Liver depression and spleen deficiency syndrome animal model evaluation method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicine, in particular to a liver depression and spleen deficiency syndrome animal model evaluation method and application thereof. The invention provides an RMS-LSCRS scale tool, which is a model recording and evaluating tool specially developed for a chronic bound stress liver depression and spleen deficiency syndrome rat model. By using an RMS-LSCRS scale tool, detailed qualitative and quantitative recording can be carried out on the modeling condition of the model developed by the rat model with the chronic constraint stress liver depression and spleen deficiency syndrome, and qualitative and quantitative evaluation can be carried out on the rat model so as to judge the success condition of modeling.

Description

TECHNICAL FIELD

[0001] The application relates to the field of traditional Chinese medicine, in particular to a liver stagnation and spleen deficiency syndrome animal model evaluation method and application thereof. BACKGROUND

[0002] The liver stagnation and spleen deficiency syndrome belongs to the range of zang-fu combined syndromes. The zang-fu combined syndrome refers to two or more than two zang-fu syndromes. Generally speaking, as long as the zang-fu organs with exterior and interior, generation and restriction, and attack and subjugation relationships, the combined syndrome is easy to occur, which indicates that the zang-fu syndromes have the internal relationship and mutual influence in pathology. The liver stagnation and spleen deficiency syndrome is a combined syndrome of liver and spleen syndromes, and the liver and spleen have the relationship of generation and restriction. The liver stagnation and spleen deficiency syndromes can influence each other. Therefore, the liver stagnation and spleen deficiency syndrome can be divided into the liver stagnation and spleen deficiency syndromes which have the internal relationship and mutual influence in pathology.

[0003] The common clinical manifestations of the liver stagnation and spleen deficiency syndrome are the liver stagnation and spleen deficiency syndromes caused by liver stagnation and spleen deficiency, the main symptoms and syndrome differentiation points of which are chest and hypochondrium distention and pain, depression of emotion, irritability, poor appetite, abdominal distension, abdominal pain and loose stool. In the New Century National Higher Medical Colleges and Universities Planning Textbook (for Chinese and Western medicine combination majors) 'Chinese Medicine Diagnosis', the liver stagnation and spleen deficiency syndrome refers to the syndrome caused by liver stagnation and spleen deficiency. The clinical manifestations are chest and hypochondrium distention and pain, depression of emotion, sighing, irritability, abdominal distension, poor appetite, abdominal pain, loose stool, and the tongue coating is white, and the pulse is string or string and slow. The chest and hypochondrium distention and pain, sighing, abdominal distension and loose stool are the syndrome differentiation points.

[0004] The liver stagnation and spleen deficiency syndrome animal model belongs to the zang-fu combined syndrome animal model, and is one of the main researches of the zang-fu combined syndrome animal model. With the development of syndrome objectification and syndrome essence research, the research of the zang-fu combined syndrome animal model appears, and the research of the liver stagnation and spleen deficiency animal model also starts at this time. The liver stagnation and spleen deficiency animal model is mainly established by simulating symptoms, chemical factors and objective indexes. At present, there are many research methods for the liver stagnation and spleen deficiency animal model, and the research methods are constantly improved.

[0005] The traditional Chinese medicine animal model is composed of disease models, syndrome models and disease syndrome combination models. The establishment of the model involves the selection of animals, modeling tools and modeling methods. Modern research shows that the pathological changes of the liver stagnation and spleen deficiency syndrome are related to the nervous system, the digestive system and the endocrine system. According to the pathological characteristics of the disease and the design principles of the animal model, the establishment of the animal model is studied.

[0006] The selection of model animals, including the selection of animal species and gender. For the selection of animal species, closed colony animals have high heterozygosity, and their average reactivity has certain stability, which can reflect the comprehensive average therapeutic effect, so closed colony animals are often used for modeling in the modeling process of liver stagnation and spleen deficiency syndrome animal models. The selection of animal gender in experiments requires the first choice of adult male animals, followed by half male and half female animals, and finally female animals and young animals. There is also this problem in the modeling of liver stagnation and spleen deficiency rat models, so the selection of model animals is different for male rats, half male and half female rats, and female rats. Among them, the most common is to select male rats for modeling. SD rats, such as Yue Lifeng, Zheng Xurui, etc. use male SD rats, Guo Zhenqiu uses half male and half female SD rats, etc.; male Wistar rats, such as Han Qiyan, Li Yanyan, Tang Yiting, Chen Jiaxu, Gu Ligang, etc. In addition, there are also studies on this syndrome modeling by selecting male and female rabbits and male mice.

[0007] The modeling method is to intervene in the model animals by physical or chemical methods, and to form a disease model or a syndrome model with specific syndrome manifestations. Traditional Chinese medicine believes that liver stagnation and spleen deficiency are mainly caused by emotional failure, anger damaging the liver, liver failing to regulate and transverse, and damaging the spleen. Or improper diet, overthinking and overwork, damaging the spleen, the spleen failing to transport, the earth damaging the wood, and the liver failing to drain. Therefore, theoretically, the modeling method can be from the liver, the spleen, or the liver and the spleen at the same time. The specific method can be divided into drug method, emotional stimulation method and combined modeling method. In the modeling method involving physical factors, the selection of modeling tools is involved. The main modeling tools are restraint frame, restraint barrel, etc. Different tools are used to restrain the rats to achieve the effect of restraint stress.

[0008] (1) Simple liver stagnation and spleen deficiency syndrome model

[0009] A simple syndrome model is an animal model established from the perspective of traditional Chinese medicine syndrome, which has the characteristics of traditional Chinese medicine syndrome and is not limited by specific diseases.

[0010] Drug modeling method: The earliest method of modeling of liver stagnation and spleen deficiency syndrome is injecting CCl4, such as the acute poisoning of CCl4 and peanut oil solution injection of mice and the acute and chronic poisoning of CCl4 and peanut oil solution injection of rats in the first affiliated hospital of Hunan Medical College. Compound Qian injection was used to treat the model animals. Xue Jingdong et al. selected ICR male mice and established the model by subcutaneous injection of CCl4. The activity, food intake, body weight and blood SG-PT were observed. Datai capsule was used for counter-therapy. Liu Shaotang et al. used female and male rabbits to replicate the animal model by using 10% CCl4 sesame oil solution 10 mL / kg body weight. The treatment method was liver and gallbladder meridian (Yanglingquan, Ququan) and spleen and stomach meridian (Yinlingquan, Zusanli) acupoint injection of Qingkailing injection and Danshen injection, and the changes of plasma sulfhydryl content, liver glycogen and ALP, AMS activity were observed.

[0011] Emotional stimulation method: This method is mainly from the influence of emotion on the liver, and then develops to the stage of liver stagnation and spleen deficiency. Tang Yiting, Yue Lifeng and Chen Jiaxu et al. used chronic restraint stress method to replicate rat model of liver stagnation and spleen deficiency, and successfully simulated the pathogenesis of "anger damaging liver" and liver stagnation and spleen. Through the observation and determination of macroscopic characterization, food intake, body weight, feces, behavior analysis, urine D-xylose excretion rate and other indicators, the success of the model was verified. At the same time, through the comparison of the treatment effect of Xiaoyao San, Sijunzi Decoction and Jinkui Shenqi Pill, it was confirmed that Xiaoyao San had the best effect. The changes of CRF mRNA content in related brain areas of rats were observed. Drug counter-therapy selected Xiaoyao San. Xu Huiran et al. used tail clamping and irritation to make them fight with other rats to replicate rat model of liver stagnation and spleen deficiency, and observed the behavior, blood rheology and platelet ultrastructure. The water decoction of liver-soothing and qi-regulating drugs (Chaihu, Baishao, Chizhi, Chuanqiong, Xiangfu and Gancao) was used for rehabilitation counter-therapy. Peng Guiying et al. selected male Kunming mice and established liver stagnation and spleen deficiency model by limiting movement, and detected the tumor weight and peritoneal macrophage function. Liver-soothing and spleen-strengthening drugs (Huangqi, Baishu, Chaihu, Zhuyangqi, Chizhi, Baishao and Dangshen) were selected for treatment counter-therapy.

[0012] Combined modeling method: This method uses drug intervention, emotional stimulation, and other interventions, while simultaneously replicating the model in the liver and spleen. For example, Han Qiuyan established a model using chronic tail-clamping irritation combined with high-concentration rhubarb gavage. General conditions such as food intake, activity level, and excitability were observed, as well as indicators such as nitric oxide (NO) and endothelin-1 (ET-1). A self-made formula, Weikang capsules, was used to treat the underlying symptoms. Li Yanyan et al. established an animal model of liver stagnation and spleen deficiency using chronic restraint stress + excessive fatigue + dietary indiscretion. Observational indicators included appearance, urinary D-xylose excretion rate, monoamine neurotransmitters, serum GAS, plasma MTL, SS, VIP, hemorheology and related regulatory factors, changes in the immune system, HPA axis, serum T3, T4, and TSH, etc. Chaihu Shugan San combined with Sijunzi Tang was used to treat the underlying symptoms. Cai Gan et al. established a rat model of liver stagnation and spleen deficiency by using male Wistar rats and administering rhubarb via gavage with electrical stimulation. Through observation of general conditions such as behavior, response, defecation, and diet, as well as analysis of the pathogenic factors, they concluded that this model reflects the characteristics and essence of liver stagnation and spleen deficiency syndrome. They also used radioimmunoassay to measure the CRF content in the hypothalamus and locus coeruleus. Chen Dexing et al. established a rat model of liver stagnation and spleen deficiency syndrome by administering rhubarb via gavage and stress restraint. They observed the rats' diet, fur, and feces, and measured the levels of gastrointestinal hormones such as somatostatin, motilin, and brain-gut peptides in the central and peripheral tissues. For drug treatment, they used Banxia Decoction (a traditional Chinese medicine formula). Peng Guiying et al. replicated a rat model of liver stagnation and spleen deficiency using a combination of restricted movement and food intake with intermittent restraint. Drug treatment employed a formula to soothe the liver and strengthen the spleen, including prepared licorice root, atractylodes macrocephala, prepared astragalus membranaceus, immature bitter orange, white peony root, and codonopsis pilosula.

