Application of methotrexate in medicine for inhibiting adenovirus

Abstract

The invention provides an application of methotrexate in a medicine for inhibiting adenovirus, relates to the technical field of medicines, and finds that nucleic acid synthesis of cells is blocked by inhibiting dihydrofolate reductase (DHFR) through antiviral experimental research on methotrexate, so that cell proliferation is inhibited. The methotrexate can significantly reduce virus replication and infection, and has dose dependence and a significant anti-adenovirus effect. The invention provides a new application of methotrexate in preparation of an anti-adenovirus product. The technical problem that in the prior art, a substance with a good inhibition effect on adenovirus is lacked is solved. The method is expected to become an important breakthrough in the field of anti-adenovirus treatment in the future.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of methotrexate in a drug for inhibiting adenovirus. Background Art

[0002] Adenoviruses are a class of widespread DNA viruses that can infect a variety of host cells, including humans, other mammals, and birds. In humans, adenovirus infections can cause a variety of diseases, ranging from mild respiratory infections to life-threatening systemic diseases. The incidence and severity of adenovirus infections are particularly high in children and patients with compromised immune systems. Adenovirus infections are not limited to specific regions or seasons and have been reported worldwide, with infection rates increasing.

[0003] Currently, the treatment options for adenovirus infection are relatively limited. Although vaccines are available to prevent certain types of adenovirus infection, their variety and coverage are far from enough. Antiviral drugs such as ribavirin and cidofovir are effective in treating adenovirus infection, but long-term use of antiviral drugs may lead to drug resistance in the virus and may also cause adverse reactions such as kidney toxicity and bone marrow suppression. Currently, no anti-adenovirus drugs have been approved clinically, and adenovirus infection treatment still uses symptomatic treatment. The development of adenovirus drugs is of great urgency.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide an application of methotrexate or a pharmaceutically acceptable salt thereof or a substance with methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting adenovirus, so as to solve the technical problem in the prior art of lacking a substance having a good inhibitory effect on adenovirus.

[0006] A second object of the present invention is to provide a use of methotrexate or a pharmaceutically acceptable salt thereof, or a substance containing methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by adenovirus infection.

[0007] The third object of the present invention is to provide a use of methotrexate or a pharmaceutically acceptable salt thereof, or a substance with methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product capable of improving symptoms caused by adenovirus infection.

[0008] A fourth object of the present invention is to provide a product.

[0009] In a first aspect, the present invention provides a use of methotrexate or a pharmaceutically acceptable salt thereof, or a substance having methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting adenovirus.

[0010] Furthermore, the inhibiting of adenovirus includes inhibiting adenovirus replication.

[0011] Furthermore, the inhibition of adenovirus comprises exerting its effect through at least one of the following pathways:

[0012] A1, inhibiting the activation of NF-κB through dihydrofolate reductase;

[0013] A2, down-regulating the expression of p65 and pp65;

[0014] C3, promotes the expression of antiviral molecules oas1, oas3 and isg15.

[0015] In a second aspect, the present invention provides a use of methotrexate or a pharmaceutically acceptable salt thereof, or a substance having methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by adenovirus infection.

[0016] In a third aspect, the present invention provides a use of methotrexate or a pharmaceutically acceptable salt thereof, or a substance having methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product capable of improving symptoms caused by adenovirus infection.

[0017] Furthermore, the adenovirus includes adenovirus type 3 or adenovirus type 7.

[0018] Furthermore, the product includes a medicine.

[0019] In a fourth aspect, the present invention provides a product, the active ingredient of which is methotrexate or a pharmaceutically acceptable salt thereof; the product has any of the following uses:

[0020] B1, inhibit adenovirus;

[0021] B2. Treatment and / or prevention of diseases caused by adenovirus infection;

[0022] B3. Improve symptoms caused by adenovirus infection.

[0023] Furthermore, the product includes a medicine.

[0024] Furthermore, the dosage form of the drug includes capsule suspension, tablet, pill, injection or emulsion;

[0025] Preferably, the dosage of the drug is once a day, 0.5-5 mg / kg body weight each time;

[0026] Preferably, the administration of the drug includes oral administration or intravenous injection.

