CN117503957A - Method for evaluating blood pressure reducing effect by using zebra fish model and application thereof - Google Patents
Method for evaluating blood pressure reducing effect by using zebra fish model and application thereof Download PDFInfo
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- CN117503957A CN117503957A CN202311389239.8A CN202311389239A CN117503957A CN 117503957 A CN117503957 A CN 117503957A CN 202311389239 A CN202311389239 A CN 202311389239A CN 117503957 A CN117503957 A CN 117503957A
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Abstract
The invention relates to the technical field of efficacy evaluation of medicines, health-care foods and medical appliances, and provides a method for evaluating blood pressure reducing efficacy by using a zebra fish model and application thereof, aiming at the problems that the existing hypertension animal model has longer experimental period and high cost and is unfavorable for evaluating the efficacy of a large number of samples. The method comprises the steps of randomly selecting zebra fish, intravenous injection of angiotensin II, detection of indexes after treatment, and evaluation and analysis of blood pressure reduction efficacy. The method utilizes the angiotensin II to establish the zebra fish juvenile fish hypertension model for the first time, has short experimental period and can complete efficacy evaluation within several hours to 7 days, and the method is a low-cost and batch-screening hypertension in-vivo model. The invention also provides application of the method in efficacy evaluation of medicines, health foods, cosmetics or medical instruments.
Description
Technical Field
The invention relates to the technical field of efficacy evaluation of medicines, health-care foods and medical appliances, in particular to a method for evaluating blood pressure reducing efficacy by using a zebra fish model and application thereof.
Background
Hypertension is a chronic disease characterized by an increase in systemic arterial blood pressure and associated with functional or organic impairment of organs such as heart, brain, kidneys, etc. The pathogenesis of the Chinese medicinal composition is complex, and mainly comprises the following aspects: 1. a change in cardiac output; 2. high-salt diet causes retention of water and sodium, and increases blood volume; 3. lesions of the renin angiotensin aldosterone system; 4. abnormal ion transport of cell membranes; 5. increased sympathetic activity; 6. increased vascular tension and thickening of the vessel wall; 7. the balance of vasodilating substances and vasoconstrictor substances is disturbed. The antihypertensive drugs commonly used in clinic mainly comprise the following five types: angiotensin Converting Enzyme Inhibitors (ACEI), angiotensin ii receptor antagonists, diuretics, beta blockers and calcium antagonists.
In order to better study the pathogenesis of hypertension and the treatment method, a plurality of hypertension experimental animal models are established by the researchers related to hypertension, and the models are specifically divided into two main types, namely a gene related hypertension animal model and a non-gene related hypertension animal model. Comprises 1. Hereditary hypertension model: spontaneous hypertension rat model and salt-sensitive hypertension genetic rat. 2. Transgenic model: AGT-REN double transgenic hypertension mice, dopamine D5F173L mutant genes, D5 normal gene transgenic mice, A142V transgenic mice, mouse kidney specific knockout Nedd4-2 or ACE, rat kidney specific knockout Hsd b2 and the like. For example, patent CN104293831a provides a method for constructing a hypertension mouse model based on CRISPR/Cas9 gene knockout technology. 3. Surgical induction model: renal artery stenotic hypertension model, abdominal aortic stenosis type hypertension model, and renal outsourcing puncture type hypertension model. 4. Drug induction model: induction of angiotensin II (Ang II), induction of N-nitro-L-arginine methyl ester, induction of deoxycorticosterone acetate, and the like.
The prior art has the following defects: 1. the existing antihypertensive drugs have a plurality of defects in terms of reducing blood pressure and improving symptoms. The exact etiology and pathogenesis of hypertension are not known, and research on the etiology and pathogenesis of hypertension is urgently needed to be enhanced, so that more effective therapeutic drugs are developed. Wherein the experimental study is not separated from the animal model of hypertension. 2. The existing rodent (mainly big and small mice) -based hypertension model has certain defects: the hereditary hypertension model has higher requirements on feeding conditions, long cultivation period, higher price, troublesome hereditary breeding and easy breaking of varieties, and has difficulty in mass use; the operation-induced hypertension model has the advantages of high difficulty, high threshold and high influence of the operation on the blood pressure and the state of model animals. 3. Common methods for detecting blood pressure in mice include tail pressure (indirect measurement) and implanted telemetry (direct measurement), which in turn can cause elevated blood pressure, typically requiring 1-2 weeks to train the animal to adapt it to the test environment. Implantable telemetry for measuring blood pressure requires surgical placement of implants into animals, and is expensive in equipment and low in throughput. 4. The existing hypertension animal model has longer experimental period, high cost and is not beneficial to evaluating the efficacy of a large amount of samples, and is especially not suitable for early drug screening. There is a need for an ideal solution.
