Application of aspirin in the preparation of drugs for treating herpes simplex virus type 1

Abstract

This invention relates to the field of pharmaceutical technology, and in particular to the application of aspirin in the preparation of drugs for treating herpes simplex virus type 1 (HSV-1). The aspirin screened from marketed drugs in this invention possesses antiviral activity. For the first time, the efficacy of 17-DMAG against HSV-1 in multiple cell lines was systematically evaluated, and a multi-level validation system was established, encompassing cell viability, viral fluorescence, gene transcription, genome replication, and protein expression. Compared with the existing drug acyclovir, 17-DMAG has significant advantages such as a novel mechanism of action (targeting host accessory proteins), low risk of drug resistance, and comprehensive and reliable efficacy, providing an important experimental basis and candidate molecules for the development of novel anti-HSV-1 drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of aspirin in the preparation of drugs for treating herpes simplex virus type 1. Background Technology

[0002] HSV-1, short for Herpes Simplex Virus Type 1, is a very common DNA virus with a lifelong latency period. Infection is mainly caused by direct contact, weakened immunity, skin or mucous membrane damage, viral reactivation, and genetic susceptibility. Herpes simplex virus type 1 belongs to the Herpesviridae family, and infection may manifest as cold sores or other symptoms, which can be treated with antiviral medications.

[0003] Currently, traditional anti-HSV-1 drugs have significant limitations. Acyclovir (ACV) is a commonly used drug for the clinical treatment of herpes simplex virus type 1 (HSV-1) infection, but long-term use can easily lead to the emergence of drug-resistant strains, and some patients cannot tolerate it due to side effects such as kidney damage. Moreover, there is a lack of novel inhibitors on the market that target the entire viral replication process. Existing drugs mainly target viral DNA polymerase (such as ACV), and their inhibitory ability on other stages of the viral life cycle (such as capsid assembly and budding) is limited, making it difficult to completely eliminate the virus. In addition, existing drug screening systems are complex and difficult to quickly assess antiviral efficacy. Conventional efficacy evaluation requires multiple independent experiments, such as plaque formation assays, viral titer determination, and gene expression analysis, which is cumbersome, time-consuming, and makes it difficult to simultaneously measure the inhibitory effects of drugs on viral proteins and nucleic acids at the cellular level.

[0004] Existing research on the anti-HSV-1 effects of Hsp90 inhibitors is insufficient. Although 17-DMAG (aspirin) has been studied as an Hsp90 inhibitor in tumor treatment, its specific anti-HSV-1 effects, concentration range, and safety (cytotoxicity) lack systematic evaluation, especially the gap in studies on differences in different cell lines (such as Vero, 293T, and A549). Summary of the Invention

[0005] The purpose of this invention is to provide the use of aspirin in the preparation of a drug for treating herpes simplex virus type 1.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Application of aspirin in the preparation of drugs for treating herpes simplex virus type 1.

[0007] Among them, the IC50 of the aspirin in uninfected cells 50The concentration is approximately 10-20 μM; under infection conditions, a concentration of 0.5-3 μM is effective against the virus without significant cytotoxicity.

[0008] The specific experimental plan is as follows: (1) Drug preparation and concentration gradient design 17-DMAG was dissolved in DMSO to prepare a 10 mM stock solution, which was then further diluted to a series of working concentrations, including 0.1 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, and 10 μM.

[0009] Acyclovir (5 μM), a traditional antiviral drug, was used as a positive control.

[0010] (2) Cell model and viral infection HSV-1-GFP (HSV-1 F strain with GFP tag) was amplified using Vero cells and infected with human-derived cells such as 293T and A549 at MOI=1.

[0011] One hour after virus adsorption, culture medium containing different concentrations of 17-DMAG was added and cultured for another hour.

[0012] (3) Multi-dimensional antiviral efficacy evaluation system Fluorescence microscopy observation: The inhibitory effect of drugs on viral proliferation can be directly characterized by utilizing the virus's own GFP signal.

[0013] CCK 8 cell viability assay: determination of the half-maximal inhibitory concentration (IC50) of 17-DMAG in different cell types. 50 ) and its cellular protective effect under infected conditions.

[0014] qPCR (RNA level): Detects the transcriptional levels of the immediate early viral gene (ICP0) and DNA polymerase gene (UL30) to evaluate the inhibitory effect of drugs on viral gene expression.

