Qi deficiency and blood stasis syndrome zebrafish model as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medical models, and discloses a qi deficiency and blood stasis syndrome zebrafish model, a preparation method and application thereof, raw materials of the model comprise an animal model and a modeling agent, the animal model is zebrafish, the animal model is treated by the modeling agent, and the modeling agent comprises verapamil and isoprenaline hydrochloride; the model can be used for evaluating and verifying clinical manifestations of venous sinus extravasated blood and pericardial edema in qi deficiency and blood stasis syndromes, has the advantages of rapidness, high efficiency and economy of in-vitro experiments, and also has the advantages of high predictability, high comparability and the like of in-vivo animal experiments.

Description

Technical Field

[0001] The invention relates to the technical field of medical models, and in particular to a zebrafish model of qi deficiency and blood stasis syndrome, a preparation method and application thereof. Background Art

[0002] Qi deficiency and blood stasis syndrome is a hallmark syndrome of cardiovascular and cerebrovascular diseases in TCM. It refers to the inability of qi to promote blood circulation, resulting in poor blood circulation or even blood stasis. The clinical manifestations of qi deficiency and blood stasis syndrome include fatigue, spontaneous sweating, lack of energy and laziness, local pain, pale purple tongue or ecchymosis, and deep and sluggish pulse. Establishing a suitable experimental animal model of qi deficiency and blood stasis syndrome is of great significance for studying the efficacy and mechanism of traditional Chinese medicine in the treatment of cardiovascular and cerebrovascular diseases.

[0003] At present, animal models of Qi deficiency and blood stasis syndrome mostly adopt rat and mouse models, and are evaluated by observing the macroscopic manifestations, limb behavior and blood rheology indicators of experimental animals that are consistent with TCM syndromes, such as a method for preparing an animal model of Qi deficiency and blood stasis syndrome disclosed in publication number CN107596388B. In recent years, the zebrafish model has been increasingly used in animal models of qi deficiency and blood stasis syndrome due to its advantages of strong visualization, convenient observation, low cost and short cycle. For example, the scientific literature "Guo Shuaijie; Construction and Evaluation of Zebrafish Model of Qi Deficiency and Blood Stasis Syndrome in Traditional Chinese Medicine [J]; World Traditional Chinese Medicine; Issue 06, 2023" used anhydrous sodium sulfite and isoproterenol hydrochloride for combined stimulation to establish a zebrafish model of qi deficiency and blood stasis syndrome; the model used a behavioral analyzer to analyze the total movement distance of the zebrafish, and used a lactate and ATP kit to determine the lactate and ATP levels in the zebrafish; used a fluorescence microscope to take pictures and analyze the fluorescence intensity of zebrafish heart apoptotic cells and myocardial cell apoptosis; and used a heartbeat blood flow analysis system to analyze the blood flow velocity and cardiac output of zebrafish. It has been verified that the combined intervention of anhydrous sodium sulfite and isoproterenol hydrochloride can successfully establish a zebrafish model of qi deficiency and blood stasis syndrome.

[0004] The corresponding clinical manifestations of Qi deficiency and blood stasis syndrome are relatively broad, and the methods for establishing animal models of Qi deficiency and blood stasis syndrome are completely different for different clinical manifestations of Qi deficiency and blood stasis syndrome. The two clinical manifestations of venous sinus congestion and pericardial edema in Qi deficiency and blood stasis syndrome cannot be evaluated and verified by the animal models of Qi deficiency and blood stasis syndrome in the existing technology. Therefore, it is necessary to establish a new animal model that can evaluate and verify venous sinus congestion and pericardial edema in Qi deficiency and blood stasis syndrome. Summary of the invention

[0005] The present invention provides a zebrafish model of qi deficiency and blood stasis syndrome, a preparation method and an application thereof. The model can evaluate and verify the clinical manifestations of venous sinus congestion and pericardial edema in qi deficiency and blood stasis syndrome, and the zebrafish model of qi deficiency and blood stasis syndrome is successfully established through the combined intervention of verapamil and isoproterenol hydrochloride.

[0006] The specific technical scheme of the present invention is: A zebrafish model of qi deficiency and blood stasis syndrome comprises raw materials including an animal model and a modeling agent. The animal model is zebrafish. The animal model is treated with a modeling agent. The modeling agent comprises verapamil and isoproterenol hydrochloride.

