Application of metformin in preparation of medicine for preventing or treating bombyx mori nuclear polyhedrosis virus disease

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of metformin in preparation of a medicine for preventing or treating bombyx mori nuclear polyhedrosis virus. Research finds that the metformin has prevention and treatment effects on bombyx mori infected with the nuclear polyhedrosis virus, and meanwhile, the metformin can also play a role in promoting the growth and development of bombyx mori. Metformin has certain biodegradability and is a green and low-pollution biological control agent.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to the application of metformin in preparing a drug for preventing or treating silkworm nuclear polyhedrosis virus disease. Background Art

[0002] The silkworm (Bombyx mori), a silk-secreting insect with important economic value among Lepidoptera, is not only a classic model organism for entomological research, but also plays an important role in the development of agricultural economy and silk industry in various countries. However, infection with Bombyx mori nucleopolyhedrovirus (BmNPV) has become one of the major diseases restricting the production of sericulture. The genome of BmNPV is composed of double-stranded circular DNA of about 128 kilobase pairs. After infecting silkworms, it often causes severe pathological processes such as swelling of the host body surface, systemic suppuration and even death. It is highly contagious and lethal. The infection process includes (1) virus particles invading midgut epithelial cells; (2) replicating and assembling new virus particles in the cell nucleus; (3) the virus spreads to other tissues such as fat body and blood cells through cell lysis or budding; (4) eventually leading to the death of the host, and forming a large number of polyhedrones in the corpse, which are released into the environment. Studies have shown that BmNPV infection not only has a significant negative impact on the growth and development of silkworms, but also achieves immune escape by inhibiting the host immune system, interfering with cell apoptosis and endoplasmic reticulum stress response. As a result, BmNPV infection has led to a large-scale outbreak of silkworm disease, and the loss of cocoon production caused by it accounts for more than 60% of the total loss caused by silkworm disease, seriously threatening the sustainable development of the sericulture industry.

[0003] At present, the prevention and control methods for silkworm BmNPV mainly include physical isolation, the use of chemical disinfectants and the application of traditional antiviral drugs. However, physical isolation is difficult to completely block the spread of the virus, the use of chemical disinfectants may have certain negative effects on the environment and the silkworms themselves, and traditional antiviral drugs have problems such as drug resistance and drug residues. Therefore, finding a safe, efficient and environmentally friendly method to fight against silkworm BmNPV has become an urgent need for current research.

[0004] Metformin, as a classic hypoglycemic drug, has been found to have a wide range of antiviral activity in recent years. For example, studies have shown that it has inhibitory effects against hepatitis C virus, rotavirus, bovine diarrhea virus, enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), and its mechanism of action involves multiple levels, including energy metabolism regulation, immune response regulation, and cell autophagy. Whether metformin can be used in silkworms to fight against Bombyx mori nuclear polyhedrosis virus disease has not been reported. Summary of the invention

[0005] After extensive screening and research, the inventors of the present invention unexpectedly discovered that metformin can prevent and inhibit nuclear polyhedrosis virus infection in silkworms, thereby completing the present invention. The technical solution of the present invention is as follows: In one aspect of the present invention, the present invention discloses the use of metformin in the preparation of a drug for preventing or treating Bombyx mori nuclear polyhedrosis virus disease.

[0006] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0007] In one embodiment, the dosage form of the drug is a liquid preparation or a wettable powder.

[0008] In one aspect of the present invention, a method for preventing or treating silkworm nuclear polyhedrosis virus disease is disclosed, the method comprising administering a preparation containing metformin to the silkworm.

[0009] In one embodiment, the preparation is administered before the silkworm is infected with BmNPV, or within 12 hours after being infected with BmNPV.

[0010] In one embodiment, the formulation is administered to silkworms by feeding them mulberry leaves sprayed with the metformin formulation.

[0011] In one aspect of the present invention, the present invention provides the use of metformin in the preparation of a drug for improving the body weight and cocoon quality of silkworms. Beneficial Effects

[0012] (1) The metformin in the present invention can inhibit the energy supply of the virus, effectively block the transmission and spread of the virus, and play a good preventive and therapeutic role in the early stage of the disease, thereby providing a new prevention and treatment approach for the prevention and treatment of Bombyx mori nuclear polyhedrosis virus disease.

[0013] (2) The metformin of the present invention can also promote the growth and development of silkworms and reduce the damage caused by viruses to the host. This effect not only helps silkworms survive in a virus-infected environment, but also may improve their silk protein synthesis ability, thereby having a positive impact on the economic benefits of the sericulture industry.

