Tx-j mouse animal model construction method based on cognitive impairment caused by intermingled phlegm and blood stasis

Abstract

The invention relates to a tx-j mouse animal model construction method based on phlegm-blood stasis syndrome cognitive impairment, and belongs to the technical field of medicine. The tx-j mouse is firstly fed with high-fat diet to make a hyperlipidemia state, then an ice-water bath method is adopted to make a'blood stasis' model, and finally the tx-j mouse animal model for the cognitive impairment of the syndrome of intermingled phlegm and blood stasis is constructed. After the model is successfully constructed, firstly observing the state and tongue color of the mouse, carrying out a water maze experiment, then detecting blood rheology indexes, blood fat indexes, tissue and serum copper contents and inflammatory factors, and observing brain pathological changes of the mouse. The high-fat feed is used for feeding to make a hyperlipidemia state, which is equivalent to'turbid phlegm 'of traditional Chinese medicine. The'blood stasis' model is manufactured by adopting an ice-water bath method on the basis of the traditional Chinese medicine theory of'cold coagulation and blood stasis', the modeling mode makes up the defects of the WD cognitive impairment model without combination of diseases and syndromes at present, and an effective experimental animal model is provided for revealing the pathogenesis of hepatolenticular degeneration cognitive impairment and evaluating the curative effect of traditional Chinese medicine.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a method for constructing a tx-j mouse animal model of cognitive impairment based on phlegm and blood stasis syndrome. Background Art

[0002] Wilson disease (WD) is a copper metabolism disorder caused by mutations in the ATPase Copper Transporting Beta (ATP7B) gene. Normally, ATP7B is located in the endoplasmic reticulum and Golgi apparatus of hepatocytes, responsible for transporting copper within the cell for incorporation into ceruloplasmin and excretion in the bile. This is the primary excretion pathway for copper. Consequently, defects in this enzyme lead to copper accumulation in the hepatocyte cytoplasm, subsequent necrosis, and release into the plasma, resulting in excessive copper deposition in the brain and other tissues and organs.

[0003] Cognitive impairment is one of the most important neurological symptoms in WD patients. Clinical studies have shown that 43 out of 72 WD patients (59.7%) have cognitive impairment. Based on the theory of traditional Chinese medicine, the research team previously divided the pathogenic factors of WD into three levels of hidden evil: the first is endowment evil, the second is metabolic evil, and the third is phlegm and blood stasis evil. Copper, as a part of normal water and blood, participates in the normal metabolism of the human body. Under pathological conditions, water metabolism is abnormal and phlegm is formed, and blood circulation is abnormal and blood stasis is formed. Phlegm can cause blood stasis, and blood stasis can also cause phlegm. The mutual binding of phlegm and blood stasis is the main cause and pathogenesis of hepatolenticular degeneration and the key to the onset of the disease. The mutual binding of phlegm and blood stasis runs through the entire course of WD patients.

[0004] The genetic defect of tx-j mice originates from a spontaneous recessive point mutation at position 2135 in exon 8 of the Atp7b gene, resulting in a missense G712D. Related studies have found that tx-j mice exhibit copper overload in the thalamus and lentiform nucleus at 3 months of age. At 12 months of age, copper concentrations in the striatum, hippocampus, and cerebellum of the mice's brains increase. As copper continues to accumulate, copper concentrations in the cerebral cortex also increase. When their behavior was analyzed, it was found that tx-j mice had mild abnormalities in their behavior, including a preference for forelimb use and clumsy movements. Therefore, tx-j mice are often used as a common animal model for studying cognitive impairment in WD.

[0005] However, there are currently no reports on animal models of WD cognitive impairment that combine disease and syndrome, and it is impossible to provide accurate experimental data for exploring the clinical efficacy of traditional Chinese medicine. Summary of the Invention

[0006] The purpose of the present invention is to propose a method for constructing a tx-j mouse animal model of cognitive impairment based on phlegm and blood stasis syndrome. For the first time, an animal model of cognitive impairment due to phlegm and blood stasis syndrome with combined symptoms is constructed, which can be easily applied in the fields of scientific research on pathogenesis, screening and evaluation of traditional Chinese medicine.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The method for constructing a tx-j mouse animal model of cognitive impairment based on phlegm and blood stasis syndrome is as follows: tx-j mice are first fed a high-fat diet to create a hyperlipidemia state, and then an ice-water bath method is used to create a "blood stasis" model, and finally a tx-j mouse animal model of cognitive impairment based on phlegm and blood stasis syndrome is constructed.

[0009] As the preferred technical solution of the present invention, the specific model construction method steps are:

[0010] Six-month-old tx-j mice were selected and fed a high-fat diet for 4 weeks. Then, starting from the 5th week, they were placed in an ice-water mixture once a day. When the mice's struggling movements significantly decreased and they were about to sink to the bottom, they were immediately fished out. The model construction lasted for 4 weeks, and finally an animal model of cognitive impairment with phlegm and blood stasis syndrome combining symptoms and functions was formed.

