Diarrhea-type irritable bowel syndrome animal model and construction and evaluation method thereof

Abstract

The invention discloses a diarrhea-type irritable bowel syndrome animal model and a construction and evaluation method thereof. A spleen-kidney-yang-deficiency IBS-D mouse model is prepared by a method of combining traditional Chinese medicine and western medicine, combining low-temperature combined constraint and tail-clamping stimulation, and a prescription Sishen pill corresponding to a spleen-kidney-yang-deficiency prescription is selected to verify the model by applying a method of testing the syndrome by using a prescription. The invention aims to provide a reference method for establishing an IBS-D spleen-kidney yang deficiency animal model which conforms to the etiology and pathogenesis of traditional Chinese medicine and is simple and easy to operate. Compared with the prior art, the method disclosed by the invention is simple to operate and short in modeling period, and the model is more in line with the spleen-kidney yang deficiency type IBS-D pathogenesis.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model preparation, and specifically relates to a diarrhea-type irritable bowel syndrome animal model and a construction and evaluation method thereof. Background Art

[0002] Irritable bowel syndrome (IBS) is a functional gastrointestinal disease with abdominal pain, bloating, stool characteristics and abnormal defecation frequency as the main clinical manifestations. The global incidence of the disease is about 9.2%, of which diarrhea-predominant irritable bowel syndrome (IBS-D) accounts for more than 70% of IBS patients. IBS-D has a long course, is difficult to cure, and is prone to relapse. It belongs to the category of "diarrhea" in traditional Chinese medicine. Among them, the incidence of spleen and kidney yang deficiency type IBS-D accounts for 13.1%, and has been on the rise in recent years. The production of a disease-syndrome combined animal model that conforms to the etiology and pathogenesis of traditional Chinese medicine is a key prerequisite for the research of traditional Chinese medicine prescriptions and treatment principles and methods. In previous studies, mother-child separation, water avoidance stress, rectal dilation combined with Chinese medicine gavage are the most commonly used methods for creating animal models of spleen and kidney yang deficiency syndrome in IBS-D. However, the operation is difficult and the modeling cycle is long, generally up to 40-75 days. Single-factor Western medicine modeling can only replicate the disease model, and it is impossible to establish a disease-syndrome combined model that conforms to the TCM etiology and pathogenesis theory.

[0003] The pathogenesis of IBS-D is complex, and the "Expert Consensus on Diagnosis and Treatment of Irritable Bowel Syndrome in Traditional Chinese Medicine (2017)" emphasizes that liver depression, spleen deficiency, and dysfunction of Qi are the key to the onset of IBS-D. The present invention intends to produce an IBS-D mouse model of spleen and kidney yang deficiency by combining Chinese and Western medicines, superimposing low temperature, combined with restraint and tail clamping stimulation, and applying the "prescription-based syndrome" method to select the "Si Shen Wan" prescription corresponding to the spleen and kidney yang deficiency syndrome to verify the model, aiming to provide a reference method for establishing an IBS-D spleen and kidney yang deficiency syndrome animal model that is consistent with the etiology and pathogenesis of traditional Chinese medicine and is simple and easy to operate. Summary of the invention

[0004] The primary purpose of the present invention is to provide a method for constructing an animal model of diarrhea-type irritable bowel syndrome. Compared with the prior art, the method of the present invention is simple to operate and has a short modeling cycle, and this model is more consistent with the pathogenesis of spleen-kidney yang deficiency type IBS-D.

[0005] The construction method comprises the following steps: intragastric administration of low-temperature adenine, restraint and tail clamping stimulation, and intragastric administration of low-temperature senna leaf decoction to jointly establish the model.

[0006] Further,

[0007] When low-temperature adenine is administered intragastrically, the adenine suspension used is 0-4°C, 50-75 mg / (kg·d) is administered intragastrically, once a day, for 10-20 consecutive days;

[0008] Preferably,

[0009] When low-temperature adenine is administered orally, the temperature of the adenine suspension used is 0°C, and 50 mg / (kg·d) is administered orally once a day for 14 consecutive days.

[0010] Further,

[0011] Restraint and tail clamp stimulation were given by centrifuge tube restraint and intermittent tail clamp stimulation for 1 h starting from the 5th to 12th day of modeling, for 7 consecutive days.

[0012] Preferably,

[0013] Restraint and tail clamp stimulation were given by centrifuge tube restraint plus intermittent tail clamp stimulation for 1 h starting from the 8th day of model establishment for 7 consecutive days.

[0014] Restraint and tail clamping stimulation are performed simultaneously.

[0015] Specific operation: At 9 am every day, the limbs of mice in the model group were restrained in centrifuge tubes to restrict their activities, and the distal 1 / 3 of the tail of the mice was clamped with a long tail clip. The restraint and intermittent tail clamping time were both 1 hour, and this lasted for 7 consecutive days.

[0016] Further: The preparation and observation of the model were carried out at the same time every day, that is, at 9 am, and the modeling time was 7 days. Restraint stimulation: The mice were restrained for 1 hour using the centrifuge tube fixation method. A 50mL centrifuge tube with a small opening at the front was used to put the mice into the centrifuge tube. The excessive length of the centrifuge tube was filled with foam blocks to limit the free movement of the mice forward and backward, but the head position was guaranteed to have airflow holes so that air could enter. Tail clamping stimulation: The distal 1 / 3 of the mouse tail was clamped with a steel clamp for 1 hour. To avoid ischemic necrosis of the mouse tail, the tail was clamped intermittently every 1-3 minutes.

[0017] Further,

[0018] Low-temperature senna leaf decoction was administered orally starting from the 8th to 10th day of modeling. The senna leaf decoction was administered orally at a dose of 8-15 g / (kg·d) at 0-4°C, once a day for 5-7 consecutive days.

[0019] Preferably,

[0020] The intragastric administration of low-temperature senna leaf decoction started from the 10th day of model establishment. The temperature of the senna leaf decoction used was 0℃, and 10g / (kg·d) was administered intragastricly once a day for 5 consecutive days.

[0021] The second object of the present invention is to provide an animal model of diarrhea-type irritable bowel syndrome constructed by the method described.

