CN113197910B

CN113197910B - Antiviral medicine box for improving antiviral activity of interferon based on aspirin and metabolite thereof

Info

Publication number: CN113197910B
Application number: CN202110528036.7A
Authority: CN
Inventors: 郑慧; 苗迎; 袁玉康
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2022-09-23
Anticipated expiration: 2041-05-14
Also published as: CN113197910A

Abstract

The invention relates to the technical field of medicines and discloses an antiviral medicine box for improving the antiviral activity of Interferon (IFN) based on aspirin and a metabolite thereof. The interferon type I is a main medicament for clinically treating various major infectious virus infections such as HBV and the like. The invention proves that sodium salicylate promotes IFN-mediated antiviral ability by promoting IFN-induced Interferon Stimulating Genes (ISGs) expression through cytology experiments and animal in vivo experiments, thereby obviously inhibiting infection of various RNA viruses and DNA viruses. The aspirin and the salicylate product thereof can be used for treating the infection of various viruses such as clinical HBV and the like by combining with IFN, and the broad-spectrum antiviral curative effect of the clinical IFN is improved.

Description

Antiviral medicine box for improving antiviral activity of interferon based on aspirin and metabolite thereof

Technical Field

The invention relates to the technical field of medicines, in particular to an antiviral medicine box for improving antiviral activity of interferon based on aspirin and metabolites thereof.

Background

Viruses are common pathogens in clinic, and have been threatening the life and health of human beings in recent years, even endangering life. Due to the diversity of virus types and high-frequency mutation of viruses, the research and development of targeted drugs are difficult, and particularly, specific drugs which can be selected are very limited aiming at the threat of sudden viruses, so that the improvement of the curative effect of the existing broad-spectrum antiviral drugs has important clinical significance.

The broad spectrum antiviral drugs in clinical use today are mainly Interferons (Interferons, IFNs). The interferon is a high-activity multifunctional glycoprotein produced by biological cells and is induced by an interferon inducer. In 1957, when Isaacs and Lindenmann et al studied the phenomenon of influenza interference using chicken embryo hair follicles, it was discovered that cells produced a protein that rendered uninfected cells resistant to subsequently invading viruses, which interfered with the replication of the infecting virus and were therefore designated interferon. Binding of interferon to different receptors activates different signaling pathways and performs multiple biological functions. Among them are the Janus kinase family and the Signal Transducer and Activator of Transcription (STAT) family pathways, i.e., the JAK-STAT pathway. Once interferon binds to its receptor, it induces tyrosine phosphorylation of the JAK (JAK1/Tyk2) family, which then activates the STATs protein. Upon activation, STATS proteins form complexes with IRF9 and enter the nucleus, bind to regulatory elements ISRE, induce transcription and expression of ISGs (IFN-stimulated genes), and finally perform a series of biological functions by these ISGs. Interferons play an important role in the innate immune process of the body, and particularly Type I Interferons (IFN-I) have been regarded by researchers for their broad spectrum antiviral function. However, because the cost is high and the clinical antiviral efficacy is not good enough, the use of the IFN-I is greatly limited, so how to improve the treatment efficiency of the IFN-I becomes a problem to be solved urgently in clinic.

Disclosure of Invention

The present invention aims to provide a new antiviral strategy, using aspirin or its product salicylate to improve the antiviral activity of clinical interferon. In order to solve the technical problems, the invention provides an application of anti-inflammatory drugs (aspirin or salicylate products thereof) in clinical adjuvant interferon antiviral treatment based on a drug development strategy of new old drugs and combined drugs.

It is a first object of the present invention to provide an antiviral kit comprising: a preparation containing aspirin or a metabolite thereof; an interferon-containing preparation.

Further, the interferon is a type I interferon.

Further, the preparation containing the aspirin or the product salicylate thereof also comprises a medicinal excipient, a carrier or a diluent.

Further, the preparation containing aspirin or the metabolite thereof is an injection or a powder injection.

Further, the interferon-containing preparation also comprises a medicinal excipient, a carrier or a diluent.

Furthermore, the preparation containing the interferon is an injection or a powder injection.

Further, the preparation containing aspirin or a metabolite thereof promotes expression of an interferon inducible gene.

Further, the preparation containing aspirin or a metabolite thereof promotes expression of an interferon-induced gene in an immune cell, a fibroblast cell, or an epithelial cell.

Further, the interferon inducible genes comprise IFIT1, Viperin, ISG15 and ISG 54.

The invention proves the improvement of the antiviral activity of the sodium salicylate to the interferon through a large number of experiments.

First, it was investigated whether sodium salicylate could inhibit viral infection. The results show that at the cellular level, the sodium salicylate can obviously inhibit the infection of the virus, including the RNA level, the viral protein level and the amount of the cell-infected virus of various RNA viruses and DNA viruses; in animal experiments, the sodium salicylate can obviously inhibit the infection amount of viruses in each organ in a mouse body.

