CN110558285B - Animal model construction method for spleen deficiency and excessive dampness syndrome research and application thereof - Google Patents
Animal model construction method for spleen deficiency and excessive dampness syndrome research and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and particularly discloses an animal model construction method for the study of spleen deficiency and excessive dampness syndrome. The animal model is constructed in two steps: firstly, selecting male rats and feeding high-fat feed, keeping a dim and quiet environment with the relative humidity of 53 +/-3%, regularly recording the color depth of the back of the tongue of the rats, taking tongue pictures, carrying out comparative analysis and quantitative classification, regularly measuring physiological and biochemical indexes, intestinal flora, tryptophan and metabolites thereof, regularly recording signs of hair color, urine, stool, mental conditions and the like of the rats until 4-6 weeks, and completing the construction of the rats in the depression and damp-heat syndrome stages; and secondly, continuously feeding high-fat feed to the rats in the depression and damp-heat syndrome stage, simultaneously increasing the relative humidity to 90 +/-3%, continuously detecting and observing the indexes until 8-10 weeks are finished, and completing the construction of the rat model in the spleen deficiency and blood stasis syndrome stage. The rat model construction has the characteristics of simple modeling method, easy operation and popularization, clinical application and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an animal model construction method for the study of spleen deficiency and excessive dampness syndrome and application thereof.
Background
As the diet type of the 'rich nutrition age' is mainly 'fat, sweet and thick taste', too much food and drink is ingested, and more people with spleen deficiency and dampness abundance are also in the syndrome. Spleen deficiency and excessive dampness not only can cause symptoms of poor appetite, diarrhea, burning pain of anus, mental fatigue and hypodynamia, but also the spleen is the acquired root and the source of qi and blood generation, and is located in the middle energizer. Therefore, spleen deficiency and excessive dampness are also associated with many visceral diseases such as liver and stomach. Due to the importance of the spleen, the development of spleen-invigorating and dampness-eliminating medicaments is also becoming more important. The establishment and evaluation of traditional Chinese medicine syndrome animal models have been the important subject of traditional Chinese medicine research, although more and more documents are about experimental research on tongue manifestation of spleen deficiency models, such as: chenxiaoyan, Zhoushejie, \20319Tong, etc. the tongue picture observation of rats of long-term spleen deficiency and heat syndrome modeling [ J ]. the university of traditional Chinese medicine of Beijing 1998,21(01): 32-34; for another example: chenxiaoye, Zhoushejie, Zhangzhi, etc. Experimental study of Long-term spleen deficiency modeling in rats [ J ] J, J.Chinese J.J.Med.Med.Med.1995, 01(01): 37-4; the method also comprises the following steps: chenxiaoyou, Zhoushejie, rat deficiency syndrome model pathological rule of tongue picture [ J ] Proc of Guiyang college of traditional Chinese medicine 2001,23(03): 54-57. However, so far, there is no quantitative evaluation standard research on the spleen deficiency animal model syndrome characterization, and there is no method for reasonably applying the traditional Chinese medicine four-diagnosis combined reference technology to make the success rate of the spleen deficiency and excessive dampness syndrome consistent with the human being high and the model stability good.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the problems that in the prior art, no quantitative evaluation standard related to the spleen deficiency animal model syndrome representation exists, the modeling is simple, and the defects of a model in the development process of the spleen deficiency and the excessive dampness of human beings are met, the animal model construction method for the research of the spleen deficiency and the excessive dampness syndrome and the application thereof are provided. The invention is to feed highThe fat feed is used for constructing a spleen deficiency rat model, so that the spleen is injured by damp heat to cause depression, damp heat, spleen deficiency and blood stasis. Observing the tongue color, the urine, the mental state and the abdominal change condition starting from the depression and the damp-heat of the rat, and carrying out the single-factor variance analysis of the tongue color according to the scoring standard to obtain the tongue color change, the urine, the mental state and the abdominal change condition of the spleen deficiency and the blood stasis syndrome caused by the rat fed with the high-fat feed, and the intestinal characteristic bacteriaAkkermansia(AKK) And tryptophan and its metabolite change characteristics. The rat model is expected to be used for the research of human depression, damp-heat, spleen deficiency and blood stasis and the development of medicaments for tonifying spleen and clearing damp.
