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

Abstract

The invention provides an application of choline in preparation of a drug for preventing or / and treating broad-spectrum virus infection. The broad-spectrum virus is a DNA (deoxyribonucleic acid) mode virus HSV-1 and an RNA (ribonucleic acid) mode virus VSV of infected people. Choline can inhibit replication and expression of a DNA mode virus HSV-1 and an RNA mode virus VSV, and an effective new way is provided for prevention or / and treatment of broad-spectrum virus infection; the choline can activate PI3K-AKT signals and promote transcription of type I interferon, and a new potential drug is provided for prevention and treatment of broad-spectrum virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of choline 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, antiviral drugs are mainly divided according to targets. For example, in 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 and transport, as well as the activation of signal pathways. 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, but compared with the conservatism of host cells, viruses, especially RNA viruses and small DNA viruses, are more prone to genetic mutations during self-replication due to the much higher nucleotide replacement rate than host cells during replication, which makes the target of the original drug treatment ineffective. In addition, there are currently no drugs on the market for broad-spectrum viral infections, only drugs for single-type viral infections, and drugs for single-type viral infections often face the challenge of drug resistance. Therefore, in-depth research on the common mechanisms of interaction between viruses and host cells and the development of new drugs against broad-spectrum viral targets are the key to preventing and treating a variety of broad-spectrum viral infectious diseases and developing corresponding drugs. Since host cells are genetically more stable than viruses, and some host cell proteins participate in regulating viral replication through conservative strategies in many types of viruses, they are of great significance in anti-broad-spectrum viral treatment. By in-depth research on the interaction between host genes and viral infection, new targets will be provided for the development of safe and effective new drugs against broad-spectrum viruses.

[0003] Choline is an important nutrient. The human body needs choline to synthesize phosphatidylcholine, which is a component of the cell membrane. In addition, the human body needs choline to synthesize an important neurotransmitter, acetylcholine. In addition, choline also has the function of providing methyl. Although the human body can synthesize choline through phosphatidylcholine in the liver, the choline naturally synthesized by the human body cannot meet the needs of the human body itself, so the human body needs to obtain choline in food. Studies have found that choline deficiency can impair the synthesis of phosphatidylcholine in the Kennedy choline metabolic pathway. In addition, when the expression of CHKA, the key rate-limiting enzyme in Kennedy choline metabolism, is low, the MAPK and PI3K-AKT signaling pathways will be inhibited. This is because the end product of Kennedy choline metabolism, phosphatidylcholine, is converted into phosphatidic acid by phospholipase D, that is, reducing the content of phosphatidic acid can directly weaken the MAPK signal. 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. These all point to the fact that choline deficiency can affect immunity by reducing downstream phospholipid signaling molecules. The PI3K-AKT signal can target and activate the transcription of type I interferon. Therefore, it is necessary to further study the effects of choline and viruses. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a nutritional additive choline for use in the preparation of drugs for preventing or / and treating broad-spectrum viral infections. The relationship between choline and DNA and RNA model viral infections is determined, and reliable technical support is provided for the preparation of drugs for preventing or / and treating broad-spectrum viral infections, thereby using choline in the prevention or treatment of broad-spectrum viral infections.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: Application of choline 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 comprises at least one of: plants containing choline, plant extracts containing choline, effective plant parts containing choline, extracted and purified choline, and chemically synthesized choline.

[0008] Furthermore, the drug for preventing and / or treating broad-spectrum viral infections comprises an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is a plant extract of choline 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 includes choline 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] Choline is poorly soluble in water and is usually added in the form of choline chloride.

[0014] The present invention mainly studies: (1) choline can inhibit the replication of DNA model virus HSV-1 and RNA model virus VSV; (2) choline can activate PI3K-AKT signaling and thus promote the expression of IFNB1.

