Construction and evaluation method of mouse model with liver depression transforming into fire type depression

Abstract

The invention belongs to the technical field of animal model construction, and particularly relates to a construction and evaluation method of a mouse model with liver depression transforming into fire type depression. According to the method, uncertainty drinking water stimulation and intraperitoneal injection lipopolysaccharide are combined to construct the liver depression fire type depression model, and the technical problem that an existing depression model cannot accurately correspond to traditional Chinese medicine dialectical typing, namely, the depression model is poor in specificity is solved. According to the method disclosed by the invention, the liver depression transforming into fire type depression model is specifically evaluated by analyzing the difference of depression behavioristics, water intake, excrement water content and anal temperature indexes before and after modeling and adopting the change of serum endogenous metabolites and the change of liver pathological tissues. The invention provides the liver depression fire type depression mouse model which is strong in specificity, high in stability, good in reproducibility and easy to popularize; meanwhile, by adopting the evaluation method disclosed by the invention, the liver depression transforming into fire type depression mouse model can be more comprehensively and systematically evaluated, and the method has the advantages of sensitivity, high efficiency, rapidness and strong specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and particularly relates to a construction and evaluation method of a liver depression-fire type depression mouse model. Background Art

[0002] Depression is a mental illness caused by persistent sadness and loss of interest. With the accelerated pace of life and increasing stress, the incidence of depression has risen rapidly and has now become the second largest "killer" of humans after cancer. According to statistics from the World Health Organization, about 1 billion people around the world are suffering from mental disorders.

[0003] Chinese medicine and Western medicine have their own theories and methods for the treatment of depression. Western medicine believes that depression is a mental illness caused by an imbalance of neurotransmitters in the brain due to biological, psychological, and social factors, and it is often treated with drugs and psychological counseling. Chinese medicine believes that depression belongs to the category of "depression syndrome" and the disease is located in the liver, so the treatment is mainly to soothe and regulate the liver, and is treated through Chinese medicine and acupuncture.

[0004] Traditional Chinese medicine has a deep understanding and comprehension of the etiology and pathogenesis of depression. Most doctors agree that depression is caused by the liver. If the liver fails to release qi, liver qi will stagnate and easily turn into fire. If qi stagnates, dampness cannot be transformed, and if dampness gathers, phlegm will be generated, which leads to stagnation of qi and phlegm. If qi is stagnant for a long time, it can also cause blood stasis. Qi stagnation can cause various depressions, and various depressions can affect the qi of the five internal organs and cause qi stagnation. Through the syndrome differentiation and treatment of traditional Chinese medicine, depression can be divided into liver depression and spleen deficiency syndrome, liver depression turning into fire syndrome, phlegm-heat disturbing the mind syndrome, heart and spleen deficiency syndrome, heart and gallbladder qi deficiency syndrome, and heart and kidney yin deficiency syndrome. Among them, as the course of the disease deepens, qi stagnation will turn from liver qi stagnation to liver depression turning into fire syndrome, and long-term medication will damage body fluids. These factors will cause depression to evolve into liver depression turning into fire syndrome, so liver depression turning into fire syndrome accounts for a high proportion in common syndrome differentiation.

[0005] In summary, the animal model of depression caused by liver depression transforming into fire not only conforms to the manifestation of qi depression transforming into fire in TCM theory in terms of syndrome type, but also conforms to the category of depression in terms of disease, and can better simulate the clinical manifestations of patients with depression caused by liver depression transforming into fire. However, although there are many studies on animal models of depression in China, there are few studies on animal models of depression caused by liver depression transforming into fire. Summary of the invention

[0006] In view of this, in order to solve the technical problem that the existing depression model cannot accurately correspond to the dialectical classification of traditional Chinese medicine, that is, the depression model has poor specificity, the present invention provides a method for constructing and evaluating a mouse model of liver depression-fire type depression.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides a method for constructing a mouse model of depression caused by liver depression transforming into fire, comprising: after training the mice with regular water feeding for 7 days, subjecting them to uncertain drinking water stimulation for 14 days, and intraperitoneally injecting lipopolysaccharide into the mice starting from the 14th day, and continuing the injection for 7 days, thereby obtaining a mouse model of depression caused by liver depression transforming into fire.

[0009] Furthermore, the specific steps are as follows: the mice are trained to be fed with water on a regular basis, with the feeding time being once in the morning and once in the evening, each time for 10 minutes, for 1 week; after the training, uncertain drinking water stimulation is given during the aforementioned two feeding time periods, maintained once / day or twice / day, for 2 weeks; starting from the 14th day, the mice are intraperitoneally injected with lipopolysaccharide for 1 week.

