Use of phosphatidic acid in the preparation of a medicine for preventing and / or treating a broad spectrum of viral infections

Abstract

The application relates to application of phosphatidic acid in preparation of medicines for preventing and / or treating broad-spectrum viral infections, and belongs to the technical field of biological medicines. The broad-spectrum viruses are DNA mode virus HSV-1 and RNA mode virus VSV infecting human beings. The application finds and determines the relationship between phosphatidic acid and infection of DNA virus HSV-1 and RNA virus VSV, that is, adding phosphatidic acid PA can inhibit the replication and proliferation of DNA virus HSV-1 and RNA virus VSV. The phenomenon of inhibiting virus replication is generated by directly activating the PI3K-AKT signal path by phosphatidic acid PA as a signal molecule, and then starting the transcription of type I interferon, so that the phosphatidic acid PA can be used in the prevention and / or treatment of broad-spectrum antiviral infections.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of phosphatidic acid (PA) in preparation of a medicine for preventing and / or treating a broad-spectrum viral infection. BACKGROUND

[0002] Viruses are simple microorganisms that must parasitize in host cells to replicate and proliferate. At present, the drugs for treating viruses are mainly divided according to the target, such as in the process of virus adsorption, internalization, transport, membrane fusion and genome release, the drugs directly targeting viruses can combine the proteins or nucleic acids of viruses to affect the entry, transcription, replication, assembly and release of viruses; and the drugs directly targeting hosts can affect the entry, transcription, replication, processing, transport and signal pathway activity of viruses. At present, the main molecular forms of the anti-viral drugs include small molecules, polypeptides, neutralizing antibodies, interferons, CRISPR-CAS systems, si / shRNA and other nucleic acid polymers, etc., but compared with the conservation of host cells, the nucleotide substitution rate in the replication process of viruses, especially RNA viruses and small DNA viruses, is much higher than that of host cells, because viruses are more likely to produce genetic material mutations when self-replicating, resulting in the failure of the original drug treatment target. In addition, there is no drug on the market for broad-spectrum viruses, only drugs for single (class) viruses, and such drugs for single (class) viruses often face the challenge of drug resistance. Therefore, in-depth study of the common mechanism of the interaction between viruses and host cells and the excavation of new anti-viral drug targets are the key problems that need to be solved for the prevention and treatment of viral infectious diseases. Since the host cells are more stable in heredity than viruses, and part of the host cell proteins participate in the regulation of viral replication through conservative strategies in multiple viruses, it is of great significance in broad-spectrum anti-viral therapy.

[0003] Phosphatidic acid (PA) is a common phospholipid and a component of cell membranes. PA can be synthesized by Kennedy choline metabolic end product-phosphatidylcholine under the action of phospholipase D. PA can act as a precursor of other lipids in cells and can also affect membrane curvature by its physical properties. In addition, PA can directly act as a signal molecule to transmit signals in cells. It has been found that the decrease of expression of the key rate-limiting enzyme CHKA in Kennedy choline metabolism can inhibit the MAPK and PI3K-AKT signaling pathways, and the decrease of PA content can directly weaken the MAPK signal. It has been found that positive-strand RNA viruses can promote the accumulation of PA in plants, thereby triggering MAPK-mediated plant anti-viral immune resistance to viral infection. In addition, it has been proved that the PI3K-AKT signal can target the transcription of type I interferon. However, there is no relevant research report on the relationship between phosphatidic acid (PA) and broad-spectrum viruses, and the influence of PA on PI3K-AKT signal and type I interferon. SUMMARY

[0004] The present application provides an application of phosphatidic acid (PA) in preparing a medicine for preventing and / or treating a broad-spectrum viral infection. The test data provided by the present application prove that phosphatidic acid (PA) can inhibit the replication of DNA mode virus HSV-1 and RNA mode virus VSV, activate PI3K-AKT signal, promote the expression of IFNB1, and can be used to provide technical support for preparing a medicine for preventing and / or treating a broad-spectrum viral infection, so as to apply phosphatidic acid to the medicine for preventing and / or treating a broad-spectrum viral infection.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The application of phosphatidic acid in preparing a medicine for preventing and / or treating a broad-spectrum viral infection.