[0022] The third object of the present invention is to provide an evaluation method for an animal model of diarrhea-type irritable bowel syndrome, which is evaluated based on the general behavioral changes, gastrointestinal motility and minimum pain threshold, serum SCFAs, 5-HT, ACTH, CORT, and D-xylose of mice; general behavioral changes include: mental state, gathering in groups, cold body and limbs, fur color, nail color, stool morphology, perianal cleanliness, stool moisture content, rectal temperature, body weight, and food intake, and then Si Shen Wan is used to test the formula.

[0023] Beneficial effects of the present invention:

[0024] The present invention successfully constructed an IBS-D spleen and kidney yang deficiency syndrome disease and syndrome animal model by using low-temperature adenine superimposed on low-temperature senna leaf gavage combined with tail clamping and centrifuge tube restraint. On the basis of detecting the macroscopic indicators of gastrointestinal motility and pain threshold reflecting visceral sensitivity, the microscopic indicators SCFAs and 5-HT that can regulate visceral hypersensitivity and the microscopic indicators ACTH, CORT, and D-xylose reflecting the yang of the spleen and kidney were combined for evaluation, and the model was verified by the prescription test method. On the basis of the macroscopic symptoms and signs related to the disease, the diversification of microscopic indicators was strengthened, and the model was verified by integrating the prescription test method, so as to construct an efficient and complete method for establishing an IBS-D spleen and kidney yang deficiency syndrome disease and syndrome combination model. The present invention takes into account both the pathogenic factors of IBS-D and the pathogenesis of spleen and kidney yang deficiency syndrome, and the model preparation time only takes 14 days, which greatly shortens the modeling cycle of the currently commonly used disease-syndrome combined model. At the same time, the model is made of mice, which is not only inexpensive, but also easier to operate. It can be said to be simple, inexpensive and effective, and is of great significance for quickly constructing an IBS-D spleen and kidney yang deficiency syndrome disease-syndrome combined animal model that is short-term effective, inexpensive and in line with the etiology and pathogenesis of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Comparison of food intake of mice in the model group and the normal group at different times during the modeling stage in Example 1;

[0026] Figure 2 : Comparison of feces moisture content of mice in the model group and normal group at different times during the modeling stage in Example 1;

[0027] Figure 3 : Comparison of the dietary intake of mice in each group at different times during the model validation phase in Example 1;

[0028] Figure 4 : Comparison of feces moisture content of mice in each group at different times during the model validation phase in Example 1;

[0029] Figure 5 : The effects of different stimuli on mouse behavior in Example 2;

[0030] Note: (A) food intake, (B) feces water content, (C) body weight growth rate, (D) rectal temperature; data are expressed as mean ± standard deviation (n=9); *P<0.05 indicates that the difference is statistically significant, **P<0.01 indicates that the difference is extremely statistically significant; NC: normal group; BM: restraint stimulation group; CTM: tail clamp stimulation group; BCTM: restraint plus tail clamp stimulation group;

[0031] Figure 6 : The effects of different stimuli in Example 2 on gastrointestinal motility, pain threshold, and visceral sensitivity indices in mice;

[0032] Note: (A) Gastric residual rate, (B) Small intestinal propulsion rate, (C) Pain threshold, (D) Serum SCFAs content, (E) Serum 5-HT content; Data are expressed as mean ± standard deviation (n=3 / 6); *P<0.05 indicates significant difference; **P<0.01, ***P<0.001 indicate extremely significant difference. DETAILED DESCRIPTION

[0033] The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention.

[0034] Embodiment 1:

[0035] 1 Materials and methods

[0036] 1.1 Animals

[0037] Sixty SPF female Kunming mice, weighing (20 ± 2) g, were provided by Hunan Slex Experimental Co., Ltd., with a quality certificate number of ZS-2023009190015. The mice were housed in the Experimental Animal Center of Hunan University of Chinese Medicine, and the experiments were conducted in a barrier environment with a room temperature of 23-25 ​​°C, a relative humidity of 50%-70%, a daily light / dark cycle of 12 h, and free access to food and water.

[0038] 1.2 Drugs

[0039] Adenine (Changsha Yaer Biotechnology Co., Ltd., batch number: EZ7890C450), 20 mg of adenine was dissolved in 0.4 ml of 0℃ sterile water to prepare a 5 mg / ml adenine suspension. Pinaverium bromide tablets (France Solvay Pharmaceuticals, batch number: HJ20163096), crushed and sieved, added with distilled water to prepare a 2.163 g / ml solution, and stored at 4℃. Senna leaves (Anhui Puren Chinese Medicine Pieces Co., Ltd., batch number: 2005302), soaked in 400 ml of boiling water for 30 min at a ratio of 100 g, then boiled for 3 min, concentrated into a decoction containing 1.0 g / ml of crude drug, and stored at 0℃.

[0040] Sishen Pills are composed of: 12 g of Psoralea corylifolia, 6 g of nutmeg, 3 g of Evodia rutaecarpa (soaked and fried), 6 g of Schisandra chinensis with vinegar, 6 g of ginger, and 6 g of jujube. The pieces were provided by Hunan Junhao Chinese Medicine Pieces Science and Trade Co., Ltd., with batch numbers of HY21012203, Hunan 20160113, 2020082806, HY21020306, 2103120084, and 170905. Weigh each Chinese medicine according to the above dosage ratio, soak it in 5 times the amount of water for 30 min, boil it over high heat, then simmer it over low heat for 30 min, filter out the liquid with gauze, add 200 ml of water to the residue, and decoct it in the same way to get the filtrate. Mix the two liquids and concentrate them into a decoction containing 1.0 g / ml of crude drug, and store it at 4 °C.

[0041] 1.3 Main reagents and instruments

[0042] ELISA kit: short-chain fatty acids (SCFAs, batch number: JM11498M1), serotonin (5-HT, batch number: JM02726M1), adrenocorticotropic hormone (ACTH, batch number: JM02837M1), corticosterone (CORT, batch number: JM02344M1), D-xylose (D-xylose, batch number: JM12962M2), Jiangsu Jingmei Biotechnology Co., Ltd.; isoflurane (batch number: 2023052902), Shandong Ante Animal Husbandry Technology Co., Ltd.; activated carbon (batch number: Q / 12HB 5029-2015), Xianshuiku Town Industrial Zone, Jinnan District, Tianjin.

