronic enteritis. Through condition optimization, 2% sodium dextran (DSS) treatment for 3 cycles + 4% DSS treatment for 1 cycle was used. While ensuring a 100% survival rate of mice, it effectively overcame the defect of the traditional DSS model that is easy to heal on its own, and achieved a 100% incidence rate in the model mice. Proteoglycan PG immune inducer (0.1-0.3mL / 20g), which is a mixture of bovine cartilage proteoglycan and complete Freund's adjuvant in a volume ratio of 0.8:1-1.2:1, was intraperitoneally injected into the model mice at 0-1 weeks, 3-4 weeks, and 6-7 weeks of modeling, respectively. The incidence rate of axial arthritis in mice reached 100%, the model was highly stable and repeatable, and the survival rate of mice reached 100%. This model mouse can simultaneously display the typical symptoms, imaging changes and pathological characteristics of both ankylosing spondylitis and chronic enteritis. It can be used to study the interaction mechanism between the two diseases and explore new therapeutic targets. It can also help evaluate the potential application of new drugs in the treatment of ankylosing spondylitis and chronic enteritis, and provide an important tool for mechanism research and drug development of such co-morbidities. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the animal model of ankylosing spondylitis and chronic enteritis;

[0015] Figure 2 Changes in body weight of mice in each group, *Compared with the blank group;

[0016] Figure 3 CV graph of coefficient of variation of body weight change of mice in each group;

[0017] Figure 4 Comparison of DAI scores of mice in each group, *Compared with the blank group, △ Compared with the AS group;

[0018] Figure 5 CV graph of coefficient of variation of DAI scores of mice in each group;

[0019] Figure 6 Micro-CT imaging results of sacroiliac joints of mice in each group;

[0020] Figure 7 Colon length of mice in each group;

[0021] Figure 8 Comparison of colon length among mice in each group;

[0022] Figure 9 Hematoxylin-eosin staining results of colon tissues of mice in each group;

[0023] Figure 10 Comparison of inflammation scores between VG1 and bovine cartilage proteoglycans;

[0024] Figure 11 Percentage change in DAI score at 1wt%, 2wt%, and 3wt%;

[0025] Figure 12 1wt%, 2wt%, 3wt% percent body weight change;

[0026] Figure 13 The percentage change of body weight when the concentration was changed to 4wt%. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially. Experimental methods without specific conditions in the examples are generally based on conditions conventional in the art or conditions recommended by the manufacturer.

[0028] Example 1 Construction of an animal model of ankylosing spondylitis and chronic enteritis.

[0029] 1. Experimental Materials

[0030] Experimental Animals

[0031] BALB / c female mice, 6 weeks old, weighing 20 ± 2 g, clean grade, were provided by the Laboratory Animal Center of Nanjing University of Chinese Medicine, with a qualification certificate number of SCXK(Su)2012-0008. The experimental design and procedures were in accordance with the guidelines and requirements of the Laboratory Animal Ethics Committee of Nanjing University of Chinese Medicine.

[0032] 1.2 Experimental Reagents

[0033] Proteoglycan PG (D8428) and complete Freund's adjuvant (F5881) were purchased from Sigma, USA; dextran sulfate sodium DSS, 36KDa-50KDa (MFCD00081551) was purchased from MP Biomedicals, USA; urine and fecal occult blood test kit (C027-1-1) was purchased from Nanjing Jiancheng Bioengineering Institute; phosphate buffered saline PBS (C10010500BT, pH 7.4) was purchased from Gibco, USA; hematoxylin-eosin staining kit (C0105S) was purchased from Shanghai Biotech.

[0034] Proteoglycan PG (proteoglycan) immune inducer: Bovine cartilage proteoglycan is used as an immune antigen. Every 100 μg of proteoglycan PG is fully dissolved in 100 μL of phosphate buffered saline (PBS), and then vortexed and mixed. The proteoglycan solution is then mixed with complete Freund's immune adjuvant in a 1:1 volume ratio and ultrasonically emulsified to produce a white emulsified suspension, namely the proteoglycan PG immune inducer.

