Construction method and application of animal blood stasis model

Abstract

The invention relates to the technical field of biology, in particular to a construction method and application of an animal blood stasis model. The nitroglycerin is utilized to successfully induce an animal blood stasis model. Experiments show that blood stasis related phenotypes of zebrafish embryos can be caused by nitroglycerin treatment, for example, blood flow velocity blocking, heart red blood cell dyeing intensity reduction, and blood coagulation factor and endothelial injury marker expression level increase, which indicates that a zebrafish blood stasis model can be successfully established by nitroglycerin treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing and applying an animal blood stasis model. Background Technology

[0002] Blood-stasis syndrome (BSS) is a common basic syndrome in traditional Chinese medicine (TCM) in East Asia. TCM believes that BSS is caused by factors such as qi stagnation, qi deficiency, cold coagulation, phlegm obstruction, blood heat, and external injury, leading to disordered blood flow or obstructed blood vessels, resulting in bleeding that fails to dissipate in time. Its main clinical manifestations include pain, lumps, bleeding, dark purple complexion or cyanosis of the lips and tongue, dry and scaly skin, and a thin, hesitant, or intermittent pulse. During the Eastern Han Dynasty, the physician Zhang Zhongjing, based on the theories of the *Huangdi Neijing* (Yellow Emperor's Inner Classic), first defined the disease name "blood stasis" and established a complete system of diagnosis and treatment for BSS in his *Shanghan Zabing Lun* (Treatise on Febrile and Miscellaneous Diseases). During the Tang and Song Dynasties, the treatment methods for BSS and blood-activating and stasis-removing therapies were further developed in terms of theory, prescriptions, and medications, providing valuable supplements to the theory of BSS. Modern medicine believes that BSS plays an important role in the pathogenesis and development of various diseases, including cardiovascular and cerebrovascular diseases, diabetes, tumors, inflammation, tissue hyperplasia, and immune dysfunction. Its essence is microcirculatory disturbance and increased blood coagulability, accompanied by various pathological changes such as degeneration, inflammation, and pathological masses. The mechanisms involved in these pathological processes include platelet activation, hypercoagulability, abnormal blood rheological properties, and imbalances in the expression of cell proliferation factors, inflammation-related factors, and pro-fibrotic factors. Thrombosis is the process by which blood components within blood vessels coagulate to form solid masses (thrombi). This is a pathological phenomenon caused by the combined effects of vascular injury, blood flow stagnation, and a hypercoagulable state. Medical experts, including Academician Chen Keji of the Chinese Academy of Sciences, after reviewing relevant literature and combining modern clinical and experimental research, believe that blood stasis can be divided into two types: narrow and broad. Narrowly defined, blood stasis refers to the stagnation of blood flow due to poor circulation. Broadly defined, blood stasis encompasses all cases where various etiologies lead to poor blood flow, either accumulating within or outside the vessels, forming thrombi, or causing abnormalities in blood-related systems, altering the function, properties, and composition of the blood. A series of clinical syndromes resulting from blood stasis are called "blood stasis syndrome."

[0003] Currently, reported animal models of blood stasis are mainly induced by various methods such as chemical induction, ice-water baths, and exhaustive swimming, primarily using blood stasis models in higher animals such as mice and rats. The modeling methods are evaluated by observing whether the experimental animals exhibit macroscopic phenotypes and limb behaviors consistent with traditional Chinese medicine syndromes, and by using hemorheological indicators. While blood stasis models based on mammals such as mice and rats can highly simulate the pathophysiological changes of human blood stasis and have irreplaceable advantages in deeply validating drug efficacy and elucidating its mechanism of action, their disadvantages are also significant. For example, they require a large number of experimental animals, have a long modeling cycle, and are costly overall, limiting their applicability to evaluating the antithrombotic effects of a few drugs or exploring mechanisms, and making them unsuitable for large-scale screening of antithrombotic drugs. For preliminary evaluation of drug activity and large-scale drug screening requiring rapid responses, they are inefficient and too costly. Therefore, developing rapid and economical novel experimental animal models of blood stasis is of great significance. However, using animals such as rats and mice to create models requires a huge number of animals, involves complex experimental procedures, has a long cycle, high experimental costs, and poor model reproducibility.

