Application of metformin hydrochloride in the preparation of anti-vitiligo syndrome drugs

Abstract

The invention belongs to the technical field of disease prevention and treatment and aquaculture, relates to the specific therapeutic activity of metformin hydrochloride, and specifically relates to the use of metformin hydrochloride in the preparation of an anti-white spot syndrome drug. The invention verifies through experiments that metformin hydrochloride can induce 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, thereby giving shrimp a certain anti-white spot syndrome virus ability.

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 metformin hydrochloride, and specifically relates to the application of metformin hydrochloride in the preparation of an anti-vitiligo syndrome drug. Background Art

[0002] White Spot Syndrome Virus (WSSV) induces White Spot Syndrome (WSS), which has the characteristics of wide spread, high mortality rate and fast death rate. It is the most serious viral pathogen affecting shrimp aquaculture, and seriously threatens the shrimp farming safety of many commercially important shrimp species such as Penaeus vannamei, Penaeus japonicus and Penaeus monodon. The large host population makes WSS widely spread and difficult to control, making WSS the main disease faced by the global shrimp aquaculture industry in recent decades.

[0003] At present, studies have shown that RNAi (RNA interference) reaction 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 directly destroy the structure of viral particles or promote the phagocytosis of viral particles by body cells to block viral infection. Therefore, if a new white spot syndrome virus drug can be developed, it 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, effectively inhibit WSSV infection, and treat white spot syndrome, which is of great significance for protecting the healthy development of shrimp aquaculture.

[0004] Metformin hydrochloride is a widely used hypoglycemic drug. It mainly reduces blood sugar by inhibiting the output of glucose in the liver, improving the sensitivity of peripheral tissues to insulin, and increasing the uptake and utilization of glucose. It is often used to reduce blood sugar, prevent diabetes, reduce weight, and relieve hyperinsulinemia. The use of metformin hydrochloride in the preparation of drugs for the treatment of white spot syndrome in shrimp has not yet been found. Summary of the invention

[0005] The purpose of the present invention is to provide an application of metformin hydrochloride in the preparation of an anti-white spot syndrome drug, thereby overcoming the deficiencies of the prior art, being able to simultaneously increase the expression levels of key members of the RNAi mechanism and key effector factors of humoral immunity, so as to improve the antiviral ability of shrimp, effectively inhibit WSSV infection, and treat white spot syndrome, and having a wide range of application scenarios in the field of healthy development of shrimp aquaculture.

[0006] In order to achieve the above object, the technical solution of the present invention is:

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

[0008] The CAS number of the metformin hydrochloride described in the present invention is 1115-70-4, 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 metformin hydrochloride or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

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

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

[0014] The hydrate described in the present invention refers to a crystal structure formed by the crystallization of metformin hydrochloride 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 in the form of an injection.

[0017] In some embodiments, the drug is administered to an aquatic animal, and specifically, the aquatic animal is a prawn.

[0018] Since drugs can also be appropriately added to feed additives, in some embodiments, the drugs are used as feed additives for aquatic animals.

[0019] In some embodiments, the drug has one or more of the following effects:

[0020] (a) Inducing the expression of Ago2 and Dicer2 genes, core components of the RNAi mechanism in aquatic animals;

[0021] (b) Inducing the expression of antiviral effector factors AlfB1 and Draper genes in aquatic animals;

[0022] (c) inhibiting the mRNA transcription of the white spot syndrome virus structural protein VP28 gene;

[0023] (d) inhibiting the protein expression of VP28, a structural protein of white spot syndrome virus;

[0024] (e) inhibiting the replication of intact virions of white spot syndrome virus;

[0025] (f) Inhibit shrimp mortality caused by white spot syndrome virus.

[0026] In the second aspect, an anti-white spot syndrome virus product comprises at least one or more of the following uses:

[0027] (a) Inducing the expression of Ago2 and Dicer2 genes, core components of the RNAi mechanism of shrimp;

[0028] (b) Inducing the expression of key antiviral effector genes AlfB1 and Draper in shrimp;

[0029] (c) inhibiting the mRNA transcription of the white spot syndrome virus structural protein VP28 gene;

[0030] (d) inhibiting the protein expression of VP28, a structural protein of white spot syndrome virus;

[0031] (e) inhibiting the replication of intact virions of white spot syndrome virus;

[0032] (f) Inhibit shrimp mortality caused by white spot syndrome virus.

