Application of cepharanthine in preparation of anti-akabane virus medicine

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly discloses application of cepharanthine in preparation of an anti-akabane virus drug. By measuring the safe concentration range of cepharanthine on MDBK cells and through RT-qPCR, WB and IFA, it is found that cepharanthine can be used as an inhibitor of AKAV viruses, and the application of cepharanthine in preparation of the anti-akabane virus drug in preparation of the anti-akabane virus drug in preparation of the anti-akabane virus drug is achieved. When the concentration of cepharanthine is 10 [mu] M, the cepharanthine has a significant inhibition effect on AKAV virus, and a new candidate drug is provided for preventing and treating AKAV virus infection.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to the application of styrax extract in the preparation of drugs against Akabane virus. Background Technology

[0002] Akabane virus (AKAV) is a phased, single-stranded, negative-sense RNA virus belonging to the Bunyaviridae family (…). Peribunyaviridae ) Genus Ortho Bunyavirus ( Orthobunyavirus The AKAV genome consists of three single-stranded negative-sense RNA segments, encoding a total of four open reading frames (ORFs). The L gene encodes the L protein, which has RNA-dependent RNA polymerase (RdRp) and endonuclease activities. The M gene encodes the glycoproteins Gc and Gn, and the non-structural protein NSm. The S gene contains two overlapping ORFs, encoding the nucleocapsid protein N and the non-structural protein NSs. AKAV is an infectious virus characterized by symptoms such as abortion, premature birth, stillbirth, congenital joint deformities, hydrocephalus, anencephaly, and encephalomyelitis in cattle and sheep. The disease caused by AKAV infection is called Akabane disease. This disease has two main endemic areas globally: East Asia, Southeast Asia, and Australia; and the Middle East and South Africa. Akabane disease is one of the legally notifiable diseases of the World Organisation for Animal Health (OIE). In my country, it is classified as a Class III animal disease and is also a mandatory disease for the entry of cattle and sheep into my country.

[0003] Cepharanthine (Cep) belongs to the class of bisbenzylisoquinoline alkaloids. Studies have confirmed that CEP, as a component with broad-spectrum antiviral activity, exerts inhibitory effects in the infection processes of various viruses through multiple mechanisms. During the viral entry and membrane fusion stages, CEP can effectively inhibit the invasion of viruses such as EqHV-8, HIV-1, and PEDV: for EqHV-8, it blocks viral membrane fusion with host cells and activates the AMPK and Nrf2 / HO-1 pathways to alleviate oxidative stress; in HIV-1 infection, CEP inhibits the viral envelope-mediated fusion process by stabilizing cell membrane fluidity; and in PEDV, CEP blocks viral replication by binding to the viral 3CL protease (Mpro) and inhibiting its activity. In terms of directly inhibiting viral replication, CEP has shown significant effects against JEV, DENV, and HSV-1. For example, it inhibits viral RNA synthesis by blocking the binding of RdRp to GTP in JEV; against DENV, it both inhibits viral replication and alleviates cytokine storm; and against HSV-1, CEP interferes with viral plaque formation and mRNA synthesis. Furthermore, CEP also plays a role by modulating host immune and inflammatory responses, such as inhibiting NF-κB activation and pro-inflammatory cytokine secretion in HIV-1 infection, and targeting the host protein CD163 to block viral replication in PRRSV. Its antioxidant and cytoprotective functions have also been demonstrated in EqHV-8 and HTNV infections, especially in HTNV infection, where CEP significantly inhibits viral replication at low micromolar concentrations. Finally, in coronaviruses including SARS-CoV-2 and GX_P2V, CEP exerts antiviral effects through multiple pathways, such as inhibiting membrane fusion or targeting viral proteases, demonstrating broad antiviral potential. In conclusion, CEP has potential in antiviral therapy.

[0004] Although vaccines against the Akabane virus have been developed abroad, they have not been widely used. Therefore, there is an urgent need to develop specific drugs against the Akabane virus. Screening FDA-approved drug libraries is an effective strategy for identifying potential antiviral drugs and has been successfully applied to novel viruses such as Zika and Ebola. Given the approval status of these drugs, identified candidate drugs have the potential to be rapidly advanced into the clinical setting as treatment strategies. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of senna extract in the preparation of drugs against Akabane virus. The mechanism of action against Akabane virus is to inhibit Akabane virus gene replication or Akabane virus protein expression. The Akabane virus gene is the NSs gene of Akabane virus, and the Akabane virus protein is the Gc protein of Akabane virus.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is the application of senna extract as the sole active ingredient in the preparation of drugs against Akabane virus.

[0007] The second technical solution provided by the present invention is a drug for treating Akabane virus, which contains 10 μM of senna extract.

[0008] Furthermore, the drug for treating Akabane virus also includes a pharmaceutically acceptable carrier and / or excipients.