[0014] (3) Advantages of biodegradability: Metformin has a certain degree of biodegradability. After entering the natural water or soil environment, it can be gradually decomposed under the action of microorganisms. This is different from some organic pollutants that are difficult to degrade. It can avoid long-term accumulation in the environment, reduce the continuous harm to the ecosystem, ensure the stability of the water and soil ecological environment, and ensure green environmental protection during production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1, Detection of toxicity of metformin on silkworm cells BmN.

[0016] Figure 2 , TUNEL staining was used to detect cell apoptosis after metformin treatment.

[0017] Figure 3 , Effects of different concentrations of Metformin on viral gene expression and replication in cells.

[0018] Figure 4 , Western blot was used to detect the expression level of viral GP64 protein by Metformin.

[0019] Figure 5 , Flow cytometry was used to quantitatively analyze the viral inhibition efficiency of BmNPV-infected cells after metformin treatment.

[0020] Figure 6 , Analysis of the inhibitory effect of metformin on silkworm infection viruses.

[0021] Figure 7 , Effects of metformin on the growth and development of silkworm. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Example 1: Detection of cytotoxicity of metformin in silkworm cells BmN

[0023] One day in advance, the silkworm BmN cells in good growth state were inoculated into a 96-well plate, with about 5×10³ cells in each well, and 100 μL of complete medium was added. The cells were cultured overnight in an incubator at 27°C and 5% CO2 to allow the cells to adhere to the wall and enter the logarithmic growth phase.

[0024] The next day, according to the concentration gradient determined in the pre-experiment (500 μM, 1 mM), the corresponding volume of metformin solution was added to each well, so that the final volume of each well was 150 μL. A blank control group containing only culture medium (without cells and drugs) and a negative control group containing only cells (without drugs) were set up, and 3-5 replicates were set up in each group. The treated cells were cultured continuously and tested at 0 h, 24 h, 48 h, 72 h, and 96 h time gradients. At each detection time point, 10 μL of CCK-8 reagent was added to each well, gently mixed and continued to culture for 2-4 hours. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an enzyme reader, and the data was recorded.

[0025] Cell survival rate (%) = (OD value of experimental group − OD value of blank control group / OD value of negative control group – OD value of blank control group) * 100%.

[0026] See also Figure 1 , 0.5mM and 1mM metformin have no toxicity to BmN cells, and can even promote cell growth to a certain extent. Example 2: Detection of the effectiveness of metformin against BmNPV virus in silkworm BmN cells 2.1 Metformin anti-apoptosis detection

[0027] The CCK8 method was used to evaluate the protective effect of metformin on cell survival at different time points (24 h, 48 h, 72 h, and 96 h), and the cell activity was expressed by the OD490 value. The MTT test results showed that at 24 h after infection (24 hpi), there was no significant difference in the OD450 values ​​between the metformin-treated group and the untreated group, indicating that metformin had little effect on cell activity in the early stage of viral infection. At 48 h (48 hpi), 72 h (72 hpi), and 96 h (96 hpi) of infection, the OD490 values ​​of the metformin-treated group were significantly higher than those of the untreated group (Control, BmNPV+). This indicates that metformin treatment can effectively increase the activity of cells in the late stage of BmNPV infection and reduce the damage of the virus to cells.

[0028] See also Figure 2, TUNEL staining was used to detect cell apoptosis after Metformin treatment, where green fluorescence represents the presence of apoptotic DNA fragments in cells, and blue is DAPI-stained nucleic acid. The results of the TUNEL staining experiment showed that: In the control group (BmNPV-) not infected with BmNPV, no green fluorescence was observed in TUNEL staining, indicating that no obvious apoptosis occurred in the cells. In the Control (BmNPV+) group, i.e. the BmNPV-infected group (untreated with Metformin), green fluorescence was clearly visible, indicating that BmNPV infection caused cell apoptosis. In the Metformin-treated infection group (Metformin (BmNPV+)), green fluorescence was significantly reduced, and the number of apoptotic cells was significantly reduced compared with the untreated group. This shows that Metformin can alleviate cell apoptosis caused by BmNPV infection.