[0013] (2) Animal model of liver stagnation and spleen deficiency combined with disease syndrome

[0014] Animal models combining disease and syndrome are an important development direction in the development of animal models of TCM syndromes in recent years. They can provide a good basis for TCM diagnosis and treatment of diseases. The replication of such models often adopts the method of combined modeling. Chen Guozhen et al. initially used 10% CCl4 peanut oil solution to inject rats to create an animal model of chronic hepatitis with liver stagnation and spleen deficiency, and observed the general manifestations, serological indicators, and histological and chemical observations of the liver and intestines. Compound Chaihu-Qi injection was selected as the treatment drug to soothe the liver, strengthen the spleen, and promote blood circulation. Gu Ligang et al. used male Wistar rats and used a method of restricting movement and food and injecting acetic acid into the colon to establish an animal model of ulcerative colitis with liver stagnation and spleen deficiency. They observed the pathological changes of the colonic mucosa and measured the NO content and myeloperoxidase activity of the colonic mucosa. The drug counter-syndrome was composed of Chaihu Guizhi Ganjiang Decoction, a formula for soothing the liver and strengthening the spleen, with added ingredients. Wang Yi et al. used male SD rats and employed a trinitrobenzenesulfonic acid and ethanol solution enema combined with restraint to replicate a rat model of ulcerative colitis with liver stagnation and spleen deficiency. They observed changes in colonic VIP and IL-10 levels. They used traditional Chinese medicine external therapy, ginger-separated moxibustion, for counter-treatment. Zhang Fangyan et al. established an animal model of ulcerative colitis with liver stagnation and spleen deficiency using electric shock, dietary indiscretion, and acetic acid perfusion, observing changes in main symptoms and signs, colonic mucosal damage index, and immunological indicators. Hu Xuguang et al. replicated an animal model of ulcerative colitis with liver stagnation and spleen deficiency using a combination of activity restriction, food restriction, restraint stress, and 5% acetic acid colonic injection, observing changes in symptoms, signs, pathological, and immunological indicators. They used a diaphoretic formula and sulfasalazine for counter-treatment. Qin Zhensheng et al. replicated an ulcerative colitis model with liver stagnation and spleen deficiency using a TNBS / ethanol solution enema combined with restraint, observing changes in body weight, colonic histology, and vasoactive intestinal peptide and L-10 levels. The counter-evidence of traditional Chinese medicine (TCM) was a self-made preparation – Shugan Jianpi Granules. Zheng Xurui et al. used male SD rats and established a rat model of liver fibrosis with liver stagnation and spleen deficiency from the perspective of etiology, namely, subcutaneous injection of carbon tetrachloride olive oil solution combined with tail clamping stimulation and intermittent fasting. The model was observed and evaluated by symptom observation, blood stasis index detection, and histopathological examination. The counter-evidence of the drug was treated with Jiawei Si Ni San. Guo Zhenqiu et al. used a high-fat diet combined with subcutaneous injection of ethanol combined with CCl4 castor oil solution to replicate a rat model of liver fibrosis with liver stagnation and spleen deficiency. The liver pathology was examined by light microscopy, and serum ALT, Alb, HYP, Fb and other indicators were measured. The counter-evidence of the drug was treated with Ganxianning, a drug for treating chronic liver disease with liver fibrosis, liver stagnation and spleen deficiency, and blood stasis obstructing the collaterals. Peng Guiying et al. used a method of restricting movement and food, intermittent restraint, and oral administration of diethylnitrosamine to replicate a rat model of liver stagnation, spleen deficiency and liver cancer. The general condition, T lymphocyte proliferation, and the ability of spleen cells to produce IL-2 and IFN-γ were observed in the model rats. Treatment with a liver-soothing and spleen-strengthening formula showed significant improvement in the symptoms.Wang Ji et al. used Kunming mice and employed a combination of tumor cell inoculation, restraint, and dietary irregularities to replicate a mouse model of transplanted ascites tumors and liver stagnation and spleen deficiency syndrome. They observed changes in the general condition of the model mice and tumor cell proliferation cycle. The treatment for this syndrome was a modified version of Si Ni San from the *Shang Han Lun* (Treatise on Cold Damage). Peng Guiying et al. used male Kunming mice and established a tumor-bearing mouse model of liver stagnation and spleen deficiency syndrome using movement restriction and S180 sarcoma cell inoculation. They also examined tumor weight and peritoneal macrophage function. They selected a liver-soothing and spleen-strengthening formula to treat this syndrome.

[0015] Shi Junjie et al. selected male SD rats and used the method of chronic restraint stress plus tail clamping stimulation to establish a rat model of chronic restraint stress with liver stagnation and spleen deficiency syndrome. They developed an evaluation scale for the rat model of irritability syndrome and observed colonic motility function, mucosal tissue pathological changes, abdominal wall reflexes and electromyography. The drugs used to confirm the diagnosis were Xiaoyao San and pinaverium bromide.

[0016] Due to the incomplete similarities between animal and human structures, physiology, and pathology, it is impossible to completely apply the traditional four diagnostic methods of Traditional Chinese Medicine to animal experimental research for model evaluation. Therefore, a systematic and applicable animal model evaluation system should be established. Zhang Hong et al. believe that objective, specific, and easily observable indicators such as symptoms and signs should be selected for observation.

[0017] Existing model evaluation methods mainly include macroscopic characterization, objective indicators, microscopic indicators, and drug-based counter-evidence.

[0018] Macroscopic observations of rats are essential in the study of traditional Chinese medicine (TCM) models. However, a unified standard and system for observation and evaluation has not yet been established. Further research and discussion should be conducted, referencing the generally accepted syndrome differentiation diagnostic criteria within the TCM community.

[0019] The selection and establishment of objective indicators often involve choosing indicators that are easy to record objectively and can be quantified, such as weight, body temperature, grip strength, food intake, and behavioral experiments, in order to facilitate recording and various analyses.

[0020] The selection of microscopic indicators is too broad, encompassing various aspects such as tissues of different organs, systems, blood, and urine, and involving various examinations. However, the research is not in-depth, the indicators are not focused, and there are few specific and sensitive indicators with weak supporting evidence. A meaningful positive indicator system should be established based on the characteristics of traditional Chinese medicine and clinical practice, making these indicators specific and sensitive, thereby creating a comprehensive microscopic indicator system.

[0021] Regarding drug-based counter-evidence in the model, three situations exist: classic prescriptions, modified classic prescriptions, and self-made prescriptions. The reliability and comparability of these self-made prescriptions require further research due to various factors such as clinical application and efficacy evaluation. Traditional prescriptions used for counter-evidence often include Xiaoyao San, Tongxie Yaofang, Banxia Xiexin Tang, Jiawei Sini San, and Chaihu Shugan San, among others. In recent years, there has been a growing trend towards the use of Xiaoyao San. Chen Jiaxu et al., through their research, concluded that Xiaoyao San, a prescription for liver stagnation and spleen deficiency, can significantly improve the behavior and central nervous system peptides in rats with a 21-day stress-induced liver stagnation and spleen deficiency model, demonstrating a good effect superior to spleen-tonifying and kidney-nourishing compound prescriptions. This indicates a close relationship between the 21-day restraint stress state and the TCM syndrome of liver stagnation and spleen deficiency. Wu Shengxian et al. also believe that the clinical efficacy of Xiaoyao San is relatively reliable. However, Fang Zhaoqin, Xiao Yun, and others believe that "due to the effects of drug efficacy, it is often difficult to define the attributes of the syndrome," and using drugs to refute the attributes of animal models is not entirely reliable and deserves further consideration. Summary of the Invention

[0022] In view of this, the present invention provides an evaluation method for an animal model of liver stagnation and spleen deficiency syndrome and its application, specifically involving the RMS-LSCRS scale tool, which is a model recording and evaluation method specifically developed for a rat model of liver stagnation and spleen deficiency syndrome under chronic restraint stress. Using the RMS-LSCRS scale tool, the modeling status of the rat model of liver stagnation and spleen deficiency syndrome under chronic restraint stress can be recorded in detail qualitatively and quantitatively, and the rat model can be evaluated qualitatively and quantitatively to determine the success of modeling.

[0023] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0024] This invention provides a method for identifying an animal model of liver stagnation and spleen deficiency syndrome, including methods based on activity status and degree of mental arousal (i.e., Figure 13 The scale indicates whether the person is mentally agitated, whether they are irritable, and their posture before being captured (i.e., mental agitation). Figure 13 The "condition before capture" in the scale), vigilance, and irritability (i.e., Figure 13 The "irritability" status in the scale, and the action response during capture (i.e.) Figure 13 The scale includes "action response during capture", degree of resistance, and degree of hunching (i.e., the degree of resistance during capture). Figure 13 The "arched back condition" in the scale, and the degree of resistance during gavage (i.e., Figure 13 The scale includes "resistance during gavage" and the degree of tongue resistance during gavage (i.e., Figure 13 The scale includes "tongue resistance during gavage" and the degree of body twisting during gavage (i.e., ...). Figure 13 The scale includes "body writhing during gavage", the animal's behavior when restrained or placed in a cage after gavage, fur luster, fur color, and fur softness (i.e., the animal's body writhing during gavage). Figure 13The "softness of fur" and the degree of messiness and uncleanliness of fur in the "scale" (i.e.) Figure 13 The scale includes criteria such as "whether the fur is disheveled and unclean," ear color, nail color, and fecal appearance (i.e.,...). Figure 13 The assessment includes the "fecal condition" section of the "scale".

[0025] In some specific embodiments of the present invention, the identification described in the above identification method is based on... Figure 13 The evaluation scale for the chronic restraint stress rat model of liver stagnation and spleen deficiency syndrome shown is used to identify the specific judgment criteria as follows:

[0026] The liver stagnation syndrome assessment was conducted starting from day 6 of animal model establishment, with continuous measurements for 4 days. The total score for liver stagnation syndrome in any measurement was ≥19.5. The spleen deficiency syndrome assessment was conducted starting from day 18 of animal model establishment, with continuous measurements for 4 days. The total score for spleen deficiency syndrome in any measurement was ≥51.5. The animal model of liver stagnation syndrome and spleen deficiency syndrome was considered to have been successfully established.

[0027] In some specific embodiments of the present invention, the animal used in the above identification method is a mouse.

[0028] The present invention also provides a method for constructing an animal model of liver stagnation and spleen deficiency syndrome, characterized by comprising: modeling, and identifying the animal model from the following aspects, and obtaining the animal model after passing the identification;

[0029] The identification was performed using the identification method described above.

[0030] In some specific embodiments of the present invention, the above-mentioned construction method is characterized in that the modeling includes the use of drug modeling, emotional stimulation and / or chronic restraint stress modeling.

