Construction method and application of senile constipation animal model

Abstract

The invention discloses a construction method and application of an animal model for treating senile constipation, and belongs to the technical field of animal model construction. According to the method, D-galactose is injected into an experimental animal and loperamide is subjected to intragastric administration, so that the animal model with senile constipation is obtained; the method has the advantages that animal aging is induced by D-galactose, constipation symptoms are induced by the aid of loperamide, accordingly, double-factor collaborative modeling can be realized, senile constipation animal models with characteristics of behavioral abnormality, small intestine propulsion rate reduction, histopathology and the like can be successfully constructed, and the method is high in stability and repeatability and can be widely applied to the field of senile constipation animal models. The method can be widely applied to senile constipation pathogenesis research and drug screening, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and particularly relates to a construction method of an elderly constipation animal model and application thereof. Background Art

[0002] Constipation in the elderly is mainly caused by decreased intestinal motility, weakened intestinal tension and insufficient force for defecation. Long-term constipation not only affects the quality of life of the elderly, but also may transmit toxic substances to the brain through blood circulation, leading to pathological changes such as neurofibrillary entanglement and nerve cell death, thereby inducing serious diseases such as Alzheimer's disease.

[0003] At present, the pathogenesis of senile constipation has not been fully elucidated, and clinical treatment lacks standardization. In the research field, the existing aging animal models usually use natural aging animal models, D-galactose-induced aging animal models, PS1 / APP double transgenic mice, and rapid aging SAMP8 mouse models; constipation models mainly include animal models caused by different factors such as drug type, vinegar + activated carbon ice water type, and rectal stenosis surgery type. These methods have problems such as large individual differences, long modeling cycle, complex operation, high cost, or poor model stability, and there is no dual-factor composite model of aging and constipation that can be widely promoted. Therefore, there is an urgent need for a low-cost, short-cycle and stable animal model of senile constipation to provide a reliable tool for studying disease mechanisms and screening drugs. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for constructing an animal model of constipation in the elderly and its application, so as to solve the problems of long modeling cycle, poor model stability and complex operation in the prior art.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a method for constructing an animal model of elderly constipation is provided, wherein the animal model of elderly constipation is obtained by injecting D-galactose into experimental animals and administering loperamide intragastrically; The specific treatment method of D-galactose injection is to subcutaneously inject D-galactose into the experimental animals from the first day of modeling, with a dosage of 130-170 mg / kg, once a day, for 4-6 weeks; The specific treatment method of oral administration of loperamide is to gavage loperamide to the experimental animals starting from the third week of modeling, with a dosage of 1-3 mg / kg, once a day, for 2-3 weeks.

[0006] The beneficial effects of the present invention are as follows: D-galactose mimics the senescent state of the body by inducing oxidative stress and chronic inflammation. Loperamide acts on the opioid receptors of intestinal smooth muscle, delays intestinal transit, reduces the frequency of defecation and increases the hardness of feces, thereby inducing constipation symptoms. At the same time, the senescent state induced by D-galactose leads to a decrease in the tension of intestinal smooth muscle and a weakening of the nerve regulation function, making the intestine more sensitive to the inhibitory effect of loperamide. On this basis, loperamide further exacerbates the inhibition of intestinal peristalsis, and the constipation symptoms are more obvious. The synergistic use of the two can successfully establish a stable animal model of senile constipation in a relatively short time.

[0007] On the basis of the above technical solutions, the present invention can be further improved as follows: Further, the experimental animal is a mouse.

[0008] Further, the mouse is a KM mouse.

[0009] Further, the dosage of the D-galactose solution is 150 mg / kg.

[0010] Further, the administration time of D-galactose lasts for 4 weeks Further, the dosage of the loperamide solution is 2 mg / kg.

[0011] Further, the administration time of loperamide lasts for 2 weeks.

[0012] The present invention also provides the application of the animal model of senile constipation constructed by the method for constructing the animal model of senile constipation as described above in the experimental research of senile constipation-related diseases, or in the preparation and / or screening of drugs for treating senile constipation.

