Application of metformin in preparing a medicament for preventing or treating Bombyx mori nuclear polyhedrosis virus disease

Abstract

The present invention belongs to the field of agricultural biotechnology. Specifically, the present invention relates to the application of metformin in the preparation of a drug for preventing or treating Bombyx mori nuclear polyhedrosis virus disease. Through research, it is found that metformin has preventive and therapeutic effects on Bombyx mori infected with nuclear polyhedrosis virus. At the same time, it can also promote 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 present invention belongs to the field of agricultural biotechnology. Specifically, the present invention relates to the application of metformin in the preparation of a medicament for preventing or treating Bombyx mori nucleopolyhedrovirus disease. Background Art

[0002] The silkworm (Bombyx mori), as a silk-secreting insect with important economic value in Lepidoptera insects, is not only a classical model organism for entomological research but also occupies an important position in the development of agricultural economy and silk industry in various countries. However, the infection of Bombyx mori nucleopolyhedrovirus (BmNPV) has become one of the main diseases restricting the production of sericulture. The genome of BmNPV consists of double-stranded circular DNA of about 128 kilobase pairs. After infecting the silkworm, it often triggers a serious pathological process of host body surface swelling, systemic suppuration and even death, with extremely high infectivity and lethality. Its infection process includes: (1) virus particles invade the midgut epithelial cells; (2) replicate and assemble new virus particles in the nucleus; (3) the virus spreads to other tissues, such as fat body, hemocytes, etc. through cell lysis or budding; (4) ultimately leads to the death of the host, and a large number of polyhedra are formed in the corpse and released into the environment. Research shows that BmNPV infection not only has a significant negative impact on the growth and development of silkworms, but also realizes viral immune escape through mechanisms such as inhibiting the host immune system, interfering with apoptosis and endoplasmic reticulum stress response. Therefore, BmNPV infection leads to large-scale outbreaks of silkworm diseases, and the loss of cocoon production caused by it accounts for more than 60% of the total loss caused by silkworm diseases, seriously threatening the sustainable development of sericulture.

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

[0004] As a classical hypoglycemic drug, metformin has been found to have broad antiviral activities in recent years. For example, some studies have shown that it has inhibitory effects on viruses such as hepatitis C virus, rotavirus, bovine diarrhea virus, enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). Its mechanism of action involves multiple levels, including energy metabolism regulation, immune response regulation and autophagy processes. Whether metformin can be used in silkworms to combat Bombyx mori nucleopolyhedrovirus disease has not been reported yet. Summary of the Invention

[0005] After extensive screening and research, the inventors of the present invention unexpectedly found that metformin can play a role in preventing and inhibiting the infection of Bombyx mori nucleopolyhedrovirus, thus completing the present invention. The technical solution of the present invention is as follows:

[0006] In one aspect of the present invention, the present invention discloses the application of metformin in the preparation of a drug for preventing or treating Bombyx mori nucleopolyhedrovirus disease.

[0007] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0008] In one embodiment, the dosage form of the drug is a liquid preparation or a wettable powder.

[0009] In one aspect of the present invention, the present invention discloses a method for preventing or treating Bombyx mori nucleopolyhedrovirus disease, the method comprising administering to Bombyx mori a preparation containing metformin.

[0010] In one embodiment, the administration time of the preparation is before the Bombyx mori is infected with BmNPV, or within 12 hours after the Bombyx mori is infected with BmNPV.

[0011] In one embodiment, the administration method of the preparation is to administer to Bombyx mori by feeding mulberry leaves sprayed with the metformin preparation.

[0012] In one aspect of the present invention, the present invention provides the application of metformin in the preparation of a drug for increasing the body weight and cocoon quality of Bombyx mori. Beneficial effects

[0013] (1) In the present invention, metformin can inhibit the energy supply of the virus, effectively block the spread and diffusion of the virus, and also play a good prevention and treatment role in the initial stage of the disease, providing a new prevention and treatment approach for the prevention and treatment of Bombyx mori nucleopolyhedrovirus disease.

[0014] (2) The metformin of the present invention can also play a role in promoting the growth and development of Bombyx mori and reducing the damage of the virus to the host. This effect not only helps Bombyx mori survive in a virus-infected environment, but also may improve its silk protein synthesis ability, thus having a positive impact on the economic benefits of the sericulture industry.

