CN114391507B - Modeling and evaluation method of mice with depression liver depression transforming into fire syndrome - Google Patents
Modeling and evaluation method of mice with depression liver depression transforming into fire syndrome Download PDFInfo
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Abstract
The invention discloses a modeling method of a mouse with depression liver depression transforming into fire, relating to the field of animal model construction methods, wherein the modeling method comprises the following steps: the healthy mice are subjected to continuous sleep deprivation stimulation for 2-30 days, and light irradiation daily stimulation and alternate-day grain stimulation are given daily at the same time, so that the obtained model mice have symptomatic expression of depression, and the model is judged to have the characteristic of liver depression transforming into fire by the traditional Chinese medicine syndrome diagnosis method. The depression mouse model established by the invention not only has the clinical liver depression fire-transforming and depression states on general signs, but also accords with the characteristics of liver depression fire-transforming of traditional Chinese medicine through the comprehensive evaluation of the weight, the total distance of the activity in the open field, the time, the frequency and the average speed of entering the center of the open field, the frequency and the time of entering the open arm in the elevated plus maze, the water intake, the forced swimming immobility time and the indexes of 5-HT, GABA, DA and ACH, and can be used for further research on the pathogenesis and treatment of depression liver depression fire-transforming syndrome.
Description
Technical Field
The invention relates to the technical field of animal model construction, in particular to a modeling method of a mouse with depression liver depression fire syndrome.
Background
The depression is a serious mental disease, which is mainly manifested by mental abnormalities such as depressed mood, interest decline, thought retardation and the like, and the world health organization predicts that the depression will rise to the first place of global disease burden by 2030.
At present, only 20-30% of depression patients are effective for drug treatment, and some patients are stopped taking the drug due to intolerable adverse reactions. Therefore, the search for effective and safe antidepressants is urgent. The pathogenesis of depression is complex, and the specific mechanism is not completely clear. Although some limitations have been overcome by using postmortem brain samples from human subjects, these samples are often influenced by factors such as age, sex, medication history, postmortem tissue collection, and the like. Therefore, a reasonable depression animal model is very important for researching the pathogenesis of depression, screening drugs and guiding clinical reasonable medication.
To date, there is an extreme lack of animal models for screening and searching for antidepressant drugs, and the main modeling approaches are: the Chronic Unpredictable Mild Stimulation (CUMS) molding is carried out, but the cycle is long, the technical requirement is high, and the required equipment is complex; the death rate of the drug molding animal is high and certain adverse reaction exists; the preparation process of the operation molding is complex; genetic modeling is expensive and has limited practical application.
The principle of traditional medicine "treatment based on syndrome differentiation" emphasizes the individual differences of diseases. The transformation of liver depression into fire is one of the traditional Chinese medicine syndromes of depression, the liver belongs to wood in five elements, and the liver of a human body is as spring ascending qi and has the characteristics of smoothness, smoothness and dredging. For instance, if liver qi stagnates and does not dredge qi, according to the theory of five elements, wood will generate fire, so it is called liver qi stagnation transforming into fire. It often causes the phenomena of poor appetite, yellow and red urine, constipation, red tongue, wiry and rapid, etc., which are consistent with the external symptoms of our model.
The method for treating depression by traditional Chinese medicine is unique, and the research on a disease model combining the symptoms and signs of depression is rare at present. So far, animal models related to different traditional Chinese medicine syndrome types of depression mainly comprise animal models for establishing traditional Chinese medicine syndrome on the basis of western medicine disease models, and on the basis of diseases, the original western medicine disease animal models are possibly changed by artificially applying intervention, and the result hardly meets the requirements of the animal models on the disease and the syndrome; many animal models of the "syndrome" of traditional Chinese medicine are caused by adverse reactions generated by western medicines.
The disease animal model with good credibility and validity is an important guarantee that research results can be smoothly transformed from a laboratory to clinic. The animal model of depression is constructed according to etiology and pathology in western medicine research, the credibility and the validity are high, but the individual difference of animals is ignored, the construction of a disease model is emphasized in the modeling process, the construction of a syndrome type is weakened, and the disease and the animal model are relatively ideal animal models at present.