Disclosure of Invention
In order to overcome the problems, the invention provides a method for evaluating the blood pressure reducing effect by using a zebra fish model and application thereof, and an in-vivo model which is quick (2-3 days), low in cost and capable of being screened in batches is established.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for evaluating blood pressure lowering effect by using zebra fish model comprises randomly selecting zebra fish, intravenous injection of angiotensin II, detecting index after treatment, evaluating and analyzing blood pressure lowering effect.
Zebra fish has been the first model organism in the biology of research development, regeneration, human disease and small molecule biological screening for the last decades, because of its in vitro fertilization, short development cycle (24 hours organ formation), optical transparency, small individuals, high number of eggs laid per time, small experimental dosages, adaptability to genetic manipulation and wide tissue regeneration capacity. Furthermore, zebra fish possess tissues, organs and systems similar to humans, and genes and signal pathways are highly conserved with humans: >87%; physiological, developmental and metabolic are highly similar to mammals, and therefore, it is possible to study the role of conserved genes in human disease using zebra fish disease models.
The renin-angiotensin-aldosterone system (RAAS) is highly conserved, with all vertebrates presenting components of the RAS to some extent. Studies have shown that almost all of the components of human RAAS are conserved in zebra fish and are generally expressed in the same cell type as the human RAAS genes, and that zebra fish have paralogues of multiple RAAS components, demonstrating their evolutionary relationship to human genes.
Adequate blood filling and cardiac ejection in the circulatory system are fundamental factors in the formation of blood pressure. In the arterial system, another factor affecting arterial blood pressure is peripheral resistance. The endocrine regulating system related to blood pressure regulation in the human body is the renin-angiotensin-aldosterone system (RAAS), which can control blood flow and peripheral resistance in the human body and can regulate the balance of blood pressure, water and electrolytes in the body. During the regulation of blood pressure by the renin-angiotensin system (RAS), angiotensin II plays a key role, mainly with a powerful vasoconstrictor effect, causing an increase in blood pressure. The mechanism is as follows: 1. the smooth muscle of the systemic arteriole is contracted, and the peripheral resistance is increased; 2. triggering adrenal gland to release aldosterone and pituitary gland to release vasopressin (antidiuretic hormone), causing water sodium retention and increased blood volume; 3. directly promote the reabsorption of sodium and water by the renal tubules; 4. causing sympathetic vasoconstriction central tonic activity in the brain, causing an increase in peripheral vascular resistance; 5. stimulating the release of epinephrine and norepinephrine from the adrenal medulla.
Preferably, the zebra fish is a vascular endothelial cell red fluorescence Tg (fli 1aep: dsRedEx) strain zebra fish, a vascular endothelial cell green fluorescence Tg (fli-1: EGFP) strain zebra fish or a cardiac green fluorescence Tg (cmlc 2: EGFP) strain zebra fish.
Preferably, the zebra fish is injected for a period of time ranging from 2 to 5 days (2 to 5 dpf) after fertilization.
Preferably, the injection dosage of the angiotensin II is 10-100mg/kg.
Preferably, the sample is treated at a temperature of 25-30deg.C for a period of 1 hour to 7 days.
Preferably, the detection indicators are cardiac output and vessel diameter. Since MAP (mean arterial pressure) =co (cardiac output) ×svr (systemic peripheral vascular resistance), when cardiac output increases, peripheral vascular resistance increases (blood vessel diameter decreases), an increase in blood pressure is indicated. The blood pressure of the juvenile zebra fish is commonly characterized by using two indexes of detectable cardiac output and intuitively-analyzable blood vessel diameter. If the sample is capable of reducing cardiac output and peripheral vascular resistance (increased vessel diameter), it is indicated that the sample has hypotensive effect.
