Tree shrew hypertension model construction method

Abstract

The invention discloses a tree shrew hypertension model construction method, which specifically comprises: (1) weighing QM385, and dissolving in a carbonate buffer solution (50mM, pH 9.0) containing 1% of Tween-80 and 0.5% of hydroxypropyl methyl cellulose (HPMC) to prepare a QM385 solution with a concentration of 0.12 mg / mL; and (2) performing intragastric administration on the tree shrew with the QM385 solution prepared in the step (1) according to the dosage of 0.8 mg / kg for 21 days, and after intragastric administration is finished, performing adaptive feeding for one week to finish modeling. The tree shrew has the characteristics of primates and rodents, has high homology with the primates in organ tissue, biochemical metabolism and genomics, and has the advantages of small size, high brain-body ratio, short breeding cycle, strong fertility, easiness in feeding and the like; compared with primates, the tree shrews are easier to obtain, lower in price, more convenient to operate and more simplified in ethical examination and verification, the stable tree shrew hypertension model can be constructed through intragastric administration of the QM385 solution with the dosage of 0.8 mg / kg for 3 weeks, and the model preparation method is simple, stable and high in model forming 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: 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

[0006] Figure 1 The results of blood pressure changes of 5 groups of tree shrews during the experiment of this application; Figure 2 These are the SPR results of 5 groups of tree shrews during the experiment of this application; Figure 3These are the BH4 results of 5 groups of tree shrews during the experiment of this application; Figure 4 The NO results of 5 groups of tree shrews during the experiment of this application; Figure 5 The results of Ang Ⅱ of 5 groups of tree shrews during the experiment of this application; Figure 6 These are the ET-1 results of 5 groups of tree shrews during the experiment of this application; Figure 7 These are the MDA results of 5 groups of tree shrews during the experiment of this application; Figure 8 These are the SOD results of 5 groups of tree shrews during the experiment of this application. DETAILED DESCRIPTION

[0007] The technical solution of the present invention will be described clearly and completely in conjunction with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of the embodiments. All other embodiments belong to the scope of protection of the present invention.

[0008] , Experimental Animals Fifteen ordinary male Burmese tree shrews weighing 120-150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023-0003] and kept in the ordinary animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003] in stainless steel cages at room temperature (24±2)℃, relative humidity (50±5)%, and light / dark cycle of 12h / 12h.

[0009] , Main Reagents Reagent name company QM385 Mce Sepilopterin Reductase (SPR) Detection Kit Wuhan Cloud Clone Tetrahydrobiopterin (BH4) Detection Kit Shanghai ELISA Nitric Oxide (NO) Detection Kit Wuhan Elerite Malondialdehyde (MDA) Detection Kit Wuhan Elerite Superoxide dismutase (SOD) detection kit Wuhan Elerite Endothelin 1 (ET-1) Detection Kit Wuhan Elerite Angiotensin II (Ang-II) detection kit Wuhan Elerite 3. Main instruments Instrument Name company Smart non-invasive blood pressure monitor Softron BP-98A Multifunctional ELISA reader Aosheng Feyond-A300 4. Preparation of reagents Preparation of QM385 solution: Weigh the required amount of QM385 and dissolve it in carbonate buffer (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.

[0010] , Experimental methods 5.1 Animal model preparation Experimental groups: ①, normal group, ②, solvent group, ③, QM385 0.2mg / kg group, ④, QM385 0.4mg / kg group, ⑤, QM385 0.8mg / kg group Group ① was gavaged with 1 mL of normal saline every day, group ② was gavaged with 1 mL of carbonate buffer solution containing 1% Tween-80 + 0.5% HPMC at the same time every day, and groups ③-⑤ were gavaged with corresponding doses of QM385 solution according to the grouping. The gavage time of the five groups lasted for 21 days. After gavage, the rats continued to be fed.

[0011] Arterial blood pressure measurement After three days of adaptive feeding of tree shrews, blood pressure measurement adaptation experiments were performed on each tree shrew at least twice in the next four days to eliminate the changes in blood pressure caused by fear. After one week of adaptive feeding, the blood pressure of tree shrews was measured at 8:30-10:30 in the morning. After all tree shrews were measured, intraperitoneal injections were performed according to the groups, and the blood pressure of tree shrews was measured at the same time every week thereafter.

