Traditional Chinese medicine composition for treating atherosclerosis and method for detecting effect of traditional Chinese medicine composition

Abstract

The invention relates to a traditional Chinese medicine composition for treating atherosclerosis and a method for detecting the effect of the traditional Chinese medicine composition. The traditional Chinese medicine composition comprises the following raw material components in parts by weight: 5-29 parts of Ziziphora bungeana, 1-20 parts of salvia miltiorrhiza, 1-15 parts of rhodiola rosea and 1-10 parts of lignum dalbergiae odoriferae. The traditional Chinese medicine composition can reduce the content of inflammatory factors in serum, inhibit inflammation and slow down AS formation, and the effect of the traditional Chinese medicine composition on prevention and treatment of atherosclerosis can be effectively verified by establishing an atherosclerosis mouse model.

Description

Technical Field

[0001] This invention belongs to the technical field of traditional Chinese medicine formulations for treating atherosclerosis, specifically relating to a traditional Chinese medicine composition for treating atherosclerosis and a method for detecting its efficacy. Background Technology

[0002] Atherosclerosis (AS) is the main pathological basis of cardiovascular and cerebrovascular diseases such as coronary heart disease and stroke. Its pathological process begins with local damage to the arterial intima, followed by lipid deposition, intimal fibrosis, and local thickening of the intima, eventually forming plaques. This leads to thickening and hardening of the blood vessel wall, narrowing or even complete occlusion of the lumen, causing serious complications such as myocardial infarction, cerebral ischemia, and peripheral tissue ischemia. Atherosclerotic cardiovascular disease has become one of the leading causes of death worldwide, with approximately 17.3 million deaths annually, a figure projected to rise to 23.6 million by 2030. AS is caused by a combination of factors, and its pathogenesis remains incompletely understood. Studies have shown that lipid metabolism disorders, vascular endothelial damage, inflammatory cell infiltration, oxidative stress, and immune dysfunction are all important factors in AS development. With recent research, AS has been proven to be a chronic inflammatory disease characterized by immune activity. Inflammation persists throughout all stages of AS, ultimately leading to thrombotic plaque complications, resulting in high rates of disability and mortality. Research indicates that the Wnt signaling pathway is closely related to the development of atherosclerotic disease, including inflammatory responses, endothelial dysfunction, and macrophage activation. Therefore, the role of the Wnt signaling pathway in the development and progression of cardiovascular disease has attracted considerable attention. Regulating the signaling pathway to inhibit the release of inflammatory factors or mediators, and reducing the inflammatory response to effectively delay plaque formation or stabilize plaques to prevent rupture, may be a key aspect of AS prevention and treatment. Summary of the Invention

[0003] To address the above problems, the purpose of this invention is to provide a traditional Chinese medicine composition with *Pterocarya stenoptera* water extract as the main ingredient, which downregulates the content of inflammatory factors in serum, inhibits inflammation, slows down the formation of atherosclerosis, and tests the effect of this composition in preventing and treating atherosclerosis by establishing a mouse model of atherosclerosis.

[0004] The objective of this invention is achieved through the following technical solution: a traditional Chinese medicine composition for treating atherosclerosis, characterized in that the weight ratio of each component of its raw materials is as follows: 5-29 parts of aromatic stachys pubescens, 1-20 parts of salvia miltiorrhiza, 1-15 parts of rhodiola rosea, and 1-10 parts of dalbergia odorifera.

[0005] A traditional Chinese medicine composition for treating atherosclerosis, with the following weight ratio of each component: 13 parts of aromatic staphylococcus aureus, 7 parts of salvia miltiorrhiza, 2 parts of rhodiola rosea, and 9 parts of dalbergia odorifera.

[0006] A traditional Chinese medicine composition for treating atherosclerosis, with the following weight ratio of each component: 5 parts of aromatic staphylococcus aureus, 1 part of salvia miltiorrhiza, 1 part of rhodiola rosea, and 1 part of dalbergia odorifera.

