Application of cucurbitacin B in preparing medicine for preventing or treating silkworm nuclear polyhedrosis virus disease

Abstract

This invention discloses the application of cucurbitacin B in the preparation of drugs for the treatment or prevention of silkworm nucleopolyhedrovirus. The research of this invention shows that the terpene compound cucurbitacin B can effectively inhibit the replication of silkworm nucleopolyhedrovirus (BmNPV) in silkworm cells and within the silkworm. By using appropriate concentrations and drug addition times, cucurbitacin B can inhibit the replication of the virus in silkworm cells and within the silkworm, providing a new approach for the prevention of BmNPV infection in silkworms and laying the foundation for the prevention and control of viral infections in sericulture.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the application of cucurbitacin B in the preparation of drugs for the prevention or treatment of silkworm nucleopolyhedrovirus disease. Background Technology

[0002] Cucurbitacin B is a tetracyclic triterpenoid natural compound extracted from plants of the Cucurbitaceae family, mainly found in the roots, stems, leaves, and fruits of melons, watermelons, cucumbers, and bitter melons. Its chemical structure is complex; it is poorly soluble in water but readily soluble in organic solvents, exhibiting significant biological activity and is widely used in medicine and agriculture. Modern research shows that cucurbitacin B possesses various biological activities, including hepatoprotective, anti-inflammatory, and antitumor effects. In the 1970s and 1980s, cucurbitacin tablets (containing cucurbitacin B and cucurbitacin E) were developed in China for adjuvant treatment of hepatitis and primary liver cancer. In recent years, cucurbitacin B has received widespread attention for its antiviral activity in humans. Studies have shown that it has inhibitory effects on various viruses, particularly demonstrating potential against hepatitis B virus (HBV), human immunodeficiency virus (HIV), and herpes simplex virus, providing an important direction for the development of novel antiviral drugs.

[0003] Silkworms, as silk-producing insects, have significant economic value. Bombyxmori nuclear polyhedrosis virus (BmNPV) is one of the common pathogenic viruses in silkworm farming. It belongs to the double-stranded DNA virus family (Baculoviridae). Silkworms, as silk-producing insects, have significant economic value. BmNPV is a baculovirus, and a key characteristic is that during its infection cycle, it produces two different types of viral particles: one nucleocapsid is released into the intercellular matrix through budding of the cytoplasm membrane, called a budding virus; the other nucleocapsid acquires an envelope in the host cell nucleus, its envelope having a lipid bilayer structure, with the virion embedded in protein crystalline inclusion bodies, called a polyhedrotic virion. There are two main routes of viral infection: oral infection and wound infection, with oral infection being the predominant route. Inclusion bodies (OBs) are ingested orally by the host and lyse in the highly alkaline environment of the midgut, releasing inclusion body-derived viruses (ODVs). ODVs cross the peritrophic membrane and bind to the microvilli of midgut epithelial cells, releasing their nucleocapsids into the cytoplasm. As the virus replicates and the nucleocapsid assembles within the nucleus or crosses directly, the nucleocapsid is transported to the matrix membrane, budding to form progeny viral particles—a process known as primary infection. Subsequently, BV infects other cells, such as tracheal cells and hemocytes, or circulates in the hemolymph, eventually causing systemic infection—a process known as secondary infection. Bombyx mori nucleopolyhedrovirus (BmNPV) causes significant economic losses to the sericulture industry annually. Currently, there are no effective methods for prevention or treatment. Therefore, research into drugs that inhibit BmNPV is of great importance. Summary of the Invention

[0004] Based on the need to prevent and control silkworm nucleopolyhedrovirus (BmNPV), this invention, through extensive research and screening, discovered the relationship between cucurbitacin B and BmNPV, and thus proposed this invention, namely, using cucurbitacin B to prevent and control silkworm nucleopolyhedrovirus.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides the use of cucurbitacin B in the preparation of a medicament for preventing infection with silkworm nucleopolyhedrovirus.

[0007] In one aspect, the present invention provides the use of cucurbitacin B in the preparation of a medicament for treating silkworm nucleopolyhedrovirus infection.