[0012] Blood pressure measurement method: Put the tree shrew head forward into the rat net, put it into the net bag, and leave the tail outside. After fixing the net bag, use electric clippers to shave the tail hair from 2 / 3 of the tail tip to the base of the tail, exposing the skin about 4 cm long at the base of the tail. Put the tree shrew's tail into the pressure sensor with a rubber membrane. After the tree shrew is quiet for 5 minutes, press the start button of the blood pressure meter to start automatic measurement. Each animal is measured again after an interval of 5 minutes, and a total of 3 measurements are taken, and the average value is taken as the final measurement value.

[0013] Sepiopterin reductase (SPR), tetrahydrobiopterin (BH4), nitric oxide (NO), endothelin-1 (ET-1), and angiotensin II (Ang II) detection Before modeling (0d), 7d, 14d, 21d, 28d, and 35d, blood was collected from tree shrews and plasma was separated after centrifugation. The levels of sepiopterin reductase (SPR), tetrahydrobiopterin (BH4), nitric oxide (NO), endothelin-1 (ET-1), and angiotensin II (AngⅡ) were detected by kits.

[0014] Redox-related factor detection Blood was collected from tree shrews before modeling (0d), 7d, 14d, 21d, 28d, and 35d, and plasma was separated after centrifugation. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by kits.

[0015] , Experimental results 6.1 Blood pressure results The blood pressure (systolic and diastolic) of tree shrews in the normal group and solvent group changed little on days 0, 7, 14, 21, 28, and 35. Compared with the normal group, the blood pressure (systolic and diastolic) of the QM385 solution group on days 7, 14, 21, 28, and 35 showed an increasing trend in a dose-dependent manner.

[0016] Table 1 Blood pressure changes of five groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 6.2 SPR results The plasma SPR levels of tree shrews in the normal group and solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma SPR levels of the QM385 solution group showed a decreasing trend on days 7, 14, 21, 28, and 35, and were dose-dependent.

[0017] Table 2 SPR results of five groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 6.3 BH4 results The plasma BH4 content of tree shrews in the normal group and solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma BH4 content of the QM385 solution group showed a decreasing trend on days 7, 14, 21, 28, and 35, and was dose-dependent.

[0018] Table 3 BH4 results of five groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 6.4 NO Result The plasma NO levels of tree shrews in the normal group and solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma NO levels of the QM385 solution group showed a decreasing trend on days 7, 14, 21, 28, and 35, and were dose-dependent.

[0019] Table 4 NO results of five groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 6.5 Ang II Results The plasma angiotensin II levels of tree shrews in the normal group and solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma angiotensin II levels in the QM385 solution group increased on days 7, 14, 21, 28, and 35 in a dose-dependent manner.

[0020] Table 5 Results of Ang Ⅱ in five groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 6.6 ET-1 Results The plasma endothelin-1 levels of tree shrews in the normal group and solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma endothelin-1 levels in the QM385 solution group showed an increasing trend on days 7, 14, 21, 28, and 35, and were dose-dependent.

[0021] Table 6 ET-1 results of five groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 6.7 MDA Results The plasma MDA content of tree shrews in the normal group and solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma MDA content of the QM385 solution group on days 7, 14, 21, 28, and 35 showed an increasing trend in a dose-dependent manner.

[0022] Table 7 MDA results of five groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 6.8 SOD Results The plasma SOD content of tree shrews in the normal group and solvent group remained basically the same on days 0, 7, 14, 21, 28, and 35, with little change. Compared with the normal group, the plasma SOD content of the QM385 solution group showed a downward trend on days 7, 14, 21, 28, and 35, and was dose-dependent.

[0023] Table 8 SOD results of 5 groups of tree shrews during the experiment ( , n=3) 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 0.2mg / 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 0.4mg / 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 0.8mg / 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 During the experiment, in addition to testing the blood pressure (systolic and diastolic) of tree shrews, key factors in the tree shrew renin-angiotensin system and oxidative stress-related indicators were also tested. The experiment found that intragastric administration of QM385 solution led to a significant increase in the blood pressure (systolic and diastolic) of tree shrews, and it was dose- and time-dependent; intragastric administration of QM385 solution led to a significant decrease in septopterin reductase (SPR) and tetrahydrobiopterin (BH4), and it was dose- and time-dependent; the content of nitric oxide (NO), a key factor in the renin-angiotensin system, decreased, and the content of endothelin-1 (ET-1) and angiotensin II (Ang II) increased, and it was dose- and time-dependent; the content of malondialdehyde (MDA), an indicator related to oxidative stress, decreased, and the content of superoxide dismutase (SOD) increased, and it was dose- and time-dependent.

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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