[0007] A traditional Chinese medicine composition for treating atherosclerosis, with the following weight ratio of each component: 29 parts of aromatic staphylococcus aureus, 20 parts of salvia miltiorrhiza, 15 parts of rhodiola rosea, and 10 parts of dalbergia odorifera.

[0008] A traditional Chinese medicine composition for treating atherosclerosis, with the following weight ratio of each component: 12 parts of aromatic staphylococcus aureus, 10 parts of salvia miltiorrhiza, 8 parts of rhodiola rosea, and 5 parts of dalbergia odorifera.

[0009] A method for detecting the efficacy of the above-mentioned traditional Chinese medicine composition for treating atherosclerosis includes the following steps: Step 1: Establishing a mouse model of atherosclerosis: Prepare 30 eight-week-old ApoE- / - mice (18±2 g, C57BL / 6J) and 10 ordinary C57 mice. After one week of acclimatization, the ApoE- / - mice were randomly divided into a model group, an atorvastatin group, and the traditional Chinese medicine combination group. The C57 mice served as a blank control group. Each group consisted of 10 mice. The control group was fed a basal diet, while the other groups were fed a high-fat diet for 8 weeks to establish the atherosclerosis model. Step 2, Specimen Collection and Preparation: The control group and model group were given 0.5 ml of distilled water by gavage once a day (standard ≤ 0.4 ml / 10 g), the statin group was given 3.0 g / (kg·d) of atorvastatin calcium tablets dissolved in 0.5 ml of distilled water by gavage once a day, and the traditional Chinese medicine combination group was given once a day. The gavage intervention was carried out for 12 consecutive weeks. All mice were fed in an SPF environment, with no restrictions on food and water, and were free to move around. Step 3, Specimen Collection and Processing: (1) After the serological specimen intervention, the mice in each group were fasted but allowed to drink water for 12 hours. After anesthesia, blood was collected from the eyeballs into Ep tubes, centrifuged at 7500 r / min for 15 min, and the supernatant was collected and stored in a -80℃ refrigerator. (2) After blood was collected from the eyeballs of mice, they were immediately euthanized by cervical dislocation. The heart was quickly exposed by opening the chest. The heart and the surrounding adipose tissue of the aorta were removed. The aortic arch, thoracic aorta, abdominal aorta and iliac aorta were separated by dissecting scissors and curved forceps. The aortic arch, thoracic aorta, abdominal aorta and iliac aorta were washed in pre-cooled 1xPBS, the PBS was blotted dry with filter paper and fixed in 4% paraformaldehyde. Step 4: Tissue embedding and slide preparation: (1) Sampling and fixation: Mouse aorta was taken and fixed with 4% paraformaldehyde at a volume ratio of 1:10 to formaldehyde for 48 h, and then placed in an embedding cassette; (2) Dehydration and clearing: Place the embedding cassette with tissue in a round beaker and rinse with running water for 10 min; then place it in 95% ethanol I for 30 min, 95% ethanol II for 30 min, anhydrous ethanol I for 30 min, and anhydrous ethanol II for 30 min in sequence; then soak gauze with alcohol on the surface of the tissue and place it in xylene I for 30 min and xylene II for 30 min. (3) Wax impregnation: Immerse in wax solution I in a constant temperature oven at 60℃ for 1 hour; then immerse in wax solution II for 1 hour. (4) Embedding: Place the wax-impregnated embedding box into the molten paraffin, place the tissue embedding box with the cap removed into a specific iron box, drip in the molten paraffin, and then place the iron box on a cooling table; (5) Sectioning: After the wax block solidifies, place it in a -20℃ refrigerator overnight; cut into 4μm continuous sections, taking 6 sections from each wax block; gently lay the sections flat on a 40℃ water surface with ophthalmic forceps, and after the wrinkles of the sections naturally flatten, quickly place the sections on a non-detachable glass slide and let them air dry naturally. Step 5: Detect serum lipid levels and cytokine levels using enzyme-linked immunosorbent assay (ELISA) sandwich method; Step 6: Observe the pathological changes in the aorta using immunohistochemistry.