[0008] Preferably, the final concentration of cucurbitacin B in inhibiting the infection of silkworm nucleopolyhedrovirus (BmNPV) cells is 0.2 μM - 0.6 μM, and the maximum effective feeding concentration in inhibiting the infection of silkworm nucleopolyhedrovirus (BmNPV) bodies is 120 μM.

[0009] In one embodiment, the drug further includes pharmaceutically acceptable excipients.

[0010] In one embodiment, the drug dosage form can be one of a variety of pharmaceutically applicable dosage forms; the preferred dosage form is granules or liquid preparations.

[0011] In one aspect, this invention discloses a method for inhibiting silkworm nucleopolyhedrovirus (BmNPV) using cucurbitacin B. By rationally utilizing cucurbitacin B to inhibit the replication of BmNPV in cells and silkworm bodies, it can be mixed into the silkworm's food (mulberry leaves) to exert a therapeutic effect when infection occurs or within 12 hours after infection; alternatively, cucurbitacin B can be added to the silkworm's food 12-24 hours before infection to inhibit BmNPV proliferation and play a preventive role against infection. Beneficial effects

[0012] This invention provides a new approach to the treatment of nucleopolyhedrovirus infection in silkworms and lays the foundation for the prevention and control of viral infections in sericulture.

[0013] The cucurbitacin B used in this invention is a tetracyclic triterpenoid natural compound extracted from Cucurbitaceae plants. It is widely used in the pharmaceutical and agricultural fields, exhibiting good safety and efficacy. Furthermore, it is inexpensive and non-toxic. Attached Figure Description

[0014] Figure 1 To detect cell viability after adding different concentrations of cucurbitacin B;

[0015] Figure 2 The effect of different concentrations of cucurbitacin B on the proliferation of BmNPV in cells after 24 h of viral infection. VP39 Genetic testing;

[0016] Figure 3 To detect the effect of different concentrations of cucurbitacin B on the proliferation of BmNPV in cells after 24 h of viral infection, with Tubulin as an internal control.

[0017] Figure 4 Fluorescence observation of the effect of different concentrations of cucurbitacin B on the proliferation of BmNPV in cells after 24 h of treatment with virus-infected cells;

[0018] Figure 5The effect of different concentrations of cucurbitacin B on BmNPV proliferation in cells 12 h after viral infection. VP39 Genetic testing;

[0019] Figure 6 The effect of cucurbitacin B treatment on the proliferation of BmNPV in silkworms VP39 Genetic testing. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0021] In this invention, cucurbitacin B was purchased from Maclean's (catalog number: C860601); CCK-8 was purchased from White Shark (catalog number: BS350A); the NovoStart SYBR qPCR SuperMix kit was purchased from Nearshore Protein (catalog number: E096); proteinase K was purchased from Beijing Tiangen (catalog number: RT403); anti-β-Tubulin antibody, Goat Anti-Rabbit IgG antibody, and Goat Anti-Mouse IgG antibody were purchased from Beijing TransGen (catalog numbers: HC101; HS101; HS201, respectively); and the anti-VP39 antiserum was prepared by Hangzhou Huaan.

[0022] Example 1: Inhibition of silkworm nucleopolyhedrovirus replication in vitro using cucurbitacin B:

[0023] To detect the cytotoxicity of cucurbitacin B, healthy silkworm BmN cells were seeded one day in advance. The next day, different concentrations of DMSO-dissolved cucurbitacin B were added to different wells to achieve final concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 μM, respectively. The control was used as the culture medium. Cells were cultured for another 48 h. Cell viability was then assessed using a CCK-8 assay kit. Figure 1 As shown, cucurbitacin B had no effect on the activity of silkworm cells at a concentration of 0.6 μM.

[0024] In the in vitro virus infection experiment, healthy silkworm BmN cells were seeded two days in advance. On the third day, 10 μL of cucurbitacin B at different concentrations were added during medium change to achieve final concentrations of 0.2, 0.4, 0.5, and 0.6 μM, respectively. The control was the same volume of DMSO. After culturing for another 24 h, the same volume of fluorescently labeled BV-EGFP virus particles was added. After 48 h of infection, viral fluorescence was observed first, and then cells were harvested for PCR and Western blotting to detect viral replication. The results are as follows: Figure 2 , 3 As shown in Figure 4, 0.2 μM cucurbitacin B can inhibit BmNPV in cells. The inhibitory activity increases with increasing concentration, showing a significant difference compared to the control group (con) (* indicates P < 0.05 compared to control, ** indicates P < 0.01 compared to control). This indicates that cucurbitacin B can prevent BmNPV infection in silkworm cells.

