New application of zebra fish homocysteine model

Abstract

The invention relates to the technical field of disease models, in particular to novel application of a zebra fish homocysteine model. The zebra fish homocysteine model provided by the invention has any one of the following purposes: (1) simulating uric acid metabolism; (2) screening drugs related to uric acid; (3) predicting diseases related to uric acid; and (4) researching the relationship between homocysteine and uric acid indexes. Based on the research basis that homocysteine has certain influence on the uric acid index, the zebra fish homocysteine model is utilized to simulate uric acid metabolism, screen drugs related to uric acid, predict diseases related to uric acid, research the relationship between homocysteine and the uric acid index, and improve the accuracy of uric acid index detection.

Description

Technical Field

[0001] The present invention relates to the technical field of disease models, and in particular to a new application of a zebrafish homocysteine ​​model. Background Art

[0002] Homocysteine ​​is a sulfur-containing amino acid and an intermediate product of methionine metabolism. Its metabolic abnormalities are closely related to a variety of diseases. By establishing a zebrafish homocysteine ​​model, we can study metabolic pathways, screen drugs for cardiovascular and neurodegenerative diseases, reveal the mechanism of action of homocysteine ​​in diseases, and provide targets for precision treatment.

[0003] Existing studies have shown that homocysteine ​​and uric acid are jointly involved in multiple diseases through oxidative stress, inflammation and metabolic interactions, and may synergistically cause disease in cardiovascular and kidney diseases. However, there are few studies on the effect of homocysteine ​​on uric acid, and it is impossible to clearly distinguish and distinguish between homocysteine ​​and uric acid, which leads to certain errors in the judgment of uric acid indicators. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect in the prior art that the relationship between homocysteine ​​and uric acid cannot be clearly defined, resulting in errors in the determination of uric acid indicators, thereby providing a new use of the zebrafish homocysteine ​​model.

[0005] To this end, the present invention provides a zebrafish homocysteine ​​model having any of the following uses: (1) simulating uric acid metabolism; (2) screening drugs related to uric acid; (3) predicting diseases related to uric acid; (4) studying the relationship between homocysteine ​​and uric acid indicators.

[0006] In some embodiments, the method for constructing the zebrafish homocysteine ​​model comprises the steps of applying a reagent containing DL-homocysteine ​​to a zebrafish culture medium, culturing, and obtaining a zebrafish homocysteine ​​model, wherein the concentration of DL-homocysteine ​​in the reagent containing DL-homocysteine ​​is less than 15 mg / L.

[0007] In some embodiments, the concentration of DL-homocysteine ​​in the reagent containing DL-homocysteine ​​is 0.4-12 mg / L.

[0008] In some embodiments, the concentration of DL-homocysteine ​​in the reagent containing DL-homocysteine ​​is 3-12 mg / L.

[0009] In some embodiments, the zebrafish is 3-6 days post fertilization.

[0010] In some embodiments, the zebrafish is a wild-type AB strain.

[0011] In some embodiments, the culture duration is 1-4 days.

[0012] In some embodiments, the reagent containing DL-homocysteine ​​further includes dilution water, and the dilution water is obtained by diluting the reagent 40 times with dilution water.

[0013] In some embodiments, the 40-fold dilution water includes 2-3 g / L sodium bicarbonate, 0.1-0.4 g / L potassium chloride, 10-15 g / L calcium chloride, and 4-6 g / L magnesium sulfate.

[0014] In some embodiments, the zebrafish culture medium includes seawater, the seawater includes 180-220 mg / L sea salt, the conductivity of the seawater is 450-550 μS / cm, and the pH value of the seawater is 5-8.5.

[0015] The technical solution of the present invention has the following advantages:

[0016] The present invention provides a zebrafish homocysteine ​​model that has any of the following uses: (1) simulating uric acid metabolism; (2) screening for uric acid-related drugs; (3) predicting uric acid-related diseases; and (4) studying the relationship between homocysteine ​​and uric acid indicators. Based on research that homocysteine ​​has a certain influence on uric acid indicators, the present invention utilizes the zebrafish homocysteine ​​model to simulate uric acid metabolism, screen for uric acid-related drugs, predict uric acid-related diseases, and study the relationship between homocysteine ​​and uric acid indicators, thereby improving the accuracy of uric acid indicator detection.