[0011] Furthermore, the high-fat diet formula used in the model construction method is: 75% basic feed + 10% cholesterol + 10% lard + 4% sucrose + 1% bile salt.

[0012] Furthermore, after the model was successfully constructed, the mouse's condition and tongue color were first observed, and a water maze experiment was conducted. Then, blood rheology indicators, blood lipid indicators, tissue and serum copper content, and inflammatory factors were tested to observe pathological changes in the mouse brain.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The model construction method employed in this invention uses a high-fat diet to induce hyperlipidemia, equivalent to "phlegm turbidity" in Traditional Chinese Medicine (TCM). Based on the TCM theory of "cold coagulation and blood stasis," an ice-water bath method is used to create a "blood stasis" model. This modeling approach addresses the shortcomings of current WD cognitive impairment models, which lack a clear link between disease and syndrome. Furthermore, this invention provides an effective experimental animal model for elucidating the pathogenesis of cognitive impairment in hepatolenticular degeneration and evaluating the efficacy of Traditional Chinese Medicine (TCM). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The tongue images of mice in each group are shown in Figure 2 .

[0016] Figure 2 Morris water maze test trajectory diagram of each group of mice.

[0017] Figure 3 Figure 3 shows the pathological staining results of mice in each group.

[0018] Figure 4 Transmission electron microscopy results of mice in each group. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with embodiments and drawings.

[0020] Example 1

[0021] Model animal selection

[0022] 1. Copper content determination

[0023] Brain and liver copper levels were measured in mice (WT and tx-j) at 1, 3, 6, 9, and 12 months of age. The results showed that brain and liver copper levels increased steadily starting at 3 months of age, reaching a peak at 6 months of age. Therefore, 6-month-old mice were selected for subsequent experiments (Table 1).

[0024] Table 1 Determination of copper content in mouse brain and liver

[0025] Age in months Brain copper (μg / g) WT Brain copper (μg / g)tx-j Liver copper (μg / g) WT Liver copper (μg / g)tx-j 1 2.37±0.19 2.28±0.5 12.72±1.79 <![CDATA[33.28±10.46 * ]]> 3 2.45±0.36 3.78±0.12 5.34±0.84 <![CDATA[242.19±48.38 * ]]> 6 3.52±0.45 <![CDATA[6.43±0.47 ** ]]> 7.99±2.07 <![CDATA[336.4±45.27 ** ]]> 9 2.75±0.29 <![CDATA[8.59±1.18 ** ]]> 4.22±0.15 <![CDATA[246.03±28.17 ** ]]> 12 3.07±0.31 <![CDATA[10.74±0.8 ** ]]> 3.78±0.29 <![CDATA[181.91±34.79 ** ]]>

[0026] Note: Compared with WT group ** P<0.01.

[0027] 2 Open field test and Barnes maze test

[0028] Mice (WT and tx-j) aged 1, 3, 6, 9, and 12 months were tested in the open field test and the Barnes maze test. The results showed that starting at 6 months of age, the total distance covered by tx-j mice and the time spent in the central area decreased (Tables 2 and 3).

[0029] Table 2 Open field test

[0030] Age in months total distance(m)WT total distance(m)tx-j ClimbingWT Climbingtx-j time in center(s)WT time in center(s)tx-j 1 22.52±1.26 20.25±3.28 29.87±3.21 37.83±5.4 118.03±6.79 100.99±7.14 3 29.19±1.61 27.81±2.12 79.25±1.87 69.54±5.75 177.7±5.96 91.92±4.74* 6 34.77±0.77 20.5±2.08** 85.32±3.54 30.35±2.16** 167.6±5.27 41.58±4.76** 9 18.96±4.24 15±3.13 61.36±5.82 23.95±3.43** 149±4.94 27.4±4.9** 12 17.16±1.49 10.31±1.24** 38.62±1.88 21.15±1.89** 137.15±9.97 19.72±1.85**

[0031] Note: Compared with WT group ** P<0.01

[0032] Table 3 Barnes maze test escape latency(s)

[0033] Age in months WT tx-j 1 121.17±4.34 130.62±1.89 3 199.3±5.45 172.55±4.26 6 59.65±1.8 192.69±6.3** 9 71.15±1.52 197.26±4.45** 12 96.29±9.11 220.85±4.91**

[0034] Note: Compared with WT group ** P<0.01

[0035] Example 2

[0036] Construction of a tx-j mouse model of cognitive impairment based on phlegm and blood stasis syndrome

[0037] 1 Materials and Methods

[0038] 1.1 Experimental Animals

[0039] Eighty six-month-old SPF tx-j mice and 20 WT mice of the same age were housed in individually ventilated cages in an SPF barrier environment at the Experimental Animal Center of Anhui Agricultural University. The temperature was (23 ± 2)°C, the humidity was 40% ± 5%, and the diet was ad libitum, with a 12-h / 12-h day / night cycle.

