Application of tetrandrine in preparation of anti-pseudorabies virus drug

Abstract

The invention discloses an application of tetrandrine in preparation of an anti-pseudorabies virus drug, tetrandrine has no significant toxicity to cells in a concentration range of 0-2 [mu] M, and tetrandrine can significantly reduce pseudorabies virus infection by inhibiting virus genome replication, protein synthesis and progeny virus titer in a concentration range of 0.5-2 [mu] M, so that the anti-pseudorabies virus drug has good anti-pseudorabies virus resistance. The tetrandrine can be used as an effective component for developing the anti-pseudorabies virus medicine, and has the advantages of safety, few toxic and side effects, low medicine residue, no pollution and the like.

Description

Technical Field

[0001] This invention generally relates to the field of biopharmaceutical technology, and specifically to the application of tetrandrine in the preparation of anti-pseudorabies virus drugs. Background Technology

[0002] Pseudorabies virus (PRV) is an enveloped linear double-stranded DNA virus belonging to the alpha herpesvirus subfamily. This virus can establish latent infection and exhibits neurotropism. Its only natural host is the pig, but it can also infect many other animals. Pseudorabies, caused by PRV infection, can lead to severe respiratory symptoms, reproductive disorders, and neurological diseases, resulting in significant economic losses to the global pig industry. Currently, prevention and control of this disease mainly rely on gE gene-deleted vaccines. However, with continuous viral mutations, especially the genotype II variants that have emerged since 2011, the protective efficacy of existing vaccines has decreased, and even immune escape has occurred, leading to increasing pressure on prevention and control. Of particular concern is the potential risk of cross-species transmission of PRV, as evidenced by recent epidemiological evidence. Clinical cases have confirmed that humans can be infected with PRV under certain conditions, with infected individuals often exhibiting viral encephalitis, visual impairment, and other neurological symptoms, and a poor prognosis. Serological surveys show that the PRV antibody positivity rate among encephalitis patients in my country is significantly higher than that in the healthy population, further highlighting its importance in the field of public health. Currently, there are no specific clinical treatments for PRV infection. Therefore, developing novel anti-PRV drugs is of urgent practical significance.

[0003] Tetrandrine (TET) is an alkaloid extracted from traditional Chinese medicine. Modern pharmacological studies have confirmed that it possesses various activities, including anti-inflammatory, immunomodulatory, and anti-fibrotic effects. Some studies have shown that TET has inhibitory effects on certain viruses, with mechanisms involving interference with viral invasion and regulation of the host immune response. However, to date, no publicly available research or literature reports whether TET has inhibitory activity against PRV, nor its application in combating PRV infection. Summary of the Invention

[0004] In view of the above-mentioned defects or deficiencies in the prior art, it is desirable to provide an application of tetrandrine in the preparation of anti-pseudorabies virus drugs.

[0005] This invention provides the application of tetrandrine in the preparation of anti-pseudorabies virus drugs.

[0006] Preferably, in the anti-pseudorabies virus drug, the concentration of tetrandrine, which is non-toxic to cells, is 0~2μM.

[0007] Preferably, in the anti-pseudorabies virus drug, the concentration of tetrandrine used to inhibit the proliferation of pseudorabies virus is 0.5~2μM.

[0008] Preferably, the tetrandrine is used to prepare a drug that inhibits the proliferation of pseudorabies virus.

[0009] Preferably, the drug comprises a pharmaceutical composition or compound preparation with tetrandrine as the active ingredient.

[0010] Preferably, the concentration of tetrandrine in the pharmaceutical composition or compound preparation is 0.5~2μM.

[0011] Preferably, the pharmaceutical composition or compound preparation further includes pharmaceutically acceptable excipients or auxiliary ingredients.

[0012] Preferably, the dosage form of the drug includes tablets, powders, granules, suspensions, emulsions, capsules, oral liquids, injections, or sustained-release formulations.