[0004] Compared to traditional mammalian models, zebrafish models exhibit several significant advantages in drug screening research. Zebrafish are small and can be cultured on a large scale in 96-well or 384-well plates, making them suitable for high-throughput, high-content automated analysis. Their rearing costs are low, they require minimal space, and the amount of drug needed is extremely small, requiring only micrograms of sample—approximately 1 / 100 to 1 / 1000 of the amount needed for mice—significantly reducing experimental costs. Furthermore, zebrafish have a high reproductive capacity and rapid embryonic development; a complete embryo forms within 24 hours of fertilization, and major organs such as the heart, brain, liver, kidneys, and blood vessels are largely formed after about 60 hours. During the first 7 days of development, the embryo is transparent, allowing for clear observation of organ morphological and functional changes, such as heartbeat, blood flow, and organ developmental abnormalities, in vivo using fluorescent labeling technology. At the genetic level, zebrafish share 85% gene homology with humans, and disease-related genes show a 99% similarity, enabling them to effectively simulate human disease processes, thus significantly shortening drug development cycles and reducing costs. Zebrafish models are particularly suitable for research on traditional Chinese medicine with complex components. The isolation and purification of components in traditional Chinese medicine (TCM) is challenging and costly, while zebrafish models require only trace samples for overall efficacy evaluation—a feature difficult to achieve with other models. Furthermore, as a live animal model, zebrafish can systematically reflect the overall effects of drugs on the body, meeting the needs of TCM research on the mechanisms of action of multiple components and targets, and effectively avoiding potential missed screening issues in in vitro screening. Therefore, the zebrafish model has unique and significant application value in the screening of active components in TCM.

[0005] Zebrafish share a high degree of similarity with humans in coagulation mechanisms and blood stasis formation, making them an ideal animal model for studying blood stasis syndrome and screening drugs that promote blood circulation and remove blood stasis. However, research in this field is still in its early stages, and existing models have limitations in simulating the complete pathological characteristics of human blood stasis. For example, the "qi deficiency and blood stasis syndrome" model established by Guo Shuaijie et al. using anhydrous sodium sulfite and isoproterenol hydrochloride, while simulating the phenotype of slowed blood flow, failed to reproduce key pathological indicators such as endothelial dysfunction and abnormal coagulation function. Therefore, developing novel zebrafish blood stasis models with comprehensive phenotypes and clearly defined mechanisms is of great value. Summary of the Invention

[0006] In view of this, the present invention provides a method for constructing an animal blood stasis model and its application.

[0007] This invention provides the application of nitroglycerin in the preparation of animal blood stasis models.

[0008] In this invention, the animal includes zebrafish. In a specific embodiment of the invention, using zebrafish as an example, an aqueous solution of nitroglycerin successfully induced a blood stasis-related phenotype characterized by microcirculatory disturbances in zebrafish embryos. Although nitroglycerin is generally considered a vasodilator, the applicant's research found that in zebrafish, nitroglycerin effectively induced a decrease in blood flow velocity, a reduction in cardiac erythrocyte staining intensity, and significantly upregulated the expression levels of coagulation-related factors and endothelial injury markers, suggesting that microcirculatory disturbances and endothelial injury jointly participate in model formation. This model not only highly resembles the formation mechanism of blood stasis in humans but also has the advantages of good stability and strong reproducibility, making it suitable for large-scale, efficient screening of drugs that promote blood circulation and remove blood stasis.

[0009] In this invention, the zebrafish includes wild-type AB strain zebrafish, Tg(gata1:DsRed) red transgenic strain zebrafish, or hybrid offspring of these two zebrafish.