[0010] Furthermore, the concentration of LPS was 0.1 mg / mL, calculated as 0.5 mg / kg per intraperitoneal injection per mouse.

[0011] Furthermore, the body weight of the mice was 18-22 g.

[0012] On the other hand, the present invention provides an evaluation method for the liver-stagnation-fire type depression mouse model constructed by the method as described above, comprising: performing pharmacodynamic evaluation, serum metabolomics evaluation and pathological evaluation on the liver-stagnation-fire type depression mouse model, wherein the pharmacodynamic evaluation includes sugar water preference, forced swimming, open field, water intake, fecal water content and rectal temperature tests, the serum metabolomics evaluation includes the test of changes in the content of endogenous metabolites in serum, and the pathological evaluation includes observation of liver pathological tissue.

[0013] Furthermore, the endogenous metabolites in serum include 9 biomarkers, namely: Choline, Hypoxanthine, Indole-3-acetaldehyde, L-Isoleucine, L-Tyrosine, Pyruvicacid, Xanthine, DL-Lacticacid, and Uricacid.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention provides an animal model of liver-stagnation-fire type depression with strong specificity, high stability, good reproducibility, and easy promotion, which provides an experimental basis for prescription screening, mechanism research, and new drug development for the treatment of liver-stagnation-fire type depression.

[0016] (2) Compared with previous model evaluations, the method of the present invention more comprehensively and systematically reflects the external differences in symptom behavior and the dynamic outline of the body in the liver-stagnation-fire type depression model mice, and also reflects the rationality and scientificity of model replication. It can provide a reliable evaluation method for the liver-stagnation-fire type depression model for new drug development and pharmacological research, and has the advantages of high efficiency, rapidity, and strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A schematic diagram of a model construction process according to an embodiment of the present invention;

[0018] Figure 2 This is a graph showing the sugar water preference rate of mice in the blank control group and the model group of an embodiment of the present invention;

[0019] Figure 3 This is a forced swimming diagram of mice in the blank control group and the model group of an embodiment of the present invention;

[0020] Figure 4 This is a graph showing the number of open field crossings of mice in the blank control group and the model group according to an embodiment of the present invention;

[0021] Figure 5 This is a graph showing the water intake of mice in the blank control group and the model group of an embodiment of the present invention;

[0022] Figure 6 This is a diagram showing the water content of feces of mice in the blank control group and the model group of an embodiment of the present invention;

[0023] Figure 7 The rectal temperature graphs of mice in the blank control group and the model group of the embodiment of the present invention;

[0024] Figure 8 This is a principal component analysis score diagram of the blank control group and the model group mice in the embodiment of the present invention;

[0025] Fig. 9 It is an orthogonal partial least squares-discriminant analysis score diagram of the blank control group and the model group of the embodiment of the present invention;

[0026] Fig.10 It is an orthogonal partial least squares-discriminant analysis loading diagram of the blank control group and the model group of the embodiment of the present invention.

[0027] Fig.11 Pathological diagram of liver tissue of the blank control group of the embodiment of the present invention.

[0028] Fig.12 This is a liver tissue pathology diagram of the model group of an embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0030] Example

[0031] like Figure 1 As shown, a method for constructing a mouse model of liver depression-fire type depression comprises the following steps:

[0032] The mice were given water for 7 days, that is, they were given water for 10 minutes every morning and evening, and the water bottles were removed at other times. After the training, the stress experiment was started. In the above two time periods, the mice were given uncertain drinking stimulation according to the experimental procedures. The stimulation was given irregularly, once a day or twice a day, for 2 weeks. From the 14th day, the mice were intraperitoneally injected with 0.5 mg / kg of lipopolysaccharide (LPS), and the injection was continued for 1 week to obtain the liver depression type depression mouse model.

[0033] A method for evaluating a mouse model of liver depression-fire transformation depression, comprising the following steps:

[0034] 1. Pharmacodynamic evaluation: After the mouse model was constructed, six indicators including sugar water preference rate, forced swimming, open field, water intake, fecal water content and rectal temperature were tested and recorded. The experimental data were statistically analyzed using SPSS, graphed using GraphPadPrism, and compared between multiple groups using t. P<0.05 indicated a significant difference, P<0.01 indicated a highly significant difference, and P<0.001 indicated an extremely significant difference. The details are as follows:

[0035] 1) Sugar water preference experiment: Before the experiment, each mouse was given a 1% sucrose solution and daily drinking water for the sugar water preference training experiment. Two water bottles were placed in the cage of each mouse (single cage). First, two bottles of 1% sucrose solution were given within the first 24 hours. Secondly, one bottle of 1% sucrose solution and another bottle of daily drinking water were given within the second 24 hours to train all mice. After the training, all mice were fasted and deprived of water for 12 hours, and then the sugar water consumption was measured within 12 hours. During the test, the mice were free to choose 1% sucrose solution or water. A baseline measurement of the sugar water preference rate was performed on day 0, and a sugar water preference experiment was performed on day 21. The weight of the water bottle before and after the experiment was measured to calculate the sugar water preference rate of the mouse. Sugar water preference rate = weight of consumed sucrose solution / (weight of consumed sucrose solution + weight of consumed daily drinking water).

