Application of phosphatidic acid in preparation of medicine for preventing and / or treating broad-spectrum virus infection

Abstract

The invention relates to application of phosphatidic acid in preparation of a medicine for preventing and / or treating broad-spectrum virus infection, and belongs to the technical field of biological medicine. The broad-spectrum virus is a DNA (Deoxyribose Nucleic Acid) mode virus HSV-1 and an RNA (Ribonucleic Acid) mode virus VSV of an infected person. Research finds and determines the relationship between phosphatidic acid and DNA virus HSV-1 and RNA virus VSV infection, that is to say, the addition of phosphatidic acid PA can inhibit replication and proliferation of DNA virus HSV-1 and RNA virus VSV, and the phenomenon of inhibiting virus replication is generated by using phosphatidic acid PA as a signal molecule to directly activate a PI3K-AKT signal channel and then start type I interferon transcription. Therefore, phosphatidic acid PA can be applied to prevention and / or treatment of broad-spectrum antiviral infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of phosphatidic acid (PA) in the preparation of drugs for preventing and / or treating broad-spectrum viral infections. Background Art

[0002] Viruses are microorganisms with simple structures that must parasitize host cells to replicate and proliferate. At present, drugs for treating viruses are mainly divided according to targets. For example, during the process of virus adsorption, internalization, transport, membrane fusion and genome release, drugs that directly target viruses can bind to viral proteins or nucleic acids to affect viral entry, transcription, replication, assembly and release; while drugs that directly target hosts can affect viral entry, transcription, replication, processing, transport and signaling pathway activity. At present, the main molecular forms of antiviral drugs include: small molecules, peptides, neutralizing antibodies, interferons, CRISPR-CAS systems, si / shRNA and other nucleic acid polymers, etc. However, compared with the conservatism of host cells, viruses, especially RNA viruses and small DNA viruses, have a much higher rate of nucleotide substitution during replication than host cells. This is because viruses are more likely to produce genetic material mutations when they replicate themselves, causing the original drug treatment targets to fail. In addition, there are currently no drugs on the market for broad-spectrum viruses, only drugs for single (type) viruses, and such drugs for single (type) viruses often face the challenge of drug resistance. Therefore, in-depth research on the common mechanisms of interaction between viruses and host cells and the discovery of new antiviral target drugs are key issues that need to be addressed in the prevention and treatment of viral infectious diseases. Since host cells are genetically more stable than viruses, and some host cell proteins participate in regulating viral replication through conservative strategies in multiple viruses, they are of great significance in broad-spectrum antiviral treatment.

[0003] Phosphatidic acid PA is a common phospholipid and a component of cell membrane. Phosphatidic acid can be synthesized by phosphatidylcholine, the end product of Kennedy choline metabolism, under the action of phospholipase D. Phosphatidic acid can serve as a precursor of other lipids in cells and can also affect membrane curvature with its physical properties. In addition, phosphatidic acid can directly act as a signal molecule to transmit signals in cells. Studies have found that the expression of CHKA, a key rate-limiting enzyme in Kennedy choline metabolism, is reduced, resulting in the inhibition of MAPK and PI3K-AKT signaling pathways, and the reduction of phosphatidic acid content can directly weaken MAPK signals. Studies have found that positive-strand RNA viruses can promote the accumulation of PA in plants, thereby triggering MAPK-mediated plant antiviral immunity to resist viral infection. In addition, studies have shown that PI3K-AKT signals can target and activate the transcription of type I interferon. However, there are no reports on the relationship between phosphatidic acid PA and broad-spectrum viruses, and the effects of PA on PI3K-AKT signals and type I interferon. Summary of the invention

[0004] The present invention is to solve the deficiencies of the prior art and provide an application of phosphatidic acid PA in the preparation of drugs for preventing and / or treating broad-spectrum viral infections. The experimental data provided by the present invention prove that phosphatidic acid PA can inhibit the replication of DNA model virus HSV-1 and RNA model virus VSV, activate PI3K-AKT signaling, and promote the expression of IFNB1, and can be used to provide technical support for the preparation of drugs that prevent and / or treat broad-spectrum viral infections, thereby applying phosphatidic acid to drugs that prevent or treat broad-spectrum viral infections.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows: Application of phosphatidic acid in the preparation of drugs for preventing and / or treating broad-spectrum viral infections.

