A method for constructing a tree shrew hypertension model

Abstract

The present invention discloses a method for constructing a tree shrew hypertension model, specifically as follows: (1) Weigh QM385 and dissolve it in a carbonate buffer solution (50 mM, pH 9.0) containing 1% Tween-80 + 0.5% hydroxypropyl methylcellulose (HPMC) to prepare a QM385 solution with a concentration of 0.12 mg / mL; (2) Administer the QM385 solution prepared in step (1) to the tree shrews by gavage at a dose of 0.8 mg / kg for 21 days. After the gavage is completed, the tree shrews are adaptively raised for one week to complete the model establishment. Tree shrews have the characteristics of both primates and rodents. Their organ tissues, biochemical metabolism, and genomics are highly homologous to those of primates. They have the advantages of small body size, high brain-body ratio, short reproductive cycle, strong fertility, and easy to raise. Moreover, compared with primates, tree shrews are easier to obtain, have a lower price, are more convenient to operate, and have a simpler ethical review. By gavage with a QM385 solution at a dose of 0.8 mg / kg for 3 weeks, a stable tree shrew hypertension model can be constructed. The preparation method of this model is simple, stable, and has a high model success rate.

Description

Technical Field

[0001] The invention belongs to the technical field of disease animal model construction, and in particular relates to a tree shrew hypertension model construction method. Background Art

[0002] Hypertension is a common chronic disease and one of the most important risk factors for cardiovascular disease, stroke, chronic kidney disease and dementia. Increased blood pressure is the main preventable risk factor for cardiovascular disease and premature death. Establishing an animal model is an important way to study the pathogenesis of human diseases and diagnostic and treatment methods. At present, rodent (rat and mouse) disease models are mostly used, but they are far from the genetic basis of humans and cannot replicate the full picture of the histological and immunological characteristics of human diseases. Primate disease models are closer to the characteristics of human disease, but they face many difficulties in practical application. Therefore, establishing an animal model that is closely related to humans, convenient and effective is of great significance for understanding the pathogenic mechanism of the disease, formulating effective prevention and control measures and developing new drugs. Summary of the invention

[0003] The purpose of the present invention is to provide a method for constructing a tree shrew hypertension model.

[0004] The object of the present invention is achieved by: (1) weighing QM385 and dissolving it in a carbonate buffer solution (50 mM, pH 9.0) containing 1% Tween-80 + 0.5% hydroxypropyl methylcellulose (HPMC) to prepare a QM385 solution with a concentration of 0.12 mg / mL; (2) administering the QM385 solution prepared in step (1) to tree shrews by gavage at a dose of 0.8 mg / kg for 21 days. After the gavage, the tree shrews are adaptively fed for one week to complete the modeling.

[0005] The beneficial effects of the present invention are:

[0006] Tree shrews have the characteristics of both primates and rodents. Their organ tissues, biochemical metabolism, and genomics are highly homologous to those of primates. They have the advantages of small size, high brain-to-body ratio, short breeding cycle, strong fertility, and easy breeding. Compared with primates, tree shrews are easier to obtain, cheaper, more convenient to operate, and have simpler ethical review. In this application, a stable tree shrew hypertension model can be constructed by gavage of QM385 solution at a dose of 0.8 mg / kg for 3 weeks. The model preparation method is simple, stable, and has a high success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The results of blood pressure changes of 5 groups of tree shrews during the experiment of this application;

[0008] Figure 2 These are the SPR results of 5 groups of tree shrews during the experiment of this application;

[0009] Figure 3 These are the BH4 results of 5 groups of tree shrews during the experiment of this application.

[0010] Figure 4 These are the NO results of 5 groups of tree shrews during the experiment of this application.

[0011] Figure 5 These are the Ang Ⅱ results of 5 groups of tree shrews during the experiment of this application.

[0012] Figure 6 These are the ET-1 results of 5 groups of tree shrews during the experiment of this application.

[0013] Figure 7 These are the MDA results of 5 groups of tree shrews during the experiment of this application.

[0014] Figure 8 These are the SOD results of 5 groups of tree shrews during the experiment of this application. Detailed implementation manners

[0015] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments fall within the scope of protection of the present invention.