[0007] Further, the broad-spectrum virus is DNA mode virus HSV-1 and RNA mode virus VSV infecting human.

[0008] Further, the medicine for preventing and / or treating a broad-spectrum viral infection includes but is not limited to at least one of the following: a plant containing phosphatidic acid, a plant extract containing phosphatidic acid, a plant effective part containing phosphatidic acid, extracted and purified phosphatidic acid, and chemically synthesized phosphatidic acid.

[0009] Further, the medicine for preventing and / or treating a broad-spectrum viral infection includes but is not limited to: an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is a plant extract of phosphatidic acid and / or a pharmaceutically acceptable salt thereof.

[0010] Further, the medicine for preventing and / or treating a broad-spectrum viral infection is a pharmaceutically acceptable oral preparation, injection, inhalation preparation or external preparation.

[0011] Further, the medicine for preventing and / or treating a broad-spectrum viral infection can contain phosphatidic acid and / or a pharmaceutically acceptable salt thereof.

[0012] Further, the medicine for preventing and / or treating a broad-spectrum viral infection acts on inhibiting the replication and proliferation of DNA mode virus HSV-1 and RNA mode virus VSV.

[0013] Further, the medicine for preventing and / or treating a broad-spectrum viral infection acts on activating PI3K-AKT signal and promoting type I interferon transcription.

[0014] Compared with the prior art, the present application has the following beneficial effects:

[0015] (1) The present application clarifies the relationship between phosphatidic acid and DNA mode virus HSV-1 and RNA mode virus VSV infection, makes phosphatidic acid as a potential target in the preparation of drugs for preventing and / or treating broad-spectrum viral infection, and provides an effective new way for the treatment of broad-spectrum viral infection by inhibiting the replication and proliferation of DNA mode virus HSV-1 and RNA mode virus VSV;

[0016] (2) The present application determines the feasibility of phosphatidic acid in the preparation of drugs for preventing and / or treating broad-spectrum viral infection at the cellular level, and provides a new potential drug for the treatment of broad-spectrum viral infection. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 For phosphatidic acid pretreatment of mouse mononuclear macrophage immune cells (RAW264.7) for 60h, inoculation of HSV-1, VSV virus for 12h, detection of ICP0 protein, VSVgB glycoprotein, and the resulting phosphatidic acid inhibits HSV-1, VSV virus in RAW264.7 cell viral protein replication chart, wherein: A is the inhibition of HSV-1 viral protein replication chart, B is the inhibition of VSV viral protein replication chart;

[0018] Figure 2 For phosphatidic acid treatment of mouse mononuclear macrophage immune cells (RAW264.7) for 48h, inoculation of HSV-1, VSV virus for 10h, detection of the number of live virus particles by TCID50 method, and the resulting phosphatidic acid inhibits HSV-1, VSV virus in RAW264.7 cell viral protein replication chart, wherein: C is the inhibition of HSV-1 viral replication chart, D is the inhibition of VSV viral replication chart;

[0019] Figure 3 E in the figure is the transcription of type I interferon IFNB1 under the stimulation of HSV-1 virus, which is 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 60h and inoculating HSV-1 virus for 12h; Figure 3 F in the figure is the transcription of type I interferon IFNB1 under the stimulation of VSV virus, which is 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 48h and inoculating VSV virus for 5h;

[0020] Figure 4Figures G and H are graphs showing the phosphorylation level of PI3K protein stimulated by HSV-1 and VSV viruses, respectively, after the mouse monocyte macrophage immune cells (RAW264.7) were treated with phosphatidic acid for 60 hours and inoculated with HSV-1 and VSV viruses for 12 hours, wherein the phosphorylation level of PI3K protein stimulated by HSV-1 virus is shown in Figure G, and the phosphorylation level of PI3K protein stimulated by VSV virus is shown in Figure H.