[0043] 5810R desktop large-capacity general-purpose refrigerated centrifuge (Eppendorf, Germany); R520IP anesthesia machine (Revod, China); SPARK multifunctional microplate reader (Tecan, Switzerland); XY1000-2C weight scale (Changzhou Electronic Equipment Co., Ltd.); YD-206 medical electronic thermometer (Wenzhou Youshang Medical Technology Co., Ltd.).

[0044] 1.4 Model establishment

[0045] During the model establishment phase, 20 SPF female Kunming mice were fed adaptively for 3 days and then randomly divided into a normal group (n=10) and a model group (n=10) according to the random number table method. Modeling method: Starting from the first day of modeling, the mice in the model group were given 0℃ adenine suspension 50 mg / (kg·d) by gavage once a day for 14 consecutive days; starting from the eighth day of modeling, they were given centrifuge tube restraint plus intermittent tail clamp stimulation, with restraint and intermittent tail clamp time of 1 h for 7 consecutive days; starting from the tenth day, they were given 0℃ senna leaf decoction 10 g / (kg·d) by gavage once a day for 5 consecutive days. The mice in the normal group were given 0.4 ml sterile water by gavage once a day for 14 consecutive days.

[0046] 1.5 Model Validation

[0047] In the model validation phase, 40 SPF female Kunming mice were used. After 3 days of adaptive feeding, they were randomly divided into a blank group of 10 mice and a modeling group of 30 mice according to the random number table method. After modeling, the modeling group was divided into a model control group, a Sishen Pill group, and a pinaverium bromide group, with 10 mice in each group. The mice in the Sishen Pill group were given a 5 g / (kg·d) decoction of Sishen Pills by gavage (the equivalent dose was calculated according to the conversion method of experimental animals and human dosage in the Methodology of Pharmacological Research of Traditional Chinese Medicine), the mice in the pinaverium bromide group were given a clinical equivalent dose of 21.63 mg / (kg·d) of pinaverium bromide solution by gavage (converted to 150 mg per day for a 60 kg adult), and the mice in the model group and the blank group were given a 0.70 ml / d distilled water by gavage. Each group was gavaged twice a day for 7 consecutive days.

[0048] 1.6 Observation indicators and methods

[0049] According to the clinical manifestations of IBS-D spleen and kidney yang deficiency syndrome, combined with the "Criteria for Diagnosis and Efficacy of Clinical Diseases and Syndromes in Traditional Chinese Medicine", "Expert Consensus on Diagnosis and Treatment of Irritable Bowel Syndrome in Traditional Chinese Medicine (2017)", "Interpretation of the <2020 Chinese Expert Consensus on Irritable Bowel Syndrome>", and "Establishment and Evaluation of IBS-D Rat Model with Spleen and Kidney Yang Deficiency Combined with Diseases and Syndromes", the macroscopic physical sign diagnostic criteria for the mouse IBS-D spleen and kidney yang deficiency syndrome model were proposed: loose stools or undigested food, poor appetite, weight loss, cold body and limbs, laziness, listlessness, and high sensitivity to pain stimuli. The following observation indicators were determined accordingly.

[0050] 1.6.1 General

[0051] During the experiment, the general behavior of mice was observed, including mental state, gathering in groups, cold body and limbs, fur color, nail color, feces morphology, anal cleanliness, etc., and the food intake was tested every day. A fixed amount of feed (600 g) was added at a fixed time every day, and the remaining feed amount was measured after 24 hours. The average daily food intake (g) = (amount of feed added - remaining amount) / number of mice.

[0052] 1.6.2 Weight and rectal temperature

[0053] The body weight and rectal temperature of mice were measured on days 1, 4, 7, 11, and 14 of the model establishment phase and on days 1, 4, and 7 of the model validation phase, and the weight growth rate was calculated. The body weight measured on the first day of model establishment or administration was taken as the initial body weight, and the weight growth rate = (actual body weight at that time - initial body weight) / initial body weight × 100%.

[0054] 1.6.3 Moisture content of feces

[0055] On the 10th to 14th day of the model establishment phase and the 1st to 7th day of drug administration, fresh feces of mice were collected at the same time every day. The samples of 5 mice in the same cage in each group were collected as one sample, and the wet weight of the sample was weighed and recorded. The sample was heated and dried at 110°C to a constant weight, and the dry weight was weighed and the feces moisture content was calculated. Feces moisture content = (wet weight - dry weight) / wet weight × 100%.

[0056] 1.6.4 Pain Threshold

[0057] On the last day of modeling and drug administration, 4 mice were selected from each group by random number table to measure pain threshold. The mice in each group were fasted but not watered for 18 h, and then anesthetized with isoflurane inhalation (the mice were placed in the anesthesia induction box of the anesthesia machine, and the concentration of isoflurane was set to 5% and the airflow rate was 0.2 L / min. The concentration was slightly adjusted by observing the state of the mice). After the mice entered a coma, a 6F catheter coated with paraffin oil and with an air bag (the catheter was connected to a syringe for injecting water into the air bag) was inserted through the anus, so that the end of the air bag was 1 cm away from the anus, and it was fixed at the base of the tail of the mouse. Then the mice were placed in a transparent plastic cage box (length × width × height = 20 cm × 6 cm × 6 cm) to restrict the mice from turning around and only moving forward and backward. After the mice were fully awake, they were first adapted to the environment for 15 min, and then the minimum amount of water injected that caused pain in the mice was recorded, that is, the pain threshold (the strong contraction of the abdominal and back muscles of the mice or the lifting of the abdomen off the ground was used as the manifestation of their pain threshold). The measurement was repeated 3 times for each group of mice, with an interval of 5 min each time. Reference: “ED AL, KAWASKI M, PASRICHA PJ. A new model of chronic visceral hypersensitivity in adult rats induced by colonirritation during postnatal development[J]. Gastroenterology, 2000, 119(5): 1276-1285” and has been adjusted.