Preferably, the detection index further comprises blood flow velocity, blood vessel wall thickness, heart remodeling, myocardial fibrosis, NO level, oxidative stress (ROS level and NADP + /NADPH ratio), inflammation. The wall thickness of the blood vessel is used for assisting in characterizing the peripheral vascular resistance of the body circulation. Myocardial cell area of cardiomyocyte fluorescent zebra fish was used to characterize hypertensive zebra fish heart remodeling.
The invention also provides application of the method for evaluating the blood pressure reducing efficacy by using the zebra fish model in efficacy evaluation of medicines, health foods, cosmetics or medical appliances.
Preferably, the zebra fish is administered at a stage of 2-5dpf.
Therefore, the invention has the beneficial effects that: the method utilizes the angiotensin II to establish the zebra fish juvenile fish hypertension model for the first time, has short experimental period and can complete efficacy evaluation within several hours to 7 days, and the method is a low-cost and batch-screening hypertension in-vivo model.
Drawings
FIG. 1 is a typical graph of the vascular diameter of example 5 treated with 3dpf zebra fish angiotensin II for 48 h;
FIG. 2 is a typical graph of blood vessel diameter of zebra fish after treatment with captopril tablet, valsartan capsule and amlodipine besylate tablet of application example 1;
FIG. 3 is a typical graph of blood vessel diameter of zebra fish after application example 2 marine phospholipid treatment;
FIG. 4 is a typical graph of the wall thickness of zebra fish blood vessels after application example 2 marine phospholipid treatment;
FIG. 5 is a representative graph of the area of cardiomyocytes in zebra fish after application example 2 marine phospholipid treatment;
FIG. 6 is a typical graph of blood vessel diameters of zebra fish after treatment with the propolis soft capsule of application example 3.
Detailed Description
The technical scheme of the invention is further described through specific embodiments.
In the present invention, unless otherwise specified, the materials and equipment used are commercially available or are commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.
The main instruments used in the examples and application examples are: dissecting microscope (SZX 7, OLYMPUS, japan); CCD camera (VertA 1, shanghai Tusen Vision technologies Co., ltd.); microinjection apparatus (IM-300, narishige, japan); needle pullers (PC-10, narishige, japan); precision electronic balances (CP 214, OHAUS, USA); heartbeat blood flow analysis System (Zebralab3.3 (PB 2084C), viewPointLifesciences, france); electrokinetically focused continuously variable magnification fluorescence microscope (AZ 100, nikon, japan); 6-well plate (Zhejiang Bei Lanba Biotechnology Co., ltd., china).
The main reagents are as follows: angiotensin ii, white powder, cat No. a107852, available from ala Ding Shiji (shanghai) limited; captopril tablet, available from the biopharmaceutical company, jinhua Kang Enbei, zhejiang; valsartan capsules, available from beijing nohua pharmaceutical limited; amlodipine besylate tablets, available from the company of the sciences pharmaceutical Co., ltd; marine phospholipids, shandong sea Dabeol Biotechnology Co., ltd; a soft capsule, FENGJIAO Soft Capsule,a health food company from Hangzhou bees; nitric oxide content detection kit (total NO measured enzymatically), product number BC1475, available from Solarbio company;reagent, cat No. 15596026, available from thermo cleaner company; />II 1st Strand cDNA Synthesis Kit (gDNA digester plus), cat number 11121ES60, available from Shanghai, inc.; />qPCR SYBR Green Master Mix (High Rox Plus), cat No. 11203ES08, available from Shanghai, inc. of Saint Biotech; NADP (H) content detection kit, product number BC5200, available from Solarbio company; CM-H2DCFDA, cat No. C6827, available from Thermofisher company.
Example 1
Wild type AB strain zebra fish 2 days after fertilization (2 dpf) were randomly selected and treated in 6-well plates with 30 zebra fish per well (experimental group) at a capacity of 3mL per well. Angiotensin II 40mg/kg and 80mg/kg were intravenously injected while a normal control group was set. The treatment is carried out at 28℃for 1h. Information is then collected and analyzed, specifically: each experimental group randomly selects 15 zebra fish, and the video is collected in a heart beat blood flow analysis system, and the cardiac output and the blood flow velocity of the zebra fish are analyzed by ViewPoint Application Manager software. Statistical treatment results are expressed in mean+ -SE. Statistical analysis was performed with SPSS26.0 software, p <0.05 indicated that the differences were statistically significant.