Disclosure of Invention
The invention aims to at least solve one of the technical problems in the prior art and provides a modeling method for a mouse with depression and liver depression transforming into fire.
The technical solution of the invention is as follows:
a modeling method for mice with depression and liver depression transforming into fire comprises selecting healthy mice, subjecting the healthy mice to continuous sleep deprivation stimulation for 2-30 days, and simultaneously giving stimulation of light irradiation for 1-24 h/day and stimulation of grains every other day.
Further, the sleep deprivation stimulation is performed in a sleep deprivation instrument configured to rotate intermittently.
Furthermore, the rotation speed of the sleep deprivation instrument is 2-50 r/min, the rotation time is 3-24 hours/day, and the interval time of intermittent rotation is 1-10 min/time.
The invention also discloses an evaluation method of the mouse model obtained by the modeling method, which comprises the following steps:
the method comprises the following steps: constructing a depression model, and judging whether the model of the depression animal is successfully made according to an open field experiment, an elevated plus maze experiment, a tail suspension experiment and a forced swimming experiment;
step two: and (3) carrying out a traditional Chinese medicine syndrome diagnosis method on the mice successfully modeled to judge whether the depressed animals have the syndrome of liver depression transforming into fire or not.
Further, in the first step, urine and body weight of the mouse are used as judgment indexes of the overall physiological state in the depression model building process.
Further, in the second step, the judgment indexes in the method for diagnosing Chinese medicine symptoms comprise: the overall behavioral indexes for evaluating the liver depression state of the model, the overall physiological indexes for evaluating the qi depression fire-transforming state of the model and the in-vivo biochemical indexes for evaluating the liver depression fire-transforming state of the model.
Further, the overall behavioral indicator is: forced swimming immobility time of the mouse, total distance of activities in the open field, time, frequency and average speed of entering the center of the open field, and frequency and time of entering the open arm in the elevated plus maze.
Further, the overall physiological index includes: tongue picture, paw color and water intake of mice.
Further, the in vivo biochemical indicators include: the 5-HT (5-hydroxytryptamine), GABA (gamma-aminobutyric acid) and DA (dopamine) contents in the blood serum of the mouse and the acetylcholine content in the brain of the mouse.
The invention has the beneficial effects that: the invention establishes a novel depression liver depression fire transformation model by sleep deprivation based on the principle of disease and syndrome combination, and provides an animal model establishing method which has short modeling time, simple and convenient operation, high success rate, economy, durability, low lethality rate and practicability for deeply understanding the pathogenesis of depression and the action mechanism of antidepressant drugs. After the depression model is successfully established, the depression model is judged to have the characteristics of the traditional Chinese medicine 'liver depression forming fire syndrome' and the liver depression forming fire syndrome type shown after the depression model is established by the traditional Chinese medicine syndrome diagnosis method. The invention abstracts and summarizes the information obtained from the animal model, and then belongs to the syndrome type. Meanwhile, whether the establishment of a classical prescription anti-syndrome model is successful or not is utilized. Based on liver qi stagnation, the liver depression transforming into fire is the essential pathogenesis of the anxiety caused by the liver depression transforming into fire, the classical prescription of Dan Zhi Xiaoyao san adjuvant model for treating the liver depression transforming into fire is selected, and animal experiments and clinical tests show that the prescription has the advantages of effective treatment of depressive disorder and small side effect.