[0040] 1.2 Main Reagents

[0041] Total cholesterol (TC), total triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) assay kits (batch numbers: BH016Z, BH017Z, BH018Z, and BH019Z) were purchased from Shanghai Toujing Life Science Co., Ltd.; the hematoxylin-eosin (HE) staining kit (batch number: G1076) was purchased from Wuhan Saiweier Biotechnology Co., Ltd.) IL-1β, TNF-α, and IL-6 assay kits (batch numbers: H002-1-2, H052-1-2, and H007-1-1) were purchased from Nanjing Jiancheng.

[0042] 1.3 Main instruments

[0043] Water maze video analysis system (Anhui); blood rheology analyzer (Beijing ScioHealth); animal automatic biochemical analyzer (USA); Noldus maze video tracking system (Netherlands).

[0044] 1.4 Experimental methods

[0045] 1.4.1 Feeding steps

[0046] Normal group: 20 WT mice of the same strain were selected as controls and fed with normal feed (Xiaohe Technology, Jiangsu, the same below) for 8 weeks.

[0047] Model group: Twenty 6-month-old tx-j mice were selected and fed with normal feed for 8 weeks without other treatments.

[0048] Phlegm syndrome group: 20 6-month-old tx-j mice were selected and fed with a high-fat diet (formula: 75% basal diet + 10% cholesterol + 10% lard + 4% sucrose + 1% bile salt, the same below) for 8 weeks to form a high-fat state.

[0049] Blood stasis group: Twenty six-month-old tx-j mice were selected and fed a normal diet for 8 weeks. Starting from the fifth week, mice were placed in a glass cylinder with a bottom diameter of 10 cm, a depth of 25 cm, and a wall thickness of 0.2 cm at a fixed time each day. The ice-water mixture was kept at a temperature of 0°C to 4°C. When the mice showed a marked decrease in struggling movements and were about to sink to the bottom, they were immediately removed. This procedure was repeated once daily for 4 weeks to establish a blood stasis model.

[0050] Phlegm and Blood Stasis Group: Twenty six-month-old tx-j mice were selected and fed a high-fat diet for 8 weeks. Starting from the fifth week, at a fixed time each day, the mice were placed in a glass cylinder with a bottom diameter of 10 cm, a depth of 25 cm, and a wall thickness of 0.2 cm. The ice-water mixture was kept at a temperature of 0°C to 4°C. The mice were removed immediately when their struggling movements in the ice-water mixture significantly decreased and they were about to sink to the bottom. This procedure was repeated once a day for 4 weeks to establish a phlegm and blood stasis model.

[0051] 1.4.2 Detection Method

[0052] (1) A TCM syndrome scoring system was developed based on the literature. The scoring system was adjusted based on the dynamic changes in mouse symptoms during the modeling process and the differences between groups. The scoring system was divided into 0-3 points. Seven aspects were evaluated, including mental state, coat color, tongue color, claw color, ear color, feces, and body weight (Table 4). The severity of the syndrome was positively correlated with the score.

[0053] Table 4 TCM syndrome scoring table for mice

[0054] Syndrome items Scoring Criteria Mental state 0 points: active, bright eyes, quick reaction; 1 point: poor spirit, less active, slow reaction; 2 points: mental fatigue, significantly reduced activity; 3 points: avoidance, mental depression, significantly reduced aggressive behavior Coat color 0 points: black, fine and shiny, soft and dense; 1 point: abnormal, black with some white hair, slightly messy, less shiny; 2 points: accompanied by hair loss, frizzy, dull; 3 points: severe hair loss, dull coat Tongue color 0 points: red tongue; 1 point: red tongue with a slightly blue base, and the lingual vein filling does not exceed 3 / 4 of the main trunk length; 2 points: red tongue with a slightly darker color, the base of the tongue is dark purple, or the lingual vein filling does not exceed 3 / 4 of the main trunk length; 3 points: the tongue is obviously dark red, the base of the tongue is dark purple, and the lingual vein filling exceeds 3 / 4 of the main trunk length Claw color 0 points: pink; 1 point: dark red; 2 points: reddish and dark; 3 points: dark red or dark purple ear color 0 points: no petechiae, moderate number of blood vessels; 1 point: 1-2 petechiae, slightly increased number of blood vessels; 2 points: 3-4 petechiae, slightly increased number of blood vessels; 3 points: >4 petechiae stool 0 points: formed stool, soft stool; 1 point: loose stool, partially unformed stool; 2 points: loose stool, completely unformed stool body mass 0 points: no significant change in body weight; 1 point: body weight increased by 20% compared to before the experiment; 2 points: body weight increased by 20% to 30% compared to before the experiment; 3 points: body weight increased by more than 30% compared to before the experiment

