Application of salvianolic acid A and its derivatives in the preparation of antiviral drugs

Abstract

The present invention provides the use of salvianolic acid A and its derivatives in the preparation of antiviral drugs, primarily for use against herpes viruses, particularly pseudorabies virus. The virus is specifically herpes virus, and the salvianolic acid A derivative is a pharmaceutically acceptable salt or a modified compound based on the salvianolic acid A. Salvianolic acid A and its derivatives can be used to prepare drugs against herpes viruses, particularly pseudorabies virus, thereby inhibiting or preventing herpes viruses, particularly pseudorabies virus.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the application of salvianolic acid A and its derivatives in the preparation of antiviral drugs. Background Art

[0002] Pseudorabies virus (PRV) is a linear, double-stranded DNA virus belonging to the genus Porcine Herpesvirus in the family Herpesviridae. It has a broad spectrum of infection, with pigs being its natural reservoir. Key symptoms include abortion in sows, reproductive failure in boars, diarrhea in piglets, respiratory distress, and neurological symptoms. It is susceptible to infection in pigs of all ages, and infected pigs can carry the virus for long periods, causing significant economic losses to the swine industry. Vaccination is the primary method for preventing the disease, but due to the recent emergence of PRV variants, vaccines are no longer able to protect pigs from infection. Therefore, the discovery of anti-PRV drugs is crucial for both prevention and treatment. Summary of the Invention

[0003] To solve the above problems, the present invention provides the use of salvianolic acid A and its derivatives in the preparation of antiviral drugs, mainly for herpes viruses, especially for pseudorabies virus.

[0004] The object of the present invention is achieved in the following manner:

[0005] The invention relates to the use of salvianolic acid A and its derivatives in the preparation of antiviral drugs, wherein the virus is specifically herpes virus, and the salvianolic acid A derivatives are pharmaceutically acceptable salts or compounds modified based on the same.

[0006] The herpes simplex virus is pseudorabies virus. The antiviral here includes antiviral itself and also includes antiviral protein alone.

[0007] The pseudorabies virus strain is at least one of PRV-QXY, PRV-GFP, PRV-HN, or PRV-ΔgE, or any of their identical genotypes. PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE are representative strains of different genotypes. SAA can inhibit PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE. Therefore, it can also inhibit strains of the same genotype as PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE.

[0008] Salvianol acid A (SAA) is a phenolic acid compound extracted and isolated from Salvia miltiorrhiza. It is a water-soluble compound, abbreviated as SAA. The structural formula of SAA is as follows:

[0009]

[0010] The present study proves that SAA has a broad-spectrum effect against multiple strains of PRV on Vero cells, and SAA has a broad-spectrum effect against multiple strains of PRV on LLC-PK cells, and proves that the virus proliferation can be effectively inhibited by SAA pretreatment and co-treatment methods. SAA has a significant inhibitory effect on the infectivity and RNA expression of multiple strains of PRV on Vero cells, SAA has a significant inhibitory effect on the protein expression of multiple strains of PRV on Vero cells, SAA has a significant inhibitory effect on the infectivity of multiple strains of PRV on LLC-PK cells, and SAA has a significant inhibitory effect on the infectivity of multiple strains of PRV on LLC-PK cells. It has a significant inhibitory effect on the protein expression of multiple strains of PRV. The SAA pretreatment and co-treatment methods can effectively inhibit the virus infection of PRV-GFP in Vero cells, and the SAA pretreatment and co-treatment methods can effectively inhibit the virus infection of PRV-QXY in Vero cells. Moreover, SAA has weak cytotoxicity to Vero and LLC-PK cells. Therefore, within a reasonable dosage range, salvianolic acid A and its derivatives can be used to prepare anti-herpes virus drugs, especially for the preparation of pseudorabies virus drugs, thereby inhibiting or preventing herpes viruses, especially pseudorabies virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the maximum non-toxic concentration of SAA on Vero and LLC-PK cells.

[0012] Figure 2 The virus TCID was measured after SAA-treated Vero cells and normal Vero cells were infected with different PRV strains. 50 and copy number expression.

[0013] Figure 3 Indirect immunofluorescence analysis of viral protein expression in SAA-treated Vero cells and normal Vero cells infected with different PRV strains. Panel A shows the inhibition of PRV-ΔgE infection in Vero cells by SAA. Anti-gB is used to label the virus (green fluorescence), and DAPI is used to stain the nuclei (blue fluorescence). Merge is an overlay of Anti-gB and DAPI images. As shown in the figure, PRV-ΔgE completely infects cells when 0 μM of the drug is added. As the SAA concentration increases (1 and 10 μM), green fluorescence decreases. At the maximum non-toxic concentration (50 μM), PRV-ΔgE infection is completely inhibited, resulting in the absence of green fluorescence. Panels B, C, and D, respectively, show the inhibition of PRV-HN, PRV-GFP, and PRV-QXY infection in Vero cells by SAA. The annotations are as in Figure A.