[0015] (3) Advantage of biodegradability: Metformin has a certain biodegradability. After entering the natural water body or soil environment, it can be gradually decomposed under the action of microorganisms, etc. This is different from some organic pollutants that are difficult to degrade, which 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 the production and application processes. Description of the drawings

[0016] Figure 1, Toxicity detection of metformin on silkworm cells BmN.

[0017] Figure 2 , Detection of apoptosis of cells after Metformin treatment by TUNEL staining.

[0018] Figure 3 , Effects of different concentrations of Metformin on viral gene expression and replication in cells.

[0019] Figure 4 , Detection of the expression level of viral GP64 protein by Western blot with Metformin.

[0020] Figure 5 , Quantitative analysis of the virus inhibition efficiency of BmNPV-infected cells after Metformin treatment by flow cytometry.

[0021] Figure 6 , Analysis of the inhibitory effect of metformin on silkworms infected with virus.

[0022] Figure 7 , Effects of metformin on the growth and development of silkworms. Specific implementation manners

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. 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 used to limit the present invention.

[0024] Example 1: Cytotoxicity detection of metformin in silkworm cells BmN

[0025] One day in advance, inoculate well-growing silkworm BmN cells into a 96-well plate, inoculate about 5×10³ cells per well, and add 100 μL of complete medium. Place the cells in an incubator at 27 °C and 5% CO2 for overnight culture to allow the cells to adhere and enter the logarithmic growth phase.

[0026] On the next day, according to the concentration gradients (500 μM, 1 mM) determined by the preliminary experiment, add the corresponding volume of metformin solution to each well to make the final volume of each well 150 μL. Set up a blank control group containing only the culture medium (without cells and drugs) and a negative control group containing only cells (without drugs), with 3 - 5 replicates in each group. Continue to culture the treated cells and perform detections at time gradients of 0 h, 24 h, 48 h, 72 h, and 96 h. At each detection time point, add 10 μL of CCK-8 reagent to each well, gently mix, and continue to culture for 2 - 4 hours. Measure the absorbance (OD value) of each well at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and record the data.

[0027] Cell survival rate (%) = (OD value of the experimental group - OD value of the blank control group / OD value of the negative control group - OD value of the blank control group) * 100%.

[0028] See Figure 1 , 0.5 mM and 1 mM of metformin have no toxicity to BmN cells and can even promote cell growth to a certain extent.

[0029] Example 2: Detection of the antiviral efficacy of metformin against BmNPV in Bombyx mori BmN cells

[0030] 2.1 Detection of metformin against cell apoptosis

[0031] The CCK8 method was used to evaluate the survival protection effect of metformin on cells at different time points (24 h, 48 h, 72 h, 96 h), and the cell viability was represented by the OD490 value. The MTT test results showed that: at 24 hours post-infection (24 h.p.i.), there was no significant difference in the OD450 values between the Metformin treatment group and the untreated group, indicating that Metformin had little effect on cell viability in the early stage of virus infection. At 48 hours post-infection (48 h.p.i.), 72 hours post-infection (72 h.p.i.), and 96 hours post-infection (96 h.p.i.), the OD490 value of the Metformin treatment group was significantly higher than that of the untreated group (Control, BmNPV+). This indicates that Metformin treatment can effectively improve the viability of cells in the late stage of BmNPV infection and reduce the damage of the virus to cells.

[0032] See Figure 2, cell apoptosis after Metformin treatment was detected by TUNEL staining. Among them, 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 (BmNPV-) group, which was the control group not infected with BmNPV, no green fluorescence was observed in the TUNEL staining, indicating that there was no obvious apoptosis in the cells. In the Control (BmNPV+) group, that is, the BmNPV-infected group (without Metformin treatment), green fluorescence was clearly visible, indicating that BmNPV infection induced cell apoptosis. In the infected group treated with Metformin (Metformin (BmNPV+)), the green fluorescence was significantly reduced, and the number of apoptotic cells decreased significantly compared with the untreated group. This indicates that Metformin can reduce cell apoptosis caused by BmNPV infection.

[0033] In summary, the CCK8 assay showed that Metformin could significantly increase the survival rate of BmNPV-infected cells. At the same time, the results of TUNEL staining showed that Metformin treatment could significantly inhibit cell apoptosis caused by BmNPV infection; these results indicate that Metformin has the effects of antiviral proliferation and protecting cell activity.