[0027] The use of methotrexate provided by the present invention in a drug for inhibiting adenovirus, through antiviral experimental studies on methotrexate, it is found that the nucleic acid synthesis of cells is blocked by inhibiting dihydrofolate reductase (DHFR), thereby inhibiting cell proliferation. Methotrexate can significantly reduce viral replication and infection, and is dose-dependent, and has a significant anti-adenovirus effect. A new use of methotrexate in the preparation of anti-adenovirus products is provided. The technical problem of the lack of a substance with a good inhibitory effect on adenovirus in the prior art is solved. It is expected to become an important breakthrough in the field of anti-adenovirus treatment in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The toxicity diagram of different concentrations of methotrexate on A549 cells and AD293 cells provided in Example 1 of the present invention;

[0030] Figure 2 This is a graph showing the effect of different concentrations of methotrexate on anti-adenovirus type 7 in A549 cells provided in Example 2 of the present invention;

[0031] Figure 3 This is a graph showing the anti-adenovirus effect of different concentrations of methotrexate in AD293 cells provided in Example 2 of the present invention;

[0032] Figure 4 A statistical graph showing the half effective viral inhibition concentration of methotrexate in A549 cells and AD293 cells provided in Example 2 of the present invention;

[0033] Figure 5 This is a diagram showing the inhibitory effect of methotrexate on adenovirus nucleic acid in A549 cells provided in Example 2 of the present invention;

[0034] Figure 6 This is a graph showing the anti-adenovirus effect of different concentrations of methotrexate in MRC-5 cells provided in Example 3 of the present invention;

[0035] Figure 7 A statistical graph showing the half effective viral inhibition concentration of methotrexate in MRC-5 cells provided in Example 3 of the present invention;

[0036] Figure 8The expression statistics of DHFR in A549 cells and MRC-5 cells provided in Example 3 of the present invention;

[0037] Fig. 9 The tertiary structure docking diagram of methotrexate and DHFR provided in Example 3 of the present invention;

[0038] Fig.10 This is a graph showing the effect of methotrexate on adenovirus under treatment with different concentrations of folic acid provided in Example 4 of the present invention;

[0039] Fig.11 A statistical graph of TRAF2 expression in cells treated with different concentrations of methotrexate provided in Example 4 of the present invention;

[0040] Fig.12 A statistical graph showing the expression of antiviral factors in cells treated with different concentrations of methotrexate provided in Example 4 of the present invention;

[0041] Fig.13 This is a graph showing the effects of different concentrations of methotrexate on protein expression provided in Example 4 of the present invention;

[0042] Fig.14 This is a graph showing the effects of different concentrations of methotrexate on cell gene levels provided in Example 5 of the present invention;

[0043] Fig.15 This is a statistical graph of cell gene expression levels under different treatments provided in Example 5 of the present invention.

[0044] Fig.16 This is a graph showing the effect of different concentrations of methotrexate on anti-type 3 adenovirus in A549 cells provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0045] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0046] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0047] In one aspect, the present invention provides the use of methotrexate or a pharmaceutically acceptable salt thereof or a substance having methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting adenovirus.

[0048] Methotrexate, chemically known as 4-amino-10-methylfolate pterin, is a known antimetabolite drug, commonly used to treat cancer, severe psoriasis and rheumatoid arthritis. The present invention, through antiviral experimental research on methotrexate, found that it inhibits cell proliferation by blocking the nucleic acid synthesis of cells by inhibiting dihydrofolate reductase (DHFR). Methotrexate can significantly reduce viral replication and infection, and is dose-dependent, with significant anti-adenovirus effect. A new use of methotrexate in the preparation of anti-adenovirus products is provided. The technical problem of the lack of substances with good inhibitory effects on adenoviruses in the prior art is solved. It is expected to become an important breakthrough in the field of anti-adenovirus treatment in the future.