[0007] Preferably, the mass ratio of verapamil to isoproterenol hydrochloride is 0.03:3.12-25.

[0008] Preferably, the concentration of the verapamil is 0.03 mg / ml, and the concentration of the isoproterenol hydrochloride is 3.12-25 mg / ml.

[0009] The invention provides a zebrafish model of qi deficiency and blood stasis syndrome, wherein verapamil and isoproterenol hydrochloride are used as modeling agents, the model has visualization, and the clinical manifestations of qi deficiency and blood stasis syndrome can be observed visually, and the model adopts verapamil and isoproterenol hydrochloride as modeling agents, and the two clinical manifestations of venous sinus congestion and pericardial edema in qi deficiency and blood stasis syndrome can be evaluated and verified.

[0010] Verapamil is a calcium ion antagonist that exerts its pharmacological action by regulating the influx of calcium ions on the cell membranes of myocardial conduction cells, myocardial contractile cells, and arterial smooth muscle cells; it can reduce myocardial contractility and atrioventricular conduction and lower the heart rate, which may cause hemodynamic changes and simulate the "blood stasis" part of qi deficiency and blood stasis; isoproterenol hydrochloride is a beta receptor agonist that has a strong stimulatory effect on both β1 and β2 receptors and almost no effect on α receptors. It acts on the cardiac β1 receptors, increasing cardiac contractility, heart rate, conduction acceleration, cardiac output and myocardial oxygen consumption; it acts on the vascular smooth muscle β2 receptors, causing significant dilation of skeletal muscle vessels, as well as varying degrees of dilation of the kidneys, mesenteric vessels and coronary arteries, and reduced peripheral vascular resistance; this drug can simulate the "Qi deficiency" part of Qi deficiency and blood stasis by enhancing the heart's contractility and heart rate, and dilating blood vessels; in addition, the combined use of verapamil and isoproterenol hydrochloride can reduce the hypotensive effect, which can further eliminate the interference of hypotension on Qi deficiency and blood stasis syndrome.

[0011] A method for preparing the above-mentioned zebrafish model of qi deficiency and blood stasis syndrome comprises the following steps: preparing a modeling agent with verapamil and isoproterenol hydrochloride, and administering the modeling agent to the zebrafish intestine in a water-soluble exposure manner to prepare a zebrafish model of qi deficiency and blood stasis syndrome.

[0012] Preferably, the zebrafish is a wild-type AB strain zebrafish.

[0013] Preferably, the water used for feeding zebrafish is reverse osmosis water and instant sea salt, the conductivity of the feeding water is 450-550 μS / cm, the pH value is 6.5-8.5, and the hardness is 50-100 mg / L CaCO3.

[0014] An application of the above-mentioned zebrafish model of qi deficiency and blood stasis syndrome in drug screening is characterized by comprising the following steps: using a water-soluble modeling agent to administer to a drug group zebrafish to prepare a zebrafish model of qi deficiency and blood stasis syndrome, then using a water-soluble drug administration method to treat the zebrafish model of the drug group, wherein the drug is Qili Qiangxin Capsule, and then testing the zebrafish heart area, venous congestion area and total movement distance of the zebrafish model of the drug group.

[0015] Preferably, the zebrafish heart area is tested using a fluorescence microscope.

[0016] Preferably, the zebrafish venous congestion area is tested using a fluorescence microscope.

[0017] Preferably, the total movement distance of the zebrafish is tested using a behavior analyzer.

[0018] Compared with the prior art, this application has the following technical effects: The present invention provides a zebrafish model of qi deficiency and blood stasis syndrome, which can evaluate and verify the clinical manifestations of venous sinus congestion and pericardial edema in qi deficiency and blood stasis syndrome. The zebrafish model of qi deficiency and blood stasis syndrome is successfully established through the combined intervention of verapamil and isoproterenol hydrochloride. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the embodiments.

[0020] The experimental animals used in the following embodiments are zebrafish, which are wild-type AB strain zebrafish. The zebrafish are bred by natural pair mating. The experimental animal use license number is SYXK (Zhejiang) 2022-0004, and the breeding and management meet the requirements of the international AAALAC certification (certification number: 001458).