[0014] Figure 4The virus TCID was determined after SAA-treated LLC-PK cells and normal LLC-PK cells were infected with different PRV strains. 50 .

[0015] Figure 5 Figure 1 shows indirect immunofluorescence analysis of viral protein expression in SAA-treated and normal LLC-PK cells infected with different PRV strains. Panel A shows the inhibition of PRV-GFP infection in LLC-PK cells by SAA. GFP is the viral marker (green fluorescence), and DAPI is the nucleus stain (blue fluorescence). Merge represents the overlay of GFP and DAPI images. As shown in the figure, PRV-GFP completely infects cells with 0 μM of the drug. As the SAA concentration increases (1 and 10 μM), green fluorescence decreases. At the maximum non-toxic concentration (50 μM), PRV-GFP infection is completely inhibited, resulting in the absence of green fluorescence. Panels B, C, and D, respectively, show the inhibition of PRV-QXY, PRV-ΔgE, and RV-HN infection in LLC-PK cells by SAA. The annotations are as in Figure A.

[0016] Figure 6 TCID 50 Indirect immunofluorescence was used to detect PRV-GFP proliferation in Vero cells after drug pre-treatment and co-treatment. Panel C shows the inhibition of PRV-GFP infection in Vero cells after SAA pre-treatment. GFP represents the viral antibody marker (green fluorescence), DAPI represents the nucleus stain (blue fluorescence), and Merge represents the overlay of GFP and DAPI images. The figure shows that PRV-GFP can fully infect cells when 0 μM of the drug is added. At the maximum non-toxic concentration (50 μM), the drug significantly inhibits PRV-GFP infection, as evidenced by a significant decrease in green fluorescence. Figure D shows that co-treatment with the drug SAA inhibits PRV-GFP infection of Vero cells; GFP is the labeled virus group (green fluorescence), DAPI is the cell nucleus staining group (blue fluorescence), and Merge is the overlapping synthesis of GFP and DAPI images; it can be seen from the figure that when the 0 μM drug group is added, PRV-GFP can completely infect the cells. As the SAA concentration increases (1, 10 μM), it can be seen that the green fluorescence decreases. When the drug is used to the maximum non-toxic concentration (50 μM), PRV-GFP infection is completely inhibited, that is, there is no green fluorescence.

[0017] Figure 7 TCID 50Indirect immunofluorescence was used to detect PRV-QXY proliferation after drug pre-treatment and co-treatment with Vero cells. Panel C shows the inhibition of PRV-QXY infection of Vero cells after SAA pre-treatment. GFP represents the viral antibody marker (green fluorescence), DAPI represents the nucleus stain (blue fluorescence), and Merge represents the overlay of GFP and DAPI images. The figure shows that PRV-QXY can fully infect cells when the drug is added at 0 μM. However, the drug significantly inhibits PRV-QXY infection at 10 and 50 μM, as evidenced by a significant decrease in green fluorescence. Figure D shows that co-treatment with the drug SAA inhibits PRV-QXY infection of Vero cells; GFP is the virus antibody group (green fluorescence), DAPI is the cell nucleus staining group (blue fluorescence), and Merge is the overlapping synthesis of GFP and DAPI images; it can be seen from the figure that when the 0 μΜ drug group is added, PRV-QXY can completely infect the cells. As the SAA concentration increases (1, 10 μΜ), it can be seen that the green fluorescence decreases. When the drug is used to the maximum non-toxic concentration (50 μΜ), PRV-QXY infection is completely inhibited, that is, there is no green fluorescence. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to specific embodiments. It is necessary to point out that this embodiment is only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art in this field can make some non-essential improvements and adjustments based on the contents of the present invention.

[0019] Implementation 1: Maximum non-toxic concentration of SAA on Vero and LLC-PK cells

[0020] 1. Experimental Materials

[0021] 1.1 Cells, compounds, and kits

[0022] SAA compounds were purchased from MedChemExpress (MCE); Vero cells and LLC-PK cells were purchased from the American Type Culture Collection (ATCC); CCK kit (ZP328) was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.; DMEM medium was purchased from Solebold (catalog number 11995); fetal bovine serum (FBS) was purchased from Gibco, a subsidiary of Thermo Fisher Scientific; DMEM medium containing 10% FBS is DMEM with FBS added at a volume ratio of 10%; 24-well cell plates were purchased from Thermo Fisher Scientific.

