Establishment method for researching zebra fish hypertension model based on Poiseuille law

Abstract

The invention discloses a method for establishing a model for researching zebra fish hypertension based on the Poiseuille law. The method comprises the following steps: randomly selecting a plurality of zebra fishes; preparing angiotensin reagents with different concentrations to carry out medicated bath on the zebrafish; determining the concentration of an angiotensin reagent; treating medicine reagents with different concentrations are prepared for treating the zebra fish in a medicated bath; measuring the concentration of angiotensin of the zebra fish; through model establishment, the zebrafish hypertension model can be used as a platform for developing hypertension drugs and testing a treatment mechanism of human related genes.

Description

Technical Field

[0001] The invention relates to the field of hypertension research, and in particular to a method for establishing a zebrafish hypertension model based on Poiseuille's law. Background Art

[0002] Hypertension refers to the phenomenon that the pressure exerted by blood on the blood vessel wall when flowing is continuously higher than normal. There are about 245 million people with hypertension in my country, and the number is still on the rise. The prevention and treatment of hypertension is an important public health strategy. Research on the causes, prevention and treatment of all forms of hypertension remains an active field in biomedicine. Establishing an animal model of hypertension is the main method to understand the mechanism of disease occurrence and to design and test new therapies.

[0003] In recent years, zebrafish (Danio rerio) has become a highly regarded model organism for the study of cardiovascular and metabolic diseases, with unique advantages for both basic research and drug discovery. Genetic conservation between zebrafish and humans, as well as high productivity and transparent juveniles, enable efficient large-scale genetic and drug-oriented screening studies. Zebrafish possess a simplified cardiovascular system similar to that of mammals, which makes them particularly suitable for modeling various aspects of cardiac development, function and disease. Visualization of zebrafish embryos enables real-time, digital, and reproducible cardiovascular dynamics, while providing insights into early embryonic events and facilitating the study of heart-related abnormalities. High reproduction rates allow large samples for credible statistical analysis and serve as advanced genetic tools, such as CRISPR / Cas9-mediated mutants and tissue-specific expression knockouts to mimic specific gene mutations associated with human diseases, enabling precise gene editing and identification of potential therapeutic targets. In addition, the permeability of zebrafish embryos to small molecules facilitates drug discovery and screening, providing a rapid and economical method for identifying compounds with therapeutic potential. In summary, the establishment of zebrafish disease models allows us to understand the pathogenesis of diseases and provides a platform for translational research and the development of new therapeutic interventions.

[0004] Rider used transgenic zebrafish and found that zebrafish affected blood circulation under high sodium content. The released renin changed the process of angiotensin I (Ang I) conversion to angiotensin II (Ang II) through angiotensin converting enzyme. The data and function of renin are similar to those of humans, and the renin-angiotensin-aldosterone system (RAAS) is one of the mechanisms for regulating blood pressure. In addition, a hypertensive nephropathy model was established by knocking out the zebrafish hypertension-related gene NOSTRIN. The mechanism is that knocking out the NOSTRIN gene will change the glomerular filtration barrier function, induce proteinuria and lead to ultrastructural morphological changes in the glomerular basement membrane of the endothelial and epithelial sides and the glomerular capillary loops. Another example is the discovery of the connection between the new gene lysine-deficient protein kinase 1 (WNK1) and hypertension and angiogenesis. When WNK1 is overexpressed, it will cause human hypertension; when WNK1 is knocked out in zebrafish, it will cause cardiovascular defects. It can be seen that the genetic similarity between zebrafish and mammals can support the establishment of a model of hypertension through gene mutation, chemical induction and dietary changes. When administering angiotensin II to zebrafish, because angiotensin II itself is an intravenous drug, it is injected into the zebrafish, and the developmental stage of the zebrafish is ignored, resulting in deviations and inaccuracies in the experimental results. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract of the specification and the title of the invention of this application to avoid blurring the purpose of this section, the abstract of the specification and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the technical problem to be solved by the present invention is that when administering angiotensin II to zebrafish, since angiotensin II itself is an intravenous drug, the zebrafish is injected and the developmental stage of the zebrafish is ignored, resulting in deviations and inaccuracies in the experimental results.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for establishing a zebrafish hypertension model based on Poiseuille's law, comprising:

[0009] Randomly select a number of zebrafish;

[0010] Prepare different concentrations of angiotensin reagents to bathe zebrafish;

[0011] Determine angiotensin reagent concentration;

[0012] Preparing therapeutic drug reagents of different concentrations to perform therapeutic drug bath on zebrafish;

[0013] Determination of angiotensin concentrations in zebrafish.