[0036] 2) Forced swimming: Place the mouse in an open container with a diameter of 14 cm and a height of 20 cm, with a water column height of 12 cm. Control the water temperature inside the container to 25±1°C, and force the mouse to swim for 6 minutes. Three different observers record the time the mouse does not swim in the last 4 minutes, and take the average of the time recorded by the three observers. After 6 minutes, take the mouse out of the container, wipe it dry, and put it back in the cage. The time the mouse keeps its head suspended above the water without obvious struggle is defined as the immobility time.

[0037] 3) Open field test: The bottom of the open field test apparatus (100cm×100cm×40cm) is divided into 25 squares and kept in a relatively quiet environment (≤60 decibels). First, place the mice in the open field test apparatus for 1 minute to adapt to the environment. Observe the number of grids crossed by the mice (at least 3 paws crossed the grid lines) and the number of upright times (both forelimbs were at least 1 cm from the ground). After acclimating to the environment for 1 minute, record the number of grids crossed by the mice within 4 minutes.

[0038] 4) Water intake: The mice were deprived of water and food for 6 hours before the experiment, and then their water intake within 12 hours was tested. The water intake measurement was used to assess whether the mice had dry mouth.

[0039] 5) Fecal water content: Collect mouse feces and measure the fecal water content. Fecal water content = (AB) / A×100%, A is the weight of fresh feces collected, and B is the weight of fresh feces after drying at 60°C for 24 hours. By measuring the fecal water content of each group of mice, it is determined whether the mice have dry stools.

[0040] 6) Rectal temperature: Place the mouse on a flat surface, gently grasp the mouse's tail with one hand to prevent the mouse from moving, and use the other hand to insert a thermometer coated with an appropriate amount of sterile lubricant into the mouse's anus to a depth of about 1-2 cm. Keep the thermometer in the anus for about 30 seconds until the digital display on the thermometer stabilizes. Take out the thermometer and record the displayed number, which is the mouse's rectal temperature.

[0041] The results of the six pharmacodynamic indicators are:

[0042] 1) Sugar water preference experiment: Compared with the blank control group, the sugar water preference rate of mice in the model group was significantly reduced (p<0.001). Figure 2 ;

[0043] 2) Forced swimming: Compared with the blank control group, the immobility time of mice in the model group was significantly prolonged (p<0.01). Figure 3 ;

[0044] 3) Open field test: Compared with the blank control group, the number of grids crossed by mice in the model group was significantly reduced (p<0.001). Figure 4 ;

[0045] 4) Water intake: Compared with the blank control group, the water intake of mice in the model group was significantly increased (p<0.001). Figure 5 ;

[0046] 5) Fecal water content: Compared with the blank control group, the fecal water content of mice in the model group was significantly reduced (p<0.01). Figure 6 ;

[0047] 6) Rectal temperature: Compared with the blank control group, the rectal temperature of mice in the model group was significantly increased (p<0.01). Figure 7 ;

[0048] 2. Serum metabolomics evaluation: After the mouse model was constructed, the serum of the blank control group and the model group mice was collected for mass spectrometry analysis to obtain the mass spectra of the blank control group and the model group mice; the changes in the endogenous metabolites in the serum of the blank control group and the model group mice were analyzed. The details are as follows:

[0049] 1) Multivariate statistical analysis methods were used to characterize the metabolic profiles, and principal component analysis was used to perform pattern recognition on the data to examine the separation of the data profiles of each group. The specific method was to collect the serum of the blank control group and the model group mice for mass spectrometry analysis after the mouse model was constructed, and obtain the mass spectra of the blank control group and the model group mice; then, multivariate statistical analysis was performed on the integral data matrix of the mass spectra of the blank control group and the model group mice to obtain the profiles of the blank control group and the model group mice; Figure 8 (The horizontal and vertical axes represent the first and second principal components, respectively. C: blank control group; M: model group) As shown: The model group was significantly separated from the blank control group, indicating that the metabolic regulatory network had changed significantly, proving that the liver depression-fire type depression model was successfully replicated. The changes in endogenous metabolites in the serum of mice in the blank control group and the model group were evaluated.