[0023] 1.2 Experimental Instruments

[0024] Varioskan Flash spectral scanning multifunctional reader (Thermo Fisher, USA).

[0025] 2. Experimental methods and results

[0026] 2.1 Cell Culture: Culture cells at 37°C in a 5% CO2 humidified incubator. Use Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. Subculture cells at a 1 / 3 to 1 / 4 ratio after reaching 90% confluence.

[0027] 2.2 Cytotoxicity assay of SAA

[0028] Vero cells were collected at 5×10 3 Cells were seeded at 100 μL / well in a 96-well cell culture plate and allowed to adhere until ready for use. DMSO-dissolved SAA was prepared in DMEM at a concentration gradient of 0 (DMSO only), 100, 50, 10, 1 μM, and 500, 100, and 50 nM. Three replicate wells were set up for each concentration gradient. 24 hours after drug treatment, 10 μL / well of CCK reagent was added to the culture supernatant. The cells were incubated in a cell incubator for 2 hours. Absorbance was read using a Varioskan Flash spectrophotometer and cell viability was calculated. The SAA cytotoxicity assay for LLC-PK cells was performed identically to that for Vero cells.

[0029] The results are as follows Figure 1 The results showed that SAA had no cytotoxicity to Vero and LLC-PK cells below 50 μM.

[0030] Implementation 2: All-treatment of Vero cells with SAA and determination of viral TCID after infection of normal Vero cells with different PRV strains 50 and copy number expression

[0031] 1. Experimental Materials

[0032] 1.1 Viruses, cells, compounds,

[0033] PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE viruses were maintained in the laboratory; Vero cell lines were purchased from the American Type Culture Collection (ATCC); SAA compounds were purchased from MedChemExpress (MCE). DNA extraction kits were purchased from Takara; the fluorescence quantitative reagent SYBR Premix Ex TapII was purchased from Takara; PEDV primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Solebro (Cat. No. 11995); fetal bovine serum (FBS) was purchased from Gibco, a Thermo Fisher Scientific subsidiary; DMEM with 10% FBS was prepared by adding FBS to DMEM at a 10% volume ratio; 24-well cell plates were purchased from Thermo Fisher Scientific; and PBS (0.01 M, pH 7.2) was purchased from Solebro.

[0034] 1.2 Experimental Instruments

[0035] QuantStudioTM6 Flex real-time fluorescence quantitative PCR instrument (Thermo Fisher, USA).

[0036] 2. Experimental methods and results

[0037] 2.1 SAA Full Processing

[0038] SAA was diluted with DMEM to different concentrations (0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2), and then the diluted SAA was added to 24-well cell plates containing cells (24-well cell plates containing cells: Vero cells cultured in DMEM medium containing 10% fetal bovine serum were added to the 24-well cell plates until they were 90% confluent, with 1×10 cells per well). 5 ), add 600 μL to each well and incubate in a 5% CO2 incubator for 4 hours. Then, wash the cells in the 24-well cell plate once with PBS to remove residual SAA from the pretreatment. Add the virus and diluted SAA to the cell plate together, adding 200 μL of the virus solution and diluted SAA solution at a virus concentration of 0.1 MOI. SAA diluted to the same concentration should always be added to the same cell well and placed in the cell incubator for 1 hour of adsorption. Replace with DMEM; after 1 hour of adsorption, wash the cells in the 24-well cell plate twice with PBS, then add DMEM containing different concentrations of SAA (0, 1, 10, and 50 μM) (dilution method as described in Implementation Plan 1, Section 2.2).

[0039] 2.2 Virus Collection

[0040] Calculate the time from the time of adding the virus. After 24-48 hours of infection, observe the cell lesions in the culture plate under a microscope. When the lesions reach 80-90%, collect the virus. Place the culture plate in the refrigerator and freeze and thaw it repeatedly twice. Use a pipette to blow the cells at the bottom of the plate, aspirate the culture medium and cells into an EP tube, centrifuge at 3900 g for 10 minutes, and dispense the supernatant into 2 mL centrifuge tubes at 120 μL / tube. Store at -80℃ for a longer time. Used for subsequent TCID 50 and fluorescence quantitative determination.