[0014] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, the randomly selected zebrafish are 3dpf wild AB type zebrafish.

[0015] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, the angiotensin reagent is a mother solution prepared by adding angiotensin to DMSO to prepare a concentration of 10 mmol / L.

[0016] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, the drug bathing of zebrafish includes: bathing 4 ml of angiotensin reagent with different concentrations for 48 hours, and changing the drug solution every 24 hours.

[0017] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, the method of determining the concentration of angiotensin reagent comprises:

[0018] Measure blood vessel diameters in zebrafish embryo images;

[0019] Measuring blood flow velocity in zebrafish embryo videos;

[0020] The angiotensin reagent concentration was determined to be 2 μM.

[0021] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, the preparation of therapeutic drug reagents of different concentrations includes: preparing a mother solution of 100 mmol / L verapamil with DMSO and preparing a mother solution of 100 mmol / L valsartan with DMSO.

[0022] As a preferred scheme of the method for establishing a zebrafish hypertension model based on Poiseuille's law described in the present invention, the therapeutic drug bathing of zebrafish includes: selecting valsartan (Val) and verapamil (Ver) as therapeutic drugs, administering 4 ml of the drug, and bathing for 48 hours.

[0023] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, it also includes:

[0024] Observe and record adverse reactions such as zebrafish embryo death, deformity, cardiotoxicity, and liver toxicity to determine the dosage concentration.

[0025] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, the determination of zebrafish angiotensin concentration includes detecting the angiotensin level in vivo by enzyme-linked immunosorbent assay (ELISA).

[0026] As a preferred solution of the method for establishing a zebrafish hypertension model based on Poiseuille's law of the present invention, blank control groups are set for the angiotensin reagents of different concentrations and the therapeutic drug reagents of different concentrations.

[0027] Beneficial effects of the present invention: By selecting zebrafish at an appropriate developmental stage and the medicinal bath method, it was found that the vascular diameter and blood flow rate of zebrafish in the model group were significantly lower than those in the blank control group (P<0.05). The vascular diameter and blood flow rate of zebrafish in the treatment group were higher than those in the model group (P<0.05); at the same time, the Ang II content in the treatment group was lower than that in the model group (P<0.05). The zebrafish hypertension model can be used as a platform for the development of hypertension drugs and testing the therapeutic mechanism of human-related genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0029] Figure 1 A schematic diagram of using software to measure blood vessel conditions in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0030] Figure 2 A schematic diagram of the effect of different doses of Ang II for 48 hours on blood vessel diameter in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0031] Figure 3 A schematic diagram of the effect of different doses of Ang II on blood flow velocity for 48 hours in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0032] Figure 4 A schematic diagram of the analysis of zebrafish blood vessel diameter after blood pressure reduction (Val) in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0033] Figure 5A schematic diagram of the analysis of zebrafish blood vessel diameter after blood pressure reduction (Ver) in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0034] Figure 6 A schematic diagram of the blood flow velocity of zebrafish after blood pressure reduction (Val) in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0035] Figure 7 A schematic diagram of the blood flow velocity of zebrafish after blood pressure reduction (Ver) in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0036] Figure 8 A schematic diagram of detecting the zebrafish Ang II content after blood pressure reduction (Val) in a method for establishing a zebrafish hypertension model based on Poiseuille's law according to an embodiment of the present invention;

[0037] Fig. 9 A schematic diagram of detecting the Ang II content in zebrafish after blood pressure reduction (Ver) in a method for establishing a zebrafish hypertension model based on Poiseuille's law as described in an embodiment of the present invention. DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0039] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0040] Secondly, the present invention is described in detail with reference to schematic diagrams. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0041] Furthermore, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0042] The main reagents and instruments used in the embodiment are: angiotensin II (Abmole, USA, catalog number: M6240); verapamil (Beijing Solebow Technology Co., Ltd., China, catalog number: SV8440); valsartan (Shanghai Yuanye Biotechnology Co., Ltd., China, catalog number: B33744); DMSO (Beijing Solebow Technology Co., Ltd., China, catalog number: D8371); zebrafish angiotensin II (Ang-II) Elisa kit (Jiangsu Jingmei Biotechnology Co., Ltd., China, catalog number: JM-08247F1); micro high-speed centrifuge (Changsha Xiangzhi Centrifuge Instrument Co., Ltd., China, model: TG16W); microplate reader (Labsystems Multiskan MS, Finland, model: 352).