[0050] 2) Based on PCA, the orthogonal partial least squares-discriminant analysis method was used to further analyze the serum of the blank control group and the model group mice to obtain the serum profiles of the blank control group and the model group mice. The results are shown in Fig. 9 (The horizontal and vertical axes represent the first and second principal components, respectively). Fig. 9 It can be seen that the separation effect of the two groups on the first axis of the principal component is obvious. Then the results of variable loading are described through the loading diagram. Fig.10:(The horizontal and vertical axes represent the first principal component and the correlation coefficient. The larger the coefficient, the greater the contribution to the grouping). Using S-plot combined with VIP value>1 and statistical p<0.05, potential biomarkers were obtained, and variables with significant differences in content changes were found from the blank control group and the model group. The metabolic pathways involved in these variables may lead to the formation of the liver-depression-fire type depression model. Then, through literature research, 9 biomarkers related to liver-depression-fire type depression were screened out to evaluate whether the liver-depression-fire type depression mouse model was successfully constructed. The 9 biomarkers are: Choline, Hypoxanthine, Indole-3-acetaldehyde, L-Isoleucine, L-Tyrosine, Pyruvicacid, Xanthine, DL-Lacticacid, Uricacid.

[0051] 3) The changes in the levels of the above 9 biomarkers were analyzed. Compared with the blank control group mice, the changes in the levels of the 9 biomarkers in the model group were as follows:

[0052] The mean integrated area of ​​Choline decreased from 0.0028326±0.0006850 in normal mice to 0.0021169±0.0003672, p<0.05;

[0053] The mean integrated area of ​​Hypoxanthine decreased from 0.0009194±0.0004371 in normal mice to 0.0000898±0.0001028, p<0.01;

[0054] The mean integrated area of ​​indole-3-acetaldehyde decreased from 0.0001320±0.0000363 in normal mice to 0.0000107±0.0000065, p<0.01;

[0055] The mean integrated area of ​​L-Isoleucine decreased from 0.0095474±0.0015559 in normal mice to 0.0079821±0.0010763, p<0.05;

[0056] The mean integrated area of ​​L-Tyrosine decreased from 0.0047992±0.0008879 in normal mice to 0.0026980±0.0006216, p<0.01;

[0057] The mean integrated area of ​​pyruvicacid decreased from 0.0005750±0.0000924 in normal mice to 0.0002432±0.0002244, p<0.01;

[0058] The mean integrated area of ​​Xanthine decreased from 0.0001338±0.0001010 in normal mice to 0.0000032±0.0000030, p<0.01;

[0059] The mean integrated area of ​​DL-LacticAcid decreased from 0.0278691±0.0026154 in normal mice to 0.0085578±0.0051526, p<0.01;

[0060] The mean integrated area of ​​uricacid increased from 0.0003369±0.0000794 in normal mice to 0.0004619±0.0000925, p<0.05;

[0061] 3. Pathological evaluation: Hematoxylin-eosin (H&E) staining: The liver was removed after dissection, and the liver tissue was fixed in 10% paraformaldehyde solution. The liver tissue was sliced ​​in 5-6 μm paraffin, then stained with H&E, and the liver tissue was pathologically evaluated by optical microscopy. The details are as follows:

[0062] The liver sections were dewaxed with xylene, then hydrated with ethanol of various concentrations, and then stained with hematoxylin for 4 minutes. The floating color was washed off with running double distilled water, and then differentiated with differentiation solution for 3 minutes until the cell nucleus was purple-blue. The sections were washed with tap water for 4 minutes for anti-blueing, and then stained with eosin stain for 2 minutes. Finally, they were washed with double distilled water, and quickly dehydrated and transparent as follows: 75% ethanol for 30 seconds, 85% ethanol for 30 seconds, 95% ethanol I for 30 seconds, 95% ethanol II for 30 seconds, anhydrous ethanol I for 30 seconds, anhydrous ethanol II for 1 minute, xylene I for 1 minute, and xylene II for 2 minutes. Neutral gum was then added to the sections and coverslips were placed for sealing.

[0063] Compared with the blank control group, the arrangement of liver cell cords in the model group was slightly scattered, the radial pattern was not obvious, and inflammatory cell infiltration was present. Figure 11 to Figure 12 , indicating that the mouse model of liver depression transforming into fire type depression was successfully established.