[0015] qPCR (DNA level): Extract total DNA from infected cells, quantify viral genome copy number, and directly reflect the blocking effect of drugs on viral DNA replication.

[0016] Western Blot (protein level): The expression levels of viral envelope and capsid proteins were detected using GFP antibody to verify the inhibitory effect of the drug on viral protein synthesis.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention provides the first systematic evaluation of the anti-HSV-1 efficacy of 17-DMAG in various cell lines, establishing a multi-level validation system encompassing cell viability, viral fluorescence, gene transcription, genome replication, and protein expression. Compared to the existing drug acyclovir, 17-DMAG exhibits significant advantages, including a novel mechanism of action (targeting host accessory proteins), low risk of drug resistance, and comprehensive and reliable efficacy, providing an important experimental foundation and candidate molecules for the development of novel anti-HSV-1 drugs.

[0018] (1) 17-DMAG significantly inhibits HSV 1 replication Fluorescence experiments showed that in 293T and A549 cells, the number of GFP-positive cells was significantly reduced after treatment with 17 DMAG (0.5~3 μM) in a concentration-dependent manner.

[0019] qPCR results showed that 17 DMAG could effectively reduce the RNA levels of HSV 1 ICP0 and UL30, as well as the viral genomic DNA copy number, with effects comparable to or better than 5 μM ACV.

[0020] Western blot results showed that the expression level of viral GFP fusion protein decreased with increasing 17 DMAG concentration, further confirming its ability to inhibit viral protein synthesis.

[0021] (2) A safe and effective concentration range has been determined. The CCK8 assay determined the IC50 of 17 DMAG in uninfected cells. 50 The concentration is approximately 10–20 μM (slightly different depending on the cell line); under infection conditions, a concentration of 0.5–3 μM is effective against the virus without significant cytotoxicity.

[0022] (3) It has broad-spectrum cell line activity Consistent inhibitory effects were observed in Vero (used for viral amplification), 293T, and A549 cells, suggesting that the drug may be suitable for host cells from different tissue sources. Attached Figure Description

[0023] Figure 1 IC for 17-DMAG processing for 12 hours 50 result.

[0024] Figure 2 Cell viability was determined by treating HSV-1-infected cells with 17-DMAG for different durations.

[0025] Figure 3 Preliminary results from fluorescence experiments of 17-DMAG in 293T and A549 cells demonstrate that 17-DMAG can inhibit HSV-1 infection.

[0026] Figure 4 To verify the experimental results of 17-DMAG inhibiting HSV-1 infection (RNA) in 293T cells alone.

[0027] Figure 5 The experimental results of 17-DMAG inhibiting HSV-1 infection (DNA) were verified in A549 cells by combining 17-DMAG with a positive control drug.

[0028] Figure 6 The experimental results of 17-DMAG inhibiting HSV-1 infection (DNA) were verified in 293T cells by combining 17-DMAG with a positive control drug.

[0029] Figure 7 This study aims to further confirm the experimental results of 17-DMAG inhibiting HSV-1 replication in 293T and A549 cells. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: HSV-1-GFP Amplification 1. Cell Culture Cell passage: Vero cells were grown to approximately 90% confluence in 10cm dishes. The old culture medium was discarded, and the cells were washed twice with 6mL PBS. 1mL trypsin was added, and the cells were incubated at 37°C for 2 minutes for digestion. Under a microscope, most cells were observed to have become rounded. The trypsin was discarded, and 1mL of 10% FBS and 1% P / S DMEM was added to stop the digestion. The cells were centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1mL of 10% FBS and 1% P / S DMEM. One-third of the cells were then passaged.

[0032] 2. Viral infection The culture medium was aspirated from the culture dish, and the cells were rinsed with 6 mL of PBS. This process was repeated twice, and then the PBS was discarded. Herpes simplex virus strain 1F (HSV-1-GFP, from Guangzhou laboratory, website: https: / / www.gzlab.ac.cn) tagged with GFP was used for infection. 100 μL of the viral stock solution was mixed with 7 mL of serum-free and antibiotic-free DMEM and added to the culture dish. After adsorption for 1.5 h, the liquid in the culture dish was discarded, and 7 mL of 2% FBS and 1% P / S DMEM was added. The viral infection was then observed under a fluorescence microscope at 12 h, 24 h, and 48 h.

