Method for constructing yang deficiency and blood stasis animal model and application thereof
The method of administering iced coffee solution via gavage simplifies the construction of an animal model of Yang deficiency and blood stasis, achieving convenient and efficient model construction that is suitable for research in traditional Chinese medicine.
Patent Information
- Application Number
- CN202511695631.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-18
- Publication Date
- 2025-12-23
AI Technical Summary
Existing technologies are complex to construct animal models of Yang deficiency and blood stasis, requiring multi-factor modeling, which is difficult to operate and subject to large interference from multiple variables, affecting the stability and success rate of the model.
Iced coffee solution was administered to model animals via gavage at a concentration of 50-100 mg/kg, a temperature of 0-4℃, a frequency of 1-2 times/day, and a cycle of 13-15 days. This single non-invasive intervention induced symptoms of spleen and kidney yang deficiency and blood stasis.
The model building process has been simplified, the operation difficulty has been reduced, the modeling efficiency and success rate have been improved, the model is closer to clinical pathology, the cost is low and it is easy to standardize, making it suitable for traditional Chinese medicine research.
Smart Images
Figure CN121176418A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal model construction technology, and in particular to a method for constructing an animal model of yang deficiency and blood stasis and its application. Background Technology
[0002] When Yang Qi is deficient, the pulse becomes constricted, blood circulation is impaired, and cold congeals and stagnates the blood, resulting in Yang deficiency and blood stasis. Yang deficiency is the root cause, while blood stasis is the manifestation. Yang deficiency can be divided into kidney Yang deficiency, spleen-kidney Yang deficiency, and heart Yang deficiency, with kidney Yang deficiency being the most common. Animal models of kidney Yang deficiency often use drug-induced models, such as intramuscular injection, gavage, subcutaneous injection, or intraperitoneal injection of hydrocortisone. Hydrocortisone is a glucocorticoid, and large doses can lead to adrenal insufficiency in experimental animals. The modeling period for hydrocortisone is generally 14 days. In addition to drug-induced models, most literature uses multifactorial models. For example, hydrocortisone combined with adrenaline can construct Yang deficiency and blood stasis; placing experimental animals in a cold environment combined with drug-induced models can also be used. Animal models of spleen-kidney Yang deficiency combine the methods used for spleen Yang deficiency and kidney Yang deficiency. Bitter and cold purgatives induce spleen Yang deficiency symptoms in animals, while injections of hydrocortisone or adenine induce kidney Yang deficiency. Combining these two methods creates a spleen-kidney Yang deficiency model. Currently, a problem with TCM syndrome differentiation is the need for single-factor or multi-factor modeling, which involves various methods and relatively complex processes. Therefore, there is an urgent need in this field for an animal model construction method with a relatively simple modeling process. Summary of the Invention
[0003] The purpose of this invention is to provide a method for constructing an animal model of Yang deficiency and blood stasis and its application. This method only requires giving the model animal iced coffee liquid to obtain an animal model of spleen and kidney Yang deficiency with simultaneous blood stasis.
[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing an animal model of yang deficiency and blood stasis by administering iced coffee to the model animals via gavage.
[0005] Preferably, the model animal is a rat.
[0006] Preferably, the concentration of the iced coffee is 50~100mg / kg, and the temperature of the iced coffee is 0~4℃.
[0007] Preferably, the frequency of gavage is 1-2 times / day, and the cycle of gavage is 13-15 days.
[0008] Preferably, after administering gavage to the model animals, model validation is required; the model validation method is index detection.
[0009] Preferably, the indicators include one or more of the following: body weight, fur condition, arched back, stool characteristics, rectal temperature, hematological indicators, organ index, tongue appearance, and mental state.
[0010] Preferably, the organ index is calculated as follows: after euthanizing the model animal, the spleen and kidney are removed, fat and fascia are removed, and the organs are weighed and the organ index is calculated.
[0011] Preferably, the formula for calculating the organ index is: Organ index = (organ mass / body weight) × 100% Equation 1.
[0012] This invention provides the application of the method in constructing an animal model of yang deficiency and blood stasis.