[0010] In this invention, the effects of the traditional Chinese medicine composition were tested using the above methods. The results showed that, compared to other groups of mice, the high-dose group of *Xintanghua* (a type of herb) exhibited reduced lipid deposition in the arteries, milder intimal damage and less significant thickening, slight deformation of the vessel wall with a few visible plaques, and slight luminal narrowing. The mice in this group also had lower body weight than the model group. This suggests that *Xintanghua* can alleviate weight gain in mice to a certain extent and can also alleviate the formation of ankylosing spondylitis (AS). The successful establishment of the mouse AS model confirms that the traditional Chinese medicine composition has the effects of regulating blood lipid levels, improving myocardial ischemia, inhibiting inflammatory responses, and protecting endothelial cells. It can effectively regulate blood lipid levels, increase serum VEGF levels, prevent vascular endothelial damage, increase vascular permeability, accelerate blood flow, prevent coagulation and thrombosis, and simultaneously promote vascular endothelial cell proliferation and angiogenesis.

[0011] Beneficial effects: The traditional Chinese medicine composition in this invention can downregulate the content of inflammatory factors in serum, inhibit inflammation, slow down the formation of atherosclerosis, and establish a mouse model of atherosclerosis to effectively verify the effect of the traditional Chinese medicine composition in preventing and treating atherosclerosis. Detailed Implementation

[0013] Example 1: A traditional Chinese medicine composition for treating atherosclerosis, wherein the weight ratio of each component of the raw materials is as follows: 5-29 parts of aromatic stachys pubescens, 1-20 parts of salvia miltiorrhiza, 1-15 parts of rhodiola rosea, and 1-10 parts of dalbergia odorifera.

[0014] Example 2: A traditional Chinese medicine composition for treating atherosclerosis, the weight ratio of each component of the raw materials is as follows: 13 parts of aromatic stachys pubescens, 7 parts of salvia miltiorrhiza, 2 parts of rhodiola rosea, and 9 parts of dalbergia odorifera.

[0015] Example 3: A traditional Chinese medicine composition for treating atherosclerosis, wherein the weight ratio of each component of the raw materials is as follows: 5 parts of aromatic stachys pubescens, 1 part of salvia miltiorrhiza, 1 part of rhodiola rosea, and 1 part of dalbergia odorifera.

[0016] Example 4: A traditional Chinese medicine composition for treating atherosclerosis, wherein the weight ratio of each component of the raw materials is as follows: 29 parts of aromatic stachys pubescens, 20 parts of salvia miltiorrhiza, 15 parts of rhodiola rosea, and 10 parts of dalbergia odorifera.

[0017] Example 5: A traditional Chinese medicine composition for treating atherosclerosis, wherein the weight ratio of each component of the raw materials is as follows: 12 parts of aromatic stachys pubescens, 10 parts of salvia miltiorrhiza, 8 parts of rhodiola rosea, and 5 parts of dalbergia odorifera.

[0018] The main representative component of *Pterocarya stenoptera* is flavonoids. Therefore, the total flavonoids of *Pterocarya stenoptera* were first extracted, purified, and identified. Rutin, diosmin, hyperoside, and styraxoside were selected for the synthesis and identification of artificial antigens. Hyperoside, which showed good conjugation performance, was used to synthesize antigens to immunize animals. Serum antibody titers and competitiveness were measured using an indirect ELISA method to detect the immunogenicity of the synthesized antigens. Monoclonal antibody-positive hybridoma cell lines were prepared, and the specificity of the antibodies was studied. Immunoaffinity chromatography columns were prepared, and samples with specific knockout of *Pterocarya stenoptera* flavonoids were developed. The content of specific knockout *Pterocarya stenoptera* flavonoids before and after knockout was determined using HPLC. The effect of total flavonoids of *Pterocarya stenoptera* on endothelial cell damage was also investigated.

[0019] The results showed that the total flavonoid content of *Pterocarya stenoptera* was 73%. Ten components were identified, namely baicalin, quercetin, apigenin, diosmin, styracin, rutin, hyperoside, quercetin 3-O-β-D-glucopyranoside, apigenin, and kaempferol.

