A method for constructing a plateau animal model of blood stasis due to deficiency of qi and its application

Abstract

The application provides a construction method and application of a plateau qi deficiency and blood stasis syndrome animal model, and belongs to the technical field of model animal preparation. The construction method of the plateau qi deficiency and blood stasis syndrome animal model comprises the following steps: placing experimental animals in a low-pressure and low-oxygen environment for 1-4 days, and performing ice water bath on the experimental animals every day; meanwhile, the experimental animals are administered with adrenaline hydrochloride 1-3 times on the first day to the fourth day, and a plateau qi deficiency and blood stasis syndrome animal model is obtained. The method is normative in operation and has a perfect evaluation system, the plateau qi deficiency and blood stasis syndrome animal model constructed is more in line with the characteristics of a traditional Chinese medicine clinical syndrome, meets the characteristics of rapid onset of the plateau qi deficiency and blood stasis syndrome, highlights the blood stasis condition of the model, and provides a scientific basis for treating the plateau qi deficiency and blood stasis syndrome by using traditional Chinese medicines.

Description

Technical Field

[0001] This invention belongs to the field of animal model preparation technology, and particularly relates to a method for constructing and applying an animal model of high-altitude qi deficiency and blood stasis syndrome. Background Technology

[0002] High-altitude Qi deficiency and blood stasis syndrome refers to a condition caused by the thin air and insufficient source of pure Qi in high-altitude areas, leading to a deficiency of vital Qi. This deficiency hinders the lungs from functioning properly and affects the heart and blood vessels, resulting in a decline in bodily functions. Combined with the cold climate of high altitudes, this easily causes the blood to become "thick, viscous, concentrated, and stagnant," exhibiting symptoms of Qi deficiency and blood stasis. The main clinical symptoms include shortness of breath, fatigue, weakness, palpitations, chest tightness, insomnia, chest pain, hypochondriac pain, cyanosis of the lips and nails, a purplish-dark tongue, and a deep, thready, and hesitant pulse. Healthy individuals entering high-altitude areas from plains will experience varying degrees of typical Qi deficiency symptoms such as chest tightness, shortness of breath, fatigue, weakness, and weakness, which are aggravated by exertion. The incidence and severity of symptoms are positively correlated with altitude gradient. Currently, there is insufficient clinical research on anti-inflammatory formulas and treatments for high-altitude Qi deficiency and blood stasis syndrome; therefore, research on effective drugs for the prevention and treatment of this syndrome is essential. The foundation of these studies lies in establishing a reproducible animal model of high-altitude qi deficiency and blood stasis syndrome with a sound evaluation system. Currently, research on animal models of high-altitude qi deficiency and blood stasis syndrome is limited, and the construction procedures are not standardized, with incomplete evaluation systems. Furthermore, existing methods for constructing models using only low pressure and hypoxia are time-consuming, generally taking at least four weeks, which does not reflect the rapid onset characteristic of this syndrome. Therefore, establishing a reproducible animal model of high-altitude qi deficiency and blood stasis syndrome with a sound evaluation system and rapid onset is a technical problem that needs to be solved at this stage. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for constructing and applying an animal model of high-altitude qi deficiency and blood stasis syndrome. The animal model of high-altitude qi deficiency and blood stasis syndrome constructed by this method is more consistent with the clinical syndrome characteristics, and the established operating procedures and evaluation standards are sound, providing a scientific basis for the treatment of high-altitude qi deficiency and blood stasis syndrome.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for constructing an animal model of high-altitude qi deficiency and blood stasis syndrome, comprising the following steps: placing experimental animals in a low-pressure, low-oxygen environment for 1-4 days and giving them an ice-water bath daily; simultaneously administering adrenaline hydrochloride to the experimental animals 1-3 times on days 1-4; wherein the oxygen partial pressure in the low-pressure, low-oxygen environment is 9-12 kPa, the oxygen concentration is 11-20%, and the temperature is 20-25℃.

[0006] Preferably, the experimental animals include rats or mice.

[0007] Preferably, the low-pressure, low-oxygen environment is a simulated plateau environment at an altitude of 5500-6500m.

[0008] Preferably, the time of day is from 9:30 a.m. to 10:30 a.m.

[0009] Preferably, the ice-water bath time is 3-6 minutes.

[0010] Preferably, the method of administering epinephrine hydrochloride includes: administering epinephrine hydrochloride for the first time between 9:30 and 10:30 a.m. on days 1-4, followed by a second administration 1.5-2.5 hours later, with an ice water bath for 3-6 minutes in between.

