Modeling method of irritable bowel syndrome animal model

Abstract

The invention relates to a modeling method of an irritable bowel syndrome animal model. An IBS model is prepared through an anoxic stress method. In the construction process, the plateau environment is simulated through the oxygen deficit box, and physiological and biochemical indexes of model mice are promoted to change; hypoxia can reduce the weight of mice, reduce the water content of excrement, increase the sensitivity of viscera and weaken intestinal motility; the hypoxia can reduce the expression of intestinal tight junction proteins ZO-1 and Occludin and weaken the mechanical barrier function of the intestinal tract; and the expression levels of SOD, GSH, MDA and Nrf2 / HO-1 pathways can be influenced, and the oxidative stress degree of the intestinal tract of the mouse can be aggravated. Carrying out hypoxia stress treatment to obtain an animal model of which the physiological characteristics, the protein expression condition and the pathogenesis are the same as those of typical irritable bowel syndrome animals; the modeling method is simple in processing method, high in modeling speed, short in period, free of human intervention factors and convenient to use.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for modeling an irritable bowel syndrome animal model. Background Art

[0002] At present, animal models of irritable bowel syndrome (IBS) are prepared through unpredictable stress, neonatal mouse letter separation, colorectal balloon dilatation, intrarectal acetic acid or mustard oil stimulation, ice water gavage, non-local inflammatory acute restraint stress, and acute water avoidance stress. The above methods are simple to operate and no pathological histological changes are observed, which are suitable for the preparation of IBS. However, the above methods are all artificial interventions and cannot explain the pathogenic factors of IBS in the natural environment. At the same time, the current studies are all conducted in plain areas and cannot fully cover the incidence of IBS in plateau environments. In addition, the modeling cycle is usually long. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for establishing an irritable bowel syndrome animal model.

[0004] The technical solution adopted by the present invention is: a method for modeling an irritable bowel syndrome animal model, wherein the model animal is placed in a hypoxic environment and the irritable bowel syndrome animal model is obtained after cultivation.

[0005] Preferably, the model animal is placed in an environment with an oxygen content of 10% and cultured in anoxic environment for at least 7 days.

[0006] Preferably, the culture conditions further include controlling the humidity at 40% to 70% and the temperature at 20 to 25°C.

[0007] The animal model is constructed by the irritable bowel syndrome animal model establishment method.

[0008] Preferably, the model animal used is a combination of one or more of C57BL / 6, BALB / c, ICR or KM mice.

[0009] Preferably, the mouse model has reduced body weight, enhanced visceral sensitivity, reduced fecal water content, and weakened intestinal motility; weakened expression of ZO-1 and Occludin; enhanced expression of IL-6 and TNF-α; weakened expression of SOD, GSH, and Nrf2 / HO-1, and enhanced expression of MDA; and enhanced expression of neurotransmitters 5-HT and VIP.

[0010] Application of animal models in verifying the efficacy of drugs for the treatment of irritable bowel syndrome.

[0011] The advantages and positive effects of the present invention are: providing a novel method for establishing an irritable bowel syndrome animal model, which, after hypoxic stress treatment, obtains an animal model whose physiological characteristics, protein expression conditions and pathogenic mechanisms are the same as those of typical irritable bowel syndrome animals; the modeling method is simple in processing method, fast in modeling speed, short in cycle, without human intervention factors, and easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Physiological tests of IBS model mice; (A) Body weight changes; (B) Fecal water content changes; (C) CRD-AWR; Compared with the normoxic group, *p<0.05, **p<0.01, ***p<0.001;

[0013] Figure 2 Colon status of IBS model mice; (A) dilated colon status; (B) colon diameter;

[0014] Figure 3 Pathological examination of colon tissues in IBS model mice;

[0015] Figure 4 Effects of hypoxia on the expression of ZO-1 and Occludin in IBS mice; (A) Expression of ZO-1 mRNA;

[0016] (B) Occludin mRNA expression; (C) ZO-1 protein expression; (D) Occludin protein expression;

[0017] Figure 5 Effects of hypoxia on the expression of SOD, GSH, MDA, and Nrf2 / HO-1 mRNA in IBS model mice; *p<0.05;

[0018] Figure 6 Effect of hypoxia on the expression of IL-6 and TNF-α in IBS model mice; *p<0.05, **p<0.01, ***p<0.001;

[0019] Figure 7 Effect of hypoxia on the expression of 5-HT and VIP in IBS model mice; *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0021] The present invention relates to a method for modeling an irritable bowel syndrome animal model, wherein an IBS model is prepared by a hypoxic stress method. During the construction process, a hypoxic box is used to simulate a plateau environment to promote changes in physiological and biochemical indicators of model mice. Dynamic monitoring of changes in C57 mouse body weight, fecal water content, visceral sensitivity, intestinal motility, pathological sections and related intestinal barrier indicators, inflammatory indicators, oxidative stress indicators, and neurotransmitter indicators confirms that hypoxia can induce IBS by damaging the intestinal barrier.

