Application of irisin in preparation of antiviral drugs

Abstract

The invention discloses application of irisin in preparation of antiviral drugs, and relates to the technical field of molecular biology. According to the application disclosed by the invention, through cytological experiments, it is found that Irisin promotes the IFN-mediated antiviral capacity by promoting expression of IFN-induced interferon stimulating genes (ISGs), so that infection of various RNA viruses and DNA viruses is remarkably inhibited. Meanwhile, the antiviral function of the irisin also depends on IFNAR1 phosphorylation, and the irisin is new ISG. The irisin disclosed by the invention can be used for treating infection of various viruses such as clinical H1N1 by being combined with IFN, and the broad-spectrum antiviral curative effect of the clinical IFN is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to the application of irisin in the preparation of antiviral drugs. Background Art

[0002] Viruses are common clinical pathogens that have been threatening human life and health, and even endangering life, in recent years. Due to the diversity of virus species and the high frequency of virus mutations, the development of targeted drugs is difficult, especially for the threat of emerging viruses, the specific drugs that can be selected are very limited, so the development of broad-spectrum antiviral drugs is of great clinical significance.

[0003] The IFN family is divided into three types based on its gene sequence, receptor specificity and chromosomal localization, namely type I (IFNα / β), type II (IFNγ) and type III (IFNλ) interferons. Currently, the IFN-I subtype is mainly used for clinical antiviral treatment, which has a targeted effect at any stage of viral replication (including entry, transcription, translation initiation, maturation, assembly and release). Interferon binding to different receptors can activate different signaling pathways and exert a variety of biological functions. Among them, the main ones are the Janus kinase family and the signal transducer and activator of transcription (STAT) family pathway, namely the JAK-STAT pathway. Once interferon binds to its receptor, it will induce tyrosine phosphorylation of the JAK (JAK1 / Tyk2) family, and the phosphorylated JAK family will then activate STATs proteins. After activation, STATs proteins form a complex with IRF9 and enter the nucleus. The interferon-stimulated regulatory element ISRE receives the stimulation signal, induces the transcription and expression of ISGs (IFN-stimulated genes), and finally performs a series of biological functions. During the long-term evolution of hosts and viruses, viruses can evade the host's immune response by limiting the antiviral activity of IFN.

[0004] The use of IFN in clinical antiviral treatment is limited by many factors, such as its high cost and poor clinical antiviral efficacy, which greatly limits its use and thus affects its clinical treatment efficiency. Therefore, exploring ways to enhance IFN-I's own signal and improve the clinical efficacy of IFN-I is an important topic in broad-spectrum antiviral research.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide the use of irisin in the preparation of antiviral drugs, thereby providing a new antiviral strategy and improving the antiviral activity of clinical interferon.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides the use of irisin in the preparation of antiviral drugs.

[0009] In a second aspect, the present invention also provides the use of irisin and interferon in the preparation of a combined antiviral drug.

[0010] The present invention has the following beneficial effects:

[0011] At present, there is a lack of broad-spectrum antiviral drugs for the treatment of viral infections. The broad-spectrum antiviral drugs commonly used in clinical practice are mainly interferons, but because IFN-I is expensive and has limited therapeutic effects, there are many limitations in its use. The inventors found through experiments on various cell lines that irisin has obvious antiviral functions, can significantly inhibit viral infection, can significantly inhibit the replication of various RNA viruses and DNA viruses, and inhibit the RNA levels of various RNA viruses and DNA viruses, the levels of viral proteins, and the amount of cell-infected viruses. Therefore, the present invention provides a new strategy for the development of broad-spectrum antiviral drugs.

[0012] By studying the antiviral mechanism of irisin, it was found that treating cells with irisin can significantly promote the IFN-I signaling pathway, including upregulating the expression of multiple interferon-induced genes (ISGs) under IFN-I stimulation, thereby enhancing antiviral ability. Irisin is expected to become a safer broad-spectrum antiviral drug for single antiviral or to improve the antiviral activity of clinical interferon.