[0015] The present invention conducted experiments on mouse mononuclear macrophage RAW264.7 to investigate the relationship between choline and DNA model virus HSV-1 and RNA model virus VSV, as well as the effect of choline on PI3K-AKT signal and type I interferon; the results showed that choline can inhibit the replication and expression of DNA model virus HSV-1 and RNA model virus VSV, and choline promotes type I interferon transcription by activating PI3K-AKT signal.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention clarifies the relationship between choline and DNA model virus HSV-1 and RNA model virus VSV infection, so that choline can be regarded as a potential target in the preparation of drugs for the prevention and / or treatment of 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 prevention and / or treatment of broad-spectrum viral infections; (2) The feasibility of choline in the preparation of drugs for the prevention and / or treatment of a wide range of viral infections was determined at the cellular level, providing new potential drugs for the prevention and / or treatment of a wide range of viral infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 After adding choline chloride to RAW264.7 cells, it can significantly inhibit the DNA virus HSV-1 ( Figure 1 A) and RNA virus VSV ( Figure 1B) Protein replication diagram in the cell; the figure shows the results of treating mouse mononuclear macrophage immune cells (RAW264.7) with choline chloride (1200μM) for 48h, and using enzyme-free sterile water as the control group, inoculating DNA virus HSV-1 (MOI=0.5) and RNA virus VSV (MOI=1) for 5h, respectively, and detecting the collected DNA virus HSV-1 gB glycoprotein and RNA virus VSV gB glycoprotein samples by protein immunoblotting (WB) respectively; Figure 2 After adding choline chloride to RAW264.7 cells, it can significantly inhibit the DNA virus HSV-1 ( Figure 2 C) and RNA virus VSV ( Figure 2 D) Virus replication diagram in the cell; the figure shows the results of detecting virus titers by ICID50 method after treating mouse mononuclear macrophage immune cells RAW264.7 cells with choline chloride (1200μM) and choline chloride (4800μM) for 48h, using enzyme-free sterile water as the control group, and inoculating DNA virus HSV-1 (MOI=0.5) and RNA virus VSV (MOI=1) for 5h and 4h respectively: Figure 3 After adding choline chloride to RAW264.7 cells, it can significantly activate the DNA virus HSV-1 ( Figure 3 E) and RNA virus VSV ( Figure 3 F) Transcription diagram of type I interferon IFNB1A gene level under stimulation; in the figure, mouse mononuclear macrophage immune cell RAW264.7 cells were treated with choline chloride (1200μM) for 48h, and enzyme-free sterile water was used as the control group. DNA virus HSV-1 (MOI=0.5) and RNA virus VSV (MOI=1) were inoculated for 10h, and the collected gene samples were tested by RT-qPCR to obtain the relative expression results of type I interferon gene; Figure 4 After adding choline chloride to RAW264.7 cells, it can significantly activate the DNA virus HSV-1 ( Figure 4 G) and RNA virus VSV ( Figure 4 (H) PI3K protein phosphorylation level under stimulation; in the figure, mouse mononuclear macrophage immune cell RAW264.7 cells were treated with choline chloride (1200μM) for 48h, and enzyme-free sterile water was used as the control group. DNA virus HSV-1 (MOI=0.5) and RNA virus VSV (MOI=1) were inoculated for 10h, and PI3K total protein and p85 activation levels at 199, 458 tyrosine phosphorylation sites were detected by RT-qPCR. In addition, β-actin protein was also detected; Figure 5 After adding choline chloride to RAW264.7 cells, it can significantly activate the DNA virus HSV-1 ( Figure 5 I) and RNA virus VSV ( Figure 5 (J) Graph of phosphorylation level of AKT protein under stimulation; in the figure, mouse mononuclear macrophage immune cells RAW264.7 cells were treated with choline chloride (1200μM) for 48h, and enzyme-free sterile water was used as the control group. DNA virus HSV-1 (MOI=0.5) and RNA virus VSV (MOI=1) were inoculated for 10h and 5h, respectively. The harvested protein samples were subjected to protein immunoblotting (WB) to detect the phosphorylation level of p-AKT (Ser473) protein. In addition, β-actin protein was also detected. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with embodiments.