Drawings
FIG. 1 is a graph of body weight change during molding of each group of mice;
FIG. 2 is a graph comparing the results of the center entry times in the open field test of the model group and the control group; FIG. 2 is a graph comparing the results of the time experiment at the center in the open field test of the model group and the control group; FIG. 2 is a graph comparing the experimental results of the model group and the control group entering the open arm in the elevated plus maze experiment;
FIG. 3 is a graph comparing the experimental results of the tail suspension immobility time of the model group and the control group; FIG. 3 is a graph comparing the experiment results of the forced swimming immobility time of the model group and the control group;
FIG. 4 is a graph comparing the results of the experiment on the time to tail suspension for the model group, the control group and the paroxetine group; FIG. 4 is a graph comparing the experimental results of the forced swimming immobility time in the model group, the control group and the paroxetine group;
FIG. 5 is a graph comparing water intake for the control group, model group and Danzhi Xiaoyao san (DZXYS) group;
FIG. 6 is a graph comparing the results of the open field test for the control group, the model group and the Danzhi Xiaoyao san (DZXYS) group;
FIG. 7 is a graph of the results of elevated plus maze tests in the control, model and Danzhi Xiaoyao san (DZXYS) groups;
FIG. 8 is a graph comparing the results of forced swimming tests in the control group, model group and Danzhi Xiaoyao san (DZXYS) group;
FIG. 9 is a graph comparing the levels of 5-HT, GABA, DA and ACH in mice of the control, model and Danzhi Xiaoyao san (DZXYS) groups;
in the figure, the position of the first and second end faces, *** (P < 0.001) and * (P is less than 0.05), and P is less than 0.05, which represents that the model group has statistical significance compared with the control group;
### (P＜0.001)、 ## (P < 0.01) and (P < 0.05) represent the paroxetine group or the Danzhi Xiaoyao group, where P < 0.05 is statistically significant compared to the model group.
Detailed Description
This section will describe in detail specific embodiments of the invention, which should not be construed as limiting the scope of the invention.
1. And (3) molding:
1. preparation of experimental animals:
60 WT adult male mice (animal production permit: SCXK (Su) 2017-0007) with a weight of 20-30 g are born for 6-8 weeks. The animal house is bred adaptively for one week at the experimental animal science and technology center of the university of traditional Chinese medicine in Jiangxi, wherein the environmental temperature of the animal house is 20-26 ℃, and the humidity is 40-60%.
Baseline body weight measurements were completed after adaptive feeding was completed.
The mice were divided into 30 groups of depression and 30 groups of depression with liver depression transforming into fire. The depression group was divided into 10 control groups, 10 model groups and 10 paroxetine groups. The group of depression with the syndrome of liver depression transforming into fire was divided into 10 control groups, 10 model groups and 10 Danzhi Xiaoyao san (DZXYS) groups. In order to avoid mutual biting among the mice in the molding process, only 1 mouse is raised in each cage.
The experimental group was modeled in a combined manner of disease and syndrome, and the control group was not treated at all.
The raising environment and mode of the experimental group and the control group are completely consistent except the stimulation of the molding behavior.
2. Modeling of depression:
the experimental groups (model group and paroxetine group) in the depression group are placed into a sleep deprivation instrument to perform continuous sleep deprivation stimulation for 15 days, the rotation speed of the sleep deprivation instrument is set to be 5rpm, and the rotation time is set to be 1200min. The sleep deprivation instrument with the animal is in an intermittent rotation state, the intermittent time is 3min, and the operation direction is positive and negative rotation. And simultaneously giving stimulation of light irradiation for 24 h/day and stimulation of grains every other day, and continuously stimulating for 15 days. The control group was not treated at all.
2.2. Method for evaluating depression
Weighing a proper amount of paroxetine (Kyoto Jiuzhou medicine Co., ltd.) in the paroxetine group in the depression group, adding water to prepare a solution with the concentration of 1.0mg/ml, carrying out intraperitoneal injection for treatment, wherein the dosage is 10mg/d/kg, and continuously carrying out 14d administration; the control group and the model group are infused with physiological saline with the same dosage.
2.3. Observation index and method for depression
The general physical signs, body weight, food intake and water intake of the mice are observed and recorded every day, the ethology of each group of mice is measured at the 16 th d of the model building, 10 mice in a control group, a model group and a paroxetine group are dissected after the model building of the depression is finished, blood and brain are collected from eye sockets, and the content of neurotransmitters 5-HT, GABA, DA and acetylcholine is measured.
3. Traditional Chinese medicine syndrome diagnosis method for liver depression transforming into fire
After the model building of the depression mouse is finished, whether the model mouse has the symptomatic expression of the liver depression transforming into fire is judged by observing and detecting the hair, urine, tongue and paw colors, behavioral experiments and detecting the contents of neurotransmitters 5-HT, GABA, DA and acetylcholine in brain and blood by adopting GC-MS (liquid chromatography-mass spectrometry).