[0013] Irisin is a new type of molecule produced by muscles with a wide range of therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 RAW264.7 cell lines were treated with different doses of Irisin and infected with VSV, SeV, and H1N1 viruses, respectively. Real-time quantitative PCR (qPCR) was used to detect the RNA levels of different viruses.

[0016] Figure 2 :HeLa cells were treated with Irisin and infected with VSV virus (with green fluorescent protein GFP tag), and the changes in the amount of cells infected with VSV-GFP virus after Irisin treatment were observed using a fluorescent inverted microscope;

[0017] Figure 3 :RAW264.7 cells were treated with different doses of Irisin and infected with VSV virus. The effect of Irisin on the amount of cells infected with VSV virus was detected by virus TCID50 assay.

[0018] Figure 4 : 2fTGH and A549 cells were treated with Irisin, then infected with VSV virus or H1N1 virus, and viral proteins VSV-G and H1N1-HA were detected by Western Blot technology;

[0019] Figure 5 :RAW264.7 cell line was pretreated with Irisin and infected with H1N1, CVB, HSV-ICP27, HSV-UC46, and SeV viruses, and the RNA levels of different viruses were detected using Realtime qPCR technology;

[0020] Figure 6 :293T and HeLa were treated with Irisin and infected with VSV virus (with green fluorescent protein GFP tag), and the changes in the amount of cells infected with VSV-GFP virus after Irisin treatment were observed using a fluorescent inverted microscope;

[0021] Figure 7 : HT1080 cells were treated with Irisin and then infected with VSV virus or H1N1 virus. The viral proteins VSV-G and H1N1-HA were detected by Western Blot technology;

[0022] Figure 8 :HT1080 cells were treated with different doses of Irisin, and the effect of Irisin on IFNβ production was detected by Realtime qPCR technology;

[0023] Fig. 9 :2fTGH and U3A were treated with Irisin and infected with VSV virus (with green fluorescent protein GFP label), and the changes in the amount of cells infected with VSV-GFP virus after Irisin treatment were observed with a fluorescent inverted microscope;

[0024] Fig.10 : HT1080 cells were treated with Irisin, stimulated with IFNα, and the proteins Viperin and p-STAT1 were detected by Western Blot technology;

[0025] Fig.11 :RAW264.7 cells were treated with Irisin, stimulated with IFNα, and protein p-STAT1 was detected by Western Blot technique;

[0026] Fig.12 :RAW264.7 cells were treated with irisin, stimulated with IFNα, and the mRNA levels of representative interferon-induced genes (ISGs) were detected by Realtime qPCR technology;

[0027] Fig.13 :RAW264.7, 2fTGH, and HT1080 cells were treated with Irisin, and the expression of proteins STAT1, p-STAT1, and p-Tyk2 were detected by Western Blot technique;

[0028] Fig.14 :The cells were treated with Irisin and infected with VSV virus (with green fluorescent protein GFP label), and the changes in the amount of cells infected with VSV-GFP virus after Irisin treatment were observed using a fluorescent inverted microscope;

[0029] Fig.15 : HT1080 cells were stimulated with IFNα, and the mRNA level of Irisin was detected by Realtime qPCR technology. DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0032] The term "virus" includes all viruses. Thus, a virus can be an RNA virus (single-stranded or double-stranded) or a DNA virus (single-stranded or double-stranded). It can also be an enveloped or non-enveloped virus. A virus can target a prokaryotic organism or a eukaryotic organism; it can be a bacteriophage or a virus that targets a plant, an animal, or a fungus. Preferably, the virus targets a eukaryotic organism, preferably an animal or a plant. Representative virus families that target animals are Parvoviridae, Papovaviridae, Adenoviridae, Brachyviridae, Reoviridae, Retroviridae, Coronaviridae, Resilviridae, Paramyxoviridae, Orthomyxoviridae, Herpesviridae, Hepadnaviridae, and Poxviridae. Representative virus families that target plants are Bromoviridae, Nematode Polyhedraviridae, Cowpea Mosavirus, Cauliflower Mosavirus, Reoviridae, Rhabdoviridae, Tobacco Mosavirus, Cucumber Mosavirus, Flavoviridae, Potato Xviridae, and Potato Yviridae. The virus can be any of these families, such as HIV, herpes simplex virus, Epstein-Barr virus, orthopoxvirus, fowlpox virus, papillomavirus, adenovirus, parvovirus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rabies virus, measles virus, hand, foot and mouth disease virus, SARS coronavirus, rhinovirus, rotavirus, rubella virus and mumps virus.