[0021] Figure 5 Figures I and J are graphs showing the phosphorylation level of AKT protein stimulated by HSV-1 and VSV viruses, respectively, after the mouse monocyte macrophage immune cells (RAW264.7) were treated with phosphatidic acid for 60 hours and inoculated with HSV-1 and VSV viruses for 12 hours, wherein the phosphorylation level of AKT protein stimulated by HSV-1 virus is shown in Figure I, and the phosphorylation level of AKT protein stimulated by VSV virus is shown in Figure J. DETAILED DESCRIPTION

[0022] The present application will be further described in conjunction with the following examples.

[0023] Those skilled in the art will appreciate that the following examples are intended to be illustrative only and should not be viewed as limiting the scope of the present application. Unless otherwise indicated, the techniques or conditions described in the examples were conducted in accordance with the techniques or conditions described in the literature or in accordance with the manufacturer's instructions. Unless otherwise indicated, the materials or equipment used were commercially available and were used in accordance with the manufacturer's instructions.

[0024] The following are the formulations of all the culture media used in the present application:

[0025] 10% DMEM complete medium: 10% fetal bovine serum (v%) was added to the total volume of commercial DMEM (Vivacell) medium;

[0026] 2% DMEM medium: 2% fetal bovine serum (v%) was added to the total volume of commercial DMEM (Vivacell) medium;

[0027] 4% DMEM medium: 4% fetal bovine serum (v%) was added to the total volume of commercial DMEM (Vivacell) medium;

[0028] Serum-free DMEM medium: commercial DMEM medium (Vivacell).

[0029] The cells involved in the present application:

[0030] Mouse monocyte macrophage immune cells RAW264.7 were purchased from Ponsay Biotech Co., Ltd.

[0031] Viruses involved in the present application;

[0032] HSV-1 virus, Strain-17; VSV virus, New Jersey strain, both provided by the Vaccine Research Lab of the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0033] Antibodies involved in the present application are shown in Table 1.

[0034] Table 1

[0035]

[0036] Reagents involved in the present application are shown in Table 2.

[0037] Table 2

[0038]

[0039] The preparation of corresponding reagents in the present application is as follows:

[0040] 1) The composition of transfer membrane buffer is shown in Table 3.

[0041] Table 3

[0042]

[0043] Add ultrapure water to 850 ml in the two components of glycine and Tris base, then add 150 ml of methanol, and store at room temperature after fully dissolving.

[0044] 2) The composition of 10x TBS buffer is shown in Table 4.

[0045] Table 4

[0046]

[0047] Dissolve the above two components into 800 ml of ultrapure water, adjust the pH value to 7.5 with concentrated hydrochloric acid, and finally dilute to 1000 ml.

[0048] 3) The composition of TBST (0.1% TWEEN 20) is shown in Table 5.

[0049] Table 5

[0050]

[0051] Mix the two components and shake well, and prepare them immediately before use.

[0052] 4) The composition of blocking solution is shown in Table 6.

[0053] Table 6

[0054]

[0055] Mix well after mixing, ready-to-use.

[0056] The English abbreviations involved in the present application and their corresponding Chinese names are shown in Table 7.

[0057] Table 7

[0058]

[0059] Example 1

[0060] The virus HSV-1, VSV respectively infects the RAW264.7 cells treated with DMSO as a control group and the RAW264.7 cells treated with phosphatidic acid PA, and the expression of virus HSV-1 immediate early gene product ICP0 protein and virus VSV gB glycoprotein is detected by protein immunoblotting, and the specific steps are as follows:

[0061] (1) Prepare cells: take well-grown RAW264.7 cells and inoculate them into 12-well plates, add 1ml of 10% DMEM complete culture medium to each well, the initial cell confluence is 30%-40%, shake uniformly, and place in a cell culture incubator at 37 degrees and 5% carbon dioxide concentration for culture;

[0062] (2) Cell treatment: after the RAW264.7 cells in step (1) adhere, treat the adherent RAW264.7 cells with phosphatidic acid PA at a concentration of 4μM (wherein the stock solution is 4mM), and treat RAW264.7 cells with DMSO at the same volume as phosphatidic acid PA as a control group, shake well, and place in a culture incubator at 37 degrees and 5% carbon dioxide concentration for culture;