[0017] The present invention verifies the synergistic effect of homocysteine ​​and uric acid levels through the constructed zebrafish homocysteine ​​model, providing a clue for subsequent research in areas such as uric acid metabolism. It may be possible to determine the accuracy of the detection index by replacing or increasing one of the indicators of homocysteine ​​or uric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 It is a line graph of homocysteine ​​and uric acid content in the zebrafish homocysteine ​​model in the experimental example of the present invention. DETAILED DESCRIPTION

[0020] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0021] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0022] The study found that during the methionine cycle, methionine is converted into adenosyl homocysteine, which is then decomposed into homocysteine ​​and adenylic acid, and adenylic acid is then decomposed into uric acid. Therefore, when higher levels of homocysteine ​​are produced, they are often accompanied by the production of a large amount of adenosine, which leads to an increase in the uric acid produced by the decomposition. This indicates that an increase in uric acid indicators is not necessarily just a problem of uric acid metabolism. It is very likely that the increase in homocysteine ​​content has an impact on the uric acid indicator. Therefore, the present invention relates to a zebrafish homocysteine ​​model that can simultaneously detect homocysteine ​​and uric acid indicators, reveal the relationship between homocysteine ​​and uric acid, and thus achieve the purpose of accurately judging uric acid indicators.

[0023] Example 1

[0024] This example provides a method for constructing a zebrafish homocysteine ​​model. The specific steps and parameters are as follows:

[0025] (1) Materials and instruments:

[0026] Wild-type AB strain zebrafish and Danio rerio were maintained in 28°C seawater (water quality: 200 mg sea salt per liter; conductivity 500 μS / cm; pH = 7);

[0027] DL-homocysteine ​​(lot number: F28OE-QE, manufacturer: TCI Chemical Industries, Ltd.);

[0028] Prepare 40x standard dilution with water: Sodium bicarbonate: 2.59g (NaHCO3, batch number: 20240123); Potassium chloride: 0.23g (KCl, batch number: 20161130); Calcium chloride: 11.76g (CaCl2·2H2O, batch number: C2430197); Magnesium sulfate: 4.93g (MgSO4·7H2O, batch number: 20240403). Mix and dilute to 1L with deionized water.

[0029] Preparation of standard dilution water: Take 25 mL of 40-fold standard dilution water and dilute to 1 L to obtain standard dilution water.

[0030] (2) Construction of the Zebrafish Homocysteine ​​Model:

[0031] Five-day-post-fertilization zebrafish were selected, and a 0.4 mg / mL DL-homocysteine ​​solution was prepared using standard dilution water. The drug was administered continuously for two days, with the solution replaced once daily. On the third day, the solution was replaced with normal saline to establish a zebrafish homocysteine ​​model.

[0032] The specific method of administering the medicine is to absorb as much liquid as possible from the zebrafish and then add 3 ml of the mixed medicine solution.

[0033] To ensure that the zebrafish were on the same day of fertilization, adult zebrafish were placed at either end of the mating tank at 4:00 PM, with a male-female ratio of 2:2. The next day, the zebrafish began spawning under light stimulation by pulling out the plate, and the eggs were collected at 10:00 AM. The collected eggs were selected and incubated at 28.5°C, and that day was counted as day 0.

[0034] Example 2

[0035] This example provides a method for constructing a zebrafish homocysteine ​​model. The specific steps and parameters are the same as those in Example 1, except that the concentration of DL-homocysteine ​​in the medicinal solution in step (2) is 3 mg / mL.

[0036] Example 3

[0037] This example provides a method for constructing a zebrafish homocysteine ​​model. The specific steps and parameters are the same as those in Example 1, except that the concentration of DL-homocysteine ​​in the medicinal solution in step (2) is 6 mg / mL.

[0038] Example 4

[0039] This example provides a method for constructing a zebrafish homocysteine ​​model. The specific steps and parameters are the same as those in Example 1, except that the concentration of DL-homocysteine ​​in the medicinal solution in step (2) is 9 mg / mL.

[0040] Example 5

[0041] This example provides a method for constructing a zebrafish homocysteine ​​model. The specific steps and parameters are the same as those in Example 1, except that the concentration of DL-homocysteine ​​in the medicinal solution in step (2) is 12 mg / mL.

[0042] Experimental example

[0043] The zebrafish homocysteine ​​model drugs prepared in Examples 1-5 were homogenized (ground using a high-speed cryogenic tissue grinder) and the homogenate was pipetted into a centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was collected and the homocysteine ​​and uric acid levels in the zebrafish homocysteine ​​models prepared in Examples 1-5 were measured using a homocysteine ​​kit (Batch No.: G020240723R01 / 02, manufacturer; Shanghai ELISA) and a uric acid kit (Batch No.: 20240125, Nanjing Jiancheng), respectively. The results are shown in Table 1.