[0013] From a Traditional Chinese Medicine perspective, coffee is considered pungent and depletes Qi (vital energy). While coffee provides a short-term stimulant effect, it depletes Yang Qi in the long run, aligning with kidney Yang deficiency. Iced coffee can lead to cold-induced damage to Yang Qi; the low temperature inhibits blood circulation and also harms the spleen and stomach, aligning with spleen Yang deficiency.
[0014] The beneficial effects of this invention are as follows: The technical solution of this invention significantly simplifies the construction process of animal models of Yang deficiency and blood stasis, with its core advantages lying in the dual improvement of operational convenience and modeling efficiency. It eliminates the need for complex interventions such as surgery and compound drug injections; the model can be induced simply by administering a high-concentration iced coffee solution—a single, non-invasive method—significantly reducing operational difficulty and the technical requirements for experimental personnel. Simultaneously, the single intervention factor reduces the interference of multiple variables on the modeling results, enabling more stable and rapid induction of simultaneous symptoms of spleen and kidney Yang deficiency and blood stasis in the model animals. This shortens the modeling cycle while also improving the success rate and reproducibility of model construction, providing an efficient and stable experimental foundation for subsequent research on related pathological mechanisms and drug screening.
[0015] The animal model constructed in this invention has stronger clinical-pathological relevance and broader application value. From a pathological mechanism perspective, the spleen and kidney yang deficiency with blood stasis induced by iced coffee is closer to the pathological development process of yang deficiency and subsequent blood stasis caused by long-term consumption of cold drinks and other unhealthy dietary habits. The model has higher clinical simulation accuracy, and the experimental data obtained have greater reference value for clinical research. In addition, this method requires no special instruments or expensive reagents, has low modeling costs, and is easy to standardize and promote. It can be widely used in the study of the mechanism of diseases related to yang deficiency and blood stasis syndrome in traditional Chinese medicine, the efficacy evaluation of candidate drugs, and safety testing, providing economical, practical, and clinically relevant animal model support for traditional Chinese medicine research. Attached Figure Description
[0016] Figure 1 The body weight of rats in each group before and after modeling was compared with that of the Control group. P<0.05, P <0.01; Figure 2 The rat rectal temperature was compared with that of the Control group. P <0.01; Figure 3 The images show the tongue surfaces of rats in each group; Figure 4 To compare with the RGB values of the rat tongue image, the Control group, P <0.05, P <0.01; Figure 5 Changes in organ indices after rat modeling. Detailed Implementation
[0017] This invention provides a method for constructing an animal model of yang deficiency and blood stasis by administering iced coffee to the model animals via gavage.
[0018] In this invention, the model animal is preferably a rat.
[0019] In this invention, the concentration of the iced coffee is preferably 50-100 mg / kg, more preferably 60-90 mg / kg, even more preferably 70-80 mg / kg, and most preferably 75 mg / kg. The temperature of the iced coffee is preferably 0-4°C, and more preferably 2°C.
[0020] In this invention, the frequency of gavage is preferably 1 to 2 times / day, more preferably 1 time / day, and the cycle of gavage is preferably 13 to 15 days, more preferably 14 days.
[0021] In this invention, after the model animal is given a gavage, it is necessary to perform model validation; the preferred method for model validation is index detection.
[0022] In this invention, the indicators preferably include one or more of the following: body weight, fur condition, arched back, stool characteristics, rectal temperature, hematological indicators, organ index, tongue appearance, and mental state.
[0023] In this invention, the preferred method for calculating the organ index is as follows: after euthanizing the model animal, the spleen and kidney are removed, fat and fascia are removed, and the organs are weighed and the organ index is calculated.
[0024] In this invention, the formula for calculating the organ index is preferably: Organ index = (organ mass / body weight) × 100% Equation 1.
[0025] This invention provides the application of the method in constructing an animal model of yang deficiency and blood stasis.
[0026] 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.
[0027] Example 1 Material Preparation
[0028] 1. Animals: Twenty-seven male SPF-grade SD rats, weighing (200g ± 20%), were selected.
[0029] 2. Medicines and reagents: Iced coffee liquid, hydrocortisone tablets, isoflurane.