[0031] In some specific embodiments of the present invention, the above-described construction method is characterized in that the modeling includes using chronic restraint stress modeling.

[0032] In some specific embodiments of the present invention, the above-mentioned construction method is characterized in that the modeling includes: restraining the animal continuously for 21 days, restraining it for 3 hours each day.

[0033] In some specific embodiments of the present invention, the above-described construction method is characterized in that the restraint includes restraining the animal to a restraint frame;

[0034] The restraint frame has grooves for placing the limbs and adhesive nylon tape for restraint.

[0035] In some specific embodiments of the present invention, the above-described construction method is characterized in that the animal is a mouse.

[0036] This application provides the RMS-LSCRS scale tool, a model recording and evaluation tool specifically developed for a rat model of chronic restraint stress with liver stagnation and spleen deficiency. Using the RMS-LSCRS scale tool, detailed qualitative and quantitative records of the model establishment process in the rat model of chronic restraint stress with liver stagnation and spleen deficiency can be made, and the rat model can be qualitatively and quantitatively evaluated to determine the success of model establishment. Macroscopic characteristics of rats are essential observation items in the study of traditional Chinese medicine models. This invention provides a unified observation and evaluation standard and system, which is of great significance. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0038] Figure 1 The comparison of food intake among the rats in each group is shown;

[0039] Figure 2 The comparison of water intake among the rats in each group is shown.

[0040] Figure 3 The comparison of rat body weights in each group is shown;

[0041] Figure 4 The comparison shows the number of times rats from each group entered the central region;

[0042] Figure 5 Comparison of dwell time in the central grid among rats in each group;

[0043] Figure 6 The comparison of the central region movement speed of rats in each group is shown;

[0044] Figure 7 The comparison of the distances the rats moved in the central region is shown in the diagram.

[0045] Figure 8 The comparison of the total movement speed of rats in each group is shown;

[0046] Figure 9 The total distance traveled by rats in each group was compared.

[0047] Figure 10 This is a technical framework diagram of the experimental process for developing the "Evaluation Scale for Rats with Chronic Restraint Stress and Liver Qi Stagnation and Spleen Deficiency Syndrome (Version 1)".

[0048] Figure 11 This is a partial technical framework diagram of the development of the "Evaluation Scale for Rats with Chronic Restraint Stress and Liver Qi Stagnation and Spleen Deficiency Syndrome (Version 1)";

[0049] Figure 12 The theoretical framework is shown, and the items in the observation table are represented (the numbers in parentheses in the figure represent the number of items).

[0050] Figure 13 Show the evaluation scale for the rat model of liver depression and spleen deficiency syndrome induced by chronic restraint stress (version 1);

[0051] Figure 14 Show the estimated marginal mean of liver depression syndrome;

[0052] Figure 15 Show the estimated marginal mean of spleen deficiency syndrome. Specific implementation manners

[0053] This application discloses a method for evaluating an animal model of liver depression and spleen deficiency syndrome and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of this invention to implement and apply the technology of this invention.

[0054] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this application are all ordinary commercially available products and can be purchased from the market.​​​​​​​​​​​​​​​​​​​​​​​(2) Drugs and reagents

[0063] The traditional Chinese medicine preparation used in the experiment was Xiaoyao San (from the Taiping Huimin Heji Jufang), with the following ingredients: Bupleurum chinense 30 g, Angelica sinensis 30 g, Paeonia lactiflora 30 g, Atractylodes macrocephala 30 g, Poria cocos 30 g, Glycyrrhiza uralensis (processed) 15 g, Zingiber officinale (processed) 10 g, and Mentha haplocalyx 10 g. The dry extract was prepared strictly according to the process flow and dissolved in deionized water before use.

[0064] D-xylose, D-xylose assay kit (batch number: 20091120, purchased from Nanjing Jiancheng Biotechnology Institute).

[0065] (3) Experimental equipment

[0066] Rat restraint frame (self-made); ACS-2EAS electronic scale (Beijing Fims Technology Development Co., Ltd.); EthoVision 3.0 analysis software (Noldus, Netherlands); WV-CP470 camera (Panasonic, Japan); open field box (self-made).

[0067] 1.2 Animal grouping design and modeling methods

[0068] (1) Group design

[0069] After one week of acclimatization, rats were initially screened, and those that were too active or too quiet were excluded. The remaining rats were randomly divided into three groups according to their body weight. Group A: normal control group, n=24; Group B: model group, n=24; Group C: traditional Chinese medicine group, n=25.

[0070] (2) Modeling method

[0071] A rat model of liver stagnation and spleen deficiency syndrome was established using chronic restraint stress.

[0072] Homemade rat restraint frame: made of wood, T-shaped structure, with a base 10 cm wide, 20 cm long and 2.8 cm thick, and a restraint platform at the top 22 cm long and 6.6 cm at its widest point. There are grooves on both sides suitable for placing the limbs and two adjustable adhesive nylon straps to fix the rat's chest and abdomen.

[0073] Rats in groups B and C were restrained and fixed on specially designed restraint frames for 3 hours daily at random times for 21 consecutive days. Meanwhile, rats in group A were fasted and deprived of water at the same time points and were allowed to roam freely in their respective cages for 3 hours / day without any restraint for 21 consecutive days.

[0074] (3) Administration method

[0075] The dosage for rats was the rat equivalent dose converted from the human dosage, with an administration volume of 3.854 g / kg·d and an oral gavage volume of 10 mL / kg body weight.

[0076] Starting from the first day of modeling, Group C was given 2 mL of Xiaoyao San effective component suspension by gavage from 8:00 to 8:30 every morning; Groups A and B were given the same volume of physiological saline as Group C.

[0077] 1.3 Indicator Observation

[0078] The selection of observation indicators was mainly to verify the success of the rat model of liver stagnation and spleen deficiency syndrome under restraint stress, and to provide a basis and support for the next step of the experiment. The experiment mainly adopted observation of general condition, food intake, weight change and gain, as well as behavioral experiments.

[0079] (1) General state observation

[0080] Observe the rats' mental state, stress state, physical appearance, and fecal condition daily.

[0081] (2) Food intake of rats

[0082] (3) Measurement of rat weight change

[0083] <1> Rat weight measurement

[0084] After the experiment began, the rats' weight was measured with an electronic scale at 7:30 a.m. every day. The dosage of medication was adjusted according to the weight and the weight gain of the rats was calculated to verify the success of the rat model of liver stagnation and spleen deficiency syndrome and to explore the daily changes in the rats' weight.

[0085] <2> Measurement of rat weight gain

[0086] The weight gain of rats in each group on days 1, 7, 14, and 21 was calculated and compared using the measured rat body weight.

[0087] Weight gain (g) = Weekly weekend weight after confinement (g) - Weekly weight before confinement (g)

[0088] (4) Behavioral experiments

[0089] The behavioral experiments employed the Open-field Test (OFT).

[0090] <1> Open field box construction

[0091] The open field test chamber used was a homemade, rectangular wooden structure measuring 100 cm × 100 cm × 40 cm. The inner walls and bottom were gray. The bottom was divided into 25 equal sections of 20 cm × 20 cm by black lines. The sections along the side walls were called the outer perimeter sections (16 in total), and the remaining sections were the central sections (9 in total), with the very center section being the center section. A camera was installed directly above the center section to record the rats' behavior over a specified period. The camera was concealed within the ceiling to avoid interfering with the rats' behavior.

[0092] <2> Experimental steps

[0093] On days 0, 7, 14, and 21 of the preliminary experiment, the experiment was conducted in a completely enclosed, quiet environment with blackout curtains, eliminating reference points and minimizing human and computer interference. Before the experiment, the rats were placed in the behavioral laboratory for 10 minutes to acclimatize. The procedure was performed by two people: one operator held the rat by the base of its tail and quickly placed it into the center compartment of the open field box, while the other operator operated the computer to simultaneously record and time the video. The observation period was 5 minutes. After observation, the rats were removed, their excrement was cleaned, and the bottom of the box was thoroughly wiped with a towel dampened with water and a low-concentration alcohol. The bottom of the box was then dried with a hairdryer to prevent any lingering odor from affecting the observation of the next rat. The experimental procedure was performed in the following order: normal group → model group → traditional Chinese medicine group → normal group → ... to avoid inconsistencies in the time points between groups and thus variations in their activity levels during measurement.

[0094] <3> Observation indicators

[0095] ① Number of times entering the central area: The number of times from when the rat has three paws entering the central area to when it has three paws leaving the area;

[0096] ② Central grid dwell time (s): The time from when the rat is placed in the central grid until it leaves the area with three paws;

[0097] ③ Central zone movement speed (cm / s): The average movement speed of the rat in the central zone;

[0098] ④ Central zone movement distance (cm): The total distance the rat moves in the central zone;

[0099] ⑤ Total moving speed (cm / s): The total moving speed of the rat in one open field experiment;

[0100] ⑥ Total distance traveled (cm): The total distance the rat traveled in one open field experiment.

[0101] 1.4 Behavioral Experiment Analysis

[0102] The EthoVision 3.0 software, an internationally recognized animal behavior activity marker analysis system developed by the Dutch company Noldus, was used to analyze and evaluate the spontaneous activity behavior of rats.

[0103] 1.5 Data Processing

[0104] All data are expressed as mean ± standard deviation ( ). Mean squared (±S). SPSS 11.5 statistical software was used to perform normality tests and homogeneity of variance tests on the obtained general data. If both tests were satisfactory, one-way ANOVA was used for statistical analysis, and the LSD-t test (Least significant difference t-test) was used for comparisons between groups. If either test was unsatisfactory, the Kruskal-Wallis H test (multiple independent samples) was used for statistical analysis in nonparametric statistics. For data obtained from continuous measurements of the same indicator, the comparison method between groups at the same time point was the same as before; for comparisons of the same treatment factor at different time points, a trend graph was used instead. P < 0.05 was used as the criterion for statistical significance. Statistical graphs were created using Excel 2003.

[0105] 2. Results

[0106] 2.1 Observation of the general condition of rats in each group

[0107] Before the experiment, the rats in each group were in good spirits, active and energetic; they reacted flexibly when caught; their fur was clean, white and glossy, their skin and mucous membranes were rosy, their eye fissures were normal, and their ears, paws and noses were light pink; their feces were grain-shaped and of moderate dryness and wetness.