[0064] In summary, if the six indicators of the blank control group and the model group mice show significant differences, the endogenous metabolic profiles show significant separation, involving 9 metabolites, and inflammatory lesions in the liver, it indicates that the liver stagnation and fire type depression mouse model is successfully established. By comparison, it can be seen that the evaluation method of the present invention can be used to more comprehensively and systematically evaluate the liver stagnation and fire type depression mouse model, which has the advantages of sensitivity, efficiency, rapidity and strong specificity.

[0065] The pathological changes in the body of mice with liver depression can lead to significant differences in symptom indicators, abnormal endogenous metabolic profiles, and inflammatory lesions in the liver. The significant changes in these symptom indicators, metabolites, and liver pathological tissues between the blank control group and the model group can be used to comprehensively evaluate the transition of mice from a normal state to a liver depression state.

[0066] In the technical solution of the present invention, 6 kinds of efficacy indices (3 antidepressant indices and 3 hepatitis hyperactivity indices) are used for pharmacodynamic evaluation. By recording the behavioral differences between the blank control group and the model group mice, and using SPSS for data analysis, the model of liver depression and fire type depression is evaluated from the pharmacodynamic aspect. The above 6 indicators are the basis for establishing the liver depression and fire type depression model, which can provide a lot of useful information for researchers and help to establish a more reliable animal model of mental and neurological related diseases. At the same time, the metabolomics technology is used to analyze the changes in endogenous metabolites in the serum of the blank control group and the model group mice, obtain the metabolic profile map, use Compound Discover (CD) software to process all the mass spectrograms to obtain the integral data, and combine the content of 9 biomarkers. Statistical analysis was performed, and it was found that the changes in the integral mean values ​​of 9 biomarkers in the serum of the blank control group and the model group mice reflected the change trend of the serum metabolite content of the liver depression and fire type depression mice to a certain extent, thereby evaluating the model of liver depression and fire type depression in a targeted manner. Metabolites are at the terminal of biological organisms. Minor changes in upstream genes and proteins will be amplified in metabolites. Therefore, metabolites can more sensitively characterize life phenomena and can faithfully reflect the microscopic changes in the regulation of the metabolic network of the body caused by external intervention. H&E staining can more intuitively show the physiological and pathological changes of tissues. By observing the structural morphology of tissues, we can improve our understanding of the disease. So far, the above method has not been used to construct a depression model of liver depression and fire transformation, and the above six indicators, metabolomics methods and H&E staining have not been used to evaluate the depression model of liver depression and fire transformation.

[0067] The above description is only for better explaining the embodiments of the present invention, and is not intended to limit the present invention. Any modification or equivalent substitution that does not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims

1. A method for constructing a mouse model of liver depression-fire type depression, characterized in that: include: After 7 days of regular water feeding training, the mice were subjected to 14 days of uncertain water drinking stimulation. From the 14th day, the mice were intraperitoneally injected with lipopolysaccharide for 7 consecutive days to obtain a mouse model of liver depression and fire transformation type depression.

2. The method for constructing a mouse model of liver stagnation and fire-transforming depression according to claim 1, characterized in that: The specific steps are as follows: mice are trained to drink water at regular intervals, once in the morning and once in the evening, for 10 minutes each time, for one week; after the training, mice are given uncertain drinking stimulation during the two drinking time periods, once or twice a day, for two weeks; The mice were intraperitoneally injected with lipopolysaccharide starting from day 14 for 1 week.

3. The method for constructing a mouse model of liver depression-fire type depression according to claim 1, characterized in that: The concentration of lipopolysaccharide was 0.1 mg / mL, calculated as 0.5 mg / kg per intraperitoneal injection per mouse.

4. The method for constructing a mouse model of liver depression-fire type depression according to claim 1, characterized in that: The body weight of mice was 18-22 g.

5. The evaluation method of the mouse model of liver depression-fire transformation type depression constructed by the method according to any one of claims 1 to 4, characterized in that: include: The pharmacodynamic evaluation, serum metabolomics evaluation and pathological evaluation were performed on the mouse model of liver depression and fire transformation type depression. The pharmacodynamic evaluation included sugar water preference, forced swimming, open field, water intake, fecal water content and rectal temperature tests; the serum metabolomics evaluation included the test of changes in the content of endogenous metabolites in serum; and the pathological evaluation included observation of liver pathological tissue.

6. The evaluation method of the mouse model of liver stagnation and fire transformation depression according to claim 5, characterized in that: Endogenous metabolites in serum include 9 biomarkers, namely: choline, hypoxanthine, indole-3-acetaldehyde, L-isoleucine, L-tyrosine, pyruvate, xanthine, DL-lactic acid, and uric acid.

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