[0033] 3. Sample collection Once 90% of the cells showed cytopathic effects, the culture dish was sealed with sealing film and frozen at -80°C for 30 minutes. It was then shaken on a shaker at room temperature for 15 minutes, and this process was repeated three times. The thawed liquid was collected and transferred to 15 mL centrifuge tubes, and centrifuged at 12,000 rpm for 15 minutes at 4°C. A 0.45 μm filter was connected to a syringe, and the supernatant was poured into the syringe. The syringe was pushed to the bottom to filter the solution. The filtrate was collected and aliquoted into 1.5 mL centrifuge tubes to obtain the first-generation virus solution (P1), which was then stored at -80°C.

[0034] 4. Second amplification of HSV-1-GFP Take 100 μL of P1 generation HSV-1-GFP stock solution and mix it with 1 mL of serum-free and antibiotic-free DMEM. Transfer the mixture to a 1.5 mL centrifuge tube for later use. Rinse Vero cells in the culture dish with 6 mL of PBS, then add 3 mL of serum-free and antibiotic-free DMEM and virus dilution buffer. Incubate for 1.5 h after infection. After incubation, add 4 mL of 10% FBS and 1% P / S DMEM and incubate overnight. Observe the virus infection status under a fluorescence microscope at 12 h, 24 h, and 48 h. Collect the sample after 90% cytopathic effect, freeze-thaw, and filter. Repeat four times.

[0035] Example 2 Plaque Emission Experiment 1. Cell Culture The steps are the same as step 1 in Example 1.

[0036] 2. Seeding cells In a 12-well plate, at 0.5 × 10 6 The cells were seeded at a density of 100 cells / well, and then the 12-well plates were placed in an incubator containing 5% CO2 at 37°C for overnight incubation.

[0037] 3.2% agar preparation Add 0.2g of agar (white shark) and 10mL of deionized water to a 50mL Erlenmeyer flask, seal the flask, and sterilize it in an autoclave. After sterilization, place it in an oven for later use.

[0038] 4. Dilution of the original virus solution Take 12 1.5 mL centrifuge tubes and add 900 μL of serum-free and antibiotic-free DMEM to each tube, labeling them for later use. Add 100 μL of 1x HSV-1-GFP stock solution to tube #1 and mix thoroughly. Then add 100 μL from tube #1 to tube #2 and mix thoroughly. Continue this process until tubes #12 contain a 10⁻¹¹ HSV-1-GFP dilution.

[0039] 5. Viral infection Remove the 12-well plate, discard the original culture medium, and wash twice with PBS. Then, add the prepared HSV-1-GFP dilutions of various concentrations to each well sequentially, allowing adsorption for 1.5 hours. Preheat 10 mL of 10% FBS 1%P / SDMEM in a 42°C water bath. Remove the agar from the oven and mix it 1:1 with the preheated DMEM. Add 1 mL of agar along the wall of each well, wait for the agar to solidify, and then return the 12-well plate to the incubator for further incubation. Observe the fluorescence count and spot formation in each well under a fluorescence microscope at 12, 24, and 48 hours after infection.

[0040] 6. Cell fixation Prepare a fixative solution by mixing 9 mL of anhydrous ethanol and 3 mL of acetic acid. Add 1 mL of fixative solution to each well and let stand at room temperature for 30 min. Discard the fixative solution, gently scrape along the well wall with a 200 μL pipette tip to remove the agar, and gently rinse any remaining agar with deionized water. Add 1 mL of crystal violet dye to each well along the wall and stain at room temperature for 30 min. After staining, recover the crystal violet, rinse off excess dye with deionized water along the wall, and then invert the plate onto absorbent paper to air dry for 2–3 h.

[0041] 7. Calculate the number of plaques. Example 3 Drug treatment 1. Preparation of Acyclovir (ACV) and Alvespimycin (17-DMAG) Prepare a 5 mM stock solution (1.13 mg / mL) by mixing 5 mg ACV with 4.4405 mL DMSO thoroughly. Aliquot the stock solution into 1.5 mL centrifuge tubes and store at -80°C protected from light. Separately, prepare a 10 mM stock solution (6.53 mg / mL) by mixing 5 mg 17-DMAG with 0.7655 mL DMSO thoroughly. Aliquot the stock solution into 1.5 mL centrifuge tubes and store at -80°C protected from light.