[0108] When initially restrained, rats in both the model and treatment groups exhibited strong resistance, hissing, desperate struggle, biting of the restraint frame, increased respiratory rate, wide-open eyes, and significantly increased feces. After gavage and release from restraint, they were restless or stood in a defensive posture in the cage.

[0109] One week later, the general condition of the normal group rats was the same as before the modeling. The model group rats were hyperactive, agitated, and highly alert; they mostly stood before being captured. They were easily irritated, reacted quickly, and resisted strongly when restrained, hissing and arching their backs. They also resisted strongly during gavage, resisting with their tongues. After gavage or release from restraint, they jumped around restlessly or stood facing each other. Their eyes were wide open or glaring; their fur was relatively clean and glossy, white, and the color of their ears, paws, and noses was acceptable. The rats in the traditional Chinese medicine group were better than the model group rats in all aspects, with a better mental state and slight agitation. When captured, their cries were softer, but they resisted more strongly, arching their backs. Their fur was clean, white, and glossy, and the color of their ears, paws, and noses was rosy.

[0110] Two weeks later, no abnormalities were observed in the normal group rats. In the model group rats, their mental state was generally normal, with reduced agitation, decreased activity level, and lower alertness; their irritability decreased, and their responses to being handled were generally sluggish or slow. When restrained, they arched their backs, and their resistance was generally weak or absent. During gavage, their resistance, tongue resistance, and writhing decreased. After gavage or release from restraint, the rats mostly moved slightly or hid in corners; their eyes were dull, their fur was disheveled or unclean, dark, slightly dry, and yellowish in color, and their ears, paws, and noses were mostly pale. Their feces began to show loose or irregular consistency. In the herbal medicine group rats, they remained active and agile, with stronger resistance than the model group. After gavage or restraint, many ran or walked; their eyes were bright, their fur was still clean and white, and their ears, paws, and noses were still rosy; their feces only occasionally showed irregular consistency or were slightly soft.

[0111] At the end of the 3-week modeling period, the general condition of the normal group rats was basically the same as at the beginning. The model group rats were lethargic, drowsy or prone to lying down, huddled together, and showed little or no alertness; they responded slightly to stimulation and handling, occasionally making sounds, resisting, and arching their backs weakly and without much resistance or effort. During gavage, they showed little or no resistance, tongue resistance, and writhing. After gavage or release from restraints, they mostly lay dormant, arched their backs, curled up, or huddled together; their eyes were dull, squinting, or closed, with occasional discharge from the corners of their eyes; their fur was messy, unclean, dull, and yellowish; their ears, paws, and noses were pale; their feces were soft, wet, or loose. The rats in the traditional Chinese medicine group were in generally good spirits, and their reactions were still relatively agile. They resisted more strongly than the model group when restrained or gavaged; their eyes were still bright, with no discharge from the corners of their eyes; their fur was still relatively clean and moist, with a slight yellowish tinge; their ears, paws, and noses were still rosy or slightly pale; their feces were only occasionally irregular in consistency or slightly soft.

[0112] 2.2 Comparison of food intake among rats in each group

[0113] Combining Table 1 and Figure 1 The results showed that before restraint, there was no statistically significant difference in food intake among the groups of rats. On day 7 of restraint, at the same time point, the food intake of the model group and the traditional Chinese medicine group was significantly different from that of the normal group (P<0.01). On day 14 of restraint, only the food intake of the model group was significantly different from that of the normal group (P<0.05), while there was no statistically significant difference in food intake between the normal group and the traditional Chinese medicine group. On day 21 of restraint, there was a significant statistically significant difference between the normal group and the model group (P<0.01), no statistically significant difference between the normal group and the traditional Chinese medicine group, but a statistically significant difference in food intake between the traditional Chinese medicine group and the model group (P<0.05).

[0114] Table 1: Changes in food intake of rats in each group ( ±S, g)

[0115]

[0116] Note: Compared with the normal group,※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0117] 2.3 Changes in water intake of rats in each group

[0118] Combine Table 2 and Figure 2 This indicates that before restraint, there was no statistically significant difference in water intake among the groups of rats; on day 7 of restraint, at the same time point, the water intake of the model group was significantly different from that of the normal group (P<0.01); on days 14 and 21 of restraint, there was no statistically significant difference in water intake among the groups, but from... Figure 2 It can be seen that on day 21 of restraint, the water intake of rats in each group showed a different trend, with the normal group > the Chinese medicine group > the model group.

[0119] Table 2: Changes in water intake of rats in each group ( ±S, g)

[0120]

[0121] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0122] 2.4 Changes in body weight of rats in each group

[0123] Table 3: Changes in body weight of rats in each group ( ±S, g)

[0124]

[0125] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0126] Combined with Table 3 and Figure 3The results showed that before restraint, there were no statistically significant differences in body weight among the groups. On day 7 of restraint, at the same time point, the body weight of rats in the model group and the traditional Chinese medicine group was significantly different from that in the normal group (P<0.01, P<0.05). On day 14 of restraint, the body weight of rats in the model group and the traditional Chinese medicine group was significantly different from that in the normal group (P<0.01). On day 21 of restraint, there were statistically significant differences between the normal group and the model group (P<0.01), between the normal group and the traditional Chinese medicine group (P<0.05), and between the traditional Chinese medicine group and the model group (P<0.05).

[0127] 2.5 Open field test results for each group of rats

[0128] (1) Comparison of the number of times rats from each group entered the central area

[0129] Table 4: Comparison of the number of times rats entered the central zone in each group ( ±S, times)

[0130]

[0131] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0132] Combine Table 4 and Figure 4 The results showed that from before restraint to day 14, the number of times rats entered the central grid decreased in all groups. Before restraint, there was no statistically significant difference in the number of times rats entered the central grid among the different groups. On day 7, the number of times rats entered the central grid decreased in all groups at the same time point, but the difference was not statistically significant. On day 14, the number of times rats in the Chinese medicine group entered the central grid was significantly different from that in the normal group and the model group (P<0.01, P<0.05). On day 21, the number of times rats entered the central grid increased significantly in the normal group, but there was no statistically significant difference among the groups.

[0133] (2) Comparison of dwell time in the central grid of rats in each group

[0134] Table 5: Comparison of dwell time in the central grid among rats in each group ( ±s, s)

[0135]

[0136] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group,▲ P<0.05, ▲▲ P<0.05.

[0137] Combined with Table 5 and Figure 5 The results showed that the central grid dwell time of rats in all groups decreased from before restraint to day 14 of restraint. However, from day 14 to day 21 of restraint, the central grid dwell time of rats in the model group continued to decrease, while that in the normal group and the traditional Chinese medicine group showed an increasing trend. There were no statistically significant differences in central grid dwell time among the groups before restraint and on days 7 and 14. On day 21 of restraint, the difference between the normal group and the model group was statistically significant (P<0.01), while there were no statistically significant differences between the normal group and the traditional Chinese medicine group, or between the traditional Chinese medicine group and the model group.

[0138] (3) Comparison of the movement speed of rats in the central area among the groups

[0139] Table 6: Comparison of moving speed of rats in the central area among different groups ( ±S, cm / s)

[0140]

[0141] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0142] Combined with Table 6 and Figure 6 The results showed that from before restraint to the end of day 21 of restraint, there was no statistically significant difference in the central grid movement speed among the rats in each group, but the movement speed showed an overall increasing trend. On day 7 of restraint, the central grid movement speed of the rats in the model group was higher than that of the normal group and the Chinese medicine group, but on days 14 and 21 of restraint, the central grid movement speed of the rats in the normal group and the Chinese medicine group was higher than that of the model group.

[0143] (4) Comparison of the distances traveled by rats in the central zone among the different groups

[0144] Table 7: Comparison of the distance traveled by rats in the central region among different groups ( ±S, cm)

[0145]

[0146] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0147] Combined with Table 7 andFigure 7 The results showed that the central zone movement distance of rats in all groups decreased from before restraint to day 7 of restraint. On day 21 of restraint, except for the model group where the central zone movement distance continued to decrease, the normal group and the traditional Chinese medicine group showed an increasing trend. There were no statistically significant differences in the central zone movement distance among the groups before restraint and on days 7 and 14. On day 21 of restraint, there was a statistically significant difference between the normal group and the model group (P<0.01), a statistically significant difference between the traditional Chinese medicine group and the model group (P<0.05), and no statistically significant difference between the normal group and the traditional Chinese medicine group.

[0148] (5) Comparison of total movement speed of rats in each group

[0149] Table 8: Comparison of total moving speed of rats in each group ( ±S, cm / s)

[0150]

[0151] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0152] Combined with Table 8 and Figure 8 The results showed that the total movement speed of rats in each group decreased from before restraint to day 14 of restraint. On day 21 of restraint, there was almost no difference in the total movement speed between the normal group and the traditional Chinese medicine group. The difference between the normal group and the model group was statistically significant (P<0.05). The difference between the traditional Chinese medicine group and the model group was not statistically significant, but the traditional Chinese medicine group was significantly higher than that of the model group.

[0153] (6) Comparison of total movement distance of rats in each group

[0154] Table 9: Comparison of total movement distance of rats in each group ( ±S, cm / s)

[0155]

[0156] Note: Compared with the normal group, ※ P<0.05, ※※ P<0.01; compared with the model group, ▲ P<0.05, ▲▲ P<0.05.

[0157] Combining Table 9 and Figure 9The results showed that the total movement distance of rats in each group decreased from before restraint to day 14 of restraint. There were no statistically significant differences between the groups before restraint and on day 7 of restraint. On day 14 of restraint, the total movement distance of the model group was significantly different from that of the normal group (P<0.05), and the total movement distance of the traditional Chinese medicine group was significantly different from that of the model group (P<0.05). On day 21 of restraint, there was almost no difference in the total movement distance between the normal group and the traditional Chinese medicine group, and both groups were greater than those of the model group, but the statistical results showed no statistical significance.

[0158] Example 2: Content and related technical route of the proposed evaluation scale

[0159] 1. Content of the proposed evaluation scale

[0160] 1.1 Name of the evaluation scale

[0161] The scale is proposed to be named: Evaluation Scale for a Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency.

[0162] 1.2 Applicable Evaluation Objects

[0163] The scale is intended to be used to assess rats with a liver-spleen deficiency syndrome induced by chronic restraint stress.

[0164] 1.3 Purpose of the scale development

[0165] To develop an evaluation scale for a rat model of chronic restraint stress with liver stagnation and spleen deficiency, which has good reliability and validity, and to provide a new and reliable evaluation method and tool for this model research through evaluation studies.