[0055] (2) Morris water maze method was used to detect behavioral changes in mice in each group. The test procedure mainly included two parts: the positioning navigation test and the spatial exploration test, which mainly tested the mice's motor ability and spatial memory ability. The positioning navigation test lasted for 5 days, with 4 training sessions per day. The latency to the platform on the last day was recorded (unit: s), and the total distance swam by each group of mice before reaching the platform or within 90 seconds was recorded (unit: cm), and the average swimming speed was calculated (unit: cm / s). The movement route of each mouse in the pool was recorded to generate a movement trajectory diagram for the positioning navigation test. After the positioning navigation test, the underwater platform in the pool was removed, and the number of times each group of mice crossed the original platform position was recorded, which was recorded as the number of platform crossings (unit: times). The movement route of each mouse in the pool was recorded to generate a movement trajectory diagram for the spatial exploration test.

[0056] (3) Open field test: The test mouse was placed in the central area of ​​an open field arena, whose length, width, and height were 50, 40, and 30 cm, respectively. The mouse's movement was monitored for 5 min using an automated video tracking system (XinRuan, Shanghai, China). Various parameters were subsequently analyzed to elucidate the specific trajectory of the mouse.

[0057] (4) Barnes maze test: Mice were subjected to a standardized experiment to assess memory. Initially, each mouse was carefully placed on the platform and guided to the shelter. Subsequently, the mice underwent a spatial acquisition phase for 4 consecutive days, during which they were allowed unrestricted access to the testing room for 3 minutes per day. If the mouse failed to reach the shelter during the allocated exploration period, manual guidance was provided. On the fifth day, a probe test was performed, in which the mouse was allowed to navigate the maze freely while all entrances, including the entrance to the shelter, were sealed. The time required for each mouse to reach the escape box was measured using the Noldus maze video tracking system (Noldus, Amsterdam, The Netherlands). After each experiment, the platform and escape box were cleaned with 70% ethanol solution to eliminate any residual odor.

[0058] (5) Copper ion content determination: The urine volume was recorded, and then the sample was centrifuged. A 1 mL aliquot was taken and diluted to 10 mL with diluent (0.5% HNO3-0.1% TritonX-100), and the mixture was vortexed to determine the copper content in urine. Blood samples were processed in the same manner. In addition, 200 mg of liver, brain, kidney, and freeze-dried feces tissue samples were microwave digested. The copper content of all treated tissue samples was determined by atomic absorption spectrometry (AAS) (Thermo, Massachusetts, USA).

[0059] (6) Hemorheological response: Blood viscosity of mice in each group was measured by removing food and fasting for 12 hours before the experiment. Water was not allowed. After a minor anesthesia operation, blood was collected from the heart and divided into two tubes, one of which was placed in an anticoagulant tube containing heparin. The high, medium, and low shear viscosities of whole blood and plasma were measured by a hemorheology analyzer.

[0060] (7) Biochemical analysis reflects the changes in blood lipids in each group of mice. Another blood sample was collected from the above group, and the serum was separated. The contents of TC, TG, HDL-C and LDL-C in the serum of each group of mice were measured using an automatic biochemical analyzer.

[0061] (8) Hematoxylin and eosin (HE), Nissl staining, and TUNEL staining: The brain fixed in 4% paraformaldehyde was immersed in wax, embedded, and sliced ​​at a thickness of 2 μm. HE and Nissl staining were performed routinely, and then observed under a microscope (BX51, OLYMPUS, JPN). The whole brain and paraffin sections were taken; TUNEL staining was performed on the paraffin sections of the brain using the In Situ Cell Death Detection Kit (Intergen, USA) according to the kit instructions. After dewaxing and hydration, the sections were permeabilized with proteinase K (20 μg / ml) at room temperature for 20 minutes. The sections were then placed in a mixture of TdT equilibration buffer, recombinant TdT enzyme, and Alexa Fluor 488-12dUTP labeling solution and reacted at 37°C for 60 minutes. The cell nuclei were stained using the DAPI method.

[0062] 1.4.3 Statistical methods

[0063] SPSS 26.0 statistical software package was used for analysis. Normally distributed quantitative data were presented as mean ± standard deviation ( ̄x ± s). Multiple-group data were compared using one-way analysis of variance (ANOVA). All data were tested for homogeneity of variance. If the variances were homogeneous, further pairwise comparisons were performed using the LSD method. If the variances were unequal, the Tamhane T2 method was used. A P value of < 0.05 was considered statistically significant.