Example 2
3dpf wild type AB strain zebra fish were randomly selected in 6 well plates, and 30 zebra fish were treated per well (experimental group) with a capacity of 3mL per well. Angiotensin II 40mg/kg and 80mg/kg were intravenously injected while a normal control group was set. The treatment is carried out at 28℃for 1h. Information was then collected and analyzed as in example 1.
Example 3
2dpf wild type AB strain zebra fish were randomly selected in 6-well plates, and 30 zebra fish were treated per well (experimental group) with a capacity of 3mL per well. Angiotensin II was administered intravenously at 20mg/kg and 40mg/kg, while setting the normal control group. And treating at 28 ℃ for 24 hours. Information was then collected and analyzed as in example 1.
Example 4
3dpf wild type AB strain zebra fish were randomly selected in 6-well plates, and 30 zebra fish were treated per well (experimental group). Angiotensin II 20mg/kg and 40mg/kg were intravenously injected, while a normal control group was set at a capacity of 3mL per well. And treating at 28 ℃ for 24 hours. Information was then collected and analyzed as in example 1.
Example 5
The 3dpf vascular endothelial cell red fluorescent labeled Tg (fli 1aep: dsRedEx) strain zebra fish was randomly selected in 6 well plates, and 30 zebra fish were treated per well (experimental group) with a capacity of 3mL per well. Angiotensin II 20mg/kg was intravenously injected while a normal control group was set. Treatment at 28℃for 24, 48 and 72h. Information was then collected and analyzed as in example 1.
Experimental results
TABLE 1 effects of zebra fish angiotensin II post-treatment in examples 1-4 (n=15, mean.+ -. SE)
P <0.05, p <0.01, p <0.001 compared to the normal control group.
From Table 1, (1) treatment of 2dpf zebra fish with angiotensin II for 1h in example 1 significantly increased cardiac output and blood flow velocity. (2) Example 2 treatment of 3dpf zebra fish with angiotensin II for 1h significantly increased cardiac output and blood flow velocity. (3) Example 3 treatment of 2dpf zebra fish with angiotensin II for 24h, cardiac output and blood flow velocity were not significantly altered. (4) Example 4 treatment of 3dpf zebra fish with angiotensin II for 24h significantly increased cardiac output and blood flow velocity.
TABLE 2 treatment of 3dpf zebra fish angiotensin II in example 5 24-72h effect (n=15, mean.+ -. SE)
P <0.001 compared to normal control group.
From Table 2 and FIG. 1, example 5 treatment of 3dpf zebra fish with angiotensin II for 24h showed no significant change in internode vessel diameter; treating for 48 hours, and obviously narrowing the internode vessel diameter; the treatment is carried out for 72 hours, and the internode vessel diameter is restored to be normal.
The invention also provides an application example for evaluating the blood pressure reducing effect of the medicine by using the zebra fish model.
Application example 1 (captopril tablet, valsartan capsule and amlodipine besylate tablet)
The 3dpf vascular endothelial cell red fluorescent labeled Tg (fli 1aep: dsRedEx) line was randomly selected in six well plates, 30 zebra fish were treated per well (experimental group), and 8 biological replicates were set. The captopril tablet, the valsartan capsule and the amlodipine besylate tablet (the concentrations are shown in table 3) were given in water-soluble form, and a normal control group and a model control group were simultaneously set, with a capacity of 3mL per well. The zebra fish hypertension model is built by injecting 20mg/kg of angiotensin II into all other groups except the normal control group. Treated to 5dpf at 28 ℃. And after the treatment is finished, testing and analyzing respectively: (1) cardiac output and blood flow velocity, (2) internode vessel diameter, (3) NO level in zebra fish body, and (4) relative expression of marker genes. The method comprises the following steps:
(1) Each experimental group randomly selects 15 zebra fish to be placed in a heartbeat and blood flow analysis system to collect video, and the cardiac output and the blood flow velocity are analyzed by ViewPoint Application Manager software. (2) 15 zebra fish from each experimental group were randomly selected and subjected to fluorescence microscopy to obtain photographs, and internode vessel diameters were analyzed using NIS-ElementsD3.20 advanced image processing software.