[0025] In addition, healthy silkworm BmN cells were seeded two days in advance. On the third day, 10 μL of the same volume of fluorescently labeled BV-EGFP virus particles were added. After 12 h, different concentrations (0.2, 0.4, 0.6 μM) of cucurbitacin B were added, with the control being the same volume of DMSO. After culturing for another 36 h, the cells were harvested to detect virus replication. The results are as follows: Figure 5 As shown in the figure. The results showed that, compared with the control group, 0.2 μM cucurbitacin B could inhibit the proliferation of the virus in cells. With increasing concentration, its inhibitory activity showed an increasing trend, and there was a significant difference compared with the control group (con) (* indicates P<0.05 compared with the control, ** indicates P<0.01 compared with the control). That is, cucurbitacin B can play a therapeutic role in the infection of silkworm cells with BmNPV.

[0026] Example 2: Inhibition of silkworm nucleopolyhedrovirus replication in vivo using cucurbitacin B:

[0027] In vivo virus infection experiments were conducted on fifth-instar silkworm larvae, who were orally fed 5 μL of 120 μM cucurbitacin B (with the same volume of DMSO as a control). After 12 h, 10 μL of inclusion body-derived virus (ODV) was added. The larvae were then fed mulberry leaves for another 48 h. Genomic DNA was extracted from the midgut of the larvae, and the viral genes were detected by quantitative real-time PCR. VP39 The replication process within the silkworm body yielded the following results: Figure 6 As shown in the figure, cucurbitacin B was able to inhibit the proliferation of the virus in silkworms compared with the control group.

[0028] In this embodiment, the method for detecting nucleopolyhedrovirus replication by real-time PCR involves extracting total DNA from samples using a genome extraction reagent, designing real-time primers based on the nucleopolyhedrovirus gene, and then using silkworms as an example. GAPDH The gene is used as an internal control, and the primers used for its quantitative fluorescence assay are as follows:

[0029] BmNPV- VP39 Forward: 5'-ACTTTTCATGATGTCACTGC-3' (SEQ ID NO.1)

[0030] BmNPV- VP39 Reverse: 5'-AGTACTTGCAAATCGACACG-3' (SEQ ID NO.2)

[0031] Bm- GAPDH Forward: 5'-CATTCCGCGTCCCTGTTGCTAAT-3' (SEQ ID NO.3)

[0032] Bm- GAPDH Reverse: 5'-GCTGCCTCCTTGACCTTTTGC-3' (SEQ ID NO.4)

[0033] The extracted DNA and fluorescent primers were prepared according to the instructions of the NovoStart SYBR qPCR SuperMix kit for quantitative PCR. The PCR reaction system was run using a CFX96 Real-Time PCR instrument (Bio-Ray). The reaction conditions were: preheating at 94℃ for 30 seconds; denaturation at 94℃ for 10 seconds; annealing and extension at 60℃ for 30 seconds, followed by fluorescence collection. This process was repeated for 40 cycles. The detection results were analyzed using a 2... -Δ Δ CT The method is used for analysis.

[0034] In this embodiment, the method for detecting nucleopolyhedrovirus replication by Western blotting involves separating the total protein of cell samples by SDS-PAGE, transferring it to a PVDF membrane, incubating it with different antibodies (primary antibody against VP39, primary antibody against β-Tubulin, and corresponding secondary antibodies), and finally detecting the expression level of proteins on the membrane by color development.

[0035] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. The application of cucurbitacin B in the preparation of drugs for preventing silkworm infection with nucleopolyhedrovirus BmNPV, characterized in that, The cucurbitacin B was used to inhibit the replication of silkworm nucleopolyhedrovirus.

2. The application of cucurbitacin B in the preparation of drugs for treating silkworm infection with nucleopolyhedrovirus BmNPV, characterized in that, The cucurbitacin B was used to inhibit the replication of silkworm nucleopolyhedrovirus.

3. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 1 or 2, characterized in that, The drug is in the form of solid granules or liquid preparation.

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