[0064] 2. Experimental results

[0065] 2.1 General condition, tongue color, and water maze test of mice in each group

[0066] 2.1.1 Changes in general status of mice in each group

[0067] Compared with the normal group, mice in all four groups showed significantly decreased activity, decreased energy, and slower reactions. Their fur became yellowish, greasy, and sparse. The mice in the stasis syndrome group and the phlegm-stasis group also had noticeable ecchymoses and petechiae. The mouse syndrome score table showed that compared with the normal group, the TCM syndrome scores of mice in the remaining groups were significantly elevated, with a particularly significant increase in the phlegm-stasis group (P < 0.01) (Table 5).

[0068] Table 5 TCM syndrome scores of mice in each group

[0069] WT tx-j tx-j phlegm syndrome group tx-j stasis syndrome group tx-j phlegm and blood stasis group 1.05±0.83 <![CDATA[5.1±1.55 # ]]> <![CDATA[9.65±1.14 ** ]]> <![CDATA[10.85±1.46 ** ]]> <![CDATA[16.55±1.93 ** ]]>

[0070] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05, ** P<0.01

[0071] 2.1.2 RGB values ​​of tongue color of mice in each group

[0072] Compared with the WT group, the tongue color of mice in the other groups was dark red, and the difference was statistically significant (P<0.05).

[0073] Compared with the mice in the tx-j group, the tongue color of mice in the phlegm syndrome group, blood stasis group, and phlegm and blood stasis group was dark red, which was statistically significant (P<0.05). Pairwise comparison of the phlegm syndrome group, blood stasis group, and phlegm and blood stasis group showed no significant difference in tongue color between the phlegm syndrome group and the blood stasis group, which was not statistically significant (P>0.05). However, there was a statistically significant difference between the phlegm syndrome group and the phlegm and blood stasis group, and between the blood stasis group and the phlegm and blood stasis group (P<0.05). Figure 1 , Table 6).

[0074] Table 6 R, G, and B values ​​of the tongue surface of mice in each group

[0075] Group R G B WT 197.84±3.57 79.98±1.16 84.92±1.69 tx-j <![CDATA[183.74±3.17 # ]]> <![CDATA[65.34±3.12 # ]]> <![CDATA[94.8±2.85 # ]]> tx-j phlegm syndrome group <![CDATA[170.27±6.46 * ]]> <![CDATA[71.25±2.01 * ]]> <![CDATA[102.32±8.74 * ]]> tx-j stasis syndrome group <![CDATA[188.85±0.82 * ]]> <![CDATA[86.18±1.82 * ]]> <![CDATA[102.22±2.44 * ]]> tx-j phlegm and blood stasis group <![CDATA[174.54±3.82 * ]]> <![CDATA[80.93±1.76 * ]]> <![CDATA[111.55±5.79 * ]]>

[0076] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0077] 2.1.3 Results of Morris water maze and novel object recognition tests in each group of mice

[0078] In the Morris water maze test, compared with the WT group, the number of crossings, the ratio of distance traveled to the original platform quadrant, and the ratio of time traveled to the original platform quadrant in the other groups were all reduced, and the differences were statistically significant (P<0.05). Compared with the tx-j group, the mice in the phlegm and blood stasis group were more obvious, and the differences were statistically significant (P<0.05). Pairwise comparisons were made between the phlegm syndrome group, blood stasis syndrome group, and phlegm and blood stasis syndrome group. There was no significant difference between the phlegm syndrome group and the blood stasis syndrome group, and there was no statistical significance (P>0.05). Figure 2 , Table 7).

[0079] In the novel object recognition experiment, all mice showed no statistically significant difference in preference for Objects A and B placed within the experimental apparatus (P>0.05), indicating that the presence of Object A did not significantly affect the mice's subsequent behavioral patterns in exploring the novel Object C. After entering the testing phase, the tx-j group mice showed a significant decrease in the time it took to recognize the novel Object C compared to the WT group (P<0.05). Compared with the tx-j group, the differences in the phlegm and blood stasis syndrome groups were not statistically significant (P>0.05), but the phlegm and blood stasis group showed statistical significance (P<0.05) (Table 8).

[0080] This shows that the mice in the phlegm and blood stasis group had more severe lesions and showed a significant decline in cognitive behavior.

[0081] Table 7 Morris water maze test results of mice in group

[0082] Group Number of times of crossing the platform Original platform quadrant distance ratio Original platform quadrant time ratio WT 3.74±0.63 28.29±1.27 31.51±1.88 tx-j <![CDATA[1.93±0.43 # ]]> <![CDATA[25.79±3.13 # ]]> <![CDATA[27.2±3.26 # ]]> tx-j phlegm syndrome group 1.81±0.46 25.36±2.32 26.59±2.99 tx-j stasis syndrome group 1.71±0.25 25.43±2.1 26.35±2.13 tx-j phlegm and blood stasis group <![CDATA[1.38±0.16 * ]]> <![CDATA[24.34±2.03 * ]]> <![CDATA[25.45±2.03 * ]]>

[0083] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0084] Table 8 Experimental results of new object recognition