[0016] 1. Experimental animals

[0017] 15 ordinary-grade male tree shrews (Tupaia belangeri chinensis) with a body mass of 120 - 150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023 - 0003] and were housed in the ordinary-grade animal house of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021 - 0003]. They were housed in stainless steel cages at a room temperature of (24 ± 2) °C, a relative humidity of (50 ± 5) %, and a light / dark cycle of 12 h / 12 h.

[0018] 2. Main reagents

[0019] Reagent Name Company QM385 Mce Sepiapterin Reductase (SPR) Detection Kit Wuhan Yunke Long Tetrahydrobiopterin (BH4) Detection Kit Shanghai Enzyme-linked Nitric Oxide (NO) Detection Kit Wuhan Elabscience Malondialdehyde (MDA) Detection Kit Wuhan Elabscience Superoxide Dismutase (SOD) Detection Kit Wuhan Elabscience Endothelin 1 (ET-1) Detection Kit Wuhan Elabscience Angiotensin II (Ang-II) Detection Kit Wuhan Elabscience

[0020] 3. Main instruments

[0021] Instrument Name Company Intelligent Non-invasive Sphygmomanometer Softron BP-98A Multifunctional Microplate Reader Ausbian Feyond-A300

[0022] 4. Preparation of reagents

[0023] Preparation of QM385 solution: Weigh the required QM385 and dissolve it in a carbonate buffer solution (50 mM, pH 9.0) containing 1% Tween-80 + 0.5% hydroxypropyl methylcellulose (HPMC) to prepare a QM385 solution with a concentration of 0.12 mg / mL.

[0024] 5. Experimental Methods

[0025] 5.1 Animal Model Preparation

[0026] Experimental Groups: ① Normal group, ② Solvent group, ③ QM385 0.2 mg / kg group, ④ QM385 0.4 mg / kg group, ⑤ QM385 0.8 mg / kg group

[0027] Group ① was gavaged with 1 mL of normal saline daily, group ② was gavaged with 1 mL of 1% Tween-80 + 0.5% HPMC carbonate buffer at the same time every day, and groups ③ - ⑤ were gavaged with the corresponding doses of QM385 solution according to the grouping. The gavage time for all 5 groups lasted for 21 days. After the gavage was completed, the animals were continued to be raised.

[0028] Measurement of Arterial Blood Pressure

[0029] After 3 days of adaptive feeding of tree shrews, a blood pressure measurement adaptation experiment was conducted on each tree shrew at least twice in the following 4 days to eliminate blood pressure changes caused by fear. After one week of adaptive feeding, the blood pressure of tree shrews was measured from 8:30 to 10:30 in the morning. After all tree shrews were measured, the blood pressure of tree shrews was measured at the same time every week thereafter.

[0030] Blood Pressure Measurement Method: The tree shrew was placed head-first into a rat cage, covered with a cage bag, and the tail was left outside. After fixing the cage bag, the tail hair was shaved clean from 2 / 3 of the tail tip to the root of the tail with an electric clipper to expose about 4 cm of skin at the root of the tail. The tail of the tree shrew was placed into a pressure sensor with a rubber membrane. After the tree shrew was quiet for 5 minutes, the start button of the sphygmomanometer was pressed to start automatic measurement. Each animal was measured again after an interval of 5 minutes, and a total of 3 measurements were taken, and the average value was taken as the final measurement value.

[0031] Detection of Sepiapterin Reductase (SPR), Tetrahydrobiopterin (BH4), Nitric Oxide (NO), Endothelin-1 (ET-1), and Angiotensin II (Ang II)

[0032] On days 0 (before modeling), 7, 14, 21, 28, and 35, blood samples were taken from tree shrews, and plasma was separated by centrifugation. The contents of sepiapterin reductase (SPR), tetrahydrobiopterin (BH4), nitric oxide (NO), endothelin-1 (ET-1), and angiotensin II (Ang II) were detected using a kit.

[0033] Detection of Redox-Related Factors

[0034] On days 0 (before modeling), 7, 14, 21, 28, and 35, blood samples were taken from tree shrews, and plasma was separated by centrifugation. The contents of malondialdehyde (MDA) and superoxide dismutase (SOD) were detected using a kit.