[0013] Beneficial effects:

[0014] This invention reveals for the first time that the traditional Chinese medicine tetrandrine possesses anti-pseudorabies virus (PRV) activity, and provides its application in the preparation of anti-PRV drugs. TET exhibits a significant inhibitory effect on PRV proliferation in host cells, demonstrating good anti-PRV efficacy. Tetrandrine shows no significant toxicity to PK-15 cells in the 0–2 μM concentration range, and significantly reduces PRV infection in the 0.5–2 μM concentration range by inhibiting viral genome replication, protein synthesis, and progeny virus titers, with the effect being dose- and time-dependent.

[0015] This invention is the first to discover and confirm that TET can effectively inhibit the proliferation of PRV in vitro. This discovery provides a novel source of compounds and action strategy for developing therapeutic drugs against PRV infection. It has important scientific value and application prospects for making up for the shortcomings of current vaccine prevention and control, responding to viral mutations, and reducing health risks to humans and animals.

[0016] Tetrandrine is safe and has few toxic side effects when used to treat pseudorabies virus. Tetrandrine is an extract from traditional Chinese medicine and is different from hormones, antibiotics, and chemically synthesized drugs, so it has no obvious toxic side effects on the body. Attached Figure Description

[0017] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The effect of tetrandrine on PK-15 cell activity provided in the embodiments of this application; Figure 2The fluorescence microscopy results (×100 μm) provided in this application embodiment are used to evaluate the infection effect of tetrandrine on PK-15 cells against PRV-GFP; wherein, Figure 2 (a) is the DMSO group. Figure 2 In (b), the concentration of tetrandrine was 0.5 μM. Figure 2 The concentration of tetrandrine in (c) was 2 μM; Figure 3 The results of absolute fluorescence quantitative PCR detection of the effect of tetrandrine on PRV genome replication provided in the embodiments of this application; Figure 4 The results of Western blot analysis of the inhibition of PRV gE protein expression by tetrandrine provided in the embodiments of this application are shown. Figure 5 The results of the inhibition test of PRV progeny virus yield by tetrandrine using the half-maximal tissue culture infection dose method provided in the embodiments of this application. Detailed Implementation

[0018] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0020] The innovation of this invention lies in the first revelation of the specific inhibitory effect of tetrandrine (TET) on PRV. Through relevant experiments, it has been confirmed that it can significantly reduce viral load and block host cell infection, providing a new strategy for the development of anti-PRV drugs and has important clinical application and industrialization prospects.

[0021] This invention provides the application of tetrandrine in the preparation of anti-pseudorabies virus drugs, wherein the structural formula of tetrandrine is shown in formula (1):

[0022] Equation (1) In this embodiment of the invention, porcine kidney-15 (PK-15) cells were used as a cell model for research. Cytotoxicity experiments showed that tetrandrine had no significant cytotoxicity in the concentration range of 0–2 μM. Within the concentration range of 0.5–2 μM, fluorescence microscopy, Western blotting (WB), and TCID assays were also effective. 50 Both qPCR results and clinical trials showed that tetrandrine significantly inhibited PRV proliferation, and the antiviral effect was positively correlated with the concentration used. Tetrandrine can be developed as a safe and effective anti-PRV drug, and developing novel anti-PRV drugs under the existing application conditions of tetrandrine has good application prospects.

[0023] The specific embodiments of the present invention will be further described in detail below with reference to examples. Unless otherwise specified, the instruments and equipment used in the examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the experimental methods involved are all conventional methods.

[0024] The main experimental materials and instruments used in the embodiments of this invention are as follows: (1) Main experimental materials The green fluorescent protein-expressing strain (PRV-GFP), wild-type strain (PRV-HeNLH / 2017), porcine kidney epithelial cells (PK-15), pEASY-Blunt-UL54 standard plasmid, and PRV gE mouse monoclonal antibody were all preserved in our laboratory. Mouse β-tubulin monoclonal antibody (Catalog No.: M20005, Abmart); tetrandrine (Catalog No.: HY-13764, MCE); CCK-8 kit (Catalog No.: CA1410-500T), dimethyl sulfoxide (Catalog No.: D8371), and high-glucose DMEM medium (Catalog No.: 14100) were purchased from Solarbio; SYBR qPCR Master Mix (Catalog No.: Q314-04 / 03), cell / tissue nucleic acid extraction kit (Catalog No.: DC104-01), and virus nucleic acid extraction kit (Catalog No.: RC311) were purchased from Novizan; electrophoresis buffer (Catalog No.: WB54001), 40× rapid transfer buffer (Catalog No.: WB4600), ultrasensitive ECL chemiluminescence kit (Catalog No.: P10300), and serum-free cell cryopreservation solution (Catalog No.: C40100) were purchased from Syntec.