[0010] The present invention also provides a method for constructing a blood stasis model, comprising: administering nitroglycerin to animals.

[0011] Furthermore, the construction method includes culturing animals in a culture medium containing nitroglycerin. In this invention, the concentration of nitroglycerin in the culture medium is 25-50 μM, specifically 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, or 50 μM. The culture conditions include culturing at 28°C for 48 hours.

[0012] In this invention, the light-dark cycle for raising adult zebrafish is 14h / 10h, that is, alternating between 14 hours of light and 10 hours of darkness per day, in order to simulate the natural physiological rhythm and provide stable environmental conditions for the normal growth and development of zebrafish embryos and the construction of the model.

[0013] In a specific embodiment of the present invention, the blood stasis model is a zebrafish blood stasis model, and its construction method includes: culturing zebrafish embryos in a culture medium containing nitroglycerin.

[0014] In this invention, the zebrafish embryo is preferably an embryo that has developed to 2-3 dpf, that is, a zebrafish embryo that is 2-3 days after fertilization.

[0015] In this invention, the zebrafish embryos are obtained by mating mature zebrafish (i.e., adult fish) at a female-to-male ratio of 1:2. The adult zebrafish are raised using reverse osmosis water containing sea salt. The preferred conductivity of the rearing water is 450-550 μS / cm, the preferred pH value is 7.0-7.5, and the rearing light / dark cycle is 14h / 10h.

[0016] This invention demonstrated that zebrafish embryos treated with nitroglycerin exhibited blood stasis-related phenotypes, such as impaired blood flow velocity, decreased cardiac erythrocyte staining intensity, and increased expression levels of coagulation factors and endothelial injury markers, indicating that nitroglycerin treatment can successfully establish a zebrafish blood stasis model.

[0017] In this invention, the staining intensity of erythrocytes in the zebrafish embryonic heart was tested using a digital microscope. The blood flow velocity in the zebrafish embryo was tested using a high-content imaging system. The expression levels of coagulation factors and endothelial damage markers in the zebrafish embryo were tested using quantitative real-time PCR.

[0018] The present invention also provides an animal model obtained by any of the above-described construction methods.

[0019] This invention also provides the application of animal blood stasis models obtained by the construction method described above in screening drugs for promoting blood circulation and removing blood stasis and / or antithrombotic drugs.

[0020] Compared with the prior art, this application has the following technical effects:

[0021] This invention utilizes nitroglycerin to induce a zebrafish blood stasis model. This model boasts advantages such as high induction success rate and stable phenotype, effectively reproducing key pathological features like microcirculation and endothelial dysfunction. Its operation is simple, facilitating high-throughput screening in microplates, and is suitable for systematic screening and efficacy evaluation of drugs that promote blood circulation and remove blood stasis. Attached Figure Description

[0022] Figure 1 This study presents cardiac erythrocyte staining imaging and blood flow velocity detection in a nitroglycerin-induced zebrafish blood stasis model. A represents zebrafish o-anisidine staining results, B represents statistical data on cardiac erythrocyte staining intensity, C represents erythrocyte fluorescence imaging results in transgenic zebrafish, and D represents the percentage of embryos with peripheral circulation obstruction (%).

[0023] Figure 2 To detect the expression levels of coagulation factors and endothelial injury markers in a nitroglycerin-induced zebrafish blood stasis model; where A~F represent the relative mRNA expression levels of f2, fgg, ptges, edn1, nos2a, and thbd, respectively.

[0024] Figure 3 This study aims to detect the expression levels of coagulation factors / endothelial injury markers by staining and imaging cardiac erythrocytes after adrenaline treatment. A represents the zebrafish o-anisidine staining results, B represents the statistical analysis of cardiac erythrocyte staining intensity, and C-H represent the relative mRNA expression levels of edn1, f2, fgg, nos2a, ptges, and thbd, respectively. Detailed Implementation

[0025] This invention provides a method for constructing an animal blood stasis model and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0026] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0027] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0028] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0029] In this article, when referring to units for data ranges, if the unit is only followed by the right endpoint, it indicates that the units for the left and right endpoints are the same. For example, 450~550 uS / cm means that the units for the left endpoint "450" ​​and the right endpoint "550" are both uS / cm.