The invention adopts the following technical scheme to achieve the purpose of the invention.
Firstly, the invention discloses an animal model construction method for spleen deficiency and excessive dampness syndrome research. The construction method comprises the construction of rats in the depression and damp-heat syndrome stages and the construction of rat models in the spleen deficiency and blood stasis syndrome stages.
The construction steps of the rat in the depression and damp-heat syndrome stage are as follows: selecting male rats with the weight of 160 +/-20 g, carrying out adaptive feeding on the male rats in a barrier system for one week, randomly dividing the animals into a normal group and a model group, respectively feeding common feed and high-fat feed, keeping free food and free drinking water, keeping the relative humidity of 53 +/-3%, maintaining a dim and quiet environment, regularly detecting and observing multiple indexes for 4-6 weeks, and completing the construction of the rats in the depression and dampness-heat syndrome stages.
The construction steps of the rat model at the spleen deficiency and blood stasis stage are as follows: and (3) continuously feeding high-fat feed to the rats in the depression and dampness-heat syndrome stage, simultaneously increasing the relative humidity to 90 +/-3%, continuously detecting and observing multiple indexes at regular time till 8-10 weeks, and completing the construction of the rat model in the spleen deficiency and blood stasis syndrome stage.
Further, the high-fat feed is obtained by adding butter, lard and cholesterol on the basis of a normal basal maintenance feed.
Further, the normal basal maintenance feed contains 10% of fat; the formula comprises the following components in parts by mass: 0.1-0.3 part of choline chloride, 20-30 parts of corn, 30-40 parts of wheat, 2-3 parts of honey yeast, 1.5-2.5 parts of calcium hydrophosphate, 0.8-1.2 parts of stone powder, 1 part of growth premix, 0.2-0.4 part of salt, 2-3 parts of oil, 15-25 parts of bean pulp, 4-6 parts of fish meal and 8-12 parts of casein.
Further, the fat content of the high-fat feed is 60%; the specific formula comprises the following components in parts by mass: 0.1-0.3 part of choline chloride, 20-30 parts of corn, 30-40 parts of wheat, 2-3 parts of honey yeast, 1.5-2.5 parts of calcium hydrophosphate, 0.8-1.2 parts of stone powder, 1 part of growth premix, 0.2-0.4 part of salt, 2-3 parts of oil, 15-25 parts of bean pulp, 4-6 parts of fish meal and 8-12 parts of casein, and 12-18 parts of butter, 10-16 parts of lard and 0.8-1.2 parts of cholesterol are additionally added.
More preferably, the high-fat feed comprises the following components in parts by mass: 0.2 part of choline chloride, 25 parts of corn, 35.5 parts of wheat, 2.5 parts of honey yeast, 2 parts of calcium hydrophosphate, 1 part of stone powder, 1 part of growth premix, 0.3 part of salt, 2.5 parts of oil, 20 parts of soybean meal, 5 parts of fish meal and 10 parts of casein, and 15 parts of butter, 13 parts of lard and 1 part of cholesterol are additionally added.
Furthermore, the body weight of the rat in the depression and damp-heat syndrome stage is more than 1.5 times of the average body weight of the control rat fed normally in the same period, the tongue color is red, urine is yellow, loose stool is produced in feces, and dyslipidemia is taken as a standard, and the rat model in the depression and damp-heat syndrome stage is judged to be successfully constructed, or some animals have water and slippery coating and is also judged to have damp syndrome.
Furthermore, the objective index of the rat model at the spleen deficiency and blood stasis stage is that insulin resistance is generated; the main signs are reddened tongue color and rising tongue color value; the auxiliary physical signs are abdominal obesity, inappetence, loose stool, dark yellow urine color, lassitude and hypodynamia, pymetrorrhagia and sleepiness, lusterless hair and squinting, and the tongue color is purple, so the auxiliary diagnosis is blood stasis which is more similar to the spleen deficiency and excessive dampness syndrome of human.
