An animal model of lung and spleen deficiency syndrome based on the theory of "mother disease affects the child" and its evaluation method

Abstract

The present invention relates to the technical field of experimental animal models, and discloses an animal model of lung and spleen qi deficiency syndrome based on the theory of "mother disease and child", and the model realization method includes the following steps: S1, material selection and grouping; S2, model replication; S3, model evaluation; S4, index detection; S5, statistical processing; S6, model verification. The present invention gives model rats a high-fat and low-protein feed, and puts them in a homemade swimming pool to swim to exhaustion after daily weight bearing, and evaluates the model after 6 weeks; the modeling method does not directly damage the lungs of rats, and the spleen qi deficiency process caused by it is very mild, and the onset characteristics of the complex deficiency syndrome course are prolonged, and at the same time, spleen qi deficiency comes first, and lung qi deficiency comes later, which is in line with the basic mechanism of the formation of lung and spleen qi deficiency syndrome in traditional Chinese medicine; the method establishes an animal model of lung and spleen qi deficiency syndrome, and the etiology and pathogenesis are accurate, and it is non-invasive, stable, and easy to evaluate, which broadens the application scope of the original modeling method and lays a foundation for the mechanism research of the method of accompanying soil to produce gold.

Description

Technical Field

[0001] The invention relates to the technical field of experimental animal models, and in particular to an animal model of lung and spleen qi deficiency syndrome established based on the "mother disease affects the child" theory and an evaluation method thereof. Background Art

[0002] "The mother's disease affects the child" is an abnormal mutual generation phenomenon in traditional Chinese medicine theory. It is a pathological phenomenon in which the mother's organs cannot nourish the child's organs normally after being damaged, resulting in abnormal functions of the child's organs. For example, the spleen is the mother and the lungs are the child; spleen deficiency leads to lung deficiency, and ultimately lung and spleen deficiency is "the mother's disease affects the child."

[0003] At present, most of the complex syndrome models of lung and spleen qi deficiency in traditional Chinese medicine remain at the stacking of spleen qi deficiency symptoms and lung qi deficiency symptoms rather than from the perspective of the true pathogenesis of the syndrome: The main methods for modeling animal models of lung deficiency caused by spleen deficiency are as follows: (1) Fumigation combined with senna leaf method: First, the rats are induced to have lung qi deficiency symptoms by fumigation, or lipopolysaccharide is instilled into the trachea to cause lung damage, and then the spleen qi deficiency model is established by cold senna leaf laxative method; (2) Fatigue combined with dietary disorder plus fumigation method: The rats are made to swim to exhaustion every day and fed with cabbage on odd days and pig fat gavage on even days to establish a spleen deficiency model, and then the lung qi deficiency model is established by fumigation; (3) Fumigation plus lipopolysaccharide method combined with senna leaf method: Lipopolysaccharide is instilled into the rat trachea on the 1st and 15th days of modeling; on the 2nd to 13th day and the 15th to 40th day of modeling, the rats are placed in a fumigation box for passive smoking for 0.5 hours, twice a day, with an interval of 4 minutes each time. h; Starting from the 20th day after modeling, senna leaves were soaked in boiling water and then precooled at 4°C to prepare a rat model of lung and spleen qi deficiency diarrhea; (4) LPS plus smoke combined with rhubarb decoction gavage: on the 1st and 14th days after modeling, tracheotomy and lipopolysaccharide instillation were performed; from the 2nd to the 13th day and the 15th to the 28th day, cigarette smoke was continuously inhaled twice a day in a homemade plexiglass box for half an hour each time; from the 7th day of modeling, rhubarb decoction was gavage for a total of 8 days.

[0004] Although these modeling methods can also produce symptoms of lung and spleen qi deficiency, the organ damage caused by the modeling methods of spleen qi deficiency and lung qi deficiency respectively is relatively isolated, and there is no continuity and correlation in the pathogenesis; at the same time, fumigation and lipoprotein tracheal instillation are originally invasion of evil spirits, and are mostly positive syndromes in the early stages. Even if the modeling time is extended, it is due to excess to deficiency, and finally manifests as a mixture of deficiency and excess. In addition, the lipoprotein instillation method itself involves invasive surgery, and the trauma of animals leads to uncertainty and complexity in its modeling. It does not accurately correspond to the etiology and pathogenesis for the study of the method of accompanying soil to produce gold in the tonic method of traditional Chinese medicine. Summary of the invention

[0005] The purpose of the present invention is to provide an animal model of lung and spleen qi deficiency syndrome based on the "mother disease affects the child" theory and an evaluation method thereof to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] An animal model of lung and spleen qi deficiency syndrome established based on the theory of "mother disease affects the child", the model implementation method comprises the following steps:

[0008] S1. Material selection and grouping: Several SPF Wistar rats were selected, half of which were male and half were female. The rats were housed in an IVC independent air-purified cage and adaptively raised for 3 days. After the rats were observed to be in good condition and without abnormalities, they were numbered and weighed using an automatic rat tail labeling instrument. Then, according to the rat weight factor, the Excel-generated random number method was used to group them into a blank group and a model group.