[0044] Table 1 Homocysteine ​​and uric acid levels in the zebrafish homocysteine ​​model

[0045]

[0046] The zebrafish homocysteine ​​model constructed by selecting embodiment 2 is added with the medicine for reducing homocysteine, administered for 24h, wherein, the medicine for reducing homocysteine ​​is formulated as concentration of 0.3mg / mL with standard dilution water, and the main raw material in the medicine for reducing homocysteine ​​is folic acid, vitamin B6 and vitamin B12, the medicine for reducing homocysteine ​​is from the Health Food Testing Center of the College of Applied Arts and Sciences of Beijing Union University, respectively, utilizing homocysteine ​​test kit (lot number: G020240723R01 / 02, manufacturer; Shanghai ELISA) and uric acid test kit (lot number: 20240125, Nanjing Jiancheng) to measure homocysteine ​​content and uric acid content in the zebrafish homocysteine ​​model after administration. After administration, homocysteine ​​content in the zebrafish homocysteine ​​model is 22.19μmol / L, and uric acid content is 14.87μmol / L.

[0047] According to Table 1 and Figure 1 The results of the zebrafish homocysteine ​​model after administration show that the homocysteine ​​content and uric acid content in the zebrafish homocysteine ​​model prepared by the present invention are positively correlated, that is, the uric acid index fluctuates with the homocysteine ​​index. The added homocysteine ​​index can be used to analyze whether the change in the uric acid index is caused by uric acid or homocysteine.

[0048] According to the model construction method of Example 2, the drug solution was replaced with a mixture of xanthine sodium salt and 10 mmol / L potassium oxonate (potassium oxonate (batch number: #K2317036, Aladdin), carboxymethyl cellulose (batch number: 20141209, Shanghai trial), xanthine sodium salt (batch number: 1003419670, Sigma), potassium oxonate was prepared with 0.1% sodium carboxymethyl cellulose to a 20 mmol / L stock solution, and xanthine sodium salt was prepared with standard dilution water to a 10 mmol / L stock solution). The steps and parameters were consistent with those of Example 2 to obtain a uric acid model, and the contents of homocysteine ​​and uric acid in the uric acid model were measured. The results are shown in Table 2.

[0049] Table 2 Contents of homocysteine ​​and uric acid in the uric acid model

[0050]

[0051] According to Table 2, the change of uric acid content in the uric acid model is only related to the uric acid index. This is because the uric acid model lacks the homocysteine ​​index, and it is impossible to accurately determine whether the uric acid content is related to the metabolism of homocysteine. Therefore, there may be errors in the test results.

[0052] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A zebrafish homocysteine ​​model having any of the following uses: (1) Simulating uric acid metabolism; (2) Screening for drugs related to uric acid; (3) Predicting diseases related to uric acid; (4) Study the relationship between homocysteine ​​and uric acid indicators.

2. The use according to claim 1, characterized in that The method for constructing the zebrafish homocysteine ​​model comprises the following steps: A reagent containing DL-homocysteine ​​is added to the culture medium of zebrafish and cultured to obtain a zebrafish homocysteine ​​model. The concentration of DL-homocysteine ​​in the reagent containing DL-homocysteine ​​is less than 15 mg / L.

3. The use according to claim 2, characterized in that The concentration of DL-homocysteine ​​in the reagent containing DL-homocysteine ​​is 0.4-12 mg / L.

4. The use according to claim 3, characterized in that The concentration of DL-homocysteine ​​in the reagent containing DL-homocysteine ​​is 3-12 mg / L.

5. The use according to claim 2, characterized in that The zebrafish are 3-6 days after fertilization.

6. The use according to claim 2, characterized in that The zebrafish were of wild-type AB strain.

7. The use according to claim 2, characterized in that The culture duration is 1-4 days.

8. The use according to claim 2, characterized in that The reagent containing DL-homocysteine ​​also includes dilution water, which is obtained by diluting the solution 40 times with dilution water.

9. The use according to claim 2, characterized in that The 40-fold dilution water includes 2-3 g / L sodium bicarbonate, 0.1-0.4 g / L potassium chloride, 10-15 g / L calcium chloride, and 4-6 g / L magnesium sulfate.

10. The use according to claim 3, characterized in that The zebrafish culture medium includes seawater, which includes 180-220 mg / L of sea salt. The conductivity of the seawater is 450-550 μS / cm, and the pH value of the seawater is 5-8.5.

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