[0166] 1.4 Item Setting and Project Classification and Evaluation Criteria

[0167] Since the scale evaluation will be conducted concurrently with the experimental procedure, the scale must be concise and the number of items must be minimized. Therefore, the scale is expected to have around 20 items, and after training, the observers are expected to complete the questionnaire in approximately 30 seconds.

[0168] Items are determined based on the following principles:

[0169] (1) Based on thinking habits;

[0170] (2) From the whole to the part, the overall information comes first;

[0171] (3) Based on observation habits, the parts are arranged from front to back, and the questions are arranged in this order according to the operation sequence;

[0172] (4) The results show that highly relevant information is grouped together;

[0173] (5) The duration of the time;

[0174] (6) The numbers start from 1 and are arranged sequentially.

[0175] Because too few item levels on the scale result in low sensitivity, while too many levels make it difficult to grasp the grading criteria, leading to low consistency, this scale uses a five-level quantification system, ranging from 4 to 0, decreasing progressively. Each level is primarily determined by symptom descriptions, allowing operators to better understand and refer to the scale during operation, thereby increasing the accuracy of the assessment.

[0176] 1.5 Information to be filled in on the scale.

[0177] Before the scale assessment, the assessors need to fill in four items: rat number, observation time, observer and experimenter.

[0178] 2. Development steps and technical approach of the evaluation scale

[0179] 2.1 Steps

[0180] After establishing the basic concepts of liver stagnation, spleen deficiency, liver stagnation and spleen deficiency, and stress, and referring to the development process of TCM clinical scales or questionnaires, combined with literature research and animal experimental methods, and following the principles and general procedures of scale development internationally, the "Evaluation Scale for a Rat Model of Chronic Restraint Stress and Liver Stagnation and Spleen Deficiency Syndrome" was developed under the guidance of TCM theoretical knowledge. The specific operating method is as follows:

[0181] (1) Based on existing literature, referring to textbooks and related books, and combining the research objectives and previous research results of this research group, we propose a theoretical framework, determine the item pool, and preliminarily envision the dimensions of this scale;

[0182] (2) Referring to the relevant diagnostic criteria for liver stagnation and spleen deficiency and the symptom descriptions of animal models in relevant literature, we further explored the overall structure and item composition of the evaluation scale for the chronic restraint stress liver stagnation and spleen deficiency rat model, and formed an item pool for measuring the model.

[0183] (3) Based on the above research, combined with expert opinions and the results of the research group discussion, the “screening symptom items” of the “Evaluation Scale for Rats with Chronic Restraint Stress and Liver Qi Stagnation and Spleen Deficiency Syndrome” were compiled.

[0184] (4) Use “screening symptom items” to conduct the first pre-observation on the model rats to determine the timing of symptom observation, and test the language description and operability of the items. Based on the results and professional knowledge, modify the language description of the items, delete items that are difficult to observe and operate, and form a “screening table”.

[0185] (5) Using the “screening table”, the model rats were observed for the second time. The observation results, the discussion results of the research group, professional knowledge and statistical results were combined to further optimize and screen the scale and items, and form the “observation table”.

[0186] (6) Using the “Observation Table”, a large-scale systematic observation of the model rats was conducted. The data of the observation results were statistically analyzed. Combining traditional Chinese medicine theory and relevant professional knowledge, various methods were used to analyze the rationality of the structure and items of the scale, further optimize the scale, and form the final “Evaluation Scale of Chronic Restraint Stress Liver Qi Stagnation and Spleen Deficiency Rats (Version 1)”. The reliability and validity of the “Scale (Version 1)” were analyzed to prepare the foundation for further improvement of the scale.

[0187] 2.2 Technical Approach

[0188] The development of the "Evaluation Scale for a Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency (Version 1)" organically combines animal experimental procedures with scale observation, forming a complementary and mutually reinforcing process. The specific technical framework is illustrated in the diagram below. Figure 10 , Figure 11 As shown.

[0189] (1) Experimental Operation Section

[0190] like Figure 10 As shown in the experimental procedure section, this is the necessary foundation for the development and evaluation of the scale. Only when the model is confirmed as the "chronic restraint stress liver stagnation and spleen deficiency rat model" can the target of the developed scale be determined to be this syndrome animal model, rather than other models.

[0191] (2) Scale Development and Evaluation Section

[0192] like Figure 11 As shown.

[0193] Example 3: Construction of the structure and items of the "Evaluation Scale for a Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency"

[0194] The structure is the basic framework of a scale, including its dimensions and individual items. A dimension consists of several items reflecting a common trait, while an item is a specific measurement question within the scale that addresses the predictive purpose; these are the core components of the scale. The rationality of the scale's framework, including its structure and items, is extremely important for its measurement capability.

[0195] Therefore, guided by traditional Chinese medicine theory, this application, based on the concepts of stress and liver stagnation and spleen deficiency syndrome and related diagnostic criteria, and drawing on psychometric techniques and clinical scale development procedures and methods, proposes a theoretical framework based on the TCM syndrome model and the previous work of this research group. Combining expert opinions and animal model observation and evaluation, the basic structure of the scale is preliminarily summarized, items under the structural dimensions are established, and finally the "Evaluation Scale for a Rat Model of Chronic Restraint Stress and Liver Stagnation and Spleen Deficiency Syndrome (Version 1)" is formed.

[0196] 1. Observe the composition of the table entries.

[0197] like Figure 12 As shown in the figure, the numbers in parentheses represent the number of items.

[0198] 2. Basis for constructing the theoretical framework

[0199] Based on existing literature, the research group initially designed the four dimensions of this scale to include the general state, stress response, physical appearance, and fecal condition involved in rat models of liver stagnation syndrome, spleen deficiency syndrome, and liver stagnation and spleen deficiency syndrome. The research group believes that in the chronic restraint stress rat model of liver stagnation and spleen deficiency, the use of chronic restraint stress during the modeling process leads to a progression from liver stagnation to spleen deficiency, ultimately resulting in liver stagnation and spleen deficiency syndrome. This can be fully reflected in the general state (e.g., mental state, alertness), stress response (e.g., resistance, arched back, resistance to gavage), physical appearance (e.g., skin, mucous membranes), and fecal condition. This aligns with the TCM theories of "anger injuring the liver" and "liver disease spreading to the spleen." The close relationship between stress and the TCM concept of "liver" can lead to "liver qi stagnation," a finding made by the research group in previous basic research.

[0200] This application also takes into account the uniqueness of the restraint stress modeling method. During its modeling process, there is a process of restraining and immobilizing the rats, which is also a stress process for the rats. Therefore, this scale considers the resistance, arching of the back and struggling behaviors during the restraint process.

[0201] The specific measurement indicators, including 13 items such as body weight, food intake, water intake, body length, tail length, tail circumference, abdominal circumference, axillary temperature, heart rate, heart rhythm, grip strength, respiratory rate, and serum D-xylose measurement, were initially planned to be included in the evaluation scale. However, considering that the measurement of objective indicators is an independent model evaluation method, and that due to the characteristics of the animal model itself, there are certain difficulties in grading and quantifying and the problem of measurement and evaluation time, they were not included in the final "symptom screening items".

[0202] 3. Basic Structure of the Scale

[0203] 3.1 Formation of the "Symptom Screening Items" in the Item Pool

[0204] Based on extensive literature review and preliminary research by the research team, and combined with expert opinions and discussion results, a pool of "symptom screening items" was formed, comprising 71 items across 4 dimensions. The specific dimensions and item composition are shown in Table 10.

[0205] Table 10: Composition and Specific Content of "Symptom Screening Items"

[0206]

[0207]

[0208]

[0209] 3.2 Formation of the screening table

[0210] Based on literature review and our research group's previous work, although the symptom items obtained are frequently observed in experimental model evaluation, their suitability for the scale's evaluation requirements still needs to be verified through practical observation. Adhering to the principle of scientific rigor, our research group did not directly apply these "symptom screening items" to model evaluation. Instead, we conducted a pre-observation and evaluated the feasibility of the items based on the results. Necessary modifications were made to items that were difficult to observe, unsuitable for the scale's requirements, or repetitive, as well as to the descriptions of specific items and options, resulting in the "Screening Scale for a Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency" (see Table 11).

[0211] Table 11: Composition and specific contents of the "Screening Scale for a Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency"

[0212]

[0213]

[0214] 3.3 Filtering by Items in the Filter Table

[0215] After the screening checklist was developed, the research team conducted continuous and dynamic observations of the animal model rats using the checklist. Based on the observation results, the selection was carried out through feasibility analysis and statistical methods. The specific methods are as follows:

[0216] (1) By observation, items that are difficult to observe are removed. The specific items to be deleted and the basis for deletion in the screening table are shown in Table 12.

[0217] Table 12: Deleted items and criteria for the "Screening Scale for Rats with Chronic Restraint Stress and Liver Qi Stagnation and Spleen Deficiency Syndrome"

[0218]

[0219] (2) Specific technical methods for item statistical screening

[0220] After removing items that were difficult to observe, the remaining items were renumbered and then screened using statistical methods. The statistical methods used were the t-test, correlation coefficient method, and Cronbach's alpha coefficient method. An item was considered for deletion if two of the three screening methods met the deletion criteria (see Table 13).

[0221] Table 13: Statistical Methods for Filtering Items in the "Filter Table"

[0222]

[0223] (3) Statistical results of the "screening table" item selection

[0224] <1> Statistical results of t-test

[0225] To examine the discrimination and importance of the scale items, a two-sample t-test was used to compare the high-scoring group (the top 27% of participants) and the low-scoring group (the bottom 27% of participants) for each item. The results are shown in Table 14.

[0226] <2> Statistical results of correlation coefficient method

[0227] Statistical analysis was performed on the correlation coefficients between the scores of each item and the total score of the scale. The specific results are shown in Table 14.

[0228] <3> Cronbach's α coefficient method

[0229] Statistical analysis yielded an overall Cronbach's α coefficient of 0.8200 for the scale. The Cronbach's α coefficient was also calculated after removing each item from the scale. The statistical results are shown in Table 14.

[0230] Table 14: Summary of Statistical Results of Item Screening in the "Screening Table"

[0231]

[0232]

[0233] Note: "*": p < 0.05; "**": p < 0.01.

[0234] In principle, if two of the three statistical methods used indicate that an entry should be deleted, then that entry should be deleted.