[0025] (2) Main instruments and equipment CO2 constant temperature cell culture incubator purchased from SHELLAB; ELISA reader purchased from BioTek Instruments; inverted fluorescence microscope purchased from OLYMPUS; electrophoresis apparatus purchased from BIO RAD; clean bench purchased from Suzhou Antai Air Technology Co., Ltd.; real-time fluorescence quantitative PCR instrument and biosafety cabinet purchased from Thermo Fisher Scientific; high-speed refrigerated centrifuge purchased from Eppendorf; blood bank-specific centrifuge purchased from Changsha Yingtai Co., Ltd.; horizontal refrigerated-freezing conversion cabinet, medical low-temperature storage box, medical refrigerator, and double-door refrigerator purchased from Haier Co., Ltd.; integrated chemiluminescence imaging analysis system purchased from Baijing Biotechnology Co., Ltd.; digital display constant temperature water bath purchased from Jiangsu Yitong Co., Ltd. Example 1

[0026] Effect of tetrandrine on PK-15 cell viability PK-15 cells in good growth condition were seeded at an appropriate density into 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator. When the cell confluence was approximately 80%, tebufenozide solution was added in serially diluted dimethyl sulfoxide (DMSO) at concentrations of 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, and 16 μM. A blank control group (Mock) was also included, with 3-4 replicates for each concentration. After 48 hours of drug treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for another 2 hours. The absorbance (OD) of each well was measured at 450 nm using a microplate reader. 450 Data processing was performed using GraphPad software, and the Student t-test was used for statistical analysis of intergroup comparisons.

[0027] The results are as follows Figure 1 As shown, compared with the Mock group, tetrandrine did not significantly inhibit the activity of PK-15 cells in the concentration range of 0-2 μM, indicating that the compound has low cytotoxicity and good biosafety in this concentration range. Example 2

[0028] Evaluation of tetrandrine against PRV-GFP infection based on fluorescence microscopy Healthy PK-15 cells were seeded into 96-well plates and cultured to approximately 95% confluence. Tetrandrine diluted with DMSO at specified concentrations (0 μM, 0.5 μM, 2 μM) (3-4 replicates per concentration) was added, and the cells were simultaneously infected with the PRV-GFP recombinant fluorescent strain at a multiplicity of infection (MOI) of 0.1. One hour after infection, the virus solution was discarded, and the cells were washed three times with phosphate-buffered saline (PBS). The solution was then replaced with maintenance medium containing the appropriate drug concentration, and the cells were cultured for another 24 hours. After culture, the medium was discarded, and after washing with PBS, 50 μL of 4% paraformaldehyde solution was added to each well for fixation at room temperature for 20 minutes. After washing again with PBS, the intensity of the green fluorescence signal was observed and recorded under an inverted fluorescence microscope to assess the viral proliferation level within the cells.

[0029] The results are as follows Figure 2 As shown, compared with the DMSO solvent control group (without tetrandrine), the fluorescence signal emitted by PRV-GFP in cells treated with 0.5 μM and 2 μM tetrandrine was significantly weakened, and the inhibitory effect at the 2 μM concentration was more obvious, proving that tetrandrine has the effect of inhibiting PRV infection, and the antiviral effect of tetrandrine increases with increasing concentration.

