Application of uracil in the preparation of drugs against vitiligo syndrome

Abstract

The present invention belongs to the field of disease prevention and control and aquaculture technology, and relates to the specific therapeutic activity of uracil, and specifically to the application of uracil in the preparation of anti-white spot syndrome drugs. The present invention verifies through experiments that uracil can give shrimp a certain ability to resist white spot syndrome virus by inducing the upregulation of the expression of Ago2 and Dicer2 genes, key members of the RNAi antiviral mechanism of shrimp, and antiviral effector factors AlfB1 and Draper genes.

Description

Technical Field

[0001] The invention belongs to the technical field of disease prevention and treatment and aquaculture, relates to the specific therapeutic activity of uracil, and specifically to the application of uracil in the preparation of an anti-white spot syndrome drug. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] White Spot Syndrome (WSS) is a serious disease faced by shrimp farming. Its pathogen is the White Spot Syndrome Virus (WSSV). WSS has a high mortality rate. After being infected with WSSV, shrimps die rapidly within 3-10 days. Globally, the economic losses caused by WSS are increasing at a rate of US$1 billion per year.

[0004] There is no effective prevention and treatment method for WSS at present. Since shrimp lack lymphocytes and antibody-based adaptive immunity, it is difficult to use vaccines for disease prevention. However, shrimp have evolved sensitive virus recognition mechanisms and a series of strategies to protect the body from viral infection, including cellular immune responses involving different types of cells and some effector molecules in humoral immunity. At present, studies have shown that RNAi (RNA interference) response is the main antiviral mechanism of shrimp, which inhibits viral replication and infection by targeting and degrading viral mRNA. The key components of this mechanism include Ago2 and Dicer2. In addition to the RNAi mechanism, there are also a series of key antiviral effector factors in shrimp humoral immunity, including AlfB1 and Draper, which can block viral infection by directly destroying the structure of viral particles or promoting the phagocytosis of viral particles by body cells. Therefore, if a new antiviral drug can be developed that can simultaneously increase the expression of key members of the RNAi mechanism and key effector factors of humoral immunity to improve the antiviral ability of shrimp, it will be able to effectively inhibit WSSV infection and treat white spot syndrome, which will be of great significance for protecting the healthy development of shrimp aquaculture. Summary of the invention

[0005] The present invention has proved through experiments that uracil can induce the expression level of the core members of the antiviral RNAi mechanism and the key antiviral direct effector factors in the shrimp, inhibit the synthesis of WSSV gene mRNA and protein and the replication of complete virus particles, and alleviate the damage of white spot syndrome to the body. This shows that uracil can give shrimp a certain antiviral ability by inducing the expression of antiviral genes. The present invention discloses the application of uracil as an antiviral drug for shrimp for the first time, and it is different from the known medicinal uses of uracil.

[0006] Based on the above research results, the present invention proposes the use of uracil in the preparation of anti-vitiligo syndrome drugs. Specifically, the technical solution provided by the present invention is as follows:

[0007] In the first aspect, a use of uracil in the preparation of an anti-vitiligo syndrome drug.

[0008] The CAS number of uracil described in the present invention is 66-22-8, and its chemical structure is as follows:

[0009] .

[0010] In some embodiments, the drug is a composition. The composition can be in different forms, such as powder, liquid preparation, etc., according to the needs of the recipient.

[0011] In some embodiments, the active ingredient of the drug is uracil or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0012] The pharmaceutically acceptable salts described in the present invention refer to salts that are pharmaceutically considered safe, effective and suitable for use in pharmaceutical preparations, such as hydrochlorides, sulfates, nitrates, acetates, oxalates, citrates, fumarates, toluenesulfonates, etc.

[0013] The solvate described in the present invention refers to a crystal structure formed by the crystallization of uracil and an organic solvent.

[0014] The hydrate described in the present invention refers to a crystal structure formed by the crystallization of uracil and water.

[0015] In some embodiments, the drug includes pharmaceutical excipients, which may be adhesives, diluents, fillers, preservatives, absorption enhancers, and the like.

[0016] In some embodiments, the drug is administered to an aquatic animal, and specifically, the aquatic animal is a prawn.

[0017] Since drugs can also be appropriately added to feed additives, in some embodiments, the drugs are used as feed additives for aquatic animals.

[0018] In some embodiments, the drug is administered by gavage.

[0019] In some embodiments, the dosage of the drug is: the dosage for one aquatic animal is (2-8)×10 -3 nmol. Specifically, the drug concentration is 80~120 nM. The unit "nM" represents nmol / L, where 1 nmol=10 -9 mol.