[0035] Preparation method of 2% or 4% dextran sodium sulfate (DSS) in mouse drinking water: Dissolve 20g or 40g DSS powder in 1000mL Millipore ultrapure water and use immediately.

[0036] 1.3 Experimental instruments

[0037] Small animal in vivo Micro CT imaging system (Quantum GX), Perkin Elmer, USA; Millipore ultrapure water instrument, Millipore, USA.

[0038] 2. Experimental Methods

[0039] 2.1 Experimental animals and groups

[0040] Thirty female BALB / c mice, 6-8 weeks old, weighing 20±2g, SPF grade, were fed adaptively for one week and randomly divided into three groups: blank group, simple ankylosing spondylitis group, and AS+chronic enteritis group, with 10 mice in each group.

[0041] Blank group: Each mouse in the model group or treatment group was intraperitoneally injected with an equal amount of normal saline at week 0-1, week 3-4, and week 6-7 of modeling.

[0042] AS group: Proteoglycan PG immune inducer (0.1-0.3 mL / 20 g), which was a mixture of bovine cartilage proteoglycan and complete Freund's adjuvant at a volume ratio of 0.8:1-1.2:1, was intraperitoneally injected into model mice at 0-1 week, 3-4 week, and 6-7 week of modeling.

[0043] AS+ chronic enteritis group: After 1 week of adaptive feeding in an SPF animal room, mice were fed with drinking water containing 2% dextran sodium sulfate (DSS) for 7 consecutive days, and then fed with normal drinking water for 14 days. This constituted one cycle, with a total of 4 cycles. The first 3 cycles used 2% DSS, and the last cycle used 4% DSS. At the same time, each model group mouse was intraperitoneally injected with proteoglycan PG immune inducer (0.2 mL / 20 g) on ​​day 1 (week 0), day 21 (week 3), and day 42 (week 6). The modeling cycle was as follows: Figure 1 As shown, before intraperitoneal injection of mice, the abdominal injection site was disinfected with alcohol to prevent infection, and after injection, a cotton swab was used to gently press the injection site to prevent a large amount of injection solution from leaking.

[0044] 2.2 Index observation and detection

[0045] 2.2.1 Observation indicators

[0046] (1) Comparison of general condition and DAI scores of mice

[0047] From week 0 to week 20 of modeling, the mice's mental state, coat condition, activity, food and water intake, body weight, stool characteristics, and blood in stool were observed and recorded. Blood in stool was assessed using a fecal occult blood test kit. The DAI scoring criteria are shown in Table 1. In the formula, S1, S2 and S3 represent the percentage score of animal weight loss, animal loose stool score and animal bloody stool score, respectively.

[0048] Table 1 DAI scoring criteria for intestinal disease activity

[0049]

[0050] (2) Mouse morphological observation and Micro CT

[0051] The activity of the mice and changes in the softness of the tail, hip joints, and spine were observed. The changes in the sacroiliac joints of the living mice were evaluated using a small animal in vivo MicroCT imaging system. The scanning parameters were set to a voltage of 60 kV, a current of 166 μA, and a resolution of 10 μm.

[0052] 2.2.2 Sample collection

[0053] (1) Changes in colon length

[0054] After successful modeling, mice were anesthetized with isoflurane and sacrificed. After sacrifice, the colon was removed and laid flat on white A4 paper. The length of the colon from the ileocecal junction to the anus was measured with a vernier caliper and photographed.

[0055] (2) Colon histopathological staining

[0056] Part of the colon tissue was removed and fixed with 4% paraformaldehyde (>48h), embedded in paraffin, sectioned, stained with hematoxylin-eosin and mounted. Inverted phase contrast microscopy was used to evaluate intestinal inflammation in each group of mice from three aspects: intestinal epithelium and crypt structure, goblet cells, and inflammatory cell infiltration.

[0057] 3. Statistical processing

[0058] SPSS 29.0 software was used for statistical analysis. The measurement data were all in accordance with the normal distribution and were expressed as mean ± standard deviation. The data were described in detail. One-way analysis of variance was used for comparison among multiple groups. P < 0.05 or P < 0.0001 was considered statistically significant.