Experimental results 1.1
TABLE 3 cardiac output, blood flow velocity and vessel diameter of zebra fish after treatment with captopril tablet, valsartan capsule and amlodipine besylate tablet (n=15, mean.+ -.SE)
Comparison between groups of model control group, ×p<0.05，**p<0.01，***p<0.001; compared with the two groups of the model control group, # p<0.05。
the hypotensive effect was evaluated by the results of statistical analysis of the cardiac output and the vessel diameter of 2 indices. Statistical treatment results are expressed in mean+ -SE. From table 3 and fig. 2, the model control group had significantly increased cardiac output and blood flow compared to the normal control group, and the internode vessel diameter was significantly reduced compared to the normal control group. After the captopril tablet, the valsartan capsule and the amlodipine besylate tablet are respectively administered for 2 days, the cardiac output of the zebra fish is obviously reduced, the internode blood vessel diameter is obviously increased, and the blood flow speed is obviously slowed down, so that the captopril tablet, the valsartan capsule and the amlodipine besylate tablet have obvious blood pressure reducing effect.
(3) And (3) collecting zebra fish samples of 30 zebra fish in each experimental group according to the instruction of the NO detection kit, and analyzing the NO level in the zebra fish by using a multifunctional enzyme-labeled instrument.
Experimental results 1.2
Table 4 NO levels after captopril tablet, valsartan capsule and amlodipine besylate tablet treatment (n=10, mean±se)
Comparison with model control groups, p <0.05, p <0.01, p <0.001.
Endothelial-derived Nitric Oxide (NO) is the most powerful vasodilator known to regulate blood pressure by promoting diastole or inhibiting systole. Ang II induction impairs NO synthesis. As can be seen from table 4, the NO levels of the model control group were significantly lower than those of the normal control group, and after 2 days of administration of the captopril tablet, the valsartan capsule and the amlodipine besylate tablet, respectively, the NO levels of zebra fish were significantly higher than those of the model control group, indicating that the captopril tablet, the valsartan capsule and the amlodipine besylate tablet can promote vasodilation by increasing the NO levels.
(4) Total RNA was extracted from 30 zebra fish per experimental group, and the relative expression levels of acta2 and col1a1a were examined (primer sequences are shown in Table 5), and 3 biological replicates were set.
TABLE 5 primer sequence information
Experimental results 1.3
TABLE 6 relative expression level of fibrosis-related marker genes after treatment of captopril tablet, valsartan capsule and amlodipine besylate tablet
(n＝3，mean±SE)
Comparison with model control groups, p <0.05, p <0.01, p <0.001.
From table 6, the fibrosis-related markers of the model control group include a-smooth muscle actin (acta 2) and collagen I (col 1a1 a) mRNA levels were significantly elevated compared to the normal control group. After 2 days of administration of captopril tablet, valsartan capsule and amlodipine besylate tablet respectively, zebra fish acta2 and col1a1a were significantly reduced, indicating that captopril tablet, valsartan capsule and amlodipine besylate tablet all improved Ang II-induced myocardial fibrosis.
Application example 2 (Marine phospholipids)
Experiment one, 5dpf vascular endothelial cell green fluorescence labeled Tg (fli-1: EGFP) lines were randomly selected in six well plates, 30 zebra fish were treated per well (experimental group), and 6 biological replicates were set. The marine phospholipids were given 62.5, 125 and 250 μg/mL in water, while the normal control and model control were set to a capacity of 3mL per well. The zebra fish hypertension model is built by injecting 40mg/kg of angiotensin II into all other groups except the normal control group. Treated to 9dpf at 28 ℃. And after the treatment is finished, testing and analyzing respectively: (1) cardiac output and blood flow velocity, (2) internode vessel diameter, (3) vessel wall thickness, (4) relative expression of marker genes. The method comprises the following steps:
(1) 10 zebra fish were randomly selected from each experimental group and placed in a heartbeat and blood flow analysis system to collect video, and the cardiac output and blood flow velocity were analyzed by ViewPoint Application Manager software. (2) 10 zebra fish were randomly selected from each experimental group and placed under a fluorescence microscope to collect photographs, and data were analyzed and collected using NIS-ElementsD3.20 advanced image processing software to analyze the zebra fish internode vessel diameters. (3) 10 zebra fish were randomly selected from each experimental group, photographs were collected under a confocal fluorescence microscope, and data were analyzed and collected using ImageJ image processing software to analyze the thickness of the zebra fish blood vessel wall.