[0235] Based on the above statistical results, combined with expert opinions, research group discussions, and professional knowledge, the deleted items in the "screening table" are summarized in Table 15.

[0236] Table 15: Items to be deleted from the "Filter Table" and the basis for deletion

[0237]

[0238]

[0239] In addition, during the modeling process, changes in "b4's vocalizations during capture" and "c1's fecal shape" did exist and will be temporarily retained for observation with a larger sample size. The decision on whether to delete them will be based on the results of the large sample observation.

[0240] Regarding the descriptions of the entries, entry "b2 (Reaction during capture)" is changed to "Action reaction during capture"; "b12 (Behavior when released from restraint)" is changed to "Behavior when placed in a cage after being restrained or gagged"; "c5 Fur condition (soft)" is changed to "Fur condition"; and "c6 Fur condition (clean)" is changed to "Fur condition".

[0241] Based on the above research results and screening criteria, the total number of items was reduced from 48 in the "screening table" to 26 through item screening and merging. After being rearranged, the "Observation Table of Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency" (hereinafter referred to as the "Observation Table") was formed.

[0242] 3.4 Specific dimensions and items of the "Observation Table for a Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency" (hereinafter referred to as the "Observation Table")

[0243] See Table 16 (each item is categorized into five levels, from 4 to 0, decreasing progressively).

[0244] Table 16: Specific Composition of Dimensions and Items in the "Observation Table"

[0245]

[0246] Example 4: Item Analysis and Item Selection of the Observation Table

[0247] In order to screen the items in the observation table, make the scale more concise and practical, and expand the sample size, the "observation table" was used for further observation and research. Through statistical analysis of the scale evaluation results, combined with relevant theories, expert opinions and the results of the research group's discussion, the items in the "screening table" were analyzed. The scale items were screened based on the results of the item analysis, and finally the "Evaluation Scale of Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency Syndrome" (hereinafter referred to as "Scale (Version 1)") was formed.

[0248] 1. Experimental Data

[0249] From April 8, 2010 to May 10, 2010, 150 male SD rats were housed in the animal facility of the Department of Traditional Chinese Medicine Diagnostics, School of Basic Medical Sciences, Beijing University of Chinese Medicine. After acclimatization, rats were induced to develop a rat model starting on April 18, 2010, and their dynamic behavior was monitored using an observation table.

[0250] 2. Research Methods

[0251] 2.1 Experimental Modeling

[0252] The rat model was established for 21 days using a restraint stress method, with restraint for 3 hours daily at random duration. The initial body weights were (211.17±9.743) g in the normal group, (210.75±10.398) g in the model group, and (213±11.280) g in the traditional Chinese medicine group (no statistically significant difference). After 21 days of modeling, the rat model was confirmed as a liver stagnation and spleen deficiency syndrome model through general observation, behavioral observation and analysis, and drug-related evidence.

[0253] 2.2 Evaluation and Screening Methods

[0254] The rat model was observed and evaluated using an "observation form". The evaluation results were summarized into scores and statistically analyzed using SPSS 11.5. The results, combined with expert opinions and discussions within the research group, were used to select scale items.

[0255] The specific statistical methods used are as follows:

[0256] (1) Factor analysis

[0257] Factor analysis is a multivariate statistical method that allows a large number of observed indicators to be categorized. This application employs principal component analysis and the orthogonal rotation method for maximum variance in factor analysis to extract items with factor loadings greater than 0.3.

[0258] (2) Independent samples t-test

[0259] The differences in the mean scores between the high-scoring group (the top 27% of participants) and the low-scoring group (the bottom 27% of participants) under each item of the scale were analyzed to examine the discriminative power of the items.

[0260] (3) Correlation coefficient method

[0261] The correlation coefficient method includes the correlation coefficient between items and scale scores, the correlation coefficient between items and dimension scores, and the correlation coefficient between items. The correlation coefficient between an item and the total scale and its corresponding dimension should be greater than 0.3. If the correlation coefficient between items is greater than 0.8, it indicates that the content measured by the two is highly consistent, and one of them can be deleted or the two can be merged based on professional knowledge or actual situation.

[0262] (4) Cronbach's α coefficient method

[0263] If removing an item increases the Cronbach's α coefficient of the scale or dimension, it indicates that the item decreases the Cronbach's α coefficient of the scale or dimension, and the item should be deleted; otherwise, it should be retained.

[0264] The criteria for removing items from the "Observation Table" are as follows:

[0265] (1) Delete entries with small factor loadings (factor loading < 0.4) in factor analysis.

[0266] (2) Delete entries with no statistically significant difference (P<0.05).

[0267] (3) Delete entries with low correlation coefficients (correlation coefficient < 0.3) between the entries and the total table and the dimension in which the entries are located.

[0268] (4) Delete the items that reduce the Cronbach's α coefficient of the scale.

[0269] (5) Delete entries that reduce the Cronbach's α coefficient of their respective dimensions.

[0270] (6) In the screening method, if two or more items meet the criteria, they will be deleted.

[0271] (7) If the correlation coefficient between items under a dimension is large (correlation coefficient > 0.8), then consider deleting or merging one item based on professional knowledge.

[0272] 4.3 Results

[0273] Based on the modeling, the rat model was observed and evaluated using an "observation table". Based on the evaluation results, relevant statistical tests were conducted to screen items and optimize the scale.

[0274] 4.3.3.1 Results of Exploratory Factor Analysis

[0275] (1) Examine whether the “observation table” is suitable for factor analysis.

[0276] Table 17: Results of the test on whether the data in the "Observation Table" are suitable for factor analysis

[0277]

[0278] The results showed that the KMO value was 0.843, which is suitable for factor analysis; the Bartley test of sphericity showed a chi-square value of 23556.083, P<0.01, indicating that the difference was statistically significant and suitable for factor analysis.

[0279] Note: Since there was virtually no data for the "eye discharge" item, factor analysis was performed after removing the item.

[0280] (2) Number of factors and contribution rate extracted from statistical results

[0281] Table 18: Eigenvalues, contribution rates, and cumulative contribution rates of factors in the "Observation Table"

[0282]

[0283] Based on the eigenvalue greater than 1, a total of 6 common factors were extracted, with a cumulative contribution rate of 62.770%.

[0284] (3) Factor loadings of each item under the factor

[0285] Table 19: Loading values ​​of items under factors

[0286]

[0287]

[0288] 3.2 Discrimination of Items under Each Dimension

[0289] Good entries should have a certain ability to distinguish. To examine the discriminative ability of the entries, an independent samples t-test was used to conduct statistical analysis on each entry. The specific results are shown in Tables 20 to 23.

[0290] Table 20: Discriminative power t-values ​​and statistical significance of items a1~a4 in the general state dimension

[0291]

[0292] Table 21: Discriminative t-values ​​and statistical significance of items b1-b9 of the stress response dimension.

[0293]

[0294] Table 22: Discriminative power t-values ​​and statistical significance of items c1~c10 in the appearance characterization dimension.

[0295]

[0296] Table 23: Discriminative power t-values ​​and statistical significance of items d1-d3 in the fecal condition dimension.

[0297]

[0298] 3.3 Correlation analysis results of items within each dimension of the "Observation Table" and their corresponding dimensions and scales.

[0299] Table 24: Correlation coefficients among items in the general state dimension and with their respective dimensions and scales.

[0300]

[0301] Note: ▲ * represents P<0.01, and * represents P<0.05.

[0302] Table 25: Correlation coefficients among items of the stress response dimension and with their respective dimensions and scales.

[0303]

[0304] Note: ▲ * represents P<0.01, and * represents P<0.05.

[0305] Table 26: Correlation coefficients among items in the appearance representation dimension and with their respective dimensions and scales

[0306]

[0307] Note: ▲ * represents P<0.01, and * represents P<0.05.

[0308] Table 27: Correlation coefficients among items in the fecal condition dimension and with their respective dimensions and scales.

[0309]

[0310] Note: ▲ * represents P<0.01, and * represents P<0.05.

[0311] 3.4 The impact of each item on the consistency of the scale and its corresponding dimension

[0312] Table 28: Scale and Cronbach's α coefficients for each dimension

[0313]

[0314] Table 29: Scale and Cronbach's α coefficients for each dimension after removing each item.

[0315]

[0316] Example 5

[0317] Based on the above analysis results, the scale items were screened, integrated, and adjusted, ultimately forming the "Evaluation Scale for a Rat Model of Chronic Restraint Stress with Liver Qi Stagnation and Spleen Deficiency (Version 1)". (See attached text.) Figure 13 .

[0318] Note: Liver Qi Stagnation and Spleen Deficiency Syndrome is composed of two syndromes: Liver Qi Stagnation Syndrome and Spleen Deficiency Syndrome. Therefore, in the rat animal model evaluation scale, Liver Qi Stagnation Syndrome and Spleen Deficiency Syndrome are identified independently and then combined to establish the diagnosis of Liver Qi Stagnation and Spleen Deficiency Syndrome.

[0319] 1. Diagnostic criteria for liver stagnation syndrome and spleen deficiency syndrome

[0320] 1.1 Items and scoring of Liver Qi Stagnation Syndrome

[0321] See Table 30.

[0322] Table 30: Items and Scoring for Liver Qi Stagnation Syndrome

[0323]

[0324] 1.2 Items and scoring for Spleen Deficiency Syndrome

[0325] See Table 31.

[0326] Table 31: Items and Scoring for Spleen Deficiency Syndrome

[0327]

[0328] 1.3 Shared items and scoring for liver stagnation syndrome and spleen deficiency syndrome

[0329] See Table 32.

[0330] Table 32: Common Items and Scoring for Liver Qi Stagnation Syndrome and Spleen Deficiency Syndrome

[0331]

[0332] 2. Scoring Instructions

[0333] 2.1 Raw scores: These are the scores marked on the scale, such as:

[0334] Item 5. Vigilance

[0335] (4) Very strong (3) Average (2) Relatively weak (1) Basically no vigilance (0) No vigilance at all

[0336] Select “(4) Very strong”, the original score is 4 points; select “(3) Average”, the original score is 3 points; select “(2) Weak”, the original score is 2 points; select “(1) Basically no vigilance”, the original score is 1 point; select “(0) No vigilance”, the original score is 0 points.

[0337] 2.2 Conversion score: The score after conversion from the original score, which is divided into two types: positive score and negative score.