[0059] 4. Experimental Results

[0060] (1) Comparison of general condition and DAI scores of mice

[0061] General situation: The mice in the blank group were energetic, had smooth and shiny hair, were agile, and had hard and oval feces. The mice in the other two groups became listless, had sparse and less shiny hair, decreased activity, and were lethargic after being stimulated by PG or DSS. They had loose stools and even occult blood and visible blood in their stools. Starting from the 5th week of modeling, the body weight of the mice in the AS and AS+ chronic enteritis groups began to show a downward trend compared with the mice in the blank group (P < 0.05). In addition, we calculated the coefficient of variation (CV) of the body weight of each group of mice from 0 to 20 weeks to evaluate the consistency of body weight changes within the group: Blank group mice: The CV value was low and the variation was small, showing a high consistency. Mice in the AS group and AS+ chronic enteritis group: After the 5th week, the CV value fluctuated slightly, but was relatively stable overall. The low and stable CV values ​​indicated that the body weight changes of the mice in each group were more consistent. See Figure 2 、 Figure 3 .

[0062] Body weight loss, stool characteristics, and blood in stool were recorded weekly, and intestinal disease activity (DAI) scores were calculated. We further calculated the coefficient of variation (CV) of DAI scores between weeks 0 and 20 for each group to assess the consistency of DAI score changes within the groups. Compared with the blank group, DAI scores increased in the AS and AS+chronic enteritis groups from weeks 0 to 20 (P < 0.05). The CV of the blank group showed missing values ​​(NaN) at multiple time points, possibly indicating that the control group values ​​were zero or very close to zero at these time points. Therefore, the control group showed no significant variation at these time points, indicating that the mice maintained a relatively stable state under normal conditions, consistent with the expected background stability. From weeks 0 to 6, DAI scores increased in the AS group, while CVs remained low, reflecting the consistent inflammatory manifestations of AS mice early in the disease course and accurately reflecting the early pathological features of the model. DAI scores in the AS+chronic enteritis group also increased, but were slightly lower than those in the AS group (P < 0.05), with a higher CV, indicating that the comorbidity model exhibits significant individual variability in the early stages. This may reflect that the model has not yet fully developed consistent comorbid symptoms in the early stages, resulting in slight differences in the course of disease between individuals, highlighting the complexity of the comorbidity model in the early stages of modeling. From week 6 to week 9 of modeling, the DAI score in the AS group decreased compared to the AS+chronic enteritis group (P < 0.05), indicating that pathological changes in the AS group slowed down during this stage. The CV in the AS group increased slightly during this stage, but remained relatively low overall, reflecting that the differences between individuals were within an acceptable range and further illustrating the differences in the pathogenesis of the AS and AS+chronic enteritis models. At week 7, mice in the AS group gradually developed fecal occult blood, while the AS+chronic enteritis group developed this symptom later (week 9). At this point, the DAI score in the AS+chronic enteritis group gradually increased and exceeded that of the AS group, reflecting the exacerbation of intestinal symptoms in the comorbidity model. The CV in the comorbidity group gradually decreased during this stage, indicating that the model phenotype gradually became consistent. Starting from week 9, the DAI score in the AS group remained stable at a low level and was significantly lower than that in the AS+chronic enteritis group (P < 0.05). The CV of both the AS group and the AS+chronic enteritis group was low during this period, which not only indicated that the AS model was highly stable in the long-term maintenance phase and the inflammatory phenotype tended to be consistent, providing a stable control for comparative studies of AS and comorbidity models, but also further demonstrated the repeatability and reliability of the AS+chronic enteritis comorbidity model. Figure 4 、 Figure 5 .