[0034] 2.2 Detection of Metformin inhibiting virus proliferation

[0035] Take Bombyx mori BmN cells in good growth state, plate them one day in advance, and add Metformin to a final concentration (0.5 mM, 1 mM) the next day. At the same time, use the same volume of medium as the control. After continuing to culture for 24 h, add 10 μL of virus particles BV (1.0×10 7 pfu / mL) to one well of a 6-well plate containing BmN cells treated with Metformin. At 24, 48, and 72 hours post-infection (h p.i.), collect the cells, extract genomic DNA, and use it for qPCR to detect the replication of virus genes GP64 and VP39. After exporting the qPCR data, calculate it through an excel worksheet, and analyze it after plotting with GraphPad Prism 9.3.1. Obtain the effective concentration of Metformin in Bombyx mori cells. Provide a reference for in vivo experiments.

[0036] See Figure 3, compared with Control, Metformin effectively inhibited viral gDNA copies with the change of time gradient, and its inhibitory effect showed a time-dependent enhancement. In the experiments with different concentrations, there were significant differences in the inhibitory effects of 0.5 mM and 1 mM Metformin on viral replication (p<0.05), and the effect of 0.5 mM was the most obvious. The viral gene expression levels in the 0.5 mM Metformin treatment group decreased by 36.8% and 74.3% at 48 h and 72 h respectively, while those in the 1 mM group only decreased by 32.5% and 51.2%. The experimental results showed that under in vitro conditions, 0.5 mM Metformin had the most significant inhibitory effect on Bombyx mori BmNPV virus, which might be because a lower concentration of Metformin was more conducive to maintaining cell viability, thus exerting a better antiviral effect.

[0037] Detection of viral GP64 protein in virus-infected cells treated with Metformin

[0038] The expression level of viral GP64 protein was detected by Western blot to evaluate the situation of viral GP64 protein in BmN cells infected with BmNPV after treatment with Metformin. 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. See Figure 4 , the intensities of β-Tubulin bands in each experimental group were basically the same, indicating that the loading amounts of the samples were uniform and the experimental results were reliable. The expression level of GP64 protein decreased significantly, and the expression intensity was significantly weakened compared with the untreated group, which indicated that Metformin treatment could effectively inhibit the proliferation of BmNPV. The Western Blot results showed that Metformin treatment could significantly reduce the expression level of GP64 protein in BmNPV-infected cells, indicating that Metformin had a strong inhibitory effect on the replication and proliferation of BmNPV.

[0039] Example 4: Quantitative analysis of the virus inhibition efficiency of BmNPV-infected cells after treatment with Metformin by flow cytometry

[0040] Flow cytometry was used in the experiment 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 evaluate the degree of virus infection because the expression of EGFP is related to the replication and infection of the virus.

[0041] The results showed that compared with the DMSO control group, there were significant differences in cell count and EGFP intensity in the Metformin treatment group at 48 hours and 72 hours. This indicates that Metformin has a significant effect on inhibiting BmNPV virus infection. The effect of the 0.5 mM Metformin treatment group was stronger than that of the 1 mM treatment group. The treatment results at 72 hours were more significant than those at 48 hours, indicating that the virus inhibition effect of Metformin increases with time. In summary, Metformin shows a significant effect on inhibiting BmNPV virus infection.

[0042] Example 5: Detection of the anti-BmNPV virus effect of metformin in the silkworm strain P50

[0043] Take well-growing 5th instar silkworms of the P50 strain and divide them into 3 groups, namely the polyhedron virus control group (Control), the PB1 group containing metformin (Ployhedra + 0.5 mM Metformin), and the PB2 group (Ployhedra + 1 mM Metformin). Each group was orally fed 5 μL of ODV-type BmNPV virus (1.0×10 7 pfu / mL) to complete virus inoculation. Then, the metformin-fed groups were fed with metformin (the control group was not supplemented with metformin but with PBS). After 24, 48, and 72 hours of rearing, blood, midgut, and fat body were collected by dissection for extracting mRNA and genomic DNA for qPCR detection of the expression levels and copy numbers of the viral genes GP64 and VP39. After exporting the qPCR data, calculations were performed using an excel worksheet, and analysis was carried out after plotting with GraphPad Prism 9.3.1. The optimal concentration for inhibiting virus replication and reducing virus expression was obtained.