[0019] 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.

[0020] All culture media used in the present invention are 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% maintenance solution: add 2% fetal bovine serum (v%) to the commercial DMEM (Vivacell) medium; 4% maintenance solution: add fetal bovine serum (v%) accounting for 4% of the total volume to commercial DMEM (Vivacell) culture medium; Serum-free culture medium: commercial DMEM culture medium (Vivacell).

[0021] The cells involved in the present invention: mouse mononuclear macrophage immune cells (RAW264.7) were purchased from Pronocell Biotechnology Co., Ltd.

[0022] The virus involved in the present invention is: HSV-1 virus, Strain-17; VSV virus, New Jersey strain; both were provided by the Vaccine Research Laboratory, Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0023] The antibodies involved in the present invention are shown in Table 1.

[0024] Table 1

[0025] The reagents involved in the present invention are shown in Table 2.

[0026] Table 2

[0027] The preparation of the corresponding reagents in the present invention is as follows: 1) The components of the transfer buffer are shown in Table 3; Table 3

[0028] Add glycine and Trisbase to ultrapure water to make up to 850 ml, then add 150 ml of methanol, and store at room temperature after fully dissolving. 2) The composition of 10×TBS buffer is shown in Table 4; Table 4

[0029] Dissolve the above ingredients in 800 ml of ultrapure water, adjust the pH to 7.5 with concentrated hydrochloric acid, and finally make up to 1000 ml; 3) TBST (0.1% TWEEN20) composition is shown in Table 5: Table 5

[0030] Mix the two ingredients and shake well, prepare before use; 4) The composition of the blocking solution is shown in Table 6; Table 6

[0031] Mix the two ingredients and shake well before use.

[0032] The English abbreviations involved in the present invention correspond to the Chinese and English names as shown in Table 7. Table 7