Secondly, it was investigated whether sodium salicylate could enhance interferon-induced antiviral effects to achieve antiviral efficacy. The results show that sodium salicylate does not increase interferon production, but significantly promotes IFN-mediated antiviral function.

And finally, whether the sodium salicylate promotes an IFN-I mediated signal channel is analyzed, and the antiviral activity of the interferon can be further improved by the sodium salicylate. The results show that sodium salicylate can significantly promote IFN-I signaling pathway, including up-regulation of the expression of multiple interferon inducible genes (ISGs) under IFN-I stimulation, by treating cells with sodium salicylate in combination with IFN-I.

Compared with the prior art, the technical scheme of the invention has the following advantages:

at present, broad-spectrum antiviral drugs are lacked for treating viral infection, and the clinically commonly used broad-spectrum antiviral drugs are mainly interferon, but IFN-I has high manufacturing cost and limited treatment effect, so that a plurality of limitations exist in the using process. The invention discloses a drug development strategy based on new old drugs and combined drugs, and explains that aspirin and a metabolite salicylate thereof can promote interferon-mediated antiviral ability by up-regulating expression of IFN-I induced ISGs, thereby providing a new strategy for the development of broad-spectrum antiviral drugs. The medicine (aspirin and its metabolite salicylate) is a common clinical medicine and has low toxicity. Aspirin and salicylate as a metabolite thereof are expected to become safer broad-spectrum antiviral drugs for improving the antiviral activity of clinical interferon.

Drawings

FIG. 1: a549 cell line is added with sodium salicylate for pretreatment, and respectively infected with H1N1, SeV, VSV and HSV viruses, and the RNA level of different viruses is detected by utilizing a real-time fluorescent quantitative PCR (real time qPCR) technology. N-4, mean ± SD, × p < 0.001.

FIG. 2: and (3) respectively taking primary cells of different tissues of the mouse, adding sodium salicylate, infecting VSV (VSV), and detecting the RNA levels of different viruses by using a Real time qPCR (quantitative polymerase chain reaction) technology. N-3, mean ± SD, <0.05, <0.01, < 0.001.

FIG. 3: a549, HCT116 and HepG2 cell lines are respectively added with different doses of sodium salicylate for treatment and infected with viruses, and the RNA levels of different viruses are detected by using Real time qPCR technology. N-4, mean ± SD, × p < 0.001.

FIG. 4: cells were treated with sodium salicylate and then infected with VSV virus or H1N1 virus, and viral proteins VSV-G and H1N1-HA were detected by Western Blot technique.

FIG. 5: HT1080 and HCT116 were treated with sodium salicylate and infected with VSV virus (with green fluorescent protein GFP tag), and changes in the amount of cells infected with VSV-GFP virus after the treatment with sodium salicylate were observed with a fluorescence inverted microscope.

FIG. 6: HT1080 cells were treated with different doses of sodium salicylate, and interferon production was stimulated with SeV, and the effect of sodium salicylate on interferon production was examined using the Real time qPCR technique.

FIG. 7: after treating Raw264.7 cells with sodium salicylate, the cells were stimulated with different doses of murine IFN β (mIFN β) and infected with H1N1 virus, and the sodium salicylate-treated group and untreated group were tested for interferon-mediated antiviral capacity changes using Real time qPCR technology. N-4, mean ± SD, × p < 0.001.

FIG. 8: after treating 2fTGH cells with sodium salicylate, the cells were stimulated with IFN α and infected with VSV virus, and interferon-mediated changes in antiviral ability between the sodium salicylate-treated and untreated groups were detected using Real time qPCR technology. N-4, mean ± SD, × p < 0.001.

FIG. 9: after treatment with sodium salicylate, different cell lines were stimulated with IFN and mRNA levels of representative interferon inducible genes (ISGs) were detected by Real time qPCR technique. N-4, mean ± SD, <0.05, <0.01, < 0.001.

FIG. 10: protein levels of representative interferon inducible genes (ISGs) were measured by Western Blot using different doses of sodium salicylate treatment and IFN addition to stimulate different cell lines.

FIG. 11: infecting a mouse by using the virus, injecting sodium salicylate into an abdominal cavity, and detecting the RNA level of the virus in each organ of the mouse by using a Real time qPCR technology. N-5, mean ± SD, <0.05, <0.01, < 0.001.

FIG. 12: sodium salicylate is used for intraperitoneal injection of mice, then interferon is used for intraperitoneal injection, and the Real time qPCR technology is used for detecting the mRNA level of interferon-induced genes (ISGs) in mouse organs. N-4, mean ± SD, [ p ] 0.01.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

Unless otherwise specified, the reagents according to the examples of the present invention are all commercially available products, and all of them are commercially available.