[0063] (3) Virus inoculation: after the RAW264.7 cells treated with phosphatidic acid PA and DMSO respectively in step (2) for 60h, discard the 10% DMEM complete culture medium, wash the cells with PBS twice, then add 1ml of serum-free DMEM medium to each well, inoculate virus HSV-1 (MOI=1) and VSV (MOI=2) respectively, 1h later, add 1ml of 4% DMEM medium to each well, then continue to culture in a culture incubator at 37 degrees and 5% carbon dioxide concentration for 12h;

[0064] (4) Harvesting protein sample: After taking out the hole plate from the incubator of step (3), the virus-containing supernatant was discarded in a safety cabinet, and then the hole plate was washed twice with PBS. Then, 100 μl of RIPA (middle) lysis solution containing PMSF and phosphatase inhibitor was added to each cell in the 12-hole plate, and the cells were lysed for 20 min. The ratio of PMSF to phosphatase inhibitor in the lysis solution was 1:1000, and the lysis process was operated on ice. The hole plate was placed on a shaker every 5 min for 10 s. After lysis, the protein solution was transferred to an Ep tube which had been labeled, and centrifuged at 15000 rpm for 5 min at 4°C. The supernatant obtained after centrifugation was transferred to a new Ep tube, and the precipitate was discarded;

[0065] (5) Western blotting was used to detect the expression of two viral proteins:

[0066] (51) After protein quantification by BCA method, the concentration of the protein sample harvested in step (4) was adjusted in advance to be consistent, so that the volume and mass added were consistent. The loading amount of ordinary protein was 10-20 ug, the loading amount of phosphorylated protein was 60 ug, and the loading amount of protein marker was 3-5 μL. For a 10-hole plate, the volume of each hole was not more than 50 μL, and for a 15-hole plate, the volume of each hole was not more than 20 μL. An appropriate amount of 5x loading buffer was added to the centrifuge tube to dilute the final concentration of the buffer to 1x. Then, the sample was vortexed for 10 s, and the mixed sample was denatured at 100°C for 5 min. Then, it was centrifuged at 5000 g for 3 min, and the supernatant was taken for use;

[0067] (52) SDS-polyacrylamide gel electrophoresis: Assemble the electrophoresis tank, add MOPS Running Buffer Liquid (1x) electrophoresis buffer (prepared by diluting MOPS Running Buffer Liquid (20x) purchased from GenScript with ultrapure water), and immerse the inside of the loading hole and the outside of the 2Gels position. Remove the comb of the precast gel (purchased from GenScript). Add protein marker and sample, cover the upper cover, connect the electrophoresis instrument, and first run the strip at 80 v, and then adjust the voltage to 120 V for 30 min. When the marker indicates that the target band has been separated, terminate the electrophoresis;

[0068] (53) Transfer: After electrophoresis, cut the PVDF membrane in advance and activate it in methanol solution for 1 min; use a crowbar to gently pry the gel plate open, and cut the gel containing the target protein according to the protein marker, then carefully lay the gel on the filter paper; place the cut PVDF membrane with markers on the corresponding gel, making sure there are no air bubbles between them; assemble the sandwich clamp, with the black (gel) side facing the black negative electrode and the transparent (membrane) side facing the red positive electrode; pour the transfer solution (wet transfer) into the transfer tank until it is full, and add ice for ice bath transfer; set the transfer current to constant current, 300 mA, and the transfer time according to the protein size, about 1 min per 1 KD;

[0069] (54) Blocking: After transfer, remove the membrane and place it in 5 ml of blocking solution (TBST containing 5% skim milk) for 1-2 h at room temperature on a horizontal shaker;

[0070] (55) Incubate the primary antibody: Dilute the primary antibody (including ICP0 (abcam), VSV gB (abcam), and β-actin (CST)) with Western primary antibody diluent (Bi Yun Tian) according to the recommended dilution ratio; after the membrane surface is dried with absorbent paper, immediately add the diluted primary antibody and place it in the refrigerator at 4°C overnight; the dilution ratio of each antibody is ICP0 (1:2000), VSV gB (1:1000), and β-actin (1:5000);

[0071] (56) Wash the membrane: After incubating the primary antibody, transfer the membrane to a square preservation box containing TBST, so that the TBST covers the PVDF membrane, and shake it on a shaker for 10 min, repeating 3 times;