[0009] S2. Model replication: Starting from the 4th day of the experiment, the blank group was fed with AIN-76A purified feed, and the model group was fed with high-fat and low-protein feed. Both groups had free access to water and food. Starting from the 4th day, the model group was placed in a homemade swimming pool with a tail weight of 30% of body weight every afternoon, so that the rats could not touch the bottom of the cylinder of the swimming pool. The rats were stopped when they entered the water and swam to exhaustion, that is, the nose tip was submerged in the water for 10 seconds. The modeling time was 6 weeks. The general condition indicators were observed on the 14th day after the start of the experiment, and the modeling situation was evaluated on the 45th day of the experiment.

[0010] S3. Model evaluation: Evaluate the animal model of lung and spleen qi deficiency syndrome and establish general condition evaluation criteria;

[0011] S4. Index detection: various indicators of rats were tested, including general condition observation, body weight detection, open field test, metabolic cage, urine D-xylose detection, lung function test, and serological index detection;

[0012] S5. Statistical analysis: Professional statistical software and R language were used to analyze the data, which were expressed as mean ± standard deviation. t-test was used to compare continuous variables between groups, and P < 0.05 was considered statistically significant.

[0013] S6. Model verification: The lung-spleen qi deficiency syndrome animal model regression equation was used to verify whether the lung-spleen qi deficiency syndrome model was successfully replicated.

[0014] As a further embodiment of the present invention: in the step S1, the rats are 4 weeks old and weigh 105±10 g.

[0015] As a further solution of the present invention: the AIN-76A purified feed in step S2 contains 0.2% choline chloride, 0.3% methionine, 5% cellulose, 20% casein and 1% multivitamin in terms of mass ratio; the high-fat and low-protein feed is prepared by reducing the casein content and increasing lard and cholesterol on the basis of the AIN-76A purified feed. The high-fat and low-protein feed contains 0.2% choline chloride, 15% lard and 0.5% cholesterol in terms of mass ratio.

[0016] As a further solution of the present invention: in step S4, the water temperature of the swimming pool is 25±2° C. and the water depth is 30 cm.

[0017] As a further solution of the present invention: the establishment of the general condition evaluation standard of rats in the S3 step is guided by the basic theory of traditional Chinese medicine, and according to the clinical manifestations of lung and spleen qi deficiency syndrome, the macroscopic physical sign diagnostic standard and objective measurement index of the rat lung and spleen qi deficiency model are formulated; and then the macroscopic and objective indicators are combined to formulate the evaluation standard of the lung and spleen qi deficiency syndrome rat model.

[0018] An evaluation method for an animal model of lung and spleen qi deficiency syndrome based on the "mother disease affects the child" theory comprises the following steps:

[0019] S11. Simulate the thinking method of comprehensively evaluating all the information obtained in clinical diagnosis of traditional Chinese medicine, and combine all the symptoms, signs and test results of the rat model into the evaluation factor domain, comprehensively evaluate the replication status of each rat model, and determine whether the modeling is successful or not;

[0020] S12. Let Z be the domain of evaluation factors of lung-qi deficiency model in rats. The domain of evaluation factors of lung-qi deficiency model is composed of the detection indicators of rats after modeling, that is: Z={thin body, squinting eyes, dry and yellow fur, pale claws, soft and loose stools, decreased spontaneous activity, decreased body weight, decreased food intake, decreased urinary D-xylose excretion rate, decreased tidal volume, decreased serum albumin, increased serum cholesterol, and decreased serum gastrin}.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides model rats with a high-fat and low-protein feed, loads a weight every day, and swims in a self-made swimming pool until exhaustion, and evaluates the model after 6 weeks; the modeling method does not directly damage the rat lungs, and the spleen qi deficiency process caused by the method is very mild, and the onset characteristics of the complex deficiency syndrome are prolonged, and spleen qi deficiency occurs first, and lung qi deficiency occurs later, which is consistent with the basic mechanism of TCM symptom formation of lung and spleen qi deficiency syndrome; the method establishes an animal model of lung and spleen qi deficiency syndrome, and the etiology and pathogenesis are accurate, and it is non-invasive, stable, and easy to evaluate, which broadens the application scope of the original modeling method and lays a foundation for the mechanism research of the method of accompanying soil to produce gold. DETAILED DESCRIPTION