[0011] Preferably, the administration is by subcutaneous injection.

[0012] Preferably, the constructed animal model of high-altitude qi deficiency syndrome is subjected to systemic behavioral analysis, coagulation function, chrnα1 levels in brain, heart, and lung tissues, and tongue appearance detection.

[0013] This invention provides the application of the animal model of high-altitude qi deficiency and blood stasis syndrome obtained by the above-described construction method in the study of the pathogenesis of high-altitude qi deficiency and blood stasis syndrome.

[0014] This invention provides the application of the animal model of high-altitude qi deficiency and blood stasis syndrome obtained by the above-described construction method in screening drugs for the treatment of high-altitude qi deficiency and blood stasis syndrome.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention, based on the TCM syndrome characteristics of altitude acclimatization difficulties, employs a combined modeling method involving low pressure, hypoxia, and drug intervention to explore the construction of an animal model of Qi deficiency and blood stasis syndrome at high altitudes. Through comparative studies, the method described in this invention represents the optimal modeling approach for this syndrome, and operational guidelines and evaluation criteria have been established. The animal model constructed using this method better reflects the clinical syndrome characteristics in TCM. This model can be used to study the pathogenesis of Qi deficiency and blood stasis syndrome at high altitudes, providing a scientific basis for TCM treatment of this syndrome. Attached Figure Description

[0017] Figure 1 The results are from the gripping force experiment.

[0018] Figure 2 These are the results of an open field experiment.

[0019] Figure 3 These are the results of the four coagulation tests.

[0020] Figure 4 The results show the detection results of chrnα1 content in heart, brain, and lung tissues.

[0021] Figure 5This is the statistical result of tongue image detection.

[0022] Figure 6 Images of the tongues of mice in each group. Detailed Implementation

[0023] This invention provides a method for constructing an animal model of high-altitude qi deficiency and blood stasis syndrome, comprising the following steps: placing experimental animals in a low-pressure, low-oxygen environment for 1-4 days, and subjecting them to daily ice-water baths; simultaneously administering epinephrine hydrochloride 1-3 times on days 1-4 to obtain the high-altitude qi deficiency syndrome animal model. The oxygen partial pressure in the low-pressure, low-oxygen environment described in this invention is 9-12 kPa, the oxygen concentration is 11-20%, and the temperature is 20-25℃. Based on the TCM syndrome characteristics of difficulty acclimatizing to high altitudes, this invention employs a combined modeling method involving low pressure, low oxygen, and drug intervention to explore the construction of an animal model of high-altitude qi deficiency and blood stasis syndrome. Compared with models constructed using a low-pressure, low-oxygen environment, the high-altitude qi deficiency and blood stasis syndrome animal model constructed by the method described in this invention more closely resembles the clinical symptoms.

[0024] In this invention, the adrenalin hydrochloride (molecular formula: C9H) is described. 14 ClNO3 (molecular weight: 219.665) is a vasoactive drug used to treat shock and anaphylactic shock. It can also be used to treat other allergic diseases (such as bronchial asthma and urticaria). When used in combination with local anesthetics, it can help stop local bleeding and prolong the drug's effect.

[0025] In this invention, the experimental animals include rats or mice, preferably mice. The mice used in this invention are preferably 8-10 weeks old and weigh 18-20g.

[0026] In this invention, the low-pressure, low-oxygen environment simulates a plateau environment at an altitude of 5500-6500m. The low-pressure, low-oxygen environment described in this invention is implemented within a low-pressure, low-oxygen chamber. The preferred actual oxygen pressure within the chamber is 9-11 kPa, the preferred actual oxygen concentration is 11-16%, and the preferred temperature is 21-24℃.

[0027] In this invention, the ice-water bath time is 3-6 minutes. The experimental animals are subjected to ice-water bath treatment in a low-pressure, low-oxygen environment from 9:30 AM to 10:30 AM daily. The preferred time for the ice-water bath treatment is 10:00 AM daily.

[0028] In this invention, the administration of epinephrine hydrochloride includes: administering the first injection of epinephrine hydrochloride to experimental animals treated with low pressure and low oxygen between 9:30 AM and 10:30 AM on days 1-4, followed by a second subcutaneous injection 1.5-2.5 hours later, with an ice-water bath for 3-6 minutes in between. Preferably, the method of administering epinephrine hydrochloride in this invention includes: administering the first injection of epinephrine hydrochloride to experimental animals treated with low pressure and low oxygen at 10:00 AM on day 3, followed by a second subcutaneous injection 2 hours later, with an ice-water bath for 3-6 minutes in between.