[0006] Furthermore, the broad-spectrum viruses are the DNA model virus HSV-1 and the RNA model virus VSV that infect humans.

[0007] Furthermore, the drug for preventing and / or treating broad-spectrum viral infections includes but is not limited to: at least one of plants containing phosphatidic acid, plant extracts containing phosphatidic acid, effective plant parts containing phosphatidic acid, extracted and purified phosphatidic acid, and chemically synthesized phosphatidic acid.

[0008] Furthermore, the drug for preventing and / or treating broad-spectrum viral infections includes, but is not limited to, active ingredients and pharmaceutically acceptable carriers; the active ingredient is a plant extract of phosphatidic acid and / or a pharmaceutically acceptable salt thereof.

[0009] Furthermore, the drug for preventing and / or treating broad-spectrum viral infections is a pharmaceutically acceptable oral preparation, injection, inhalation preparation or external preparation.

[0010] Furthermore, the drug for preventing and / or treating broad-spectrum viral infections may contain phosphatidic acid and / or a pharmaceutically acceptable salt thereof.

[0011] Furthermore, the drug for preventing and / or treating broad-spectrum viral infections acts to inhibit the replication and proliferation of DNA-model virus HSV-1 and RNA-model virus VSV.

[0012] Furthermore, the drug for preventing and / or treating broad-spectrum viral infections acts to activate PI3K-AKT signaling and promote type I interferon transcription.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention clarifies the relationship between phosphatidic acid and DNA model virus HSV-1 and RNA model virus VSV infection, making phosphatidic acid a potential target in the preparation of drugs for preventing and / or treating broad-spectrum viral infections, and is used to inhibit the replication and proliferation of DNA model virus HSV-1 and RNA model virus VSV, providing an effective new approach for the treatment of broad-spectrum viral infections; (2) The feasibility of phosphatidic acid in the preparation of drugs for the prevention and / or treatment of broad-spectrum viral infections was determined at the cellular level, providing new potential drugs for the treatment of broad-spectrum viral infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results are shown in Figure 1. The results are shown in Figure 2. The results are shown in Figure 3. The results are shown in Figure 4. The results are shown in Figure 5. The results are shown in Figure 6. The results are shown in Figure 7. The results are shown in Figure 8. The results are shown in Figure 9. The results are shown in Figure 10. The results are shown in Figure 11. The results are shown in Figure 12. Figure 2 The figure shows the inhibition of viral protein replication of HSV-1 and VSV viruses in RAW264.7 cells by phosphatidic acid after treating mouse mononuclear macrophage immune cells (RAW264.7) with phosphatidic acid for 48 hours and then inoculating HSV-1 and VSV viruses for 10 hours. C shows the inhibition of HSV-1 virus replication, and D shows the inhibition of VSV virus replication. Figure 3 E in the figure is the transcription map of type I interferon IFNB1 under HSV-1 virus stimulation, obtained by detecting the relative expression of type I interferon gene by RT-qPCR after treating mouse mononuclear macrophage immune cells (RAW264.7) with phosphatidic acid for 60 hours and inoculating HSV-1 virus for 12 hours; Figure 3 F in the figure is the transcription map of type I interferon IFNB1 under VSV virus stimulation, obtained by detecting the relative expression of type I interferon gene by RT-qPCR after treating mouse mononuclear macrophage immune cells (RAW264.7) with phosphatidic acid for 48 hours and inoculating VSV virus for 5 hours; Figure 4 The phosphorylation level of PI3K protein was detected by Western blotting (WB) after the mouse mononuclear macrophage immune cells (RAW264.7) were treated with phosphatidic acid for 60 hours and inoculated with HSV-1 and VSV viruses for 12 hours. The phosphorylation level of PI3K protein under the stimulation of phosphatidic acid activated viruses HSV-1 and VSV is shown in Figure 1, where: G is the phosphorylation level of PI3K protein under the stimulation of HSV-1 virus, and H is the phosphorylation level of PI3K protein under the stimulation of VSV virus; Figure 5 The phosphorylation level of AKT protein was detected by protein immunoblotting (WB) after mouse mononuclear macrophage immune cells (RAW264.7) were treated with phosphatidic acid for 60 hours and inoculated with HSV-1 and VSV viruses for 12 hours. Among them, I is the phosphorylation level of AKT protein under HSV-1 virus stimulation, and J is the phosphorylation level of AKT protein under VSV virus stimulation. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below in conjunction with embodiments.

