Hyperuricemia animal model constructed by utilizing epinephrine and construction method of hyperuricemia animal model

Abstract

The invention belongs to the technical field of construction of animal models, and particularly relates to a hyperuricemia animal model constructed by utilizing epinephrine and a construction method of the hyperuricemia animal model. The method for constructing the animal model by injecting low-dose epinephrine comprises the following steps: A, injecting low-dose epinephrine into an experimental animal every day; b, normal diet and free water drinking; and C, the molding time is at least 14 days. The hyperuricemia animal model can be successfully constructed by injecting low-dose epinephrine, and the hyperuricemia animal model can also be successfully constructed in cooperation with a modeling method of high-purine diet, a uricase inhibitor and a urate transporter inhibitor. And considering from the aspects of economy, cost and the like, the hyperuricemia animal model can still be successfully constructed by only injecting low-dose epinephrine without additional auxiliary modeling conditions, so that a brand new choice is provided for the animal model construction method.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing animal models, and in particular relates to a hyperuricemia animal model constructed by utilizing adrenaline and a construction method thereof. Background Art

[0002] Hyperuricemia is an independent risk factor for chronic kidney disease, hypertension, cardiovascular and cerebrovascular diseases, diabetes and other diseases, and is an independent predictor of premature death. It is particularly important to construct an animal model of hyperuricemia that conforms to the pathogenesis of modern hyperuricemia in order to screen active ingredients that lower uric acid.

[0003] The currently recognized method for modeling hyperuricemia is mainly to achieve high uric acid levels in mice by giving them a high-purine diet, uricase inhibitors, or uric acid excretion inhibitors. The currently recognized method for modeling hyperuricemia is mainly to achieve high uric acid levels in mice by giving them a high-purine diet, uricase inhibitors, or uric acid excretion inhibitors (uric acid transporter inhibitors). Commonly used hyperuricemia models are mainly modeled by a high-purine diet, or a high-purine diet combined with a uricase inhibitor and a uric acid transporter inhibitor, see Table 1.

[0004] Table 1 Common hyperuricemia modeling methods

[0005]

[0006] The above methods have problems such as short model maintenance time (uric acid can return to normal levels 2-6 hours after drug withdrawal) and high incidence of liver and kidney damage in mice. In particular, the hyperuricemia model is short-lived, which makes it difficult to use this model for pharmacological evaluation. There are also reports on uricase gene knockout mice, that is, the Uox gene is knocked out using transcription activator-like effector nuclease technology, but this method has a low mouse survival rate and is expensive, and is not suitable for drug screening experiments.

[0007] In summary, the inventors of the present invention intend to provide a new hyperuricemia animal model constructed using adrenaline. Summary of the invention

[0008] The technical problem solved by the present invention is to provide a hyperuricemia animal model constructed by using adrenaline.

[0009] The animals that can be used to construct the animal model of the present invention include mammals and birds.

[0010] Further preferably, the mammals include primates, rodents, artiodactyls, canines, and felines;

[0011] Still further preferably, the primate is selected from monkeys and gorillas; the rodent is selected from mice, rats, rabbits, and guinea pigs; the artiodactyl is selected from donkeys, sheep, cattle, horses, and pigs; the canine is selected from dogs, wolves, jackals, and foxes; the feline is selected from cats;

[0012] Further preferably, the poultry animals are chickens, ducks, and geese;

[0013] Based on economic efficiency, rats or mice are preferably used as experimental animals, and mice are most preferably used as experimental animals.

[0014] The animal model constructed in the present invention adopts a method of injecting a low dose of adrenaline to create a model, comprising the following steps:

[0015] A. Experimental animals were injected with low doses of adrenaline daily;

[0016] B. Eat normally and drink water freely;

[0017] C. The modeling time is at least 14 days.

[0018] In the above technical solution:

[0019] The maximum range of the low dose of adrenaline in step A is 0.05-1.0 μg / g, preferably 0.1-0.5 μg / g, and the optimal value is 0.2 μg / g.

[0020] The animal model constructed in the present invention can also be constructed by injecting a low dose of adrenaline in combination with existing methods for constructing hyperuricemia animal models, such as:

[0021] (1) Modeling by injecting low doses of adrenaline combined with a high-purine diet, including the following steps:

[0022] A. Experimental animals were injected with low doses of adrenaline daily;

[0023] B. The diet is mixed feed with dry yeast powder or adenine, and drinking water is free;

[0024] C. The modeling time is at least 14 days.

[0025] In step B, the dry yeast powder content in the mixed feed is 5-10%.

[0026] Preferably, in step B, the dry yeast powder content in the mixed feed is 10%.