[0043] Statistical methods of the embodiment: GraphPad Prism 10 software was used for statistical analysis and plotting. Data are presented as mean ± standard error (SEM). All data were tested for homogeneity of variance and normality, and the experimental data all conformed to normal distribution. One-way ANOVA was used, followed by Dunnett's t test to analyze the differences between the groups.

[0044] A p value of <0.05 was considered statistically significant.

[0045] # or *: p < 0.05;

[0046] ## or **: p < 0.01;

[0047] ### or ***: p<0.001.

[0048] Reference Figures 1 to 9 This embodiment provides a method for establishing a zebrafish hypertension model based on Poiseuille's law, comprising the following steps:

[0049] Randomly select a number of zebrafish;

[0050] Prepare different concentrations of angiotensin reagents to bathe zebrafish;

[0051] Determine angiotensin reagent concentration;

[0052] Preparing therapeutic drug reagents of different concentrations to perform therapeutic drug bath on zebrafish;

[0053] Determination of angiotensin concentrations in zebrafish.

[0054] Among them, a number of wild AB zebrafish of 3 dpf (3 days after fertilization) were randomly selected. The zebrafish were obtained as follows: the zebrafish were raised in a centralized circulation system at 28°C, with fish water (water quality: 200 mg of instant sea salt was added to each 1L of reverse osmosis water, the conductivity was 480-510 μS / cm, the pH was 6.9-7.2, and the hardness was 53.7-71.6 mg / L CaCO3), and the light cycle was a 14-hour light cycle and a 10-hour dark cycle. After the dark cycle, within 1 hour after the start of the light cycle, the fertilized eggs were collected after spawning and raised in embryo water (0.137 mol / L sodium chloride, 5.4 mol / L potassium chloride, 0.25 mol / L disodium hydrogen phosphate, 0.44 mol / L potassium hydrogen phosphate, 1.3 mol / L calcium chloride, 1.0 mol / L magnesium sulfate and 4.2 mol / L sodium bicarbonate), and the embryo water was changed regularly.

[0055] After 3 days, the zebrafish were randomly assigned to 12-well plates with 4 mL of fish water in each well, and 20 zebrafish were placed in each well. The procedures for anesthesia and killing of zebrafish were in accordance with the American Veterinary Medical Association (AVMA) standard requirements for animal anesthesia and killing.

[0056] The reason why 72 hpf (3 dpf) zebrafish were selected as the hypertension model is that zebrafish at this developmental stage are relatively stable. Zebrafish at 48 hours have not fully hatched and their blood vessels have not yet completed development.

[0057] It includes 2 aspects:

[0058] 1. Starting at 51h, the shrinking yolk sac makes the pericardial cavity prominent. The strong beating of the heart makes it easy to see the bending of the heart chamber, which moves the atrium to the dorsal side of the ventricle. Nomarski light microscopy shows that the six pairs of large arterial arches are very clear at this time, but usually not all of them can carry blood flow during the long-pectoral fin stage. In general, the arterial arches closer to the front, especially the arterial arches 1-3, start blood circulation earlier. At this time, blood circulation is very active in most of the blood vessels along the trunk and tail. Each segment of the blood vessel is connected by a single channel, but tracking the blood flow channel test shows that the blood vessels from the dorsal aorta in the blood vessel segment are replaced by veins in the connecting blood vessel segment, and the axial vein is forward. Blood circulation does not occur in the pectoral fin bud until 52h, and it is more continuous at 54h. At this site, the subclavian vein can be seen looping along the base of the fin bud, and the arterial and venous sides of the loop cannot be clearly distinguished. The common cardinal vein on the yolk bulb is fan-shaped, narrow at the beginning and wider toward the abdomen. At 60 hours, the heart is prominent, pulsating strongly, and filled with circulating blood, which flows through all arterial arches and circulates under the clavicle. The main vein is no longer fan-shaped, but it is still narrow and straight, reaching most of the yolk in the front and the heart in the back. After 72 hours, it enters the early juvenile stage, and the blood vessels throughout the body are fully developed.