[0042] 2. Acyclovir (ACV) dilution Remove the aliquoted 5mM ACV from the -80℃ freezer and thaw in an ice box. Take seven 1.5mL centrifuge tubes and add 900μL of 2% FBS-DMEM to each tube. Then add 0, 0.2μL, 1μL, 2μL, 4μL, 10μL, and 20μL of ACV stock solution to each tube to prepare a 5μM ACV dilution for later use.

[0043] 3. Alvespimycin (17-DMAG) dilution Remove the aliquoted 10mM 17-DMAG from the -80℃ freezer and thaw it in an ice box. First, mix 10μL of the stock solution with 90μL LDMSO, at which point the stock solution concentration becomes 1mM. Take six 1.5mL centrifuge tubes, add 1mL of 2% FBS 1% P / SDMEM to each tube, and then add 0μL, 0.1μL, 0.5μL, 1μL, 3μL, 5μL, and 10μL of the 1mM 17-DMAG stock solution respectively, thus preparing 17-DMAG dilutions with concentrations of 0μM, 0.1μM, 0.5μM, 1μM, 3μM, 5μM, and 10μM for later use.

[0044] Example 4: CCK8 experiment to determine the IC of 17-DMAG 50 1. Cell Culture The steps are the same as step 1 in Example 1.

[0045] 2. Seeding cells In 96, with 0.5 × 10 4 Cells were seeded at a density of 1000 cells / well, and then the 96-well plates were incubated overnight at 37°C in an incubator containing 5% CO2.

[0046] 3. Chemical treatment The concentrations of 17-DMAG in 293T cells were 1 μM, 5 μM, 10 μM, 20 μM, and 50 μM, respectively; each concentration of the drug was added to a 96-well plate after 12 h.

[0047] 4. OD value measurement Add 10 μL of CCK8 solution to each well of a 96-well plate and incubate for 40-60 min. After incubation, measure the absorbance at 450 nm using a microplate reader. Results are as follows. Figure 1 As shown.

[0048] Example 5: CCK8 assay to determine the IC50 of 17-DMAG under HSV-1-GFP infection. 50 1. Cell Culture The steps are the same as step 1 in Example 1.

[0049] 2. Seeding cells The steps are the same as step 2 in Example 4.

[0050] 3. Viral infection Infection was performed using HSV-1-GFP with MOI=1. The 96-well plate was removed, and the DMEM in each well of the experimental group was aspirated. The plate was washed twice with PBS, and then 100 μL of diluted virus solution was added. After adsorption for 1 hour, the DMEM in the experimental group wells was aspirated, and 1 mL of DMEM containing 2% FBS and 1% P / S was added to each well for further incubation. The control group did not require virus infection.

[0051] Twelve hours after viral infection, the prepared 17-DMAG at various concentrations was added to the control and experimental groups at a rate of 1 mL per well according to the time points, and the wells were placed in an incubator for further incubation.

[0052] 4. Chemical treatment 17-DMAG concentrations of 5 μM, 10 μM, 50 μM, and 100 μM were added to 96-well plates at 48 h, 24 h, 12 h, and 0 h.

[0053] 5. OD value measurement Add 10 μL of CCK8 solution to each well of a 96-well plate and incubate for 40-60 min. After incubation, measure the absorbance at 450 nm using a microplate reader. Results are as follows. Figure 2 As shown.

[0054] Example 6: Fluorescence Assay for Alvespimycin (17-DMAG) 1. Cell Culture The steps are the same as step 1 in Example 1.

[0055] 2. Seeding cells The steps are the same as step 2 in Example 2.

[0056] 3. Viral infection Infection was performed using HSV-1-GFP with MOI=1. The 12-well plate was removed, and the DMEM in each well of the experimental group was aspirated. The plate was washed twice with PBS, and then 500 μL of diluted virus solution was added. After adsorption for 1 hour, the DMEM in the experimental group wells was aspirated, and 1 mL of DMEM containing 2% FBS and 1% P / S was added to each well for further incubation. The control group did not require virus infection.

[0057] 4. Chemical treatment The positive control group had an ACV concentration of 5 μM and 17-DMAG concentrations of 0.1 μM, 0.5 μM, 1 μM, and 3 μM. Twelve hours after viral infection, 800 μL of drug diluent was added to each well. Wells containing HSV-GFP were not treated with the drug, and the experiment was repeated.