[0029] In summary, CCK8 detection showed that Metformin could significantly increase the survival rate of BmNPV-infected cells. At the same time, TUNEL staining results showed that Metformin treatment could significantly inhibit cell apoptosis caused by BmNPV infection; these results indicate that Metformin has the effect of anti-viral proliferation and protecting cell activity. 2.2 Detection of metformin inhibiting viral proliferation

[0030] Take the silkworm BmN cells with good growth status, plate them one day in advance, add metformin to the final concentration (0.5mM, 1mM) the next day, and use the same volume of culture medium as a control. After continuing to culture for 24h, add 10μL virus particles BV (1.0×10 7 pfu / mL) and added to one well of a 6-well plate with BmN cells treated with metformin. At 24, 48, and 72 h after virus infection (h pi), cells were harvested and genomic DNA was extracted for qPCR detection of viral gene GP64 and VP39 replication. After exporting qPCR data, the data were calculated using an excel worksheet and analyzed using GraphPad Prism 9.3.1. The effective concentration of metformin in silkworm cells was obtained. This provides a reference for in vivo experiments.

[0031] See also Figure 3Compared with the Control, Metformin effectively inhibited viral gDNA copies as the time gradient changed, and its inhibitory effect showed a time-dependent enhancement. In the experiments with different concentrations, there were significant differences in the inhibitory effects of 0.5mM and 1mM Metformin on viral replication (p<0.05), and the effect of 0.5mM was the most obvious. The viral gene expression levels of the 0.5mM Metformin-treated group at 48h and 72h decreased by 36.8% and 74.3%, respectively, while the 1mM group only decreased by 32.5% and 51.2%. The experimental results show that under in vitro conditions, 0.5mM Metformin has the most significant inhibitory effect on the silkworm BmNPV virus, which may be because lower concentrations of Metformin are more conducive to maintaining cell activity, thereby exerting a better antiviral effect. Effect of metformin treatment on detection of viral GP64 protein in virus-infected cells

[0032] The expression level of viral GP64 protein was detected by Western blot to evaluate the effect of metformin drug treatment on viral GP64 protein in BmN cells infected with BmNPV. GP64 is the main envelope protein of BmNPV, and its expression level can reflect the replication and proliferation of the virus in host cells. β-Tubulin was used as an internal reference to verify the consistency of the loading amount of each sample. Figure 4 The intensity of β-Tubulin bands in each experimental group was basically the same, indicating that the loading amount of the samples was uniform and the experimental results were reliable. The expression level of GP64 protein was significantly reduced, and the expression intensity was significantly weakened compared with the untreated group, indicating that Metformin treatment can effectively inhibit the proliferation of BmNPV. Western Blot results showed that Metformin treatment can significantly reduce the expression level of GP64 protein in BmNPV-infected cells, indicating that Metformin has a strong inhibitory effect on the replication and proliferation of BmNPV. Example 4: Quantitative analysis of the viral inhibition efficiency of BmNPV infected cells after metformin treatment by flow cytometry

[0033] The experiment used flow cytometry to detect the effects of different concentrations (0.5 mM and 1 mM) of Metformin on cells infected with BmNPV-EGFP. The control group was treated with DMSO. The detection time points were 48 hours and 72 hours after infection. The expression level of EGFP can be used to assess the extent of viral infection, because the expression of EGFP is related to viral replication and infection.

[0034] The results showed that the cell counts and EGFP intensity at 48 and 72 hours in the Metformin-treated group showed significant differences compared with the DMSO control group. This indicates that Metformin has a significant effect in inhibiting BmNPV viral infection. The effect of the 0.5mM Metformin-treated group was stronger than that of the 1 mM-treated group. The results of the 72-hour treatment were more significant than those of the 48-hour treatment, indicating that the viral inhibitory effect of Metformin increased over time. In summary, Metformin showed a significant effect in inhibiting BmNPV viral infection. Example 5: Detection of metformin against BmNPV virus in P50 strain of silkworm

[0035] The 5th-instar well-grown P50 strain silkworms were divided into three groups: the polyhedrosis virus control group (Control), the PB1 group containing metformin (Ployhedra + 0.5mM Metformin), and the PB2 group (Ployhedra + 1mMMetformin). Each group was orally fed with 5μL ODV-type BmNPV virus (1.0×10 7 pfu / mL) to complete the addition of the virus, and then the drug-added group licked metformin and fed (the control group did not add metformin, but added PBS). After 24, 48, and 72 hours of feeding, blood, midgut, and fat body were obtained by dissection to extract mRNA and genomic DNA for qPCR detection of the expression and copy number of viral genes GP64 and VP39. After exporting the qPCR data, it was calculated through an excel worksheet and analyzed after drawing with GraphPad Prism 9.3.1. The optimal concentration for inhibiting viral replication and proliferation and reducing viral expression was obtained.