[0063] (2) Mouse morphological observation and Micro CT

[0064] Starting from the 7th week of modeling, mice in the AS and AS+chronic enteritis groups showed limited movement. Starting from the 12th week of modeling, mice in the AS+chronic enteritis group showed decreased tail flexibility (12th week), decreased hind paw grip strength (13th week), significant hip abduction (14th week), scoliosis of the lower spine (15-16th week), and scoliosis of the lower and upper spine (17-20th week). Starting from the 12th week of modeling, mice in the AS group showed decreased tail flexibility (12th week), decreased hind paw grip strength (13-15th week), significant hip abduction (16-18th week), and scoliosis of the lower spine (19-20th week).

[0065] The sacroiliac joints of mice in the blank group were normal, with no bone erosion and sclerosis. The sacroiliac joints of mice in the AS group were significantly abnormal, with bone erosion and sclerosis on the joint surface, and narrowing of the joint space. The sacroiliac joints of mice in the AS+chronic enteritis group were significantly abnormal, with bone erosion and sclerosis on the joint surface, narrowing and roughening of the joint space, and worm-eaten changes. Figure 6 .

[0066] In addition, imaging examinations confirmed that all AS model mice developed sacroiliitis, with an incidence rate (10 / 10) of 100%. All experimental mice remained alive after modeling (10 / 10), with a survival rate of 100%, ensuring the validity of the experiment and the integrity of the data.

[0067] (3) Changes in colon length

[0068] This study found that the colon length of mice in the AS group was longer than that in the blank group (P < 0.0001), while the colon of mice in the AS+chronic enteritis group was atrophied to varying degrees compared with the blank group and the AS group (P < 0.0001). Figure 7 、 Figure 8 .

[0069] (4) Colon histopathological staining

[0070] Microscopic examination of the intestinal mucosal tissue of the blank group mice showed that the colon was intact, the epithelial cells were normal, the crypt structure was clear, and the goblet cells were obvious. The research results in the existing literature on the easy self-healing of the traditional DSS model showed that in the traditional DSS modeling process, intestinal inflammation usually gradually subsided within 2-3 weeks after modeling, the self-healing rate was high, and the time range of self-healing and the specific pathological manifestations had certain commonalities. In our improved DSS model, intestinal tissue pathological examination found that the inflammatory manifestations of the intestinal tissue of the mice did not significantly improve during the observation period after the end of the modeling (12 to 20 weeks). Microscopic examination of the intestinal mucosal tissue of the AS group mice showed that some colon intestinal epithelial cells were destroyed, the glands were arranged in a disordered manner, some crypt structures were destroyed, the goblet cells were reduced, and inflammatory cells were infiltrated. Microscopic examination of the intestinal mucosal tissue of the AS+ chronic enteritis group mice showed that the colon intestinal epithelial structure was relatively intact, the crypt structure was destroyed to a certain extent, a small amount of inflammatory cells were infiltrated, and Figure 9.

[0071] In addition, histopathological examination of AS and AS+ chronic enteritis model mice confirmed that all of them had chronic enteritis pathological changes, with an incidence rate (10 / 10) of 100%. All experimental mice remained alive after modeling (10 / 10), with a survival rate of 100%, ensuring the validity of the experiment and the integrity of the data.

[0072] The present invention provides a concept and method for an animal model of ankylosing spondylitis and chronic enteritis. There are numerous methods and approaches for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

[0073] Example 2 Screening process of proteoglycan types.

[0074] During the experiment, different types of proteoglycans were screened, such as VG1 and bovine cartilage proteoglycan, which were mixed with complete Freund's immune adjuvant for induction experiments, and clinical inflammation scores were performed on the two groups of mice. The results showed that the inflammatory response induced by VG1 was weak, resulting in unstable incidence of mice; while the incidence of axial arthritis in mice using bovine cartilage proteoglycan as an inducer reached 100%, and the inflammatory phenotype was more obvious (P < 0.05). Therefore, bovine cartilage proteoglycan was finally selected to ensure the stability and consistency of the model. Figure 10 .

[0075] Example 3 Optimization experiment on concentration selection and change timing for AS combined with chronic enteritis mouse model.