[0029] In this invention, the constructed animal model of high-altitude qi deficiency and blood stasis syndrome was subjected to systemic behavioral analysis, coagulation function, levels of human nicotinic cholinergic receptor α1 (chrnα1) in brain, heart, and lung tissues, and tongue appearance detection. The systemic behavioral analysis employed grip force testing and the open field test (OFT). The grip force test reflects the "qi deficiency" in the high-altitude qi deficiency and blood stasis syndrome animals; the OFT is primarily used to study the motor ability and anxiety-like behavior of mice, and both the grip force test and the grip force test can serve as indicators of the degree of "qi deficiency." The coagulation function test reflects the "blood stasis" in the syndrome model. The chrnα1 levels in brain, heart, and lung tissues reflect the "qi deficiency" in the high-altitude qi deficiency and blood stasis syndrome animals; tongue appearance detection, as a characteristic indicator of traditional Chinese medicine, reflects the typical "dark purple tongue" phenomenon of qi deficiency and blood stasis.

[0030] The present invention also provides the application of the animal model of high-altitude qi deficiency and blood stasis syndrome obtained by the above construction method in screening drugs for treating high-altitude qi deficiency and blood stasis syndrome or studying the pathogenesis of high-altitude qi deficiency and blood stasis syndrome.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] The experimental animals prepared for this embodiment are as follows:

[0034] SPF-grade male C57BL / 6J mice, 8–10 weeks old, weighing 18–20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The basic diet for these mice was provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0035] The ambient temperature was 23-25℃ and the humidity was (45±10)%. The laboratory was kept quiet and well-ventilated. The experimental animals were housed in cages under 12h light / dark conditions. The animals were kept in standard mouse conditions and were acclimatized for 7 days before the start of the experiment. The cages were cleaned once a day to keep them clean.

[0036] The method for constructing an animal model of Qi deficiency and blood stasis syndrome at high altitudes is as follows:

[0037] Prepared mice were placed in a hypobaric chamber and raised to a simulated altitude of 6000m at a speed of 20m / s. The actual oxygen pressure in the chamber was 9.5-9.7kPa, and the oxygen concentration was 11-20%. The treatment lasted for 3 days. On the first and second days, the mice were given an ice-water bath for 3-6 minutes at 10:00 AM, and then returned to the hypobaric chamber. On the third day, at 10:00 AM, the mice were subcutaneously injected with 0.65mg / kg of epinephrine hydrochloride (purchased from Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd., catalog number: H44020575). Two hours later, a second subcutaneous injection of 0.65mg / kg of epinephrine hydrochloride was given, with an ice-water bath for 3-6 minutes in between. The mice were then returned to the hypobaric chamber after the above procedures were completed. The entire treatment process for this method was 3 days, which constituted the complete model establishment.

[0038] Example 2

[0039] The preparation of experimental animals is the same as in Example 1.

[0040] The method for constructing an animal model of Qi deficiency and blood stasis syndrome at high altitudes is as follows:

[0041] Prepared mice were placed in a hypobaric, hypoxic chamber and raised to a simulated altitude of 5500m at a speed of 20m / s for 2 days. On day 1, at 9:30 AM, the mice were subjected to an ice-water bath for 3-6 minutes. On day 2, at 9:30 AM, the mice were subcutaneously injected with 0.55mg / kg of epinephrine hydrochloride for the first time, followed by a second subcutaneous injection of 0.55mg / kg of epinephrine hydrochloride 1.5 hours later, with an ice-water bath for 3-6 minutes in between. The mice were then returned to the hypobaric, hypoxic chamber after the above procedures were completed. The entire treatment time for this method was 2 days, i.e., the model was established.

[0042] Example 3

[0043] The preparation of experimental animals is the same as in Example 1.

[0044] The method for constructing an animal model of Qi deficiency and blood stasis syndrome at high altitudes is as follows:

[0045] Prepared mice were placed in a hypobaric chamber and raised to a simulated altitude of 6500m at a speed of 20m / s for 4 days. From day 1 to day 3, mice were given an ice-water bath for 3-6 minutes at 10:30 AM each day. On day 4, at 10:30 AM, mice were subcutaneously injected with 0.75mg / kg epinephrine hydrochloride for the first time, followed by a second subcutaneous injection of 0.75mg / kg epinephrine hydrochloride 2.5 hours later, with an ice-water bath for 3-6 minutes in between. After these procedures, the mice were returned to the hypobaric chamber. The entire treatment time for this method was 2 days, completing the model establishment.