[0072] (57) Incubate the secondary antibody: Dilute the secondary antibody (including goat anti-mouse IgG (Proteintech)) with Bi Yun Tian secondary antibody diluent at a ratio of 1:10000 and incubate at room temperature for 1-2 h;

[0073] (58) Wash the membrane: After incubating the secondary antibody, transfer the membrane to a square preservation box containing TBST, so that the TBST covers the PVDF membrane, and shake it on a shaker for 10 min, repeating 3 times;

[0074] (59) Development: Prepare the developing solution (mix the developing A and B solutions from the Bi Yun Tian ultra-sensitive ECL chemiluminescence kit in a ratio of 1:1), during development, immerse the membrane in the developing solution to ensure that the developer is submerged, avoid light for 1 min, then place it in the UVP instrument to expose the protein band and take a photo;

[0075] (6) The experimental results are as follows Figure 1A, B, respectively, after RAW264.7 cells were infected with two viruses for 12 hours and harvested after DMSO and phosphatidic acid PA treatment for 60 hours, protein immunoblotting was performed to detect viral protein expression, and the results showed that phosphatidic acid PA treatment significantly inhibited the replication of DNA virus HSV-1 and RNA virus VSV.

[0076] Example 2

[0077] 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, and the amount of live virus in the cell supernatant was detected by half tissue culture infectious dose (TCID50), and the specific steps were as follows:

[0078] (1) Preparation of cells: take well-grown RAW264.7 cells, inoculate the cells into a 12-well plate, add 1 ml of 10% DMEM complete culture medium to each well, the initial cell confluence is about 30%~40%, shake evenly, and place in a 37 degree, 5% carbon dioxide concentration incubator for culture;

[0079] (2) Cell treatment: after the cells in step (1) adhere, treat the cells with phosphatidic acid PA at a concentration of 4 μM (stock solution is 4 mM), and treat RAW264.7 cells with DMSO at the same volume as phosphatidic acid PA as a control group, shake well, and place in a 37 degree, 5% carbon dioxide concentration incubator for culture;

[0080] (3) Virus inoculation: after RAW264.7 cells treated with phosphatidic acid PA and DMSO in step (2) for 48 hours, discard the 10% serum-containing DMEM culture medium, wash the cells with PBS twice, add 1 ml of serum-free DMEM culture medium to each well, inoculate virus HSV-1 (MOI=0.5) and VSV (MOI=1) respectively, 1 hour later, add 1 ml of 4% serum-containing DMEM culture medium to each well, and then continue to culture the cells in a 37 degree, 5% carbon dioxide concentration incubator for 10 hours:

[0081] (4) Virus supernatant was harvested at 10 hours, 1 ml of culture supernatant was carefully mixed and taken into a 1.5 ml centrifuge tube, and stored at -80 degrees Celsius;

[0082] (5) CCID50 method for determining virus titer:

[0083] a. Preparation of Vero cells: mix Vero cells with 4% maintenance liquid (4% fetal bovine serum, v%) and plate in a 96-well plate, with a liquid volume of 100 μl per well, containing about 5×10 4 cells;

[0084] b. Dilution of the virus to be tested: HSV-1 virus was taken out from the -80 degree Celsius refrigerator, and allowed to melt. The virus stock solution was diluted 10 times in an EP tube using serum-free medium, i.e. 100 μl of virus stock solution was mixed with 900 μl of serum-free medium to form 10 -1 dilution gradient, i.e. 100 μl of virus solution of the 10-1 dilution gradient was mixed with 900 μl of serum-free medium to form 10 -2 dilution gradient, and 10 virus gradients were formed in sequence, i.e. 100 μl of virus solution of the 10-1 dilution gradient was mixed with 900 μl of serum-free medium to form 10 -1 -10 -10 ;

[0085] c. Inoculation: 100 μl of the diluted virus solution was added to the 96-well plate with Vero cells, 8 wells for each dilution gradient, 100 μl was added to each well, and the high dilution was added first, followed by the low dilution. The remaining 8 wells were added with 100 μl of serum-free medium as blank control, and were placed in a carbon dioxide incubator at 37 degree Celsius and a carbon dioxide concentration of 5% for 5-7 days.