Experimental results 2.1
TABLE 7 blood flow velocity, vessel diameter and vessel wall thickness (n=10, mean.+ -. SE) of zebra fish after marine phospholipid treatment
P <0.05, p <0.01, p <0.001 compared to model control.
From table 7 and fig. 3 and 4, the cardiac output and blood flow velocity of the model control group were significantly increased as compared with those of the normal control group, the internode blood vessel diameter was narrowed, and the blood vessel wall thickness was increased. After the marine phospholipid is dissolved in water for 2 days, the cardiac output and the blood flow speed of the zebra fish are obviously reduced, the internode blood vessel diameter is obviously thickened, and the blood vessel wall thickness is thinned, so that the marine phospholipid has obvious blood pressure reducing effect.
(4) Total RNA was extracted from 30 zebra fish per experimental group, and the relative expression amounts of vegfaa, angpt2b (ang 2) and ppar. Gamma. (primer sequences are shown in Table 8) were examined, and 3 biological replicates were set. The hypotensive effect was evaluated by the results of statistical analysis of the cardiac output and the vessel diameter of 2 indices. Statistical treatment results are expressed in mean+ -SE.
TABLE 8 primer sequence information
Experimental results 2.2
TABLE 9 relative Gene expression following Marine phospholipid treatment (n=3, mean.+ -. SE)
P <0.05, p <0.01, p <0.001 compared to model control.
Vascular endothelium is not only a mechanical barrier, but also receives signals related to blood, such as hormone levels, stress levels, shear forces, and related cytokines, inflammatory mediators, etc., which cause endothelial cells to secrete different bioactive substances to regulate endothelial cell function and thus regulate the integrity of local vascular functions. The microcirculation of hypertensive patients to large blood vessels all have a varying degree of endothelial damage, thereby adversely affecting the vasomotor function of the blood vessels. The interaction of local inflammation of blood vessels and endothelial dysfunction of patients suffering from hypertension is involved in the formation of hypertension and the damage of relevant target organs. VEGF, an important cytokine for vascular endothelial survival, maintains the integrity of vascular endothelial function, but when the VEGF content in the body serum is too high, it causes excessive mitosis of locally microcirculation endothelial cells, which in turn causes impairment of endothelial function.
Angiotensin ii is a strong vasoconstrictor that constricts the systemic arteriole and increases blood pressure, playing an important role in the pathogenesis of hypertension.
Expression of PPARs can influence the occurrence of hypertension from multiple aspects such as genes, endocrine and the like; the different subtypes have different roles in inhibiting proliferation of Vascular Smooth Muscle Cells (VSMC) in a hypertensive state, wherein pparα and pparγ both inhibit VSMC phenotype transformation, preventing vascular remodeling. It is found that individuals with PPARgamma gene deletion mutation can develop serious early-onset hypertension; pparγ is involved in regulating the circadian rhythm of human blood pressure and heart rate by regulating brain and muscle aromatic receptor nuclear transport-like protein 1 levels. Meanwhile, PPARgamma can also improve hypertension vascular remodeling and endothelial function by inhibiting the conversion of VSMC to an undifferentiated phenotype, and can reduce the hypertension induced by AngII perfusion by blocking inflammatory factor expression, thereby correcting vascular remodeling.
From Table 9, the relative expression levels of the model control groups vegfaa and angpt2b were increased, the relative expression level of the ppar gamma gene was decreased, and after administration of marine phospholipids for 4 days, the relative expression levels of the vegfaa and angpt2b genes were decreased, and the relative expression level of the ppar gamma gene was increased, which was helpful for promoting arteriole dilation, regulating vascular stability, correcting vascular remodeling, and lowering blood pressure.