[0046] Comparative Example 1

[0047] The preparation of experimental animals is the same as in Example 1.

[0048] The method for constructing an animal model of Qi deficiency and blood stasis syndrome at high altitudes is as follows:

[0049] Prepared mice were placed in a hypobaric chamber and raised to a simulated altitude of 6000m at a speed of 20m / s for 3 days. Simultaneously, they were injected intraperitoneally with cyclophosphamide 80mg / kg once daily at 10:00 AM. After treatment, the mice were returned to the hypobaric chamber. The entire treatment process for this method was a full 3 days, signifying successful model establishment.

[0050] Comparative Example 2

[0051] The preparation of experimental animals is the same as in Example 1.

[0052] The method for constructing an animal model of Qi deficiency and blood stasis syndrome at high altitudes is as follows:

[0053] Prepared mice were placed in a hypobaric, hypoxic chamber and raised to a simulated altitude of 6000m at a speed of 20m / s for 3 days. Simultaneously, at 10:00 AM on the third day, the mice were subcutaneously injected with 0.65mg / kg of epinephrine hydrochloride for the first time, followed by a second subcutaneous injection of 0.65mg / kg of epinephrine hydrochloride 2 hours later. The mice were then returned to the hypobaric, hypoxic chamber after the above procedures were completed. The entire treatment time for this method was a full 3 days, indicating successful model establishment.

[0054] Experimental Example 1

[0055] 1. Grouping

[0056] Eight SPF-grade male C57BL / 6J mice (8-10 weeks old, 18-20g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were used as the normal group. They were not treated and were fed in accordance with normal feeding methods.

[0057] Eight animals with high-altitude qi deficiency and blood stasis syndrome constructed in Comparative Example 1 were selected as the cyclophosphamide group.

[0058] Eight animals with high-altitude qi deficiency and blood stasis syndrome constructed in Comparative Example 2 were selected as the adrenaline group.

[0059] Eight animals with high-altitude qi deficiency and blood stasis syndrome constructed in Example 1 were used as the adrenaline + ice bath group.

[0060] 2. Detection of indicators in each group of mice

[0061] Mice in the above groups were subjected to grip strength test, open field test, coagulation test, chrnα1 content in heart, brain and lung tissue and tongue appearance test.

[0062] The gripping force testing procedure is as follows: Using the YLS-13A rat and mouse gripping force measuring instrument, the mouse is placed on the gripping force testing induction grid plate and then dragged to measure the gripping force of each group of experimental animals.

[0063] The open field test procedure was as follows: An open field test chamber was used, divided into four equal-sized boxes (50cm × 50cm × 40cm). The outer walls of each box were black. The area near the bottom of the box formed the peripheral activity zone, and the remaining area formed the central activity zone. Before the test, mice were placed in the open field test chamber for acclimatization for 15 seconds. At the start of the experiment, the mice were placed in the center of the open field. Simultaneously with the release of the mice, a camera above the test chamber began synchronously tracking and recording for 5 minutes. The evaluation metrics for this open field test were the total distance traveled by the mice (peripheral area + central area), the distance traveled in the central area, and the time spent in the central area.

[0064] The steps for the coagulation panel test are as follows: Before the test, whole blood containing anticoagulants is irradiated at 3000 rpm. -1 After centrifugation for 15 minutes, plasma was obtained. Subsequently, the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) levels were measured according to the coagulation analyzer operating procedures.

[0065] Detection of chrnα1 levels in brain, heart, and lung tissues: Mice in each group were tested using ELISA kits according to the manufacturer's instructions. The ELISA kits were provided by Jiangsu Enzyme Immunosorbent Assay Co., Ltd. (Nanjing, China). Optical density was measured at 450 nm using a microplate reader (MULTISKANEX, Thermoscientific). The levels of chrnα1 in brain, heart, and lung tissues were calculated based on the standard curve.

[0066] The tongue image examination procedure is as follows: Before sampling, mice were anesthetized with isoflurane and placed on A4 white paper or a dedicated macro photography stage for digital photography. The RGB values ​​of the tongue surface and underside were analyzed using ImageJ.

[0067] 3. Results Analysis

[0068] The grip strength test results are as follows Figure 1 As shown, compared with the normal group, the grip strength of the cyclophosphamide group, adrenaline group, and adrenaline + ice bath group showed a decreasing trend, which was statistically significant.

