Application of tetrandrine in inhibiting porcine reproductive and respiratory syndrome virus

Abstract

The invention provides an application of tetrandrine in inhibiting porcine reproductive and respiratory syndrome virus and also provides a product for resisting the porcine reproductive and respiratory syndrome virus, which comprises the tetrandrine. Wherein the dosage of the tetrandrine or the derivative of the tetrandrine on the PAM cell is 4-12.5 mu M; and the molecular weight on MARC-145 cells is 7-25 [mu] M. The antagonistic action and the action mechanism of the tetrandrine on the PRRSV are found for the first time, when the concentration of the tetrandrine reaches 7 mu M or above, virus proliferation can be effectively inhibited, and meanwhile, the concentration does not cause obvious influence on cell activity. The tetrandrine provided by the invention has important significance in application of inhibiting the PRRSV in the field of resisting the PRRSV, and a new alternative medicine is provided for preventing and treating the PRRSV.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to the application of tetrandrine in inhibiting porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen that has a serious impact on the pig industry. The virus is characterized by strain diversity, high variability, and the ability to cause immunosuppression. Vaccines against PRRSV have poor cross-protection against heterologous strains and the risk of virulence reversion in live vaccines, making it difficult for current commercial vaccines to meet the needs of prevention and control. There is an urgent need to develop other anti-PRRSV measures.

[0003] Natural products are widely available, diverse, and easily accessible, making them a valuable resource for finding anti-PRRSV components. Based on the mechanisms of virus-host interaction, and targeting the characteristics of viral infection processes such as adsorption, entry into cells, replication and proliferation, diffusion, and intercellular transmission, effective components that can inhibit viral infection and proliferation are screened from natural drug libraries, peptides, and small molecule compounds. This not only reduces the direct harm caused by viral infection but also further reduces secondary bacterial infections due to immunosuppression, decreases antibiotic use, and reduces bacterial resistance. Furthermore, many natural products are extracts of substances with medicinal and edible properties, and their safety has been evaluated, making them a promising solution to the current challenges in PRRSV prevention and control. While current research has identified some natural monomers with inhibitory effects on PRRSV, the effective inhibitory concentrations are generally high, making it difficult to achieve the corresponding inhibitory concentrations in animals, thus limiting clinical feasibility or making the cost prohibitive. Therefore, continued screening of safe and effective anti-PRRSV natural monomers is crucial for developing new drugs for PRRSV prevention and control. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides the application of tetrandrine in inhibiting porcine reproductive and respiratory syndrome virus.

[0005] In a first aspect, the present invention provides the application of tetrandrine in inhibiting porcine reproductive and respiratory syndrome virus.

[0006] The present invention further provides the use of tetrandrine in the preparation of a drug for inhibiting porcine reproductive and respiratory syndrome virus.

[0007] Secondly, the present invention provides a product for combating porcine reproductive and respiratory syndrome virus, including tetrandrine.

[0008] Furthermore, the dosage of the tetrandrine or its derivatives is 4-12.5 μM in PAM cells and 7-25 μM in MARC-145 cells.

[0009] Furthermore, the product is a drug or an inhibitor.

[0010] This invention is the first to discover the antagonistic effect of tetrandrine against PRRSV and its mechanism of action. At concentrations above 7 μM, tetrandrine can effectively inhibit viral proliferation without significantly affecting cell viability. The tetrandrine provided by this invention has significant implications for the inhibition of PRRSV in the field of anti-PRRSV treatment, offering a new alternative drug for the prevention and treatment of PRRSV. Attached Figure Description

[0011] Figure 1 The figure shows the experimental results of determining the cytotoxicity of tetrandrine using the CCK-8 assay in Example 1 of this invention; where A is the chemical structural formula of tetrandrine, B is the toxicity test evaluation data of tetrandrine in MARC-145 cells, and C is the toxicity test evaluation data of tetrandrine in PAM cells. Figure 2 In Example 2 of this invention, the experimental results of determining the anti-PRRSV activity of tetrandrine using PRRSV activity tagged with luciferase were presented. Luciferase activity was used to reflect the proliferation of PRRSV virus, and the decrease in fluorescence intensity was used to reflect the drug's effect on inhibiting PRRSV proliferation. A is a schematic diagram showing the results of determining the anti-PRRSV activity of tetrandrine in MARC-145 cells based on luciferase; B is a schematic diagram showing the results of determining the anti-PRRSV activity of tetrandrine in PAM cells based on luciferase. Figure 3 The use of half-cell infection dose TCID provided in Example 3 of the present invention 50 A schematic diagram illustrating the activity of tetrandrine in inhibiting PRRSV replication, based on the determination of viral titer and viral N protein expression levels; where A is a schematic diagram of the anti-PRRSV activity of tetrandrine in MARC-145 cells based on viral titer determination; and B is a schematic diagram of the inhibitory effect of different concentrations of tetrandrine on the synthesis of porcine reproductive and respiratory syndrome virus N protein in cells based on Western Blot analysis. Detailed Implementation