[0058] 5. Observation of lesion results Twelve hours after drug treatment, the cytopathic effect was observed under a fluorescence microscope, and photographs were taken and recorded. The results are as follows: Figure 3 As shown.

[0059] Example 7 Q-PCR Experiment (1) 1. RNA extraction RNA was extracted using the Trizol method from 12-well plates used in the 17-DMAG fluorescence experiment, including Mock, HSV0, H-17-DMAG-1, H-17-DMAG-5, and H-17-DMAG-10. The experiment was repeated.

[0060] The concentration of each sample was detected and recorded using Nanodrop 2000. After reverse transcription (conditions: 50℃, 5 min; 85℃, 2 min; 12℃), a total of 10 cDNA samples were obtained and stored at -20℃.

[0061] 2. Q-PCR system preparation (1) cDNA dilution Take two rows of eight-tube strips, add 3 μL of cDNA and 40 μL of DEPC water to each tube, label them, and store them at -20°C for later use.

[0062] (2) Mix system configuration Take three 1.5 mL sterile centrifuge tubes and label them. Add 15 μL of HSV-ICP0-F and 15 μL of HSV-ICP0-R to the centrifuge tubes, then add 150 μL of SYBR and mix well. The amounts of primers UL30 and GAPDH added are the same as those for ICP0. 3. Spotting The prepared mixture of three different primers was added to an eight-tube strip at a rate of 6 μL per well. Then, 4 μL of diluted cDNA was added to each well. Each cDNA sample was processed in triplicate, and the mixture was centrifuged to mix.

[0063] 4. Q-PCR The Q-PCR reaction procedure is as follows: The results are as follows Figure 4 As shown.

[0064] Example 8 Q-PCR Experiment (2) 1. DNA extraction DNA was extracted using the Kangwei Century kit and its accompanying RNases, including Mock, HSV0, H-ACV-5, H-17-DMAG-1, H-17-DMAG-5, and H-17-DMAG-10. The experiment was repeated.

[0065] The concentration of each sample was detected and recorded using Nanodrop 2000, and the 12 DNA samples obtained were stored in a -20°C freezer.

[0066] 2. Q-PCR system preparation (1) DNA dilution Take two rows of eight-tube strips, add 3 μL of DNA and 40 μL of DEPC water to each tube, label them, and store them at -20°C for later use.

[0067] (2) Mix system configuration (plates 1 and 2, 6 samples per plate) Take three 1.5 mL sterile centrifuge tubes and label them. Add 9 μL of HSV-ICP0-F and 9 μL of HSV-ICP0-R to the centrifuge tubes, then add 90 μL of SYBR and mix well. The amounts of primers UL30 and GAPDH added are the same as for ICP0; the mixing system for the second plate is prepared in the same way.

[0068] 3. Spotting Add 6 μL of the prepared mixture of three different primers to each of the eight tubes. Then add 4 μL of diluted DNA. Perform three technical replicates for each DNA sample and centrifuge to mix.

[0069] 4. Q-PCR The Q-PCR reaction procedure is the same as step 4 in Example 7.

[0070] The results are as follows Figure 5-6 As shown.

[0071] Example 9 WB Experiment 1. Cell Culture The steps are as described in step 1 of Example 1.

[0072] 2. Seeding cells The steps are as described in step 2 of Example 2.

[0073] 3. Viral infection The steps are as described in step 3 of Example 6.

[0074] 4. Chemical treatment The steps are as described in step 4 of Example 6.

[0075] 5. Observation of lesion results The steps are the same as step 5 in Example 6.

[0076] 6. Protein sample collection Protein collection was performed entirely on ice. 12 μL of COCIK Tail (a protease inhibitor, brand: Targetmo) was mixed with 1.2 mL of Promega 5x passive lysis buffer, and 100 μL was added to each well of a 12-well plate. Lysis was performed on ice for 5-10 min. After complete cell lysis, the samples were transferred to centrifuge tubes and centrifuged at 12,000 rpm for 15 min at 4°C. After centrifugation, the supernatant was collected, and 20 μL of pre-thawed 6x loading buffer was added to each tube. The mixture was vortexed to thoroughly mix, followed by a 98°C metal bath for 10 min. After the metal bath, all 18 samples were stored at -20°C.