[0069] The results of the open field experiment are as follows Figure 2As shown, compared with the normal group, the cyclophosphamide group, adrenaline group and adrenaline + ice bath group showed a decreasing trend in central zone distance, number of entries and total distance, but the adrenaline + ice bath group showed a more significant decrease in central zone distance and number of entries (P < 0.001).

[0070] The results of the four coagulation tests are as follows Figure 3 As shown, compared with the normal group, the FIB levels in the adrenaline group and the adrenaline + ice bath group were significantly increased (P < 0.05), while the cyclophosphamide group showed no increasing trend.

[0071] Experimental results of chrnα1 content in heart, brain, and lung tissues are as follows: Figure 4 As shown, compared with the normal group, the grip strength of the model group showed a downward trend, which was statistically significant; the chrnα1 content in the adrenaline group and the adrenaline + ice bath group showed an upward trend in the heart, brain and lung tissues.

[0072] Tongue image detection test results are as follows Figures 5-6 As shown, compared with the normal group, the tongue color of the model group was dull, and the RGB values ​​of the tongue under the tongue, analyzed by ImageJ, showed a decreasing trend.

[0073] The overall statistical results of the above results are shown in Table 1. The results show that the model mice in the adrenaline + ice bath group have statistical significance in terms of the main symptoms, concurrent symptoms and TCM characteristic indicators. This indicates that the animal model of high-altitude qi deficiency and blood stasis syndrome obtained by the method of this invention is more consistent with the manifestations of high-altitude qi deficiency and blood stasis syndrome and the clinical syndrome characteristics of TCM.

[0074] Table 1. Evaluation index results of each group of mice

[0075]

[0076] Note: "√" indicates that the model has statistical significance in meeting the expected results of the plateau qi deficiency and blood stasis syndrome model for this indicator, while "×" indicates that it has no statistical significance.

[0077] In summary, the experimental results show that by establishing a model in mice for 3 days and then subcutaneously injecting epinephrine hydrochloride on the third day of modeling, compared with the normal group and other model groups, the mice in the low-pressure hypoxia combined with epinephrine plus ice bath group showed a significant decrease in total distance, distance and number of times they entered the central region, and a significant increase in the four coagulation parameters (FIB). This indicates that the animal model of high-altitude qi deficiency and blood stasis syndrome obtained by the method of this invention is more consistent with the clinical syndrome characteristics of traditional Chinese medicine, and provides a scientific basis for the treatment of high-altitude qi deficiency and blood stasis syndrome with traditional Chinese medicine.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made 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. A method for constructing an animal model of high-altitude qi deficiency and blood stasis syndrome, characterized in that, Includes the following steps: The experimental animals were kept in a low-pressure, low-oxygen environment for 1-4 days, and were given an ice-water bath for 3-6 minutes every day from 9:30 to 10:30 am. On the last day of feeding, the mice were given the first dose of epinephrine hydrochloride at 0.65 mg / kg from 9:30 to 10:30 am, and the second dose of epinephrine hydrochloride was given after an interval of 1.5-2.5 hours, with an ice-water bath for 3-6 minutes in between. The mice were then returned to the low-pressure, low-oxygen chamber after the above operations were completed. The oxygen partial pressure in the low-pressure, low-oxygen environment is 9-12 kPa, the oxygen concentration is 11-20%, and the temperature is 20-25℃. The experimental animal was a mouse; The animal model of high-altitude qi deficiency and blood stasis was tested for its overall behavioral status, coagulation function, chrnα1 levels in brain, heart and lung tissues, and tongue appearance. The whole-body state behavioral analysis was conducted using grip force detection and open field test. In the grip force detection, the grip force showed a decreasing trend. In the open field test, the distance traveled in the central area, the number of entries, and the total distance traveled all showed a decreasing trend. The coagulation function showed a significant increase in FIB; the level of chrnα1 in the brain, heart, and lung tissues showed an upward trend; and the tongue appearance showed a dull tongue color and a downward trend in the RGB value of the tongue base.

2. The construction method as described in claim 1, characterized in that, The low-pressure, low-oxygen environment simulates a plateau environment at an altitude of 5500-6500m.

3. The construction method as described in claim 1, characterized in that, The administration is by subcutaneous injection.

4. The application of the animal model of high-altitude qi deficiency and blood stasis syndrome obtained by the construction method described in any one of claims 1-3 in the study of the pathogenesis of high-altitude qi deficiency and blood stasis syndrome.

5. The application of the animal model of high-altitude qi deficiency and blood stasis syndrome obtained by the construction method according to any one of claims 1-3 in screening drugs for the treatment of high-altitude qi deficiency and blood stasis syndrome.

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