[0013] The present invention has the following beneficial effects: 1. The present invention has successfully constructed an animal model of senile constipation with behavioral abnormalities, reduced small intestine propulsion rate, electrophysiological changes in the colon and histopathological characteristics, which has high stability and repeatability, can be widely used in the research of the pathogenesis of senile constipation and drug screening, and has good application prospects.

[0014] 2. The present invention induces animal senescence by D-galactose and combines with loperamide to induce constipation symptoms, realizing the synergistic modeling of two factors. It only takes 4-6 weeks to successfully construct an animal model of senile constipation, greatly shortening the modeling time, improving the experimental efficiency, and having simple operation and low cost. Brief Description of the Drawings

[0015] Figure 1 It is a graph showing the changes in the water maze experiment trajectories of the control group mice and the model group mice. Among them, the left figure is the control group and the right figure is the model group; Figure 2These are optical microscopic images of HE-stained brain tissue sections of mice in the control group, where the left image is 100 times and the right image is 400 times; Figure 3 The optical microscope images of HE-stained brain tissue sections of mice in the model group, where the left image is 100 times and the right image is 400 times; Figure 4 The optical microscopic images of AQP3 protein immunohistochemical sections of colon tissues of mice in the control group, where the left image is 100 times and the right image is 400 times; Figure 5 These are optical microscopic images of AQP3 protein immunohistochemical sections of colon tissue of mice in the model group, where the left image is 100 times and the right image is 400 times.

[0016] Figure 6 The results of Klotho protein immunoblotting in the brain tissues of mice in the control group and the model group are shown in the figure, where the left column is the control group and the right column is the model group; Figure 7 These are the immunoblot results of c-Kit, SCF and Klotho proteins in the colon tissues of mice in the control group and the model group, where the left column is the control group and the right column is the model group. DETAILED DESCRIPTION

[0017] The experimental animals used in the examples of the present invention are normal male KM mice weighing about 20 g and aged 6 to 8 weeks, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; if the specific conditions are not specified in the examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0018] The principles and features of the present invention are described below in conjunction with embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0019] Embodiment 1: This embodiment provides a method for constructing an elderly constipation animal model, comprising the following steps: 1. 20 KM mice were randomly divided into a model group and a control group, with 10 mice in each group, and raised under standard conditions with controlled temperature, sufficient water and food; 2. D-galactose was prepared into a 15 mg / mL solution with sterile saline. The model group was subcutaneously injected with 150 mg / kg D-galactose using a 1 mL syringe, and the control group was subcutaneously injected with an equal amount of saline once a day for 4 weeks. 3. At the third week after the injection of D-galactose, loperamide was prepared into a 0.2 mg / mL solution with sterile saline. The model group was gavaged with a 1 mL syringe connected to a gavage needle at a dose of 2 mg / kg loperamide, and the control group was gavaged with an equal amount of saline once a day for 2 weeks.

[0020] Experimental Example 1 The animal model of elderly constipation established in the above Example 1 was subjected to the following evaluation tests to detect the modeling effect of the elderly constipation mouse model.

[0021] 1. Behavioral Observation 1. Experimental methods The Morris water maze test was performed on the elderly constipation mouse model constructed by the present invention, and memory training was performed for 4 days. The mouse was placed vertically in the water with its face facing the pool wall, and the mouse was observed to see whether it could find the platform within 60 seconds. If the mouse could not find the platform smoothly, the experimenter guided it to the platform and stayed on the platform for 10 seconds, then took out the mouse, wiped it dry, and carried out training in the next quadrant. After the memory training was completed, the platform was removed and the memory ability of the mouse was tested. The mouse was placed in the water in the quadrant opposite to the quadrant where the original platform was located, and the number of times the mouse reached the quadrant where the platform was located within 60 seconds, the latency period to reach the quadrant where the platform was located, and the activity trajectory were recorded.

[0022] 2. Experimental results Water maze trajectory diagram Figure 1 (The left picture is the control group, and the right picture is the model group). From the trajectory diagram, it can be observed that the mice in the control group were able to reach the quadrant where the original platform was located many times, showing good learning and memory abilities; while the movement trajectories of the mice in the model group were relatively simple and did not show significant goal orientation, reflecting a significant decline in their spatial memory abilities.