[0022] The model animals were placed in an anaerobic chamber simulating a 5000m plateau (10% O2-90% N2), with a 10% O2-90% N2 mixed gas flowing in the chamber. The anaerobic chamber can control oxygen concentration, temperature and humidity. The temperature range in the chamber is 20-25°C, the relative humidity is 40%-70%, the light rhythm is 12h / 12h, and food and water are freely available. After 7 days of culture, an IBS animal model was obtained.

[0023] The constructed IBS mouse model was tested and found that compared with the normoxic group, hypoxia could reduce the weight of mice, reduce the water content of feces, increase visceral sensitivity, and weaken intestinal motility; the anatomical observation of the mouse colon tissue showed that the colon diameter was significantly increased and the colon was obviously bloated. The expression of related proteins in the colon tissue was tested and it was found that hypoxia could reduce the expression of intestinal tight junction proteins ZO-1 and Occludin, weaken the intestinal mechanical barrier function; and hypoxia could affect the expression levels of SOD, GSH, MDA and Nrf2 / HO-1 pathways, and aggravate the degree of intestinal oxidative stress in mice. In addition, compared with the normoxic group, it was found that hypoxic stress could increase the expression levels of IL-6 and TNF-α, aggravate intestinal inflammation in IBS mice; hypoxia could also increase the expression levels of neurotransmitters 5-HT and VIP, causing brain-intestinal neural regulation disorders in IBS mice.

[0024] Compared with traditional unpredictable stress, neonatal mouse letter separation, colorectal balloon dilatation, intrarectal acetic acid or mustard oil stimulation, ice water gavage, non-local inflammatory acute restraint stress, and acute water avoidance stress methods, the animal model obtained by hypoxia stress modeling is simple in modeling method, short in cycle, and more uniform and stable in animal model, and can be used to study the incidence of IBS in plateau environment. It can be used to verify the effects of various IBS drugs.

[0025] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0026] Example 1: Construction of IBS model mice by hypoxia induction method

[0027] Forty C57BL / 6J mice were randomly divided into five groups: normoxic group, hypoxic 1d group, 3d group, 5d group and 7d group, with 8 mice in each group. The normoxic group was placed in a room with an oxygen concentration of 21%, a temperature range of 20-25℃, a relative humidity of 40%-70%, a light rhythm of 12h / 12h, and free access to food and water; the mice in each hypoxic group were placed in a hypoxic box simulating a 5000m plateau (10% O2-90% N2), which can control oxygen concentration, temperature and humidity, so that the temperature range in the box is 20-25℃, the relative humidity is 40%-70%, the light rhythm is 12h / 12h, and free access to food and water; after 1d, 3d, 5d, and 7d of culture, the hypoxic 1d, 3d, 5d, and 7d groups were obtained respectively.

[0028] Example 2: Detection of Physiological Indexes of IBS Model Mice

[0029] The weight changes, fecal moisture content and capacity threshold of mice in the normoxic and hypoxic groups were tested and statistically analyzed. Figure 1 As shown. Figure 1 As shown in (A), compared with the normoxic group, the body weight of mice decreased on the 5th day of hypoxia (p<0.05), and decreased significantly on the 7th day of hypoxia (p<0.001); Figure 1 As shown in (B), compared with the normoxic group, the fecal water content of mice decreased on the 5th day of hypoxia (p<0.05), and decreased significantly on the 7th day of hypoxia (p<0.001); Figure 1 As shown in (C), compared with the control group, the capacity threshold of mice in the hypoxia group gradually decreased, and the capacity threshold of mice was significantly different after 7 days of hypoxia (p<0.001), indicating that the visceral sensitivity of mice increased significantly after 7 days of hypoxia.

[0030] Observation of the colon of mice revealed that the colon of mice was significantly bloated after hypoxia. Figure 2 (A) shows the colon diameter, and then the colon diameter is measured. Figure 2 As shown in (B), compared with the normoxic group, the colon diameter of mice increased significantly after 7 days of hypoxia (p<0.01), indicating that the intestinal motility of mice was weakened after hypoxia.

[0031] Colon tissue sections of mice in the normoxic group and the hypoxic group with different hypoxia durations were stained and observed. Figure 3Colonic histopathology showed that the intestinal mucosa in the control group was intact, goblet cells were intact and visible, without ulcers, bleeding or necrosis. The gap below the upper layer of the intestinal mucosa in the 3-day hypoxia group was slightly enlarged, without ulcers, bleeding or necrosis. In the 5-day hypoxia group, local mucosal cells necrotized and fell off, and no inflammatory infiltration was observed. In the 7-day hypoxia group, a large number of mucosal epithelial cell layers necrotized and fell off, the gap below the upper layer of the mucosa was significantly enlarged, and a small amount of focal inflammatory infiltration was observed. These results show that hypoxia can induce IBS by reducing the body weight of mice, reducing the water content of feces, increasing visceral sensitivity, destroying the intestinal barrier, and slowing down intestinal motility, and IBS can be constructed after hypoxia induction.