[0033] Example 1 The RAW264.7 cells as the control group and the RAW264.7 cells treated with choline chloride were infected with the viruses HSV-1 and VSV, respectively, and the expression of HSV-1 gB glycoprotein and RNA virus VSV gB glycoprotein was detected by protein immunoblotting, respectively. 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% complete medium to each well. The initial cell confluence is 30%-40%. Shake evenly and place in an incubator at 37 degrees and 5% carbon dioxide concentration for subculture. (2) Cell treatment: After the RAW264.7 cells in step (1) adhere to the wall, the adherent cells are treated with 1200 μM choline chloride (the mother solution is 1200 mM), and RAW264.7 cells are added to an equal volume of enzyme-free sterile water as the choline chloride as a control group. After shaking evenly, the cells are placed in an incubator at 37 degrees and 5% carbon dioxide for culture; (3) Virus inoculation: After culturing the RAW264.7 cells treated with choline chloride and enzyme-free sterile water for 48 h, discard the 10% complete culture medium, wash the cells twice with PBS, add 1 ml of serum-free culture medium to each well, and inoculate the viruses HSV-1 (MOI = 0.5) and VSV (MOI = 1), respectively. After 1 h, add 1 ml of culture medium containing 4% serum to each well, and then place in an incubator at 37 degrees and 5% carbon dioxide concentration for 5 h; (4) Harvest protein samples; after removing the well plate from the incubator, 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 minutes, where the ratio of PMSF to phosphatase inhibitors in the lysis buffer is 1:1000. The lysis process is performed on ice, and the well plate is placed on a shaker for 10 seconds every 5 minutes. After the lysis is completed, the protein solution is transferred to a labeled Eppendorf tube and centrifuged at 15,000 g for 5 minutes in a centrifuge at 4°C. The supernatant obtained by centrifugation is transferred to a new Eppendorf tube and the precipitate is discarded; (5) Protein immunoblotting was used to detect the expression of two viral proteins, as follows: (5.1) After protein quantification by BCA method, adjust the concentration of protein samples harvested in step (4) in advance to make the added volume and mass consistent: the loading amount of common protein is 10-20ug, the loading amount of phosphorylated protein is 60ug, and the loading amount of protein marker is 3-5 μL; for 10-well plate, the loading volume per well should not exceed 50 μL, and for 15-well plate, the loading volume per well should not exceed 20 μL. Add an appropriate amount of 5× loading buffer to the centrifuge tube to dilute the final concentration to 1×, then vortex the sample for 10 seconds, place the mixed sample in an iron bath at 100℃ for 5 minutes to denature the protein, and then centrifuge at 5000g for 3 minutes in a centrifuge, and take the supernatant for use; (5.2) SDS-polyacrylamide gel electrophoresis: Assemble the electrophoresis tank, add MOPS Running Buffer Liquid (1×) electrophoresis buffer (MOPS Running Buffer Liquid (20×) purchased from GenScript and prepared with ultrapure water), with the inner side covering the sample loading hole and the outer side reaching the 2 Gels position. Pull out the comb of the precast gel (purchased from GenScript), add protein marker and the supernatant sample of step (51), keep the volume consistent, cover the top cover, connect the electrophoresis instrument, first run the bands at 80V, then adjust the voltage to 120V and run for 30min. When the marker indicates that the target bands have been separated, stop the electrophoresis. (5.3) Transfer: After electrophoresis, cut the PVDF membrane in advance and activate it in methanol solution for 1 minute. Pry open the gel plate, cut the gel containing the target protein with the reference protein marker, and then lay it flat on the filter paper. Cover the cut PVDF membrane with the marker on the corresponding gel. Be careful not to have 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. Inject 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 300mA. The transfer time is determined according to the size of the protein, and the transfer time is 1 minute per 1KD. (5.4) 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 to 2 hours; (5.5) Incubate the primary antibody; dilute the primary antibody with Western primary antibody diluent (Biyuntian) according to the dilution multiple recommended in the antibody manual (the primary antibodies used include: ICP0 (abcam), VSV gB (abcam), β-actin (CST)); after the blocking solution on the membrane surface is blotted dry with absorbent paper, add the diluted primary antibody immediately and place in a 4°C refrigerator overnight. The dilution ratio of each antibody is: ICP0 (1:2000), VSV gB (1:1000), β-actin (1:5000); (5.6) Washing the membrane: After incubation, transfer the membrane to a fresh-keeping box containing TBST, so that the TBST covers the PVDF membrane. Shake on a shaker for 10 min and repeat the washing process three times. (5.7) Incubation with secondary antibody: dilute the secondary antibody (including goat anti-mouse IgG (Proteintech)) at a ratio of 1:10000 with secondary antibody diluent (produced by Bio-Tech) and incubate at room temperature for 1-2 hours; (5.8) Washing the membrane: After incubation, transfer the membrane to a fresh-keeping box containing TBST, so that the TBST covers the PVDF membrane. Shake on a shaker for 10 min and repeat washing three times. (5.9) Development: Prepare the developer (mix the developer A and B in the Bio-Tech Ultra-Sensitive ECL Chemiluminescence Kit in a 1:1 ratio). Immerse the membrane in the developer to ensure that the developing instrument covers the membrane. After reacting in the dark for 1 minute, 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, the results showed that choline chloride treatment inhibited the replication of DNA virus HSV-1 and RNA virus VSV.