[0030] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0031] The following are the experimental materials used in the examples:

[0032] 1. Experimental Materials

[0033] 1.1 Laboratory animals:

[0034] Wild-type AB strain zebrafish and Tg(gata1:DsRed) red strain transgenic zebrafish reproduce by natural pair mating.

[0035] 1.2 Experimental Apparatus

[0036] Digital microscope (Shenzhen Ruiward Life Science Technology Co., Ltd., model: DOM-1001); Precision electronic balance (Shanghai Zhuojing Electronic Technology Co., Ltd., model: BSM-220.4); Constant temperature biochemical incubator (Shanghai Jinghong Experimental Equipment Co., Ltd., model: GPN-9080); Ultra-low temperature freezer (Thermo Scientific, USA, model: Thermo 905); Purified water system (Milli-Q Plus, Germany); Multifunctional microplate reader (Molecular Devices, USA, model: Flex station 3); Clean bench (Sujing Group, model: Sujing SW-CJ-2F); High content imaging analysis system (Meigu Molecular Instruments Co., Ltd., model: ImageXpress HCS. ai Advanced); Centrifuge (Beijing Baiyang Medical Instrument Co., Ltd., model: BY-R20); Real-time PCR instrument (Roche, LightCycler 480II).

[0037] 1.3 Drugs and Reagents

[0038] Nitroglycerin (National Institutes for Food and Drug Control, catalog number: 100236); Sodium carboxymethyl cellulose (Shanghai Institute of Biotechnology, catalog number: 30189328); 4% tissue cell fixative (Beijing Solarbio Science & Technology Co., Ltd., catalog number: P1110); Dimethoxybenzidine (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D104001); 3-ethoxyaniline methanesulfonate (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: E107465); Tween-20 (Shanghai Institute of Biotechnology, catalog number: 30036328); PBS (Nanjing Shenghang Biotechnology Co., Ltd., catalog number: BC-BPBS-01); FastPure Cell / Tissue Total RNA Isolation Kit V2 (Nanjing Novozymes Biotechnology Co., Ltd., catalog number: RC112), HiScript III RT SuperMix for gPCR (+gDNA wiper) kit (Nanjing Novozymes Biotechnology Co., Ltd., catalog number: R323) and Tag ProUniversal SYBR qPCR Master Mix reagent kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: Q712).

[0039] The present invention will be further illustrated below with reference to the embodiments:

[0040] Example 1: Preparation of the animal model of the present invention

[0041] Experimental methods: The water used for raising adult zebrafish was reverse osmosis water and instant sea salt. The conductivity of the water was 450~550 uS / cm, the pH was 7.0-7.5, and the light / dark cycle was 14h / 10h.

[0042] Healthy, sexually mature zebrafish were selected and placed in a mating tank at a female-to-male ratio of 1:2. Embryos were obtained the following morning, washed with E3 medium to remove dead eggs, and placed in a light-controlled incubator at 28°C. The E3 medium containing PTU was changed twice daily. When the embryos reached 3 days post-fertilization (dpf), wild-type AB / transgenic Tg(gata1:DsRed) zebrafish embryos were randomly selected and placed in 6-well plates, with 30 embryos per well and a volume of 4 mL per well. Both the control and experimental groups had 3 replicates. The control group received no modeling agent, while the experimental group received 25 μM nitroglycerin as a modeling agent for 48 h at an incubation temperature of 28°C to induce a zebrafish blood stasis model.