[0033] Antiviral means: preventing, improving or treating diseases related to viral infection or inhibiting viral infection in vitro, including but not limited to: inhibiting viral replication, RNA level, viral protein level and infectivity. The term "treating" a disease or condition means eliminating, inhibiting, alleviating or alleviating the disease or condition, and the term "preventing" means avoiding and preventing a disease or condition or preventing the disease or condition from occurring or appearing.

[0034] Irisin is a newly discovered myokine and adipokine, which is formed by proteolysis of fibronectin type IlI domain-containing protein 5 (FNDC5). Exercise can significantly increase the protein expression level of irisin in skeletal muscle and peripheral tissues. In terms of biological function, irisin can promote the transformation of white adipose tissue to brown adipose tissue in the body, and contribute to the specific expression of mitochondrial uncoupling protein 1 (UCP1) and brown adipose tissue genes. Brown adipose tissue is the main source of fat energy consumption in the body. The increase in its content contributes to the energy consumption of the human body. Therefore, it can be used to inhibit the occurrence of obesity and insulin resistance, thereby improving the prevention, treatment and diagnosis of metabolic diseases at the genetic level. In recent years, irisin has been found to be a potential target for exercise hormones in the treatment of metabolic diseases. At present, the research on irisin in the field of disease treatment is becoming more and more extensive, but there are few reports on whether it has certain antiviral functions.

[0035] In a first aspect, the present invention provides the use of irisin in the preparation of antiviral drugs.

[0036] At present, there is a lack of broad-spectrum antiviral drugs for the treatment of viral infections. The broad-spectrum antiviral drugs commonly used in clinical practice are mainly interferons, but because IFN-I is expensive and has limited therapeutic effects, there are many limitations in its use. The inventors found through experiments on various cell lines that irisin has obvious antiviral functions, can significantly inhibit viral infection, can significantly inhibit the replication of various RNA viruses and DNA viruses, and inhibit the RNA levels of various RNA viruses and DNA viruses, the levels of viral proteins, and the amount of cell-infected viruses. Therefore, the present invention provides a new strategy for the development of broad-spectrum antiviral drugs.

[0037] In a preferred embodiment of the present invention, the virus includes but is not limited to RNA virus and / or DNA virus.

[0038] In a preferred embodiment of the present invention, RNA viruses include but are not limited to positive-strand RNA viruses, negative-strand RNA viruses, double-strand RNA viruses or retroviruses; DNA viruses include but are not limited to double-strand DNA viruses or single-strand DNA viruses.

[0039] In a preferred embodiment of the present invention, the negative-strand RNA virus includes but is not limited to at least one virus from the Rhabdoviridae, Paramyxoviridae, Bunyavirales and Orthomyxoviridae families;

[0040] In a preferred embodiment of the present invention, the virus in the Rhabdoviridae family is VSV virus; and the virus in the Paramyxoviridae family is Sendai virus (SeV).

[0041] Positive-strand RNA viruses include but are not limited to the Picornaviridae family;

[0042] In a preferred embodiment of the present invention, the virus of the Picornaviridae family is a Coxsackie virus;

[0043] In a preferred embodiment of the present invention, the double-stranded DNA virus includes but is not limited to the Herpesviridae family. In a preferred embodiment of the present invention, the DNA virus is selected from hepatitis B virus, adenovirus, herpes simplex virus, vaccine virus, etc.