[0086] d. Observation and statistics: the cytopathic effect was observed under a microscope every day during the culture process, and the number of cytopathic wells at each gradient was recorded until the number of cytopathic wells was no longer increased in the continuous observation, and the results were counted.

[0087] e. Calculation of results: the virus titer was calculated by Karber method, i.e. lgCCID50 / ml = negative logarithm of the highest dilution of total cytopathic effect + sum of cytopathic wells of each dilution without total cytopathic effect x 0.125 + 0.5 + 1.

[0088] (6) The experimental results are shown in Table 1, and the results show that the addition of phosphatidic acid PA (4 μM) in RAW264.7 cells can inhibit the expression and replication of virus HSV-1. Figure 2

[0089] Example 3

[0090] The virus HSV-1 and VSV were respectively infected into RAW264.7 cells treated with DMSO as a control group and RAW264.7 cells treated with phosphatidic acid PA, and the transcription of type I interferon IFNB1 was detected by real-time fluorescent quantitative reverse transcription PCR (RT-qPCR), and the specific steps were as follows:

[0091] ​(1) Preparation of cells: Take well-grown RAW264.7 cells, inoculate the cells into a 12-well plate, add 1 ml of 10% DMEM complete culture medium to each well, the initial cell confluence is about 30%~40%, shake uniformly, and place in a 37-degree, 5% carbon dioxide concentration incubator for culture;

[0092] (2) Drug treatment: After the cells in step (1) adhere, treat the cells with 4 mM concentration of phosphatidic acid PA (stock solution is 4 mM), and treat RAW264.7 cells with the same volume of DMSO as phosphatidic acid PA as a control group, shake uniformly, and place in a 37-degree, 5% carbon dioxide concentration incubator for culture;

[0093] (3) Virus inoculation: After the RAW264.7 cells treated with phosphatidic acid PA and DMSO for 60 h, discard the DMEM medium containing serum, wash the cells with PBS twice, add 1 ml of serum-free DMEM medium to each well, inoculate the virus HSV-1 (MOI=1) and VSV (MOI=1) respectively, 1 h later, add 1 ml of DMEM medium containing 4% serum to each well, then continue to culture the HSV-1 inoculated cells in a 37-degree, 5% carbon dioxide concentration incubator for 12 h; continue to culture the VSV inoculated cells in a 37-degree, 5% carbon dioxide concentration incubator for 5 h;

[0094] (4) Harvesting of gene samples: After taking the plate out of 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, and the whole process is operated at room temperature; lyse the cells for about 10 min, after the lysis is completed, transfer the lysis solution to the labeled Ep tube, and start extracting RNA;

[0095] (5) Extract RNA, reverse transcription:

[0096] The total RNA extraction steps are as follows:

[0097] a. Harvest the RNA gene sample;

[0098] b. Add 1 / 5 volume of chloroform to the RNA gene sample (i.e. add 100 μl of chloroform to each 0.5 ml of Trizol), mix thoroughly under the condition of 4°C, 12000g centrifugation for 20 min;

[0099] c. Take 40% of the Trizol volume, add 1.5 times the volume of isopropanol to the water layer during extraction and mix thoroughly, or directly centrifuge, or the sample is precipitated overnight at -40°C, then centrifuge at 4°C at 12000g for 10 min, discard the supernatant, and retain the white precipitate at the bottom of the tube;

[0100] d. Add 0.8 ml of 75% ethanol pre-cooled at -40°C, mix well by inverting repeatedly, centrifuge at 4°C, 12000 g for 5 min;

[0101] e. Discard the supernatant, use a 10 ul syringe to aspirate the liquid as much as possible without touching the RNA precipitate, ensure that the remaining ethanol can be naturally dried at room temperature; when the ethanol is completely volatilized, the original white RNA precipitate will turn transparent, indicating that the liquid has been completely removed;

[0102] f. To dissolve the RNA, 20-40 μl of DEPC water needs to be added; to facilitate the dissolution of RNA, the syringe tip can be used to gently aspirate several times;