[0021] The instruments, consumables and reagents used in the following examples are: Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); Ultrapure water machine (EPED-S2-30D, Nanjing E-Pure Technology Co., Ltd., China); Biochemical incubator (SPX-250B-Z, Shanghai Boxun Medical Biological Instrument Co., Ltd., China); Behavior analyzer (Zebra Lab 3.11, ViewPoint, France); 6-well plate (lot number DB1000305, Zhejiang Bellanbo Biotechnology Co., Ltd., China); 96-well plate (lot number 080723BL01, Nest Biotech, China); Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); Isoproterenol hydrochloride (Tokyo Chemical Industry Co., Ltd., Japan, batch number: VNVFK-OR).

[0022] Embodiment 1: A method for preparing a zebrafish model of qi deficiency and blood stasis syndrome comprises the following steps: Wild-type AB zebrafish fertilized for three days were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 3.12 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were prepared into a modeling agent. Wild-type AB zebrafish fertilized for three days were treated with the modeling agent for 1 day at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. A control group model was set up, and no modeling agent was added to the control group. The Qi-deficiency and blood-stasis syndrome model and the control group model were verified and analyzed. Ten zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded. Ten zebrafish were randomly selected and photographed under a fluorescence microscope. NIS-Elements D 3.20 advanced image processing software was used to analyze the heart area and venous congestion area of ​​the zebrafish. The results were statistically analyzed using SPSS26.0 software. p<0.05 indicated that the difference was statistically significant.

[0023] Embodiment 2: A method for preparing a zebrafish model of qi deficiency and blood stasis syndrome comprises the following steps: Wild-type AB zebrafish fertilized for three days were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 6.25 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were prepared into a modeling agent. Wild-type AB zebrafish fertilized for three days were treated with the modeling agent for 1 day at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. A control group model was set up, and no modeling agent was added to the control group. The Qi-deficiency and blood-stasis syndrome model and the control group model were verified and analyzed. Ten zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded. Ten zebrafish were randomly selected and photographed under a fluorescence microscope. NIS-Elements D 3.20 advanced image processing software was used to analyze the heart area and venous congestion area of ​​the zebrafish. The results were statistically analyzed using SPSS26.0 software. p<0.05 indicated that the difference was statistically significant.

[0024] Embodiment 3: A method for preparing a zebrafish model of qi deficiency and blood stasis syndrome comprises the following steps: Wild-type AB zebrafish fertilized for three days were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 12.5 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were prepared into a modeling agent. Wild-type AB zebrafish fertilized for three days were treated with the modeling agent for 1 day at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. A control group model was set up, and the control group was not treated with modeling agents; The Qi deficiency and blood stasis syndrome model and the control group model were verified and analyzed; 10 zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded; 10 zebrafish were randomly selected and placed under a fluorescence microscope for photography, and the heart area and venous congestion area of ​​the zebrafish were analyzed using NIS-Elements D 3.20 advanced image processing software. The results were statistically analyzed using SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0025] Embodiment 4: A method for preparing a zebrafish model of qi deficiency and blood stasis syndrome comprises the following steps: Wild-type AB zebrafish fertilized for three days were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 25.0 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were prepared into a modeling agent. Wild-type AB zebrafish fertilized for three days were treated with the modeling agent for 1 day at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. A control group model was set up, and no modeling agent was added to the control group. The Qi-deficiency and blood-stasis syndrome model and the control group model were verified and analyzed. Ten zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded. Ten zebrafish were randomly selected and photographed under a fluorescence microscope. NIS-Elements D 3.20 advanced image processing software was used to analyze the heart area and venous congestion area of ​​the zebrafish. The results were statistically analyzed using SPSS26.0 software. p<0.05 indicated that the difference was statistically significant.

[0026] Comparative Example 1: Compared with Comparative Example 1, the modeling agent in Comparative Example 3 is only isoproterenol hydrochloride and water with an equal dose of verapamil, and the other conditions are the same as those in Comparative Example 1.

[0027] Comparative Example 2: Compared with Comparative Example 1, the modeling agent in Comparative Example 4 is only verapamil and water with an equal dosage of isoproterenol hydrochloride, and the other conditions are the same as those in Comparative Example 1.