[0036] The results are as follows Figure 6 It was shown that metformin had a significant inhibitory effect on viral expression. Compared with the control group (Control), metformin could effectively inhibit the viral gDNA copy number as the time gradient changed, which further showed that metformin had a good inhibitory effect on the replication of the silkworm BmNPV virus. The study found that the 0.5mM concentration gradient had the best inhibitory effect on the virus, and could effectively inhibit the replication of Bombyx mori nuclear polyhedrosis virus (BmNPV) in cells and silkworms. The results showed that compared with the control group, metformin could inhibit the proliferation of BmNPV virus in silkworms. Figure 6In the figure, A is the number of viral mRNA copies in the midgut; B is the number of viral gDNA in the midgut; C is the number of viral mRNA copies in the hemolymph; D is the number of viral gDNA in the hemolymph; E is the number of viral mRNA copies in the fat body; F is the number of viral gDNA in the fat body. Example 6: Silkworm feeding experiment

[0037] Take 5-year-old well-grown silkworms, add poison, and feed the control group orally with PBS sprayed with mulberry leaves. At the same time, start feeding the mulberry leaves sprayed with the optimal concentration of metformin to the drug-adding group (0.5mM Metformin). At the same time, record the data of larval weight, silkworm pupa weight, mortality, activity rate, cocoon rate, cocoon weight, cocoon layer rate, and cocoon shell weight of each group, and draw a box plot for effect analysis. Analyze the effect of metformin treatment on inhibiting BmNPV virus after the outbreak of the virus, and explore whether metformin can improve the quality and yield of silk cocoons.

[0038] The results are as follows Figure 7 As shown in the figure, through careful observation of the silkworm phenotype, it can be seen that the 0.5 mM concentration gradient will have a specific effect on the growth of the silkworm itself. In the cell experiment, this concentration gradient not only showed the most significant virus inhibition effect, but also promoted the growth and development of the silkworm itself. When metformin was added, the weight of the silkworm increased, the cocoon weight increased accordingly, and the food intake increased significantly. Among them, A is the weight of the larvae, B is the weight of the silkworm pupae, C is the mortality rate, D is the activity rate, E is the cocoon rate, F is the weight of the cocoon, G is the cocoon layer rate, and H is the healthy pupa rate.

[0039] In addition, studies on the mechanism have shown that in silkworms (Bombyx mori), metformin significantly reduces the energy state (ATP level) in cells by activating the AMP-activated protein kinase (AMPK) signaling pathway, thereby inhibiting the energy supply required for viral replication and blocking the spread and spread of the virus. In terms of promoting the growth and development of silkworms, metformin may promote their growth and development to a certain extent by regulating the AMPK signaling pathway and optimizing the energy metabolism of silkworms. At the same time, metformin may also improve the silkworm's tolerance to environmental stress (such as viral infection) by enhancing its immunity and anti-stress ability. This effect not only helps silkworms survive in a viral infection environment, but also may improve their ability to synthesize silk protein, thereby having a positive impact on the economic benefits of the sericulture industry.

[0040] At the molecular level, metformin may also play an antiviral role by affecting the expression of genes related to viral infection in silkworm cells, such as interferon (IFN) and interferon-stimulated genes (ISGs). Studies have shown that metformin can upregulate the expression of some genes with antiviral functions, such as genes involved in immune response, thereby enhancing the immune defense ability of silkworms themselves; at the same time, it can also downregulate the expression of some host genes that virus replication depends on, interfering with the life cycle of the virus. For example, metformin may block the replication process of the virus by inhibiting the transcription and translation of host genes (such as DNA polymerase and early expression genes) required for BmNPV virus replication. These mechanisms of action provide a solid theoretical basis for the application of metformin in the prevention and control of BmNPV virus in silkworms.

[0041] Metformin may also induce cell autophagy to clear intracellular infected virus particles, thereby further inhibiting viral replication and spread. Autophagy, as a cellular self-protection mechanism, plays an important role in antiviral immunity. Metformin activates autophagy-related pathways and enhances the ability of silkworm cells to clear virus particles, thus providing silkworms with an endogenous antiviral defense mechanism.

[0042] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the scope of protection determined by the claims submitted for the present invention.

Claims

1. The use of metformin in the preparation of drugs for preventing or treating silkworm nuclear polyhedrosis virus disease.

2. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The dosage form of the medicine is liquid preparation or wettable powder.

4. A method for preventing or treating silkworm nuclear polyhedrosis virus disease, the method comprising administering a preparation containing metformin to silkworms.

5. The method according to claim 4, wherein the preparation is administered before the silkworm is infected with BmNPV, or within 12 hours after being infected with BmNPV.

6. According to claim, the administration method of the preparation is to feed the silkworms with mulberry leaves sprayed with the metformin preparation.

7. The application of metformin in the preparation of drugs for increasing the weight and cocoon quality of silkworms.

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