[0009] Furthermore, the dosage form of the drug is one of tablets, sprays, granules, capsules, oral liquids, injections, or suspensions.

[0010] Compared with existing technologies, this invention determines the safe concentration range of gentianin on MDBK cells and discovers through RT-qPCR, WB, and IFA that gentianin can act as an inhibitor of AKAV virus. A concentration of gentianin of 10 μM has a significant inhibitory effect on AKAV virus, providing a new candidate drug for the prevention and treatment of AKAV virus infection. Attached Figure Description

[0011] Figure 1 This is a graph showing the cytotoxicity assay of styrax tectorum against MDBK cells.

[0012] Figure 2 This diagram illustrates the inhibitory effect of senna extract on the AKAV genome copy number.

[0013] Figure 3 The diagram shows the inhibitory effect of styrax extract on the expression of AKAV Gc and N proteins.

[0014] Figure 4 The image shows the results of immunofluorescence observation of AKAV inhibition by senna. Detailed Implementation

[0015] 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. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0016] The bovine kidney cells (MDBK) used in the following examples were preserved by the Guangdong Provincial Key Laboratory of Animal Molecular Design and Precision Breeding. MDBK cells are a cell line derived from adult bovine kidneys. This cell line can spontaneously differentiate into a monolayer of epithelial-like cells, making it the optimal in vitro model for studying drugs against Akabane virus. Vero cells were also preserved by the Guangdong Provincial Key Laboratory of Animal Molecular Design and Precision Breeding. Unless otherwise specified, all other reagents used were commercially available, and all methods involved were known methods.

[0017] The compound used, Cep (hereinafter referred to as Cep), was purchased from Selleck and its chemical molecular structure is shown below: .

[0018] The Akabane virus strain used was Akabane virus AKAV_FS202301, with accession number CCTCC NO:V2024119 and sequence number PQ567126-PQ567128. It was passaged in Vero cells and tested on MDBK cells.

[0019] To verify whether Cep can be used as the sole active ingredient in the preparation of a drug against Akabane virus, the following experiments were conducted: 1. Determination of safe concentration of Cep in MDBK cells The 10 mM Cep solution dissolved in 30 μL of DMSO was serially diluted with basal DMEM medium to obtain Cep dilutions with concentrations ranging from 0.5 to 20 μM.

[0020] MDBK cells were loaded at a rate of 1×10 5 Cells / well were evenly distributed in a 96-well cell plate to obtain a cell plate containing MDBK. The safe concentration of Cep for MDBK cells was determined as follows: The experimental wells were set up as follows: Cep dilutions of 0.5 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, and 20 μM were added to cell plates containing MDBK cells, with each Cep dilution repeated in four replicates (one column in a 96-well plate); blank wells were set to contain only culture medium; experimental wells were set to contain cells, culture medium, and Cep dilution; and control wells were set to not be treated with Cep.

[0021] After culturing cells (37℃, 5% CO2) for 24 h, 10 μL of CCK-8 solution was added to each well. After incubating the culture plate in an incubator for 4 hours, the absorbance at 450 nm was measured using a microplate reader, and the cell viability (%) was calculated according to Formula I.

[0022] Formula I: Cell viability (%) = [(OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells)] × 100; The results of Cep's safe concentration assay for MDBK cells are as follows: Figure 1 As shown, by Figure 1 It can be seen that Cep at concentrations of 20 μM and below has no effect on the viability of MDBK cells.

[0023] 2. Determination of the effect of Cep on the gene copy number of AKAV virus in cells MDBK cells (2 × 10⁻⁶) were cultured in 12-well plates in DMEM + 10% FBS medium. 6 / well), the experimental group was seeded with AKAV virus (MOI=0.1) and treated with different concentrations (2.5, 5, 10 μM) of Cep, while the control group was treated with AKAV (MOI=0.1) and 10 μM DMSO. The blank control was treated with cells without AKAV virus and culture medium. The cell plates were incubated at 37℃ and 5% CO2 for 24 h, and then total RNA was extracted from the cells using an RNA extraction kit and reverse transcribed into cDNA. Then, the relative copy number of the viral genome was detected by real-time PCR using the NSs gene of AKAV virus as the target and GAPDH as the internal reference gene. The total volume of the real-time PCR reaction system was 20 μL, including 10 μL of SYBR Green I (2×), 1 μL of forward and reverse primers, 1 μL of template, and sterile deionized water to 20 μL. The primer sequences are shown in Table 1.

[0024] Table 1 ; Finally, the effect of Cep on inhibiting AKAV NSs in MDBK cells was calculated using quantitative real-time PCR data.

