Construction method and application of ankylosing spondylitis and chronic enteritis co-disease animal model

Abstract

The invention discloses a construction method of an animal model for coexistence of ankylosing spondylitis and chronic enteritis. The animal model for simulating coexistence of ankylosing spondylitis and chronic enteritis from symptoms, iconography change and pathological characteristics is established through a proteoglycan PG immune induction method and a dextran sodium sulfate chemical induction method. The model mouse not only shows imaging changes such as sacroiliac articular surface bone erosion, sclerosis, joint space stenosis, roughness and worm-eaten sample change similar to human ankylosing spondylitis, but also shows intestinal epithelial cell destruction, disordered gland arrangement, crypt structure destruction, goblet cell reduction and chronic enteritis similar to human chronic enteritis. The compound can be used for researching the interaction mechanism between the two diseases and exploring a new treatment target, is beneficial to evaluating the potential application of a new medicine in treating the ankylosing spondylitis and the chronic enteritis, and provides an important tool for mechanism research and medicine development of the common diseases.

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 a comorbid animal model for studying human ankylosing spondylitis combined with chronic enteritis. Background Art

[0002] Ankylosing spondylitis (AS) is a common immune-mediated chronic inflammatory disease that mainly affects the spine and sacroiliac joints. Chronic enteritis (colitis) is a disease involving long-term (chronic) inflammation of the digestive tract tissues. Its pathological characteristics are a large number of lymphocytes and plasma cells infiltrating the intestinal mucosal lamina propria, and abnormal mucosal structure, including changes in crypt structure, metaplastic changes, increased basal plasma cells, inflammatory polyps, etc. Chronic enteritis is the histological basis of inflammatory bowel disease (IBD). Recent studies have shown that 70% of AS patients have intestinal inflammation, and about 5-10% of them will develop IBD. Bradtzaeg first proposed the concept of the "intestinal-joint axis" in 1997, believing that immune cells home from the intestine to the site of synovial inflammation, forming a communication between intestinal immunity and joint disease. In recent years, many studies have further revealed its mechanism, including increased intestinal barrier permeability, dysregulated intestinal flora and its metabolites inducing autoimmunity, and intestinal immune cells and immune molecules migrating to the joints. Mendelian randomization also found a positive causal relationship between IBD and AS, and IBD increased the risk of AS, which once again confirmed from a genetic perspective that intestinal inflammation is closely related to AS. However, there is currently a lack of an animal model that simulates the coexistence of ankylosing spondylitis and chronic enteritis in terms of symptoms, imaging changes, and pathological characteristics, which limits the study of the pathogenesis of this type of co-morbidity and the development of therapeutic drugs.

[0003] Since it is difficult to obtain human tissues in the pathological process of AS, the construction of experimental animal models is particularly important for AS research. Experimental animal models of AS can be roughly divided into four categories: HLAB27 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 mainly used for the study of AS genes. This model has the disadvantages of long modeling time, technical difficulties, complex operation, and high price. The ankylosing enthesitis animal model and other animal models have the disadvantages that the onset is greatly affected by environmental factors and requires high breeding conditions. The inflammation-related animal model of the proteoglycan PG immune-induced BALB / c mouse model focuses on the study of immune inflammation. It is not only very similar to the development of clinical AS diseases, but also very valuable for studying the autoimmunity of spondylitis and locating the genetic loci of spondyloarthritis. There are many animal models of chronic enteritis, including chemical method, immune method, gene modification method, and composite method. The latter three are not widely accepted at home and abroad due to their low success rate and high price. The disadvantage of acetic acid and oxazolidinone modeling in the chemical method is the lack of a chronic relapse process similar to that of humans after a short-term acute attack. Trinitrobenzene sulfonic 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 easy self-healing, 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 and explore the correlation between the two. It has 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 provide an animal model of co-morbidity of ankylosing spondylitis and chronic enteritis in view of the deficiencies in the prior art. An animal model simulating the coexistence of ankylosing spondylitis and chronic enteritis is established by 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 the feeding 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, 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 Balb / c mouse, female, 6 to 8 weeks old.

[0008] Preferably, the molecular weight of the sodium dextran sulfate is 36,000 to 50,000, the concentration of the first three cycles is 2wt%, and the concentration of the last cycle is 4wt%. When the concentration is changed each time, the drinking water should be changed, and it should be prepared and used immediately to ensure the use of fresh solution. Studies have shown that the stability of sodium dextran sulfate is poor, so it is necessary to change the drinking water every day.

[0009] Preferably, the preparation method of the proteoglycan immune inducer is: after selecting 100 μg of proteoglycan PG and dissolving it 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 concentration of 0.8-1.2 μg / μL and a dosage of 0.1-0.3 mL / 20 g. The administration method is intraperitoneal injection. Preferably, the administration concentration 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, narrow and rough joint space, and worm-eaten changes. The pathological manifestations of chronic enteritis include destruction of intestinal epithelial cells, disordered glandular arrangement, destruction of crypt structure, reduction of goblet cells, and infiltration of inflammatory cells. Other evaluation criteria include general condition of mice, body weight of mice, DAI score of intestinal disease activity of mice, length of mouse colon, and morphological observation of mice.