Further, the timing detection and observation multiple indexes are as follows: recording the back color of the tongue of the rat, taking a tongue picture, comparing, analyzing and quantitatively grading, regularly measuring physiological and biochemical indexes, intestinal flora, tryptophan and metabolites thereof, regularly recording the signs of hair color, urine, stool, mental status and the like of the rat.
Furthermore, the tongue picture is captured by a digital camera, a uniform fixed internal light source is adopted during the Image capture, the placing positions, the shooting angles and the shooting personnel of the rat tongue and the digital camera are fixed, the analysis of Densityrween and Densitybluen is carried out by an Image-plus medical Image analysis system after the tongue picture is captured, and the comparative analysis of the control group rat and the model group rat is carried out, so as to further verify the accuracy of the scoring system.
Furthermore, the quantitative grading is divided into 1 part of light white tongue, 2-4 parts of red tongue group and 5-6 parts of purple tongue group according to the tongue color.
Secondly, the invention discloses an application of a rat model with spleen deficiency and excessive dampness syndrome.
The rat model is used for the research of human depression, damp-heat, spleen deficiency and blood stasis and the development of medicines for reinforcing the spleen and eliminating dampness.
Furthermore, the research of human stagnation, damp-heat, spleen deficiency and blood stasis combines some clinical characteristics of a rat model with the syndrome of spleen deficiency and excessive dampness with the diagnosis standard of human spleen deficiency to determine the assistant diagnosis of human stagnation, damp-heat, spleen deficiency and blood stasis by the symptoms of abdominal obesity, inappetence, loose stool, dark yellow urine color, lassitude and hypodynamia, pygmy gomphosis, lusterless hair, squinting, reddened tongue or purplish tongue and the like of the rat, and by the characteristic bacteria in intestinal tractAkkermansia(AKK) The abundance is increased and the ratio of kynurenine to tryptophan is increased to aid in the diagnosis of spleen deficiency and blood stasis in humans.
Furthermore, the study on human depression, damp-heat, spleen deficiency and blood stasis shows that after the positive medicine simvastatin, rosiglitazone and the traditional Chinese medicine Gegenqinlian decoction are used for intervening the model, the tongue color of the rat in the model group can be recovered to be normal, and the positive medicine simvastatin enables the characteristic bacteriaAkkermansia(AKK) The abundance is reduced remarkably, and the ratio of kynurenine to tryptophan is in a downward trend. The marker is an auxiliary diagnosis index of spleen deficiency and blood stasis, and is also helpful for distinguishing spleen deficiency and blood stasis of human beings.
Furthermore, the research of the spleen tonifying and dampness eliminating medicine takes a rat model of spleen deficiency and excessive dampness syndrome as a research object, and the spleen tonifying and dampness eliminating medicine is applied to the spleenThe rat with deficiency and excessive dampness syndrome is obtained by observing tongue picture, stool, mental state and abdomen change of rat, and detecting physiological and biochemical indexes and intestinal characteristic bacteriaAkkermansia(AKK)And the ratio of kynurenine to tryptophan, and determining the screening and treatment effects of the spleen-tonifying and dampness-eliminating prescription.
Has the advantages that:
(1) the invention constructs a rat model with spleen deficiency and excessive dampness by feeding high-fat feed and improving the environmental humidity of the organism, and guides the construction of the model by the traditional Chinese medicine theory that the mixture of internal and external dampness is easy to cause the spleen deficiency and the excessive dampness. The spleen deficiency rat model construction has the characteristics of simple modeling method, high success rate, easy operation and popularization, closer approach to the spleen deficiency and excessive dampness syndrome of human, and the like. According to the invention, the tongue color change of the spleen deficiency and excessive dampness syndrome caused by high-fat feed feeding rats and increase of the environmental humidity of the organism, the change of physiological and biochemical and intestinal flora and the index of special metabolites are obtained by observing the tongue color condition starting from the depression and damp-heat of the rats and carrying out statistics on the tongue color according to the scoring standard. The evaluation method fills the defect that no quantitative evaluation standard related to the spleen deficiency animal model condition characterization exists in the prior art, and also fills the defect that no method for assisting the high success rate and good model stability of the spleen deficiency and excessive dampness syndrome meeting the human condition by reasonably applying the traditional Chinese medicine four-diagnosis combined reference technology is available.