[0035] 6. Experimental Results

[0036] 6.1 Blood Pressure Results

[0037] The blood pressure (systolic and diastolic) of tree shrews in the normal group and the solvent group changed little at 0d, 7d, 14d, 21d, 28d, and 35d. Compared with the normal group, the blood pressure (systolic and diastolic) in the QM385 solution group showed an increasing trend at 7d, 14d, 21d, 28d, and 35d, and was dose-dependent.

[0038] Table 1 Blood pressure change results of 5 groups of tree shrews during the experiment ( , n = 3)

[0039] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Systolic Blood Pressure Normal Group 131.67±10.41 132.67±6.11 130.67±9.29 128.67±10.50 133.00±11.36 130.67±7.51 Solvent Group 129.67±10.41 132.33±8.39 128.33±7.51 130.00±6.56 131.00±10.15 129.00±14.18 QM385 0.2 mg / kg Group 129.33±8.62 131.00±9.54 134.33±9.71 138.00±10.00 139.33±5.13 140.00±3.61 QM385 0.4 mg / kg Group 130.00±7.81 134.67±7.51 140.00±7.81 145.67±9.61 147.00±6.93 147.33±4.51 QM385 0.8 mg / kg Group 129.67±8.74 134.67±10.97 142.33±17.01 152.33±9.02 157.33±4.93 158.33±7.02 Diastolic Blood Pressure Normal Group 81.33±3.06 82.00±6.24 83.00±6.93 80.33±5.51 82.33±4.51 82.67±3.51 Solvent Group 81.33±2.52 83.00±4.00 82.33±6.03 82.00±6.08 83.67±5.03 82.00±7.00 QM385 0.2 mg / kg Group 80.33±9.02 82.00±3.61 84.33±1.53 89.67±2.08 87.67±4.73 88.33±4.51 QM385 0.4 mg / kg Group 82.00±8.19 86.33±4.04 90.33±2.08 95.67±2.52 96.33±4.51 95.00±4.58 QM385 0.8 mg / kg Group 82.00±9.54 89.33±6.11 98.00±5.29 112.67±4.73 113.33±5.03 115.00±4.00

[0040] 6.2 SPR Results

[0041] The SPR content in the plasma of tree shrews in the normal group and the solvent group remained basically the same at 0d, 7d, 14d, 21d, 28d, and 35d, with little change. Compared with the normal group, the plasma SPR content in the QM385 solution group showed a decreasing trend at 7d, 14d, 21d, 28d, and 35d, and was dose-dependent.

[0042] Table 2 SPR results of 5 groups of tree shrews during the experiment ( , n = 3)

[0043] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Normal Group 38.98±4.13 40.46±3.38 39.39±4.50 39.27±3.34 39.62±3.46 39.96±5.36 Solvent Group 40.39±4.99 38.68±4.60 39.33±3.79 38.85±1.48 38.55±3.41 39.59±5.21 QM385 0.2 mg / kg Group 39.88±4.10 38.68±3.29 36.93±3.14 32.12±2.13 31.88±3.37 31.52±3.68 QM385 0.4 mg / kg Group 39.60±2.24 37.32±3.02 33.69±3.12 27.58±2.19 26.77±2.92 27.38±3.22 QM385 0.8 mg / kg Group 39.61±3.86 33.00±4.20 26.71±3.48 18.79±1.49 17.6±2.27 18.06±3.13

[0044] 6.3 BH4 Results

[0045] The BH4 content in the plasma of tree shrews in the normal group and the solvent group remained basically the same at 0d, 7d, 14d, 21d, 28d, and 35d, with little change. Compared with the normal group, the plasma BH4 content in the QM385 solution group showed a decreasing trend at 7d, 14d, 21d, 28d, and 35d, and was dose-dependent.