[0032] From the above experimental results, we can see that hypoxia can cause mice to lose weight, reduce the water content of feces, increase visceral sensitivity, and weaken intestinal motility.

[0033] Embodiment 3:

[0034] 3.1 Protein expression in colon tissue of IBS model mice

[0035] Colon tissues of mice in the normoxic group and hypoxic groups with different hypoxia durations were collected to measure the levels of ZO-1 and Occludin. The expression levels of ZO-1 and Occludin in colon tissues were determined by qPCR (n=6). Figure 4 As shown in (A, B). Compared with the control group, the mRNA levels of ZO-1 and Occludin in the IBS group mice were significantly decreased (p<0.05). WB was used to detect the expression levels of ZO-1 and Occludin (CD) in colon tissue (n=3). Figure 4 As shown in (C, D), after hypoxia, the protein levels of both decreased significantly. The values ​​represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. It can be seen that hypoxia can reduce the expression of intestinal tight junction proteins ZO-1 and Occludin and weaken the intestinal mechanical barrier function.

[0036] 3.2 Expression levels of SOD, GSH, MDA and Nrf2 / HO-1 pathway in colon tissue of IBS model mice

[0037] Hypoxia can lead to oxidative stress. SOD, GSH, and MDA are important indicators of the body's oxidative stress level. The nuclear factor e2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) pathway plays an important role in anti-oxidation. The expression of SOD, GSH, MDA, and Nrf2 / HO-1 pathway-related proteins in the colon tissues of mice in the normoxic group and the hypoxic group with different hypoxia durations was detected; the results are shown in Figure 2. Figure 5 shown.

[0038] The levels of SOD, GSH and MDA in colon tissue were determined by microassay. Figure 5 As shown in (A, B), compared with the control group, the SOD and GSH of mice in the IBS group were decreased (p<0.05); Figure 5 As shown in (C), the MDA level of mice in the IBS group was increased compared with that in the control group (p<0.05). qPCR was used to detect the mRNA expression levels of Nrf2 and HO-1 in colon tissue (n=6). Figure 5 As shown in (D, E), compared with the control group, the expression of Nrf2 / HO-1 mRNA in the IBS group mice was decreased (p<0.05), indicating that hypoxia can inhibit the expression of this pathway; In summary, hypoxia can aggravate the degree of intestinal oxidative stress in mice by affecting the expression levels of SOD, GSH, MDA, and Nrf2 / HO-1.

[0039] 3.3 Expression levels of IL-6 and TNF-α in colon tissue of IBS model mice

[0040] The expression of IL-6 and TNF-α in the colon tissue of mice in the normoxic group and the hypoxic groups with different hypoxia durations was detected. ELISA was used to detect the expression levels of IL-6 (A) and TNF-α (B) in the colon tissue (n=6). The results are shown in Figure 6 As shown in (A, B). Compared with the control group, the IL-6 level of mice in the IBS group was significantly increased (p<0.01); the TNF-α level was also significantly increased (p<0.05). It can be seen that hypoxic emergency treatment can increase the expression levels of IL-6 and TNF-α and aggravate intestinal inflammation in IBS mice.

[0041] 3.4 Expression levels of neurotransmitters 5-HT and VIP in colon tissue of IBS model mice

[0042] The levels of 5-HT and VIP in colon tissue were measured by ELISA. Compared with the control group, the 5-HT in the IBS group mice increased significantly (p<0.05), and the VIP level in the IBS group mice increased (p<0.001). It can be seen that hypoxic stress treatment can affect the expression levels of 5-HT and VIP, disrupt the secretion of intestinal neurotransmitters, slow intestinal motility, increase visceral sensitivity, and cause brain-intestinal nerve regulation disorders in IBS mice.

[0043] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for establishing an irritable bowel syndrome animal model, characterized in that: The model animals were placed in a hypoxic environment and cultured to obtain an irritable bowel syndrome animal model.

2. The method for establishing an irritable bowel syndrome animal model according to claim 1, characterized in that: The model animals were placed in an environment with an oxygen content of 10% and cultured in hypoxia for at least 7 days.

3. The method for establishing an irritable bowel syndrome animal model according to claim 2, characterized in that: The culture conditions also include controlling the humidity at 40% to 70% and the temperature at 20 to 25°C.

4. An animal model constructed by the method for establishing an irritable bowel syndrome animal model according to any one of claims 1 to 3.

5. The animal model according to claim 4, characterized in that: The model animals used were one or more of C57BL / 6, BALB / c, ICR or KM mice.

6. The animal model according to claim 5, characterized in that: The mouse model had reduced body weight, enhanced visceral sensitivity, reduced fecal water content, and weakened intestinal motility; the expression of ZO-1 and Occludin was weakened; the expression of IL-6 and TNF-α was enhanced; the expression of SOD, GSH, and Nrf2 / HO-1 was weakened, and the expression of MDA was enhanced; the expression of neurotransmitters 5-HT and VIP was enhanced.

7. Use of the animal model described in any one of claims 4 to 6 in verifying the efficacy of drugs for treating irritable bowel syndrome.

Images