(2) The spleen deficiency rat model is applied to the research of human stagnation, damp-heat, spleen deficiency and blood stasis and the development of spleen tonifying and dampness eliminating medicines. The novel model is provided for normatively, standardly and scientifically evaluating the research of the traditional Chinese medicine for tonifying spleen and eliminating dampness, has good practicability, strong conformity, simplicity and objectivity, conforms to the guidance of the traditional Chinese medicine syndrome theory, and has wide popularization and application prospect.
Drawings
FIG. 1 is a graph showing the change of the tongue color of a rat with time.
FIG. 2 shows a ratAkkermansia(AKK)Bacterial change result chart. Wherein: comparison of representations with blank groupsP<0.05, # denotes comparison with the model setP<0.05。
FIG. 3 is a graph showing the results before and after administration of kynurenine to tryptophan ratio in rats. Wherein: c represents the normal group, OB represents the model group, and SIM represents the positive drug simvastatin group.
Detailed Description
The present invention will be further described with reference to specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Example 1: construction of rat model in depression and dampness-heat syndrome stage
(1) Grouping and feeding animals: 100 SPF SD male rats weighing 160 + -20 g provided by SCXK 2018 and 0003 at the scientific and technological center of Experimental animals of Jiangxi university of traditional Chinese medicine. Animal feeding conditions: naturally illuminating according to circadian rhythm, and feeding in SPF-level experimental animal barrier system at laboratory temperature of 23 + -2 deg.C and relative humidity of 53 + -3%. Animals were freely fed water and food, and after one week of acclimatization, they were randomly divided into a model group (90) and a normal group (10), and fed with a high fat diet (60% fat content) and a normal basal maintenance diet (10% fat content), respectively.
(2) Feed:
the normal basal maintenance feed comprises the following components: 0.2% of choline chloride, 30% of corn, 25.5% of wheat, 2.5% of honey yeast, 2% of calcium hydrophosphate, 1% of stone powder, 1% of growth premix, 0.3% of salt, 2.5% of oil, 20% of soybean meal, 5% of fish meal and 10% of casein;
high-fat feed: is prepared by adding butter and cholesterol based on the basic maintenance feed. The method comprises the following specific steps of: 0.2 part of choline chloride, 25 parts of corn, 35.5 parts of wheat, 2.5 parts of honey yeast, 2 parts of calcium hydrophosphate, 1 part of stone powder, 1 part of growth premix, 0.3 part of salt, 2.5 parts of oil, 20 parts of soybean meal, 5 parts of fish meal and 10 parts of casein, and 15 parts of butter, 13 parts of lard and 1 part of cholesterol are additionally added.
(3) Body weight and body length measurement: body weight and length data were collected once a week.
(4) Physiological biochemical and non-target metabolite index detection: blood and feces were collected once every 2 weeks for physiological, biochemical and non-target metabolite index detection.
(5) Collecting tongue color information: the rat is caught to expose the tongue body of the rat to the same light more, and the force is exerted on the upper and lower teeth of the rat by the stomach irrigation needle, so that the tongue of the rat can be naturally exulted as much as possible. After the tongue surface condition of the rat can be clearly seen, the color depth of the back surface of the tongue and the record of the excrement condition, the weight, the food intake, the mental condition, the hair and the like of the rat at the moment are recorded, meanwhile, the digital camera shoots the tongue Image, a unified fixed internal light source is adopted during shooting, the placing positions, the shooting angles and the shooting personnel of the rat tongue and the digital camera are fixed, and after the tongue Image is shot, the analysis of Densityred | Densitygreen | Densityblue and Densitymean is carried out by an Image-plus medical Image analysis system, and the comparison analysis between a control group and a model group rat is carried out so as to further verify the accuracy of a scoring system and better judge the modeling condition of the rat.