[0020] Artificial antigens and artificial coating antigens of rutin, diosmin, hyperoside, and strychnoside were successfully synthesized and identified. The antiserum titer reached 1:32000 using indirect ELISA.

[0021] The AA9-well cell line screening showed a 100% fusion rate and a positive rate of 4.12%. Monoclonal antibodies were prepared in large quantities using the ascites induction method, with ascites titers exceeding 1:64000.

[0022] The purity of the ascites fluid purified by the ammonium octanoate sulfate method was 92%; the concentration of monoclonal antibody ascites protein before purification by the protein G column method was 4.28 mg / mL, and the concentration after purification was 2.16 mg / mL; the antibody titer and competition and cross-reactivity before and after purification were determined by ELISA, and the titer reached more than 1:64000, and no cross-reactivity was found.

[0023] The purified hyperoside monoclonal antibody showed a 99.3% conjugation rate with the gel, with a conjugation ratio of 3.4 mg / mL. External standard method analysis revealed that approximately 90% of the hyperoside in the original 27-sample sample of *Pyrrosia lingua* was knocked out. The affinity column recovery rate reached 97.2% based on the hyperoside content in the eluent. Total flavonoids from *Pyrrosia lingua* acted on H2O2-induced endothelial cells, increasing SOD, inhibiting MDA, and decreasing LDH, thus protecting against endothelial cell damage.

[0024] Example 6: The present invention also provides a method for detecting the effect of the above-mentioned traditional Chinese medicine composition for treating atherosclerosis, comprising the following steps: Step 1: Establishing a mouse model of atherosclerosis: Thirty 8-week-old ApoE- / - mice (18±2 g, C57BL / 6J) and ten ordinary C57 mice were prepared. After one week of acclimatization, the ApoE- / - mice were randomly divided into a model group, an atorvastatin group, and the herbal combination group using a random number table. The C57 mice served as a blank control group, with 10 mice in each group. The control group was fed a basal diet, while the other groups were fed a high-fat diet for 8 weeks to establish the atherosclerosis model. Step 2, Specimen Collection and Preparation: The control group and model group were given 0.5 ml of distilled water by gavage once a day (standard ≤ 0.4 ml / 10 g), the statin group was given 3.0 g / (kg·d) of atorvastatin calcium tablets dissolved in 0.5 ml of distilled water by gavage once a day, and the traditional Chinese medicine combination group was given once a day. The gavage intervention was continued for 12 weeks. All mice were fed in an SPF environment, with no restrictions on food and water, and were free to move. Step 3, Specimen Collection and Processing: (1) Serological specimens: After the intervention, mice in each group were fasted but allowed to drink water for 12 hours. Blood was collected from the eyeballs after anesthesia and put into Ep tubes. After centrifugation at 7500 r / min for 15 min, the supernatant was collected and stored in a -80℃ refrigerator. (2) Histological specimens: After blood was collected from the eyeballs of mice, they were immediately euthanized by cervical dislocation. The heart was quickly exposed by opening the chest. The heart and the surrounding adipose tissue of the aorta were removed. The aortic arch, thoracic aorta, abdominal aorta and iliac aorta were separated by dissecting scissors and curved forceps. The aortic arch, thoracic aorta, abdominal aorta and iliac aorta were washed in pre-cooled 1xPBS, the PBS was blotted dry with filter paper, and the aortic arch was fixed in 4% paraformaldehyde. Step 4: Tissue embedding and slide preparation: (1) Sampling and fixation: Mouse aorta was taken and fixed with 4% paraformaldehyde at a volume ratio of 1:10 to formaldehyde for 48 h, and then placed in an embedding cassette; (2) Dehydration and clearing: Place the embedding cassette with tissue in a round beaker and rinse with running water for 10 min; then place it in 95% ethanol I for 30 min, 95% ethanol II for 30 min, anhydrous ethanol I for 30 min, and anhydrous ethanol II for 30 min in sequence; then soak gauze with alcohol on the surface of the tissue and place it in xylene I for 30 min and xylene II for 30 min. (3) Wax impregnation: Immerse in wax solution I in a constant temperature oven at 60°C for 1 hour; then immerse in wax solution II for 1 hour. (4) Embedding: Place the wax-impregnated embedding box into the molten paraffin, place the tissue embedding box with the cap removed into a specific iron box, drip in the molten paraffin, and then place the iron box on a cooling table; (5) Sectioning: After the wax block solidifies, place it in a -20°C refrigerator overnight; cut into 4μm continuous sections, taking 6 sections from each wax block; gently lay the sections flat on a 40°C water surface with ophthalmic forceps, and after the wrinkles of the sections naturally flatten, quickly place the sections on a non-detachable glass slide and let them air dry naturally; Step 5: Detect serum lipid levels and cytokine levels using enzyme-linked immunosorbent assay (ELISA) sandwich method; Step 6: Observe the pathological changes in the aorta using immunohistochemistry.