[0028] The test results of Examples 1 to 4 and Comparative Example 1 are shown in Table 1. In Table 1, compared with the control group, * means p<0.05, which means there is a difference, but the difference is not significant; ** means p<0.01, which means a significant difference; *** means p<0.001, which means an extremely significant difference; Table 1 Test results of Examples 1 to 4 As shown in Table 1, Examples 1 to 4 are zebrafish models of qi deficiency and blood stasis prepared by using isoproterenol hydrochloride and verapamil as modeling agents. The results of Examples 1 to 4 and the control group show that after zebrafish are treated with isoproterenol hydrochloride and verapamil as modeling agents, the venous congestion area of ​​zebrafish is significantly increased, the heart area is significantly increased, and the total movement distance is significantly reduced. The venous congestion area can correspond to the clinical manifestations of venous sinus congestion in qi deficiency and blood stasis syndrome, the heart area can correspond to the clinical manifestations of pericardial edema in qi deficiency and blood stasis syndrome, and the total movement distance can correspond to the clinical manifestations of fatigue and exertion in qi deficiency and blood stasis syndrome. The above results show that the zebrafish model of qi deficiency and blood stasis syndrome provided by the present invention is successful, and isoproterenol hydrochloride and verapamil can be used as modeling agents to successfully establish a zebrafish model of qi deficiency and blood stasis syndrome.

[0029] In addition, Examples 1 to 4 also studied the modeling dose, and the results showed that with the increase in the dosage of isoproterenol hydrochloride, the venous congestion area of ​​the zebrafish model of qi deficiency and blood stasis syndrome showed a gradual downward trend, the heart area showed a gradual downward trend, and the total movement distance also showed a gradual downward trend. When the ratio of isoproterenol hydrochloride to verapamil was 3.12:0.03, the zebrafish model of qi deficiency and blood stasis syndrome had significant differences in venous congestion area, heart area and total movement distance compared with the control group.

[0030] In Comparative Example 1, the hypotensive animal model treated with only isoproterenol hydrochloride showed a significant increase in heart area, while the total movement distance and venous congestion area did not change significantly. In Comparative Example 2, the hypotensive animal model treated with only verapamil showed a significant increase in venous congestion area, while the total movement distance and heart area did not change significantly.