[0044] The results are as Figure 6 shown. Metformin has a significant inhibitory effect on virus expression. Compared with the control group (Control), with the change of the time gradient, Metformin can effectively inhibit the copy number of viral gDNA, which further indicates that Metformin has a good inhibitory effect on the replication of the BmNPV virus in silkworms. The study found that the inhibitory effect on the virus was the best at a concentration gradient of 0.5 mM, which can effectively inhibit the replication of the Bombyx mori nucleopolyhedrovirus (BmNPV) in cells and silkworms. The results showed that compared with the control group, metformin can inhibit the proliferation of the BmNPV virus in silkworms. Figure 6Among them, A is the replication quantity of viral mRNA in the midgut; B is the quantity of viral gDNA in the midgut; C is the replication quantity of viral mRNA in the hemolymph; D is the quantity of viral gDNA in the hemolymph; E is the replication quantity of viral mRNA in the fat body; F is the quantity of viral gDNA in the fat body.

[0045] Example 6: Experiment on feeding silkworms with medicine

[0046] Take well - growing silkworms at the 5th instar. After virus inoculation, in the control group, mulberry leaves sprayed with PBS were fed orally, and at the same time, mulberry leaves sprayed with the optimal concentration of metformin (0.5 mM Metformin) were fed to the medicine - added group. At the same time, data such as the weight of larvae, the weight of silkworm pupae, the mortality rate, the activity rate, the cocoon - forming rate, the weight of cocoons, the cocoon layer rate, and the weight of the cocoon shell were recorded, and box plots were drawn for effect analysis. Analyze the therapeutic effect of metformin on inhibiting BmNPV virus after virus outbreak, and explore whether metformin can improve the quality and yield of silkworm cocoons.

[0047] The results are as Figure 7 shown. Through careful observation of the phenotypes of silkworms, it can be seen that the 0.5 mM concentration gradient has a specific impact on the growth of silkworms themselves. In cell experiments, this concentration gradient not only shows the most significant virus - inhibiting effect but also has a promoting effect on the growth and development of silkworms themselves. When metformin is added, the weight of silkworms increases, the weight of cocoons formed accordingly increases, and the food intake significantly increases. Among them, A is the weight of larvae, B is the weight of silkworm pupae, C is the mortality rate, D is the activity rate, E is the cocoon - forming rate, F is the weight of cocoons, G is the cocoon layer rate, and H is the rate of healthy pupae.

[0048] In addition, research on the mechanism shows that in the silkworm (Bombyx mori), metformin significantly reduces the intracellular energy state (ATP level) by activating the AMP - activated protein kinase (AMPK) signaling pathway, thereby inhibiting the energy supply required for virus replication, and then blocking the spread and diffusion of the virus. In promoting the growth and development of silkworms, metformin optimizes the energy metabolism of silkworms by regulating the AMPK signaling pathway, and may promote its growth and development to a certain extent. At the same time, metformin may also enhance the immunity and stress resistance of silkworms, improving their tolerance to environmental stresses (such as virus infection). This effect not only helps silkworms survive in a virus - infected environment but may also improve their silk - protein synthesis ability, thus having a positive impact on the economic benefits of the sericulture industry.

[0049] At the molecular level, metformin may also play an antiviral role by affecting the gene expression related to virus infection in silkworm cells, such as interferon (IFN) and interferon-stimulated genes (ISGs). Research has shown that metformin can upregulate the expression of some genes with antiviral functions, such as genes involved in immune responses, thereby enhancing the immune defense ability of silkworms themselves; at the same time, it can also downregulate the expression of some host genes on which virus replication depends, interfering with the virus life cycle. For example, metformin may block the virus replication process by inhibiting the transcription and translation of host genes required for BmNPV virus replication (such as DNA polymerase and early expression genes). These mechanisms of action provide a solid theoretical basis for the application of metformin in the prevention and control of BmNPV virus in silkworms.

[0050] Metformin may also further inhibit virus replication and spread by inducing autophagy to clear virus particles infected in cells. Autophagy, as a cell self-protection mechanism, plays an important role in antiviral immunity. Metformin activates the autophagy-related pathway, enhancing the ability of silkworm cells to clear virus particles, thereby providing an endogenous antiviral defense mechanism for silkworms.

[0051] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope determined by the claims submitted for the present invention.

Claims

1. Use of metformin in the preparation of a medicament for preventing or treating Bombyx mori nuclear polyhedrosis virus disease.

2. The application according to claim 1, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

3. The application according to claim 1, characterized in that The dosage form of the medicament is a liquid preparation or a wettable powder.

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