[0023] In an embodiment of the present invention, an animal model of lung and spleen qi deficiency syndrome is established based on the theory of "mother disease affects the child", and the method for realizing the model comprises the following steps:

[0024] S1. Material selection and grouping: Several SPF Wistar rats were selected, half of which were male and half were female. The rats were housed in an IVC independent air-purified cage and adaptively raised for 3 days. After the rats were observed to be in good condition and without abnormalities, they were numbered and weighed using an automatic rat tail labeling instrument. Then, according to the rat weight factor, the Excel-generated random number method was used to group them into a blank group and a model group.

[0025] S2. Model replication: Starting from the 4th day of the experiment, the blank group was fed with AIN-76A purified feed, and the model group was fed with high-fat and low-protein feed. Both groups had free access to water and food. Starting from the 4th day, the model group was placed in a homemade swimming pool with a tail weight of 30% of body weight every afternoon, so that the rats could not touch the bottom of the cylinder of the swimming pool. The rats were stopped when they entered the water and swam to exhaustion, that is, the nose tip was submerged in the water for 10 seconds. The modeling time was 6 weeks. The general condition indicators were observed on the 14th day after the start of the experiment, and the modeling situation was evaluated on the 45th day of the experiment.

[0026] S3. Model evaluation: Evaluate the animal model of lung and spleen qi deficiency syndrome and establish general condition evaluation criteria;

[0027] S4. Index detection: various indicators of rats were tested, including general condition observation, weight detection, open field experiment, metabolic cage, urine D-xylose detection, lung function test, and serological index detection. Reagents needed for index detection: rat gastrin (Gas) ELISA Kit, rat biochemical kit, D-xylose determination kit (colorimetric method), D (+) xylose, animal isoflurane (Isoflurane), experimental instruments needed: I5 type IVC independent supply and return air purification cage; homemade swimming pool, specifications: diameter 150cm, depth 80cm; rat open field test box, specifications (length × width × height): 100cm × 100cm × 40cm; TKDX-d rat metabolic cage; AMR-100 multifunctional fully automatic enzyme labeler; UV-2802 ultraviolet visible spectrophotometer; BS-420 fully automatic biochemical analyzer; Buxco whole body plethysmography (WBP) system;

[0028] 1) Observation of general condition: Observe demeanor, bowel movements, hair and other general conditions every day, and record the observation results in the Symptom Scoring Form 1 every week.

[0029] Table 1 Symptom scoring criteria for rats with lung and spleen deficiency syndrome

[0030]

[0031] 2) Body weight: The rats were weighed using an electronic balance at 5:30 pm once a week;

[0032] 3) Open field experiment: Before the experiment begins, make sure that the experimental box is clean and odorless, especially pay attention to cleaning the feces and urine left by the animals in the previous experiment at the bottom of the experimental box; the experimenter sets the corresponding parameters in the software; quickly place the experimental animal in the central area of ​​the experimental box and leave immediately; the measurement time is 6 minutes; after the experiment, put the experimental animal in other cages prepared in advance; spray the instrument with alcohol to remove odor and wipe it dry with paper towels. Record the activity distance of the rat within 6 minutes.

[0033] 4) Metabolic cage: Clean the metabolic cage, measure the weight of the rat's food and water, place the rat in the metabolic cage at 8:00 in the morning, and take the rat out at 8:00 the next morning. Measure and record the rat's 24-hour food intake, water intake, urine output, and feces output.

[0034] 5) Urine D-xylose: After fasting for 12 hours, gavage with 13% D-xylose, 4 ml / animal, and collect urine for 5 hours. Do not drink water during the fasting period, and then strictly follow the instructions of the D-xylose kit.

[0035] 6) Pulmonary function: The pulmonary function test uses the Buxco whole body plethysmography (WBP) system. Before the experiment begins, make sure that the experimental box is clean and odorless, adjust the system parameters, and record the animal number, start time and other information. The experimenter leaves immediately and the rats are allowed to adapt to the environment in the breathing chamber for 10 minutes, and the measurement time is 21 minutes. After the experiment, the experimental animals are placed in other cages prepared in advance. The breathing chamber is cleaned and dried with pure water and experimental paper. The rats' tidal volume (TV), respiratory rate (f), minute ventilation (MV) and other related data are recorded. With time as the horizontal axis and MV and f as the vertical axis, the screening is carried out to select the period with small fluctuations and stable breathing, and the average TV value in this period is calculated to represent the respiratory tidal volume of this period.