[0085] Group Preference coefficient Discrimination coefficient WT 85.8±10.27 49.34±5.44 tx-j 117.44±40.67 <![CDATA[9.32±10.4 # ]]> tx-j phlegm syndrome group 146.4±41.35 8.48±9.02 tx-j stasis syndrome group 123.64±31.22 8±7.29 tx-j phlegm and blood stasis group 149.33±45.06 <![CDATA[6.43±3.72 * ]]>

[0086] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0087] 2.2 Changes in laboratory indicators of mice in each group

[0088] 2.2.1 Hemorheological parameters of mice in each group

[0089] Compared with the WT group, mice in the tx-j group had increased whole blood viscosity and plasma viscosity (P < 0.05). Compared with the tx-j group, mice in the phlegm syndrome group had increased whole blood viscosity and plasma viscosity, but the difference was not statistically significant (P > 0.05). The whole blood viscosity and plasma viscosity of mice in the stasis syndrome group and the phlegm-stasis group were significantly increased, with statistical significance (P < 0.05). Compared with the tx-j group, the whole blood viscosity and plasma viscosity of mice in the phlegm syndrome group were increased to varying degrees, but the difference was not statistically significant (P > 0.05). The whole blood viscosity and plasma viscosity of mice in the stasis syndrome group and the phlegm-stasis group were significantly increased, with statistical significance (P < 0.05). This indicates that ice-water bath treatment can significantly alter the hemorheology of mice and effectively simulate the state of "stasis" (Table 9).

[0090] Table 9 Comparison of whole blood viscosity and plasma viscosity of mice in each group

[0091] Group Ice water bath treatment Whole blood viscosity high shear (200 / S) Whole blood viscosity median cut (50 / S) Whole blood viscosity low shear (1 / S) Plasma viscosity WT - 4.8±0.09 7.26±0.11 15.55±0.35 1.33±0.08 tx-j - <![CDATA[5.94±0.04 # ]]> <![CDATA[8.2±0.13 # ]]> <![CDATA[16.02±0.13 # ]]> <![CDATA[1.96±0.05 # ]]> tx-j phlegm syndrome group - 6.07±0.06 8.3±0.26 17.6±0.21 2.02±0.06 tx-j stasis syndrome group + <![CDATA[6.34±0.11 * ]]> <![CDATA[9.2±0.29 * ]]> <![CDATA[22.67±0.39 * ]]> <![CDATA[3.49±0.33 * ]]> tx-j phlegm and blood stasis group + <![CDATA[6.66±0.52 * ]]> <![CDATA[9.09±0.63 * ]]> <![CDATA[23.68±0.52 * ]]> <![CDATA[3.67±0.05 * ]]>

[0092] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0093] 2.2.2 Blood lipid detection of mice in each group

[0094] Compared with WT mice, all other groups showed statistically significant differences in blood lipid indices (P < 0.05). Compared with the tx-j group, the blood lipid indices in the stasis syndrome group were not significantly different (P > 0.05). Blood lipid indices in the phlegm syndrome group and the phlegm-stasis group were significantly elevated, with statistically significant differences (P < 0.05). This suggests that a high-fat diet can alter blood lipid indices in mice and effectively mimic the pathological state of "phlegm" (Table 10).

[0095] Table 10 Comparison of serum TC, TG, HDL-C, and LDL-C levels in mice of each group

[0096] Group Food type TC TG HDL-C LDL-C WT Ordinary feed 2.05±0.27 1.34±0.17 1.7±0.41 0.18±0.04 tx-j Ordinary feed <![CDATA[3.14±0.1 # ]]> <![CDATA[1.88±0.53 # ]]> <![CDATA[1.01±0.25 # ]]> <![CDATA[0.46±0.04 # ]]> tx-j phlegm syndrome group high-fat feed <![CDATA[8.53±2.1 * ]]> <![CDATA[4.79±0.71 * ]]> <![CDATA[0.74±0.06 * ]]> <![CDATA[1.93±0.07 * ]]> tx-j stasis syndrome group Ordinary feed 3.08±0.73 1.96±0.46 0.96±0.11 0.51±0.04 tx-j phlegm and blood stasis group high-fat feed <![CDATA[9.97±1.04 * ]]> <![CDATA[6.04±1.3 * ]]> <![CDATA[0.71±0.17 * ]]> <![CDATA[1.02±0.22 * ]]>

[0097] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0098] 2.2.3 Detection of copper content in tissues and serum of mice in each group

[0099] Compared with the WT group, the copper levels in the brain, liver, and kidney tissues of the mice in the other groups increased, while the copper levels in serum, urine, and feces decreased, and the differences were statistically significant (P < 0.05). Compared with the tx-j group, the tissue copper content in the phlegm-stasis group increased (P < 0.05) and the metabolic copper content decreased (P < 0.05), indicating that the phlegm-stasis group had greater tissue copper deposition and more severe pathological conditions (Tables 11 and 12).