[0027] In step B, the adenine content in the mixed feed is 0.05-0.1%.

[0028] Preferably, in step B, the adenine content in the mixed feed is 0.1%.

[0029] The daily feeding amount is based on the body weight of the experimental animals, adenine 125-250 mg / kg, dry yeast powder 12.5-25 g / kg.

[0030] Preferably, the daily feeding amount is 250 mg / kg adenine and 25 g / kg dry yeast powder based on the body weight of the experimental animals.

[0031] (2) Injection of low doses of epinephrine combined with uricase inhibitors to create a model, including the following steps:

[0032] A. Experimental animals were injected with low doses of adrenaline daily;

[0033] B. Eat normally and drink water freely;

[0034] C. The modeling time is at least 14 days, and potassium oxonate is injected intraperitoneally in the last 1-2 weeks of modeling.

[0035] In step C, potassium oxonate is injected intraperitoneally, once a day, and the injection amount is based on the body weight of the experimental animal, potassium oxonate 150-300 mg / kg.

[0036] Preferably, in step C, potassium oxonate is intraperitoneally injected, and the injection amount is once a day based on the body weight of the experimental animal, potassium oxonate 300 mg / kg.

[0037] (3) Injection of low doses of epinephrine combined with uric acid transporter inhibitors to establish a model, including the following steps:

[0038] A. Experimental animals were injected with low doses of adrenaline daily;

[0039] B. Eat normally and drink water freely;

[0040] C. The modeling period is at least 14 days, and pyrazinamide is administered by gavage in the last 1-2 weeks of modeling.

[0041] In step C, pyrazinamide is administered by oral gavage once a day, and the oral gavage amount is based on the body weight of the experimental animal, 200-400 mg / kg of pyrazinamide.

[0042] Preferably, pyrazinamide is administered by oral gavage once a day, with the oral gavage amount being 400 mg / kg of pyrazinamide based on the body weight of the experimental animal.

[0043] The reason why the present invention constructs an animal model of hyperuricemia is that when the inventors investigated the effects of chronic restraint stress simulating a high-pressure environment on experimental animals, they found that the adrenaline level increased with the extension of the restraint time when measuring the adrenaline level. They then evaluated that after the adrenal glands of the experimental animals were removed, even after the restraint experiment, as well as the existing high-purine diet, uricase inhibitors, or uric acid excretion inhibitor modeling methods, the experimental animals did not develop hyperuricemia. Further, after injecting a low dose of adrenaline into the experimental animals whose adrenal glands had been removed, it was found that the uric acid level increased significantly. Based on this discovery, the inventors completed the technical solution of constructing an animal model of hyperuricemia with adrenaline. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Diagram of the experimental scheme. DETAILED DESCRIPTION

[0045] Below in conjunction with embodiment, the scheme of the present invention will be explained and described.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if no specific technology or condition is indicated, it is carried out according to the technology or condition described in the document in this area or according to the product specification.The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained by commercial purchase.

[0046] The following describes specific embodiments of the present invention but does not limit the present invention.

[0047] 1. Experimental plan: see Figure 1 .

[0048] 2. Experimental process

[0049] (1) Epinephrine group

[0050] After 7 days of adaptive feeding, C57BL / 6J mice were randomly divided into a blank group and an adrenaline group. The adrenaline group was then injected with 0.2 μg / g adrenaline, and the animals were sampled 7, 14, and 21 days later to measure the blood uric acid level.

[0051] Sampling method: Remove the eyeballs of mice to collect blood, let the blood sample stand at room temperature for 2 hours, centrifuge at 3500r / min for 10 minutes, take the supernatant, and freeze it at -20℃ for later use. Then kill the mice by cervical dislocation, and take the liver and kidney tissues. Cut an appropriate amount of liver tissue, add 9 times the weight of the liver to the homogenization medium, use a homogenizer to homogenize at a low temperature environment, then centrifuge at 3500r / min for 10 minutes, take the supernatant, and freeze it at -20℃ for later use. The left kidney was fixed with 4% paraformaldehyde solution, and the right kidney and the remaining liver tissue were stored in a -80℃ refrigerator for later use.

[0052] (2) Epinephrine + high-purine diet group

[0053] The method of adrenaline injection was the same as above. The high-purine diet was as follows: dry yeast powder and adenine were evenly mixed into the feed to make the dry yeast powder and adenine content reach 10% and 0.1% respectively. The daily feeding amount was based on the weight of the experimental animals, adenine 250mg / kg, dry yeast powder 25g / kg. During the modeling period, the mice were fed with high-purine feed and free access to water.