[0058] 1.6.5 Gastric residual rate and small intestinal propulsion rate (i.e. gastrointestinal motility test)

[0059] After fasting for 24 hours, each mouse was gavaged with 0.4 ml of semisolid nutrient paste with 1% carbon powder. After 20 minutes, the mice were killed by cervical dislocation, the pylorus, cardia and ileocecal part were ligated, the mesentery was separated, the whole stomach and small intestine were cut, and the total weight of the stomach and its contents was weighed. The removed stomach was cut open, the attached paste was washed off, and it was placed on absorbent paper to dry after washing, and the net weight of the stomach was weighed. The length of the removed small intestine and the propulsion length from the pylorus to the front of the black semisolid paste were measured, and the gastric residual rate and small intestine propulsion ratio were calculated. Gastric residual rate = (total stomach weight - stomach net weight) / mass of carbon powder semisolid paste × 100%; small intestine propulsion ratio = (propulsion length / total length of small intestine) × 100%.

[0060] 1.6.6 ELISA for detection of serum SCFAs, 5-HT, ACTH, CORT, and D-xylose

[0061] Under sterile conditions, orbital blood was collected from the remaining 6 mice in each group, and the serum was separated. The ELISA kit was used to detect the contents of SCFAs, 5-HT, ACTH, CORT, and D-xylose in the serum, and the detection was performed strictly according to the operating procedures of the kit.

[0062] 1.7 Statistical methods

[0063] SPSS 24.0 software was used for statistical analysis. Measurement data were normal distribution and were expressed as mean ± standard deviation ( ±s), independent sample t test was used for comparison between two groups with equal variance, one-way analysis of variance was used for comparison between multiple groups, and LSD test was used for comparison between two groups; Welch's approximate F test was used for comparison between multiple groups with unequal variance, and Dunnett's T method was used for comparison between two groups. If the data did not conform to the normal distribution, the median and interquartile range [M (Q1, Q3)] were used for comparison between two groups, and the Mann-Whitney test was used for comparison between two groups, and the Kruskal-Wallis test was used for comparison between multiple groups. The test level α = 0.05.

[0064] 2 Results

[0065] 2.1 IBS-D spleen and kidney yang deficiency modeling results

[0066] 2.1.1 General conditions of mice during the modeling phase

[0067] During the modeling period, the mice in the normal group were in good spirits, flexible, with smooth hair, normal nail color, clean perianal area, moderately soft and hard feces, dark brown in color, not deformed or stained by tweezers, and not easily deformed by pressing the feces on filter paper, with no obvious water stains through the paper. The mice in the model group were sleepy and lazy, liked to gather in groups, curled up and arched their backs, had slow reactions, sparse and falling hair, lighter nail color, dirty perianal hair, increased humidity of feces, yellow-brown in color, easily deformed or stained by tweezers, and easily deformed by pressing the feces on filter paper, with obvious water stains through the paper.

[0068] 2.1.2 Comparison of food intake between the two groups of mice at different times during the modeling phase

[0069] Figure 1 The results showed that compared with the normal group during the same period, the food intake of the model group showed an overall downward trend (F=13.99, P<0.001).

[0070] 2.1.3 Comparison of fecal water content between the two groups of mice on days 10 to 14 during the modeling phase

[0071] Figure 2 The results showed that compared with the normal group during the same period, the fecal water content in the model group showed an overall upward trend (F=7.765, P=0.024).

[0072] 2.1.4 Comparison of weight growth rates of the two groups of mice at different times during the modeling phase

[0073] Table 1 shows that compared with the normal group during the same period, the weight growth rate of mice in the model group increased on the 4th and 7th days of modeling (P<0.05), and the weight growth rate of mice in the model group decreased on the 14th day of modeling (P<0.01).

[0074] Table 1 Comparison of body weight growth rate of two groups of mice at different time after modeling [%, (`x ± s) / M (Q1, Q3)]

[0075]

[0076] Note: a) Compared with the normal group at the same time, P<0.05; b) Compared with the normal group at the same time, P<0.01.

[0077] 2.1.5 Comparison of rectal temperature of two groups of mice at different times during the modeling stage

[0078] Table 2 shows that compared with the normal group during the same period, the rectal temperature of mice in the model group was significantly reduced on the 4th, 7th, and 14th days of modeling (P<0.05 or P<0.01).

[0079] Table 2 Comparison of rectal temperature of two groups of mice after modeling [℃, (`x ± s) / M (Q1, Q3)]

[0080]

[0081] Note: a) Compared with the normal group at the same time, P<0.01; b) Compared with the normal group at the same time, P<0.05.

[0082] 2.1.6 Comparison of gastrointestinal motility and pain threshold between the two groups of mice during the modeling phase

[0083] Table 3 shows that compared with the normal group, the small intestinal propulsion rate of mice in the model group was increased (P<0.01), and there was no statistically significant difference in gastric residual rate and pain threshold (P>0.05).

[0084] Table 3 Comparison of gastric residual rate, small intestinal propulsion rate and pain threshold between the two groups of mice 14 days after modeling [x ± s / M (Q1, Q3)]

[0085]

[0086] Note: a) Compared with the normal group, P < 0.01.

[0087] 2.1.7 Comparison of serum SCFAs, 5-HT, ACTH, CORT and D-xylose levels between the two groups of mice during the modeling phase

[0088] Table 4 shows that compared with the normal group, the serum SCFAs and 5-HT levels in the model group were increased, and the ACTH, CORT, and D-xylose levels were decreased (P<0.05 or P<0.01).

[0089] Table 4 Comparison of serum SCFAs, 5-HT, ACTH, CORT and D-xylose levels in the two groups of mice 14 days after modeling [(`x ±s) / M (Q1, Q3)]

[0090]

[0091] Note: SCFAs, short-chain fatty acids; 5-HT, serotonin; ACTH, adrenocorticotropic hormone; CORT, corticosterone.

[0092] a) Compared with the normal group, P<0.05; b) Compared with the normal group, P<0.01.