[0041] 2.3 TCID 50 Determination

[0042] The cells were divided into 1×10 4 Cells / well were seeded in a 96-well plate, 100 μL / well; 12 clean, sterile centrifuge tubes were prepared; 900 μL DMEM medium was added to each centrifuge tube; the virus was diluted 10 times -1 -10 -12 ; Take 100 μL of virus stock solution and add it to the first centrifuge tube, mix well, and record it as 10 -1 Dilution: Take 100 μL of the dilution solution in the first centrifuge tube and add it to the second tube, mix well, and record it as 10 -2 Dilution: Take 100 μL of the dilution solution from the second centrifuge tube and add it to the third tube, and then make 12 gradient dilutions of the virus to 10 -12 Discard the old culture medium and add 100 μL / well of the virus dilution solution to the corresponding cell wells; incubate in a 37°C, 5% CO2 incubator; discard the diluted virus solution after 1 hour and add 150 μL / well of DMEM medium; incubate in a 37°C, 5% CO2 incubator; observe and record the number of diseased wells and dilution after 4-5 days, and calculate the TCID 50 Calculation method: Find two columns with more than 50% and less than 50% lesions, and then calculate the ratio. For example: 7 columns with 6 lesion holes, 8 columns with 3 lesion holes, calculate TCID 50 is 10 7 + specific distance / 0.1mL, specific distance=(greater than 50%-50%) / (greater than 50%-less than 50%)= (6 / 8-4 / 8) / (6 / 8-3 / 8)=2 / 3.

[0043] 2.4 Total DNA extraction and real-time quantitative reverse transcription PCR

[0044] The total DNA of the sample collected in Implementation Plan 2.2 was extracted using the Tiangen Total DNA Extraction Kit, and the DNA concentration of the sample was measured using an ultra-micro spectrophotometer.

[0045] 2.5 Fluorescence quantitative PCR

[0046] Use the designed PRV fluorescent quantitative PCR primers to amplify the target gene. Reaction system:

[0047]

[0048] The reaction conditions were: pre-denaturation at 95°C for 10 min, amplification at 95°C for 10 s, and amplification at 60°C for 34 s for 40 cycles, and fluorescence signal collection. The Ct value corresponding to the viral gene copy number of each sample was calculated using a standard curve.

[0049] The results are as follows Figure 2 It was shown that 1, 10, and 50 μM SAA could significantly inhibit the TCID of PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE. 50 In addition, different concentrations of SAA could significantly inhibit viral copy number.

[0050] Implementation Plan 3: All-treatment of Vero cells with SAA and indirect immunofluorescence assay of viral protein expression after infection of normal Vero cells with different PRV strains

[0051] 1. Experimental Materials

[0052] 1.1 Viruses, cells, reagents,

[0053] PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE viruses were maintained in the laboratory; Vero cell lines were purchased from the American Type Culture Collection (ATCC); SAA compounds were purchased from MedChemExpress (MCE); monoclonal antibodies against PRV gB protein were prepared in our laboratory; goat anti-mouse FITC was purchased from Thermo Fisher Scientific; DAPI nuclear stain was purchased from Hyclone; 4% paraformaldehyde was purchased from Hyclone; Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Solebro (Cat. No. 11995); fetal bovine serum (FBS) was purchased from Gibco, a subsidiary of Thermo Fisher Scientific; DMEM with 10% FBS was prepared by adding FBS to DMEM at a 10% volume ratio; 24-well cell plates were purchased from Thermo Fisher Scientific; and PBS (0.01 M, pH 7.2) was purchased from Solebro.

[0054] 1.2 Experimental Instruments

[0055] LSM800 laser confocal microscope (ZEISS).

[0056] 2. Experimental methods and results

[0057] 2.1 SAA processing (full processing)

[0058] SAA was diluted with DMEM to different concentrations (0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2), and then the diluted SAA was added to 24-well cell plates containing cells (24-well cell plates containing cells: Vero cells cultured in DMEM medium containing 10% fetal bovine serum were added to the 24-well cell plates until they were 90% confluent, with 1×10 cells per well). 5 ), add 600 μL to each well and incubate in a 5% CO2 incubator for 4 hours. Then, wash the cells in the 24-well plate once with PBS to remove residual SAA from the pretreatment. Add the virus and diluted SAA to the plate together, adding 200 μL of the virus solution and diluted SAA at a 0.1 MOI. SAA diluted to the same concentration should always be added to the same well and adsorbed in the cell incubator for 1 hour. Replace with DMEM; after 1 hour of adsorption, wash the cells in the 24-well plate twice with PBS and add DMEM containing different concentrations of SAA (0, 1, 10, and 50 μM) (dilution method as described in Implementation Plan 1, Section 2.2).