[0338] 2.2.1 Positive scoring: This method involves scoring based on the original scores marked on the scale. The formula is: Positive score = Original score, for example:

[0339] Item 5. Vigilance

[0340] (4) Very strong (3) Average (2) Relatively weak (1) Basically no vigilance (0) No vigilance at all

[0341] Select “(4) Very strong” for 4 points; select “(3) Average” for 3 points; select “(2) Weak” for 2 points; select “(1) Basically no vigilance” for 1 point; select “(0) No vigilance” for 0 points.

[0342] 2.2.2 Reverse Scoring: This method involves rescoring the scale based on the original scores marked on the scale. The formula is: Reverse Score = 4 - Original Score, for example:

[0343] Item 5. Vigilance

[0344] (4) Very strong (3) Average (2) Relatively weak (1) Basically no vigilance (0) No vigilance at all

[0345] Select “(4) Very strong” for 0 points; select “(3) Average” for 1 point; select “(2) Weak” for 2 points; select “(1) Basically no vigilance” for 3 points; select “(0) No vigilance” for 4 points.

[0346] 2.3 Total score: Divided into total score for liver stagnation syndrome and total score for spleen deficiency syndrome.

[0347] 2.3.1 Total score for Liver Qi stagnation syndrome: It is the sum of the converted scores of each item of Liver Qi stagnation syndrome. The formula is: Total score for Liver Qi stagnation syndrome = Converted score (GY1) + Converted score (GY2) + ... + Converted score (GY10).

[0348] 2.3.2 Total score for spleen deficiency syndrome: It is the sum of the conversion scores of each item of spleen deficiency syndrome. The formula is: Total score for spleen deficiency syndrome = conversion score (PX1) + conversion score (PX2) + ... + conversion score (PX18).

[0349] 3. Determination of Evidence Type

[0350] 3.1 Diagnosis of Liver Qi Stagnation Syndrome

[0351] See Figure 14 .

[0352] (1) The total score of liver stagnation peaked on day 9, at 20.375±3.021;

[0353] (2) ROC results (Day 9) showed: AUC=0.706, 95% CI (0.553-0.859); Cut-off value=19.5.

[0354] (3) Recommendation: Start the assessment of liver stagnation syndrome from day 6 and measure for 4 consecutive days. If the total score of liver stagnation syndrome is ≥19.5 in any measurement, it can be determined as liver stagnation syndrome.

[0355] 3.2 Determination of Spleen Deficiency Syndrome

[0356] See Figure 15 .

[0357] (1) The total score for spleen deficiency peaked on Day 19, at 56.875±5.195;

[0358] (2) ROC results (Day 19) showed: AUC=0.830, 95% CI (0.708-0.952); Cut-off value=51.5.

[0359] (3) Recommendation: Start the assessment of spleen deficiency syndrome from day 18 and measure for 4 consecutive days. If the total score of spleen deficiency syndrome is ≥51.5 in any measurement, it can be determined as spleen deficiency syndrome.

[0360] 3.3 Diagnosis of Liver Qi Stagnation and Spleen Deficiency Syndrome:

[0361] The determination of liver stagnation and spleen deficiency syndrome = determination of liver stagnation syndrome (total score of liver stagnation syndrome ≥ 19.5) + determination of spleen deficiency syndrome (total score of spleen deficiency syndrome ≥ 51.5).

[0362] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for identifying an animal model of liver stagnation and spleen deficiency syndrome, characterized in that, The identification includes the following aspects: Activity state; Degree of mental hyperactivity; Whether restless; Posture before capture; Alertness; Degree of irritability; Motion reaction during capture; Degree of resistance; Degree of hunchback; Degree of resistance during gavage; Degree of tongue resistance during gavage; Degree of body twisting during gavage; Performance after being put into a cage after being bound or gavaged; Skin luster; Skin color; Degree of skin softness; Degree of skin disorder; Ear luster; Claw luster; Fecal form.

2. The method of authentication of claim 1, wherein, The rules of the identification include: Starting from the 6th day of establishing the animal model, the total score of liver stagnation syndrome is measured for 4 consecutive days, and if the total score of liver stagnation syndrome is greater than or equal to 19.5 in any one measurement, and starting from the 18th day of establishing the animal model, the total score of spleen deficiency syndrome is measured for 4 consecutive days, and if the total score of spleen deficiency syndrome is greater than or equal to 51.5 in any one measurement, it is determined that the liver stagnation and spleen deficiency syndrome animal model is successfully established; The total score of liver stagnation syndrome = GY1 + GY2 + GY3 + GY4 + GY5 + GY6 + GY7 + GY8 + GY9 + GY10; The total score of spleen deficiency syndrome = PX1 + PX2 + PX3 + PX4 + PX5 + PX6 + PX7 + PX8 + PX9 + PX10 + PX11 + PX12 + PX13 + PX14 + PX15 + PX16 + PX17 + PX18; Wherein, GY1 is valued according to the degree of mental hyperactivity: "extremely hyperactive" is valued as 4, "comparatively hyperactive" is valued as 3, "not too hyperactive" is valued as 2, "very not hyperactive" is valued as 1, and "not hyperactive" is valued as 0; GY2 is valued according to whether the animal is restless: "very restless" is valued as 4, "obviously" is valued as 3, "has" is valued as 2, "slightly" is valued as 1, and "none" is valued as 0; GY3 is valued according to the alertness of the animal: "very strong" is valued as 4, "general" is valued as 3, "relatively weak" is valued as 2, "basically no alertness" is valued as 1, and "no alertness" is valued as 0; GY4 is valued according to the degree of irritability of the animal: "extremely irritable" is valued as 4, "relatively irritable" is valued as 2, "not too irritable" is valued as 2, "very not irritable" is valued as 1, and "no reaction to stimulation" is valued as 0; GY5 is valued according to the degree of resistance of the animal: "strong" is valued as 4, "relatively strong" is valued as 3, "general" is valued as 2, "weak" is valued as 1, and "no resistance" is valued as 0; GY6 is valued according to the degree of hunchback of the animal: "strong" is valued as 4, "relatively strong" is valued as 3, "general" is valued as 2, "slightly" is valued as 1, and "none" is valued as 0; GY7 is valued according to the degree of resistance of the animal during gavage: "strong resistance leading to gavage failure" is valued as 4, "relatively strong resistance, but gavage is forced to succeed" is valued as 3, "general resistance, but has little effect on gavage" is valued as 2, "slight resistance, which has no effect on gavage" is valued as 1, and "no resistance" is valued as 0; GY8 according to the tongue resistance of the animal when gavage: "strong tongue resistance, gavage needle can not enter" is valued at 4, "tongue resistance, gavage needle has an impact" is valued at 3, "although there is resistance, but gavage needle can still be relatively smooth into" is valued at 2, "slightly resistant, but no impact on gavage" is valued at 1, "no resistance" is valued at 0; GY9 according to the degree of body twisting of the animal when gavage: "strong body twisting, leading to difficult to grasp or gavage failure" is valued at 4, "strong body twisting, but gavage success with difficulty" is valued at 3, "body twisting, but no too much impact on gavage" is valued at 2, "although there is body twisting, but no impact on gavage" is valued at 1, "no body twisting" is valued at 0; GY10 according to the performance of the animal when put into the cage after restraint or gavage: "bouncing and uneasy or confrontation" is valued at 4, "running or standing but no confrontation" is valued at 3, "running or running and standing, wall lying" is valued at 2, "walking or walking and standing, wall lying" is valued at 1, "hibernation and hunchback or huddle" is valued at 0; PX1 according to the activity state of the animal: "slobbering and lazy" is valued at 4, "lying and less active" is valued at 3, "not too active" is valued at 2, "slightly active" is valued at 1, "active and active" is valued at 4; PX2 according to the posture of the animal before capture: "standing" is valued at 0, "active" is valued at 1, "lying" is valued at 2, "crawling" is valued at 3, "huddling" is valued at 4; PX3 according to the alertness of the animal: "very strong" is valued at 0, "general" is valued at 1, "relatively weak" is valued at 2, "basically no alertness" is valued at 3, "no alertness" is valued at 4; PX4 according to the degree of irritability of the animal: "very easy to irritate" is valued at 0, "relatively easy to irritate" is valued at 1, "not too easy to irritate" is valued at 2, "very difficult to irritate" is valued at 3, "no response to stimulation" is valued at 4; PX5 according to the action response of the animal when caught: "very sensitive" is valued at 0, "general" is valued at 1, "not sensitive" is valued at 2, "dull" is valued at 3, "no response" is valued at 4; PX6 according to the degree of resistance of the animal: "strong" is valued at 0, "relatively strong" is valued at 1, "general" is valued at 2, "weak" is valued at 3, "no resistance" is valued at 4; PX7 according to the degree of arching of the animal: "strong" is valued at 0, "relatively strong" is valued at 1, "general" is valued at 2, "slightly" is valued at 3, "none" is valued at 4; PX8 according to the degree of resistance of the animal when gavage: "strong resistance, leading to gavage failure" is valued at 0, "strong resistance, but gavage success with difficulty" is valued at 1, "general resistance, but no too much impact on gavage" is valued at 2, "although there is resistance, but no impact on gavage" is valued at 3, "no resistance" is valued at 4; PX9 according to the tongue resistance of the animal when gavage, "strong tongue resistance, gavage needle cannot enter" is valued as 0, "tongue resistance has influence on gavage needle entering" is valued as 1, "although there is resistance, but gavage needle can still enter smoothly" is valued as 2, "slightly resistant, but has no influence on gavage" is valued as 3, "no resistance" is valued as 4; PX10 according to the degree of body twisting of the animal when gavage, "strong body twisting, leading to difficult to grasp or gavage failure" is valued as 0, "strong body twisting, but gavage is successful with difficulty" is valued as 1, "body twisting, but has no great influence on gavage" is valued as 2, "although there is body twisting, but has no influence on gavage" is valued as 3, "no body twisting" is valued as 4; PX11 according to the performance of the animal when put into the cage after being bound or gavage, "bouncing and unstable or confrontation" is valued as 0, "struggling or standing but no confrontation" is valued as 1, "running or standing after running, wall lying" is valued as 2, "walking or standing after walking, wall lying" is valued as 3, "hibernation, arching back or huddling" is valued as 4; PX12 according to the luster of the fur of the animal, "bright luster" is valued as 0, "still luster" is valued as 1, "slightly dark" is valued as 2, "dim" is valued as 3, "dim and black" is valued as 4; PX13 according to the color of the fur of the animal, "white color" is valued as 0, "yellowish" is valued as 1, "slightly yellow in small part" is valued as 2, "most of the fur is yellow" is valued as 3, "dry yellow" is valued as 4; PX14 according to the softness of the fur of the animal, "soft and clean" is valued as 0, "still soft" is valued as 1, "slightly dry" is valued as 2, "slightly dry" is valued as 3, "dry" is valued as 4; PX15 according to whether the fur of the animal is scattered and unclean, "clean" is valued as 0, "still clean" is valued as 1, "slightly scattered or unclean" is valued as 2, "obvious scattered or unclean" is valued as 3, "messy and dirty" is valued as 4; PX16 according to the color of the pinna of the animal, "light pink" is valued as 0, "color is acceptable" is valued as 1, "color is light" is valued as 2, "light white" is valued as 3, "pale" is valued as 4; PX17 according to the color of the claw of the animal, "ruddy" is valued as 0, "still ruddy" is valued as 1, "color is light" is valued as 2, "light white" is valued as 3, "pale" is valued as 4; PX18 according to the shape of the feces of the animal, "dry and hard feces" is valued as 0, "normal" is valued as 1, "soft and wet feces, but good shape" is valued as 2, "soft and wet feces, but good shape" is valued as 3, "wet and soft or loose" is valued as 4.