[0049] In some specific embodiments, said inhibiting adenovirus comprises inhibiting adenovirus replication.

[0050] The antiviral mechanism of methotrexate involves multiple levels. On the one hand, methotrexate blocks the nucleic acid synthesis pathway required for viral replication by inhibiting dihydrofolate reductase (DHFR). On the other hand, methotrexate can inhibit the activation of NF-κB, which is a key intracellular transcription factor involved in regulating inflammation and immune responses. By downregulating the expression of p65 and pp65, methotrexate reduces the host cell factors required for viral replication. On the one hand, methotrexate can also promote the expression of antiviral molecules oas1, oas3 and isg15. Oas1, oas3 and isg15 molecules are components of the natural immune response of host cells and can directly inhibit viral replication. In some specific embodiments, the inhibition of adenovirus includes exerting its effect through at least one of the above pathways.

[0051] According to another aspect of the present invention, there is also provided the use of methotrexate or a pharmaceutically acceptable salt thereof or a substance having methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by adenovirus infection.

[0052] The antiviral effect of methotrexate is not limited to reducing viral load, but also includes reducing cell damage and inflammatory response caused by viral infection, and has dual anti-adenovirus and anti-inflammatory effects. Methotrexate can act on diseases caused by adenovirus infection and play a positive role in treatment or prevention. A new use of methotrexate in the treatment and / or prevention of diseases caused by adenovirus infection is provided.

[0053] Among them, diseases caused by adenovirus infection include respiratory tract infection, conjunctivitis, gastroenteritis, urinary tract infection, nervous system disease, skin infection, hepatitis or myocarditis. Respiratory tract infection can be bronchitis or pneumonia.

[0054] According to another aspect of the present invention, there is also provided the use of methotrexate or a pharmaceutically acceptable salt thereof or a substance having methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product capable of improving symptoms caused by adenovirus infection.

[0055] Among them, the symptoms caused by adenovirus infection include symptoms caused by diseases caused by adenovirus infection, such as coughing, sneezing, rash and fever caused by respiratory tract infections; redness and swelling of the eyes and increased secretions caused by conjunctivitis; diarrhea, nausea or vomiting caused by gastroenteritis; and rash symptoms caused by skin infections.

[0056] In some specific embodiments, the adenovirus comprises adenovirus type 3 or adenovirus type 7.

[0057] In some specific embodiments, the product comprises a drug.

[0058] Methotrexate has very low toxicity to most cells and has good safety. Methotrexate has been approved for clinical use by the Food and Drug Administration. The present invention has discovered a new use for methotrexate and added a new therapeutic area for methotrexate. A new treatment regimen is provided for adenovirus. Through its unique mechanism of action, it can effectively inhibit the replication and infection of adenovirus, while reducing the inflammatory response caused by viral infection, providing a new option for the treatment of adenovirus infection.

[0059] According to another aspect of the present invention, there is also provided a product, the active ingredient of which is methotrexate or a pharmaceutically acceptable salt thereof; the product has any of the following uses:

[0060] B1, inhibit adenovirus;

[0061] B2. Treatment and / or prevention of diseases caused by adenovirus infection;

[0062] B3. Improve symptoms caused by adenovirus infection.

[0063] In some specific embodiments, the product comprises a drug.

[0064] In some specific embodiments, the dosage form of the drug includes capsule suspension, tablet, pill, injection or emulsion.

[0065] In some specific embodiments, the dosage of the drug is once a day, 0.5-5 mg / kg body weight each time. The specific dosage needs to be adjusted according to the specific conditions of the patient.

[0066] In some specific embodiments, the administration of the drug includes oral administration or intravenous injection.

[0067] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] Adenovirus type 3 strain and adenovirus type 7 strain were both from the National Institute of Respiratory Diseases, the First Affiliated Hospital of Guangzhou Medical University.

[0069] Methotrexate (MTX): purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number WKQ-0002591-500mg.

[0070] Example 1 Toxic effects of methotrexate on A549 cells and AD293 cells

[0071] A good antiviral effect of methotrexate was observed in A549 cells and AD293 cells. In order to further explore the antiviral effect of the drug, the cytotoxicity of methotrexate was tested on different cells.

