Method for constructing a water-deficient animal model under a moving state

Abstract

The present invention provides a method for constructing an animal model of water deficiency under exercise state, and the method includes: (1) maintaining the experimental animal in a state of water deficiency; (2) making the experimental animal exercise, and continuously maintaining the state of water deficiency during the exercise process to obtain the animal model. The animal model of water deficiency under exercise state constructed by the method of the present invention is in a state of water deficiency under exercise without pathological damage, and the normal physiological state can be restored by supplementing liquid supplements such as water. It can be used to determine the recovery effect of food or medicine on the body after supplementing liquid supplements under exercise state and screen food or medicine for supplementing liquid supplements under exercise state, and has high application value.

Description

Technical Field

[0001] The present invention relates to the field of biology. Specifically, the present invention relates to a method for constructing an animal model of water shortage under exercise conditions. Background Art

[0002] During exercise, due to the intensification of physical function activities, the human body will continuously sweat, so that the water and electrolytes in the human body will be continuously lost. If water and electrolytes are not replenished in time, the physiological balance in the human body will be lost, thus affecting human health. Sports drinks are formulated according to the characteristics of physiological consumption during exercise, and can specifically supplement the nutrients lost during exercise, playing a role in maintaining and improving exercise ability and accelerating the elimination of fatigue after exercise.

[0003] In order to understand the loss of water, electrolytes, etc. in the animal body under exercise conditions and develop foods or drugs for supplementing supplements under exercise conditions, it is necessary to conduct research and screening on animal models. However, there is currently no research report on an animal model of water shortage under exercise conditions, and its construction method remains to be developed. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0005] It should be noted that the present invention is completed based on the following discoveries of the inventors:

[0006] When constructing an animal model of water shortage under exercise conditions, the inventors of the present application initially made rats be in high-intensity exercise states and long-term exercise states respectively. Since the metabolic characteristics of rats are significantly different from those of humans, for example, the sweat glands of rats are extremely underdeveloped, with only a small number of sweat glands on the soles of the feet. During high-intensity exercise, the tail is its main heat dissipation organ and cannot secrete a large amount of sweat, resulting in rats being unable to be in a good water shortage state even until exhaustion during high-intensity exercise. Similarly, rats are also unable to be in a good water shortage state until exhaustion during long-term exercise. Furthermore, through in-depth research, the inventors found that by pre-maintaining the rats in a water shortage state for a period of time and then exercising, an acute water shortage model under exercise conditions can be obtained. Further, the inventors optimized and screened the time for maintaining the water shortage state and the exercise conditions in the early stage to obtain a method for constructing an animal model, so as to take into account water shortage under exercise conditions and no pathological damage to the body function, and the physiological state can return to normal after water replenishment, which is helpful for studying the loss of water, electrolytes, etc. in the animal body under exercise conditions, the recovery effect after supplementing supplies, and developing foods or drugs for supplementing liquid supplies under exercise conditions, with high application value.

[0007] To this end, in one aspect of the present invention, the present invention proposes a method for constructing an animal model of water shortage under a moving state. According to an embodiment of the present invention, the method includes: (1) maintaining the experimental animal in a water shortage state; (2) making the experimental animal perform exercise, and continuing to maintain the water shortage state during the exercise process. After the exercise ends, an animal model is obtained.

[0008] In the method according to the embodiment of the present invention, the rats are pre-maintained in a water shortage state for a period of time and then exercised, so as to obtain an acute water shortage model under a moving state, which helps to study the loss of body water, electrolytes, etc. in animals under a moving state, the recovery effect after supplementing the supply, and the development of foods or drugs for supplementing liquid supplements under a moving state, and has high application value.

[0009] According to an embodiment of the present invention, in step (1), the time for maintaining the water shortage state is 50-68 h. The inventors obtained the above preferred time through a large number of experiments. Thus, the body water shortage state can be achieved, and the normal physiological state can be restored after water replenishment, and pathological damage is not likely to occur. Among them, when the time is 60 h, the effect is better.

[0010] According to an embodiment of the present invention, in step (2), the initial exercise speed of the exercise is 8-12 m / min, and it is accelerated to 18-22 m / min within 8-12 min, and the total exercise time is 28-32 min.

[0011] The inventors found through a large number of experiments that too low exercise intensity cannot achieve the purpose of water shortage, and too high exercise intensity will cause the rats to be overly exhausted and affect the physiological state. When the above preferred exercise conditions are reached, the body can be further in a water shortage state, and the normal physiological state can be restored after water replenishment, and pathological damage is not likely to occur. Among them, when the initial exercise speed of the exercise is 10 m / min, it is accelerated to 20 m / min within 10 min, and the total exercise time is 30 min, the effect is better.