[0034] Example 2

[0035] The virus HSV-1 and VSV were used to infect RAW264.7 cells as a control group and RAW264.7 cells treated with choline chloride, 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 them into 12-well plates. Add 1 ml of 10% complete medium to each well. The initial cell confluence is about 30%-40%. Shake evenly and place in an incubator at 37°C and 5% carbon dioxide concentration for culture. (2) Cell treatment: After the RAW264.7 cells in step (1) adhere to the wall, treat the cells with 1200 μM concentration and 4800 μM choline chloride (the stock solution is 1200 mM). At the same time, add an equal volume of enzyme-free sterile water to the cells in the control group and shake them evenly. Then, culture them in an incubator at 37°C and 5% carbon dioxide concentration: (3) Virus inoculation: After the RAW264.7 cells were treated with choline chloride and enzyme-free sterile water for 48 h, the serum-containing medium was discarded, the cells were washed twice with PBS, 1 ml of serum-free 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 medium containing 4% serum was added to each well, and the cells inoculated with HSV-1 and VSV were then placed in an incubator at 37 ° C and 5% carbon dioxide concentration for 5 h and 4 h: (4) Harvesting the virus 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°C; (5) The virus titer was determined by CCID50 method as follows: (5.1) Preparation of Vero cells: Vero cells were mixed with 4% maintenance medium (4% fetal bovine serum, v%) and plated on a 96-well plate. The volume of each well was 100 μl, containing 5×10 4 Cells: (5.2) Diluting the virus solution to be tested: Take out the HSV-1 and VSV viruses harvested in step (4) from the -80°C refrigerator and wait for them to thaw; dilute the HSV-1 and VSV virus stock solutions in EP tubes at a 10-fold ratio with serum-free culture medium, i.e., take 100 μl of the HSV-1 and VSV virus stock solutions and mix them with 900 μl of serum-free culture medium to make 10 -1 Dilution gradient: 100 μl of 10-1 dilution gradient virus solution was mixed with 900 μl serum-free medium to make 10 -2 Dilution gradient; 10 virus gradients are formed in sequence, i.e. 10 -1 -10 -10 ; (5.3) Inoculation: Take a 96-well plate with Vero cells and add the diluted virus solution in step (52) to the 96-well plate. Add 8 wells for each dilution gradient and add 100 μl to each well. Add the high dilution first and then the low dilution. Add 100 μl of serum-free culture medium to the remaining 8 wells as a blank control. Incubate in a carbon dioxide incubator at 37°C and 5% carbon dioxide for 5-7 days. (5.4) Observation and statistics: Observe the cytopathic effect under a microscope every day during the culture process, and record the number of cytopathic wells at each gradient until the number of cytopathic wells no longer increases after continuous observation, and then make statistics; (5.5) 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 in C and D, choline chloride (1200 μM) and choline chloride (4800 μM) were added to RAW264.7 cells, and HSV-1 (MOI=0.5) and VSV (MOI=1) were infected 48 hours later. 5 and 4 hours after infection, the cell supernatant was collected and the virus titer was determined by the CCID50 method. The results showed that the addition of choline chloride to RAW264.7 cells can inhibit the expression and replication of HSV-1 and VSV viruses.