[0059] 2.2 IFA detection of target protein expression

[0060] PRV-GFP strain: Virus-infected cells were further grown in the presence of SAA until 20 hours post-infection. The culture medium was then discarded and cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were then washed five times with PBS. After nuclear staining with DAPI for 15 minutes, the cells were washed five times with PBS, the bottom surface was covered with PBS, and the cells were photographed under a fluorescence microscope.

[0061] Other strains: Virus-infected cells were further grown in the presence of SAA until 20 hours post-infection. The original culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, incubated in 1% bovine serum albumin (BSA) for 30 minutes, and washed five times with PBS. Cells were then incubated with a mouse monoclonal antibody specific for the gB protein at 37°C for 1 hour, washed five times with PBS, and incubated with a FITC-conjugated goat anti-mouse secondary antibody at 37°C in the dark for 30 minutes, followed by five washes with PBS. After nuclear staining with DAPI for 15 minutes, the cells were washed five times with PBS, the bottom surface covered with PBS, and photographed under a fluorescence microscope.

[0062] The results are as follows Figure 3 The green fluorescence of the viruses PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE in the SAA-treated group decreased, indicating that the expression of viral proteins was reduced after SAA treatment.

[0063] Implementation Plan 4: All-treatment of LLC-PK cells with SAA and normal LLC-PK cells infected with different PRV strains and then measuring viral TCID50

[0064] 1. Experimental Materials

[0065] 1.1 Viruses, cells, compounds,

[0066] PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE viruses were maintained in the laboratory; the LLC-PK cell line was purchased from the American Type Culture Collection (ATCC); and SAA compounds were purchased from MedChemExpress (MCE). DNA extraction kits were purchased from Takara; the fluorescence quantitative reagent SYBR Premix Ex Tap II was purchased from Takara; and PEDV primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Dulbecco's Modified Eagle's Body Medium (DMEM) was purchased from Solebro (Cat. No. 11995); fetal bovine serum (FBS) was purchased from Gibco, a Thermo Fisher Scientific subsidiary; DMEM supplemented with 10% FBS was prepared by adding FBS to DMEM at a 10% volume ratio. 24-well cell plates were purchased from Thermo Fisher Scientific; and PBS (0.01 M, pH 7.2) was purchased from Solebro.

[0067] 2. Experimental methods and results

[0068] 2.1 SAA processing (full processing)

[0069] SAA was diluted to different concentrations (0, 1, 10, 50 μM) using DMEM (the dilution method is as in Implementation Plan 1, Section 2.2). The diluted SAA was then added to 24-well cell plates containing cells (LLC-PK cells were cultured in DMEM containing 10% fetal bovine serum until they were 90% confluent, and then LLC-PK cells were evenly added to the 24-well cell plates, i.e., the number of cells per well was 1×10 5 ), add 600 μL to each well and incubate in a 5% CO2 incubator for 4 hours. Then, wash the cells in the 24-well cell plate once with PBS to remove residual serum and SAA from pretreatment. Add the virus and diluted SAA to the cell plate together, adding 200 μL of the virus solution and diluted SAA at a 0.1 MOI. SAA diluted to the same concentration should always be added to the same well and adsorbed in the cell incubator for 1 hour. Replace with DMEM; after 1 hour of adsorption, wash the cells in the 24-well cell plate twice with PBS, then add DMEM containing different concentrations of SAA (0, 1, 10, and 50 μM) (dilution method as described in Implementation Plan 1, Section 2.2).

[0070] 2.2 Virus collection and TCID 50 Determination

[0071] Calculate the time from the time of adding the virus. After 24-48 hours of infection, observe the cell lesions in the culture plate under a microscope. When the lesions reach 80-90%, collect the virus. Place the culture plate in the refrigerator and freeze and thaw it twice. Use a pipette to blow the cells at the bottom of the plate, aspirate the culture medium and cells into an EP tube, centrifuge at 3900 g for 10 minutes, and dispense the supernatant into 2 mL centrifuge tubes at 120 μL / tube. Store at -80°C for a longer time. Determine the TCID after infection with different strains 50 .

[0072] The results are shown in the figure Figure 4 It was shown that treatment of LLC-PK cells with 1, 10, and 50 μM SAA could significantly inhibit the TCID of PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE. 50 proliferation.