[0012] According to an embodiment of the present invention, the experimental animal is a rat. Specifically, the weight of the rat is 240-280 g.

[0013] In another aspect of the present invention, the present invention provides a method for constructing an animal model of water-deficient animals in a moving state. According to an embodiment of the present invention, the method includes: maintaining the experimental animals in a water-deficient state for 60 h, then performing exercise, with an initial exercise speed of 10 m / min, accelerating to 20 m / min within 10 min, and exercising for a total of 30 min to obtain an animal model. Thus, the animal model of water-deficient animals in a moving state obtained by using the method according to the embodiment of the present invention takes into account both water deficiency in the moving state and no pathological damage to the body functions. After replenishing water, the normal physiological state can be restored, which is helpful for studying the loss of body water, electrolytes, etc. in animals in a moving state, the recovery effect after replenishing liquid supplies, and developing foods or drugs for replenishing liquid supplies in a moving state, and has high application value.

[0014] In still another aspect of the present invention, the present invention provides a method for determining the recovery effect of the body after replenishing liquid supplies in a moving state by a food or drug. According to an embodiment of the present invention, the method includes: constructing an animal model by using the method for constructing an animal model of water-deficient animals in a moving state described above; applying the food or drug to be tested to the animal model; detecting relevant indicators of the animal model and analyzing the detection results.

[0015] As described above, the animal model constructed by using the method according to the embodiment of the present invention is water-deficient in a moving state but shows no pathological damage. By replenishing liquid supplies such as water and electrolytes, the recovery effect of the body can be studied, which has important scientific research value.

[0016] According to an embodiment of the present invention, after applying the food or drug to be tested to the animal model, the animal model is allowed to recover in a natural state for 50 - 70 min, preferably 60 min, and then the relevant indicators of the animal model are detected. The inventors found that when the body is water-deficient in a moving state, the body basically returns to a stable physiological state 50 - 70 min after replenishing the food or drug to be tested, and the accuracy and stability of the indicators detected at this time are higher.

[0017] In the present invention, the term "natural state" can also be referred to as "free state", that is, the state in which the experimental animals are located without external intervention.

[0018] According to an embodiment of the present invention, the relevant indicators include at least one of the following: chloride ion content, urea nitrogen content, creatinine content, appearance state, and behavior state in the serum. Thus, the recovery effect of the above relevant indicators can be accurately obtained.

[0019] According to an embodiment of the present invention, the food is selected from sports drinks.

[0020] According to an embodiment of the present invention, the liquid supplies include at least one of the following: water, minerals, and electrolytes.

[0021] In another aspect of the present invention, the present invention provides a method for screening foods or drugs for supplementing liquid replenishment during exercise. According to an embodiment of the present invention, the method includes: constructing an animal model by using the method for constructing an animal model lacking water during exercise described above; applying the food or drug to be tested to the animal model; detecting relevant indicators of the animal model; and determining whether the food or drug to be tested is a target food or drug based on the detection results.

[0022] As described above, the animal model constructed by using the method according to the embodiment of the present invention is lacking water during exercise but shows no pathological damage. By supplementing different components or different amounts of liquid replenishment, it can return to different states at different times. When a better liquid replenishment is selected, it can return to the normal physiological state in a short time. Therefore, the method according to the embodiment of the present invention can accurately screen out foods or drugs for supplementing liquid replenishment during exercise.

[0023] According to an embodiment of the present invention, the relevant indicators include at least one of the following: chloride ion content, urea nitrogen content, creatinine content, appearance state, and behavior state in the serum; the food to be tested is selected from sports drinks; and the liquid replenishment includes at least one of the following: water, minerals, and electrolytes.

[0024] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0026] Figure 1 Shows the chloride ion content in the serum of rats at different water deprivation times;

[0027] Figure 2 Shows the urea nitrogen content in the serum of rats at different water deprivation times;

[0028] Figure 3 Shows the creatinine content in the serum of rats at different water deprivation times;

[0029] Figure 4 Shows the white blood cell content in the serum of rats at different water deprivation times;

[0030] Figure 5 Shows the chloride ion content in the serum of rats at different recovery times;

[0031] Figure 6 Shows the urea nitrogen content in the serum of rats at different recovery times;

[0032] Figure 7 Shows the graph of the serum creatinine content in rats at different recovery times;

[0033] Figure 8 Shows the graph of the appearance status score of rats at different recovery times;