[0076] 1. Optimization of Dextran Sulfate Sodium Concentration Selection

[0077] The DAI scores and weight change percentage data of mice in the 1wt%, 2wt%, and 3% DSS groups were compared. The data showed that the DAI scores of mice in the 1wt% DSS group fluctuated less, and their weight remained basically stable, indicating mild inflammation, which is suitable for research models of long-term low-grade chronic inflammation. The DAI scores and weight changes in the 2wt% group were moderate, indicating stable chronic inflammation, which is suitable for research models of sustained chronic inflammation. The 3wt% group showed a higher DAI score, and the DAI score and weight changes were larger, especially in the fourth cycle, indicating acute inflammation, which is suitable for research on acute inflammation models (P < 0.05). Therefore, it is preferred to use a 2wt% dextran sulfate sodium concentration in the first three cycles, see Figure 11 , Figure 12 .

[0078] 2. Optimization of concentration change timing

[0079] During the course of the disease, chronic inflammation sometimes turns into a more serious state. Because we further optimized the timing of the change to a high concentration of 4wt% DSS to simulate the process of worsening or recurrence of the disease. In the experiment, it was observed that when the 4wt% DSS was changed in the 1st, 2nd and 3rd cycles, the weight loss of the mice not only increased significantly, but also had a cumulative effect, and the mice had a strong stress response, which made the experiment unable to proceed smoothly. Therefore, this experiment chose to use a lower concentration of 2wt% in the first 3 cycles, and increased it to 4wt% in the 4th cycle. Figure 13 .

[0080] In summary, three 2wt% cycles + one 4wt% cycle not only induced a more significant inflammatory response in mice, but also kept the weight loss within an acceptable range. The design of strengthening the inflammatory response not only ensured the stability of the model but also simulated the process of disease worsening, successfully constructing an animal model that meets the characteristics of comorbidity.

[0081] The present invention provides a concept and method for an animal model of ankylosing spondylitis and chronic enteritis. There are numerous methods and approaches for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for constructing an animal model of ankylosing spondylitis and chronic enteritis, characterized in that: Mice were fed with drinking water containing sodium dextran sulfate for 6-8 days, and then given normal drinking water and fed for 13-15 days as a treatment cycle. This feeding cycle was repeated for 4 cycles. At the same time, proteoglycan PG immune inducer was injected into the mice at 0-1 weeks, 3-4 weeks, and 6-7 weeks of modeling, respectively. An animal model of ankylosing spondylitis and chronic enteritis co-morbidity was obtained after 12-24 weeks of modeling.

2. The construction method according to claim 1, characterized in that The mice were female Balb / c mice, 6 to 8 weeks old.

3. The construction method according to claim 1, characterized in that The molecular weight of the dextran sodium sulfate is 36,000-50,000. The concentration of the dextran sodium sulfate in the first three cycles is 2 wt %, and the concentration of the dextran sodium sulfate in the last cycle is 4 wt %.

4. The construction method according to claim 1, characterized in that The proteoglycan PG immune inducer is a mixture of a proteoglycan solution and complete Freund's immune adjuvant, wherein the volume ratio of the proteoglycan solution to the complete Freund's immune adjuvant is 0.8:1 to 1.2:1, and the concentration of the proteoglycan solution is 0.8 to 1.2 μg / μL.

5. The construction method according to claim 4, characterized in that: The proteoglycan solution is obtained by dissolving proteoglycan PG in phosphate buffered saline (PBS).

6. The construction method according to claim 5, characterized in that: The proteoglycan PG is bovine cartilage proteoglycan.

7. The construction method according to claim 1, characterized in that The dosage of the proteoglycan PG immune inducer is 0.1-0.3 mL / 20 g, and the administration method is intraperitoneal injection.

8. Use of the animal model of ankylosing spondylitis and chronic enteritis co-morbidity constructed by the construction method according to any one of claims 1 to 7 in studying the co-morbidity mechanism of ankylosing spondylitis and chronic enteritis.

9. Use of the animal model of ankylosing spondylitis and chronic enteritis co-morbidity constructed by the construction method according to any one of claims 1 to 7 in screening drugs for treating ankylosing spondylitis and chronic enteritis.

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