[0025] The relative expression of the AKAV NSs gene was calculated after MDBK was treated with different concentrations of Cep following AKAV virus infection, as shown in the following figures. Figure 2 As shown, by Figure 2 It was found that in MDBK cells, compared with the control group, the mRNA level of the AKAV virus NSs gene gradually decreased with increasing Cep concentration. This indicates that Cep can inhibit AKAV virus gene replication.

[0026] (3) Effect of Cep on Gc protein expression of AKAV virus MDBK cells (2 × 10⁻⁶) were cultured in 12-well plates in DMEM + 10% FBS medium. 6( / well), the experimental group was seeded with AKAV virus (MOI=0.1) and treated with different concentrations (2.5, 5, 10 μM) of Cep, while the control group was treated with AKAV (MOI=0.1) and 10 μM DMSO. The blank control was treated with cells without AKAV virus and culture medium. Cell culture plates were incubated at 37°C and 5% CO2 for 24 h. The cell culture supernatant was discarded, and the cells were washed three times with PBS. After adding 1×SDS-PAGE loading buffer, protein denaturation was performed (100°C, 10 min), followed by centrifugation for 10 min (12,000 r / min). After homogenization of the protein sample, it was added to the sample well of the gel and electrophoresed (120 V, 30 min). After the bromophenol blue dye descended to the edge of the gel, the sample was transferred to a 0.2 μm nitrocellulose membrane (NC membrane) (100 V, 1 h). The membrane was blocked with 5% skim milk powder (1 h). After discarding the skim milk powder, the NC membrane was washed with 1×TBST, and the primary antibody was added and the membrane was incubated overnight at 4°C. The NC membrane was washed with 1×TBST (5 times / 5 min), and the corresponding secondary antibody was added (incubated for 1 h). The membrane was washed with 1×TBST (5 times / 5 min). Finally, the membrane was exposed using ECL luminescent solution to detect the expression of AKAV Gc protein.

[0027] The expression results of AKAV virus Gc protein after treatment with different concentrations of Cep are as follows: Figure 3 As shown, by Figure 3 It was found that in MDBK cells, compared with the control group, the expression level of AKAV virus Gc protein gradually decreased with increasing Cep concentration. This indicates that Cep can inhibit AKAV replication.

[0028] (4) Effect of Cep on the immunofluorescence of N protein of AKAV virus in cells MDBK cells (2 × 10⁻⁶) were cultured in 12-well plates in DMEM + 10% FBS medium. 6Cells were seeded with AKAV virus (MOI=0.1) in the experimental group and treated with different concentrations (2.5, 5, 10 μM) of Cep. The infection control group was treated with AKAV (MOI=0.1) mixed with 10 μM DMSO and designated as the DMSO group. The blank control group was treated with no AKAV virus and no culture medium and designated as the Mock group. Cell plates were incubated at 37°C and 5% CO2 for 24 h. The cell culture supernatant was discarded, and the cells were washed three times with PBS and fixed with 4% formaldehyde for 30 min. Permeabilization was then performed with 0.3% Triton for 10 min. Blocking: Cells were blocked with 5% BSA at room temperature for 1 h. The blocking solution was then discarded, and a 1:200 dilution of the primary antibody (AKAV virus N protein monoclonal antibody) prepared with 5% BSA was added directly, and the cells were incubated overnight at 4°C (protected from light). Finally, the cells were incubated with FITC-labeled fluorescent secondary antibody at 37°C for 1 h. After incubation, the antibody was discarded, the sample was washed three times with PBST, and then observed under an inverted fluorescence microscope.

[0029] Following AKAV virus infection, MDBK was treated with different concentrations of Cep. The fluorescence results of the N protein of AKAV virus were observed as follows: Figure 4 As shown, the DAPI group uses DPAI staining solution to stain the nuclei of MDBK cells; the Anti-N group uses fluorescein isothiocyanate (FITC) to label the N protein; and the Merge group is a composite diagram of the DAPI and Anti-N groups. Figure 4 It was found that in MDBK cells, compared with the control group, the fluorescence of the N protein of AKAV virus significantly decreased with increasing Cep concentration. The results indicate that Cep can inhibit AKAV virus replication.

[0030] In summary, Cep can be used as the sole active ingredient to prepare a drug against Akabane virus. Of course, the prepared drug contains Cep, as well as a pharmaceutically acceptable carrier and / or excipients; the dosage form of the drug is one of the following: tablets, sprays, granules, capsules, oral liquids, injections, or suspensions.

[0031] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Application of senna extract as the sole active ingredient in the preparation of drugs against Akabane virus.

2. A drug for treating Akabane virus, characterized in that, It contains 10 μM of senna extract.

3. The drug for treating Akabane virus according to claim 2, characterized in that, It also contains pharmaceutically acceptable carriers and / or excipients.

4. The antiviral drug according to claim 2 or 3, characterized in that: The dosage form of the drug is one of the following: tablets, sprays, granules, capsules, oral liquids, injections, or suspensions.

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