[0013] Beneficial effects: The invention provides an optimized animal model for simulating the co-morbidity of ankylosing spondylitis and chronic enteritis. Through condition optimization, 2% sodium dextran (DSS) was used for 3 cycles + 4% DSS for 1 cycle. While ensuring a 100% survival rate of mice, the defect of the traditional DSS model that it is easy to heal itself was effectively overcome, and a 100% incidence rate of model mice was achieved. Proteoglycan PG immune inducer (0.1-0.3mL / 20g) mixed with bovine cartilage proteoglycan and complete Freund's adjuvant in a volume ratio of 0.8:1-1.2:1 was injected intraperitoneally into the model mice at 0-1 weeks, 3-4 weeks, and 6-7 weeks of modeling, respectively, so that the incidence 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 helps to evaluate the potential application of new drugs in the treatment of ankylosing spondylitis and chronic enteritis, and provides an important tool for mechanism research and drug development of such comorbidities. 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 diagram 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 diagram 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] Fig. 9 Hematoxylin-eosin staining results of colon tissues of mice in each group;

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

[0024] Fig.11 Percentage change of DAI score at 1wt%, 2wt%, and 3wt%;

[0025] Fig.12 1wt%, 2wt%, 3wt% body weight change percentage;

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

[0027] The present invention will be further described in detail below in conjunction with 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; the experimental methods in the examples where specific conditions are not specified are generally based on conventional conditions 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] 1.1. Experimental animals

[0031] BALB / c mice, female, 6 weeks old, 20 ± 2 g, clean grade, were provided by the Experimental Animal Center of Nanjing University of Chinese Medicine, certificate number: SCXK (Su) 2012-0008. The experimental design and experimental procedures were in accordance with the guidelines and requirements of the Experimental 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 phosphate buffered saline (PBS), then vortexed and mixed. The proteoglycan solution is mixed with complete Freund's immune adjuvant in a volume ratio of 1:1 and then ultrasonically emulsified to prepare 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: fully dissolve 20g or 40g DSS powder in 1000mL Millipore ultrapure water and use it 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, 20±2g, SPF grade, were fed adaptively for one week and randomly divided into three groups: blank group, simple ankylosing spondylitis group, 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 the 0th to 1st week, the 3rd to 4th week, and the 6th to 7th week 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 the mice were adaptively raised in the SPF animal room for 1 week, they 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 was one cycle, and there were 4 cycles in total. 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 (0.2mL / 20g) on ​​the 1st day (week 0), 21st day (week 3), and 42nd day (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 avoid 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 out.

[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] The mental state, coat condition, activity, food and water intake, body weight, stool characteristics and blood in stool of mice were observed and recorded from week 0 to week 20 of modeling. The blood in stool of mice was evaluated 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, the animal loose stool score and the 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 activities of mice and the softness of their tails, hip joints, and spine were observed. The small animal in vivo MicroCT imaging system was used to evaluate the changes in the sacroiliac joints of living mice. The scanning parameters were set as 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 the model was successfully established, the mice were anesthetized with isoflurane and killed. After the animals were killed, the colons of the animals were taken out 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. An inverted phase contrast microscope 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 analysis

[0058] SPSS 29.0 software was used for statistical analysis. The measured data were all in accordance with the normal distribution and were expressed as mean ± standard deviation. 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 were listless, had sparse and less shiny hair, decreased activity, and lethargy after being stimulated by PG or DSS. They had loose stools and even occult blood and visible blood in the stools. Starting from the 5th week of modeling, compared with the mice in the blank group, the body weight of the mice in the AS and AS+chronic enteritis groups began to decrease (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 lower and the variation was smaller, showing a higher 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 lower and more stable CV values ​​indicated that the body weight changes of the mice in each group were more consistent. See Figure 2 , Figure 3 .

