Use of harringtonine in the preparation of an agent against a senecavirus

Abstract

The application provides an application of harringtonine in preparation of an anti-Senecavirus preparation and belongs to the technical field of preparation of antiviral drugs. The application provides a new use of harringtonine, the harringtonine can effectively inhibit replication of Senecavirus and reduce activity of the Senecavirus, and can be used for preparation of an anti-Senecavirus preparation.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug preparation technology, specifically relating to the application of a homoharringtonine in the preparation of an anti-Seneca virus formulation. Background Technology

[0002] In existing technologies, Seneca Valley Virus (SVV) belongs to the small RNA virus family and exhibits typical characteristics of small RNA viruses. The virus particles are spherical with icosahedral symmetry and lack an envelope. The SVV genome is a single-stranded positive-sense RNA, approximately 7.2 kb in length, containing two non-coding regions (5' UTR and 3' UTR) and an open reading frame. Pigs are the only natural host for SVV infection, which causes Seneca virus disease, manifested as vesicular lesions on the snout, coronary band, and interdigital spaces. Its clinical symptoms are highly similar to other swine vesicular diseases (such as foot-and-mouth disease and swine vesicular disease), making them difficult to distinguish. Furthermore, SVV is associated with sudden mortality in newborn piglets. The disease is spreading globally, particularly in regions with concentrated pig farming industries such as the United States and Brazil, where multiple outbreaks have caused severe economic losses to the pig farming industry. Currently, there are no safe and effective vaccines or drugs for Seneca virus disease; therefore, the development of safe and effective antiviral drugs is of great significance for epidemic prevention and control.

[0003] Harringtonine (HT) is a natural alkaloid first isolated from plants of the Cephalotaxus genus in 1963. HT possesses various biological functions. It can inhibit protein synthesis by blocking peptide bond formation and aminoacyl-tRNA binding; promote apoptosis by activating the caspase pathway and disrupting mitochondrial membrane potential; induce cell arrest in the G1 phase and inhibit cell proliferation; and play an important role in anti-tumor activity, and can be used to treat acute and chronic myeloid leukemia. This invention newly discovers that HT possesses anti-Seneca virus activity, which can be used to prepare anti-Seneca virus preparations. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for thiamethoxam alkaloids in the preparation of anti-Seneca virus agents.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A drug for treating Seneca virus disease contains homoharringtonine.

[0007] Application of holohartene in the preparation of anti-Seneca virus agents.

[0008] The application described above is characterized in that the effective concentration of the homoharringtonine in the anti-Seneca virus preparation is 1~30 μM.

[0009] The application described above is characterized in that the effective concentrations of the homoharringtonine in the anti-Seneca virus preparation are 1 μM, 10 μM, and 20 μM.

[0010] The beneficial effects of this invention are as follows:

[0011] This invention provides a novel use for homoharringtonine in the in vitro treatment of Seneca virus. Homoharringtonine can effectively inhibit the replication of Seneca virus and reduce its activity, and can be used to prepare anti-Seneca virus agents. Attached Figure Description

[0012] Figure 1 The effect of different concentrations of homoharringtonine on the viability of BHK-21 cells;

[0013] Figure 2 The effect of different concentrations of homoharringtonine on the viability of PK-15 cells;

[0014] Figure 3 The effect of different concentrations of holoharbine on the proliferation of recombinant SVV strain rSVV-eGFP;

[0015] Figure 4 To detect the effect of different concentrations of homoharringtonine on SVV replication (expression of viral capsid protein VP3) using Western blot (BHK-21 cells);

[0016] Figure 5 To detect the effect of different concentrations of homoharringtonine on SVV replication (expression of viral capsid protein VP3) using Western blot (PK-15 cells);

[0017] Figure 6 The effect of different concentrations of homoharringtonine on SVV titer was detected using TCID50 assay (BHK-21 cells).

[0018] Figure 7 The effect of different concentrations of homoharringtonine on SVV titers was detected using TCID50 assay (PK-15 cells).

[0019] Figure 8 To detect the effect of homoharringtonine on the expression level of viral capsid protein VP3 at different time points after SVV infection using Western blot (BHK-21 cells).

[0020] Figure 9 To detect the effect of homoharringtonine on the expression level of viral capsid protein VP3 at different time points after SVV infection using Western blot (PK-15 cells).

[0021] Figure 10 The effect of TCID50 on the proliferation curve of SVV (BHK-21 cells) was detected.

[0022] Figure 11 The effect of TCID50 on the proliferation curve of SVV (PK-15 cells) was detected. Detailed Implementation

[0023] This invention investigates the regulatory effect of homoharringtonine on SVV replication in vitro and finds that homoharringtonine can be used to prepare drugs against Seneca virus.