[0073] Implementation Plan 5: Indirect immunofluorescence assay of viral protein expression in LLC-PK cells and normal LLC-PK cells infected with different PRV strains after SAA all-treatment

[0074] 1. Experimental Materials

[0075] 1.1 Viruses, cells, and reagents

[0076] PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE viruses were maintained in the laboratory; the LLC-PK cell line was purchased from the American Type Culture Collection (ATCC); all SAA compounds were purchased from MedChemExpress (MCE); the monoclonal antibody against PRV gB protein was prepared in our laboratory; goat anti-mouse FITC was purchased from Thermo Fisher Scientific; DAPI nuclear stain was purchased from Hyclone; 4% paraformaldehyde was purchased from Hyclone; Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Solebro (Cat. No. 11995); fetal bovine serum (FBS) was purchased from Gibco, a subsidiary of Thermo Fisher Scientific; DMEM with 10% FBS is DMEM supplemented with FBS at a 10% volume ratio; 24-well cell plates were purchased from Thermo Fisher Scientific; and PBS (0.01 M, pH 7.2) was purchased from Solebro.

[0077] 1.2 Experimental Instruments

[0078] LSM800 laser confocal microscope (ZEISS).

[0079] 2. Experimental methods and results

[0080] 2.1 SAA processing (full processing)

[0081] SAA was diluted with DMEM to different concentrations (0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2), and then the diluted SAA was added to 24-well plates containing cells (LLC-PK cells were cultured in DMEM medium containing 10% fetal bovine serum to 90% fusion, and then LLC-PK cells were evenly added to the 24-well cell plate, i.e., the number of cells per well was 1×10 5 ), add 600 μL to each well and incubate in a 5% CO2 incubator for 4 hours. Then, wash the cells in the 24-well plate once with PBS to remove residual serum and SAA from pretreatment. Add the virus and diluted SAA to the plate together, adding a total of 200 μL of virus solution and diluted SAA at a 0.1 MOI. SAA diluted to the same concentration should always be added to the same well and adsorbed in the cell incubator for 1 hour. Replace with DMEM; after 1 hour of adsorption, wash the cells in the 24-well plate twice with PBS, then add DMEM containing different concentrations of SAA (0, 1, 10, and 50 μM) (dilution method as described in Implementation Plan 1, Section 2.2).

[0082] 2.2 IFA detection of target protein expression

[0083] PRV-GFP strain: Virus-infected cells were further grown in the presence of SAA until 20 hours post-infection. The culture medium was then discarded and cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were then washed five times with PBS. After nuclear staining with DAPI for 15 minutes, the cells were washed five times with PBS, the bottom surface was covered with PBS, and the cells were photographed under a fluorescence microscope.

[0084] Other strains: Virus-infected cells were further grown in the presence of SAA until 20 hours post-infection. The original culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, incubated in 1% bovine serum albumin (BSA) for 30 minutes, and washed five times with PBS. Cells were then incubated with a mouse monoclonal antibody specific for gB protein at 37°C for 1 hour, washed five times with PBS, and incubated with a FITC-conjugated goat anti-mouse secondary antibody at 37°C in the dark for 30 minutes, followed by five washes with PBS. After nuclear staining with DAPI for 15 minutes, the cells were washed five times with PBS, the bottom surface covered with PBS, and photographed under a fluorescence microscope.

[0085] The results are as follows Figure 5 The results showed that the green fluorescence of viruses PRV-QXY, PRV-GFP, PRV-HN, and PRV-ΔgE in the LLC-PK cell group treated with SAA decreased, indicating that the expression of viral proteins was reduced after SAA treatment.

[0086] Implementation Plan 6: TCID 50Indirect immunofluorescence detection of PRV-GFP proliferation after drug pre-treatment (Pre-treatment) and co-treatment (Co-treatment) of Vero

[0087] 1. Experimental Materials

[0088] 1.1 Viruses, cells, compounds,

[0089] PRV-GFP was maintained in the laboratory; Vero cell lines were purchased from the American Type Culture Collection (ATCC); SAA compounds were purchased from MedChemExpress (MCE). Dulbecco's Modified Eagle Medium (DMEM) was purchased from Solebro (Cat. No. 11995); fetal bovine serum (FBS) was purchased from Gibco, a Thermo Fisher Scientific subsidiary; DMEM with 10% FBS was prepared by adding FBS to DMEM at a 10% volume ratio; 24-well cell plates were purchased from Thermo Fisher Scientific; and PBS (0.01 M, pH 7.2) was purchased from Solebro.

[0090] 1.2 Experimental Instruments

[0091] LSM800 laser confocal microscope (ZEISS).