[0072] A549 and AD293 cells were cultured at 7×10 4 Cells were seeded into 96-well plates at a density of 10 cells / ml (100 μL) and incubated at 37 , 5% CO2 incubator for 12 hours. Wait until the cell density reaches 80%, take out the MTX mother solution, and dissolve it in the dark at room temperature. Use virus maintenance solution to dilute MTX to a concentration gradient (50μM, 100μM, 200μM, 400μM, 800μM, 1600μM and 3200μM). Aspirate the original culture medium and add different concentrations of MTX to a 96-well plate. Add virus maintenance solution (DMEM containing 1% FBS) to the control group, and set up more than 6 replicate wells for each concentration treatment. Each cell was cultured with the corresponding concentration of MTX solution for 48 hours. Observe under a microscope and take pictures to record the toxic effects of the drug on the cells. Specifically, Figure 1 As shown, A is A549 cells, B is AD293 cells, and Mock is the control group. The cytotoxic effects of methotrexate on A549 cells and AD293 cells are manifested as: cell breakage and shedding, increase in cytoplasmic particles, changes in cell morphology, and enlargement of cell volume.

[0073] Combination Figure 1 It can be seen that the half-toxic concentration of methotrexate for A549 cells and AD293 cells is usually above 800 μM. The concentration range of methotrexate within 800 μM is a safe and non-toxic parameter range for subsequent antiviral studies.

[0074] Example 2 Antiviral effect of methotrexate at the cellular level

[0075] A549 and AD293 cells were collected at 1×10 5 Cells / ml (100 μL) were inoculated into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 12 hours. Wait for the cell density to reach 80%. Take out the mother solution of MTX and dissolve it in a dark place at room temperature. Use the virus maintenance solution to dilute the MTX ratio to a concentration gradient (1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM and 50 μM), and mix it with the same volume of 200 TCID50 type 7 adenovirus to obtain 0.78125 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM methotrexate and 100 TCID50 type 7 adenovirus mixture as the treatment group. The culture medium in the 96-well plate was aspirated and 100 μL was added to each well of the mixture according to the concentration gradient. Virus maintenance solution (DMEM containing 1% FBS) was added to the control group, and 100 TCID50 adenovirus type 7 was added to the positive control / virus control. Six replicate wells were set for each action mode to avoid deviation between wells. , 5% CO2 incubator for 48 hours. Virus replication can be indicated by the fluorescence observed under a fluorescence microscope. Figure 2 and Figure 3 The fluorescence area and fluorescence intensity of the virus shown were significantly inhibited. By adding drugs and viruses at the same time, a very good antiviral effect was monitored. The fluorescence range was calculated using Image J software to represent the number of cells infected by the virus, and the fluorescence range data was analyzed using Graphpad Prism software to calculate the semi-effective viral inhibition concentration of MTX, as shown in the following figure: Figure 4 As shown, Figure 4 The left figure in the middle shows the half-effective viral inhibition concentration of MTX in A549 cells. Figure 4 The middle right figure shows the half-effective viral inhibition concentration of MTX in AD293 cells, showing that methotrexate at 0.9931 μM and 0.9725 μM can inhibit half of the adenovirus in A549 cells and AD293 cells, respectively;

[0076] The inhibition of methotrexate on viral nucleic acid was tested by PCR. A549 cells were cultured at 1×10 5Cells were inoculated at a density of 10 μM MTX / ml (10 mL) in a 10 cm dish, and the cells were treated with a mixture of 10 μM MTX and 200 TCID50 adenovirus type 7 (10 mL); control group: virus maintenance solution (DMEM containing 1% FBS) (10 mL); virus control group: 100 TCID50 adenovirus type 7 (10 mL) as a positive control; drug control group: 10 μM MTX (10 mL). The cells were cultured in an incubator at 37°C and 5% CO2 for 48 hours, and RNA was extracted using the traditional Trizol method and the BeyoFast TM RNA amplification was performed according to the instructions of the SYBR Green One-Step qRT-PCR Kit. The adenovirus type 7 front primer was 5'-GAGGAGCCAGATATTGATATGGAATT-3', and the adenovirus type 7 rear primer was 5'-AATTGACATTTTCCGTGTAAAGCA3-'.