3. The identification method as described in claim 2, characterized in that, The animal is a rat.

4. The method for constructing an animal model of liver stagnation and spleen deficiency syndrome, characterized in that, It comprises: modeling, and identifying the animal model from the following aspects of the animal: identifying qualified animal model after identification: activity state; degree of mental excitement; whether restless; attitude before capture; alertness; degree of irritability; action response when caught; degree of resistance; degree of arching back; degree of resistance when gavage; degree of tongue resistance when gavage; degree of body twisting when gavage; performance when put into the cage after being bound or gavage; fur luster; fur color; fur softness; degree of scattered and unclean fur; color of pinna; color of claw; shape of feces. The animal is a rat.

5. The construction method of claim 4, wherein, The identified rules include: The liver stagnation syndrome total score is evaluated from the 6th day of establishing the animal model, continuously measured for 4 days, and any one measurement of the liver stagnation syndrome total score is greater than or equal to 19.5, and the spleen deficiency syndrome total score is evaluated from the 18th day of establishing the animal model, continuously measured for 4 days, and any one measurement of the spleen deficiency syndrome total score is greater than or equal to 51.5, and it is determined to be qualified for identification; The liver stagnation syndrome total score = GY1 + GY2 + GY3 + GY4 + GY5 + GY6 + GY7 + GY8 + GY9 + GY10; The spleen deficiency syndrome total score = PX1 + PX2 + PX3 + PX4 + PX5 + PX6 + PX7 + PX8 + PX9 + PX10 + PX11 + PX12 + PX13 + PX14 + PX15 + PX16 + PX17 + PX18; GY1 is valued according to the degree of mental hyperactivity: "extremely hyperactive" is valued as 4, "comparatively hyperactive" is valued as 3, "not too hyperactive" is valued as 2, "very not hyperactive" is valued as 1, and "not hyperactive" is valued as 0; GY2 is valued according to whether the animal is restless: "very restless" is valued as 4, "obviously" is valued as 3, "has" is valued as 2, "slightly" is valued as 1, and "none" is valued as 0; GY3 is valued according to the alertness of the animal: "very strong" is valued as 4, "general" is valued as 3, "relatively weak" is valued as 2, "basically no alertness" is valued as 1, and "no alertness at all" is valued as 0; GY4 is valued according to the degree of irritability of the animal: "extremely irritable" is valued as 4, "relatively irritable" is valued as 2, "not too irritable" is valued as 2, "very not irritable" is valued as 1, and "no reaction to stimulation" is valued as 0; GY5 is valued according to the degree of resistance of the animal: "strong" is valued as 4, "relatively strong" is valued as 3, "general" is valued as 2, "weak" is valued as 1, and "no resistance" is valued as 0; GY6 is valued according to the degree of arching of the animal: "strong" is valued as 4, "relatively strong" is valued as 3, "general" is valued as 2, "slightly" is valued as 1, and "none" is valued as 0; GY7 is valued according to the degree of resistance of the animal when gavage: "strong resistance leading to gavage failure" is valued as 4, "relatively strong resistance, but gavage is successful with difficulty" is valued as 3, "general resistance, but gavage is not greatly affected" is valued as 2, "slightly resistant, and gavage is not affected" is valued as 1, and "no resistance" is valued as 0; GY8 is valued according to the resistance of the tongue of the animal when gavage: "strong resistance of the tongue, and the gavage needle cannot enter" is valued as 4, "resistance of the tongue, and the gavage needle is affected" is valued as 3, "although there is resistance, the gavage needle can still enter smoothly" is valued as 2, "slightly resistant, but gavage is not affected" is valued as 1, and "no resistance" is valued as 0; GY9 According to the degree of body twisting of the animal when it is gavaged: "strong body twisting, leading to difficulty in gripping or gavage failure" is valued at 4, "strong body twisting, but gavage is successful with difficulty" is valued at 3, "body twisting, but has little effect on gavage" is valued at 2, "although there is body twisting, but it has no effect on gavage" is valued at 1, "no body twisting" is valued at 0; GY10 According to the performance of the animal when it is put into the cage after being restrained or gavaged: "bouncing and uneasy or confronting" is valued at 4, "running or standing but no confrontation" is valued at 3, "running or standing after running, wall lying" is valued at 2, "walking or standing after walking, wall lying" is valued at 1, "hibernation, arching or huddling" is valued at 0; PX1 According to the activity state of the animal: "slobbering and lazy" is valued at 4, "lying and less active" is valued at 3, "not very active" is valued at 2, "slightly active" is valued at 1, "active and active" is valued at 4; PX2 According to the posture of the animal before being caught: "standing" is valued at 0, "active" is valued at 1, "lying" is valued at 2, "crawling" is valued at 3, "huddling" is valued at 4; PX3 According to the alertness of the animal: "very strong" is valued at 0, "general" is valued at 1, "relatively weak" is valued at 2, "basically no alertness" is valued at 3, "no alertness" is valued at 4; PX4 According to the degree of irritability of the animal: "extremely irritable" is valued at 0, "relatively easy to irritate" is valued at 1, "not easy to irritate" is valued at 2, "very difficult to irritate" is valued at 3, "no response to stimulation" is valued at 4; PX5 According to the action response of the animal when it is caught: "very sensitive" is valued at 0, "general" is valued at 1, "not sensitive" is valued at 2, "dull" is valued at 3, "no response" is valued at 4; PX6 According to the degree of resistance of the animal: "strong" is valued at 0, "relatively strong" is valued at 1, "general" is valued at 2, "weak" is valued at 3, "no resistance" is valued at 4; PX7 According to the degree of arching of the animal: "strong" is valued at 0, "relatively strong" is valued at 1, "general" is valued at 2, "slightly" is valued at 3, "none" is valued at 4; PX8 According to the degree of resistance of the animal when it is gavaged: "strong resistance, leading to gavage failure" is valued at 0, "strong resistance, but gavage is successful with difficulty" is valued at 1, "general resistance, but has little effect on gavage" is valued at 2, "although there is resistance, but it has no effect on gavage" is valued at 3, "no resistance" is valued at 4; PX9 According to the resistance of the tongue of the animal when it is gavaged: "tongue resistance is strong, and the gavage needle cannot enter" is valued at 0, "tongue resistance has an effect on gavage needle insertion" is valued at 1, "although there is resistance, but the gavage needle can still enter smoothly" is valued at 2, "slightly resistant, but has no effect on gavage" is valued at 3, "no resistance" is valued at 4; PX10 according to the degree of body twisting of the animal when it is gavaged: "strong body twisting, leading to difficulty in gripping or gavage failure" is valued as 0, "strong body twisting, but gavage is successful with difficulty" is valued as 1, "body twisting, but has little effect on gavage" is valued as 2, "although there is body twisting, it has no effect on gavage" is valued as 3, "no body twisting" is valued as 4; PX11 according to the performance of the animal when it is put into a cage after being restrained or gavaged: "bouncing and restless or confronting" is valued as 0, "struggling or standing but no confrontation" is valued as 1, "running or standing after running, wall lying" is valued as 2, "walking or standing after walking, wall lying" is valued as 3, "hibernation, arching or huddling" is valued as 4; PX12 according to the luster of the fur of the animal: "bright luster" is valued as 0, "still luster" is valued as 1, "slightly dark" is valued as 2, "dim" is valued as 3, "dim and black" is valued as 4; PX13 according to the color of the fur of the animal: "white" is valued as 0, "yellowish" is valued as 1, "slightly yellow in a small part" is valued as 2, "most of the fur is yellow" is valued as 3, "dry yellow" is valued as 4; PX14 according to the degree of softness of the fur of the animal: "soft and clean" is valued as 0, "still soft" is valued as 1, "slightly dry" is valued as 2, "slightly dry" is valued as 3, "dry" is valued as 4; PX15 according to whether the fur of the animal is scattered and unclean: "clean" is valued as 0, "still clean" is valued as 1, "slightly scattered or unclean" is valued as 2, "obvious scattering or unclean" is valued as 3, "messy and dirty" is valued as 4; PX16 according to the color of the pinna of the animal: "light pink" is valued as 0, "color is acceptable" is valued as 1, "color is light" is valued as 2, "light white" is valued as 3, "pale" is valued as 4; PX17 according to the color of the claw of the animal: "ruddy" is valued as 0, "still ruddy" is valued as 1, "color is light" is valued as 2, "light white" is valued as 3, "pale" is valued as 4; PX18 according to the shape of the feces of the animal: "dry and hard feces" is valued as 0, "normal" is valued as 1, "soft and wet feces, but good shape" is valued as 2, "soft and wet feces, but good shape" is valued as 3, "wet and soft or watery" is valued as 4.

6. The construction method of claim 4, wherein, The modeling comprises a drug modeling method, an emotional stimulation method and / or a chronic restraint stress modeling.

7. The construction method of claim 4, wherein, The modeling comprises a chronic restraint stress modeling.

8. The construction method of claim 4, wherein, The modeling comprises continuously restraining the animal for 21 days, 3 hours per day.

9. The construction method of claim 8, wherein, The restraint comprises restraining the animal on a restraint frame; The restraint frame has grooves for placing the limbs and adhesive nylon soft adhesive tape for restraint.

10. The construction method of claim 4, wherein, The animal is a rat.

Images