Method for constructing diabetic encephalopathy mouse model through combined induction of cadmium and fructose

Abstract

The invention discloses a method for constructing a diabetic encephalopathy mouse model through combined induction of cadmium and fructose. According to the method, the diabetes contamination dosage is promoted by adjusting cadmium; the condition that the fasting blood glucose of the mouse is greater than or equal to 11.1 mmol / L, urine glucose inspection is positive, and mine field experiments, new object recognition experiments and water maze mouse learning and memory experiments verify that the cognitive dysfunction of the mouse successfully induces the diabetic encephalopathy. According to the method, through combined induction of the fructose solution and cadmium, the modeling condition is met, and the cost is reduced. The model manufacturing process has little harm to animal bodies and conforms to the international and domestic animal welfare principle.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical experimental animal model construction, in particular to a method for constructing a mouse model of diabetic encephalopathy induced by cadmium and fructose and application thereof. BACKGROUND

[0002] Diabetic encephalopathy (DE) is a central nervous system complication of diabetes, which is manifested as cognitive dysfunction, learning and memory impairment, and can progress to dementia in severe cases. Its pathological features include neuronal damage, brain atrophy, neuroinflammation and blood-brain barrier destruction. DE has become an important dangerous disease threatening human health and life quality safety in the 21st century, and with the rising incidence of diabetes, diabetic encephalopathy has become an important cause of cognitive impairment after Alzheimer's disease and vascular dementia. However, there is currently a lack of specific diagnostic markers and targeted treatment methods in clinical practice. Therefore, the establishment of an ideal diabetic encephalopathy model plays a key role in studying the pathogenesis of this disease, evaluating drug efficacy and developing therapeutic drugs.

[0003] The existing preparation of diabetic encephalopathy models is usually induced by diet and drug reagents or single drug, but the existing models still have significant limitations in terms of pathological process simulation, clinical consistency and translation value. How to better simulate the human diabetic pathogenesis process and avoid the instability of drug injection and the low modeling rate are the main aspects that limit its application. SUMMARY

[0004] The present application aims to solve the technical problems of the prior art and provides a method for constructing a diabetic encephalopathy mouse model induced by cadmium and fructose. The method uses a combination of drinking fructose solution and cadmium intraperitoneal injection to induce a diabetic encephalopathy model, which is different from the traditional STZ intraperitoneal injection method for establishing a diabetic encephalopathy model and is more consistent with reality.

[0005] To solve the above technical problems, the present application discloses a method for constructing a diabetic encephalopathy mouse model induced by cadmium and fructose, comprising the following steps:

[0006] (1) Obtain male third-generation Kunming mice by self-breeding, wherein the first generation is normal mice, which are adaptively fed for two weeks, and then bred and fed under the condition of free feeding of ordinary feed and fructose aqueous solution as drinking water until male third-generation Kunming mice are obtained;

[0007] (2) Select male third-generation Kunming mice with a body weight of 30±2g, free feeding of ordinary feed, and fructose solution as drinking water, and intraperitoneally inject CdCl2 solution 3 times a week, and continuously feed to induce diabetic encephalopathy symptoms in mice.

[0008] The adaptive feeding condition is that the animals are raised in an animal room under the conditions of 12h light / 12h dark cycle, 40-60% humidity, 23±2°C environment, and adaptive feeding for 2 weeks.

[0009] The concentration of the CdCl2 solution is 1.3 mg / kg.

[0010] The concentration of the fructose solution is 10%.

[0011] The diabetic encephalopathy symptoms appear after 6-7 weeks of continuous feeding.

[0012] The application further provides application of the above-mentioned mouse model of diabetic encephalopathy in studying the mechanism of diabetic encephalopathy and in screening drugs for treating diabetic encephalopathy.