[0093] 2.2 Validation results of the IBS-D spleen and kidney yang deficiency model

[0094] 2.2.1 General conditions of mice in each group during the model validation phase

[0095] During the model validation period, the blank group mice had normal mental state, autonomous activities, good perianal cleanliness, and normal fecal characteristics. Compared with the blank group, the model control group mice had poor mental state, drowsiness, lazy movement, liked to gather in groups, curled up and arched backs, slow reaction, sparse hair shedding, lighter nails, wet and sticky perianal fur, increased feces humidity, yellow-brown, easy to deform and stick to tweezers, easy to deform when pressed on filter paper, and obvious water stains. Compared with the model control group, the mental state and autonomous activities of the Sishenwan group mice recovered, the smoothness of the hair improved, and the phenomenon of drowsiness, lazy movement, gathering and curling up and arching back was improved. The above symptoms and signs in the pinaverium bromide group did not recover significantly. The perianal fur of the mice in the Sishen Pills and Pinaverium Bromide groups recovered to dryness, the moisture content of the feces decreased and became dark brown. The feces of the mice in the Sishen Pills group did not deform or stick to the tweezers when picked up with tweezers, and the feces were not easily deformed when pressed on filter paper, and no obvious water stains were found on the paper; the feces of the mice in the Pinaverium Bromide group were slightly deformed and slightly stuck to the tweezers when picked up with tweezers, and the feces were slightly deformed when pressed on filter paper, and no obvious water stains were found on the paper.

[0096] 2.2.2 Comparison of food intake of mice in each group at different times during the model validation phase

[0097] Figure 3 Compared with the blank group, the food intake of the model control group showed an overall decreasing trend with the increase of administration time; compared with the model control group, the food intake of the Sishen Pill group and the Pinaverium Bromide group showed an overall increasing trend with the increase of administration time (F=15.622, P<0.01). After 7 days of administration, the food intake of the Sishen Pill group was closer to that of the blank group.

[0098] 2.2.3 Comparison of fecal water content of mice in each group at different times during the model validation phase

[0099] Figure 4 It showed that compared with the blank group, the fecal moisture content of the model control group showed an overall upward trend; compared with the model control group, the fecal moisture content of the Si Shen Wan group and the Pinaverium Bromide group showed an overall downward trend (F=10.127, P=0.001). After 7 days of administration, the fecal moisture content of the Si Shen Wan group was closer to that of the blank group.

[0100] 2.2.4 Comparison of weight change rates of mice in each group at different times during the model validation phase

[0101] Table 5 shows that compared with the blank group at the same time, the weight growth rate of mice in the model control group decreased on the 1st, 4th and 7th days of administration (P<0.05 or P<0.01); compared with the model control group at the same time, the weight growth rate of mice in the Sishen Pill group and the Pinaverium Bromide group was significantly reduced on the 1st day of administration, and significantly increased on the 4th day of administration (P<0.05 or P<0.01). There was no statistically significant difference in the weight growth rate of mice between the Sishen Pill group and the Pinaverium Bromide group at each time point (P>0.05).

[0102] Table 5 Comparison of body weight growth rate of mice in each group at different time after administration (%, `x ± s)

[0103]

[0104] Note: a) Compared with the blank group at the same time, P<0.01; b) Compared with the blank group at the same time, P<0.05; c) Compared with the model control group at the same time, P<0.05; d) Compared with the model control group at the same time, P<0.01.

[0105] 2.2.5 Comparison of rectal temperature of mice in each group at different times during the model validation phase

[0106] Table 6 shows that compared with the blank group at the same time, the rectal temperature of mice in the model control group, pinaverium bromide group and Sishen Pills group was significantly reduced on the 1st, 4th and 7th days of administration (P<0.01); compared with the model control group at the same time, the rectal temperature of mice in the Sishen Pills group was significantly increased on the 4th and 7th days of administration, and the pinaverium bromide group was significantly increased on the 4th day of administration (P<0.05 or P<0.01); on the 7th day of administration, the rectal temperature of mice in the pinaverium bromide group was significantly lower than that in the Sishen Pills group (P<0.05).

[0107] Table 6 Comparison of rectal temperature of mice in each group at different times after administration [℃, (`x ± s) / M (Q1, Q3)]

[0108]

[0109] Note: a) compared with the blank group at the same time, P<0.01; b) compared with the blank group at the same time, P<0.05; c) compared with the model control group at the same time, P<0.01; d) compared with the model control group at the same time, P<0.05; e) compared with the pinaverium bromide group at the same time, P<0.05.

[0110] 2.2.6 Comparison of gastrointestinal motility and visceral sensitivity of mice in each group during the model validation phase

[0111] Table 7 shows that there were no statistically significant differences in gastric residual rate, small intestinal propulsion rate and pain threshold among the mice in each group (P>0.05).

[0112] Table 7 Comparison of gastric residual rate, small intestinal propulsion rate and pain threshold of mice in each group after 7 days of administration [`x ± s / M (Q1, Q3)]

[0113]

[0114] 2.2.7 Comparison of serum SCFAs, 5-HT, ACTH, CORT and D-xylose levels in each group of mice during the model validation phase

[0115] Table 8 shows that compared with the blank group, the serum SCFAs of mice in the model control group increased, and the contents of ACTH, CORT and D-xylose were significantly decreased (P<0.01); the serum SCFAs of mice in the pinaverium bromide group increased, and the contents of 5-HT and D-xylose were decreased (P<0.05 or P<0.01), and the 5-HT content in the Sishenwan group decreased (P<0.05); compared with the model control group, the serum SCFAs and 5-HT contents of mice in the Sishenwan group decreased, and the contents of ACTH, CORT and D-xylose were increased (P<0.05 or P<0.01); the serum 5-HT content of mice in the pinaverium bromide group decreased, and the CORT content increased (P<0.05 or P<0.01); compared with the pinaverium bromide group, the serum SCFAs content of mice in the Sishenwan group decreased, and the ACTH content increased (P<0.05 or P<0.01).

[0116] Table 8 Comparison of serum SCFAs, 5-HT, ACTH, CORT and D-xylose levels in mice in each group after 7 days of administration[(`x ±s / M(Q1,Q3)]

[0117]

[0118] Note: SCFAs, short-chain fatty acids; 5-HT, serotonin; ACTH, adrenocorticotropic hormone; CORT, corticosterone.

[0119] a) Compared with the blank group, P<0.01; b) Compared with the blank group, P<0.05; c) Compared with the model control group, P<0.05; d) Compared with the model control group, P<0.01; e) Compared with the pinaverium bromide group, P<0.01; f) Compared with the pinaverium bromide group, P<0.05.