[0077] Specific results such as Figure 5 As shown, use BeyoFast TM SYBR GreenOne-StepqRT-PCR kit was used to measure the viral titer of the virus control group and the 10μM MTX treatment group. Student's t test was used for statistical analysis (two-tailed distribution, heteroscedasticity). Error bars represent the standard error of the mean (SEM). **p≤0.01, ***p≤0.001, ***p≥0.0001. The PCR method tested that 10μM methotrexate treatment also had a very significant inhibitory effect on viral nucleic acid.

[0078] Example 3: No antiviral effect was observed in MRC-5 cells, based on their DHFR expression

[0079] According to 6×10 4 MRC-5 cells were inoculated into 96-well plates at a density of 10 cells / ml (100 μL) and cultured in a 37°C, 5% CO2 incubator for 12 hours. Wait until the cell density reaches 80%, then take out the MTX mother solution and prepare it with virus maintenance solution at concentrations of 200 μM and 100 μM, respectively. A mixture of MTX and 100 TCID50 type 7 adenovirus was added to the wells according to the concentration gradient as the treatment group. The control group used virus maintenance solution (DMEM containing 1% FBS), and the virus control group was 100 TCID50 type 7 adenovirus as a positive control. Each operating mode is equipped with 6 wells to avoid bias. Culture in an incubator at 37°C and 5% CO2 for 48 hours. The replication of the virus can be represented by the amount of fluorescence observed under a fluorescence microscope. Specifically, Figure 6As shown, Mock is the control group and ADV-7 is the virus control group, indicating that MTX does not have the expected antiviral effect on MRC-5 cells.

[0080] ImageJ software was used to calculate the fluorescence range to represent the number of cells infected with the virus, and GraphPadPrism software was used to analyze the fluorescence area data to calculate the half-effective viral inhibition concentration of MTX. Figure 7 As shown, the ideal inhibitory effect of MTX on viral replication in MRC-5 cells was not observed by calculating the fluorescence area.

[0081] MTX has been reported to be a competitive inhibitor of DHFR, so this example tested the expression of DHFR, the target of methotrexate, in these two cells. The mRNA was extracted from each group using the traditional Trizol method, and the mRNA level of DHFR in A549 cells and MRC-5 cells was measured, with β-actin as the internal reference gene. The specific results are shown in Figure 2. Figure 8 As shown, compared with A549 cells, the expression of DHFR in MRC-5 cells is very low. Figure 6 and Figure 7 This confirms that DHFR, as a target protein of methotrexate, plays an indispensable role in antiviral therapy. In a cell model lacking DHFR, methotrexate cannot exert its antiviral effect. Fig. 9 The tertiary structures of MTX and DHFR shown were successfully docked by Autodock.

[0082] Example 4 Verification that DHFR is a key protein for methotrexate to exert its antiviral effect

[0083] Dihydrofolate reductase (DHFR) reduces dihydrofolate (DHF) to tetrahydrofolate (THF). It is a key enzyme for the de novo synthesis of purine and thymidylate and plays an important role in cell growth. As the downstream of the DHFR metabolic pathway, the production of tetrahydrofolate and folic acid depends on the expression of DHFR. If DHFR is inhibited, the expression of folic acid will also decrease. MTX has been reported as a competitive inhibitor of DHFR to resist folic acid production. Therefore, in this embodiment, high concentrations of folic acid are added under the condition of adding methotrexate. A mixture of 80 μM, 160 μM, and 320 μM folic acid (Folic acid) and 10 μM MTX and 100 TCID50 type 7 adenovirus is obtained using virus maintenance liquid. The mixture is added to the cells according to the concentration gradient as the treatment group; the control group is added with virus maintenance liquid (DMEM containing 1% FBS), and the virus control is 100 TCID50 type 7 adenovirus as a positive control. Each operation mode is equipped with 6 wells to avoid deviation. At 37 , 5% CO2 incubator for 48 hours. Observe the virus fluorescence under a fluorescence microscope. Fig.10 As shown. It was found that high concentrations of folic acid can reverse the antiviral effect of methotrexate. High concentrations of folic acid can interrupt the antiviral effect of methotrexate, so the DHFR-folate pathway is the key pathway inhibited when methotrexate takes effect.