[0020] In some embodiments, the drug has one or more of the following effects:

[0021] (a) Inducing the expression of Ago2 and Dicer2 genes, core components of the RNAi mechanism in aquatic animals;

[0022] (b) Inducing the expression of antiviral effector factors AlfB1 and Draper genes in aquatic animals;

[0023] (c) inhibiting the mRNA transcription of the white spot syndrome virus structural protein VP28 gene;

[0024] (d) inhibiting the protein expression of VP28, a structural protein of white spot syndrome virus;

[0025] (e) inhibiting the replication of intact virions of white spot syndrome virus;

[0026] (f) Inhibit shrimp mortality caused by white spot syndrome virus.

[0027] In the second aspect, an anti-white spot syndrome virus product comprises at least one or more of the following uses:

[0028] (a) Inducing the expression of Ago2 and Dicer2 genes, core components of the RNAi mechanism of shrimp;

[0029] (b) Inducing the expression of key antiviral effector genes AlfB1 and Draper in shrimp;

[0030] (c) inhibiting the mRNA transcription of the white spot syndrome virus structural protein VP28 gene;

[0031] (d) inhibiting the protein expression of VP28, a structural protein of white spot syndrome virus;

[0032] (e) inhibiting the replication of intact virions of white spot syndrome virus;

[0033] (f) Inhibit shrimp mortality caused by white spot syndrome virus.

[0034] The anti-white spot syndrome product can be a feed additive or a medicine;

[0035] In a third aspect, a method for preventing or treating white spot syndrome in shrimp is provided, the method comprising: applying the above product to shrimp.

[0036] The beneficial effects of the present invention are:

[0037] The present invention analyzes the expression of Ago2, Dicer2, AlfB1 and Draper in the shrimp after feeding uracil. The results show that uracil can lead to an increase in the antiviral genes of the shrimp; and uracil can inhibit WSSV in mRNA transcription, protein expression, replication of complete virus particles, and death of shrimp caused by virus infection, indicating that uracil can enhance the ability of shrimp to resist WSSV. Therefore, uracil can be used as a drug for shrimp to resist white spot syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0039] Figure 1 This is a result diagram of the effect of uracil in Example 1 of the present invention on the expression of Ago2 gene and Dicer2 gene, the core components of the RNAi mechanism of shrimp.

[0040] Figure 2 This is a result diagram of the effect of uracil of Example 2 of the present invention on the expression of the key antiviral effector factors AlfB1 and Draper genes of shrimp.

[0041] Figure 3 This is a result diagram of the effect of uracil on the transcription of the white spot syndrome virus structural protein VP28 gene at the mRNA level in Example 3 of the present invention.

[0042] Figure 4 This is a result diagram of the effect of uracil of Example 4 of the present invention on the protein expression of white spot syndrome virus structural protein VP28.

[0043] Figure 5 This is a result diagram of the effect of uracil of Example 5 of the present invention on the replication of complete viral particles of white spot syndrome virus.

[0044] Figure 6 This is a graph showing the effect of uracil of Example 6 of the present invention on the death of shrimp caused by white spot syndrome virus infection. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0046] Example 1

[0047] Experimental animal treatment: Uracil (A610564-0500; Sangon Biotech) was dissolved in DMSO and used as a stock solution. The stock solution was diluted to 100 nM (nmol / L) when used. Group 1: 50 μL of uracil dilution was orally fed into the shrimp intestine; Group 2: 50 μL of ultrapure water containing 10% DMSO was orally fed, and intestinal tissue samples were extracted 3 hours later.

[0048] After 3 h of culture, RNA extraction solution was used to obtain RNA from the shrimp intestine, and then reverse transcription kit (R323-01; Vazyme) was used to reverse transcribe RNA into cDNA. Finally, qRT-PCR technology was used to detect the expression levels of Ago2 and Dicer2 genes. The primer sequences for detecting Ago2 and Dicer2 genes were Ago2-RTF: 5'-GACGCGCTGAAGCAGAGATA-3' (as shown in SEQ ID NO.1), Ago2-RTR: 5'-CACACTGGTCCTGACTGCAT-3' (as shown in SEQ ID NO.2); Dicer2-RTF: 5'-ACCGGGTCAGGCAAAACTT-3' (as shown in SEQ ID NO.3), Dicer2-RTR: 5'-TCTCGAATCTGATGCCCTAGCT-3' (as shown in SEQ ID NO.4). β-actin was used as an internal reference, and the primer sequences were β-actin-RTF: 5'-AGTAGCCGCCCTGGTTGTAGAC-3' (as shown in SEQ ID NO.5), β-actin-RTR: 5'-TTCTCCATGTCGTCCCAGT-3' (as shown in SEQ ID NO.6). The qRT-PCR program was set to run at 94℃ for 5 min, 94℃ for 10 s, and 60℃ for 1 min, for a total of 40 cycles. The final data were obtained using 2 −ΔΔCT The data were processed by the method and the experiment was repeated three times independently. Figure 1 As shown, uracil can induce the expression of Ago2 and Dicer2 genes, the core components of the shrimp RNAi mechanism.