[0030] 3. Instruments and equipment: Bioinformatics acquisition system (MadLab Bioinformatics Medical Signal Acquisition and Processing System, Model: MadLab-4C / 5H), blood rheology analyzer (fully automatic blood rheology detector, Product Model: HL-5000), digital camera, RGB color chart (Japan CASMATCH Image Correction Tone Size Diagnostic Color Chart 10×10mm 15 pieces), small animal anesthesia machine, temperature sensor (for measuring rat rectal temperature, Model: MadLab-4C / 5H).
[0031] Example 2 Method
[0032] 1. Animal grouping and model preparation
[0033] Rats were divided into three groups: iced coffee group (HCIC group); hydrocortisone group (HC group); and blank group (Control group). There were 9 rats in each group, for a total of 3 groups.
[0034] Rats in the iced coffee group were given iced coffee solution at 2°C at a dose of 90 mg / kg per rat at 10:00 AM every day. Rats in the hydrocortisone group were given 25 mg / kg of hydrocortisone solution dissolved in 5 ml of purified water, and each rat was given 1.25 ml by gavage. The control group was given purified water by gavage. All groups were administered the solution by gavage for 14 days.
[0035] 2. Obtain materials
[0036] After modeling and behavioral testing, rats were anesthetized with an air anesthesia machine, and tongue images were captured using a digital camera. RGB color charts were placed at the same level as the rat's tongue, and the R, G, and B values at three fixed points (tip, middle, and tail) were analyzed using Adobe Photoshop 2022 software. Subsequently, the rat's abdomen was dissected, and heparinized blood was collected via the abdominal aorta for subsequent hematological testing. The spleen and kidneys of the rats were then harvested and weighed.
[0037] 3. Detection indicators
[0038] 3.1 Macroscopic observation of the animal's general condition and symptoms
[0039] Observe the color of hair, claws, and nails, as well as the mental state of rats in each group under resting conditions. Weigh the rats daily, and the results are shown in the table below. Figure 1 The rectal temperature of rats was measured at 0d, 3d, 6d, 9d, 12d, and 14d. The results are shown in the table below. Figure 2 The TCM syndrome score evaluation criteria for rats with Yang deficiency and blood stasis were established based on factors such as fur condition and nail color (see Table 1); the TCM syndrome score results for each treatment group are shown in Table 3.
[0040] 3.2 Tongue Image Collection
[0041] After anesthetizing rats, tongue images were captured using a digital camera. An RGB colorimetric chart was placed at the same horizontal level as the rat's tongue. After photographing, the R, G, and B values at three fixed points—the tip, middle, and tail of the tongue—were analyzed using Adobe Photoshop 2022 software. The results are shown below. Figure 3 and Figure 4 .
[0042] 3.3 Hematological index testing
[0043] The hemorheological properties of rats were examined using a hemorheology analyzer, and the results are shown in Table 2.
[0044] 3.4. Evaluation of spleen and kidney function in rats using organ indexes
[0045] After sacrificing the rats, the spleen and kidneys were harvested, and fat and fascia were removed. The rats were weighed, and organ indices were calculated. The results are shown below. Figure 5 .
[0046] Formula: Organ Index = (Organ Mass / Body Weight) × 100% (Formula 1)
[0047] Example 3: Model Scoring
[0048] 1. Model scoring criteria
[0049] A TCM syndrome scoring standard was established. This scoring scale, ranging from 0 to 3 points, assesses seven aspects: body weight, fur condition, arched back, stool characteristics, rectal temperature, tongue appearance, and mental state. The scoring scale was adjusted appropriately based on individual differences among rats. The severity of the syndrome was positively correlated with the score. The TCM syndrome scoring standard for rats with Yang deficiency and blood stasis is shown in Table 1.
[0050] Table 1 Evaluation criteria for TCM syndrome scores in rats with Yang deficiency and blood stasis
[0051] 2. Rat body weight
[0052] Figure 1 The results showed that body weight increased in all five groups over time. During the modeling period, the HCIC and HC groups showed slower weight gain compared to the Control group. After the modeling period, the body weight of the HCIC and HC groups was significantly lower than that of the Control group. p <0.05). The modeling results were as expected and consistent with the scoring criteria for Yang deficiency and blood stasis syndrome.