[0084] A549 cells were collected at 1×10 5 Cells were inoculated at a density of 100 cells / ml (10 ml) in a 10 cm culture dish. The treatment group: 10 ml MTX and virus mixture. Control group: virus maintenance solution (DMEM containing 1% FBS); virus control group: 100 TCID7 adenovirus, as a positive control; drug control group: 10 μM MTX concentration. Cultured in an incubator at 37°C and 5% CO2 for 48 hours, RNA was extracted using the traditional Trizol method, and the BeyoFast TM RNA amplification was performed according to the instructions of the SYBR Green One-Step qRT-PCR Kit. Fig.11 As shown, the treatment group (MTX+AdV7) upregulated the mRMA levels of antiviral factors such as TNF-α, IL-1β, and MMP9 at a high level.

[0085] A549 cells, 10 5 Cells / ml (10ml), placed in a 10cm culture dish, and cultured in a 37°C, 5% CO2 incubator for 12 hours. According to the treatment group: 200 TCID50 adenovirus type 7 mixed with 20μM drug solution. Control group: virus maintenance solution (DMEM containing 1% FBS), virus control group: 100 TCID50 adenovirus type 7, as a positive control; drug control group: 50μM MTX. Cultured in a 37°C, 5% CO2 incubator for 48 hours. Add 300μL of cell lysis solution (RIPA strength: PI:PMSF = 100:1:2) to each culture dish and break the ice for 10 minutes. Subsequently, centrifuge at 4 degrees Celsius for 30 minutes using a low-temperature high-speed centrifuge at 10000rpm. Take the supernatant as a protein sample and normalize the protein concentration according to the instructions of the BCA assay kit. Obtain protein blot samples.

[0086] like Fig.12As shown, Western Blot experiments observed upregulation of p53 and STAT proteins, and MTX inhibited NF-κB activation by releasing adenosine. The partial reversal of methotrexate-induced NF-κB inhibition by thymidine and folinic acid indicated the role of the thymidylate synthase pathway, and MTX inhibited NF-κB activation by releasing adenosine, contributing to the anti-inflammatory, immunomodulatory, and antiproliferative effects of MTX, while downregulation of p65 and pp65 was observed.

[0087] Example 5 Addition of methotrexate causes upregulation of antiviral factor mRNA levels

[0088] A549 cells were collected at 1×10 5 Cells were inoculated at a density of 1 μM / ml (10 ml) in a 10 cm culture dish, and a mixture of 2 μM, 20 μM and 100 μM MTX and 200 TCID50 adenovirus type 7 was added to the cells as the treatment group. Control group: virus maintenance solution (DMEM containing 1% FBS), virus control group: 100 TCID50 adenovirus type 7, as a positive control; drug control group: 1 μM, 10 μM and 50 μM MTX concentrations. Cultured in an incubator at 37°C and 5% CO2 for 48 hours, RNA was extracted using the traditional Trizol method, and the BeyoFast TM RNA amplification was performed using the SYBR Green One-Step q RT-PCR kit.