Aspirin is a mild antipyretic analgesic drug, has strong anti-inflammatory, antirheumatic and antiplatelet aggregation effects, and is one of the most common clinical drugs in the world. Aspirin is absorbed in the stomach and small intestine following administration and is hydrolyzed in the gastric mucosa, plasma and liver, and finally functions as a salicylate. Therefore, the invention uses Sodium Salicylate (SA) as an effective component to carry out cytology and zoology experiments. Both aspirin and sodium salicylate are non-steroidal anti-inflammatory drugs (NSAIDs).

Example 1: sodium salicylate significantly inhibits replication of multiple RNA and DNA viruses in cells

To investigate whether sodium salicylate could inhibit viral infection, A549 cells were plated on 12-well cell culture plates (approximately 0.5X 10) ⁶ Per well), pretreated for 12H with sodium salicylate (600 μ g/ml), infected with H1N1, SeV, VSV, HSV (MOI ═ 1) virus, respectively, and incubated for 20H at 37 ℃ in an incubator. Cells were lysed using Trizol, after which total RNA in the cells was extracted and reverse transcribed into cDNA, and RNA levels of different viruses were detected using fluorescent quantitative PCR (Real-time qPCR) technology. The results obtained are shown in FIG. 1.

Wild type C57BL/6 mice (6-8 weeks) were treated with lung, spleen, heart, kidney and liver tissue, ground to obtain primary cells, plated in 12-well cell culture plates, pretreated with sodium salicylate (600. mu.g/ml) for 12 hours, infected with VSV (MOI ═ 1) virus, and incubated at 37 ℃ in an incubator for 20 hours. Cells were lysed using Trizol, after which total RNA in the cells was extracted and reverse transcribed into cDNA, and RNA levels of VSV virus were detected using the fluorescent quantitative PCR (Real-time qPCR) technique. The results are shown in FIG. 2.

A549, HCT116, HepG2 cells were plated on 12-well cell culture plates (about 0.5X 10) ⁶ Per well), pre-treated for 12H with different doses of sodium salicylate (100, 200, 300 μ g/mL) added and infected with VSV or H1N1 virus, incubated at 37 ℃ for 20H in an incubator. Cells were lysed using Trizol, after which total RNA in the cells was extracted and reverse transcribed into cDNA, and the RNA level of the detected virus was detected using the fluorescent quantitative PCR (Real-time qPCR) technique. The results are shown in FIG. 3.

A549 and MEF cells were plated on 12-well cell culture plates (about 0.5X 10) ⁶ Per well), pre-treated for 12h with different doses of sodium salicylate added, infected with VSV virus, and incubated for 24h at 37 ℃ in an incubator. Lysing the cells with NP-40 lysate, preparing a protein sample, and electrophoresing the protein sample with SDS-PAGEThen transferring to PVDF membrane, sealing with 5% skimmed milk powder at room temperature for 1h, adding primary antibody for incubation at 4 ℃ overnight, washing PBST, adding secondary antibody for incubation at room temperature for 1h, washing PBST, adding luminescent substrate for exposure, development and fixation. The results are shown in FIG. 4.

HT1080 and HCT116 cells were plated on 12-well cell culture plates (approximately 0.5X 10) ⁶ Per well), pre-treated with different doses of sodium salicylate for 12h, infected with VSV-GFP virus (with green fluorescent protein GFP tag), cells infected with VSV-GFP virus were observed with a fluorescence inverted microscope, and the change in the number of cells infected with virus after sodium salicylate treatment was analyzed. The results are shown in FIG. 5.

The results of fig. 1-5 show that sodium salicylate can significantly inhibit replication of various RNA viruses and DNA viruses in cells, including down-regulating viral RNA levels, viral protein levels, and the amount of cells infected with viruses.

Example 2: sodium salicylate promotes IFN-I mediated antiviral ability

To analyze whether the broad-spectrum antiviral potency of sodium salicylate was through the modulation of IFN-I mediated antiviral signaling, HT1080 cells were plated onto 12-well cell culture plates (approximately 0.5X 10) ⁶ Per well), cells were treated with different doses of sodium salicylate (100, 300, 600 μ g/mL) and infected with sendai virus SeV (MOI ═ 1) to stimulate interferon production. Cells were lysed using Trizol, total RNA was then extracted from the cells and reverse transcribed into cDNA, and IFN β mRNA levels were detected using fluorescent quantitative PCR (Real-time qPCR) technology. The results are shown in FIG. 6.