[0023] The specific data of the water maze test are shown in Table 1: Table 1 Effects of Morris water maze spatial exploration test in mice ( , n=10)

[0024] Note: Compared with the control group, * P <0.05, ** P <0.01, *** P <0.001.

[0025] From the data in Table 1, we can see that the number of times the mice in the control group stayed in the quadrant where the platform was located was 1.75±0.96, while that in the model group was only 0.2±0.05, and the difference was significant (P<0.001), indicating that the model group mice had a significantly reduced memory ability for the location of the platform; the latency for the control group mice to reach the quadrant where the platform was located was 16.80±15.95 seconds, while that in the model group was significantly extended to 44.43±5.43 seconds (P<0.001), further verifying that the significant decline in the spatial exploration ability and memory ability of the mice in the model group was a manifestation of aging.

[0026] 2. Observation of constipation 1. Experimental methods The mice were allowed to eat freely, and the morphology, weight and number of feces produced by the mice for 24 hours were observed before sampling.

[0027] 2. Experimental results The specific experimental results are shown in Table 2: Table 2 24-hour fecal morphology, weight, and number of fecal particles in mice ( , n=10)

[0028] Note: Compared with the control group, * P <0.05, ** P <0.01, *** P <0.001.

[0029] From the data in Table 2, we can see that the feces of the mice in the control group were "large and wet", indicating a normal defecation state, while the feces of the mice in the model group were "small and dry", clearly showing symptoms of constipation, which indicated that the intestinal motility and defecation of the mice in the model group were significantly affected; compared with the mice in the control group, the amount of feces excreted by the mice in the model group was greatly reduced, further verifying their constipation state; the number of feces particles in the control group mice in 24 hours was 12.80±2.53 particles, while that in the model group was significantly reduced to 7.25±0.97 particles, and the difference was also highly significant (P<0.001), indicating that the defecation frequency of the mice in the model group decreased, consistent with the symptoms of constipation.

[0030] 3. Intestinal Transit Function Test 1. Experimental methods 30 min before sampling, mice in the control group and the model group were gavaged with 0.1 ml of ink per mouse. After the animals were killed by carbon dioxide overdose, the abdominal cavity was opened to separate the mesentery, and the intestinal tube from the pylorus at the upper end to the ileocecal part at the lower end was cut. The small intestine was gently pulled into a straight line, and the length of the intestinal tube was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was the "ink advancement length".

[0031] The small intestinal propulsion rate was calculated according to the following formula: Small intestine propulsion rate = ink propulsion length / total small intestine length × 100% 2. Experimental results The specific experimental results are shown in Table 3: Table 3 Mouse intestinal transit function test ( , n=10) Grouping Intestinal transit percentage (%) Control group 59.30±7.29 Model Group <![CDATA[48.68±9.74 ** ]]> Note: Compared with the control group, * P <0.05, ** P <0.01, *** P <0.001.

[0032] The intestinal transit percentage reflects the peristalsis ability of the small intestine. From the data in Table 3, it can be seen that the intestinal transit percentage of the mice in the model group was significantly lower than that in the control group, indicating that the intestinal transit function of the mice in the model group was significantly weakened, which is consistent with the symptoms of constipation.

[0033] 4. Histopathological examination of brain tissue and colon 1. Experimental methods The animals in the control group and the model group were anesthetized and fixed in position. The chest cavity was cut open with sterilized surgical instruments to expose the heart, and 50 mL of normal saline was taken for apical perfusion. After the blood cells were perfused cleanly, 50 mL of 4% paraformaldehyde solution was taken for perfusion tissue fixation. After the animal was perfused, the brain tissue and colon tissue were removed and immersed in 4% paraformaldehyde solution at 4°C overnight, followed by graded alcohol dehydration, paraffin embedding after xylene treatment, and 4 μm continuous sections were made using a microtome. Brain tissue sections were stained with HE, and colon tissue sections were stained with immunohistochemistry.