[0103] g. After the dissolution of RNA is completed, its concentration needs to be determined, and once the concentration determination is completed, the RNA can be stored at -40°C or directly used for subsequent reverse transcription experiments;

[0104] h. Real-time fluorescent quantitative PCR (qPCR)

[0105] When performing real-time fluorescent quantitative PCR experiments, relative quantitative analysis is performed according to the operation guide of GoTaq® qPCR MasterMix kit provided by Promega company; in order to ensure the accuracy and reliability of the experiment, a 10 μl reaction system is prepared according to the standard process, and each component in this system is 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:

[0106] Prepare a 10 μl reaction system:

[0107]

[0108] Each sample is repeated twice, and the reaction program is: 95°C, 5 min; 95°C, 15 s; 50-60°C, 30 s; 72°C, 30 s; 35 cycles;

[0109] Melting curve 65°C-95°C, 0.5°C / cycle, 5 s; detect according to the reaction program on Bio-Rad real-time fluorescent quantitative PCR instrument:

[0110] The primer sequences used in real-time fluorescent quantitative PCR are as follows:

[0111]

[0112] (6) The experimental results are as follows: Figure 3E, F, and the RAW264.7 cells treated with DMSO and treated with phosphatidic acid PA after being infected with two viruses, and the RNA was extracted after a certain time for real-time fluorescent quantitative reverse transcription PCR to detect the expression of the IFNB1 gene. The results show that the phosphatidic acid PA treatment activates the INFB1 transcription under the stimulation of the DNA virus HSV-1 and the RNA virus VSV.

[0113] Example 4

[0114] The viruses HSV-1 and VSV were respectively infected into the RAW264.7 cells treated with DMSO as a control group and the RAW264.7 cells treated with phosphatidic acid PA, and the PI3K signal was detected by Western blotting. The specific steps are as follows:

[0115] (1) Preparation of cells: take well-grown RAW264.7 cells, inoculate the cells into a 12-well plate, add 1 ml of 10% DMEM complete culture medium to each well, the initial cell confluence is about 30%~40%, shake uniformly, and place in a 37-degree, 5% carbon dioxide concentration incubator for culture;

[0116] (2) Cell treatment: after the cells in step (1) adhere, treat the cells with phosphatidic acid PA at a concentration of 4 μM (the mother liquor is 4 mM), and treat the RAW264.7 cells with DMSO at the same volume as the phosphatidic acid PA as a control group, shake uniformly, and place in a 37-degree, 5% carbon dioxide concentration incubator for culture;

[0117] (3) Virus inoculation: after the phosphatidic acid PA and DMSO treated RAW264.7 cells are cultured for 60 h, discard the DMEM culture medium containing serum, wash the cells with PBS twice, add 1 ml of serum-free DMEM culture medium to each well, inoculate the viruses HSV-1 (MOI=1) and VSV (MOI=2) respectively, 1 h later, add 1 ml of DMEM culture medium containing 4% serum to each well, and then continue to place the cells in a 37-degree, 5% carbon dioxide concentration incubator for culture for 12 h;

[0118] (4) Harvesting protein sample: After taking out the hole plate from the incubator, the virus-containing supernatant is discarded in a safety cabinet, and after being washed twice with PBS, 100 μl of RIPA (medium) lysis solution containing PMSF and phosphatase inhibitor is added to each cell in the 12-hole plate, and the cells are lysed for about 20 min, wherein: the PMSF and phosphatase inhibitor in the lysis solution are added to the RIPA (medium) lysis solution (now use now) in a ratio of 1:1000 in advance, and the lysis process is operated on ice; wherein every 5 min, the hole plate is placed in the shaker for about 10 s, and after the lysis is completed, the protein solution is transferred to an Ep tube which has been labeled, and placed in a 4°C centrifuge, 15000g, 5 min, the obtained supernatant is transferred to a new Ep tube, and the precipitate is discarded;

[0119] (5) Protein immunoblotting is used to detect the expression of two viral proteins, and the method used is the same as that in Example 1;

[0120] (6) The experimental results are shown in G and H of Figure 4 , and the protein expression of the two viruses is detected by protein immunoblotting after 12 hours of RAW264.7 cell infection with DMSO treatment and 60 hours of phosphatidic acid PA treatment, and the results show that the PI3K signal is activated under the stimulation of DNA virus HSV-1 and RNA virus VSV after phosphatidic acid PA treatment.