[0016] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0017] The whole culture medium formula used in the present invention is as follows: 10% DMEM complete medium: Add fetal bovine serum (v%) to commercial DMEM (Vivacell) medium, accounting for 10% of the total volume of the medium; 2% DMEM medium: Add 2% fetal bovine serum (v%) to commercial DMEM (Vivacell) medium; 4% DMEM medium: Add 4% fetal bovine serum (v%) to commercial DMEM (Vivacell) medium; Serum-free DMEM medium: commercial DMEM medium (Vivacell).

[0018] The cell involved in the present invention is: Mouse mononuclear macrophage immune cells RAW264.7 were purchased from Pronocell Biotechnology Co., Ltd.

[0019] The present invention relates to viruses; HSV-1 virus, Strain-17; VSV virus, New Jersey strain, were provided by the Vaccine Research Laboratory, Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0020] The antibodies involved in the present invention are shown in Table 1.

[0021] Table 1

[0022] The reagents involved in the present invention are shown in Table 2.

[0023] Table 2

[0024] The preparation of the corresponding reagents in the present invention is as follows: 1) The components of transfer buffer are shown in Table 3.

[0025] Table 3

[0026] Add ultrapure water to glycine and Trisbase to make up to 850 ml, then add 150 ml of methanol, and store at room temperature after fully dissolving.

[0027] 2) The composition of 10× TBS buffer is shown in Table 4.

[0028] Table 4

[0029] The above two components were fully dissolved in 800 ml of ultrapure water, the pH value was adjusted to 7.5 with concentrated hydrochloric acid, and the volume was finally fixed to 1000 ml.

[0030] 3) The composition of TBST (0.1% TWEEN20) is shown in Table 5.

[0031] Table 5

[0032] Mix the two ingredients and shake well, then prepare immediately before use.

[0033] 4) The composition of the blocking solution is shown in Table 6.

[0034] Table 6

[0035] Shake well after mixing and prepare before use.

[0036] The corresponding Chinese and English names of the English abbreviations involved in the present invention are shown in Table 7.