[0059] 2. Vascular endothelial growth factor receptor 2 (VEGFR2) is a marker of angiogenesis. Studies have found that VEGFR2 in zebrafish is on the rise from 12h to 72h, and decreases significantly after 72h. This indicates that zebrafish vascular development reaches maturity at 72h.

[0060] The angiotensin reagent is prepared by adding angiotensin to DMSO to prepare a 10mmol / L stock solution. Keep it in a dark place and refrigerate it. When using it, dilute it with embryo water.

[0061] 4 ml of angiotensin reagent with different concentrations was used for drug bath treatment for 48 hours, and the solution was changed every 24 hours.

[0062] Determination of angiotensin reagent concentrations involves:

[0063] Measure blood vessel diameters in zebrafish embryo images;

[0064] Measuring blood flow velocity in zebrafish embryo videos;

[0065] The angiotensin reagent concentration was determined to be 2 μM.

[0066] Specifically: zebrafish embryos were randomly divided into 12-well plates, 20 per well. Zebrafish embryos were divided into 6 groups, and different concentrations of angiotensin II (0.25, 0.5, 1, 2, 3 μM) and 0.1% DMSO were used as blank controls. 4 ml of drug bath was applied for 48 hours, and the drug solution was changed every 24 hours. Figure 1 , and then take pictures and shoot videos under a microscope for 10 seconds. FijiimageJ software was used to measure the blood vessel diameter of zebrafish embryo pictures, and DanioScope software was used to measure the blood flow velocity of zebrafish embryo videos. The measurement parts were all the same dorsal aorta. The experimental results represent the results of three independent experiments.

[0067] Reference Figures 2-3 In the process of inducing hypertension model, compared with the blank control group, different concentrations of Ang II (angiotensin II) had a significant contraction effect on the diameter of zebrafish blood vessels (p<0.001); in terms of blood flow rate, 0.25μM was lower than the blank group, but not significantly, 0.5μM and 1μM had a significant effect of reducing blood flow rate (p<0.05), 2μM and 3μM had a significant effect of reducing blood flow rate (p<0.001). According to the data of blood vessel diameter and blood flow rate, the optimal modeling concentration of Ang II was determined to be 2μM.

[0068] Reference Figures 2-3 , are representative graphs showing the effects of different concentrations of Ang II on the diameter of blood vessels and blood flow rate in zebrafish embryos. The standard is = 200 μm. Figure 2To statistically analyze the effects of different doses of Ang II on vascular diameter for 48 h, n=20, the experiment was repeated 3 times. Figure 3 To statistically analyze the effects of different doses of Ang II on blood flow velocity for 48 h, the larger the percentage, the faster the blood flow velocity, n = 20, and the experiment was repeated 3 times.

[0069] It should be noted that zebrafish can absorb through the skin, gills, digestive system, etc., which is simpler to operate and saves reagents than traditional drug administration. Zebrafish can absorb the test compound through the gills, mouth and skin. The sample can be absorbed by simply dissolving it in the zebrafish culture medium. However, only the development of zebrafish organs at 72hpf (3dpf) meets the drug bath conditions.

[0070] It should be noted that if injection is used, the injection method will lead to uneven concentration of angiotensin II in zebrafish due to individual differences in injection site and dosage, thus affecting the accuracy and repeatability of the experimental results. The "drug bath" method can make zebrafish uniformly exposed to angiotensin II in the solution, ensuring that each zebrafish receives a relatively consistent drug dose, making the experimental results more stable and reliable, which is conducive to improving the quality of experimental data and more accurately evaluating the antihypertensive effect of drugs or substances. In addition, the physiological structure of some special zebrafish strains or specific developmental stages may be relatively fragile, and the injection method may cause a greater stress response or even damage to them, affecting the normal progress of the experiment. As a non-invasive drug administration method, the "drug bath" method can avoid direct damage to these special zebrafish, expand the range of zebrafish samples that can be used for experiments, and provide a possibility for studying hypertension models of different types of zebrafish, which helps to understand hypertension-related issues more comprehensively.

[0071] After determining that the optimal modeling concentration of Ang II was 2 μM, this concentration was used as the dosing condition, and valsartan (Val) and verapamil (Ver) were selected as therapeutic drugs.

[0072] The preparation of therapeutic drug reagents of different concentrations includes: preparing a mother solution of 100 mmol / L verapamil with DMSO, and preparing a mother solution of 100 mmol / L valsartan with DMSO.