Raw264.7 cells were plated on 12-well cell culture plates (about 0.5X 10) ⁶ Per well), cells were treated with different doses of sodium salicylate (100, 300, 600 μ g/mL) and infected with H1N1(MOI ═ 1). The cells were lysed using Trizol, after which the total RNA in the cells was extracted and reverse transcribed into cDNA and the RNA level of the H1N1 virus was detected using the fluorescent quantitative PCR (Real-time qPCR) technique. The results are shown in FIG. 7.

2fTGH cells were plated on 12-well cell culture plates (approx. 0.5X 10) ⁶ Per well), cells were treated with sodium salicylate (600 μ g/mL) and infected with VSV (MOI ═ 1).Cells were lysed using Trizol, after which total RNA in the cells was extracted and reverse transcribed into cDNA, and RNA levels of VSV virus were detected using the fluorescent quantitative PCR (Real-time qPCR) technique. The results are shown in FIG. 8.

The results in fig. 6 to 8 show that sodium salicylate does not up-regulate interferon production, but significantly promotes the antiviral function of interferon, suggesting that sodium salicylate may function by promoting interferon signaling pathway.

Example 3: sodium salicylate increases expression of interferon inducible genes (ISGs)

To further analyze whether sodium salicylate up-regulates IFN-I mediated signaling pathways, cells were plated on 12-well cell culture plates (approximately 0.5X 10) ⁶ /well), treated with sodium salicylate, and then cells stimulated with IFN α. Cells were lysed using Trizol, after which total RNA in the cells was extracted and reverse transcribed into cDNA, and mRNA levels of representative ISGs genes were detected using the fluorescent quantitative PCR (Real-time qPCR) technique. The experiment was verified on immune cells (THP1, U937), fibroblasts (HT1080), epithelial cells (HeLa, a549, 293T). The results are shown in FIG. 9.

Raw264.7 cells were plated on 12-well cell culture plates (about 0.5X 10) ⁶ Per well), cells were treated with different doses of sodium salicylate (100, 300, 600 μ g/mL) and then stimulated with mfnp for 24 h. Cell is cracked by NP-40 lysate, protein sample is prepared, after SDS-PAGE electrophoresis, the protein sample is transferred to PVDF membrane, 5% skimmed milk powder is sealed for 1h at room temperature, primary antibody is added for incubation overnight at 4 ℃, after PBST is washed, secondary antibody is added for incubation for 1h at room temperature, after PBST is washed, luminescent substrate is added for exposure, development and fixation. Experiments in which sodium salicylate promoted protein levels of interferon-induced ISGs were verified in THP1, U937, 2 fTGH. The results are shown in FIG. 10.

Example 4: sodium salicylate is effective in reducing viral infection in mice

The C57BL/6 wild-type mice used in this example were purchased from the Suzhou university animal center and housed in the Suzhou university SPF environment.

To analyze the antiviral function of sodium salicylate in animals, infection was performed in miceVSV(1x10 ⁸ PFU) virus for 12h, then injecting sodium salicylate into the abdominal cavity, and respectively taking heart, liver, spleen, lung and kidney tissues of the mouse for grinding after 24 h. Trizol is used for cracking, then total RNA of each tissue is extracted and is reversely transcribed into cDNA, and the RNA level of VSV virus of each organ of the mouse is detected by utilizing a fluorescent quantitative PCR (Real-time qPCR) technology. The results obtained are shown in FIG. 11.

In order to analyze whether the sodium salicylate can up-regulate interferon-induced ISGs in animals, the sodium salicylate is intraperitoneally injected into mice for 12h, then IFN is intraperitoneally injected, and lung tissues of the mice are taken for grinding. The interferon-induced mRNA level of IFIT1 in mouse lung tissue was detected using Trizol lysis followed by extraction and reverse transcription of lung tissue total RNA into cDNA using the fluorescent quantitative PCR (Real-time qPCR) technique. The results are shown in FIG. 12.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. Use of a kit for preparing a medicament for increasing the antiviral activity of interferon-type i, said kit comprising: a preparation containing aspirin or a metabolite thereof; an interferon-containing formulation; the metabolite of the aspirin is sodium salicylate; the interferon is a type I interferon.

2. Use of a kit according to claim 1 in the manufacture of a medicament for increasing the antiviral activity of type one interferon, wherein said formulation comprising aspirin or a metabolite thereof further comprises a pharmaceutically acceptable pharmaceutical excipient.

3. Use of a kit according to claim 2 in the manufacture of a medicament for increasing the antiviral activity of type one interferon, wherein said formulation comprising aspirin or a metabolite thereof is an injection.

4. Use of a kit according to claim 1 for the manufacture of a medicament for increasing the antiviral activity of a type one interferon, wherein said interferon-containing formulation further comprises a pharmaceutically acceptable pharmaceutical excipient.

5. Use of a kit according to claim 4 for the manufacture of a medicament for increasing the antiviral activity of type I interferon, wherein said interferon-containing formulation is an injection.