[0044] The negative strand RNA virus may be a segmented RNA virus or a non-segmented RNA virus. The segmented RNA virus includes but is not limited to at least one virus in the Bunyavirales and Orthomyxoviridae; the non-segmented RNA virus is selected from the group consisting of hepatitis A virus, hepatitis C virus, measles virus, Newcastle disease virus, coxsackie virus, and the like.

[0045] In a preferred embodiment of the present invention, the negative-strand RNA virus includes at least one virus of the Phenuiviridae, Nairoviridae and Arenaviridae families under the Bunyavirales order;

[0046] In a preferred embodiment of the present invention, the negative-strand RNA virus includes but is not limited to at least one of Severe fever withthrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV) and Guertu virus (GTV) under the Leucoviridae family. The viruses of the Leucoviridae family include but are not limited to the genus Gekkovirus, the genus Parsivirus, the genus Termitovirus and the genus Tenuivirus.

[0047] In a preferred embodiment of the present invention, the negative-strand RNA virus includes but is not limited to at least one of Songling virus (SGLV) and Crimean Congohemorrhagic fever virus (CCHFV) under the Nairoviridae family.

[0048] In a preferred embodiment of the present invention, the negative-strand RNA virus includes at least one virus selected from the group consisting of lymphocytic choriomeningitis virus LCMV, Machupo virus (MACV) and Lassa virus under the Arenaviridae family;

[0049] In a preferred embodiment of the present invention, the negative-strand RNA virus includes at least one virus of the genus Influenza A virus, the genus Influenza B virus, the genus Influenza C virus and the genus Togotovirus under the family Orthomyxoviridae.

[0050] By studying the antiviral mechanism, it was found that treating cells with irisin can significantly promote the IFN-I signaling pathway, including upregulating the expression of multiple interferon-induced genes (ISGs) under IFN-I stimulation, thereby enhancing antiviral ability. Irisin is expected to become a safer broad-spectrum antiviral drug for single antiviral or to improve the antiviral activity of clinical interferon.

[0051] In a preferred embodiment of the present invention, the drug has at least one of the following uses:

[0052] (1) Inhibit viral replication;

[0053] (2) Down-regulate viral RNA levels, viral protein levels, and the number of cells infected by the virus;

[0054] (3) Promote the expression of multiple interferon-induced genes;

[0055] In a preferred embodiment of the present invention, the interferon-induced gene is selected from IFIT1, Viperin, ISG15 or ISG54;

[0056] (4) Activating the IFN signaling pathway; in a preferred embodiment of the present invention, activating the tyrosine phosphorylation of the JAK (JAK1 / Tyk2) family; in a preferred embodiment of the present invention, relying on IFNAR1 phosphorylation to exert antiviral effects;

[0057] (5) Upregulate the levels of STATs proteins.

[0058] In a preferred embodiment of the present invention, the drug further comprises pharmaceutically acceptable excipients, which include but are not limited to conventional pharmaceutical excipients, carriers or diluents.

[0059] The pharmaceutical composition also includes pharmaceutically acceptable excipients, including but not limited to fillers, lubricants, disintegrants, binders, glidants, etc.

[0060] In the preferred technical scheme of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, hydroxypropyl methyl cellulose, starch and its derivatives, polyethylene glycol and its derivatives, lactose, lactose starch complex, lactose cellulose complex, sucrose, mannitol, mannitol starch complex, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, povidone, copovidone, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvidone, magnesium stearate, sodium stearyl fumarate, talc, and stearic acid, one or more of them or a combination thereof.

[0061] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of injection), such as solution, suspension and gel usually contain liquid carrier, such as water and / or pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include pH adjusting agents, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (such as methylcellulose), such as defined above. The medicine may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the medicine may be regulated by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be regulated by pharmaceutically acceptable thickeners such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the selected agent.

[0062] In a preferred embodiment of the present invention, the drug is in the form of an injection or an oral preparation.

[0063] In a second aspect, the present invention also provides the use of irisin and interferon in the preparation of a combined antiviral drug. Irisin can directly activate the IFN-I-mediated signaling pathway, and thus can be used in combination with IFN to treat clinical H1N1 and other viral infections, thereby improving the broad-spectrum antiviral efficacy of clinical IFN.