3.2 evaluation method of liver depression transforming into fire
The model group and the Danzhi Xiaoyao powder group in the group of syndrome of liver depression transforming into fire are subjected to the steps of model making for depression. The Danzhi Xiaoyao powder is prepared from Xiamen herbal Zhenyuan biological medicine technology company. Specifically, each medicinal material comprises 10g of bighead atractylodes rhizome, 12g of white paeony root, 12g of poria cocos, 12g of angelica sinensis, 3g of honey-fried licorice root, 12g of wine-fried radix bupleuri, 3g of mint, 8g of scorched gardenia and 12g of moutan bark, and the raw materials are as follows: 1 part of water: 10 (g: ml) adding water, adding 10 doses of the original prescription, soaking for 30 minutes, decocting for 2 hours, freeze-drying to obtain 213g of dry powder, and gavage to give 16.8 mg/kg of Danzhi Xiaoyao powder with equivalent dose for adults -1 ·d -1 14d, consecutively; the control group was gavaged with the same amount of physiological saline.
3.3. Observation index and method for depression liver depression transforming into fire
The general physical signs, the body weight, the food intake and the water intake of the mice are observed and recorded every day, the tongue, the paw and the urine of each group of mice are respectively shot at the 1 st d after the model building is finished, the ethology is measured, 10 mice in a control group, a model group and a Danzhi Xiaoyao san group are respectively dissected after the model building of the depression is finished, blood is taken from the eye sockets, and the neurotransmitters 5-HT, GABA, DA and acetylcholine are measured from the brain.
2. Evaluation index
1. Indices of depression
Open field experiments, elevated plus maze experiments, tail suspension experiments and forced swimming experiments are commonly used indexes for evaluating animal depression models.
The experiment evaluates the depression degree of a model mouse by using the liveness and average speed in an open field experiment and an elevated plus maze experiment, and the tail suspension and the forced swimming immobility time.
2. Index of liver depression transforming into fire
The experiment takes the contents of neurotransmitters 5-HT, GABA, DA and acetylcholine in urine, tongue, paw, brain and blood as indexes for evaluating the transformation of liver depression into fire.
The appearance, tongue and paw of the mice were photographed the first day after molding, and urine was taken and compared with the control group, and the results are shown in table 1.
3. Analysis of results
1. After the depression is modeled, the open field experiment, the elevated plus maze experiment and the tail suspension and forced swimming test are carried out on the mice of the model group and the control group, and the results are shown in figures 2 and 3, and can be seen in figures 2 and 3: the model building of the depression reduces the speed and time of entering the center of the mouse, the movement of the mouse is limited, the immobility time in a tail suspension experiment is prolonged, the struggling time is abandoned and prolonged during forced swimming, and an hopeless depression state is presented, and the control group of mice has no change, which indicates that the model building of the model is successful.
2. After modeling, tail suspension and forced swimming tests are carried out on mice of a model group and a paroxetine group in the depression group, the results are shown in figure 4, and the immobility time in the tail suspension and forced swimming tests of the mice of the model group is obviously reduced, which shows that the depression despair behavior of the modeled mice is relieved. The paroxetine can effectively improve the depression despair behavior of depressed mice, and further proves that the establishment of depression mouse models is successful.