[0046] Table 3 BH4 results of 5 groups of tree shrews during the experiment ( , n = 3)

[0047] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Normal Group 42.26±5.55 40.79±8.99 41.77±6.06 42.14±6.06 42.98±4.90 45.00±6.97 Solvent Group 43.56±6.91 42.13±5.98 43.23±3.08 43.93±6.35 42.37±6.76 45.18±6.42 QM385 0.2 mg / kg Group 42.14±6.03 41.07±3.97 36.49±4.17 31.03±4.21 29.65±4.50 29.82±5.44 QM385 0.4 mg / kg Group 43.80±7.76 39.32±5.51 32.38±4.53 25.98±4.10 26.16±4.91 26.06±6.16 QM385 0.8 mg / kg Group 41.96±5.55 35.20±5.34 23.87±2.91 19.60±3.63 20.63±2.93 18.51±3.24

[0048] 6.4 NO Results

[0049] The NO content in the plasma of tree shrews in the normal group and the solvent group remained basically the same at 0d, 7d, 14d, 21d, 28d, and 35d, with little change. Compared with the normal group, the plasma NO content in the QM385 solution group showed a decreasing trend at 7d, 14d, 21d, 28d, and 35d, and was dose-dependent.

[0050] Table 4 Results of NO in 5 groups of tree shrews during the experiment ( , n = 3)

[0051] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Normal Group 3.72±0.25 3.65±0.16 3.69±0.49 3.70±0.38 3.84±0.57 3.75±0.17 Solvent Group 3.75±0.40 3.72±0.52 3.66±0.45 3.70±0.36 3.79±0.53 3.72±0.36 QM385 0.2 mg / kg Group 3.76±1.23 3.71±0.38 3.63±0.24 3.33±0.22 3.39±0.29 3.33±0.23 QM385 0.4 mg / kg Group 3.72±0.37 3.61±0.43 3.49±0.37 3.01±0.34 3.08±0.46 3.04±0.27 QM385 0.8 mg / kg Group 3.74±0.38 3.49±0.34 2.89±0.49 2.07±0.22 2.11±0.25 1.98±0.16

[0052] 6.5 Results of AngⅡ

[0053] The contents of angiotensin Ⅱ in the plasma of tree shrews in the normal group and the solvent group were basically the same at 0d, 7d, 14d, 21d, 28d, and 35d, with little change. Compared with the normal group, the contents of angiotensin Ⅱ in the plasma of the QM385 solution group showed an increasing trend at 7d, 14d, 21d, 28d, and 35d, and had a dose-dependence.

[0054] Table 5 Results of Ang Ⅱ in 5 groups of tree shrews during the experiment ( , n = 3)

[0055] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Normal Group 518.54±64.51 513.58±43.76 518.49±30.19 519.99±62.22 507.49±35.70 503.90±54.63 Solvent Group 518.99±46.32 491.09±48.37 506.35±17.15 514.33±52.24 517.13±37.59 508.11±48.63 QM385 0.2 mg / kg Group 527.33±27.42 532.08±40.00 542.80±19.21 572.75±28.42 576.98±26.99 579.74±57.49 QM385 0.4 mg / kg Group 519.51±46.25 547.16±48.76 629.91±34.92 667.82±65.30 673.97±40.90 674.13±62.48 QM385 0.8 mg / kg Group 527.64±35.29 592.72±61.93 709.68±75.64 793.67±66.76 808.74±34.61 796.48±49.20

[0056] 6.6 Results of ET-1

[0057] The contents of endothelin-1 in the plasma of tree shrews in the normal group and the solvent group were basically the same at 0d, 7d, 14d, 21d, 28d, and 35d, with little change. Compared with the normal group, the contents of endothelin-1 in the plasma of the QM385 solution group showed an increasing trend at 7d, 14d, 21d, 28d, and 35d, and had a dose-dependence.

[0058] Table 6 Results of ET-1 in 5 groups of tree shrews during the experiment ( , n = 3)

[0059] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Normal Group 20.09±1.47 21.00±3.80 21.17±4.25 20.93±2.38 19.95±1.12 19.28±2.10 Solvent Group 20.61±2.07 19.84±0.85 20.12±4.48 18.75±2.89 19.52±1.53 21.87±2.73 QM385 0.2 mg / kg Group 19.98±1.77 21.03±2.01 23.10±3.80 26.63±4.40 27.58±3.05 25.89±5.06 QM385 0.4 mg / kg Group 20.23±1.55 22.89±3.08 27.34±4.67 32.46±4.46 34.40±7.54 33.54±5.11 QM385 0.8 mg / kg Group 19.63±2.00 25.46±4.45 33.07±5.06 45.51±3.62 46.87±4.49 47.51±3.26

[0060] 6.7 Results of MDA

[0061] The contents of MDA in the plasma of tree shrews in the normal group and the solvent group were basically the same at 0d, 7d, 14d, 21d, 28d, and 35d, with little change. Compared with the normal group, the contents of MDA in the plasma of the QM385 solution group showed an increasing trend at 7d, 14d, 21d, 28d, and 35d, and had a dose-dependence.