Experiment two, 5dpf cardiac green fluorescence Tg (cmlc 2: EGFP) strain was randomly selected in six well plates, and 30 zebra fish were treated in each well (experimental group). The marine phospholipids were given 62.5, 125 and 250 μg/mL in water, while the normal control and model control were set to a capacity of 3mL per well. The zebra fish hypertension model is built by injecting 40mg/kg of angiotensin II into all other groups except the normal control group. Treated to 9dpf at 28 ℃. 10 zebra fish from each experimental group were randomly selected and subjected to fluorescence microscopy to collect photographs, which were analyzed and data collected using NIS-ElementsD3.20 advanced image processing software, and analyzed for zebra fish heart area (cardiomyocyte area). And evaluating the heart reconstruction improving efficacy according to the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE.
Experimental results 2.3
TABLE 10 area of zebra fish hearts after marine phospholipid treatment (n=10, mean.+ -.SE)
P <0.05, < p <0.001 compared to model control.
From Table 10 and FIG. 5, the model control atrium and ventricle were significantly larger than the normal control, which exhibited an increase in green fluorescent cardiomyocyte area. After 4 days of water-soluble administration of the marine phospholipid, the atrium and the ventricle of the zebra fish are obviously reduced (the area of green fluorescent myocardial cells is reduced), which shows that the marine phospholipid has the effect of improving myocardial reconstruction.
Application example 3 (propolis soft capsule)
Experiment one, a 4dpf vascular endothelial cell green fluorescence labeled Tg (fli-1: EGFP) strain was randomly selected in six well plates, and 30 zebra fish were treated per well (experimental group). The propolis soft capsules were given 12.5, 25.0 and 50.0 μg/mL in water-soluble form, and a normal control group and a model control group were simultaneously set at a capacity of 3mL per well. The zebra fish hypertension model is built by injecting 20mg/kg of angiotensin II into all other groups except the normal control group. Treated to 6dpf at 28 ℃. And after the treatment is finished, testing and analyzing respectively: (1) The blood pressure lowering efficacy is evaluated according to the statistical analysis results of the 2 indexes of the cardiac output and the blood flow velocity and (2) the blood vessel diameter, and the statistical treatment results are expressed by mean+/-SE. The method comprises the following steps:
(1) 10 zebra fish were randomly selected from each experimental group and placed in a heartbeat and blood flow analysis system to collect video, and the cardiac output and blood flow velocity were analyzed by ViewPoint Application Manager software. (2) 10 zebra fish were randomly selected from each experimental group and placed under a fluorescence microscope to collect photographs, and data were analyzed and collected using NIS-ElementsD3.20 advanced image processing software to analyze the zebra fish internode vessel diameters.
Experimental results 3.1
TABLE 11 zebra fish cardiac output, blood flow velocity and blood vessel diameter after propolis soft capsule treatment (n=10, mean.+ -. SE)
P <0.05, p <0.01, p <0.001 compared to model control.
From Table 11 and FIG. 6, the cardiac output and blood flow velocity of the model control group were significantly increased compared with those of the normal control group, the blood vessel diameter was significantly narrowed, and after administration of the propolis soft capsule for 2 days, the cardiac output and blood flow velocity of the zebra fish were significantly reduced, and the blood vessel diameter was significantly increased. The propolis soft capsule has obvious antihypertensive effect.
Experiment two, randomly selecting 4dpf wild type AB strain in six well plates, treating 30 zebra fish per well (experimental group), and setting 7 biological replicates. 12.5, 25.0 and 50.0 mug/mL of propolis soft capsule is given by water dissolution, and a normal control group are simultaneously arrangedModel control group, 3mL per well capacity. The zebra fish hypertension model is built by injecting 20mg/kg of angiotensin II into all other groups except the normal control group. Treated to 5dpf at 28 ℃. And after the treatment is finished, testing and analyzing respectively: (1) NADP (NADP) + NADPH ratio, (2) ROS level. The method comprises the following steps:
(1) 30 zebra fish of each experimental group are collected according to the specification of the NADP (H) content detection kit, and the NADP in the zebra fish is analyzed by a multifunctional enzyme-labeled instrument + NADPH ratio, 3 biological replicates were set.