(6) Grading the tongue color: after a large amount of observation and analysis comparison of the tongue color of the rat, quantitative grading is carried out on the back color depth of the tongue, and a tongue back color depth grading scoring method is preliminarily formulated. The color of the back of the tongue is graded as follows: light white is 1 point, light red is 2 points, red is 3 points, dark red is 4 points, light purple is 5 points, and dark purple is 6 points. The tongue is divided into a light white tongue group (1), a red tongue group (2-4) and a purple tongue group (5-6) according to the tongue color, and the table is shown in table 1.
(7) Obese rats (with depression, damp-heat syndrome): the model group rats are continuously fed with the high-fat feed, the weight of the model group rats is larger than or equal to the standard deviation of the average weight of the normal group rats by more than + 1.5 times, the tongue color is red, urine is yellow, excrement is loose, and dyslipidemia is taken as a standard, the model is judged to be successfully constructed in the depression and damp-heat stage rat model (or some animals have watery slippery coating and are also judged to be damp-heat), the weight result is shown in table 2, the number of the successfully constructed rats is 73, the success rate of the model is 81%, and is higher than that of the existing purely obese model, such as the literature ' gingerbing, soluble dietary fiber pectin, the protection effect and mechanism research of the metabolism and intestinal barrier of the diet induced obese rats, doctor ' position, Nanjing university, 2016 ' are only 33%.
Example 2: construction of rat model in spleen deficiency and blood stasis stage
(1) Grouping and feeding animals: the normal group (10) rats and the depressed and damp-heat syndrome-compliant model group (73) rats in example 1 were continuously maintained in the barrier system of SPF-grade laboratory animals in example 1, during which the animals in both groups were maintained in 2 independent laboratory rooms, the normal group (10) rats were maintained under the same conditions as in example 1, and the normal basal maintenance diet (10% of fat content) was continuously maintained, while the model group (73) rats were maintained under the conditions that the relative humidity was increased to 90 + -3% and the high-fat diet (60% of fat content) was continuously maintained.
(2) Detection of indexes: the same as in example 1.
(3) Rats with spleen deficiency and blood stasis syndrome: the clinical characteristics of the rat are combined with the diagnosis standard of the deficiency of spleen qi of human beings, and the symptoms of inappetence (appetite reduction), loose stool (frequency of tail-pulling and defecation increase, loose stool and anus pollution), lassitude and hypodynamia (hanging empty tail-pulling resistance is weakened), pyemesis and lethargy, lusterless hair, squinting and the like of the rat are determined to assist in diagnosing the spleen deficiency. The spleen taught by traditional Chinese medicine is equivalent to the spleen and pancreas in western medicine anatomy. The spleen governs transportation and transformation, which is equivalent to the internal and external secretion function of pancreas, and the spleen nourishes the viscera organs of the whole body by ascending clear, which is equivalent to the endocrine function of pancreas, and the material basis for realizing the function is insulin, so that patients with spleen deficiency have insulin resistance to a great extent, and hyperglycemia caused by insulin resistance and insulin resistance can be improved by invigorating spleen and reducing phlegm. Therefore, whether the rats generate insulin resistance is taken as an objective index to measure the generation of the rats with spleen deficiency, the insulin resistance index of the rats in the model group at the 6 th week is found to be obviously greater than that of the rats in the normal group (P < 0.05), the table 3 shows that the tongue color is purple, and the assistant diagnosis of stasis is carried out. As a result, the number of successfully molded rats was 50, and the molding success rate was 56% (based on the number of starting animals in the model group, which was 90).
Tongue color change results for the model animals of examples 1 and 2 above: in 6 tongue color observations, the color value of the model group rat tongue color is 1.8 and mainly light red, while the color value of the normal group tongue color is 3.8 and mainly red tongue color, and the single-factor variance analysis between the groups is carried out, wherein the color of the model group tongue and the normal group tongue color is significantly different (P < 0.05). With the continuous feeding of high-fat feed, the model rats gradually change from light red tongue after obesity to red tongue of spleen deficiency and dampness-heat syndrome, the color values of the tongue colors of the normal rats and the model rats are close to 2.0 (see table 4) in the later molding period, and the tongue colors of the rats tend to be light white probably due to the decline of the body constitution of the rats. The result of the change of the tongue color in the natural aging process of the rat is approximately similar to the aging result of human, so that the model is suitable for the research of the spleen deficiency and excessive dampness syndrome of human and the research of the spleen tonifying and dampness eliminating medicine.