[0036] Example 3

[0037] The viruses HSV-1 and VSV were used to infect RAW264.7 cells and RAW264.7 cells treated with choline chloride, and the transcription of type I interferon IFNB1 was detected by real-time fluorescence quantitative reverse transcription PCR (RT-qPCR), as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate them into 12-well plates. Add 1 ml of 10% 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 subculture. (2) Cell treatment: After the RAW264.7 cells in step (1) adhere to the wall, treat the cells with 1200 μM choline chloride (the stock solution is 1200 mM). At the same time, add an equal volume of enzyme-free sterile water to the cells in the control group. After shaking evenly, place the cells in an incubator at 37°C and 5% carbon dioxide for culture. (3) Virus inoculation: After the RAW264.7 cells were treated with choline chloride and enzyme-free sterile water for 48 h, the serum-containing medium was discarded, the cells were washed twice with PBS, 1 ml of serum-free 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 medium containing 4% serum was added to each well, and the cells inoculated with HSV-1 and VSV were then placed in an incubator at 37°C and 5% carbon dioxide for 10 h. (4) Sample harvesting: After removing the well plate from the incubator, discard the virus-containing supernatant in the safety cabinet, wash twice with PBS, add 400 μl of Trizol to each well of the 12-well plate, and lyse the cells for about 10 minutes. This process is performed at room temperature. After lysis is completed, transfer the lysate to a labeled Eppendorf tube to extract RNA; (5) Extract RNA and reverse transcription: The steps for total RNA extraction are as follows: (5.1) Harvesting the RNA gene extracted in step (4) as a sample; (5.2) Add 1 / 5 volume of chloroform to the sample (i.e., add 100 µl of chloroform to every 0.5 ml of Trizol), shake thoroughly to mix, and centrifuge at 12,000 g, 4°C, for 20 min. (5.3) Take 40% of the volume of Trizol and add 1.5 times the volume of isopropanol to the aqueous layer during the extraction process and mix thoroughly. Centrifuge or place the sample at -40°C overnight to precipitate. Then, centrifuge at 12,000 g for 10 minutes at 4°C, discard the supernatant, and retain the white precipitate at the bottom. (5.4) Add 0.8 ml of 75% ethanol precooled at -40°C to the white precipitate, mix by repeated inversion, and centrifuge at 12,000 g for 5 min at 4°C; (5.5) Discard the supernatant and use a 10ul pipette to remove the liquid without touching the RNA precipitate as much as possible, ensuring that the remaining ethanol can evaporate 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; (5.6) To dissolve RNA, add 20 μl to 40 μl of DEPC water. To promote RNA dissolution, gently pipette several times. (5.7) After the RNA is dissolved, measure its concentration. After the concentration is measured, store it at -40°C or use it directly in subsequent reverse transcription experiments. (5.8) Real-time fluorescence quantitative PCR (qPCR) When conducting real-time fluorescence quantitative PCR experiments, relative quantitative analysis was accurately 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 as shown in Table 8: Table 8

[0038] 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°C-95°C, 0.5°C / cycle, 5 s, and detected on a Bio-Rad real-time fluorescence quantitative PCR instrument according to the reaction program; The primer sequences used for real-time fluorescence quantitative PCR are shown in Table 9: Table 9

[0039] (6) Experimental results are as follows Figure 3 As shown in E and F, after the two viruses infected RAW264.7 cells treated with enzyme-free sterile water and choline chloride, the genes were collected after a certain period of time to extract RNA for real-time fluorescence quantitative reverse transcription PCR to detect the expression of IFNB1 gene. The results showed that choline chloride treatment activated the transcription of DNA virus HSV-1 and RNA virus stimulated INFB1.

[0040] Example 4 The RAW264.7 cells as the control group and the RAW264.7 cells treated with choline chloride were infected with HSV-1 and VSV, respectively, and the PI3K signal was detected by protein immunoblotting, as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate them into 12-well plates. Add 1 ml of 10% complete medium to each well. The initial cell confluence is about 30%-40%. Shake evenly and place in an incubator at 37°C and 5% carbon dioxide concentration for subculture. (2) Cell treatment: After the RAW264.7 cells in step (1) adhere to the wall, treat the cells with 1200 μM choline chloride (the stock solution is 1200 mM). At the same time, add an equal volume of enzyme-free sterile water to the cells in the control group. Shake evenly and place in an incubator at 37°C and 5% carbon dioxide concentration; (3) Virus inoculation: After the RAW264.7 cells were treated with choline chloride and enzyme-free sterile water for 48 h, the serum-containing culture medium was discarded, the cells were washed twice with PBS, 1 ml of serum-free culture 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 culture medium containing 4% serum was added to each well, and the cells were then placed in an incubator at 37°C and 5% carbon dioxide for 10 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, and place the well plate on a shaker for 10 seconds every 5 minutes. This process is performed on ice throughout. The PMSF and phosphatase inhibitors in the lysis buffer are added to the RIPA (medium) lysis buffer in a ratio of 1:1000 in advance (prepared before use). After lysis, transfer the protein solution to a labeled Eppendorf tube, place it in a 4°C centrifuge, 15,000 g, 5 minutes, transfer the obtained supernatant to a new Eppendorf tube, and discard the precipitate; (5) Protein immunoblotting to detect the expression of two viral proteins: the method used was the same as that in Example 1; (6) Experimental results are as follows Figure 4 As shown in G and H, RAW264.7 cells treated with enzyme-free sterile water and choline chloride for 60 hours were infected with the two viruses, and the proteins were collected 10 hours later for protein immunoblotting to detect the expression of viral proteins. The results showed that choline chloride treatment activated the activation of PI3K signaling under the stimulation of DNA virus HSV-1 and RNA virus VSV.