[0120] During the model establishment stage of the present invention, the mice in the model group had poor mental state, slow reaction, liked to gather together, dirty hair around the anus, increased feces humidity, obvious water stains on paper, and other symptoms and signs of spleen and kidney yang deficiency IBS-D. Compared with the normal group, the diet of the model group was significantly reduced, and the weight growth rate gradually decreased, indicating that the modeling significantly inhibited the food intake and body weight growth of the mice, which is consistent with the "poor appetite and weight loss" of IBS-D spleen and kidney yang deficiency syndrome. The fecal water content is an objective indicator for evaluating diarrhea. The present invention found that with the increase of modeling time, the fecal water content of the model group increased significantly, reflecting the pathological manifestation of "loose stools" of IBS-D spleen and kidney yang deficiency syndrome; the rectal temperature test results showed that compared with the normal group, the rectal temperature of the mice in the model group was significantly reduced on the 4th, 7th, and 14th days of modeling, which is consistent with the pathological state of "cold body and cold limbs" of IBS-D spleen and kidney yang deficiency syndrome. Visceral hypersensitivity and gastrointestinal motility abnormalities are the key common features of various types of IBS. Pain threshold is often used to evaluate visceral hypersensitivity. The lower the pain threshold, the higher the visceral sensitivity. Gastric residual rate and intestinal propulsion rate can be used to evaluate gastrointestinal motility. Related studies have found that SCFAs are involved in visceral hypersensitivity and show an increasing trend in IBS-D, while 5-HT can affect visceral sensitivity by regulating gastrointestinal secretion and peristalsis. The experimental results of the present invention show that the serum SCFAs and 5-HT levels in the model group increased, the pain threshold of mice decreased, and the gastric residual rate and intestinal propulsion rate increased, indicating that modeling increased the visceral sensitivity of mice and abnormal gastrointestinal motility, which may be the reason for the clinical IBS-D manifestations of "abdominal distension, abdominal pain, diarrhea, and incomplete digestion". Serum ACTH and CORT levels can be used to reflect the abnormal kidney function of kidney yang deficiency, and serum D-xylose value can reflect the transportation and transformation function of spleen yang. The research results of the present invention show that the serum ACTH, CORT, and D-xylose levels of mice in the model group were significantly decreased, indicating that the spleen and kidney functions of mice in the model group were damaged and the yang of the spleen and kidney was insufficient. The above results show that the modeling of low-temperature adenine plus low-temperature senna gavage combined with tail clamping and centrifuge tube restraint not only conforms to the characteristics of IBS-D disease such as visceral hypersensitivity, abdominal pain, and diarrhea, but also conforms to the signs and objective indicators of spleen and kidney yang deficiency.

[0121] The results of model validation showed that after administration, the mental state and autonomous activities of mice in the Sishen Pills group were restored, the hair smoothness was improved, the phenomenon of drowsiness, laziness, gathering and curling up was improved, and the rectal temperature gradually increased. The above symptoms and signs in the Pinaverium bromide group did not recover significantly. The weight growth rate and food intake of mice in the Sishen Pills and Pinaverium bromide groups gradually increased, and the fecal water content decreased, among which the Sishen Pills group was closer to the blank group. Compared with the model control group, the gastric residual rate and small intestinal propulsion rate of mice in the Sishen Pills and Pinaverium bromide groups showed a decreasing trend, the pain threshold showed an increasing trend, and the 5-HT content was significantly reduced. The SCFAs content was significantly reduced only in the Sishen Pills group. At the same time, the administration of Sishen Pills can significantly increase the serum ACTH, CORT and D-xylose levels of model mice, which are close to the blank group, while Pinaverium bromide only increases the serum CORT content of model mice. Clinically, the pinaverium bromide group can be used to improve the symptoms of IBS-D, but it has no effect of warming and tonifying the spleen and kidney yang. Sishen Pills is a classic prescription for treating diarrhea caused by spleen and kidney yang deficiency. The prescription (Sishen Pills) corresponds to the syndrome (spleen and kidney yang deficiency syndrome). Modern studies have found that Sishen Pills are clinically effective in treating IBS-D with spleen and kidney yang deficiency syndrome. In this invention, both Sishen Pills and pinaverium bromide have a tendency to improve gastrointestinal motility and pain tolerance in mice to a certain extent, and can improve diarrhea and visceral hypersensitivity by reducing fecal water content and 5-HT content, thereby playing a role in treating IBS-D. At the same time, Sishen Pills can significantly improve the symptoms and signs of spleen and kidney yang deficiency in model mice, and can also effectively promote the recovery of spleen and kidney yang indicators (ACTH, CORT, D-xylose) in mice, making them close to the blank group, while the symptoms and signs of spleen and kidney yang deficiency such as lethargy, aggregation, and decreased anal temperature in the pinaverium bromide group mice were not significantly improved, and only the serum CORT content of the model mice was increased in the spleen and kidney yang indicators. This indicates that the pinaverium bromide in the present invention has a significant effect on the IBS-D disease of the model, while the TCM syndrome and spleen and kidney yang index of the model have not been effectively restored due to the improvement of the disease symptoms, which verifies that the TCM syndrome corresponding to the model will not be effectively improved due to the recovery of the disease, and the "disease" and "syndrome" of the corresponding model of Si Shen Wan are both significantly effective, indicating that the application of the prescription (Si Shen Wan) corresponding to the (spleen and kidney yang deficiency) syndrome can simultaneously improve the disease symptoms and corresponding TCM syndrome of the model, which verifies that the TCM syndrome of the model corresponds to the "syndrome" of Si Shen Wan. Therefore, based on the correspondence between the prescription (Si Shen Wan) and the syndrome (spleen and kidney yang deficiency syndrome), the present invention verifies that the model produced in the study meets the IBS-D spleen and kidney yang deficiency syndrome through the "prescription-based syndrome test" method.

[0122] Example 2: Partial exploration of the IBS model method of the present invention

[0123] This example compares three stimulation methods, namely restraint, tail clamping, and restraint plus tail clamping, to explore the superior method of establishing an animal model that conforms to the clinical etiology of irritable bowel syndrome (IBS).

[0124] 1 Materials and methods

[0125] 1.1 Animals

[0126] Thirty-six SPF female Kunming mice, weighing 20 ± 2 g, were provided by Hunan Slex Experimental Co., Ltd., and the animal quality certificate number is ZS-202307240020. The mice were housed in the Experimental Animal Center of Hunan University of Chinese Medicine, and the experiments were conducted in a barrier environment with air conditioning at room temperature of 23-25 ​​°C, relative humidity of 50%-70%, daily light / dark cycle of 12 h, and free access to food and water.