[0100] Table 11 Copper content in tissues of mice in each group (μg / g)

[0101] Group liver brain kidney WT 9.57±4.09 3.01±0.11 5.67±1.01 tx-j <![CDATA[204.64±28.27 # ]]> <![CDATA[5.94±0.4 # ]]> <![CDATA[8.91±1.19 # ]]> tx-j phlegm syndrome group 236.34±39.33 7.5±2.61 9.97±1.13 tx-j stasis syndrome group <![CDATA[240.8±30.2 * ]]> 5.98±1.17 <![CDATA[10.95±0.8 * ]]> tx-j phlegm and blood stasis group <![CDATA[264.33±39.53 * ]]> <![CDATA[12.41±2.04 * ]]> <![CDATA[11.18±2.04 * ]]>

[0102] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0103] Table 12 Metabolic copper content in tissues of mice in each group (μg / ml)

[0104] Group serum Urine stool WT 1.09±0.15 0.4±0.1 79.98±11.21 tx-j <![CDATA[0.5±0.11 # ]]> <![CDATA[0.22±0.02 # ]]> <![CDATA[59.02±16.49 # ]]> tx-j phlegm syndrome group <![CDATA[0.38±0.13 * ]]> 0.21±0.01 63.34±13.53 tx-j stasis syndrome group 0.44±0.16 <![CDATA[0.2±0.02 * ]]> 60.18±6.14 tx-j phlegm and blood stasis group <![CDATA[0.18±0.07 * ]]> <![CDATA[0.1±0.01 * ]]> <![CDATA[38.07±6.07 * ]]>

[0105] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0106] 2.2.4 Detection of inflammatory factors in each group of mice

[0107] Compared with the WT group, all other groups showed significantly elevated hippocampal inflammatory markers, with statistically significant differences (P < 0.05). This suggests an inflammatory response within the hippocampus and cognitive pathological changes. Compared with the tx-j group, the phlegm and stasis syndrome groups showed slight differences in inflammatory indices, but these differences were not statistically significant (P > 0.05). However, the phlegm and stasis syndrome group showed significantly elevated inflammatory and pathological markers, with statistically significant differences (P < 0.05). This suggests that mice simulating both phlegm and stasis pathological states exhibited more severe pathological changes and more pronounced brain neuroinflammation (Table 13).

[0108] Table 13 Comparison of inflammatory factor levels in hippocampal tissue of mice in each group

[0109] Group TNF-α IL-1β IL-6 WT 101.37±9.05 20.08±2.14 24.08±3.35 tx-j <![CDATA[166.33±32.09 # ]]> <![CDATA[37.07±5.67 # ]]> <![CDATA[48.55±2.61 # ]]> tx-j phlegm syndrome group 176.11±27.68 <![CDATA[48.76±8.79 * ]]> <![CDATA[53.69±4.13 * ]]> tx-j stasis syndrome group 184.55±22.7 <![CDATA[56.51±10.39 * ]]> <![CDATA[67.67±7.72 * ]]> tx-j phlegm and blood stasis group <![CDATA[215.07±21.43 * ]]> <![CDATA[77.26±15.54 * ]]> <![CDATA[79.83±9.22 * ]]>

[0110] Note: Compared with WT group # P < 0.05; compared with tx-j group * P<0.05

[0111] 2.3 Pathological changes of mice in each group

[0112] 2.3.1 Pathological staining of brains of mice in each group

[0113] HE staining showed that hippocampal neurons in the WT group were regularly arranged, with round nuclei and smooth morphology. Compared with the control group, neurons in the CA1 and CA3 regions of the hippocampus in the other groups showed disorganized arrangement, cellular reduction, edema, vacuolization, degeneration, cell body necrosis, and nuclear fixation. Compared with the tx-j group, hippocampal neuronal morphology in the phlegm syndrome and stasis syndrome groups remained unchanged, while neurons in the phlegm and stasis syndrome group showed significant cellular reduction, edema, and necrosis. Nissl staining revealed a significant decrease in Nissl bodies in the other groups compared with the WT group. Pairwise comparisons of the other groups showed no significant changes between the tx-j group and the phlegm syndrome group, the tx-j group and the stasis syndrome group, and the phlegm syndrome group and the stasis syndrome group. A significant decrease in Nissl bodies was observed between the tx-j group and the phlegm and stasis syndrome group, the phlegm syndrome group and the phlegm and stasis syndrome group, and the stasis syndrome group and the phlegm and stasis syndrome group.

[0114] 2.3.2 Transmission electron microscopy of the brains of mice in each group

[0115] Transmission electron microscopy revealed that neurons in the WT group had normal cell structure, with distinct mitochondria, clear cristae, and intact endoplasmic reticulum. Cells in the other groups showed swelling, decreased mitochondria, and significant cristae fragmentation, dissolution, and vacuolization. Compared with the tx-j group, mitochondrial dissolution and vacuolization were observed in the phlegm syndrome group, the stasis syndrome group, and the phlegm and blood stasis group, with the phlegm and blood stasis group showing more severe mitochondrial dissolution and vacuolization.