The depression mice produced by sleep deprivation molding are judged to have the syndrome of liver depression transforming into fire by a traditional Chinese medicine syndrome diagnosis method, namely the index of the mice after molding is judged: the tongue and paw are red, urine is yellow obviously, and water intake is increased, so that the tongue and paw are used for evaluating the state of qi depression transforming into fire on the whole physiological indexes. The total walking distance, the time and the frequency of entering the center and the average speed are obviously reduced in the open field experiment, the air bubble is basically not entered in the elevated plus maze experiment, the arm opening and forced swimming immobility time is obviously increased, and the air bubble is used for evaluating the liver depression state expressed on the overall behavioral indexes; the 5-HT, GABA and DA in the serum of the mouse are obviously reduced, and the Acetylcholine (ACH) in the brain is obviously increased, so that the method is used for evaluating the characterization that the liver depression is transformed into fire on the biochemical indexes in the body. The overall physiological indexes, the field opening experiment, the elevated plus maze and the forced swimming experiment of the mice and the content tests of the neurotransmitters 5-HT, GABA, DA and acetylcholine in the brain and blood of the mice are carried out on the model group of the depression liver depression forming fire syndrome group and the Danzhi Xiaoyao san group, and the specific test results are as follows:
TABLE 1 test results of appearance, tongue color and paw color of each group of mice
It can be seen from table 1 that the hair of the model group in the depression liver depression transforming into fire syndrome group is messy, withered yellow and lusterless, slow in response, dry and scorched auricle, and redder in tongue color and paw color compared with the control group. The rat is prompted to show obvious abnormality in body and mind functions, and the mucosa is dry and similar to the liver depression, the pathogenic fire is transformed into heat, and the heat is excessive and damages the essence and consumes the liquid. The state of the mice in the Danzhi Xiaoyao powder group is recovered after the dry disease treatment by the Danzhi Xiaoyao powder, and the tongue color and the paw color are close to those of the control group.
It is also apparent from table 1 that the model group urine is significantly yellowish. It is fit for the condition of liver depression transforming into fire with yellowish or reddish urine. After the dry prognosis is treated by the Danzhi Xiaoyao powder (DZXYS), the urine color of the Danzhi Xiaoyao powder (DZXYS) group becomes lighter, and is closer to that of the control group.
Fig. 1 is a graph showing the weight change of each group of mice during molding, as can be seen from fig. 1, the weight change is not obvious 4 days before molding, the weight of the mice in the model group is remarkably reduced from the 5d of molding, and the weights of the Danzhi Xiaoyao san (DZXYS) group and the control group are wholly increased, which accords with the common symptom that the weight is remarkably reduced in the syndrome of liver depression transforming into fire.
FIG. 5 is a comparison of water intake of the control group and the model group of depression liver depression transforming into fire syndrome, which shows that the water intake of the depression liver depression transforming into fire syndrome model group is obviously increased compared with the control group, and the water intake of the Danzhi Xiaoyao powder (DZXYS) group is reduced and obviously improved after the treatment of the Danzhi Xiaoyao powder.
Fig. 6 is a comparison graph of the results of the open field test of the control group, the model group and the danzhi xiaoyao (dzxyz) group, which shows that the total walking distance, the time and the times of entering the center of the mice in the depression liver depression-transforming fire syndrome model group in the open field test are obviously reduced compared with the average speed of the mice in the control group, and the total distance, the average speed and the times of entering the center of the mice are obviously recovered after the danzhi xiaoyao powder treatment.
Fig. 7 is a comparison graph of the results of the elevated plus maze test in the control group, the model group and the danzhi xiaoyao powder (dzxyz) group, which shows that in the elevated plus maze test, the mouse in the depression liver depression transforming into fire syndrome model group does not enter the open arm basically, the activity range is in the closed arm basically, and has significant difference compared with the mouse in the control group, while the dry prognosis of the treatment with the danzhi xiaoyao powder, the activity of the mouse in the danzhi xiaoyao powder (dzxyz) group is enhanced, and the time of opening the arm and closing the arm is close to that of the mouse in the control group, which shows that there is significant recovery.
FIG. 8 is a comparison graph of the results of the forced swimming test conducted on the control group, the model group and the Danzhi Xiaoyao san (DZXYS) group, which shows that the immobility time of the mice in the model group of depression liver depression transforming into fire is significantly increased in the forced swimming test compared with the control group, and the Danzhi Xiaoyao san (DZXYS) group is significantly recovered after the drying treatment by the Danzhi Xiaoyao san.
FIG. 9 is a graph comparing the 5-HT, GABA, DA and ACH contents of mice in the control group, the model group and the Danzhi Xiaoyao powder (DZXYS) group, which shows that the 5-HT, GABA and DA (DBA in the graph) of the depression liver depression forming fire syndrome model group are obviously higher than those of the control group, and the 5-HT, GABA and DA contents of the Danzhi Xiaoyao powder (DZXYS) group are obviously increased after the dry prognosis of the Danzhi Xiaoyao powder treatment; the ACH of the mice in the depression liver depression fire transformation model group is obviously higher than that of the control group, and the DZXY group is obviously recovered and improved after the intervention treatment of the Danzhi Xiaoyao powder.