[0025] Example 7, as a further optimization of Example 6, in step 6, the immunohistochemical method specifically includes the following detection steps: 1. Slide baking: Place the glass slide in a 60℃ constant temperature oven for 2 hours; 2. Dewaxing and dehydration: 100% toluene for 10 min / time × 2 times, 100% ethanol for 4 min / time × 2 times, 95% ethanol for 4 min / time × 2 times, 90% ethanol for 4 min / time × 2 times, double distilled water for 5 min, PBS wash for 4 min / time × 3 times; 3. Antigen retrieval: Boil in 0.01M citrate buffer (pH 6.0) to 270°C, then boil in 0.01M citrate buffer (pH 6.0) to 140°C for 2 minutes each, and then cool naturally to below 50°C. 4. Removal of peroxidase: Wash 3 times with 1xPBS, 4 min each time; wash once with 10% hydrogen peroxide, 15 min each time; wash 3 times with 1xPBS, 4 min each time. 5. Incubation: Add primary antibodies (Wnt1, β-catenin, Dvl) and incubate overnight (4℃). Keep in an incubator for 1 hour, and rinse with 1xPBS (7.2-7.6) for 4 minutes each time × 3 times. 6. Secondary antibody: Add secondary antibody enhancer, incubate in a constant temperature incubator (37℃) for 30 min, and wash with 1xPBS for 4 min each time × 3 times. Add secondary antibody, incubate in a constant temperature incubator (37℃) for 35 min, and wash with 1xPBS for 4 min each time × 3 times; 7. Color development: Using the DAB color development kit, add 5 μl of reagents A, B, and C from the kit to a clean centrifuge tube containing 1 ml of distilled water. Mix well and then add the mixture to the sample for color development at room temperature. Control the color development time under a microscope until the cell cytoplasm shows brownish-yellow granules, while the rest of the staining is relatively light (15 min). Wash away excess color development agent with double-distilled water to terminate the reaction. 8. Staining: Hematoxylin staining, double-distilled water washing, routine dehydration, clearing, and mounting with neutral resin; 9. Radiography: Select typical fields of view for radiography. For each tissue slide, select 5 non-overlapping AS plaques as the center of the field of view. Wnt1, β-catenin, and Dvl were observed using the CIAS-1000 image analysis system to detect the gray value of each field of view. The mean was taken as the gray value of the index. The higher the gray value, the lighter the positive staining and the lower the protein expression.

[0026] In step 1, mice were fed a high-fat diet and then gavaged with distilled water. The model group was used as the standard for successful model establishment. Macroscopic observation showed that the mice in this group were sluggish, lethargic, with dry, dull, and sparse fur, and their body weight was significantly increased compared to other groups. Pathological examination under a microscope revealed fibrotic thickening and calcification of the aortic intima, significant luminal narrowing, disordered smooth muscle cell arrangement, lipid deposition, cholesterol crystals, or atherosclerotic plaques. These findings confirmed the successful establishment of the mouse AS model.