[0054] (3) Epinephrine + uricase inhibitor group

[0055] The method of adrenaline injection is the same as above, and the regimen of uricase inhibitor is as follows: Potassium oxonate is a uricase inhibitor, which can prepare a hyperuricemia model by inhibiting uricase activity. However, its long-term administration will have strong adverse reactions such as peritoneal sclerosis and ascites. Therefore, potassium oxonate (300 mg / kg) was given only in the last 2 weeks of modeling, intraperitoneally, once a day.

[0056] (4) Epinephrine + uric acid transporter inhibitor group

[0057] The method of adrenaline injection is the same as above. The scheme of uric acid transporter inhibitor is as follows: Pyrazinamide is a uric acid transporter inhibitor, which can prepare a hyperuricemia model by inhibiting uric acid excretion. However, its long-term administration will cause liver and kidney damage. Therefore, pyrazinamide (400 mg / kg) was only given in the last 2 weeks of modeling, and was administered by gavage once a day.

[0058] 3. Experimental Results

[0059] (1) Epinephrine group

[0060] The inventors first observed the effect of adrenaline injection (7-21d) on uric acid levels. As shown in Table 2, after adrenaline (0.2 μg / g) was given to mice for 7-21 days, the mice began to show an increase in uric acid levels on the 14th day (p<0.05). The results showed that after 14 days of adrenaline injection, mice could develop hyperuricemia.

[0061] Table 2 Effect of adrenaline injection on blood uric acid level in mice (μmol / L)

[0062] Grouping Uric acid levels Blank group 198±3.15 Adrenaline injection 7d group 203.03±19.04 Adrenaline injection 14d group 340.99±23.00** Adrenaline injection 21d group 343.784±3.12**

[0063] *, compared with the blank group, p<0.05; **, compared with the blank group, p<0.01

[0064] (2) Epinephrine + high-purine diet group

[0065] In order to further verify the feasibility of the combined modeling of the hyperuricemia model and the high-purine diet, the inventors further investigated the effect of the combined application of adrenaline injection and the high-purine diet on the uric acid level of mice after 14 days of modeling. The results in Table 3 show that adrenaline injection + high-purine diet can also significantly increase the blood uric acid level of mice (p < 0.01), replicating the hyperuricemia model of mice.

[0066] Table 3 Effects of adrenaline injection + high-purine diet on blood uric acid levels in mice (μmol / L)

[0067] Grouping Uric acid levels Blank group 197.05±3.75 Adrenaline injection + high-purine diet 324.39±25.08**

[0068] **, compared with the blank group, p<0.01

[0069] (3) Epinephrine + uricase inhibitor group

[0070] In order to further verify the feasibility of the combined modeling of the hyperuricemia model and uricase inhibitor, the effect of adrenaline injection combined with uricase inhibitor on the uric acid level of mice after 14 days of modeling was further investigated. The results in Table 4 show that adrenaline injection + uricase inhibitor can also significantly increase the blood uric acid level of mice (p<0.01), replicating the hyperuricemia model of mice.

[0071] Table 4 Effects of adrenaline injection + uricase inhibitor on blood uric acid levels in mice (μmol / L)

[0072] Grouping Uric acid levels Blank group 191.99±8.00 Epinephrine injection + uricase inhibitor 344.00±4.75**

[0073] **, compared with the blank group, p<0.01

[0074] (4) Epinephrine + uric acid transporter inhibitor group

[0075] In order to further verify the feasibility of the combined modeling of the hyperuricemia model and uric acid transporter inhibitors, the effect of adrenaline injection combined with uric acid transporter inhibitors on the uric acid level of mice after 14 days of modeling was further investigated. The results in Table 5 show that adrenaline injection + acid transporter inhibitors can also significantly increase the blood uric acid level of mice (p<0.01), replicating the hyperuricemia model of mice.

[0076] Table 5 Effects of adrenaline injection + uric acid transporter inhibitor on blood uric acid levels in mice (μmol / L)

[0077] Grouping Uric acid levels Blank group 205.05±17.03 Epinephrine injection + urate transporter inhibitor 348.407±7.413**

[0078] **, compared with the blank group, p<0.01

[0079] The above experiments show that the injection of low-dose adrenaline can successfully construct a hyperuric acid animal model, and the modeling method combined with a high-purine diet, uricase inhibitors, and uric acid transporter inhibitors can also successfully construct a hyperuric acid animal model. In addition, within the time range of this experiment, it was not found that low-dose adrenaline combined with a high-purine diet, uricase inhibitors, and uric acid transporter inhibitors would have more advantages. This may be related to the short modeling time and the rapid metabolism of modeling methods such as high-purine diet and uric acid metabolic enzyme inhibitors after 3 days of stopping the modeling. It can be seen that from the perspective of economy and cost, only injecting low-dose adrenaline without additional auxiliary modeling conditions can still successfully construct a hyperuric acid animal model, which provides a new option for the construction method of animal models.