[0127] 1.2 Main reagents and instruments

[0128] ELISA kits: short-chain fatty acids (SCFAs, batch number: JM11498M1), serotonin (5HT, batch number: JM02726M1), Jiangsu Jingmei Biotechnology Co., Ltd. Isoflurane (batch number 2023052902), Shandong Ante Animal Husbandry Technology Co., Ltd.; activated carbon (batch number: Q / 12HB 5029-2015), Xianshuiku Town Industrial Zone, Jinnan District, Tianjin.

[0129] 5810R desktop large-capacity general-purpose refrigerated centrifuge (Eppendorf, Germany); R520IP anesthesia machine (Revod, China); SPARK multifunctional microplate reader (Tecan, Switzerland); XY1000-2C weight scale (Changzhou Electronic Equipment Co., Ltd.); YD-206 medical electronic thermometer (Wenzhou Youshang Medical Technology Co., Ltd.).

[0130] 1.3 Grouping and modeling

[0131] (1) Animal grouping

[0132] After 3 days of adaptive feeding, 36 SPF female Kunming mice were randomly divided into 4 groups, namely, normal (NC), restraint model (BM), tail clamp model (CTM), restraint plus tail clamp model (BCTM), with 9 mice in each group. The normal group was fed as usual without stimulation.

[0133] (2) Animal modeling

[0134] The modeling method of the restraint plus tail clamping model (BCTM) group was the same as that of Example 1. The modeling method of the restraint model (BM) group was only restrained as in Example 1 but without tail clamping stimulation. The tail clamping model (CTM) group was only tail clamped as in Example 1 but without restraint treatment.

[0135] 1.4 Observation indicators and methods

[0136] 1.4.1 General behavioral signs of mice

[0137] Combined with the clinical manifestations of IBS, with reference to the interpretation of the "2020 Chinese Irritable Bowel Syndrome Expert Consensus", the macroscopic signs diagnostic criteria for the mouse irritable bowel syndrome model were formulated: changes in stool characteristics or appearance, irritability and aggressive behavior. The irritable state caused by stress was evaluated by whether irritability and aggressive behavior occurred. The food intake, weight gain and loss, and rectal temperature of each group of mice under stress were measured by food intake, body weight, and rectal temperature. The characteristics of the feces of each group of mice were measured according to the water content of the feces. During the experiment, the general behavior of the mice was observed, including the mental and emotional state, hair shape and color, and feces shape and color; the daily food intake of each group of mice was observed and recorded; fresh feces were collected at 3 pm on the 1st to 7th day of modeling, and the feces water content was calculated; and the weight and rectal temperature of each group of mice were measured at 10 am on the 0th, 4th, and 7th days of modeling. The methods are the same as the detection method in Example 1.

[0138] 1.4.2 Detection of minimum pain threshold

[0139] Three mice were selected from each group using a random number table method, for a total of 12 mice, to determine the pain threshold, using the same detection method as in Example 1.

[0140] 1.4.3 Gastric residual rate and small intestinal propulsion rate (gastrointestinal motility test)

[0141] After the pain threshold of the 12 mice in 1.4.2 was measured, they continued to fast but not to drink water for 24 hours, and then underwent gastrointestinal motility testing in the same manner as in Example 1.

[0142] 1.4.4 HE staining to observe the morphological changes of mouse small intestine and colon tissue

[0143] The detection method is the same as that of Example 1.

[0144] 1.4.5 ELISA detection of changes in serum SCFAs and 5-HT levels

[0145] The detection method is the same as that of Example 1.

[0146] 1.4.6 Statistical methods

[0147] Statistical analysis was performed using Graphpad Prism10.1.2 and SPSS24.0 software. The measured data of each group were expressed as mean±standard deviation. One-way analysis of variance was used for samples with normal distribution and equal variance, and LSD test was used for inter-group comparison. Nonparametric rank sum test was used for samples with normal distribution or unequal variance. p<0.05 indicated a significant statistical difference, and p<0.01 indicated an extremely significant statistical difference.

[0148] 2. Results and Analysis

[0149] 2.1 Effects of different stimulation methods on mouse behavior

[0150] 2.1.1 Observation of the general condition of mice

[0151] The mice in the normal group moved normally, their hair was smooth and flat, and their feces were moderately dry and dark brown. On the 5th day of modeling, the mice in the restraint stimulation group and the tail clamping stimulation group showed that their forelimbs were raised off the ground and they were in a defensive standing posture. The mice in the restraint and tail clamping stimulation group were observed to fight and scream. The feces of the mice in the three stimulation groups gradually became dry and hard. Seven days after modeling, the mice in the three stimulation groups showed different degrees of aggression, liked to fight and climb the cage, and had dry and hard feces. Among them, the above symptoms of the mice in the restraint and tail clamping stimulation group were the most obvious, and they also showed IBS irritability symptoms such as restlessness and increased resistance to gavage.

[0152] 2.1.2 Changes in diet of mice

[0153] Compared with the normal group, the food intake of mice in the restraint stimulation group, tail clamp stimulation group, restraint plus tail clamp stimulation group showed a significant decrease trend. Figure 5 A (diet amount analysis F=11.539, P=0.007).

[0154] 2.1.3 Changes in the weight growth rate of mice

[0155] Compared with the normal group in the same period, on the 4th day of modeling, the weight growth rate of mice in the restraint stimulation group, tail clamping stimulation group, and restraint combined with tail clamping stimulation group showed a downward trend (P>0.05); on the 7th day of modeling, the weight growth rate of mice in the restraint stimulation group, tail clamping stimulation group, and restraint combined with tail clamping stimulation group also showed a downward trend (P>0.05; P>0.05; P<0.01). It can be seen that at the end of modeling, only the weight growth rate of the restraint combined with tail clamping stimulation group decreased significantly, as shown in Figure 2. Figure 5 C.

[0156] 2.1.4 Changes in water content of mouse feces

[0157] By comparing the fecal moisture content, it was found that compared with the normal group, the fecal moisture content of the mice in the restraint stimulation group, tail clamping stimulation group, and restraint combined with tail clamping stimulation group showed an overall downward trend with the increase of modeling time (F=8.766, P=0.006). Figure 5 B.