[0092] 2. Experimental methods and results

[0093] 2.1 SAA Processing

[0094] Pre-treatment: SAA was diluted with DMEM to different concentrations (0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2), and then the diluted SAA was added to 24-well cell plates containing cells (24-well cell plates containing cells: LLC-PK cells were cultured in DMEM medium containing 10% fetal bovine serum to a fusion of 90%, and then LLC-PK cells were evenly added to the 24-well cell plates, i.e., the number of cells per well was 1×10 5 ) was added to each well and incubated in a 5% CO2 incubator. After 4 hours of pretreatment, virus solution was added to allow for proper viral adsorption. A total of 200 μL of virus solution and SAA diluted to various concentrations (0, 1, 10, and 50 μM) (dilution method as described in Implementation Plan 1, Section 2.2) were added to achieve a virus solution concentration of 0.1 MOI. After 1 hour, the cells were replaced with DMEM. SAA diluted to the same concentration was always added to the same well.

[0095] Co-treatment: Wash the 24-well cell plate containing cells with PBS (24-well cell plate containing cells: LLC-PK cells cultured in DMEM medium containing 10% fetal bovine serum to 90% fusion, then LLC-PK cells were evenly added to the 24-well cell plate, that is, the number of cells per well was 1×10 5 Add the virus solution and SAA (0, 1, 10, 50 μM) diluted in DMEM (dilution method as described in Implementation Plan 1, Section 2.2) to a 24-well cell plate. Add a total of 200 μL of virus solution and diluted SAA at a virus concentration of 0.1 MOI. Allow the cells to adsorb for 1 h. Wash the cells in the 24-well cell plate twice with PBS. Add DMEM culture medium containing different concentrations of SAA (0, 1, 10, 50 μM) (dilution method as described in Implementation Plan 1, Section 2.2) to the corresponding wells. Always add SAA diluted to the same concentration to the same well.

[0096] 2.2 TCID 50 Determination

[0097] The virus-infected cells treated with SAA according to the treatment steps were further infected for 20 h, and the original culture medium or culture fluid was discarded, and the infected cells were collected and TCID 50 Determination.

[0098] 2.3 IFA detection of target protein expression

[0099] Virus-infected cells treated with SAA according to the treatment steps were further grown in the presence of SAA until 20 hours post-infection. The original culture medium or fluid was discarded, and the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and washed five times with PBS. After nuclear staining with DAPI for 15 minutes, the cells were washed five times with PBS, the bottom surface was covered with PBS, and the cells were photographed under a fluorescence microscope.

[0100] The results are as follows Figure 6 The results show that after Vero cells were pretreated with 50 μM SAA, the viral TCID 50 Vero cells were co-treated with SAA 50, 10, and 1 μM, and the virus TCID 50 and fluorescence were reduced.

[0101] Implementation Plan 7: TCID 50 Indirect immunofluorescence detection of PRV-QXY proliferation after drug pre-treatment (Pre-treatment) and co-treatment (Co-treatment) of Vero

[0102] 1. Experimental Materials

[0103] 1.1 Viruses, cells, compounds,

[0104] PRV-QXY was maintained in the laboratory; Vero cell lines were purchased from the American Type Culture Collection (ATCC); and SAA compounds were purchased from MedChemExpress (MCE). Monoclonal antibodies against PRV gB protein were generated in our laboratory; goat anti-mouse FITC was purchased from Thermo Fisher Scientific; DAPI nuclear stain was purchased from Hyclone; and 4% paraformaldehyde was purchased from Hyclone. Dulbecco's modified Eagle's Membrane Medium (DMEM) was purchased from Solebro (Cat. No. 11995). Fetal bovine serum (FBS) was purchased from Gibco, a subsidiary of Thermo Fisher Scientific; and 24-well cell plates were purchased from Thermo Fisher Scientific. DMEM with 10% FBS was prepared by adding FBS to DMEM at a 10% volume ratio. PBS (0.01 M, pH 7.2) was purchased from Solebro.

[0105] 1.2 Experimental Instruments

[0106] LSM800 laser confocal microscope (ZEISS).

[0107] 2. Experimental methods and results

[0108] 2.1 SAA Processing

[0109] Pre-treatment: SAA was diluted with DMEM to different concentrations (0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2), and then the diluted SAA was added to 24-well cell plates containing cells (24-well cell plates containing cells: Vero cells were cultured in DMEM medium containing 10% fetal bovine serum until they were 90% confluent, and then the Vero cells were evenly added to the 24-well cell plates, i.e., the number of cells per well was 1×10 5 ) was added to each well and incubated in a 5% CO2 incubator. After 4 hours of pretreatment, virus solution was added to allow for proper viral adsorption. A total of 200 μL of virus solution and SAA diluted to various concentrations (0, 1, 10, and 50 μM) (dilution method as described in Implementation Plan 1, Section 2.2) were added, resulting in a virus solution concentration of 0.1 MOI. After 1 hour, the cells were replaced with DMEM. SAA diluted to the same concentration was always added to the same well.