[0031] Embodiment 5: An application of a zebrafish model of qi deficiency and blood stasis syndrome in drug screening comprises the following steps: Wild-type AB zebrafish of 3 days old were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 3.12 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were used to prepare a modeling agent. The zebrafish in the drug group were treated with the modeling agent for 1 day at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. The zebrafish model of Qi deficiency and blood stasis syndrome was treated with 15.6 mg / mL Qili Qiangxin Capsule water solution at 28°C for 1 day to prepare the zebrafish model of the drug group. A control group was set up, which was not treated with Qili Qiangxin Capsule and modeling agent; A model control group was set up, and the drug control group was not treated with Qili Qiangxin Capsule; The Qi deficiency and blood stasis syndrome model and the control group model were verified and analyzed; 10 zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded; 10 zebrafish were randomly selected and placed under a fluorescence microscope for photography, and the heart area and venous congestion area of ​​the zebrafish were analyzed using NIS-Elements D 3.20 advanced image processing software. The results were statistically analyzed using SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0032] Embodiment 6: An application of a zebrafish model of qi deficiency and blood stasis syndrome in drug screening comprises the following steps: Wild-type AB zebrafish of 3 days old were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 3.12 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were used to prepare a modeling agent. The modeling agent was used to treat zebrafish in the drug group for 2 days at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. 31.2 mg / mL of Qili Qiangxin Capsule Water-soluble was used to treat zebrafish model of qi deficiency and blood stasis syndrome at a temperature of 28°C for 1 day to prepare a zebrafish model of the drug group. A control group was set up, which was not treated with Qili Qiangxin Capsule and modeling agent; A model control group was set up, and the drug control group was not treated with Qili Qiangxin Capsule; The Qi deficiency and blood stasis syndrome model and the control group model were verified and analyzed; 10 zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded; 10 zebrafish were randomly selected and placed under a fluorescence microscope for photography, and the heart area and venous congestion area of ​​the zebrafish were analyzed using NIS-Elements D 3.20 advanced image processing software. The results were statistically analyzed using SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0033] Embodiment 7: An application of a zebrafish model of qi deficiency and blood stasis syndrome in drug screening comprises the following steps: Wild-type AB zebrafish of the three-day-old fertilization were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 3.12 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were used to prepare a modeling agent. The zebrafish in the drug group were treated with the modeling agent for 2 days at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. The zebrafish model of Qi deficiency and blood stasis syndrome was treated with 62.5 mg / mL Qili Qiangxin Capsule water solution at 28°C for 1 day to prepare the zebrafish model of the drug group. A control group was set up, which was not treated with Qili Qiangxin Capsule and modeling agent; A model control group was set up, and the drug control group was not treated with Qili Qiangxin Capsule; The Qi deficiency and blood stasis syndrome model and the control group model were verified and analyzed; 10 zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded; 10 zebrafish were randomly selected and placed under a fluorescence microscope for photography, and the heart area and venous congestion area of ​​the zebrafish were analyzed using NIS-Elements D 3.20 advanced image processing software. The results were statistically analyzed using SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0034] Embodiment 8: An application of a zebrafish model of qi deficiency and blood stasis syndrome in drug screening comprises the following steps: Wild-type AB zebrafish of 3 days old were randomly selected and placed in a 6-well plate, with 30 fish in each well and a capacity of 3 mL in each well. 3.12 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were used to prepare a modeling agent, and the modeling agent was used to treat zebrafish in the drug group for 2 days at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. 125 mg / mL of Qili Qiangxin Capsule Water-soluble was used to treat zebrafish model of qi deficiency and blood stasis syndrome at a temperature of 28°C for 1 day to prepare a zebrafish model of the drug group. A control group was set up, which was not treated with Qili Qiangxin Capsule and modeling agent; A model control group was set up, and the drug control group was not treated with Qili Qiangxin Capsule; The Qi deficiency and blood stasis syndrome model and the control group model were verified and analyzed; 10 zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded; 10 zebrafish were randomly selected and placed under a fluorescence microscope for photography, and the heart area and venous congestion area of ​​the zebrafish were analyzed using NIS-Elements D 3.20 advanced image processing software. The results were statistically analyzed using SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0035] Embodiment 9: An application of a zebrafish model of qi deficiency and blood stasis syndrome in drug screening comprises the following steps: Wild-type AB zebrafish of the three-day-old fertilization were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 3.12 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were used to prepare a modeling agent. The zebrafish in the drug group were treated with the modeling agent for 2 days at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. The zebrafish model of Qi deficiency and blood stasis syndrome was treated with 250 mg / mL Qili Qiangxin Capsule water solution at 28°C for 1 day to prepare the zebrafish model of the drug group. A control group was set up, which was not treated with Qili Qiangxin Capsule and modeling agent; A model control group was set up, and the drug control group was not treated with Qili Qiangxin Capsule; The Qi deficiency and blood stasis syndrome model and the control group model were verified and analyzed; 10 zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded; 10 zebrafish were randomly selected and placed under a fluorescence microscope for photography, and the heart area and venous congestion area of ​​the zebrafish were analyzed using NIS-Elements D 3.20 advanced image processing software. The results were statistically analyzed using SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0036] Example 10 An application of a zebrafish model of qi deficiency and blood stasis syndrome in drug screening comprises the following steps: Wild-type AB zebrafish of the three-day-old fertilization were randomly selected and placed in a 6-well plate, with 30 fish in each well and a volume of 3 mL in each well. 3.12 mg / mL of isoproterenol hydrochloride and 0.03 mg / mL of verapamil were used to prepare a modeling agent. The zebrafish in the drug group were treated with the modeling agent for 2 days at a temperature of 28°C to prepare a zebrafish model of qi deficiency and blood stasis syndrome. The zebrafish model of Qi deficiency and blood stasis syndrome was treated with 500 mg / mL Qili Qiangxin Capsule water solution at 28°C for 1 day to prepare the zebrafish model of the drug group. A control group was set up, which was not treated with Qili Qiangxin Capsule and modeling agent; A model control group was set up, and the drug control group was not treated with Qili Qiangxin Capsule; The Qi deficiency and blood stasis syndrome model and the control group model were verified and analyzed; 10 zebrafish were randomly selected and placed under a behavior analyzer for testing, and the total movement distance of the zebrafish was recorded; 10 zebrafish were randomly selected and placed under a fluorescence microscope for photography, and the heart area and venous congestion area of ​​the zebrafish were analyzed using NIS-Elements D 3.20 advanced image processing software. The results were statistically analyzed using SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0037] The test results of Examples 5 to 10 are shown in Table 2. In Table 2, compared with the control group, * means p<0.05, indicating a difference, but the difference is not significant; ** means p<0.01, indicating a significant difference; *** means p<0.001, indicating an extremely significant difference; Table 2 Test results of Examples 5 to 10 As shown in Table 2, when screening the positive drug Qili Qiangxin Capsule for Qi deficiency and blood stasis syndrome, it was found that when the dosage of Qili Qiangxin Capsule reached more than 125 mg / mL, the zebrafish model died and the screening test could not be performed. The results of the control group and the model group showed that the zebrafish model of Qi deficiency and blood stasis syndrome was successfully established. After analyzing the results of venous congestion area, it was found that Qili Qiangxin Capsule could significantly reduce the venous congestion area of ​​zebrafish, and had an extremely significant reference therapeutic effect on the treatment of venous sinus congestion in Qi deficiency and blood stasis syndrome; after analyzing the results of heart area, it was found that Qili Qiangxin Capsule could increase the heart area of ​​zebrafish, and it had a reference effect on aggravating pericardial edema in Qi deficiency and blood stasis syndrome; after analyzing the results of total movement distance, it was found that Qili Qiangxin Capsule could increase the total movement distance, and it had a reference therapeutic effect on alleviating fatigue and weakness in Qi deficiency and blood stasis syndrome.