[0037] Table 7

[0038] Example 1 The virus HSV-1 and VSV were infected with RAW264.7 cells treated with DMSO as the control group and RAW264.7 cells treated with phosphatidic acid PA, and the expression of the virus HSV-1 immediate early gene product ICP0 protein and the virus VSVgB glycoprotein were detected by protein immunoblotting. The specific steps are as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate them into 12-well plates. Add 1 ml of 10% DMEM complete medium to each well. The initial cell confluence is 30%-40%. Shake evenly and place in a cell culture incubator at 37 degrees and 5% carbon dioxide concentration for culture. (2) Cell treatment: After the RAW264.7 cells in step (1) adhere to the wall, treat the adhered RAW264.7 cells with 4 μM phosphatidic acid PA (wherein: the mother solution is 4 mM), and treat the RAW264.7 cells with an equal volume of DMSO as the phosphatidic acid PA as a control group, shake well, and place in an incubator at 37 degrees and 5% carbon dioxide for culture; (3) Virus inoculation: After the RAW264.7 cells were treated with phosphatidic acid PA and DMSO for 60 h in step (2), the 10% DMEM complete medium was discarded, the cells were washed twice with PBS, and then 1 ml of serum-free DMEM medium was added to each well, and the viruses HSV-1 (MOI = 1) and VSV (MOI = 2) were inoculated respectively. After 1 h, 1 ml of 4% DMEM medium was added to each well, and then the cells were placed in an incubator at 37 degrees and 5% carbon dioxide for 12 h; (4) Harvesting protein samples: After removing the well plate from the incubator in step (3), discard the virus-containing supernatant in the safety cabinet, wash twice with PBS, add 100 μl of RIPA (medium) lysis buffer containing PMSF and phosphatase inhibitors to each well of the 12-well plate, and lyse the cells for 20 min, wherein: the ratio of PMSF to phosphatase inhibitors in the lysis buffer is 1:1000, and the lysis process is performed on ice, and the well plate is placed on a shaker for 10 s every 5 min. After the lysis is completed, the protein solution is transferred to a labeled Eppendorf tube and centrifuged at 15,000 rpm for 5 min in a centrifuge at 4°C. The supernatant obtained after centrifugation is transferred to a new Eppendorf tube and the precipitate is discarded; (5) Protein immunoblotting to detect the expression of two viral proteins: (51) After protein quantification by the BCA method, adjust the concentration of the protein sample harvested in step (4) in advance to make the added volume and mass consistent: the loading amount of ordinary protein is 10-20ug, the loading amount of phosphorylated protein is 60ug, and the loading amount of protein marker is 3-5μL. For a 10-well plate, the loading volume per well should not exceed 50μL, and for a 15-well plate, the loading volume per well should not exceed 20μL. Add an appropriate amount of 5× loading buffer to the centrifuge tube until the final concentration of the buffer is diluted to 1×, then vortex the sample for 10s, and place the mixed sample in an iron bath at 100℃ for 5 minutes to denature the protein. Then centrifuge at 5000g for 3min and take the supernatant for use. (52) SDS-polyacrylamide gel electrophoresis: Assemble the electrophoresis tank, add MOPS Running Buffer Liquid (1×) electrophoresis buffer (MOPS Running Buffer Liquid (20×) purchased from GenScript prepared with ultrapure water), the inner side covers the sample wells, and the outer side reaches the 2 Gels position. Pull out the comb of the precast gel (purchased from GenScript); add protein marker and sample, cover the top cover, connect the electrophoresis instrument, first run the bands at 80 V, then adjust the voltage to 120 V and run for 30 min. When the marker indicates that the target bands have separated, stop the electrophoresis. (53) Transfer: After electrophoresis, cut the PVDF membrane in advance and activate it in methanol solution for 1 min. Set aside. Use a crowbar to pry open the gel plate, cut the gel containing the target protein with the reference protein marker, and then carefully spread the gel on the filter paper. Cover the cut PVDF membrane with the marker on the corresponding gel, making sure there are no bubbles between the two. Assemble the sandwich plate, with the black (gel) facing the black negative electrode and the transparent (membrane) facing the red positive electrode. Pour the transfer solution (wet transfer solution) until the entire transfer tank is filled, add ice cubes and transfer the membrane in an ice bath. Set the transfer current to a constant current of 300 mA. The transfer time is determined according to the size of the protein, and the transfer time is 1 min per 1 kD. (54) Blocking: After transfer, remove the membrane and place it in 5 ml of blocking solution (TBST containing 5% skim milk) and block it on a horizontal shaker at room temperature for 1–2 h. (55) Incubation with primary antibody: dilute the primary antibody (the primary antibodies used include: ICP0 (abcam), VSV gB (abcam), β-actin (CST)) with Western primary antibody diluent (Biyuntian) according to the dilution multiple recommended in the antibody manual; after the blocking solution on the membrane surface is blotted dry with absorbent paper, add the diluted primary antibody immediately and place in a refrigerator at 4°C overnight; the dilution ratio of each antibody is: ICP0 (1:2000), VSV gB (1:1000), β-actin (1:5000); (56) Washing the membrane: After the primary antibody incubation is completed, transfer the membrane into a square storage box containing TBST, so that the TBST covers the PVDF membrane, shake it on a shaker for 10 min, and repeat the washing three times; (57) Incubation with secondary antibody: dilute the secondary antibody (including goat anti-mouse IgG (Proteintech)) at a ratio of 1:10,000 with Biotech secondary antibody diluent and incubate at room temperature for 1–2 h. (58) Washing the membrane: After the secondary antibody incubation is completed, transfer the membrane into a square storage box containing TBST, so that the TBST covers the PVDF membrane, shake it on a shaker for 10 min, and repeat the washing three times; (59) Development: Prepare the developer (mix the developer A and B in the Bio-Yun Tian Extreme Ultra-Sensitive ECL Chemiluminescence Kit in a 1:1 ratio). When developing, immerse the membrane in the developer to ensure that the developer submerges the membrane. After reacting in the dark for 1 min, place the membrane in a UVP instrument to expose the protein bands and take photos for recording. (6) Experimental results are as follows Figure 1 As shown in A and B, RAW264.7 cells treated with DMSO and phosphatidic acid PA for 60 hours were infected with the two viruses 12 hours later, and proteins were harvested for protein immunoblotting to detect the expression of viral proteins. The results showed that the replication of DNA virus HSV-1 and RNA virus VSV was significantly inhibited after treatment with phosphatidic acid PA.