[0012] The tetrandrine (NSC-77037; d-Tetrandrine) used in this invention is a diphenylisoquinoline alkaloid extracted from the Menispermaceae plant Stephania tetrandra, which inhibits voltage-gated calcium ion channels (ICa) and Ca2+. 2+ Activated potassium ion channels.

[0013] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0014] Example 1 1. Materials and Methods 1.1 Viruses and Cells The clinical isolate JXwn06 of PRRSV was previously isolated and preserved by the Key Laboratory of Animal Epidemiology of China Agricultural University (hereinafter referred to as "this laboratory"). It was isolated in 2006 from a pig farm in Jiangxi Province where a highly pathogenic type of PRRS occurred. It is a highly pathogenic strain, and its GenBank accession number is EF641008.

[0015] PRRSV Saves the Poison RvJX-Nsp2 325 -HiBiT was constructed in the laboratory in the early stage. It is a recombinant virus carrying the complementary luciferase small subunit HiBiT. An invention patent has been applied for, application number 202110369372.1.

[0016] Both MARC-145 cells and porcine primary alveolar macrophages (PAMs) were used for PRRSV proliferation culture. The MARC-145 cells were monkey-derived kidney cell lines preserved in our laboratory, while the PAMs were obtained by alveolar lavage of specific pathogen-free (SPF) piglets.

[0017] 1.2 Molecular Biology Reagents DMEM culture medium and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific; the SuperKine™ high-sensitivity cell proliferation assay kit (CCK-8) was purchased from Acine Biotech Ltd. (Wuhan, China). The natural small molecule compound tetrandrine was purchased from Selleck Biotechnology Co., Ltd. (China Branch).

[0018] 1.3 Cell Culture Reagents DMEM cell culture medium: DMEM powder (Invitrogen, CA, USA) is dissolved in water, and the pH is adjusted to about 7.3-7.4 with HEPES buffer and NaHCO3. The medium is then filtered through a 0.22 μM filter for sterilization and stored at 4 ℃ for later use.

[0019] 1640 cell culture medium: DMEM powder (Invitrogen, CA, USA) dissolved in water, pH adjusted to approximately 7.3-7.4 with HEPES buffer and NaHCO3, sterilized by filtration through a 0.22 μM filter, and stored at 4°C for later use.

[0020] Fetal bovine serum (FBS); a product of Invitrogen (Invitrogen, CA, USA).

[0021] Preparation of 1000× bispecific antibody: penicillin (1×10) 7 Dissolve U / mL and streptomycin (10g / mL) in 100mL of ultrapure water, filter sterilize using a 0.22 filter, aliquot and store at -20℃ for later use.

[0022] Cell culture medium (10%): Add 10% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) to DMEM cell culture medium and store at 4 ℃.

[0023] DMEM cell maintenance medium (2%): Add 2% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) to DMEM cell culture medium and store at 4°C.

[0024] 1640 cell maintenance medium (2%): 2% fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml) were added to the 1640 cell culture medium and stored at 4°C.

[0025] 0.25% Trypsin: Weigh 17.80 g NaCl, 6.4 g Na2HPO4·12H2O, 0.45 g Na2EDTA, 0.45 g KCl, and 0.45 g KH2PO4 and dissolve them in 2 L of sterile deionized water. Then add 5.56 g of trypsin at a 1:250 ratio. After complete dissolution, add ultrapure water to bring the volume to 2.5 L. Filter and dispense the solution, and store at -20℃ for later use.

[0026] PBS: Weigh 8.00 g NaCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 and 0.20 g KCl respectively. After complete dissolution, add ultrapure water to make up to 1000 mL. Autoclave at 121℃ for 15 min and store at 4℃ for later use.