[0043] A control group and a model group were set up. The control group did not receive nitroglycerin, while the model group received 25 μM nitroglycerin. Validation analyses were performed on the model and control groups. Ten zebrafish embryos were randomly selected for high-content imaging. The number of flowing red blood cells in the aortic region of the control group embryos was calculated, and the average value was taken. A number of flowing red blood cells in the model group lower than 70% of that in the control group was considered as obstructing blood flow. The experiment was repeated three times. Twenty-five zebrafish embryos were randomly selected for O-Dianisidine staining and microscopic imaging. ImageJ software was then used to analyze the staining intensity of cardiac red blood cells. The expression levels of coagulation factors and endothelial injury markers were detected in another twenty-five zebrafish embryos. Statistical analysis was performed using GraphPad Prism 8.4 software. p < 0.05 indicated statistical significance.

[0044] Example 2: Preparation of the animal model of the present invention

[0045] Experimental methods: The water used for raising adult zebrafish was reverse osmosis water and instant sea salt. The conductivity of the water was 450~550 uS / cm, the pH was 7.0-7.5, and the light / dark cycle was 14h / 10h.

[0046] Healthy, sexually mature zebrafish were selected and placed in a mating tank at a female-to-male ratio of 1:2. Embryos were obtained the following morning, washed with E3 medium to remove dead eggs, and placed in a light-controlled incubator at 28°C. The E3 medium containing PTU was changed twice daily. When the embryos reached 3 days post-fertilization (3dpf), wild-type AB / transgenic Tg(gata1:DsRed) zebrafish embryos were randomly selected and placed in 6-well plates, with 30 embryos per well and a volume of 4 mL per well. Both the control and experimental groups had 3 replicates. The control group received no modeling agent, while the experimental group received 50 μM nitroglycerin as a modeling agent for 48 h at an incubation temperature of 28°C to induce a zebrafish blood stasis model.

[0047] A control group and a model group were set up. The control group did not receive nitroglycerin, while the model group received 50 μM nitroglycerin. Validation analyses were performed on the model and control groups. Ten zebrafish embryos were randomly selected for high-content imaging. The number of flowing red blood cells in the aortic region of the control group embryos was calculated, and the average value was taken. A number of flowing red blood cells in the model group lower than 70% of that in the control group was considered as obstructing blood flow. The experiment was repeated three times. Twenty-five zebrafish embryos were randomly selected for O-Dianisidine staining and microscopic imaging. ImageJ software was then used to analyze the staining intensity of cardiac red blood cells. The expression levels of coagulation factors and endothelial injury markers were detected in another 25 zebrafish embryos. Statistical analysis was performed using GraphPad Prism 8.4 software. p < 0.05 indicated statistical significance.

[0048] The test results of Examples 1 and 2 are shown below. Figure 1 and Figure 2 .

[0049] The results showed that, through analysis of blood stasis-related indicators such as cardiac erythrocyte staining intensity and blood flow velocity, the experimental group, after treatment with nitroglycerin, exhibited significantly reduced cardiac erythrocyte staining intensity and blood flow velocity compared to the control group. The 50 μM nitroglycerin concentration group (NTG-50 μM) showed superior modeling effect. Furthermore, RNA expression analysis of blood stasis-related genes such as f2, fgg, ptges, edn1, nos2a, and thbd revealed significant differences in gene expression in zebrafish embryos after nitroglycerin treatment compared to the control group. In the 50 μM nitroglycerin concentration group, the expression differences of these genes were greater and more statistically significant (e.g., ptges, nos2a, thbd, f2, and fgg showed *** or ** highly significant differences, and edn1 showed * significant differences), better highlighting the pathological molecular characteristics of the blood stasis model and providing a more stable and representative model basis for subsequent research on blood stasis mechanisms or drug intervention experiments.

[0050] Comparative Example 1: Preparation of a Traditional Animal Model

[0051] Construction of an adrenaline-induced blood stasis model in zebrafish

[0052] Experimental methods: The zebrafish were raised using reverse osmosis water and instant sea salt. The conductivity of the water was 450-550 uS / cm, the pH was 7.0-7.5, and the light / dark cycle was 14h / 10h.