[0036] 7) Serological indicators: After fasting for 12 hours, blood was collected from the inferior vena cava under isoflurane anesthesia, centrifuged, and serum was prepared and stored at low temperature. All were measured using an automatic biochemical analyzer. The operation method was carried out according to the instructions of the kit. Measurement indicators: TP, TC, ALB, HDL-C, LDL-C and Gas.

[0037] S5. Statistical analysis: Professional statistical software and R language were used to analyze the data, which were expressed as mean ± standard deviation. t-test was used to compare continuous variables between groups, and P < 0.05 was considered statistically significant.

[0038] S6. Model verification: The lung-spleen spleen deficiency model was verified by using the regression equation of the animal model of lung-spleen spleen deficiency syndrome. The 15 indicators in Table 2 were selected as independent variables, and the dependent variable Y was whether the lung-spleen spleen deficiency syndrome was present or not. , that is, when Y=1, it represents the model group, and when Y=0, it represents the control group.

[0039] Table 2 Independent variable identification table of lung and spleen qi deficiency rat model

[0040]

[0041] The PLS regression model of the animal model of lung and spleen qi deficiency syndrome is:

[0042]

[0043] in, is a constant term, are the coefficients of the partial least squares model;

[0044] Using the plsr function in the pls package of R language for PLS analysis, the regression equation is as follows:

[0045]

[0046] According to the symptom scoring standard in Table 1 and the independent variable identification table in Table 2, the rats with lung and spleen qi deficiency model were scored; among them, the positively correlated variables were symptom scores (body shape, demeanor, coat, claw color, tongue image, feces), respiratory rate, serum albumin and low-density lipoprotein cholesterol; the negatively correlated variables were body weight, activity distance, urine volume, feces volume, urine D-xylose test, tidal volume, minute ventilation, gastrin, total protein, total cholesterol, and high-density lipoprotein cholesterol. Among them, the specific indicators with judgment function were: the rats with lung and spleen qi deficiency had a thin body shape, withered and dull coat color, light red claw color, soft or unformed and small stool, reduced urination, reduced activity distance, reduced gastrin, decreased gastrointestinal absorption function, reduced tidal volume and ventilation, increased respiratory rate, reduced serum protein, and disordered blood lipid metabolism; the score result was greater than -60, and the lung and spleen qi deficiency model was successfully replicated.

[0047] Preferably, in step S1, the age of the rats is 4 weeks and the weight is 105±10 g.

[0048] Preferably, the AIN-76A purified feed in step S2 contains 0.2% choline chloride, 0.3% methionine, 5% cellulose, 20% casein and 1% multivitamin in terms of mass ratio; the high-fat and low-protein feed is prepared by reducing the casein content and increasing lard and cholesterol on the basis of the AIN-76A purified feed. The high-fat and low-protein feed contains 0.2% choline chloride, 15% lard and 0.5% cholesterol in terms of mass ratio.

[0049] Preferably, in step S4, the water temperature of the swimming pool is 25±2° C. and the water depth is 30 cm.

[0050] Preferably, the establishment of the general condition evaluation standard of rats in step S3 is guided by the basic theory of traditional Chinese medicine, and according to the clinical manifestations of lung and spleen qi deficiency syndrome, the macroscopic physical sign diagnostic standard and objective measurement index of the rat lung and spleen qi deficiency model are formulated; and then the macroscopic and objective indicators are combined to formulate the evaluation standard of the lung and spleen qi deficiency syndrome rat model. The general condition evaluation standard of the model rats is shown in Table 1. The score increases, and the symptoms worsen.

[0051] An evaluation method for an animal model of lung and spleen qi deficiency syndrome based on the "mother disease affects the child" theory comprises the following steps:

[0052] S11. Simulate the thinking method of comprehensively evaluating all the information obtained in clinical diagnosis of traditional Chinese medicine, and combine all the symptoms, signs and test results of the rat model into the evaluation factor domain, comprehensively evaluate the replication status of each rat model, and determine whether the modeling is successful or not;

[0053] S12. Let Z be the domain of evaluation factors of lung-qi deficiency model in rats. The domain of evaluation factors of lung-qi deficiency model is composed of the detection indicators of rats after modeling, that is: Z={thin body, squinting eyes, dry and yellow fur, pale claws, soft and loose stools, decreased spontaneous activity, decreased body weight, decreased food intake, decreased urinary D-xylose excretion rate, decreased tidal volume, decreased serum albumin, increased serum cholesterol, and decreased serum gastrin}.