[0089] like Fig.13 Compared with the pure virus group, the TRAF2 in the treatment group showed a steady increase in mRMA with increasing concentration; Fig.14 As shown, since TRAF2 is also a downstream protein of DHFR, TRAF2 is also inhibited when methotrexate is added compared to the virus control group. TRAF2 front primer 5`-ACTGCAAGAGTCAGGTTCCG-3`, TRAF2 rear primer 5`-CTGGCTGTAAAGGGACAGGG-3`, adenovirus type 7 front primer 5`-GAGGAGCCAGATATTGATATGGAATT-3`, adenovirus type 7 rear primer 5`-AATTGACATTTTCCGTGTAAAGCA3-`. DHFR front primer 5`-ATGGTTCGTCCGCTGAACTG-3`, rear primer 5`-AGAGGACGCCTGGGTATTCT-3`. IFN-γ front primer 5`-CCTCTCTGGCTGTTACTGCC-3`, rear primer 5`-TCAGCACCGACTCCTTTTCC-3`. GAPDH front primer 5`-GAAGGTCGGTGTGAACGGAT-3`, rear primer 5`-AGTGATGGCATGGACTGTGG-3`. Fig.15 As shown, with the increase of MTX concentration, the up-regulation of related factors at the gene level gradually increased. Among them, the jak-stat pathway, as the downstream of il-6, almost all showed up-regulation of genes at the same level as il-6; compared with the related antiviral molecules oas1, oas3 and isg15, the expression levels in the treatment group gradually increased in a concentration-dependent manner.

[0090] Example 6 Antiviral effect of methotrexate against adenovirus type 3 at the cellular level.

[0091] A549 cells were collected at 1×10 5 Cells / ml (100 μL) were inoculated into 96-well plates and cultured in a 37°C, 5% CO2 incubator for 12 hours. Wait for the cell density to reach 80%. Take out the mother solution of MTX and dissolve it in a dark place at room temperature. Use the virus maintenance solution to dilute the MTX ratio to a concentration gradient (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM), and mix it with the same volume of 200 TCID50 type 3 adenovirus to obtain 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM methotrexate and 100 TCID50 type 3 adenovirus mixture as the treatment group. Aspirate the culture medium from the 96-well plate, add 100 μL to each well of the mixture according to the concentration gradient, add virus maintenance solution (DMEM containing 1% FBS) to the control group, and add 100 TCID50 type 3 adenovirus to the positive control / virus control. Each mode of action is set up with 6 replicate wells to avoid deviation between wells. Culture in an incubator at 37°C and 5% CO2 for 48 hours. Virus replication can be represented by the amount of fluorescence observed under a fluorescence microscope. The specific results are shown in Figure 2. Fig.16 The fluorescence area and fluorescence intensity of the virus shown were significantly inhibited, and a very good antiviral effect was monitored by adding drugs and viruses at the same time.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of methotrexate or a pharmaceutically acceptable salt thereof or a substance with methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for inhibiting adenovirus.

2. The use according to claim 1, characterized in that: The inhibiting of adenovirus includes inhibiting adenovirus replication.

3. The use according to claim 2, characterized in that: The inhibition of adenovirus comprises acting through at least one of the following pathways: A1, inhibiting the activation of NF-κB through dihydrofolate reductase; A2, down-regulating the expression of p65 and pp65; C3, promotes the expression of antiviral molecules oas1, oas3 and isg15.

4. Use of methotrexate or a pharmaceutically acceptable salt thereof or a substance containing methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating and / or preventing diseases caused by adenovirus infection.

5. Use of methotrexate or a pharmaceutically acceptable salt thereof or a substance with methotrexate or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product capable of improving symptoms caused by adenovirus infection.

6. The use according to any one of claims 1 to 5, characterized in that: The adenovirus includes adenovirus type 3 or adenovirus type 7.

7. The use according to claim 6, characterized in that: The products include pharmaceuticals.

8. A product, the active ingredient of which is methotrexate or a pharmaceutically acceptable salt thereof; the product has any of the following uses: B1, inhibit adenovirus; B2. Treatment and / or prevention of diseases caused by adenovirus infection; B3. Improve symptoms caused by adenovirus infection.

9. The product according to claim 8, characterized in that The products include pharmaceuticals.

10. The product according to claim 9, characterized in that The dosage form of the drug includes capsule suspension, tablet, pill, injection or emulsion; Preferably, the dosage of the drug is once a day, 0.5-5 mg / kg body weight each time; Preferably, the administration of the drug includes oral administration or intravenous injection.

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