[0053] Healthy, sexually mature zebrafish were selected and placed in a mating tank at a female-to-male ratio of 1:2. Embryos were obtained the following morning, washed with embryo culture water to remove dead eggs, and placed in a light-controlled incubator at 28°C. The embryo culture water was changed twice daily. When the embryos reached 3 days post-fertilization (3dpf), wild-type AB strain zebrafish embryos were randomly selected and placed in 6-well plates, with 30 embryos per well and a volume of 4 mL per well. Both the control and experimental groups had 3 replicates. The control group received no modeling agent, while the experimental group received 5 mM adrenaline solution for 24 hours at an incubation temperature of 28°C to induce a zebrafish blood stasis model.

[0054] A control group and a model group were set up. The control group did not receive adrenaline, while the model group received 5 mM adrenaline. Validation analyses were performed on the model and control groups. Twenty zebrafish embryos were randomly selected for O-Dianisidine staining and microscopic imaging, followed by imageJ software analysis of cardiac erythrocyte staining intensity. Twenty-five zebrafish embryos were randomly selected for the detection of coagulation factors and endothelial injury markers. Statistical analysis was performed using GraphPad Prism 8.4 software, with p < 0.05 indicating statistical significance. Results are shown below. Figure 3 The results showed that the staining intensity of erythrocytes in the heart of zebrafish treated with adrenaline, the expression levels of coagulation factors and endothelial injury markers did not change significantly compared with the control group.

[0055] Depend on Figures 1-3 It can be seen that the zebrafish blood stasis model induced by nitroglycerin in this invention showed significant statistical differences in blood stasis indicators (including cardiac erythrocyte staining intensity, blood flow velocity, coagulation factors, and expression levels of endothelial damage markers such as F2, PTGES, GFG, NOS2A, and EDN1) compared to the control group, and the trend of these differences was stable. Furthermore, the nitroglycerin-induced model had a wider time window, and even with prolonged treatment, the zebrafish embryos maintained a high survival rate, effectively avoiding experimental data deviations caused by embryonic death and ensuring the accuracy and verifiability of the experimental results. In contrast, the zebrafish blood stasis model constructed using adrenaline in the traditional method showed slightly lower cardiac erythrocyte staining intensity than the control group, but without significant difference. The expression levels of coagulation factors and endothelial damage markers, such as PTGES, GFG, and NOS2A, were comparable to or slightly higher than the control group, while the expression level of EDN1 was even lower than the control group, all without significant differences. This indicates that the stability and reproducibility of the traditional blood stasis model are relatively weak, and its effectiveness is significantly inferior to the blood stasis model constructed in this invention.

[0056] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of nitroglycerin in the preparation of animal blood stasis models.

2. The application according to claim 1, characterized in that, The animals mentioned include zebrafish.

3. The application according to claim 2, characterized in that, The zebrafish include wild-type AB strain zebrafish or Tg(gata1:DsRed) red strain transgenic zebrafish.

4. A method for constructing a blood stasis model, characterized in that, Administer nitroglycerin to the animal.

5. The construction method according to claim 4, characterized in that, The administration of nitroglycerin to animals includes: culturing animals in a culture medium containing nitroglycerin.

6. The construction method according to claim 5, characterized in that, The concentration of nitroglycerin in the culture medium is 25-50 μM.

7. The construction method according to claim 4, characterized in that, The light / dark cycle for the rearing was 14h / 10h.

8. The construction method according to any one of claims 4 to 7, characterized in that, include: Zebrafish embryos were cultured in a medium containing nitroglycerin.

9. The construction method according to claim 8, characterized in that, The zebrafish embryos mentioned are those that have developed to 2-3 days postpartum (dpf).

10. The use of the animal blood stasis model obtained by the construction method according to any one of claims 4 to 9 in screening blood-activating and stasis-removing drugs and / or antithrombotic drugs.

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