[0013] Beneficial effects: the method of the application simulates the pathogenesis process of the human metabolic system, and the prepared animal model has typical symptoms such as central nervous system symptoms, and is suitable for mechanism research and screening and application of treatment methods for the disease. The induction by drinking a fructose solution can highly simulate the human pathogenesis process, and the injection of CdCl2 can also prove that the risk factor of heavy metals existing in life can also cause the occurrence of diabetic encephalopathy, and the result has high reliability, and the problems of adverse consequences and low film formation rate caused by injection of the induction drug are excluded. The application adjusts the cadmium-induced diabetic dose, and the diabetic encephalopathy is successfully induced by verifying the cognitive dysfunction of mice by the fasting blood glucose of mice being equal to or greater than 11.1 mmol / L, the positive urine sugar test, the mine field experiment, the new object recognition experiment, the water maze mouse learning and memory experiment. In addition, the combined induction of the fructose solution and cadmium not only meets the modeling conditions, but also reduces the cost. The model preparation process of the application causes less harm to the animal body, and meets the international and domestic animal welfare principles. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The figure is the fasting blood glucose change curve of the two groups of experimental mice in the examples, and *: compared with the control group (P<0.05), **: compared with the control group (P<0.01);

[0015] Figure 2 and Figure 3 The figure is the water maze experiment result of the two groups of experimental mice;

[0016] Figure 4 The figure is the mine field experiment-center area residence time (s / min) of the two groups of experimental mice;

[0017] Figure 5 The figure is the new object recognition experiment of the two groups of experimental mice;

[0018] Figure 6 The figure is the blood glucose change over time with different concentrations of drug doses;

[0019] Figure 7 The dose of different concentrations is modeled as a percentage. DETAILED DESCRIPTION

[0020] The above and / or other aspects of the present application will become more apparent by describing in detail the preferred embodiments thereof with reference to the attached drawings.

[0021] In the following examples, the CdCl2 concentration is calculated as a percentage of molecules, and the concentration is 1.3-1.5 mg / kg, and the concentration of fructose is 10%. The validation method of the animal model includes fasting blood glucose, urine glucose, and behavioral verification.

[0022] Example 1

[0023] Test materials: self-bred male third-generation Kunming mice, the steps are as follows: the first generation is normal mice, purchased from Sperof Bio-technology Co., Ltd. (Beijing), and the test animal batch number is SCXK (Jing) 2019-0010. The first generation of mice is adaptively fed for 2 weeks under the following conditions: 12 h light / 12 h dark cycle, 40-60% humidity, and 23±2℃ environment. Subsequently, the mice are bred under the conditions of free feeding of ordinary feed and 10% fructose aqueous solution as drinking water until male third-generation Kunming mice are obtained, and the mice are continuously fed, and 65 male third-generation Kunming mice with a body weight of 30±2 g are selected as a model group, and 17 normal mice are taken as a control group. Among them, the mice in the model group are fed by freely taking ordinary feed and 10% fructose solution as drinking water, and 1.3 mg / kg CdCl2 solution is injected intraperitoneally 3 times a week, and the amount of the administered drug is matched according to the body weight range. The mice in the control group drink tap water and freely take ordinary feed. During the administration, the fasting blood glucose in the tail vein is detected once every 2 weeks, the fasting blood glucose is stably increased, and the urine is collected in the metabolic cage after 6 weeks. After 6 weeks of administration, the brain function is verified by behavioral experiments to determine whether the pathological changes occur. After the data is obtained, the results are analyzed.

[0024] Figure 1 The fasting blood glucose change curve of the two groups of experimental mice is shown in the graph, and compared with the control group (Control), the blood glucose value of the model group (Model) is significantly increased (P<0.01), the fasting blood glucose is ≥11.1 mmol / L, and there is a statistically significant difference. Compared with the control group, the urine glucose index of the mice in the model group is positive, as shown in Table 1.

[0025] Table 1 Analysis of urine index

[0026]

[0027] The water maze experiment is used to verify the spatial learning and memory ability of the mice, Figure 2 and Figure 3The water maze test results of the two groups of experimental mice are shown in the figure. Compared with the control group, the platform stay times of the mice in the model group were significantly reduced (P<0.01); compared with the control group, the platform stay times of the mice in the model group were significantly shortened (P<0.01), indicating that the spatial learning and memory ability of the mice in the model group was impaired.