[0024] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0025] Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art. Unless otherwise specified, all reagents used in the examples are analytical grade or higher and can be commercially available products. For example, DMEM culture medium and fetal bovine serum can be purchased from Thermo Fisher Scientific; trypsin can be purchased from Amresco; goat anti-mouse IgG (H+L) HRP can be purchased from Solarbio; goat anti-rabbit IgG (H+L) HRP and nitrocellulose membrane (NC membrane) can be purchased from Bio-Rad; FITC-labeled goat anti-mouse IgG (H+L) can be purchased from Merck; and protein...

[0026] Marker is available from Yamei Co., Ltd., SDS-PAGE Buffer (5×) is available from Kangwei Century Co., Ltd., methanol is available from Fuyu Fine Chemical Co., Ltd., skim milk powder, tris-hydroxymethylaminomethane (Tris-Base), peptone, yeast powder, agarose, sodium dodecyl sulfate (SDS), 50×

[0027] TAE buffer, green fluorescent nucleic acid dye, 5× loading buffer, and transfection reagent lip2000 can be purchased from MCE Ltd., RNA extraction kit can be purchased from Yisheng Biotechnology Co., Ltd., reverse transcription kit can be purchased from Lamborghide Biotechnology Co., Ltd., and SDS-PAGE gel preparation kit can be purchased from Yamei Co., Ltd.

[0028] Example 1

[0029] 1. Materials

[0030] The BHK-21 (hamster kidney cells), PK-15 (pig kidney cells), and SVV CHhb17 strain used in this invention were provided by the High Technology Research Laboratory of Animal Biological Agents, Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. The recombinant SVV virus rSVV-eGFP expressing green fluorescent protein was donated by Professor Liu Fuxiao of Qingdao Agricultural University.

[0031] The main chemical reagents and preparation methods used to prepare the solutions are shown in Table 1.

[0032] Table 1. Main Chemical Reagents and Preparation Methods

[0033]

[0034] 2. Methods and Results

[0035] 2.1 Cell Culture

[0036] This study primarily used BHK-21 and PK-15 cells as host cells for SVV infection.

[0037] 2.1.1 Cell resuscitation

[0038] Prepare a 37°C constant temperature water bath in advance and quickly place the cells in it to thaw. The entire thawing process should be controlled within about 1 minute, until the cryopreservation solution in the tube is completely thawed. Centrifuge the cells horizontally at 1000 rpm for 3 minutes, then carefully aspirate the supernatant with a pipette, add culture medium to 5 mL, and mix well by pipetting. Gently shake the cell culture flask to ensure that the cells are evenly distributed, and then place it in an incubator for culture.

[0039] 2.1.2 Cell passage

[0040] Cell passage can be performed when the cell density reaches 90% or higher. Discard the old culture medium, gently add phosphate-buffered saline (PBS) to the cell wells to wash the cells, then digest with 0.25% trypsin. Once cell detachment is observed, discard the supernatant, and vigorously tap the side of the cell culture flask to allow the cells to fall off like quicksand. Then add an appropriate amount of culture medium, gently pipette the cells evenly, and transfer them to a cell plate for subsequent experiments.

[0041] 2.1.3 Cell cryopreservation

[0042] Cells were collected by centrifugation into centrifuge tubes, and freshly prepared cell cryopreservation solution was added. The cells were first placed in a -80°C freezer for 24 hours, and then transferred to a liquid nitrogen tank for long-term storage.

[0043] 2.2 Assay for the cytotoxicity of homoharringtonine

[0044] To determine the cytotoxic effects of homoharringtonine, this experiment treated cells with homoharringtonine at final concentrations of 0 μM (Mock), 1 μM, 10 μM, and 20 μM, and cell viability was assessed using the CCK-8 assay. Specifically, cells were seeded at an appropriate density into 96-well culture plates, and the plates were gently agitated to ensure even cell distribution before incubation. After 24 h, 1 μM, 10 μM, and 20 μM homoharringtonine were added, respectively. Following 24 h, the CCK-8 reagent was added, taking care to avoid air bubbles. After incubation for 1 h, the absorbance at 450 nm was measured, and cell viability was calculated.

[0045] The viability results of BHK-21 cells are as follows: Figure 1 As shown, the viability results of PK-15 cells are as follows: Figure 2 As shown in the figure, the activity of BHK-21 and PK-15 cells treated with these three concentrations of homoharringtonine was not different from that of the control group, indicating that homoharringtonine at concentrations of 20 μM and below had no significant effect on the activity of either cell type.