[0041] Example 5 The RAW264.7 cells as the control group and the RAW264.7 cells treated with choline chloride were infected with HSV-1 and VSV, respectively, and the AKT signal was detected by protein immunoblotting as follows: (1) Prepare cells: Take well-growing RAW264.7 cells and inoculate them into 12-well plates. Add 1 ml of 10% complete medium to each well. The initial cell confluence is about 30%-40%. Shake evenly and place in an incubator at 37°C and 5% carbon dioxide concentration for subculture. (2) Cell treatment: After the RAW264.7 cells in step (1) adhere to the wall, treat the cells with 1200 μM choline chloride (the stock solution is 1200 mM). At the same time, add an equal volume of enzyme-free sterile water to the cells in the control group. After shaking evenly, place the cells in an incubator at 37°C and 5% carbon dioxide concentration. (3) Virus inoculation: After the RAW264.7 cells were treated with choline chloride and enzyme-free sterile water for 48 h, the serum-containing culture medium was discarded, the cells were washed twice with PBS, 1 ml of serum-free culture 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 culture medium containing 4% serum was added to each well, and the cells were then placed in an incubator at 37 ° C and 5% carbon dioxide for 10 h and 5 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, and place the well plate on an oscillator for 10 seconds every 5 minutes. This process is performed on ice throughout. The PMSF and phosphatase inhibitors in the lysis buffer are added to the RIPA (medium) lysis buffer in a ratio of 1:1000 in advance (prepared immediately before use). After lysis, transfer the protein solution to a labeled Eppendorf tube, place it in a 4°C centrifuge, 15,000 g, 5 minutes, transfer the obtained supernatant to a new Eppendorf tube, and discard the precipitate. (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 5 As shown in I and J, RAW264.7 cells treated with enzyme-free sterile water and choline chloride for 48 hours were infected with the two viruses, and the proteins were collected 10 and 5 hours later for protein immunoblotting to detect the expression of viral proteins. The results showed that choline chloride treatment activated the activation of AKT signaling under the stimulation of DNA virus HSV-1 and RNA virus VSV.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. The use of choline in the preparation of drugs for preventing and / or treating broad-spectrum viral infections.

2. The use of choline according to claim 1 in the preparation of a drug for preventing or / and 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 choline according to claim 1 in the preparation of a drug for preventing or / and treating a broad spectrum of viral infections, characterized in that The drug for preventing and / or treating broad-spectrum viral infections comprises at least one of plants containing choline, plant extracts containing choline, effective plant parts containing choline, extracted and purified choline and chemically synthesized choline.

4. The use of choline 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 medicine for preventing and / or treating broad-spectrum viral infection comprises an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is a plant extract of choline and / or a pharmaceutically acceptable salt thereof.

5. The use of choline according to claim 1 in the preparation of a drug for preventing or / and 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. The use of choline 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 comprises choline and / or a pharmaceutically acceptable salt thereof.

7. The use of choline 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 functions to inhibit the replication and proliferation of DNA-model virus HSV-1 and RNA-model virus VSV.

8. The use of choline 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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