The tongue has been studied in traditional Chinese medicine for a long time, the onychomycosis unearthed from the ruins has been recorded as "diseased and diseased tongue", and for the spleen deficiency syndrome, the traditional Chinese medicine is characterized by a pale tongue with little coating, while the damp-heat syndrome is characterized by a red tongue with greasy coating. In the related works of Chen Xiao Ye, Zhoushejie, rat deficiency syndrome model tongue manifestation pathological law [ J ]. Guiyang Chinese medicine college, 2001,23(03):54-57, it was found that the spleen deficiency rat model made by the method of consuming qi and breaking qi had a pale and tender tongue with little coating. However, there is no research on the quantitative evaluation criteria of the tongue color of the animal model with the spleen deficiency and excessive dampness. The invention combines the researches on spleen deficiency syndrome, damp-heat syndrome and tongue color, and evaluates the tongue color of rats with spleen deficiency and damp-abundance syndrome by recording the light white color of the back of the tongue as 1 point, the light red as 2 points, the red as 3 points, the deep red as 4 points, the light purple as 5 points and the deep purple as 6 points.
According to the traditional Chinese medicine theory that after eating, the patient is fat, sweet, thick and greasy, and the patient is easy to get internal dampness and the patient is easy to have spleen deficiency and excessive dampness due to the mixture of internal and external dampness, the traditional Chinese medicine compound feed meets the spleen deficiency and excessive dampness syndrome of people in southern China through feeding high-fat feed and a spleen deficiency rat model caused by humidity increase.
In the experimental process, the light sources of the illuminating bodies with different standards can cause the deviation of tongue diagnosis data collection, so that the rat feeding environment is set to be a dim environment, and only one fixed light source is provided to prevent too large error. And because the mouse moves at night, the mouse is more excited at night, so that the tongue color is prevented from being observed at night, and the living mouse is caught. For the observation of the tongue color of the rat, the rat tongue color is found to be instantly purple along with the change of emotion, such as panic, fear and the like. Therefore, the experimenter is required to skillfully catch the rat, the action is mild, when the abnormal tongue color is found, the rat is allowed to rest for a moment, and observation is started after the rat is calm, so as to avoid generating larger errors.
(4) Positive drug (simvastatin and rosiglitazone) and traditional Chinese medicine (gegen qinlian decoction high-medium low dose group) intervention model results:
the rats in the model group were randomly divided into a low, medium and high group of Gegen Qinlian Decoction (GQD), a simvastatin group, a rosiglitazone group and a model group, each group contained 8 rats, and the blank group was randomly selected from 8 rats in the 10 normal groups in example 1. The corresponding solutions were administered by gavage at 10 weeks. The color of the administration group is found to be darker at 14 weeks, which is different from that of the model group without administration (P < 0.05), the color of the GQD with low dose is darker as the positive medicines simvastatin and rosiglitazone, and the tongue color is darker as the concentration of the GQD is increased, and is darker than that of the positive medicine group. However, by 16 weeks, the effect of the GQD drug group on the tongue color was significantly stronger than that of the positive drug group, and at 21 weeks, the effect of each drug group on the tongue color was almost consistent, but the effect of the low dose GQD group on the tongue color was most significant, as shown in table 4 below. The result shows that the positive medicine simvastatin, rosiglitazone and the traditional Chinese medicine GQD can restore the normal tongue color of the rats in the model group, but the influence of the traditional Chinese medicine GQD is more obvious. Meanwhile, the traditional Chinese medicine GQD has better influence on body weight, blood fat and blood sugar than the positive medicine group. Although GQD can relieve excessive spleen deficiency and excessive dampness caused by overeating fat, sweet and thick taste and in vitro dampness to a certain extent, GQD is a bitter cold heat-clearing formula, has the effect of stomach poisoning after long-term administration and is easy to cause spleen deficiency, and needs to be used with caution clinically.