[0077] 7. Gel preparation and protein loading electrophoresis Two 10% separating gels were prepared (1.9 mL deionized water, 1.7 mL 30% acrylamide, 1.3 mL 1.5 mol / L Tris-HCl (pH=8.8), 0.05 mL 10% SDS, 0.05 mL 10% APS, 0.004 mL TEMED). The mixtures were poured into gel plates and sealed with anhydrous ethanol, allowing them to solidify for 30 min. Simultaneously, two 5% stacking gels were prepared (3.4 mL deionized water, 0.83 mL 30% acrylamide, 0.63 mL 1.0 mol / L Tris-HCl (pH=6.8), 0.05 mL 10% SDS, 0.05 mL 10% APS, 0.005 mL TEMED). After the lower gel solidified, the anhydrous ethanol was discarded, the upper gel was added, a comb was inserted, and the gels were allowed to solidify at room temperature for 1 h.

[0078] After the upper gel solidifies, remove the comb, fix the gel plate to the electrophoresis clamp, and pour 1x electrophoresis buffer (900mL deionized water mixed with 100mL 10x electrophoresis buffer) into the electrophoresis tank. Divide the 18 protein samples into 3 groups. The loading order for the first group is: Mock, HSV-0, H-ACV-5, H-17-DMAG-0.5, H-17-DMAG-1, H-17-DMAG-3; the loading order for the second and third groups is the same. Cover the tank, adjust the electrophoresis voltage to 80V, and after 30 minutes, adjust the voltage to 120V and continue electrophoresis for 40 minutes.

[0079] 8. Transfer and sealing Activate the PVDF membrane by adding methanol and shaking for 3 minutes at room temperature. After activation, wash away the methanol by shaking with deionized water for another 3 minutes. After electrophoresis, remove the gel plate, pry open the short glass plate, cut off the excess gel, and then transfer the gel to pre-chilled transfer buffer (700 mL deionized water, 200 mL methanol, 100 mL 10x transfer buffer). Place the gel and membrane in the order of filter paper-membrane-gel-filter paper, close the transfer clamp, and place it in the transfer tank. Fill the tank completely with transfer buffer, ensuring it does not submerge the transfer clamp. Transfer at a constant voltage of 100V for 80 minutes under ice-water bath conditions. After transfer, transfer the membrane to a clean antibody incubation box, pour in pre-prepared skim milk (1 g milk powder + 20 mL TBST, vortex thoroughly), and block on a shaker at room temperature for 1 hour.

[0080] 9. Antibody incubation Discard the skim milk, add 1x TBST to cover the bottom of the membrane, and wash three times at room temperature with shaking, 10 min each time. After washing, add the primary antibody (Wuhan Sanying Biotechnology Co., Ltd., Cat No. 50430-2-AP GFP tag, host-rabbit; 1:2000 dilution), and incubate overnight at 4°C with shaking. After the primary antibody incubation, recover the primary antibody, add TBST again to cover the bottom of the filter paper, wash for 10 min at room temperature with shaking, discard the TBST, and wash the membrane again, repeating three times. Then add the secondary antibody (HRP, goat anti-rabbit IgG; 1:5000 dilution), and incubate at room temperature for 1 h.

[0081] 10. Development After incubation, the secondary antibody was recovered, and the membrane was washed three times with TBST for 10 min each time. After washing the membrane, the two chemiluminescent solutions (200 μL) were poured into the antibody incubation box at a 1:1 ratio. The TBST on the membrane was blotted dry with absorbent paper, and then the membrane was fully immersed in the chemiluminescent solution. The exposure results were observed using a chemical imaging system.

[0082] 11. Adjustment of sample loading amount Based on the first WB result and the imagej software, adjust the sample loading amount and repeat the above steps twice until the bands are clear and neat.

[0083] The results are as follows Figure 7 As shown.

[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Application of aspirin in the preparation of drugs for treating herpes simplex virus type 1.

2. The use of aspirin according to claim 1 in the preparation of a drug for treating herpes simplex virus type 1, characterized in that: The aspirin in uninfected virus-containing cells at IC50 50 The concentration is 10~20μM; under the condition of herpes simplex virus type 1 infection, the concentration of 0.5~3μM can effectively fight the virus without significant cytotoxicity.

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