Experimental results 3.2
TABLE 12 Whole NADP of zebra fish after propolis soft capsule treatment + NADPH ratio (n=3, mean±se)
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P <0.01 and p <0.001 compared to model control. (2) 30 zebra fish of each experimental group are transferred into a 96-well plate, added with an oxygen free radical fluorescent probe CM-H2DCFDA, incubated overnight at 28 ℃, placed in an enzyme-labeled instrument for data collection, and analyzed for the ROS level of the zebra fish. The antioxidation efficacy was evaluated based on the statistical analysis result of the index. Statistical treatment results are expressed in mean+ -SE.
Experimental results 3.3
Table 13 Whole ROS levels (n=10, mean.+ -. SE) in zebra fish after propolis soft capsule treatment
P <0.01 and p <0.001 compared to model control.
Oxidative stress exacerbates mitochondrial injury and promotes Vascular Smooth Muscle Cell (VSMC) proliferation as one of the important mechanisms responsible for vascular remodeling in hypertension. Reactive Oxygen Species (ROS) are important signaling molecules in VSMC, low concentration ROS exert vascular function regulation and vascular integrity maintenance under physiological conditions, elevated ROS levels, reduced vascular antioxidant activity, oxygen under pathophysiological conditionsChemo-stress occurs, ultimately leading to vascular remodeling. In addition to vascular remodeling, ROS can also contribute to related pathological mechanisms such as vascular reactivity, vascular inflammation, endothelial dysfunction, and the like. Ang II exerts its various pathophysiological effects by stimulating ROS production through activation of vascular NADPH Oxidase (NOX) activity. In addition, ROS adversely affect redox-sensitive signaling molecules. NADPH, the primary provider of subcellular reducing equivalents, can be oxidized to NADP by NADPH oxidase + . Thus, NADP + NADPH is generally used to react the activation of NADPH oxidase and subsequent generation of ROS.
From tables 12 and 13, the model control group zebra fish NADP + The ratio of NADPH is increased, and after administration of propolis soft capsule for 2 days, NADP is added + the/NADPH ratio decreases. Further detection of ROS levels, the model control group had elevated levels of zebra fish ROS and propolis soft capsule dry prognosis decreased ROS levels compared to the normal control group. The above studies indicate that propolis soft capsules can reduce ROS levels by inhibiting NADPH oxidase activation, thereby inhibiting Ang II-induced oxidative stress.
The present invention is not limited to the above-mentioned embodiments, but is intended to be limited to the following embodiments, and any modifications, equivalent changes and variations in the above-mentioned embodiments can be made by those skilled in the art without departing from the scope of the present invention.
Claims (9)
1. A method for evaluating the blood pressure reducing effect by using a zebra fish model is characterized in that zebra fish is randomly selected, angiotensin II is injected intravenously, and indexes are detected and evaluated after treatment to analyze the blood pressure reducing effect.
2. The method for evaluating blood pressure lowering efficacy by using a zebra fish model according to claim 1, wherein the zebra fish is a vascular endothelial cell red fluorescence Tg (fli 1aep: dsRedEx) strain zebra fish, a vascular endothelial cell green fluorescence Tg (fli-1: EGFP) strain zebra fish or a cardiac green fluorescence Tg (cmlc 2: EGFP) strain zebra fish.
3. A method for assessing blood pressure lowering efficacy using a zebra fish model according to claim 1 or 2, wherein the zebra fish is injected for a period of time of 2-5dpf.
4. The method for evaluating blood pressure lowering efficacy using a zebra fish model according to claim 1, wherein the injection dose of angiotensin ii is 10-100mg/kg.
5. The method for evaluating blood pressure lowering efficacy using a zebra fish model according to claim 1 or 4, wherein the treatment is performed at a temperature of 25-30 ℃ for a period of 1 hour to 7 days.
6. The method of claim 1, wherein the test index comprises cardiac output and vessel diameter.
7. The method of claim 6, wherein the test index further comprises at least one of blood flow velocity, blood vessel wall thickness, heart remodeling, myocardial fibrosis, NO levels, oxidative stress, and inflammation.
8. Use of the method for evaluating blood pressure lowering efficacy using zebra fish model according to any one of claims 1 to 7 in efficacy evaluation of drugs, health foods, cosmetics or medical devices.
9. The use according to claim 8, wherein the zebra fish is administered at a stage of 2-5dpf.
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