[0027] 1.4 Instruments and Equipment PB-21 pH meter: Sartorius GmbH, Germany ULTRA GENETIC Ultrapure Water System: ELGA (UK) Nikon Eclipse Ti-U Inverted Fluorescence Microscope: Nikon Corporation, Japan Thermo Scientific Forma 905 refrigerator: ThermoFisher, USA HERAcell i CO2 Cell Incubator: ThermoFisher, USA SterilGARD Ⅲ Advnance Biosafety Cabinet: Baker Company, USA Spark® Multifunctional Microplate Reader: TECAN, Switzerland 2. Cytotoxicity assay of tetrandrine 2.1 Assay of the cytotoxicity of tetrandrine against MARC-145 cells MARC-145 cells were seeded in 96-well plates and, after reaching a confluent monolayer, different concentrations of tetrandrine (100 μM / L, 50 μM / L, 25 μM / L, 12.5 μM / L, 6.25 μM / L, 3.125 μM / L) were added and incubated for 48 h. Then, CCK-8 solution (10 μL per well) was added and incubated at 37 ℃ for 1 h. The cells were then shaken and mixed at room temperature for 1 min. Finally, the absorbance at 450 nm was measured. Zeroing wells (2% maintenance solution) and control wells (DMSO with the same concentration of the drug) were also included.

[0028] 2.2 Assay of the cytotoxicity of tetrandrine against PAM cells PAM cells were seeded in 96-well plates. After the cells had settled completely, different concentrations of tetrandrine (200 μM / L, 100 μM / L, 50 μM / L, 25 μM / L, 12.5 μM / L, 6.25 μM / L) were added and incubated for 48 h. Then, CCK-8 solution (10 μL per well) was added and incubated at 37 ℃ for 1 h. The mixture was then shaken at room temperature for 1 min. Finally, the absorbance at 450 nm was measured. Zeroing wells (2% maintenance solution) and control wells (DMSO with the same concentration of the drug) were also set up.

[0029] The absorbance values ​​were determined by Figure 1 It was found that different concentrations of tetrandrine had different effects on MARC-145 cells and PAM cells; cell viability decreased with increasing tetrandrine concentration. At a tetrandrine concentration of 43.06 μM, the viability of MARC-145 cells was 50%; at a tetrandrine concentration of 15.57 μM, the viability of PAM cells was 50%.

[0030] Example 2 This embodiment provides 1. Materials and Methods 1.1 Viruses and Cells Same as Example 1.

[0031] 1.2 Molecular Biology Reagents The Nano-Glo® HiBiT Lytic Detection System reagents were purchased from Promega Corporation (Wisconsin, USA); the other reagents were the same as in Example 1.

[0032] 1.3 Cell Culture Reagents Same as Example 1.

[0033] 1.4 Instruments and Equipment Same as Example 1.

[0034] 2. Determination of the anti-PRRSV activity of tetrandrine based on luciferase activity RvJX-Nsp2 325 HiBiT was seeded at MOI=0.1 into 24-well plates containing a confluent monolayer of MARC-145 cells or PAMs. After 1 h of incubation, the medium was discarded, and cell maintenance medium containing different concentrations (20 μM / L, 15 μM / L, 10 μM / L, 7.5 μM / L, 5 μM / L, 2.5 μM / L) of tetrandrine was added. After 36 h of culture, the cells were collected and cooled to -80°C. After two freeze-thaw cycles, 50 μL of the virus solution was added to each well of a black 96-well plate, followed by an equal volume of Nano-Glo® HiBiT LyticReagent. The plates were thoroughly mixed at room temperature for 10 min, and the readings were recorded using a Spark® multi-plate reader.

[0035] The results of the determination of the half-maximal inhibitory concentration of tetrandrine based on luciferase activity are as follows: Figure 2 As shown, tetrandrine exhibited highly significant anti-PRRSV activity in both PAMs and MARC-145 cells, with half-maximal inhibitory concentrations (IC50) of 3.95 μM and 7.04 μM, respectively, indicating that tetrandrine is an inhibitory factor against PRRSV within cells. Combined with cytotoxicity data of tetrandrine in PAMs and MARC-145 cells, it can be concluded that the safe and effective concentration range of tetrandrine in PAM cells is 4-12.5 μM, and in MARC-145 cells it is 7-25 μM. Regarding safety, the safe and effective concentration range of this invention in MARC-145 cells is 2.5-25 μM, which, compared to other components with PRRSV antiviral effects, demonstrates lower cytotoxicity and greater safety.

[0036] Example 3 This embodiment provides 1. Materials and Methods 1.1 Viruses and Cells Same as Example 1.

[0037] 1.2 Molecular Biology Reagents The GAPDH antibody was purchased from Selleck Biotechnology Co., Ltd. (China Branch); the PRRSV N protein monoclonal antibody N35 was prepared in our laboratory, with accession number CGMCC NO.3238, and is disclosed in Chinese patent CN101661042B; the other reagents were the same as in Example 1.