[0034] Figure 9 Shows the graph of the behavioral status score of rats at different recovery times;

[0035] Figure 10 Shows the graph of the exhaustion time of rats;

[0036] Figure 11 Shows the graph of the body weight change trend of rats;

[0037] Figure 12 Shows the graph of the change in the serum white blood cell content after 1 hour of water supplementation;

[0038] Figure 13 Shows the graph of the change in the serum creatinine content after 1 hour of water supplementation. Detailed implementation method

[0039] The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0040] Example 1 Determination of water shortage conditions

[0041] The water shortage time is crucial for constructing a water shortage rat model. Too low a water shortage time will result in insufficient water shortage, and too high a water shortage time will cause pathological damage to the rats. Therefore, the water shortage time was determined through the following experiment:

[0042] In this experiment, male SD rats with a body weight of 240 - 280 g were used. 49 rats were randomly divided into 7 groups: 0 h group, 24 h group, 36 h group, 48 h group, 60 h group, and 72 h group. The feeding method of the 0 h group was free drinking water and diet, and the other groups were free diet with water cut off for 24 - 72 h. After the water cut-off ended, they exercised according to the exercise conditions (initial exercise speed 10 m / min, increased to 20 m / min within 10 minutes, and exercised for a total of 30 min).

[0043] After the experiment ended, the rats were sacrificed, and whole blood of the rats was taken. After separating the serum, the chloride ion content in the serum was detected, and the results were as Figure 1 ; the urea nitrogen (BUN) content in the serum was detected, and the results were as Figure 2; Detect the creatinine (CRE) content in the serum, and the results are as Figure 3 ; Detect the white blood cell content in the whole blood, and the results are as Figure 4 .

[0044] As Figure 1 shown, the role of chloride ions is to maintain the body's electrolyte, acid-base balance and osmotic pressure balance. Water shortage will cause an increase in chloride ions. Under normal circumstances, the longer the water shortage time, the higher the chloride ion content. At 0 h, that is, without water shortage, compared with the control group at 60 h, the chloride ion content increased significantly. At 72 h, there was a decrease, which may be due to too long water shortage time, resulting in pathological damage. Therefore, when the water shortage time is 60 h, the water shortage effect is the best.

[0045] As Figure 2 shown, blood urea nitrogen (BUN) increases when renal insufficiency decompensates, which is an index reflecting glomerular filtration function. Both exercise and water shortage can increase BUN. When the lack of necessary drinking water continues for a certain period of time, it can reduce renal blood circulation, resulting in a sharp decrease in urine output and a disorder in the function of the body's toxic metabolites excreted through the kidneys. Compared with the control group at 60 h, the blood urea nitrogen content increased significantly. At 72 h, there was a decrease, which may be due to too long water shortage time, resulting in pathological damage. Therefore, when the water shortage time is 60 h, the water shortage effect is the best.

[0046] As Figure 3 shown, creatinine (CRE) reflects the damage of renal function. Exercise muscle metabolism produces endogenous creatinine. Exercise + water shortage has an adverse effect on the kidneys and will lead to an increase in creatinine levels. Compared with the control group at 60 h, the creatinine content increased significantly. At 72 h, there was an abnormal decrease, which may be due to too long water shortage time, resulting in pathological damage. Therefore, when the water shortage time is 60 h, the water shortage effect is the best.

[0047] As Figure 4 shown, white blood cells (WBC) reflect the ability of immune response. A decrease in the number of blood white blood cells induced by stress represents an adaptive response, which may increase the immune surveillance / response of white blood cells in the transported organs during stress. Therefore, the stress caused by water shortage will lead to a decrease in white blood cells. Compared with the control group at 60 h, the number of white blood cells decreased significantly. At 72 h, there was an abnormal increase, which may be due to too long water shortage time, resulting in pathological damage. Therefore, when the water shortage time is 60 h, the water shortage effect is the best.

[0048] Example 2 Determine the detection time

[0049] Experiment on the best detection time for water replenishment after water shortage. After the water shortage model is constructed, it can be used to explore the water replenishment ability of different substances on rats. In this experiment, by detecting different recovery times after gavage of rats, the best detection time is explored. The specific experiment is as follows:

[0050] 1. Male Sprague-Dawley rats weighing 240 - 280 g were used. 19 rats were randomly divided into 4 groups: a control group, a 20-min group, a 40-min group, and a 60-min group. The control group was fed and watered ad libitum, while the other groups were fed ad libitum and deprived of water for 60 h. After the water deprivation ended, they exercised according to the exercise conditions (same as in Example 1).