[0034] 2. Experimental results (1) HE-stained pathological sections of brain tissue Control group: The experimental results are as follows Figure 2 As shown (left picture, 200 times; right picture, 400 times), it can be seen from the picture that the brain tissue structure of the mice in the control group is basically normal. The neuronal cells in the cerebral cortex area in the field of view are arranged regularly and tightly, with normal cell structure, clear nuclei, rich cytoplasm, and no degeneration or necrosis. The tissue structure in the brain parenchyma is tight, and no edema or glial cell infiltration is observed.

[0035] Model group: Experimental results are as follows Figure 3As shown in the figure (left, 200 times; right, 400 times), it can be seen that the brain tissue structure of mice in the model group is slightly abnormal, the neurons in the cortical area of ​​the visual field are arranged more closely, a small number of neurons are degenerated, the nuclei are condensed, darkly stained, and basophilicity is enhanced; the tissue structure in the brain parenchyma is tight, and no edema and glial cell infiltration are observed.

[0036] (2) AQP3 protein immunohistochemistry sections of colon tissue AQP3 plays an important role in regulating colon water transport and is mainly distributed in intestinal mucosal epithelial cells. Yellow-brown color indicates positive expression after immunohistochemical staining. Figure 4-5 As shown, Figure 4 The AQP3 protein immunohistochemical sections of the colon tissue of the control group mice (left, 100 times; right, 400 times). Figure 5 The following are sections of AQP3 protein immunohistochemistry in the colon tissue of mice in the model group (left, 100 times; right, 400 times). It can be seen from the figures that compared with the control group, the mucus area in the colon tissue of mice in the model group was significantly reduced, the number of goblet cells was significantly reduced, the expression of AQP3 was significantly increased, the water content of feces was significantly reduced, and the colon absorbed water excessively.

[0037] 4. Changes in serum SOD, MDA, and GSH-Px levels 1. Experimental methods Before blood collection, mice were anesthetized and fixed to make the retroorbital venous plexus congested, and blood was collected by piercing the bottom of the orbit with a capillary glass tube. After blood collection, the pressure on the mouse was released and the capillary tube was pulled out, and the bleeding was stopped by pressing with a dry cotton ball. The collected blood was allowed to stand for 3 hours and then centrifuged at 3000 r / min for 15 minutes. The serum was aspirated and packaged, and the levels of SOD, MDA, and GSH-Px in the serum were detected according to the enzyme-linked immunosorbent assay kit method.

[0038] 2. Experimental results The specific experimental results are shown in Table 4: Table 4 Serum SOD, MDA, and GSH-Px levels in mice ( , n=10)

[0039] Note: Compared with the control group, * P <0.05, ** P <0.01, *** P <0.001.

[0040] Increased oxidative stress is a major feature of aging, which is reflected in the increase of malondialdehyde (MDA) content, the decrease of glutathione peroxidase (GSH-Px) content and the decrease of superoxide dismutase (SOD) activity. From the data in Table 3, it can be seen that compared with the control group mice, the serum content of antioxidant indicators SOD and GSH-Px decreased in the model group mice (P < 0.001), and the serum content of oxidative stress product MDA increased (P < 0.001), indicating that the antioxidant capacity of the model group mice decreased and the model group showed aging.

[0041] 5. Detection of Klotho protein expression in brain tissue 1. Experimental methods Mice were killed by overdose of carbon dioxide, and brain tissues were obtained. The homogenized supernatant was collected by centrifugation for protein quantitative detection, and the expression level of Klotho protein in brain tissues was detected by immunoblotting.

[0042] 2. Experimental results The specific experimental results are shown in Table 5: Table 5 Relative expression of Klotho protein in hippocampus ( , n=4) Grouping Klotho Control group 1.30±0.17 Model Group <![CDATA[0.78±0.15 ** ]]> Note: Compared with the control group, * P <0.05, ** P <0.01, *** P <0.001.