[0038] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A zebrafish model of Qi deficiency and blood stasis syndrome, characterized in that: The raw materials include an animal model and a modeling agent. The animal model is zebrafish. The animal model is treated with a modeling agent. The modeling agent includes verapamil and isoproterenol hydrochloride. The mass ratio of verapamil to isoproterenol hydrochloride is 0.03:3.12-25.

2. The zebrafish model of Qi deficiency and blood stasis according to claim 1, characterized in that: The concentration of verapamil is 0.03 mg / ml.

3. The zebrafish model of Qi deficiency and blood stasis according to claim 1, characterized in that: The concentration of the isoproterenol hydrochloride is 3.12-25 mg / ml.

4. A method for preparing a zebrafish model of Qi deficiency and blood stasis according to any one of claims 1 to 3, characterized in that: The following steps are involved: Verapamil and isoproterenol hydrochloride were made into a modeling agent, which was then administered into the intestine of zebrafish in a water-soluble exposure manner to create a zebrafish model of qi deficiency and blood stasis syndrome.

5. The preparation method according to claim 4, characterized in that: The zebrafish is a wild-type AB strain zebrafish.

6. The preparation method according to claim 4 or 5, characterized in that: The water used for feeding the zebrafish is reverse osmosis water and instant sea salt. The conductivity of the water used for feeding the zebrafish is 450-550 μS / cm, the pH value is 6.5-8.5, and the hardness is 50-100 mg / L CaCO3.

7. Use of the zebrafish model of qi deficiency and blood stasis according to any one of claims 1 to 3 in drug screening, characterized in that: The following steps are involved: The zebrafish model of qi deficiency and blood stasis syndrome was established by administering the modeling agent in the drug group to the zebrafish. The zebrafish model in the drug group was then treated with the drug in the form of water-soluble drug administration. The drug was Qili Qiangxin Capsule. The heart area, venous congestion area and total movement distance of the zebrafish model in the drug group were tested.

8. The use according to claim 7, characterized in that: The zebrafish heart area was tested using a fluorescence microscope.

9. The use according to claim 7, characterized in that: The zebrafish venous congestion area is tested using a fluorescence microscope.

10. The use according to claim 7, characterized in that: The total movement distance of the zebrafish was tested using a behavior analyzer.

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