[0033] The anti-white spot syndrome product can be a feed additive or a medicine;

[0034] 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.

[0035] Beneficial effects of the present invention:

[0036] After injecting metformin hydrochloride into shrimp, the expression levels of Ago2, Dicer2, AlfB1 and Draper in the shrimp are analyzed, and the results show that metformin hydrochloride can lead to an increase in the antiviral genes of shrimp; and metformin hydrochloride can inhibit the replication level of WSSV virus particles, the mRNA transcription level of WSSV structural protein VP28, the protein expression level, and the death of shrimp caused by virus infection, indicating that metformin hydrochloride can enhance the ability of shrimp to resist WSSV. Therefore, metformin hydrochloride can be used as a drug for shrimp to resist white spot syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1 This is a diagram showing the confirmation result of the optimal dosage of metformin hydrochloride in shrimp according to Example 1 of the present invention;

[0039] Figure 2 This is a result diagram showing the effect of metformin hydrochloride in Example 2 of the present invention on the expression of Ago2 gene and Dicer2 gene, core components of the RNAi mechanism of shrimp;

[0040] Figure 3 This is a result diagram of the effect of metformin hydrochloride on the expression of the key antiviral effector factors AlfB1 and Draper genes of shrimp in Example 3 of the present invention;

[0041] Figure 4 This is a result diagram of the effect of metformin hydrochloride on the replication of complete virions of white spot syndrome virus according to Example 4 of the present invention;

[0042] Figure 5 This is a result diagram of the effect of metformin hydrochloride in Example 5 of the present invention on the transcription of the white spot syndrome virus structural protein VP28 gene at the mRNA level;

[0043] Figure 6 This is a result diagram of the effect of metformin hydrochloride on the protein expression of white spot syndrome virus structural protein VP28 according to Example 6 of the present invention;

[0044] Figure 7 This is a result diagram showing the effect of metformin hydrochloride in Example 7 of the present invention on the death of shrimp caused by white spot syndrome virus infection;

[0045] in Figure 2-Figure 7 The control group was injected intramuscularly with 50 μL artificial seawater, and the metformin hydrochloride group was injected intramuscularly with 50 μL diluent of metformin hydrochloride. DETAILED DESCRIPTION

[0046] Example 1

[0047] The optimal dosage of metformin hydrochloride is:

[0048] Metformin hydrochloride (A506198-0025; Sangon Biotech) was dissolved in artificial seawater for use as a stock solution, and the stock solution was diluted to 100 μg / mL before use.

[0049] 125 shrimps (average weight 8.3 g) were divided into 5 groups, with 25 shrimps in each group. Each shrimp in each group was given 0, 5, 25, 50, and 100 μg of metformin hydrochloride, and the survival rate was recorded every 12 hours. Figure 1 As shown, p<0.0001 (0, 100 μg), p=0.0003 (0, 50 μg), p=0.0123 (0, 25 μg), p>0.1 (0, 5 μg). Figure 1 It can be seen that within 12 hours, there was no significant difference in the survival rate of shrimp between the drug administration group (5, 25, 50, 100 μg) and the control group (0 μg). As the administration time increased, the survival rate of shrimp in the drug administration group (5, 25, 50, 100 μg) was gradually lower than that of the control group (0 μg). However, there was no significant difference in the survival rate of shrimp in the 5 μg administration group compared with the control group (0 μg administration) within 24 hours, indicating that the administration of 5 μg of metformin hydrochloride would not affect the survival of shrimp within 24 hours. Therefore, the optimal dosage of metformin hydrochloride was set at 5 μg.

[0050] Example 2

[0051] Experimental animal handling

[0052] Metformin hydrochloride was dissolved in artificial seawater as a stock solution, and the stock solution was diluted to 100 μg / mL when used. 50 shrimps (average weight 8.3 g) were divided into 2 groups, with 25 shrimps in each group. The first group: intramuscular injection of 50 μL of metformin hydrochloride dilution (labeled as metformin hydrochloride); the second group: intramuscular injection of 50 μL of artificial seawater (labeled as control), and blood tissue samples of shrimps in the first and second groups were extracted 3 hours later.

[0053] After 3 h of culture, RNA extraction solution was used to obtain RNA from shrimp hemocytes, 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), D ice r2 - 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 and the test was repeated three times independently. Figure 2 As shown. Figure 2 It can be seen that the mRNA transcription levels of Ago2 and Dicer2 genes in the metformin hydrochloride group were significantly upregulated compared with those in the control group. Therefore, metformin hydrochloride can induce the expression of Dicer2 genes, the core components of the RNAi mechanism of shrimp, in the Ago2 gene.