In addition, the model group has characteristic bacteria of intestinal tract compared with blank groupAkkermansia(AKK) Increased abundance and a significant increase in the kynurenine to tryptophan ratio (P)<0.05), whereas the positive drug simvastatin is dry to cause the characteristic bacteriaAKKThe abundance decreased significantly, while the kynurenine to tryptophan ratio tended to decrease (see figures 2 and 3 for details).AKKThe kynurenine-tryptophan ratio metabolic pathway is enhanced and is related to inflammation and cardiovascular diseases. Therefore, the temperature of the molten metal is controlled,AKKand the ratio of kynurenine to tryptophan can be used as an auxiliary diagnosis index for spleen deficiency and blood stasis, and is also helpful for distinguishing spleen deficiency and blood stasis of human beings.
Example 3: application of rat model with spleen deficiency and excessive dampness syndrome
The first is that some clinical characteristics of the spleen deficiency rat are combined with the diagnosis standard of the spleen deficiency of human beings, and the symptoms of abdominal obesity, inappetence, loose stool, dark yellow urine color, purple tongue color or water-slippery coating, lassitude and hypodynamia, withered and sleepy, lusterless hair, squinting and the like of the rat are determined to assist in diagnosing the human stagnation, damp-heat, spleen deficiency and blood stasis. Furthermore, the study on spleen deficiency and excessive dampness syndrome uses intestinal characteristic bacteriaAkkermansia(AKK) The abundance is increased and the ratio of kynurenine to tryptophan is increased to aid in the diagnosis of spleen deficiency and blood stasis in humans. Furthermore, in the study of spleen deficiency and excessive dampness syndrome, after positive medicines simvastatin, rosiglitazone and traditional Chinese medicine (Gegen Qinlian decoction) intervene in the model, the tongue color of the rat in the model group can be recovered to be normal, and the medicine simvastatin enables the characteristic bacteria to be normalAkkermansia(AKK) The abundance is reduced remarkably, and the ratio of kynurenine to tryptophan is in a downward trend.
Second, it is a model of rats with spleen deficiency and excessive dampness syndromeThe preparation is a research object, the medicine for tonifying spleen and eliminating dampness is applied to rats with spleen deficiency and excessive dampness syndrome, and the change of tongue picture, stool, mental state and abdomen of the rats is observed, and physiological and biochemical indexes and intestinal characteristic bacteria are detectedAkkermansia(AKK)And the ratio of kynurenine to tryptophan, and determining the screening and treatment effects of the spleen-tonifying and dampness-eliminating prescription.
Excessive eating of food is rich in flavor, sweet and thick, and abnormal change in climate, so spleen deficiency and damp-turbidity phlegm-fluid retention are common in human body. The spleen deficiency can cause abdominal distension, poor appetite, loose stool, edema, chronic bleeding, viscera prolapse and other diseases, and the damp turbidity and phlegm retention can block qi and blood, consume yang qi, and cause cough, expectoration, dizziness, heaviness of head, loose stool, turbid urine and other manifestations. Therefore, the research on the prescription for tonifying spleen and eliminating dampness is more and more important, for the research on the medicine, the establishment of an animal model is very important, and the tongue body is the only tissue which is exposed outside and can quickly change correspondingly under the influence of the environment in the organism, so the observation of the tongue is of great significance for the research on the model and the medicine.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the above-described embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent alterations and modifications are intended to be included within the scope of the invention, without departing from the spirit and scope of the invention.