Claims

1. A method for constructing a hyperuricemia animal model using adrenaline, characterized in that: The model was established by injecting low doses of adrenaline, including the following steps: A. Experimental animals were injected with low doses of adrenaline daily; B. Eat normally and drink water freely; C. The modeling time is at least 14 days.

2. The method for constructing a hyperuricemia animal model using adrenaline according to claim 1, characterized in that: The low dose of adrenaline in step A is 0.05-1.0 μg / g based on the body weight of the experimental animal; Preferably, the low dose of adrenaline in step A is 0.1-0.5 μg / g based on the body weight of the experimental animal. Most preferably, the low dose of adrenaline in step A is 0.2 μg / g based on the body weight of the experimental animal.

3. The method for constructing a hyperuricemia animal model using adrenaline according to claim 1 or 2, characterized in that: The model was established by injecting low-dose adrenaline combined with a high-purine diet, uricase inhibitors or uric acid transporter inhibitors.

4. The method for constructing a hyperuricemia animal model using adrenaline according to claim 3, characterized in that: The model was established by injecting low doses of adrenaline and combining it with a high purine diet, which included the following steps: A. Experimental animals were injected with low doses of adrenaline daily; B. The diet is mixed feed with dry yeast powder or adenine, and drinking water is free; C. The modeling time is at least 14 days.

5. The method for constructing a hyperuricemia animal model using adrenaline according to claim 4, characterized in that: At least one of the following is met: In step B, the dry yeast powder content in the mixed feed is 5-10%; Or preferably, in step B, the dry yeast powder content in the mixed feed is 10%; Or in step B, the adenine content in the mixed feed is 0.05-0.1%; Or preferably, in step B, the adenine content in the mixed feed is 0.1%; Or the daily feeding amount is based on the weight of the experimental animals, adenine 125-250mg / kg, dry yeast powder 12.5-25g / kg; Or preferably, the daily feeding amount is 250 mg / kg of adenine and 25 g / kg of dry yeast powder based on the body weight of the experimental animals.

6. The method for constructing a hyperuricemia animal model using adrenaline according to claim 3, characterized in that: The model was established by injecting low doses of epinephrine combined with uricase inhibitors, including the following steps: A. Experimental animals were injected with low doses of adrenaline daily; B. Eat normally and drink water freely; C. The modeling time is at least 14 days, and potassium oxonate is injected intraperitoneally in the last 1-2 weeks of modeling; More preferably, at least one of the following is satisfied: In step C, potassium oxonate is intraperitoneally injected once a day, and the injection amount is based on the body weight of the experimental animal, potassium oxonate 150-300 mg / kg; Preferably, in step C, potassium oxonate is intraperitoneally injected, and the injection amount is once a day based on the body weight of the experimental animal, potassium oxonate 300 mg / kg.

7. The method for constructing a hyperuricemia animal model using adrenaline according to claim 3, characterized in that: The model was established by injecting low doses of epinephrine combined with uric acid transporter inhibitors, including the following steps: A. Experimental animals were injected with low doses of adrenaline daily; B. Eat normally and drink water freely; C. The modeling time is at least 14 days, and pyrazinamide is administered by gavage in the last 1-2 weeks of modeling; More preferably, at least one of the following is satisfied: In step C, pyrazinamide is administered by oral gavage once a day, and the oral gavage amount is based on the body weight of the experimental animal, 200-400 mg / kg of pyrazinamide. Preferably, pyrazinamide is administered by oral gavage once a day, with the oral gavage amount being 400 mg / kg of pyrazinamide based on the body weight of the experimental animal.

8. The method for constructing a hyperuricemia animal model using adrenaline according to any one of claims 1 to 7, characterized in that: The animals used to construct the animal model include mammals and birds; Further preferably, the mammals include primates, rodents, artiodactyls, canines, and felines; Still further preferably, the primate is selected from monkeys and gorillas; the rodent is selected from mice, rats, rabbits, and guinea pigs; the artiodactyl is selected from donkeys, sheep, cattle, horses, and pigs; the canine is selected from dogs, wolves, jackals, and foxes; the feline is selected from cats; Further preferably, the poultry animals are chickens, ducks, and geese; Preferably, rats or mice are used as experimental animals; Most preferably, mice are used as experimental animals.

9. The hyperuricemia animal model obtained by the method for constructing a hyperuricemia animal model using adrenaline according to any one of claims 1 to 8.

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