[0028] The mine field experiment is to verify the anxiety and motor ability of the mice, Figure 4 The mine field experiment results of the two groups of experimental mice are shown in the figure. Compared with the control group, the center area stay time of the mice in the model group was significantly decreased (P<0.01), and the anxiety and motor ability of the mice in the model group was impaired.

[0029] The new object experiment is to verify the cognitive and recognition memory ability of the mice, Figure 5 The new object recognition experiment results are shown in the figure. As Figure 5 shown, compared with the control group, the cognitive index of the mice in the model group was significantly reduced (P<0.01). The model group mice had impaired cognitive function.

[0030] The test results show that: compared with the control group, the fasting blood glucose of the model group is significantly higher, and the urine sugar detection of the model group is positive. After observing the behavior experiment, the model group has obvious brain recognition dysfunction.

[0031] Example 2 Determination of the optimal drug concentration of the cadmium-promoted diabetic encephalopathy model.

[0032] In this study, different concentrations of CdCl2 solution were used to determine the optimal drug concentration for constructing the cadmium-promoted diabetic encephalopathy model. Figure 6 The results show that the blood glucose concentration of the 1.5mg / kg group rises slowly, and shows a downward trend after the 2nd week, and a significant decrease in blood glucose after the 4th week; the blood glucose of the 1.4mg / kg group decreases after the 4th week; the blood glucose concentration of the 1.3mg / kg group shows a gradual upward trend.

[0033] Table 2 Number of models at different drug concentrations

[0034]

[0035] At the same time, Table 2 and Figure 7 Data show that the model rate of the 1.3mg / kg group is the highest, reaching 60%.

[0036] Based on the above results, the study concludes that the optimal CdCl2 solution concentration for constructing the cadmium-promoted diabetic encephalopathy model is 1.3mg / kg.

[0037] The application provides a method for constructing a mouse model of diabetic encephalopathy by using cadmium and fructose in combination, and there are many methods and approaches for realizing the technical scheme, and the above description is only the preferred embodiment of the application, and it should be pointed out that, for ordinary skilled in the art, some improvements and refinements can be made without departing from the principle of the application, and these improvements and refinements should also be regarded as the protection scope of the application. The components not explicitly described in the embodiment can be realized by using the prior art.

Claims

1. A method for constructing a mouse model of diabetic encephalopathy induced by cadmium combined with fructose, characterized in that, It comprises the following steps: (1) obtaining male third generation Kunming mice by self-breeding, wherein the first generation is normal mice, which are adaptively bred for two weeks, and then are bred and reproduced under the condition of free feeding of ordinary feed and fruit sugar solution as drinking water until male third generation Kunming mice are obtained; (2) screening male third generation Kunming mice with a body weight of 30±2 g, free feeding of ordinary feed, fruit sugar solution as drinking water, and intraperitoneal injection of CdCl2 solution three times a week, to continuously feed and induce the mice to produce symptoms of diabetic encephalopathy.

2. The method of claim 1, wherein, The adaptive breeding condition is that the animals are bred in an animal room under the condition of 12 h light and then 12 h dark cycle, 40-60% humidity, and 23±2℃ environment.

3. The method of claim 1, wherein, The concentration of the CdCl2 solution is 1.3 mg / kg.

4. The method of claim 1, wherein, The concentration of the fruit sugar solution is 10%.

5. The method of claim 1, wherein, The symptoms of diabetic encephalopathy appear after 6-7 weeks of continuous feeding.

6. Application of the diabetic encephalopathy mouse model prepared by the method of any one of claims 1-5 in the study of the mechanism of diabetic encephalopathy.

7. Application of the diabetic encephalopathy mouse model prepared by the method of any one of claims 1-5 in the screening of drugs for treating diabetic encephalopathy.

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