[0039] Example 2 The virus HSV-1 and VSV were used to infect RAW264.7 cells treated with DMSO as the control group and RAW264.7 cells treated with phosphatidic acid PA, and the amount of live virus in the cell supernatant was detected using half tissue culture infection dose (TCID50). The specific steps are as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate the cells into 12-well plates. Add 1 ml of 10% DMEM complete medium to each well. The initial cell confluence is about 30%-40%. Shake evenly and place in an incubator at 37 degrees and 5% carbon dioxide concentration for culture. (2) Cell treatment: After the cells in step (1) adhere to the wall, treat the cells with phosphatidic acid PA at a concentration of 4 μM (the mother solution is 4 mM). At the same time, treat RAW264.7 cells with DMSO of the same volume as phosphatidic acid PA as a control group. Shake well and place in an incubator at 37 degrees and 5% carbon dioxide for culture; (3) Virus inoculation: After the RAW264.7 cells were treated with phosphatidic acid PA and DMSO for 48 h in step (2), the DMEM medium containing 10% serum was discarded, the cells were washed twice with PBS, 1 ml of serum-free DMEM medium was added to each well, and the viruses HSV-1 (MOI = 0.5) and VSV (MOI = 1) were inoculated respectively. After 1 h, 1 ml of DMEM medium containing 4% serum was added to each well, and the cells were then placed in an incubator at 37 degrees and 5% carbon dioxide for 10 h: (4) After 10 hours, harvest the viral supernatant, carefully mix the culture supernatant, take out 1 ml and put it into a 1.5 ml centrifuge tube, and store it at -80 degrees Celsius; (5) Determination of virus titer by CCID50 method: a. Prepare Vero cells: Mix Vero cells with 4% maintenance solution (4% fetal bovine serum, v%) and plate them in a 96-well plate. The volume of each well is 100 μl, containing approximately 5×104 cells; b. Dilute the virus solution to be tested: Take the HSV-1 virus out of the -80 degrees Celsius refrigerator, wait for it to melt, and dilute the virus stock solution 10 times with serum-free medium in an EP tube, that is, take 100 μl of the virus stock solution and mix it with 900 μl of serum-free medium to make 10 -1 Dilution gradient: take 100 μl of 10-1 dilution gradient virus solution and mix it with 900 μl serum-free medium to make 10 -2 Dilution gradient, sequentially forming 10 virus gradients, i.e. 10 -1 -10 -10 ; c. Inoculation: Take a 96-well plate with Vero cells, add the diluted virus solution to the 96-well plate, add 8 wells for each dilution gradient, add 100μl to each well, add high dilution first and then low dilution; add 100μl serum-free culture medium to the remaining 8 wells as blank control, place in a carbon dioxide incubator at 37°C and 5% carbon dioxide concentration for 5-7 days; d. Observation and statistics: Observe the cytopathic condition under a microscope every day during the culture process, and record the number of cytopathic wells in each gradient until the number of cytopathic wells no longer increases after continuous observation, and then make statistics; e. Calculation results: The virus titer was calculated using the Karber method, i.e., lgCCID50 / ml = the negative logarithm of the highest dilution of complete lesions + the sum of the lesion wells of each dilution of incomplete lesions × 0.125 + 0.5 + 1; (6) Experimental results are as follows Figure 2 As shown, PA (4 μM) was added to RAW264.7 cells, and HSV-1 (MOI=0.5) and VSV (MOI=1) were infected 48 hours later. The cell supernatant was collected 10 hours after infection and the virus titer was determined by the CCID50 method. The results showed that the addition of phosphatidic acid PA in RAW264.7 cells can inhibit the expression and replication of viral HSV-1.