[0062] Table 7 Results of MDA in 5 groups of tree shrews during the experiment ( , n = 3)

[0063] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Normal Group 5.07±0.27 4.93±0.78 4.91±0.38 4.94±0.58 4.86±0.43 5.09±0.83 Solvent Group 5.08±0.55 5.11±0.39 4.91±0.75 4.89±0.40 5.10±0.41 4.98±0.23 QM385 0.2 mg / kg Group 5.05±0.37 5.23±0.47 5.61±0.49 6.21±0.35 6.28±0.38 6.22±0.25 QM385 0.4 mg / kg Group 5.10±0.45 5.71±1.03 6.49±1.11 7.64±1.17 7.71±1.23 7.73±0.31 QM385 0.8 mg / kg Group 5.05±0.78 5.93±0.62 7.74±0.91 10.33±0.95 10.10±1.35 10.47±1.40

[0064] 6.8 Results of SOD

[0065] The SOD content in the plasma of tree shrews in the normal group and the solvent group remained basically the same at 0d, 7d, 14d, 21d, 28d, and 35d, with little change. Compared with the normal group, the plasma SOD content in the QM385 solution group showed a downward trend at 7d, 14d, 21d, 28d, and 35d, and was dose-dependent.

[0066] Table 8 SOD results of 5 groups of tree shrews during the experiment ( , n = 3)

[0067] Grouping (n = 3) 0d 7d 14d 21d 28d 35d Normal Group 261.57±25.17 268.53±30.03 255.89±23.96 260.52±29.38 271.88±23.25 265.37±23.64 Solvent Group 263.46±30.98 266.76±11.80 268.45±20.58 255.16±33.96 260.23±31.42 269.19±9.57 QM385 0.2 mg / kg Group 261.92±31.71 242.13±30.45 221.83±29.76 196.85±24.46 207.51±24.57 186.27±34.77 QM385 0.4 mg / kg Group 261.99±24.39 232.26±23.37 195.60±22.12 157.60±52.48 154.51±10.15 150.08±33.35 QM385 0.8 mg / kg Group 268.99±21.55 224.03±17.34 170.26±25.26 96.38±22.05 101.52±16.39 91.53±17.23

[0068] During the experiment, in addition to measuring the blood pressure (systolic blood pressure and diastolic blood pressure) of tree shrews, key factors in the renin-angiotensin system and oxidative stress-related indicators of tree shrews were also measured. The experiment found that after gavage with the QM385 solution, the blood pressure (systolic blood pressure and diastolic blood pressure) of tree shrews increased significantly, and showed dose- and time-dependence; gavage with the QM385 solution led to a significant decrease in sepiapterin reductase (SPR) and tetrahydrobiopterin (BH4), and showed dose- and time-dependence; the content of the key factor nitric oxide (NO) in the renin-angiotensin system decreased, while the contents of endothelin-1 (ET-1) and angiotensin II (Ang II) increased, and showed dose- and time-dependence; the content of the oxidative stress-related indicator malondialdehyde (MDA) decreased, and the content of superoxide dismutase (SOD) increased, and showed dose- and time-dependence.

Claims

1. A method for constructing a tree shrew hypertension model, characterized in that: The steps include: (1) Weigh QM385 and dissolve it in carbonate buffer containing 1% Tween-80 + 0.5% hydroxypropyl methylcellulose to prepare a QM385 solution with a concentration of 0.12 mg / mL; (2) The QM385 solution prepared in step (1) was administered orally to tree shrews at a dose of 0.8 mg / kg for 21 days. After the administration, the tree shrews were adaptively fed for one week to complete the modeling.

2. A method for constructing a tree shrew hypertension model according to claim 1, characterized in that: The tree shrew is a common male Burmese tree shrew.

3. The method for constructing a tree shrew hypertension model according to claim 1, characterized in that: The tree shrew has a body weight of 120-150 g, and is kept at a room temperature of 24±2° C., a relative humidity of 50±5%, and a light / dark cycle of 12 h / 12 ​​h.

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