[0027] In this invention, the effects of the traditional Chinese medicine composition were tested using the above methods. The results showed that, compared to other groups of mice, the high-dose group of *Xintanghua* (a type of herb) exhibited reduced lipid deposition in the arteries, milder intimal damage and less significant thickening, slight deformation of the vessel wall with a few visible plaques, and slight luminal narrowing. The mice in this group also had lower body weight than the model group, suggesting that *Xintanghua* can alleviate weight gain in mice to a certain extent and can also alleviate the formation of ankylosing spondylitis (AS). The successful establishment of the mouse AS model confirms that the traditional Chinese medicine composition has the effects of regulating blood lipid levels, improving myocardial ischemia, inhibiting inflammatory responses, and protecting endothelial cells. It can effectively regulate blood lipid levels, increase serum VEGF levels, prevent vascular endothelial damage, increase vascular permeability, accelerate blood flow, prevent coagulation and thrombosis, and simultaneously promote vascular endothelial cell proliferation and angiogenesis.

[0028] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A traditional Chinese medicine composition for treating atherosclerosis, characterized in that, The weight ratio of each component of the raw materials is as follows: Xintahua 5-29 parts, Danshen 1-20 parts, Hongtian 1-15 parts, Jiangxiang 1-10 parts.

2. The traditional Chinese medicine composition for treating atherosclerosis as described in claim 1, characterized in that, The weight ratio of each component of the raw materials is as follows: Xintahua 5-29 parts, Danshen 1-20 parts, Hongtian 1-15 parts, Jiangxiang 1-10 parts.

3. The traditional Chinese medicine composition for treating atherosclerosis as described in claim 1, characterized in that, The weight ratio of each component of the raw materials is as follows: Xintahua 5-29 parts, Danshen 1-20 parts, Hongtian 1-15 parts, Jiangxiang 1-10 parts.

4. The traditional Chinese medicine composition for treating atherosclerosis as described in claim 1, characterized in that, The weight ratio of each component of the raw materials is as follows: Xintahua 5-29 parts, Danshen 1-20 parts, Hongtian 1-15 parts, Jiangxiang 1-10 parts.

5. The traditional Chinese medicine composition for treating atherosclerosis as described in claim 1, characterized in that, The weight ratio of each component of the raw materials is as follows: Xintahua 5-29 parts, Danshen 1-20 parts, Hongtian 1-15 parts, Jiangxiang 1-10 parts.

6. A method for detecting the effect of the above-mentioned traditional Chinese medicine composition for treating atherosclerosis, comprising the following steps: Step 1, establishing a mouse atherosclerosis model: 8-week-old ApoE- / - mice (18±2 g, C57BL / 6J) 30, ordinary C57 mice 10, after adaptive feeding for one week, according to the random number table method, the ApoE- / - mice were divided into model group, atorvastatin group, the traditional Chinese medicine composition group, and C57 mice as blank control group, 10 in each group, the control group was fed with basic feed, and the rest of each group was fed with high-fat feed for 8 weeks to establish AS model; Step 2, specimen collection preparation: the control group and the model group were given 0.5 ml distilled water by gavage once a day (standard ≤0.4 ml / 10g), the statin group was given atorvastatin calcium tablets 3.0 g / (kg·d) dissolved in 0.5 ml distilled water by gavage once a day, and the traditional Chinese medicine composition group was given once a day, and the continuous gavage intervention lasted for 12 weeks, all mice were fed in SPF environment, and the diet and water were not limited, and the activity was free; Step 3, specimen collection and processing: (1) After the intervention, the serum specimens of each group of mice were fasted for 12 h, and the eyeball blood was taken into the Ep tube after anesthesia, and the supernatant was taken after centrifugation at 7500 r / min for 15 min, and was stored in-80℃ refrigerator; (2) After the eyeball blood of the mouse was taken, the mouse was immediately sacrificed by cervical dislocation, the heart was quickly exposed by thoracotomy, the heart was removed together with the surrounding fatty tissue, the aortic arch, thoracic aorta, abdominal aorta and iliac aorta were separated by using dissecting scissors and curved forceps, washed in pre-cooled 1xPBS, the PBS was absorbed by filter paper, and was placed in 4% paraformaldehyde for fixation; Step 4, tissue embedding and sectioning: (1) The mouse aorta was taken and fixed in 4% paraformaldehyde according to the volume ratio of formaldehyde 1:10 for 48 h, and was placed in an embedding box; (2) Dehydration and transparency: place the embedding box with the tissue in a round beaker and wash with running water for 10 min; sequentially place in 95% ethanol I for 30 min, 95% ethanol II for 30 min, anhydrous ethanol I for 30 min, and anhydrous ethanol II for 30 min; absorb the alcohol on the surface of the tissue with gauze, and place in xylene I for 30 min and xylene II for 30 min; (3) Wax immersion: maintain in a 60℃ constant temperature box for 1 h in wax immersion liquid I; and 1 h in wax immersion liquid II; (4) Embedding: Put the embedding box soaked in wax into the melted paraffin, and put the tissue embedding box without cover into a special iron box. After dropping the melted paraffin, place the iron box on the cooling table; (5) Slicing: After the wax block solidifies, place it in a-20℃ refrigerator overnight; slice continuously at 4μm, and take 6 slices from each wax block; gently place the slices on the surface of 40℃ water with an ophthalmic forceps, and then place the slices on a non-stick slide after the wrinkles of the slices are naturally flattened; and then naturally dry the slices; Step 5, detect the serum lipid level and the content of cytokines by enzyme-linked immunosorbent double-antibody sandwich method; Step 6, observe the pathological changes of the aorta by immunohistochemical method.