[0073] The method of treating zebrafish with a medicinal bath comprises: selecting valsartan (Val) and verapamil (Ver) as therapeutic drugs, administering 4 ml of the drugs, and performing a medicinal bath treatment for 48 hours.

[0074] Observe and record adverse reactions such as zebrafish embryo death, deformity, cardiotoxicity, and liver toxicity to determine the dosage concentration.

[0075] Specifically, zebrafish embryos were randomly divided into 12-well plates, with 20 in each well. The concentrations of Val were (50, 25, 12.5, 10, 7.5 μM), the concentrations of Ver were (60, 40, 20, 10, 5 μM) and 0.1% DMSO was used as the blank group, 4 ml was administered, and the drug bath was treated for 48 hours. Under a microscope, the adverse reactions such as death, deformity, cardiotoxicity, and liver toxicity of zebrafish embryos were observed and recorded at 4h, 12h, 24h, and 48h, respectively, to determine the dosage concentration. The experimental results represent the results of three independent experiments.

[0076] After 48 hours, Val did not cause death at a concentration below 50μM, but the heart rate slowed down significantly, pericardial sac edema was obvious, and a large number of escape reactions occurred; at concentrations of 25μM and 12.5μM, 5 and 2 animals had pericardial sac edema, respectively, and most of them had a significant decrease in heart rate; at concentrations of 10μM and 7.5μM, no cardiotoxicity occurred. Ver did not cause death at concentrations of 60μM and 40μM, but the heart rate was significantly reduced to less than 60 beats / min. In addition, pericardial sac edema was obvious, and pericardial hematoma was occasionally seen; when the concentration was 20μM, 10μM, and 5μM, no cardiotoxicity was observed, but the heart rate at 20μM was lower than that of the blank control group. According to the results of microscopic observation, the high and low dose groups of Val were given 10μM and 7.5μM concentrations, respectively; the high and low dose groups of Ver were given 10μM and 5μM concentrations, respectively.

[0077] After determining the conditions for Ang II administration, antihypertensive drugs Val and Ver were given for antihypertensive treatment, and zebrafish embryos were randomly divided into: blank group, model group (Ang II group), Val high-dose group (Ang II + Val high-dose group), Val low-dose group (Ang II + Val low-dose group), Ver high-dose group (Ang II + Ver high-dose group), Ver low-dose group (Ang II + Ver low-dose group). After 24 hours, photos and videos were taken under a microscope for 10 seconds to complete the measurement of vascular diameter and blood flow rate. The experimental results represent the results of three independent experiments.

[0078] Reference Figures 4 to 7 , the blood vessel diameter and blood flow velocity of zebrafish after blood pressure reduction were statistically analyzed. In terms of blood vessel diameter, compared with the model group, both the high-dose Val and low-dose Ver treatment groups had significant dilation effects (p<0.001); in terms of blood flow velocity, compared with the model group, the high-dose Val treatment group had significant (p<0.001), the high-dose Ver treatment group had significant (p<0.01), and the low-dose Val and Ver treatment groups had an accelerating effect.

[0079] The determination of zebrafish angiotensin concentration includes: detecting the level of angiotensin in vivo by enzyme-linked immunosorbent assay (ELISA). 60 zebrafish were collected from each group, and the AngⅡ content was detected according to the operating steps of the ELISA kit instructions. The experimental results represent the results of three independent experiments.

[0080] Finally, refer to Figures 8-9 Elisa detected the Ang II content in zebrafish and found that compared with the model group, both the high- and low-dose treatment groups of Val and Ver were significantly reduced (p<0.001).

[0081] Application examples:

[0082] The blood pressure of zebrafish was measured before and after the above experiment using Poiseuille's law.