[0121] Example 5

[0122] The viruses HSV-1 and VSV are respectively infected with DMSO-treated RAW264.7 cells as a control group, and phosphatidic acid PA-treated RAW264.7 cells, and the AKT signal is detected by protein immunoblotting, and the specific steps are as follows:

[0123] (1) Prepare cells: take well-grown RAW264.7 cells, inoculate cells into a 12-hole plate, add 1 ml of 10% DMEM complete culture medium to each hole, the initial cell confluence is about 30%~40%, shake evenly, and place in a 37-degree, 5% carbon dioxide concentration incubator for culture;

[0124] (2) Cell treatment: after the cells in step (1) adhere, treat the cells with phosphatidic acid PA at a concentration of 4 μM (stock solution is 4 mM), and treat RAW264.7 cells with DMSO at the same volume as phosphatidic acid PA as a control group, shake well, and place in a 37-degree, 5% carbon dioxide concentration incubator for culture;

[0125] (3) Virus inoculation: After the RAW264.7 cells were treated with phosphatidic acid PA and DMSO for 60 hours, the DMEM medium containing serum was discarded, the cells were washed twice with PBS, 1 ml of DMEM medium without serum was added to each well, and the virus HSV-1 (MOI = 1) and VSV (MOI = 2) were inoculated, respectively, 1 hour later, 1 ml of DMEM medium containing 4% serum was added to each well, and then the cells were continuously cultured in a 37-degree, 5% carbon dioxide concentration incubator for 12 hours;

[0126] (4) Protein sample harvesting: After the plate was taken out of the incubator, the supernatant containing the virus was discarded in a safety cabinet, and the cells were washed twice with PBS. 100 μl of RIPA (medium) lysis solution containing PMSF and phosphatase inhibitor was added to each well of the 12-well plate, and the cells were lysed for about 20 minutes. The PMSF and phosphatase inhibitor in the lysis solution were added to the RIPA (medium) lysis solution (used immediately) in a ratio of 1:1000 in advance, and the lysis process was operated on ice. Every 5 minutes, the plate was placed in a shaker for about 10 seconds, and after the lysis was completed, the protein solution was transferred to an Ep tube which had been labeled, and was placed in a 4-degree centrifuge at 15000g for 5 minutes. The obtained supernatant was transferred to a new Ep tube, and the precipitate was discarded;

[0127] (5) Western blotting was used to detect the expression of two viral proteins. The method used was the same as that in Example 1;

[0128] (6) The experimental results are shown in I and J of Figure 5 After the RAW264.7 cells were infected with two viruses, treated with DMSO, and treated with phosphatidic acid PA for 60 hours, the protein was collected after 12 hours, and Western blotting was used to detect the expression of viral proteins. The results showed that the AKT signal was activated under the stimulation of DNA virus HSV-1 and RNA virus VSV after phosphatidic acid PA treatment.

Claims

1. Use of phosphatidic acid and / or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and / or treatment of a broad spectrum of viral infections, characterized in that, The broad-spectrum viral infection drug comprises an active ingredient, and the active ingredient is phosphatidic acid and / or a pharmaceutically acceptable salt thereof; the broad-spectrum virus is a DNA mode virus HSV-1 and an RNA mode virus VSV infecting human.

2. Use of the phosphatidic acid and / or a pharmaceutically acceptable salt thereof according to claim 1 for the manufacture of a medicament for the prevention and / or treatment of a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infection comprises an active ingredient and a pharmaceutically acceptable carrier.

3. Use of the phosphatidic acid and / or a pharmaceutically acceptable salt thereof according to claim 1 for the manufacture of a medicament for the prevention and / or treatment of a broad spectrum of viral infections, characterized in that: The drug for preventing and / or treating broad-spectrum viral infection is a pharmaceutically acceptable oral preparation, injection preparation, inhalation preparation or external preparation.

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