[0051] After exercise, except for the control group, 5 mL of pure water was intragastrically administered to the rats in each group. They were sacrificed after 20, 40, and 60 min of recovery respectively. Whole blood of the rats was taken, and after separating the serum, the chloride ion content in the serum was detected. The results are as Figure 5 shown; the urea nitrogen content in the serum was detected. The results are as Figure 6 shown; the creatinine content in the serum was detected. The results are as Figure 7 shown.

[0052] As Figure 5 shown, with the increase of the recovery time, the chloride ion content in the serum of the rats gradually decreased and tended to be stable.

[0053] As Figure 6 shown, with the increase of the recovery time, the urea nitrogen content in the serum of the rats gradually decreased and tended to be stable.

[0054] As Figure 7 shown, with the increase of the recovery time, the creatinine content in the serum of the rats gradually decreased and tended to be stable.

[0055] 2. Male Sprague-Dawley rats weighing 240 - 280 g were used. 21 rats were randomly divided into 4 groups: a negative control (NC) group, a mock (M) group, a pure water (PW) group, and an electrolyte water (EW) group. The NC group was fed and watered ad libitum, and the M group was fed ad libitum and deprived of water for 60 h. After the water deprivation ended, they exercised according to the exercise conditions (same as in Example 1). The PW and EW groups were fed ad libitum and deprived of water for 60 h. After the water deprivation ended, they exercised according to the exercise conditions. Immediately after exercise, 10 mL of pure water or electrolyte water was intragastrically administered. After the intragastric administration, recovery was carried out. The appearance state and behavioral state of the rats were scored according to the following scoring criteria. The results are as Figure 8 and Figure 9 shown. At 60 min of recovery time, there were significant differences, and the electrolyte water was more significant for the recovery of the skin and behavioral state. Therefore, 60 min after intragastric administration was selected as the time for index determination.

[0056] Table 1 Appearance Scoring Criteria

[0057] Appearance status Score Normal skin tension and status 1 Back skin shows relaxation 2 Body curls up, hair stands on end, moderate skin tension relaxation 3 Eyes are sunken, hair stands on end, severe skin relaxation 4 Unable to control oneself 5

[0058] Table 2 Behavior Scoring Criteria

[0059] Behavior status Score Behavior is active before and during treatment 1 Activity level decreases, responsive to treatment 2 Lethargic, unresponsive to treatment 3 No response, only moves when touched 4 Does not move when touched 5

[0060] Example 3 Exhaustion Experiment

[0061] In this experiment, male SD rats weighing 240 - 280 g were used. Twenty - one rats were randomly divided into 4 groups: negative control (NC) group, mock (M) group, pure water (PW) group, and electrolyte water (EW) group. The feeding method of the NC group was free access to water and food. The feeding method of the M group was free access to food and water was withheld for 60 h. After the water - withholding ended, they exercised according to the exercise conditions. The feeding methods of the PW and EW groups were free access to food and water was withheld for 60 h. After the water - withholding ended, they exercised according to the exercise conditions (the same as in Example 1). Immediately after exercise, 10 mL of pure water or electrolyte water was administered by gavage. After the gavage ended, they were allowed to recover for 60 min and then the following exhaustion conditions were implemented:

[0062] Start running on a treadmill at a speed of 25 m / min with a slope of 10°. Reach a slope of 15° within 20 minutes.

[0063] When the rats stayed on the circuit board for more than 10 seconds; lost the righting reflex, when the rats were lying on their backs and could not adjust themselves, they were evaluated as exhausted.

[0064] The results are as Figure 10 shown. After supplementing with electrolyte water, the time for the rats to reach exhaustion was prolonged, indicating that the effect of supplementing electrolyte water was better.

[0065] Example 4 Determine Whether the Water - Deprived Animal Model Has the Ability to Restore the Indicators of Normal Rats

[0066] 1. In this experiment, male SD rats weighing 240 - 280 g were used. The feeding method of 8 rats was free access to food. After water was withheld for 60 h, after the water - withholding ended, they exercised according to the exercise conditions (the same as in Example 1). After the exercise was completed, water supply was restored. After the experiment ended, the body weights of the rats were measured. The results are as Figure 11 shown.

[0067] As Figure 11 shown, the body weight data was normalized with 0 h as the reference. During the water - withholding period, as the water - withholding time increased, the body weight of the rats gradually decreased and reached the minimum body weight at 60 h. After restoring water intake, as time increased, the body weight of the rats gradually recovered and exceeded the original body weight at 96 h, proving that it has the ability to restore to the normal physiological state.