By the technical method, the obtained immobility time of tail suspension and forced swimming of the model mouse is obviously increased, the activity distance in the open field, the time, the times and the average speed of entering the center of the open field and the times and the time of entering the open arm in the elevated plus maze are obviously reduced, and the model group shows an obvious depression state on the overall behavioral indexes; meanwhile, the model group mice have disordered fur, and are accompanied with symptoms of squinting, lassitude and the like, the response to external stimulation is slow, the mice struggle without force and have short time, and the weight is obviously reduced. The urine color is obviously yellow, the water intake is obviously increased, and the tongue color and the paw color are obviously reddish, thus indicating that the model mouse has the symptomatic expression of the depression liver depression fire syndrome. The disease model mouse has the clinical phenotype of successfully simulating depression diseases and liver depression fire transformation symptoms, accords with the pathogenesis of impatience and irritability, chest distress and distending pain and the like caused by emotional disorder or unsmooth depression and long-term fire transformation of depression, and can be used for further research on pathogenesis and treatment of depression liver depression fire transformation symptoms.
The invention provides a modeling and evaluating method for depression liver depression transforming into fire syndrome according with clinical syndrome by combining the characteristics of the disease and the syndrome. According to the modern discipline theory and the scientific knowledge of relevant experimental animals, the disease is copied to the animal body by using the modern medicine, and the manifestation of the syndrome of the traditional Chinese medicine on the animal model is observed by using factors such as the cause of the disease and the like. The purpose of doing so is to make the "disease and syndrome combined" animal model not only have the characteristics of western medicine diseases, but also have the characteristics of traditional Chinese medicine syndromes, provide a research basis for individualized and accurate medical treatment, and be favorable for more comprehensively and intuitively referring to the traditional Chinese medicine clinical to carry out scientific research and the experimental results can be applied clinically in time.
The above description is only a preferred embodiment of the present invention, and the technical solutions that achieve the objects of the present invention by substantially the same means are within the protection scope of the present invention.
Claims (3)
1. A model building method for mice with depression, liver depression and fire transformation syndrome diseases is characterized in that healthy mice are selected, continuous sleep deprivation stimulation is performed on the healthy mice for 2-30 days, and light irradiation stimulation for 1-24 h/day and food stimulation every other day are given; the sleep deprivation stimulation is performed in a sleep deprivation instrument set to intermittently rotate;
also included is a method for evaluating a mouse model obtained by the modeling method:
the method comprises the following steps:
the method comprises the following steps: constructing a depression model, and judging whether the molding of the depression animal is successful or not according to an open field experiment, an elevated plus maze experiment, a tail suspension experiment and a forced swimming experiment;
step two: carrying out a traditional Chinese medicine syndrome diagnosis method on the mice successfully modeled to judge whether the depressed animals have the syndrome of liver depression transforming into fire;
in the second step, the judgment indexes in the traditional Chinese medicine syndrome diagnosis method comprise: the overall behavioral indexes for evaluating the liver depression state of the model, the overall physiological indexes for evaluating the qi depression fire-transforming state of the model and the in-vivo biochemical indexes for evaluating the liver depression fire-transforming state of the model;
the overall behavioral indicator is as follows: forced swimming immobility time of the mouse, total moving distance of the open field, time, times and average speed of entering the center of the open field, and times and time of entering the open arm in the elevated plus maze;
the overall physiological index comprises: tongue picture, paw color and water intake of the mouse;
the in vivo biochemical indexes comprise: the 5-HT, GABA and DA contents in mouse serum and the acetylcholine content in brain.
2. The molding method according to claim 1, wherein the rotation speed of the sleep deprivation instrument is 2 to 50 revolutions/min, the rotation time is 3 to 24 hours/day, and the interval time of the intermittent rotation is 1 to 10 min/time.
3. The modeling method according to claim 1, wherein in the first step, urine and body weight of the mouse are used as judgment indexes of the overall physiological state in the process of constructing the depression model.
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