[0158] 2.1.5 Changes in rectal temperature of mice

[0159] On the first day of modeling, the rectal temperature of mice in the four groups was stable at around 37°C. Compared with the normal group, on the fourth day of modeling, the rectal temperature of mice in the restraint stimulation group, tail clamping stimulation group, and restraint superimposed tail clamping stimulation group showed an upward trend (P>0.05; P>0.05; P<0.01); on the seventh day of modeling, the rectal temperature of mice in the restraint stimulation group, tail clamping stimulation group, and restraint superimposed tail clamping stimulation group also showed an upward trend (P>0.05; P>0.05; P>0.05), and only on the fourth day of modeling, the rectal temperature of mice in the restraint superimposed tail clamping stimulation group increased significantly. Figure 5 D.

[0160] 2.2 Effects of different stimuli on visceral sensitivity and gastrointestinal motility

[0161] Depend on Figure 6 AB showed that compared with the normal group, the gastric residual rates of mice in the restraint stimulation group, tail clamping stimulation group, and restraint superimposed tail clamping stimulation group were all increased (P>0.05; P>0.05; P<0.05); the small intestinal propulsion rates were also increased (P>0.05; P>0.05; P>0.05), and only the gastric residual rate in the restraint superimposed tail clamping stimulation group was significantly increased.

[0162] Compared with the normal group, the pain thresholds of mice in the restraint stimulation group, tail clamp stimulation group, and restraint combined with tail clamp stimulation group were all lower (P>0.05; P<0.05; P<0.001). Figure 6 C.

[0163] As shown in Figure 6D-E, compared with the normal group, the serum SCFAs content of mice in the restraint stimulation group was increased, while the serum SCFAs content of mice in the tail clamping stimulation group and the restraint superimposed tail clamping stimulation group was decreased, and the differences were not significant (P>0.05). The serum 5-HT content of mice in the restraint stimulation group, the tail clamping stimulation group, and the restraint superimposed tail clamping stimulation group were all increased (P>0.05; P>0.05; P<0.01), and only the 5-HT content in the restraint superimposed tail clamping stimulation group was significantly increased.

[0164] 3.IBS model compliance analysis

[0165] Gastrointestinal dysmotility and visceral hypersensitivity are key to the onset of IBS. The pain threshold can reflect visceral sensitivity. The lower the pain threshold, the worse the body's tolerance to pain and the higher the visceral sensitivity. The fecal moisture content is used to measure the characteristics of the feces of each group of mice. SCFAs, as an important metabolite of intestinal flora, has multiple functions such as regulating intestinal immunity, anti-inflammation, and protecting the intestinal mucosal barrier. It tends to increase in diarrhea-type irritable bowel syndrome and decrease in constipation-type irritable bowel syndrome. 5-HT may affect visceral sensitivity by regulating gastrointestinal secretion and peristalsis, while SCFAs may induce visceral hypersensitivity and change stool characteristics in IBS-D patients by increasing 5-HT levels in the intestine. The results of this experiment showed that 7 days after modeling, compared with the normal group, the mice in the restraint stimulation group, the tail clamping stimulation group, and the restraint plus tail clamping stimulation group all showed varying degrees of emotional irritability, accompanied by IBS symptoms and signs such as decreased food intake, changes in stool characteristics, and decreased fecal water content. The mice in the restraint plus tail clamping stimulation group showed the most obvious emotional irritability and irritability symptoms and signs. At the same time, the gastric residual rate and 5-HT content of the mice in the restraint plus tail clamping stimulation group were significantly increased, and the tolerance to pain was significantly reduced, which is consistent with the gastrointestinal motility disorder and increased visceral sensitivity of IBS. This shows that restraint plus tail clamping stimulation has advantages among the three stimulation methods and can successfully construct an IBS mouse model.

Claims

1. A method for constructing an animal model of diarrhea-type irritable bowel syndrome, characterized in that: The following steps are involved: The model was established by intragastric administration of low-temperature adenine, restraint and tail clamping stimulation, and intragastric administration of low-temperature senna leaf decoction.

2. The construction method according to claim 1, characterized in that: When low-temperature adenine is administered orally, the adenine suspension used is at 0-4°C, 50-75 mg / (kg·d) is administered orally once a day for 10-20 consecutive days.

3. The construction method according to claim 2, characterized in that: When low-temperature adenine is administered orally, the temperature of the adenine suspension used is 0°C, and 50 mg / (kg·d) is administered orally once a day for 14 consecutive days.

4. The construction method according to claim 1, characterized in that: Restraint and tail clamp stimulation were given by centrifuge tube restraint plus intermittent tail clamp stimulation for 1 h from the 5th to 12th day of modeling for 7 consecutive days.

5. The construction method according to claim 4, characterized in that: Restraint and tail clamp stimulation were given by centrifuge tube restraint plus intermittent tail clamp stimulation for 1 h starting from the 8th day of model establishment for 7 consecutive days.

6. The construction method according to claim 1, characterized in that: Low-temperature senna leaf decoction was administered orally starting from the 8th to 10th day of modeling. The senna leaf decoction was administered orally at a dose of 8-15 g / (kg·d) at 0-4°C, once a day for 5-7 consecutive days.

7. The construction method according to claim 6, characterized in that: The intragastric administration of low-temperature senna leaf decoction started from the 10th day of model establishment. The temperature of the senna leaf decoction used was 0℃, and 10 g / (kg·d) was administered intragastricly once a day for 5 consecutive days.

8. The diarrhea-predominant irritable bowel syndrome animal model constructed by the method according to any one of claims 1 to 7.

9. A method for evaluating an animal model of diarrhea-type irritable bowel syndrome, characterized in that: The evaluation was conducted based on the general behavioral changes, gastrointestinal motility and pain threshold of mice, serum SCFAs, 5-HT, ACTH, CORT, and D-xylose indicators; general behavioral changes included mental state, gathering in groups, cold body and limbs, fur color, nail color, stool morphology, perianal cleanliness, stool moisture content, rectal temperature, body weight, and food intake, and then Si Shen Wan was used to test the symptoms.

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