[0054] Example 3

[0055] The experimental animal treatment and detection methods were the same as those in Example 2. The primer sequences for detecting AlfB1 and Darper 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'- TGT CC TGAAGGGAAATGGG - 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 Figure 2. Figure 3 As shown. Figure 3 It can be seen that the mRNA transcription levels of AlfB1 and Draper genes in the metformin hydrochloride group were significantly upregulated compared with those in the control group. Therefore, metformin hydrochloride can induce the expression of AlfB1 and Draper genes, the key antiviral effector factors in shrimp.

[0056] Example 4

[0057] The experimental animal treatment and detection methods were the same as those in Example 2. 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 DNA samples of shrimp blood cells in the two groups were extracted using the QIAamp DNA Blood Mini Kit (51104; Qiagen), and the DNA changes of WSSV were detected by qRT-PCR technology. The test results are shown in Figure 4 As shown. Figure 4 It can be seen that after infection with white spot syndrome virus, the copy number of complete virus particles in the metformin hydrochloride group shrimp was significantly reduced compared with that in the control group shrimp. Therefore, metformin hydrochloride can inhibit the replication of complete virus particles of white spot syndrome virus.

[0058] Example 5

[0059] The experimental animal treatment and detection methods were the same as those in Example 2. The two groups of shrimp were cultured for 3 h after treatment. Each group of shrimp was injected with 50 μL of WSSV (10 6Virus particles), after 24 h and 48 h of culture, RNA extraction reagent was used to obtain RNA samples of two groups of shrimp blood cells, and then the RNA was reversed into cDNA using a reverse transcription kit. The 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 Figure 5 As shown. Figure 5 It can be seen that the mRNA transcription level of the white spot syndrome virus structural protein VP28 gene in the metformin hydrochloride group shrimp was significantly downregulated compared with that in the control group shrimp. Therefore, metformin hydrochloride can inhibit the mRNA transcription of the white spot syndrome virus structural protein VP28 gene.

[0060] Example 6

[0061] The experimental animal treatment and detection methods were the same as those in Example 2. The two groups of shrimp were cultured for 3 hours after treatment. Each group of shrimp was injected with 50 μL of WSSV (10 6 Virus particles), after 24h and 48h of culture, RIPA lysis buffer (P0013C; Beyotime) was used to obtain the total protein of shrimp blood cells, and after being treated with SDS-PAGE protein loading buffer (P0015L; Beyotime), Western Blot technology was used to characterize and detect the WSSV structural protein VP28 in the two groups of shrimp samples. The test results are shown in Figure 6 ImageJ software was used to analyze the grayscale of the images, and the grayscale of anti-VP28 was divided by the grayscale of anti-β-actin to obtain the value of each group. Figure 6 The values ​​1.0, 0.6, 1.5 and 1.1 in the figure represent the multiples of the values ​​of each group relative to the 24h control. Specifically, the gray scale of VP28 in the 24h control group is 1.0, the gray scale of VP28 in the 24h metformin hydrochloride group is 0.6, the gray scale of VP28 in the 48h control group is 1.5, and the gray scale of VP28 in the 48h metformin hydrochloride group is 1.1. The above values ​​represent the relative expression of VP28 protein.

[0062] from Figure 6 It can be seen that the protein level of VP28, a structural protein of white spot syndrome virus, in the metformin hydrochloride group was significantly downregulated compared with that in the control group. Therefore, metformin hydrochloride can inhibit the expression of VP28, a structural protein of white spot syndrome virus.

[0063] Example 7

[0064] The experimental animal treatment and detection methods were the same as those in Example 2. 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), the survival rate of each group of shrimp was recorded every 12 hours. Figure 7 As shown, p = 0.0044 (metformin hydrochloride, control). Figure 7 It can be seen that the mortality rate of shrimp in the metformin hydrochloride group after infection with white spot syndrome virus was significantly reduced compared with that in the control group. Therefore, metformin hydrochloride can inhibit the death of shrimp caused by white spot syndrome virus.

[0065] 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 metformin hydrochloride 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 medicine includes pharmaceutical excipients.

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

5. The use according to claim 4, characterized in that The aquatic animal is a prawn.

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

7. The use according to claim 1, characterized in that The dosage form of the medicine is injection.

8. 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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