7. The method for detecting the effect of the Chinese medicinal composition for treating atherosclerosis according to claim 6, wherein: In step 6, the immunohistochemical method specifically includes the following detection steps: Step 1, baking: place the glass slide in a 60℃ constant temperature box for 2h; Step 2, deparaffinization and dehydration: 100% toluene 10min / time x 2 times, 100% ethanol 4min / time x 2 times, 95% ethanol 4min / time x 2 times, 90% ethanol 4min / time x 2 times, double distilled water 5min, PBS washing 4min / time x 3 times; Step 3, antigen repair: boiling in 0.01M citric acid buffer (pH 6.0) to 270℃, and then boiling in 0.01M citric acid buffer (pH 6.0) to 140℃, each for 2min, and then naturally cooling to below 50℃; Step 4, remove peroxidase: 1xPBS washing 3 times, each for 4min; 10% hydrogen peroxide washing 1 time, 15min; 1xPBS washing 3 times, each for 4min; Step 5, incubation: drop the first antibody (Wnt1, β-catenin, Dvl) into the refrigerator for incubation overnight (4℃), and then place the incubation box in a constant temperature box for 1h, and then wash with 1xPBS (7.2-7.6) for 4min / time x 3 times; Step 6, secondary antibody: drop the secondary antibody enhancer into a constant temperature box (37℃) for incubation for 30min, and then wash with 1xPBS for 4min / time x 3 times; drop the secondary antibody into a constant temperature box (37℃) for incubation for 35min, and then wash with 1xPBS for 4min / time x 3 times; Step 7, color development: use DAB color development kit, add 5μl of reagent A, B and C in the kit into a clean centrifuge tube containing 1ml of distilled water, mix well, and then drop the sample into the upper chamber for color development at room temperature, control the color development time under a microscope until the cytoplasm of cells presents brown-yellow particles, and the rest of the staining is light (15min), and then wash the excess color developing agent with distilled water to terminate the reaction; Step 8, staining: hematoxylin staining, double distilled water washing, conventional dehydration, transparency, and neutral resin sealing; Step 9, photographing: select typical fields for photographing, and select 5 non-overlapping AS plaques as the center of each field, and then use CIAS-1000 image analysis system to detect the gray value of each field, and then take the average as the gray value of the index, and the higher the gray value, the lighter the positive color, and the lower the protein expression.

8. The method for detecting the effect of the Chinese medicinal composition for treating atherosclerosis according to claim 6, wherein: In step 1, the mice are fed with high-fat feed, and after 12 days of intragastric administration (1 time / day), the model group mice are used as the standard of successful modeling.