[0038] 1.3 Cell Culture Reagents Same as Example 1.

[0039] 1.4 Instruments and Equipment Protein electrophoresis apparatus and transfer apparatus: Bio-Rad Systems, USA FlourChemE Imager: ProteinSimple, USA The remaining instruments and equipment are the same as in Example 1.

[0040] 2. Assay of the anti-PRRSV activity of tetrandrine Virus titer: JXwn06 cells were inoculated with a confluent monolayer of MARC-145 cells at MOI=0.1, incubated for 1 hour, the culture medium was discarded, and cell maintenance medium containing different concentrations (20 μM / L, 10 μM / L, 5 μM / L, 2.5 μM / L) of tetrandrine was added. After culturing for 36 hours, the cell plates were collected and cooled to -80°C. After two freeze-thaw cycles, the virus solution was collected and serially diluted 10-fold with 2% maintenance medium. 10 -1 ~10 -6 The dilutions were inoculated into 96-well plates containing a monolayer of MARC-145, 100 μL per well, with 8 wells in parallel for each dilution. After incubation at 37 °C for 48 h, indirect immunofluorescence was performed, and the results were observed and recorded under a fluorescence microscope. The viral titer was calculated using the Reed-Muench method.

[0041] Western Blot: (1) Preparation of protein samples: Discard the cell culture medium, wash twice with pre-cooled PBS, and remove the residual liquid with a pipette. Add protein lysis buffer NP40 containing 1 mM PMSF, lyse on ice for 30 min, collect in a 1.5 EP tube, centrifuge and collect the supernatant. Determine the protein concentration by BCA method, add 5× loading buffer according to the calculated amount, boil in a water bath for 10 min, collect the sample and store at -20 ℃.

[0042] (2) SDS-Page: Prepare separating gel and stacking gel according to protein size. Spot 20ug of sample into each well, add electrophoresis buffer, and adjust the voltage. Set the stacking gel to 80 V for 30 min and the separating gel to 120 V for 90 min. Stop electrophoresis when the bromophenol blue indicator runs out.

[0043] (3) Transfer: Before transfer, prepare a PVDF membrane of similar size to the protein gel and activate it with methanol for 2 min. Soak the filter paper and sponge pad in the pre-cooled transfer solution. Then, arrange them in the following order: negative electrode clamp - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - positive electrode clamp. After removing air bubbles, place them in the transfer tank and transfer at 100 V for 2 h.

[0044] (4) Sealing: Take out the PVDF membrane and seal it overnight at 4 ℃ with 5% skim milk or at room temperature for 1 h.

[0045] (5) Antibody incubation: Wash the blocked PVDF membrane with PBST for 5 min, dilute the corresponding primary antibody and incubate overnight at 4 °C. Take out the PVDF membrane bound to the primary antibody and wash it 5 times with PBST for 5 min each time. Incubate the HRP-labeled secondary antibody at room temperature for 1 h, and then wash it 5 times with PBST for 5 min each time.

[0046] (6) Exposure: Mix equal volumes of solution A and solution B to prepare an appropriate amount of film exposure solution, and evenly drop it onto the PVDF membrane. Develop color in the dark for 2 minutes, and then chemically expose it in a gel imaging system. Save the image and analyze the results.

[0047] The test results are as follows Figure 3 As shown, at the titer level, tetrandrine at 2.5 μM effectively inhibited PRRSV replication in MARC-145 cells; Western blot results showed that it had a significant inhibitory effect on PRRSV in MARC-145 cells, manifested by a significant reduction in viral N protein expression, and the inhibitory effect was dose-dependent.

[0048] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of tetrandrine in inhibiting porcine reproductive and respiratory syndrome virus.

2. Application of tetrandrine in the preparation of products for inhibiting porcine reproductive and respiratory syndrome virus.

3. A product for combating porcine reproductive and respiratory syndrome virus, characterized in that, The product contains a pharmacologically effective concentration of tetrandrine.

4. The article of claim 3, characterized in that, The pharmacologically effective concentration is 4-25 μM.

5. The article of claim 3, characterized in that, The pharmacologically effective concentration mentioned above is 4-12.5 μM when treating PAM cells.

6. The article of claim 3, characterized in that, The pharmacologically effective concentration mentioned above is 7-25 μM when treating MARC-145 cells.

7. The article of claim 3, characterized in that, The product described is a drug or an inhibitor.

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