[0030] As can be seen from Examples 1 and 2 above, in the anti-pseudorabies virus drug provided in this application, the concentration of tetrandrine that is non-toxic to cells is 0~2μM, and the concentration of tetrandrine that inhibits the proliferation of pseudorabies virus is 0.5~2μM. This indicates that the anti-pseudorabies virus drug provided in this application can take effect at a safe dose, and tetrandrine can be used in the preparation of drugs that inhibit the proliferation of pseudorabies virus. Example 3

[0031] Detection of the effect of tetrandrine on PRV genome replication by absolute real-time PCR PK-15 cells were seeded in 24-well plates and cultured to near monolayer confluence (approximately 95%). Cells were then treated with media containing 0 μM, 0.5 μM, and 2 μM tetrandrine, respectively, and simultaneously infected with wild-type PRV at an MOI of 0.01. After 1 hour of adsorption, the viral load was removed, and the cells were washed with PBS and then cultured in maintenance medium containing the corresponding drug concentration. Cells were collected at 24 and 36 hours post-infection, and total DNA was extracted according to the nucleic acid extraction kit instructions. Absolute quantitative PCR was performed using primers targeting PRV-specific genes to determine viral genome copy number, thus evaluating the effect of the drugs on viral replication.

[0032] The results are as follows Figure 3As shown, compared with the control group, the PRV genome copy number of tetrandrine was significantly reduced at both 24 and 36 hours under the treatment concentration conditions (0.5 μM and 2 μM). Moreover, the viral copy number of tetrandrine at the 2 μM concentration was significantly lower than that at the 0.5 μM concentration, and the inhibitory effect at 36 h was more significant than that at 24 h. The degree of inhibition increased with increasing drug concentration and duration of action, indicating that tetrandrine inhibits PRV genome replication in a dose- and time-dependent manner, further confirming that tetrandrine can inhibit PRV replication in cells. Example 4

[0033] The inhibitory effect of tetrandrine on PRV gE protein expression was analyzed by Western blotting. PK-15 cells were seeded in 24-well plates and cultured to an appropriate density. Medium containing 0 μM, 0.5 μM, and 2 μM tetrandrine was added, and the cells were simultaneously infected with PRV wild-type virus at an MOI of 0.01. After 1 hour, the medium was replaced with maintenance medium containing the corresponding drug concentration. Cells were collected at 24 and 36 hours to prepare whole-cell lysates. Proteins were separated by SDS-PAGE and transferred to a membrane. The membrane was incubated with anti-PRV gE mouse monoclonal antibody and anti-β-tubulin mouse monoclonal antibody to detect the expression of gE protein and the internal control β-tubulin.

[0034] The results are as follows Figure 4 As shown, compared with the DMSO control group, tetrandrine significantly reduced the expression level of PRV gE protein; and the inhibitory effect of 2 μM tetrandrine on gE protein expression was better than that of 0.5 μM tetrandrine. At the same concentration, the longer the treatment time, the more significant the inhibitory effect, indicating that the inhibition of PRV gE protein expression by tetrandrine is dose- and time-dependent. Example 5

[0035] The effect of tetrandrine on PRV progeny production was determined by half-maximal tissue culture infectious dose method. PK-15 cells were seeded in 24-well plates and cultured to approximately 95% confluence. They were then treated with media containing 0 μM, 0.5 μM, and 2 μM tetrandrine, respectively, and inoculated with a PRV wild-type virus strain at an MOI of 0.01. One hour later, the medium was replaced with maintenance medium containing the corresponding drug concentration for continued culture. Culture plates were collected at 24 and 36 hours post-infection, and after three freeze-thaw cycles, the supernatant was collected by centrifugation to obtain virus samples. The virus was analyzed using the half-maximal tissue culture infectious dose (TCID50). 50 The titer of infectious viral particles in each sample was determined on PK-15 cells using the PCR method.

[0036] The results are as follows Figure 5 As shown, compared with the DMSO control group, the progeny virus titers of samples treated with tetrandrine (using concentrations of 0.5 μM and 2 μM) were significantly reduced. Furthermore, the viral titer at the 2 μM concentration was significantly lower than that at the 0.5 μM concentration, and the inhibitory effect at 36 h was stronger than that at 24 h. The degree of reduction in viral titer was positively correlated with drug concentration and treatment time, further confirming that tetrandrine can inhibit PRV proliferation in cells in a dose- and time-dependent manner. Therefore, tetrandrine can be used as a drug for inhibiting pseudorabies virus proliferation in vitro.