[0046] 2.3 Effect of harzianum base on SVV replication

[0047] 2.3.1 Effects of different doses of homoharringtonine on SVV replication

[0048] To evaluate the effects of different doses of homoharringtonine on SVV replication, this experiment first used fluorescence microscopy to observe the effects of 0 μM, 1 μM, 10 μM, and 20 μM treatments on eGFP-labeled recombinant SVV virus. Specifically, BHK-21 cells were seeded in 6-well plates using DMEM medium containing 10% fetal bovine serum and cultured at 37 ℃ and 5% CO2. When the cells reached 70-80% confluence, recombinant SVV virus expressing green fluorescent protein (rSVV-eGFP) was inoculated at a multiplicity of infection (MOI) of 5. Homoharringtonine was then added to achieve final concentrations of 0 μM, 1 μM, 10 μM, and 20 μM in the cell slurry, respectively. Samples were collected 12 h post-infection, and fluorescence intensity was analyzed using fluorescence microscopy.

[0049] The results are as follows Figure 3 As shown, the results indicate that the rSVV-eGFP fluorescence signal gradually decreased with increasing dose of homoharringtonine, suggesting that viral replication was inhibited.

[0050] To further determine the effects of different doses of homoharringtonine on SVV replication, Western blot and TCID50 assays were used to analyze the effects of different doses of homoharringtonine on the expression level of capsid protein and viral titer of wild-type SVV CHhb17. Specifically, BHK-21 and PK-15 cells were seeded in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ and 5% CO2. When the cells reached 70-80% confluence, SVV was inoculated at an MOI of 1, and homoharringtonine was added at final concentrations of 0 μM, 1 μM, 10 μM, and 20 μM, respectively. Cells were collected 12 h post-infection, and the expression level of viral capsid protein and viral titer were analyzed using Western blot and TCID50 assays.

[0051] Western blot results of BHK-21 cells are as follows: Figure 4 As shown, the Western blot results of PK-15 cells are as follows: Figure 5 As shown, the TCID50 results of BHK-21 cells are as follows: Figure 6 As shown, the TCID50 results of PK-15 cells are as follows: Figure 7 As shown, from Figure 4-7 As can be seen, treatment of cells with 1 μM, 10 μM, and 20 μM homoharringtonine can inhibit the expression of viral capsid protein VP3 12 h after SVV infection. Figure 4 and Figure 5 ) and viral titer ( Figure 6 and Figure 7 ).

[0052] 2.3.2 Effects of Homoharringtonine on SVV Replication at Different Stages

[0053] Based on the above experiments, the effects of homoharringtonine on SVV replication at different stages of infection were further analyzed. Cells were collected at different time points after viral infection, and the effects of homoharringtonine on the expression level of capsid protein and viral titer of wild-type SVV CHhb17 were analyzed using Western blot and TCID50 assays. Specifically, BHK-21 and PK-15 cells were seeded in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ and 5% CO2. When the cells reached 70-80% confluence, SVV CHhb17 was inoculated at an MOI of 1, and homoharringtonine was added to a final concentration of 20 μM. Cells were collected at 0 h, 6 h, 9 h, and 12 h post-infection, and the expression level of viral capsid protein VP3 (β-actin was used as an internal reference protein) and viral titer were analyzed using Western blot and TCID50 assays, respectively.

[0054] Western blot results are as follows Figure 8 and Figure 9 As shown, the results indicated that the cephalotaxine treatment group, compared with the solvent control DMSO treatment group, could inhibit the expression of viral protein VP3 at 9 h and 12 h post-infection; the viral titer detection results are as follows. Figure 10 and Figure 11 As shown in the figure, *** indicates significant differences (P<0.01). The results indicate that treatment with homoharringtonine significantly inhibited SVV replication at 6 h, 9 h and 12 h after infection, with a 10-fold decrease in viral titer at 6 h, an 8.91-fold decrease at 9 h and a 3.76-fold decrease at 12 h.

[0055] In summary, homoharringtonine can significantly inhibit SVV replication and exhibits anti-SVV activity. Homoharringtonine can be used to prepare anti-SVV drugs.

Claims

1. Application of holoharbine in the preparation of anti-Seneca virus agents.

2. The application according to claim 1, characterized in that, The effective concentration of the homoharringtonine in the anti-Seneca virus preparation is 1~30 μM.

3. The application according to claim 1, characterized in that, The effective concentrations of the homoharringtonine in the anti-Seneca virus preparation are 1 μM, 10 μM, and 20 μM.

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