[0043] Klotho protein levels decrease with age. The expression and changes of Klotho in the hippocampus are closely related to aging-related cognitive impairment. Hippocampal neurons need Klotho to maintain health and normal function. Figure 6 The results of Klotho protein immunoblotting in the brain tissues of mice in the control group and the model group (left: control group, right: model group), combined with the data in Table 5 and Figure 6 It can be seen that compared with the control group, the Klotho protein level of the mice in the model group was significantly decreased (P < 0.01), indicating that the mice in the model group were in an aging state and may have cognitive impairment.

[0044] 6. Detection of c-Kit, SCF, and Klotho protein expression in colon tissue 1. Experimental methods Colon tissues of mice were obtained, homogenized and centrifuged to obtain the supernatant for protein quantitative detection. The expression levels of c-Kit, SCF and Klotho proteins in colon tissues were detected by immunoblotting.

[0045] 2. Experimental results The specific experimental results are shown in Table 6: Table 6 Relative expression of c-Kit, SCF and Klotho proteins in colon tissue ( , n=4)

[0046] Note: Compared with the control group, * P <0.05, ** P <0.01, *** P <0.001.

[0047] Abnormal interstitial cells of Cajal (ICC) is one of the pathogenesis of constipation. ICC exists between the enteric nervous system and smooth muscle, and plays an important role in the transmission of gastrointestinal electrical activity and the transduction of gastrointestinal neurotransmitter signals. c-kit is a transmembrane glycoprotein located on the cell membrane of ICC. It can be used as a specific marker of gastrointestinal ICC and can directly reflect the number and density of ICC. c-kit is a receptor for SCF, and the combination of the two can initiate a series of signal transduction. The SCF / c-kit pathway can regulate the growth, development and phenotypic maintenance of ICC, and plays a key role in the polarization of ICC cell membrane and pacemaker activity. In addition, the reduction of the expression of related proteins in the SCF / c-kit pathway is closely related to the reduction of ICC. Figure 7 The results of immunoblotting of c-Kit, SCF and Klotho proteins in the colon tissues of mice in the control group and model group (left: control group, right: model group), combined with the data in Table 6 and Figure 7 It can be seen that compared with the control group, the expression levels of c-kit and SCF proteins in the colon tissue of the model group mice were significantly reduced (P < 0.001, P < 0.05), indicating that the constipation model of the model group mice was successfully established. In addition, the expression level of Klotho protein in the colon tissue of the model group mice was significantly decreased, indicating that the colon tissue also showed aging, and the constipation and aging models of the model group mice were successfully established at the same time.

[0048] The above experimental results prove that the animal model of elderly constipation constructed in the present invention is significant and reliable in terms of aging and constipation characteristics, and provides a scientific basis for further studying the mechanism of elderly constipation and related drug screening.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model of elderly constipation, characterized in that: The animal model of elderly constipation was obtained by injecting D-galactose and gavaging loperamide into the experimental animals. The specific treatment method of injecting D-galactose is to subcutaneously inject D-galactose into the experimental animals starting from the first day of modeling, with a dosage of 130-170 mg / kg, once a day, for 4-6 weeks; The specific treatment method of intragastric administration of loperamide is to intragastricly administer loperamide to the experimental animals starting from the third week of modeling, with a dosage of 1-3 mg / kg, once a day, for 2-3 weeks.

2. The method for constructing an animal model of elderly constipation according to claim 1, characterized in that: The experimental animals are mice.

3. The method for constructing an animal model of elderly constipation according to claim 2, characterized in that: The mice are KM mice.

4. The method for constructing an animal model of elderly constipation according to claim 1, characterized in that: The dosage of D-galactose is 150 mg / kg.

5. The method for constructing an animal model of elderly constipation according to claim 4, characterized in that: The administration period of D-galactose lasted for 4 weeks.

6. The method for constructing an animal model of elderly constipation according to claim 1, characterized in that: The dosage of loperamide is 2 mg / kg.

7. The method for constructing an animal model of elderly constipation according to claim 6, characterized in that: The administration period of loperamide lasted for 2 weeks.

8. Use of the animal model of elderly constipation constructed by the method for constructing an animal model of elderly constipation according to any one of claims 1 to 7 in experimental research on diseases related to elderly constipation, or in the preparation and / or screening of drugs for treating elderly constipation.

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