[0083] Blood flows in blood vessels. According to Poiseuille's law, when a viscous fluid flows steadily in a uniform circular tube, its flow rate is:

[0084] Q=πr4Δp / (8ηL)=Δp / R

[0085] Among them, Q is the flow rate, that is, the cardiac output per minute; r is the inner radius of the circular tube, that is, the radius of the blood vessel; L is the length of the circular tube, that is, the length of the blood vessel; ΔP is the pressure difference between the two ends of the tube, that is, the pressure difference between the artery and the vein; η is the viscosity coefficient of the liquid, that is, the blood viscosity; R is the flow resistance, that is, the peripheral vascular resistance. It can be seen that when a viscous liquid flows in a horizontal uniform blood vessel, the flow rate of the liquid is proportional to the pressure difference between the two ends of the tube and the fourth power of the inner radius of the tube, and is inversely proportional to the viscosity of the liquid and the length of the tube. Among them, the blood pressure near the heart at the end of the venous pressure is almost zero, and ΔP is the value of the arterial blood pressure. Blood is a non-Newtonian fluid, and the blood viscosity is not constant. Non-Newtonian fluids have different apparent viscosities at different shear rates. This is a major feature of non-Newtonian fluids. The blood content of zebrafish embryos is very small, and it can be assumed to be a constant value after the blood flows for a period of time. The number of the inner radius of the tube needs to be raised to the fourth power and plays the most important role. Assuming that the inner radius of the blood vessel is reduced by 1 times, and other conditions remain unchanged, the blood flow resistance will expand from 4 to 16 times the original. In layman's terms, if the blood vessels constrict even a little bit, the blood pressure will rise significantly.

[0086] When the human body is healthy, changes in blood pressure are regulated by nerves and hormones. When blood pressure rises in normal people, it slows down the heart rate and causes vasodilation, which reduces both the output Q and peripheral resistance R, thereby maintaining arterial pressure within the physiological range and being relatively stable. However, in patients with hypertension, the increase in arterial blood pressure does not cause a decompression reflex. Once the hypertension lasts for a long time, the myocardium will inevitably increase its output in order to maintain blood supply to tissues and organs, which will increase the load and cause the myocardium to be stretched, resulting in cardiac hypertrophy and dilatation. When the myocardium is stretched beyond a certain limit, its contractile force will decrease, and eventually heart failure will occur, cardiac output will decrease, and blood flow will slow down. In addition, when blood vessels are narrowed, the initial blood flow speed will increase, the pressure will decrease, and cavitation will occur

[25] . Endothelial cells will be damaged, atherosclerotic plaques will form, vascular elasticity will decrease, and the lumen will shrink, resulting in increased peripheral vascular resistance, increased flow resistance, decreased blood flow, and slower flow rate.

[0087] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for establishing a zebrafish hypertension model based on Poiseuille's law, characterized in that: include, Randomly select a number of zebrafish; Prepare different concentrations of angiotensin reagents to bathe zebrafish; Determine angiotensin reagent concentration; Preparing therapeutic drug reagents of different concentrations to perform therapeutic drug bath on zebrafish; Determination of angiotensin concentrations in zebrafish.

2. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 1, characterized in that: The randomly selected zebrafish are 3 dpf wild AB type zebrafish.

3. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 2, characterized in that: The angiotensin reagent is prepared by adding angiotensin to DMSO to prepare a stock solution with a concentration of 10 mmol / L.

4. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 2 or 3, characterized in that: The drug bathing of zebrafish includes: bathing with 4 ml of angiotensin reagents of different concentrations for 48 hours, and changing the drug solution every 24 hours.

5. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 4, characterized in that: Determining the concentration of angiotensin reagent comprises: Measure blood vessel diameters in zebrafish embryo images; Measuring blood flow velocity in zebrafish embryo videos; The angiotensin reagent concentration was determined to be 2 μM.

6. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 1 or 5, characterized in that: The preparation of therapeutic drug reagents with different concentrations includes: preparing a mother solution with a concentration of 100 mmol / L of verapamil using DMSO, and preparing a mother solution with a concentration of 100 mmol / L of valsartan using DMSO.

7. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 6, characterized in that: The method of treating zebrafish with a medicinal bath comprises: selecting valsartan (Val) and verapamil (Ver) as therapeutic drugs, administering 4 ml of the drugs, and performing a medicinal bath treatment for 48 hours.

8. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 7, characterized in that: Also includes: Observe and record adverse reactions such as zebrafish embryo death, deformity, cardiotoxicity, and liver toxicity to determine the dosage concentration.

9. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to any one of claims 5, 7 and 8, characterized in that: The method for determining the zebrafish angiotensin concentration comprises: detecting the angiotensin level in vivo by enzyme-linked immunosorbent assay adsorption experiment.

10. The method for establishing a zebrafish hypertension model based on Poiseuille's law according to claim 9, characterized in that: Blank control groups are set up for the angiotensin reagents of different concentrations and the therapeutic drug reagents of different concentrations.

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