[0053] 3. Changes in rat rectal temperature
[0054] Figure 2 The results showed that after modeling, compared with the control group, the rectal temperature in the other two groups decreased with the increase of the modeling days, and the rectal temperature values were all lower than those in the control group, which was statistically significant. P <0.01); This aligns with the "aversion to cold and cold limbs" syndrome of Yang deficiency, suggesting a decrease in the rat's basal metabolic rate.
[0055] 4. Changes in the tongue appearance of rats
[0056] Figure 3 and Figure 4 The results showed that, compared with the Control group, the R, G, and B values of the tongue surface were significantly reduced in both the HC and HCIC groups. The R, G, and B values of the tongue surface in the HCIC group were slightly darker than those in the Control group. P <0.05; the tongue surface color was darker in the HC group than in the Control group ( P <0.01). The dark color of the rat's tongue indicates insufficient Yang energy and blood stasis, resulting in a purplish-dark appearance.
[0057] 5. Hematological indicators
[0058] The results in Table 2 show that, compared with the Control group, the low-shear, medium-shear, and high-shear whole blood viscosity of the HC and HCIC groups were significantly increased, which was statistically significant. P <0.01, P <0.05).
[0059] Table 2. Results of hemorheological analysis in rats of each group (Mean±SD, n=9)
[0060] Note: Compared to the Control group, P <0.05, P <0.01; compared with the HC group, △ P <0.05, △△ P <0.01.
[0061] 6. Organ Index
[0062] Figure 5 The results showed that there was no statistically significant difference between the kidney index and spleen index among the groups. P >0.05), indicating that the model did not cause immune system disorders or nephrotoxicity in rats.
[0063] 7. Traditional Chinese Medicine Syndrome Scoring Table
[0064] The results in Table 3 show that, compared with the Control group, the syndrome scores of the other two groups were significantly higher, indicating that the modeling results are consistent with the characteristics of Yang deficiency and blood stasis syndrome.
[0065] Table 3. TCM syndrome score table for rats with Yang deficiency and blood stasis (Mean±SD, n=9)
[0066] Compared to the Control group, P <0.05, P <0.01.
[0067] As shown in the above embodiments, this invention provides a method for constructing an animal model of Yang deficiency and blood stasis, and its application. The method involves administering iced coffee to the model animals via gavage. The technical solution of this invention significantly simplifies the construction process of the Yang deficiency and blood stasis animal model, with its core advantages lying in the dual improvement of operational convenience and modeling efficiency. It eliminates the need for complex interventions such as surgery or compound drug injections; the model can be induced simply by administering a high-concentration iced coffee solution—a single, non-invasive method—significantly reducing operational difficulty and the technical requirements for experimental personnel. Simultaneously, the single intervention factor reduces the interference of multiple variables on the modeling results, enabling more stable and rapid induction of symptoms of spleen and kidney Yang deficiency and blood stasis in the model animals. This shortens the modeling cycle and improves the success rate and reproducibility of model construction, providing an efficient and stable experimental foundation for subsequent research on related pathological mechanisms and drug screening.
[0068] 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 Yang deficiency and blood stasis, characterized in that, Iced coffee was administered to the model animals via gavage.
2. The method according to claim 1, characterized in that, The model animal was a rat.
3. The method according to claim 2, characterized in that, The concentration of the iced coffee is 50~100mg / kg, and the temperature of the iced coffee is 0~4℃.
4. The method according to claim 3, characterized in that, The frequency of gavage is 1-2 times / day, and the cycle of gavage is 13-15 days.
5. The method according to claim 1, characterized in that, After administering gavage to the model animals, model validation is required; the method for model validation is index detection.
6. The method according to claim 5, characterized in that, The indicators include one or more of the following: body weight, coat condition, arched back, stool characteristics, rectal temperature, hematological indicators, organ indices, tongue appearance, and mental state.
7. The method according to claim 6, characterized in that, The organ index is calculated as follows: after euthanizing the model animal, the spleen and kidney are removed, fat and fascia are removed, and the organs are weighed and the organ index is calculated.
8. The method according to claim 7, characterized in that, The formula for calculating the organ index is as follows: Organ index = (organ mass / body weight) × 100% Equation 1.
9. The application of the method according to any one of claims 1 to 8 in constructing an animal model of yang deficiency and blood stasis.