[0054] What is described above is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An animal model of lung and spleen deficiency syndrome based on the theory of "mother's disease affects the child", characterized in that: The model implementation method includes the following steps: S1. Material selection and grouping: Several SPF Wistar rats were selected, half of which were male and half were female. The rats were raised in independent air-purified cages for 3 days. After the rats were observed to be in good condition and without abnormalities, they were numbered and weighed using an automatic rat tail numbering instrument. Then, according to the rat weight factor, the rats were grouped using the Excel method to generate random numbers, and divided into a blank group and a model group; S2. Model replication: Starting from the fourth day of the experiment, the blank group was fed with AIN-76A purified feed, and the model group was fed with high-fat and low-protein feed. Both groups had free access to water and food. Starting from the fourth day, the model group was placed in a homemade swimming pool with a tail weight of 30% of body weight every afternoon, so that the rats could not touch the bottom of the cylinder of the swimming pool. The rats were stopped when they entered the water and swam to exhaustion, that is, when the nose tip was submerged in the water for 10 seconds. The modeling time was 6 weeks. The general condition indicators were observed on the 14th day after the start of the experiment, and the modeling situation was evaluated starting from the 45th day of the experiment. S3. Model evaluation: Evaluate the animal model of lung and spleen qi deficiency syndrome and establish general condition evaluation criteria; S4. Index detection: various indicators of rats were tested, including general condition observation, body weight detection, open field test, metabolic cage, urine D-xylose detection, lung function test, and serological index detection; S5. Statistical analysis: Professional statistical software and R language were used to analyze the data, which were expressed as mean ± standard deviation. t-test was used to compare continuous variables between groups, and P < 0.05 was considered statistically significant. S6. Model verification: The lung-spleen qi deficiency syndrome animal model regression equation was used to verify whether the lung-spleen qi deficiency syndrome model was successfully replicated.

2. The animal model of lung-spleen deficiency syndrome based on the "mother disease affects the child" theory according to claim 1, characterized in that: In the S1 step, the rats were 4 weeks old and weighed 105±10 g.

3. The animal model of lung-spleen deficiency syndrome based on the "mother disease affects the child" theory according to claim 1, characterized in that: The AIN-76A purified feed in step S2 contains 0.2% choline chloride, 0.3% methionine, 5% cellulose, 20% casein and 1% multivitamin in terms of mass ratio; the high-fat and low-protein feed is prepared by reducing the casein content and increasing lard and cholesterol on the basis of the AIN-76A purified feed. The high-fat and low-protein feed contains 0.2% choline chloride, 15% lard and 0.5% cholesterol in terms of mass ratio.

4. The animal model of lung-spleen deficiency syndrome based on the "mother disease affects the child" theory according to claim 1, characterized in that: The water temperature of the homemade swimming pool in step S2 is 25±2° C. and the water depth is 30 cm.

5. The animal model of lung-spleen deficiency syndrome based on the "mother disease affects the child" theory according to claim 1, characterized in that: The establishment of the general condition evaluation standard of rats in step S3 is guided by the basic theory of traditional Chinese medicine. According to the clinical manifestations of lung and spleen qi deficiency syndrome, the macroscopic physical sign diagnostic standard and objective measurement index of the rat lung and spleen qi deficiency model are formulated; and then the macroscopic and objective indicators are combined to formulate the evaluation standard of the lung and spleen qi deficiency syndrome rat model.

6. A method for evaluating the animal model of lung-spleen deficiency syndrome based on the "mother disease affects the child" theory as described in claim 1, characterized in that: The following steps are involved: S11. Simulate the thinking method of comprehensively evaluating all the information obtained in clinical diagnosis of traditional Chinese medicine, and combine all the symptoms, signs and test results of the rat model into the evaluation factor domain, comprehensively evaluate the replication status of each rat model, and determine whether the modeling is successful or not; S12. Let Z be the domain of evaluation factors of lung-qi deficiency model in rats. The domain of evaluation factors of lung-qi deficiency model is composed of the detection indicators of rats after modeling, that is: Z={thin body, squinting eyes, dry and yellow fur, pale claws, soft and loose stools, decreased spontaneous activity, decreased body weight, decreased food intake, decreased urinary D-xylose excretion rate, decreased tidal volume, decreased serum albumin, increased serum cholesterol, and decreased serum gastrin}.

Images