[0062] The weight loss, fecal characteristics, and blood in the stool of mice were recorded weekly, and the intestinal disease activity DAI score of mice was calculated. We further calculated the coefficient of variation (CV) of the DAI score of each group of mice from 0 to 20 weeks to evaluate the consistency of the DAI score changes within the group. Compared with the blank group, the DAI scores of mice in the AS and AS+chronic enteritis groups increased from 0 to 20 weeks of modeling (P < 0.05). The CV of the blank group was shown as missing (NaN) at multiple time points, which may indicate that the values ​​of the control group were zero or very close to zero at these time points. Therefore, there was no significant variation in the control group at these time points, indicating that the mice maintained a relatively stable state under normal conditions, which was consistent with the expected background stability. From 0 to 6 weeks of modeling, the DAI score of the AS group increased, and the CV remained at a low level, reflecting that the inflammatory manifestations of AS mice in the early stage of the disease were more consistent and could more accurately reflect the early pathological characteristics of the model. The DAI score of the AS+chronic enteritis group also increased, but was slightly lower than that of the AS group (P < 0.05), and the CV was higher, indicating that the comorbidity model showed large individual differences in the early stage. This may reflect that the model has not yet fully formed consistent comorbid symptoms in the early stage, and the course of disease between individuals is slightly different, showing the complexity of the comorbid model in the early stage of modeling. In the 6th to 9th week of modeling, the DAI score of the AS group decreased compared with the AS+chronic enteritis group (P<0.05), indicating that the pathological changes in the AS group slowed down at this stage. The CV of the AS group at this stage increased slightly, but the overall level remained low, reflecting that the differences between individuals were still within an acceptable range, and further explained the differences in the pathogenesis of the AS and AS+chronic enteritis models. In the 7th week, mice in the AS group gradually developed fecal occult blood, while this symptom in the AS+chronic enteritis group was later (9th week). At this time, the DAI score of mice in the AS+chronic enteritis group gradually increased and exceeded that of the AS group, reflecting the aggravation of intestinal symptoms in the comorbid model. The CV of the comorbid group gradually decreased at this stage, indicating that the model phenotype gradually became consistent. From the 9th week, the DAI score of the AS group remained stable at a low level and was significantly lower than that of 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 stage and the inflammatory phenotype tended to be consistent, providing a stable control for the comparative study of AS and comorbidity models, but also further demonstrated the repeatability and reliability of the comorbidity model of AS+chronic enteritis, see Figure 4 , Figure 5 .

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

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

[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 narrow 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, narrow and rough 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 data integrity.

[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 mice in the blank group 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 on the easy self-healing of the traditional DSS model in the existing literature showed that in the traditional DSS modeling process, intestinal inflammation usually gradually subsided within 2-3 weeks after modeling, and the self-healing rate was high. The time range of self-healing and the specific pathological manifestations had certain commonalities. In our improved DSS model, intestinal tissue pathological examination showed that the inflammatory manifestations of the intestinal tissue of 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 mice in the AS group 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 mice in the AS+chronic enteritis group showed that the intestinal epithelial structure of the colon was relatively intact, the crypt structure was destroyed to a certain extent, and a small amount of inflammatory cells were infiltrated. Fig. 9.

[0071] In addition, histopathological examination of AS and AS+chronic enteritis model mice confirmed that all of them had pathological changes of chronic enteritis, 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 an idea and method for an animal model of ankylosing spondylitis and chronic enteritis. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by 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 for 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. Fig.10 .

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

[0076] 1. Optimization of the concentration selection of dextran sulfate sodium

[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 body weight changes in the 2wt% group were moderate, indicating stable chronic inflammation, which is suitable for research models of persistent chronic inflammation. The 3wt% group showed a higher DAI score, and the DAI score and body 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 Fig.11 , Fig.12 .

[0078] 2. Optimization of concentration change timing

[0079] During the course of disease progression, chronic inflammation can sometimes turn into a more severe state. We further optimized the timing of the change to a high concentration of 4wt% DSS to simulate the process of disease aggravation or relapse. In the experiment, we observed that when the 4wt% DSS was changed in the 1st, 2nd, and 3rd cycles, the weight loss of mice not only increased significantly, but also had a cumulative effect, and the mice had a strong stress response, which prevented the experiment from proceeding smoothly. Therefore, this experiment chose to use a lower concentration of 2wt% in the first 3 cycles, and only increased it to 4wt% in the 4th cycle. Fig.13 .

[0080] In summary, 3 cycles of 2wt% + 1 cycle of 4wt% not only induced a more significant inflammatory response in mice, but also kept the weight loss within an acceptable range, thereby strengthening the design of the inflammatory response, which not only ensured the stability of the model but also simulated the process of disease aggravation, successfully constructing an animal model that met the characteristics of comorbidities.

[0081] The present invention provides an idea and method for an animal model of ankylosing spondylitis and chronic enteritis. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for constructing an animal model of ankylosing spondylitis and chronic enteritis, characterized in that: The 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 week, 3-4 week, and 6-7 week of modeling, and 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 are Balb / c mice, female, 6 to 8 weeks old.

3. The construction method according to claim 1, characterized in that: The molecular weight of the sodium dextran sulfate is 36,000-50,000, the concentration of the sodium dextran sulfate in the first three cycles is 2 wt %, and the concentration of the sodium dextran 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. Application of the ankylosing spondylitis and chronic enteritis comorbidity animal model constructed by the construction method according to any one of claims 1 to 7 in studying the comorbidity mechanism of ankylosing spondylitis and chronic enteritis.

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

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