[0110] Co-treatment: Wash the 24-well cell plate containing cells with PBS (24-well cell plate containing cells: Vero cells cultured in DMEM medium containing 10% fetal bovine serum to 90% confluence, and then add Vero cells evenly into the 24-well cell plate, that is, the number of cells per well is 1×10 5), add the virus solution and SAA diluted in DMEM (0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2) to a 24-well cell plate. Add 200 μL of virus solution and SAA diluted to different concentrations (0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2) at a virus concentration of 0.1 MOI. Allow adsorption for 1 h. Wash the cells in the 24-well cell plate twice with PBS, and add DMEM culture medium containing different concentrations of SAA (DMSO, i.e., 0, 1, 10, 50 μM) (dilution method as in Implementation Plan 1, 2.2) accordingly. SAA diluted to the same concentration is always added to the same cell well.

[0111] 2.2 TCID 50 Determination

[0112] The virus-infected cells treated with SAA were further infected for 20 h, the original culture medium or fluid was discarded, and the infected cells were collected and TCID 50 Determination.

[0113] 2.3 IFA detection of target protein expression

[0114] Following SAA treatment, virus-infected cells were further grown in the presence of SAA until 20 hours post-infection. The original culture medium or fluid was then discarded and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The cells were incubated in 1% bovine serum albumin (BSA) for 30 minutes and washed five times with PBS. The cells were then incubated with a mouse monoclonal antibody specific for gB protein at 37°C for 1 hour, washed five times with PBS, and incubated with a FITC-conjugated goat anti-mouse secondary antibody at 37°C in the dark for 30 minutes. The cells were then washed five times with PBS. After nuclear staining with DAPI for 15 minutes, the cells were washed five times with PBS, the bottom surface covered with PBS, and photographed under a fluorescence microscope.

[0115] The results are as follows Figure 7 It shows that after Vero cells were pretreated with 50 and 10 μM SAA, the virus TCID 50 Vero cells were co-treated with SAA 50, 10, and 1 μM, and the virus TCID 50 and fluorescence were reduced.

[0116] In the second, third, fourth and fifth embodiments of the present invention, full-treatment (all-treatment) was performed on Vero cells and LLC-PK cells, i.e., drug addition was performed throughout the entire process to verify the effects of drugs on different strains (PRV-QXY, PRV-GFP, PRV-HN, PRV-ΔgE). In the second and fourth embodiments, TCID 50To detect the effect of SAA on viral infectivity and the effect of fluorescent quantitative detection on viral RNA expression, implementation plans three and five use indirect immunofluorescence to detect the effect of SAA on viral protein expression.

[0117] Plans 6 and 7 performed pre-treatment and co-treatment on Vero cells. Pre-treatment involved adding the drug beforehand, followed by no drug addition; co-treatment involved adding the drug and virus simultaneously, without pre-treatment. These tests examined the inhibitory effects of the drugs on selected representative virus strains (PRV-GFP vaccine strain and PRV-QXY clinical field strain). Plans 6 and 7 also tested key viral infectivity and protein expression.

[0118] The purpose of implementation plans two, three, four, and five is to verify the broad-spectrum effects of drugs on PRV and their impact on all stages of PRV (RNA expression, viral infectivity, viral proteins), so multiple strains and multiple methods are selected.

[0119] The main purpose of implementation plans six and seven is to verify whether the drug has the potential to prevent and treat the disease.

[0120] Through various embodiments of the present invention, the present invention used SAA compounds as research objects and found that SAA can inhibit the proliferation of different PRV strains in Vero and LLC-PK cells. Therefore, it is inferred that SAA and its derivatives can be used to fight herpes viruses and even most enveloped viruses.

[0121] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. It should be pointed out that for those skilled in the art and any technician familiar with this technical field, without departing from the overall concept of the present invention, the technical solution and the inventive concept of the present invention are equivalently replaced or changed, and several changes and improvements are made, which should also be regarded as the scope of protection of the present invention.

Claims

1. The use of salvianolic acid A and its derivatives in the preparation of antiviral drugs, characterized in that: The virus is specifically pseudorabies virus, and the salvianolic acid A derivative is a pharmaceutically acceptable salt of salvianolic acid A.

2. The use of salvianolic acid A and its derivatives in the preparation of antiviral drugs according to claim 1, characterized in that: The pseudorabies virus strain is one of PRV-QXY, PRV-GFP, PRV-HN, PRV-ΔgE, or one of the same genotypes thereof.

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