[0049] Example 2

[0050] The experimental animal treatment and detection methods were the same as those in Example 1. The primer sequences for detecting AlfB1 and Draper genes were AlfB1-RTF: 5'-CGGTGGTGGCCCTGGTGGCACTCTTCG-3' (as shown in SEQ ID NO.7), AlfB1-RTR: 5'-GACTGGCTGCGTGTGCTGGCTTCCCCTC-3' (as shown in SEQ ID NO.8); Draper-RTF: 5'-TGTCCTGAAGGGAAATGGG-3' (as shown in SEQ ID NO.9), Draper-RTR: 5'-GTTCTGACAGCGACACCGA-3' (as shown in SEQ ID NO.10). The detection results are shown in the attached figure. Figure 2 As shown, uracil can induce the expression of AlfB1 and Draper genes, which are key antiviral effector factors in shrimp.

[0051] Example 3

[0052] The experimental animal treatment and detection methods were the same as in Example 1. The two groups of shrimp were cultured for 3 h after treatment. Each group of shrimp was injected with 50 μL of WSSV (10 6 Virus particles), after 24 h and 48 h of culture, RNA samples of shrimp intestinal tissue were obtained using RNA extraction reagents, and then reversed into cDNA using a reverse transcription kit. qRT-PCR technology was used to detect the changes in WSSV mRNA. The primers involved were VP28-RTF: 5'-AGCTCCAACACCTCCTCCTTCA-3' (as shown in SEQ ID NO.11), VP28-RTR: 5'-TTACTCGGTCTCAGTGCCAGA-3' (as shown in SEQ ID NO.12). The test results are shown in the attached Figure 3 As shown, uracil can inhibit the mRNA transcription of the VP28 gene, a structural protein of the white spot syndrome virus.

[0053] Example 4

[0054] The experimental animal treatment and detection methods were the same as in Example 1. The two groups of shrimp were cultured for 3 h after treatment. Each group of shrimp was injected with 50 μL of WSSV (10 6 Virus particles), after 24 h and 48 h of culture, the total protein of shrimp intestinal tissue was obtained using RIPA lysis buffer (P0013C; Beyotime), and after being treated with SDS-PAGE protein loading buffer (P0015L; Beyotime), the structural protein VP28 of WSSV in the sample was characterized by Western Blot technology. The test results are shown in the attached Figure 4As shown, uracil can inhibit the protein expression of VP28, a structural protein of white spot syndrome virus.

[0055] Example 5

[0056] The experimental animal treatment and detection methods were the same as in Example 1. The two groups of shrimp were cultured for 3 h after treatment. Each group of shrimp was injected with 50 μL of WSSV (10 6 Virus particles), cultured for 24 h and 48 h later, the total DNA samples of intestinal tissue were extracted using the QIAamp DNA Blood Mini Kit (51104; Qiagen), and the DNA changes of WSSV were detected by qRT-PCR. The test results are shown in the attached Figure 5 As shown, uracil can inhibit the replication of complete virions of white spot syndrome virus.

[0057] Example 6

[0058] The experimental animal treatment and detection methods were the same as in Example 1. The two groups of shrimp were cultured for 3 h after treatment. Each group of shrimp was injected with 50 μL of WSSV (10 6 Virus particles), and record the survival rate of each group of shrimp. The test results are as attached. Figure 6 As shown, uracil can inhibit the mortality of shrimp caused by white spot syndrome virus.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of uracil in the preparation of an anti-vitiligo syndrome drug.

2. The use according to claim 1, characterized in that: The medicine is a composition.

3. The use according to claim 1, characterized in that: The active ingredient of the drug is uracil or a pharmaceutically acceptable salt thereof.

4. The use according to claim 1, characterized in that: The medicine includes pharmaceutical excipients.

5. The use according to claim 1, characterized in that: The drug is administered to aquatic animals.

6. The use according to claim 5, characterized in that: The aquatic animal is a prawn.

7. The use according to claim 1, characterized in that: The medicine is used as a feed additive for aquatic animals.

8. The use according to claim 1, characterized in that: The drug is administered by gavage.

9. The use according to claim 1, characterized in that: The dosage of the drug is: the dosage for one aquatic animal is (2-8)×10 -3 nmol.

10. The use according to claim 1, characterized in that: The drug has one or more of the following effects: (a) Inducing the expression of Ago2 and Dicer2 genes, core components of the RNAi mechanism in aquatic animals; (b) Inducing the expression of antiviral effector factors AlfB1 and Draper genes in aquatic animals; (c) inhibiting the mRNA transcription of the white spot syndrome virus structural protein VP28 gene; (d) inhibiting the protein expression of VP28, a structural protein of white spot syndrome virus; (e) inhibiting the replication of intact virions of white spot syndrome virus; (f) Inhibit shrimp mortality caused by white spot syndrome virus.

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