[0040] Example 3 The virus HSV-1 and VSV were used to infect RAW264.7 cells treated with DMSO as a control group and RAW264.7 cells treated with phosphatidic acid PA, respectively, and real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) was used to detect the transcription of type I interferon IFNB1. The specific steps are as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate the cells into 12-well plates. Add 1 ml of 10% DMEM complete medium to each well. The initial cell confluence is about 30%-40%. Shake evenly and place in an incubator at 37 degrees and 5% carbon dioxide concentration for culture. (2) Drug treatment: After the cells in step (1) adhere to the wall, treat the cells with phosphatidic acid PA at a concentration of 4 μM (the mother solution is 4 mM). At the same time, treat RAW264.7 cells with DMSO in an equal volume to that of phosphatidic acid PA as a control group. Shake well and place in an incubator at 37 degrees and 5% carbon dioxide for culture. (3) Virus inoculation: After the RAW264.7 cells were treated with phosphatidic acid PA and DMSO for 60 h, the serum-containing DMEM medium was discarded, the cells were washed twice with PBS, 1 ml of serum-free DMEM medium was added to each well, and the viruses HSV-1 (MOI=1) and VSV (MOI=1) were inoculated respectively. After 1 h, 1 ml of DMEM medium containing 4% serum was added to each well, and the cells inoculated with HSV-1 were continued to be cultured in an incubator at 37 degrees and 5% carbon dioxide for 12 h; the cells inoculated with VSV were continued to be cultured in an incubator at 37 degrees and 5% carbon dioxide for 5 h; (4) Harvesting gene samples: After taking out the well plate from the incubator, discard the virus-containing supernatant in the safety cabinet, wash twice with PBS, and add 400 μl of Trizol to each well of the 12-well plate. This process is performed at room temperature. Lyse the cells for about 10 minutes. After the lysis is complete, transfer the lysate to the labeled Eppendorf tube and start extracting RNA. (5) RNA extraction and reverse transcription: The steps for total RNA extraction are as follows: a. Harvest RNA gene samples; b. Add 1 / 5 volume of chloroform to the RNA gene sample (i.e., add 100µl of chloroform to every 0.5ml Trizol), centrifuge at 4°C and 12000g for 20min, and shake thoroughly to mix; c. Take 40% Trizol volume and add 1.5 times volume of isopropanol to the water layer during extraction and mix thoroughly. Centrifuge directly or place the sample at -40℃ overnight to precipitate. Then, centrifuge at 12000g for 10 minutes at 4℃, discard the supernatant and keep the white precipitate at the bottom of the tube. d. Add 0.8 ml of 75% ethanol precooled at -40°C, mix by repeated inversion, and centrifuge at 12000 g for 5 min at 4°C; e. Discard the supernatant and use a 10ul pipette to remove as much liquid as possible without touching the RNA precipitate, ensuring that the remaining ethanol can dry naturally at room temperature; when the ethanol is completely evaporated, the original white RNA precipitate will turn transparent, indicating that the liquid has been fully removed; f. To dissolve RNA, add 20μl to 40μl of DEPC water. To promote RNA dissolution, use the tip of a pipette to gently pipette several times. g. After the RNA is dissolved, its concentration needs to be determined. Once the concentration is determined, the RNA can be stored at -40°C or used directly in subsequent reverse transcription experiments; h. Real-time fluorescence quantitative PCR (qPCR) When conducting real-time fluorescence quantitative PCR experiments, relative quantitative analysis was performed according to the operating instructions of the GoTaq® qPCR MasterMix kit provided by Promega; in order to ensure the accuracy and reliability of the experiment, a 10μl reaction system was prepared according to the standard process. Each component in this system was accurately prepared according to the recommended ratio of the kit to ensure the smooth progress of the PCR reaction and the accuracy of the results: Prepare 10 μl reaction system:

[0041] Each sample was repeated twice, and the reaction program was: 95°C, 5 min; 95°C, 15 s; 50-60°C, 30 s; 72°C, 30 s; 35 cycles; Melting curve 65℃-95℃, 0.5℃ / cycle, 5s; detection was performed on the Bio-Rad real-time fluorescence quantitative PCR instrument according to the reaction program: The primer sequences used for real-time fluorescence quantitative PCR are as follows:

[0042] (6) Experimental results are as follows Figure 3 As shown in E and F, after the two viruses infected RAW264.7 cells treated with DMSO and phosphatidic acid PA, the genes were collected after a certain period of time and RNA was extracted for real-time fluorescence quantitative reverse transcription PCR to detect the expression of IFNB1 gene. The results showed that phosphatidic acid PA treatment activated INFB1 transcription under the stimulation of DNA virus HSV-1 and RNA virus VSV.

[0043] Example 4 The virus HSV-1 and VSV were used to infect RAW264.7 cells treated with DMSO as a control group and RAW264.7 cells treated with phosphatidic acid PA, respectively, and the PI3K signal was detected by protein immunoblotting. The specific steps are as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate the cells into 12-well plates. Add 1 ml of 10% DMEM complete medium to each well. The initial cell confluence is about 30%-40%. Shake evenly and place in an incubator at 37 degrees and 5% carbon dioxide concentration for culture. (2) Cell treatment: After the cells in step (1) adhere to the wall, treat the cells with phosphatidic acid PA at a concentration of 4 μM (the mother solution is 4 mM). At the same time, treat RAW264.7 cells with DMSO of the same volume as phosphatidic acid PA as a control group. Shake well and place in an incubator at 37 degrees and 5% carbon dioxide for culture; (3) Virus inoculation: After the RAW264.7 cells were treated with phosphatidic acid PA and DMSO for 60 h, the serum-containing DMEM medium was discarded, the cells were washed twice with PBS, 1 ml of serum-free DMEM medium was added to each well, and the viruses HSV-1 (MOI = 1) and VSV (MOI = 2) were inoculated respectively. After 1 h, 1 ml of DMEM medium containing 4% serum was added to each well, and the cells were then placed in an incubator at 37 degrees and 5% carbon dioxide for 12 h. (4) Harvesting protein samples: After removing the well plate from the incubator, discard the virus-containing supernatant in the safety cabinet, wash twice with PBS, and add 100 μl of RIPA (medium) lysis buffer containing PMSF and phosphatase inhibitors to each well of the 12-well plate. Lyse the cells for about 20 minutes, wherein: PMSF and phosphatase inhibitors in the lysis buffer are added to the RIPA (medium) lysis buffer (prepared immediately before use) in advance at a ratio of 1:1000, and the lysis process is performed on ice; the well plate is placed on a shaker for about 10 seconds every 5 minutes. After the lysis is completed, the protein solution is transferred to a labeled Eppendorf tube, placed in a 4°C centrifuge, 15,000 g, 5 minutes, the supernatant obtained is transferred to a new Eppendorf tube, and the precipitate is discarded; (5) Western blotting was used to detect the expression of the two viral proteins, using the same method as in Example 1; (6) Experimental results are as follows Figure 4 As shown in G and H, RAW264.7 cells treated with DMSO and phosphatidic acid PA for 60 hours were infected with the two viruses 12 hours later, and the protein was collected for protein immunoblotting to detect the expression of viral proteins. The results showed that PI3K signaling was activated under the stimulation of DNA virus HSV-1 and RNA virus VSV after phosphatidic acid PA treatment.