[0116] 3 Conclusion

[0117] Currently, the main animal models of WD are TX mice, tx-j mice, ATP7B - / - Mice and LEC rats. TX mice show early and prominent liver damage without neurological manifestations; tx-j mice show early copper deposition and mild neurological symptoms, making them suitable for studies of copper metabolism accompanied by neurological symptoms. ATP7B - / - Mouse liver copper accumulation is early and high, with no neurological symptoms, making it suitable for efficacy evaluation. However, it is expensive and not widely used. LEC rats have mild neurological symptoms and rapidly progressive liver disease, making them suitable for interventional studies, but they have a high mortality rate. Therefore, tx-j mice are the optimal animal model for studying WD with cognitive impairment.

[0118] The present invention uses tx-j mice as model mice and congenic WT mice as control mice. First, the age of the mice was selected. From 6 months of age, the brain copper content of mice reached a peak and the cognitive level declined. Therefore, 6-month-old tx-j mice were selected for subsequent experiments.

[0119] Comparison of TCM syndrome scores and tongue color across the different mouse models revealed changes in corresponding TCM syndrome types in the phlegm syndrome, stasis syndrome, and phlegm-stasis syndrome groups. Water maze and novel object recognition tests revealed more severe cognitive changes in the phlegm-stasis syndrome group. Further analysis using laboratory parameters revealed significant increases in whole blood viscosity and plasma viscosity in the stasis syndrome and phlegm-stasis syndrome groups compared with the tx-j group (P<0.05), with the increase being more pronounced in the phlegm-stasis syndrome group. Furthermore, blood lipid profiles revealed significant increases in the phlegm syndrome and phlegm-stasis syndrome groups compared with the tx-j group (P<0.05), with the increase being more pronounced in the phlegm-stasis syndrome group. This demonstrates the feasibility of using an ice water bath and a high-fat diet to establish models for phlegm and stasis syndromes.

[0120] Copper levels were measured in mice from each group. Compared with the tx-j group, the phlegm-stasis group showed decreased metabolic copper levels, while copper levels in various tissues increased. Inflammatory cytokines were measured in the hippocampi of mice from each group. Compared with the tx-j group, inflammatory cytokines were elevated in the hippocampi of mice in the phlegm-syndrome, stasis-syndrome, and phlegm-stasis groups to varying degrees, with the phlegm-stasis group showing more pronounced inflammatory cytokines. HE, Nissl, and electron microscopy revealed that compared with the tx-j group, mice in the phlegm-syndrome, stasis-syndrome, and phlegm-stasis groups all showed varying degrees of damage, with the phlegm-stasis group showing more pronounced damage.

[0121] The above results confirm that the animal model of combined disease and syndrome may lead to a decrease in metabolic copper, thereby further depositing copper in the tissues and causing more serious pathological changes.

Claims

1. A method for constructing a tx-j mouse animal model of cognitive impairment based on phlegm and blood stasis syndrome, characterized in that: tx-j mice were first fed a high-fat diet to create a hyperlipidemia state, and then an ice-water bath method was used to create a "blood stasis" model. Finally, a tx-j mouse animal model of cognitive impairment caused by phlegm and blood stasis syndrome was established.

2. The method for constructing a tx-j mouse animal model of cognitive impairment based on phlegm-stasis syndrome according to claim 1, characterized in that: Six-month-old tx-j mice were selected and fed a high-fat diet for 4 weeks. Then, starting from the 5th week, they were placed in an ice-water mixture once a day. When the mice's struggling movements significantly decreased and they were about to sink to the bottom, they were immediately fished out. The model construction lasted for 4 weeks, and finally an animal model of cognitive impairment with phlegm and blood stasis syndrome combining symptoms and functions was formed.

3. The method for constructing a tx-j mouse animal model of cognitive impairment based on phlegm-stasis syndrome according to claim 2, wherein: The high-fat feed formula used in the high-fat diet feeding is: 75% basic feed + 10% cholesterol + 10% lard + 4% sucrose + 1% bile salt.

4. The method for constructing a tx-j mouse animal model of cognitive impairment based on phlegm-stasis syndrome according to claim 2, wherein: After the model was successfully constructed, the mouse's condition and tongue color were first observed, and a water maze experiment was conducted. Then, blood rheology indicators, blood lipid indicators, tissue and serum copper content, and inflammatory factors were tested, and pathological changes in the mouse brain were observed.

5. Application of the tx-j mouse animal model of cognitive impairment caused by phlegm and blood stasis syndrome constructed according to any one of the methods of claims 1 to 4 in scientific research on pathogenesis, screening and evaluation of traditional Chinese medicine.

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