Claims (4)
1. An animal model construction method for the study of spleen deficiency and excessive dampness syndrome is characterized in that: the construction method comprises the steps of constructing the rats in the depression and damp-heat syndrome stages and constructing the rat models in the spleen deficiency and blood stasis syndrome stages;
the construction steps of the rat in the depression and damp-heat syndrome stage are as follows: selecting male rats with the weight of 160g +/-20 g, carrying out adaptive feeding on the male rats in a barrier system for one week, randomly dividing the animals into a normal group and a model group, respectively feeding normal basal maintenance feed and high-fat feed, keeping free eating and drinking water, keeping the relative humidity of 53 +/-3%, maintaining a dim and quiet environment, detecting and observing multiple indexes at regular time till 4-6 weeks, and completing the construction of the rats in the depression and dampness-heat syndrome stages;
the construction steps of the rat model at the spleen deficiency and blood stasis stage are as follows: continuously feeding high-fat feed to the rats in the depression and dampness-heat syndrome stage, simultaneously increasing the relative humidity to 90 +/-3%, continuously detecting and observing multiple indexes at regular time till 8-10 weeks, and completing the construction of the rat model in the spleen deficiency and blood stasis syndrome stage;
the timing detection and observation multiple indexes are as follows: recording the back color of the tongue of the rat, taking a tongue picture, comparing, analyzing and quantitatively grading, regularly measuring physiological and biochemical indexes, intestinal flora, tryptophan and metabolites thereof, and regularly recording the hair color, urine, stool and mental conditions of the rat;
the signs of the rats in the depression and damp-heat syndrome stage are as follows: the weight of the rat exceeds 1.5 times of the average weight of the control rats fed normally in the same period, and the standards of red tongue color, yellow urine, loose stool generation of feces and dyslipidemia are adopted, so that the rat model is judged to be successfully constructed in the depression and damp-heat syndrome stage;
the objective index of the rat model at the spleen deficiency and blood stasis stage is that insulin resistance is generated; the main signs are reddened tongue color and rising tongue color value; the auxiliary physical signs are abdominal obesity, inappetence, loose stool, dark yellow urine color, lassitude and hypodynamia, pymetrorrhagia and sleepiness, lusterless hair and squinting, and the tongue color is purple, so the auxiliary diagnosis is blood stasis which is more similar to the spleen deficiency and excessive dampness syndrome of human.
2. The method for constructing an animal model for studying spleen deficiency and excessive dampness syndrome according to claim 1, wherein the method comprises the following steps: the high-fat feed is obtained by adding butter, lard and cholesterol on the basis of a normal basic maintenance feed.
3. The method for constructing an animal model for studying spleen deficiency and excessive dampness syndrome according to claim 2, wherein the method comprises the following steps: the fat content of the normal basal maintenance feed is 10 percent; the formula comprises the following components in parts by mass: 0.1-0.3 part of choline chloride, 20-30 parts of corn, 30-40 parts of wheat, 2-3 parts of honey yeast, 1.5-2.5 parts of calcium hydrophosphate, 0.8-1.2 parts of stone powder, 1 part of growth premix, 0.2-0.4 part of salt, 2-3 parts of oil, 15-25 parts of bean pulp, 4-6 parts of fish meal and 8-12 parts of casein; the fat content of the high-fat feed is 60 percent; the specific formula comprises the following components in parts by mass: 0.1-0.3 part of choline chloride, 20-30 parts of corn, 30-40 parts of wheat, 2-3 parts of honey yeast, 1.5-2.5 parts of calcium hydrophosphate, 0.8-1.2 parts of stone powder, 1 part of growth premix, 0.2-0.4 part of salt, 2-3 parts of oil, 15-25 parts of bean pulp, 4-6 parts of fish meal and 8-12 parts of casein, and 12-18 parts of butter, 10-16 parts of lard and 0.8-1.2 parts of cholesterol are additionally added.
4. Use of the method of constructing an animal model according to any one of claims 1 to 3, wherein: is used for preparing the medicine for reinforcing the spleen and eliminating dampness.
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| CN113016714B (en) * | 2021-03-04 | 2023-08-22 | 湖南中医药大学 | Preparation method of animal model with diarrhea and food stagnation gastrointestinal symptoms |
| CN116236551B (en) * | 2023-03-18 | 2024-03-01 | 新疆医科大学 | Traditional Chinese medicine composition for treating spleen deficiency and excessive dampness syndrome type spleen stomach diseases and preparation method thereof |
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