[0068] 2. In this experiment, male SD rats weighing 240 - 280 g were used. 19 rats were randomly divided into 3 groups: a control group, a 60 - h group, and a 60 - h pure - water group. The feeding method of the control group was free access to water and food, while the feeding method of the other groups was free access to food, with water deprivation for 60 h. After the water deprivation ended, they exercised according to the exercise conditions (the same as in Example 1). After the exercise ended, the 60 - h pure - water group was gavaged with 5 mL of pure water and then recovered for 1 h.

[0069] After the experiment ended, the rats were sacrificed, and whole blood was taken from the rats to measure the white blood cell content. The results are as Figure 12 ; After separating the serum, the creatinine content was measured. The results are as Figure 13 .

[0070] As Figure 12 shown, after exercise, the white blood cell content decreased significantly. After rehydration, the white blood cell content showed an upward trend and was close to that of the control group, proving its ability to recover to the normal physiological state.

[0071] As Figure 13 shown, after exercise, the creatinine content decreased significantly. After rehydration, the creatinine content showed a downward trend and was close to that of the control group, proving its ability to recover to the normal physiological state.

[0072] Although the embodiments of the present invention have been shown and described above, it can be understood that the above - mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above - mentioned embodiments within the scope of the present invention.

Claims

1. A method for constructing an animal model of water deficiency in a moving state, characterized in that, Comprising: (1) Maintaining the experimental animals in a dehydrated state; (2) Making the experimental animals perform exercise, during which the dehydrated state is continuously maintained, and after the exercise ends, an animal model is obtained; In step (1), the time for maintaining the dehydrated state is 50 - 68 h; In step (2), the initial exercise speed of the exercise is 8 - 12 m / min, and it accelerates to 18 - 22 m / min within 8 - 12 min, and the total exercise time is 28 - 32 min; The experimental animals are mice.

2. The method according to claim 1, characterized in that, In step (1), the time for maintaining the dehydrated state is 60 h.

3. The method according to claim 1, characterized in that, In step (2), the initial exercise speed of the exercise is 10 m / min, and it accelerates to 20 m / min within 10 min, and the total exercise time is 30 min.

4. A method for constructing a water-deficient animal model in a moving state, characterized in that, Comprising: Maintaining the experimental animals in a dehydrated state for 60 h, then performing exercise with an initial exercise speed of 10 m / min, accelerating to 20 m / min within 10 min, and exercising for a total of 30 min to obtain an animal model; The experimental animals are mice.

5. A method for determining the recovery effect of a food or drug on the body after replenishing a liquid supplement during exercise, characterized in that, Comprising: Constructing an animal model by using the method for constructing a dehydrated animal model in a moving state according to any one of claims 1 - 4; Applying the food or drug to be tested to the animal model; Detecting the relevant indicators of the animal model and analyzing the detection results.

6. The method for determining the recovery effect of a food or drug on the body after replenishing a liquid replenisher in a moving state according to claim 5, characterized in that After applying the food or drug to be tested to the animal model, allowing the animal model to recover in a natural state for 50 - 70 min, and then detecting the relevant indicators of the animal model.

7. The method for determining the recovery effect of a food or drug on the body after replenishing a liquid supplement during exercise according to claim 5, characterized in that, After applying the food or drug to be tested to the animal model, allowing the animal model to recover in a natural state for 60 min, and then detecting the relevant indicators of the animal model.

8. The method for determining the recovery effect of the body after replenishing a liquid supplement during exercise for a food or drug according to claim 5, characterized in that, The relevant indicators include at least one of the following: chloride ion content, urea nitrogen content, creatinine content, appearance state, and behavior state in the serum.

9. The method for determining the recovery effect of a food or drug on the body after replenishing a liquid supplement during exercise according to claim 5, characterized in that, The food is selected from sports drinks; The liquid replenisher includes at least one of the following: water and electrolytes.

10. A method for screening foods or drugs for supplementing liquid supplements during exercise, characterized in that, Comprising: Constructing an animal model by using the method for constructing a dehydrated animal model in a moving state according to any one of claims 1 - 4; Applying the food or drug to be tested to the animal model; Detecting the relevant indicators of the animal model; Based on the detection results, determining whether the food or drug to be tested is the target food or drug.

11. The method for screening a food or drug for supplementing a liquid replenisher during exercise according to claim 10, characterized in that, The relevant indicators include at least one of the following: chloride ion content, urea nitrogen content, creatinine content, appearance state, and behavior state in the serum; The food to be tested is selected from sports drinks; The liquid replenisher includes at least one of the following: water and electrolytes.

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