[0037] In summary, the tetrandrine provided in this application has no significant toxicity to PK-15 cells in the range of 0-2 μM. In particular, tetrandrine at 0.5-2 μM (such as 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, etc.) significantly reduces PRV infection by inhibiting viral genome replication, protein synthesis and progeny virus titer, and the effect is dose- and time-dependent.

[0038] This invention provides the application of the traditional Chinese medicine tetrandrine in the in vitro inhibition of pseudorabies virus (PRV) proliferation. This marks the first discovery of tetrandrine as a potential antiviral drug for inhibiting PRV proliferation in vitro. Anti-pseudorabies virus drugs are those containing tetrandrine as the sole active ingredient, or pharmaceutical compositions containing tetrandrine. Anti-pseudorabies virus drugs refer to drugs used to prevent and treat PRV infection. This invention verifies the antiviral activity of tetrandrine by infecting PK-15 cells with PRV. At a safe concentration (≥0.5 μM), TET significantly reduces the intracellular viral genomic DNA copy number, viral titer, and expression levels of viral structural proteins, showing statistically significant differences compared to the untreated control group. These results indicate that TET can effectively inhibit PRV proliferation in vitro and therefore can be used to prepare anti-PRV drugs.

[0039] In some embodiments, the anti-pseudorabies virus drugs provided in this application include pharmaceutical compositions or compound preparations with tetrandrine as the active ingredient.

[0040] In some embodiments, the concentration of tetrandrine in the pharmaceutical composition or compound preparation is 0.5~2 μM.

[0041] In some embodiments, the pharmaceutical composition or compound preparation may further include pharmaceutically acceptable excipients or auxiliary ingredients. Excipients may include diluents (such as lactose, microcrystalline cellulose), disintegrants (sodium carboxymethyl starch), lubricants (magnesium stearate), pH adjusters (sodium citrate), etc.

[0042] In some embodiments, the dosage form of the anti-pseudorabies virus drug includes tablets, powders, granules, suspensions, emulsions, capsules, oral solutions, injections, or sustained-release formulations.

[0043] In some embodiments, the anti-pseudorabies virus drug can be administered via known drug injection methods, including but not limited to subcutaneous, intramuscular, and intravenous injection, such as enteric, oral (e.g., pills, tablets, sublingual, disintegrant, capsules, films, liquid solutions or suspensions, powders, solid crystals or liquids), rectal (e.g., suppositories, enemas), via injection (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal), via inhalation (e.g., intrabronchial), surface, vaginal, skin, or intranasal administration of the anti-pseudorabies virus drug of the present invention.

[0044] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. Application of tetrandrine in the preparation of anti-pseudorabies virus drugs.

2. The application according to claim 1, characterized in that, In the aforementioned anti-pseudorabies virus drug, the concentration of tetrandrine, which is non-toxic to cells, is 0-2 μM.

3. The application according to claim 1, characterized in that, In the aforementioned anti-pseudorabies virus drug, the concentration of tetrandrine used to inhibit the proliferation of pseudorabies virus is 0.5~2 μM.

4. The application according to claim 1, characterized in that, The tetrandrine is used to prepare a drug that inhibits the proliferation of pseudorabies virus.

5. The application according to claim 1, characterized in that, The drug includes a pharmaceutical composition or compound preparation with tetrandrine as the active ingredient.

6. The application according to claim 5, characterized in that, The concentration of tetrandrine in the pharmaceutical composition or compound preparation is 0.5~2 μM.

7. The application according to claim 5, characterized in that, The pharmaceutical composition or compound preparation also includes pharmaceutically acceptable excipients or auxiliary ingredients.

8. The application according to claim 5, characterized in that, The dosage forms of the drug include tablets, powders, granules, suspensions, emulsions, capsules, oral liquids, injections, or sustained-release formulations.

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