[0044] Example 5 The virus HSV-1 and VSV were used to infect RAW264.7 cells treated with DMSO as a control group and RAW264.7 cells treated with phosphatidic acid PA, respectively, and the AKT signal was detected by protein immunoblotting. The specific steps are as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate the cells into 12-well plates. Add 1 ml of 10% DMEM complete medium to each well. The initial cell confluence is about 30%-40%. Shake evenly and place in an incubator at 37 degrees and 5% carbon dioxide concentration for culture. (2) Cell treatment: After the cells in step (1) adhere to the wall, treat the cells with phosphatidic acid PA at a concentration of 4 μM (the mother solution is 4 mM). At the same time, treat RAW264.7 cells with DMSO of the same volume as phosphatidic acid PA as a control group. Shake well and place in an incubator at 37 degrees and 5% carbon dioxide for culture; (3) Virus inoculation: After the RAW264.7 cells were treated with phosphatidic acid PA and DMSO for 60 h, the serum-containing DMEM medium was discarded, the cells were washed twice with PBS, 1 ml of serum-free DMEM medium was added to each well, and the viruses HSV-1 (MOI = 1) and VSV (MOI = 2) were inoculated respectively. After 1 h, 1 ml of DMEM medium containing 4% serum was added to each well, and the cells were then placed in an incubator at 37 degrees and 5% carbon dioxide for 12 h. (4) Harvesting protein samples: After removing the well plate from the incubator, discard the virus-containing supernatant in the safety cabinet, wash twice with PBS, and add 100 μl of RIPA (medium) lysis buffer containing PMSF and phosphatase inhibitors to each well of the 12-well plate. Lyse the cells for about 20 minutes, wherein: PMSF and phosphatase inhibitors in the lysis buffer are added to the RIPA (medium) lysis buffer (prepared immediately before use) in advance at a ratio of 1:1000, and the lysis process is performed on ice; the well plate is placed on a shaker for about 10 seconds every 5 minutes. After the lysis is completed, the protein solution is transferred to a labeled Eppendorf tube, placed in a 4°C centrifuge, 15,000 g, 5 minutes, the supernatant obtained is transferred to a new Eppendorf tube, and the precipitate is discarded; (5) Protein immunoblotting to detect the expression of two viral proteins. The method used is the same as that in Example 1; (6) Experimental results are as follows Figure 5 As shown in I and J, 12 hours after the two viruses infected RAW264.7 cells treated with DMSO and phosphatidic acid PA for 60 hours, the proteins were collected and immunoblotted to detect the expression of viral proteins. The results showed that AKT signaling was activated under the stimulation of DNA virus HSV-1 and RNA virus VSV after phosphatidic acid PA treatment.

Claims

1. Application of phosphatidic acid in the preparation of drugs for preventing and / or treating broad-spectrum viral infections.

2. The use of phosphatidic acid according to claim 1 in the preparation of a drug for preventing and / or treating a broad spectrum of viral infections, characterized in that The broad-spectrum viruses are the DNA model virus HSV-1 and the RNA model virus VSV that infect humans.

3. The use of phosphatidic acid according to claim 1 in the preparation of a drug for preventing and / or treating a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infections includes but is not limited to: at least one of plants containing phosphatidic acid, plant extracts containing phosphatidic acid, effective plant parts containing phosphatidic acid, extracted and purified phosphatidic acid and chemically synthesized phosphatidic acid.

4. The use of phosphatidic acid according to claim 1 in the preparation of a drug for preventing and / or treating a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infections includes, but is not limited to, active ingredients and pharmaceutically acceptable carriers; the active ingredients are plant extracts of phosphatidic acid and / or pharmaceutically acceptable salts thereof.

5. The use of phosphatidic acid according to claim 1 in the preparation of a drug for preventing and / or treating a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infections is a pharmaceutically acceptable oral preparation, injection, inhalation preparation or external preparation.

6. Use of phosphatidic acid according to claim 1 in the preparation of a drug for preventing and / or treating a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infections contains phosphatidic acid and / or a pharmaceutically acceptable salt thereof.

7. Use of phosphatidic acid according to claim 1 in the preparation of a drug for preventing and / or treating a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infections is used to inhibit the replication and proliferation of DNA-model virus HSV-1 and RNA-model virus VSV.

8. Use of phosphatidic acid according to claim 1 in the preparation of a drug for preventing and / or treating a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infections acts to activate PI3K-AKT signaling and promote the transcription of type I interferon.

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