[0064] In a preferred embodiment of the present invention, the virus is selected from RNA virus and / or DNA virus, and the interferon is IFN-I subtype.

[0065] In a preferred embodiment of the present invention, the RNA virus is selected from a positive-strand RNA virus, a negative-strand RNA virus, a double-strand RNA virus or a retrovirus; the DNA virus is selected from a double-strand DNA virus or a single-strand DNA virus.

[0066] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0067] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0068] Example 1

[0069] This example explores the antiviral function of Irisin through experiments.

[0070] In order to explore whether Irisin can inhibit viral infection, RAW264.7 cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), pretreated with different doses of Irisin for 12h, infected with VSV, SeV or H1N1 virus, and incubated at 37℃ for 20h. The cells were lysed with Trizol, and then total RNA was extracted from the cells and reverse transcribed into cDNA. Real-time qPCR was used to detect the RNA level of the virus. The results are shown in the figure. Figure 1 The results showed that the RNA levels of VSV, SeV or H1N1 viruses infected were significantly reduced after Irisin treatment.

[0071] HeLa cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), added different doses of Irisin for pretreatment for 12 hours, infected with VSV-GFP virus (with green fluorescent protein GFP label), observed the cells infected with VSV-GFP virus with a fluorescent inverted microscope, and analyzed the changes in the number of cells infected with the virus after Irisin treatment. The results are shown in Figure 2 The results showed that after Irisin treatment, the number of cells infected with VSV-GFP virus was significantly reduced.

[0072] RAW264.7 cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), pretreated with different doses of Irisin for 12h, infected with VSV virus, and incubated in a 37°C incubator for 20h. The effect of Irisin on the amount of cells infected with VSV virus was detected by virus TCID50 assay. The results are shown in Figure 3 The results showed that after Irisin treatment, the amount of cells infected with VSV virus was significantly reduced.

[0073] 2fTGH and A549 cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), pretreated with different doses of Irisin for 12h, infected with VSV or H1N1 virus, and incubated in a 37℃ incubator for 24h. The cells were lysed with NP-40 lysis buffer, protein samples were prepared, and after SDS-PAGE electrophoresis, they were transferred to PVDF membranes, blocked with 5% skim milk powder at room temperature for 1h, and incubated overnight at 4℃ with primary antibody. After washing with PBST, secondary antibody was added and incubated at room temperature for 1h. After washing with PBST, luminescent substrate was added for exposure, development and fixation. The results are shown in Figure 4 The results showed that after Irisin treatment, the level of viral protein in cells infected with VSV or H1N1 virus was significantly decreased.

[0074] RAW264.7 cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), transfected with FNDC5, and then infected with H1N1, CVB, HSV-ICP27, HSV-UC46, and SeV (MOI = 1) viruses, respectively, and incubated at 37°C for 20 hours. The cells were lysed with Trizol, and then total RNA was extracted from the cells and reversely transcribed into cDNA. Real-time qPCR was used to detect the RNA levels of different viruses. The results are shown in the figure. Figure 5 The results showed that after Irisin treatment, the viral RNA levels in cells infected with H1N1, CVB, HSV-ICP27, HSV-UC46, and SeV viruses decreased significantly.

[0075] 293T and HeLa cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), transfected with FNDC5, and then infected with VSV-GFP virus (with green fluorescent protein GFP tag). The cells infected with VSV-GFP virus were observed with a fluorescent inverted microscope, and the changes in the number of cells infected with virus after Irisin treatment were analyzed. The results are shown in Figure 6 The results showed that the number of cells infected with the virus decreased significantly after Irisin treatment.

[0076] HT1080 and A549 cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), pretreated with different doses of Irisin for 12h, infected with VSV or H1N1 virus, and incubated in a 37℃ incubator for 24h. The cells were lysed with NP-40 lysis buffer, protein samples were prepared, and after SDS-PAGE electrophoresis, they were transferred to PVDF membranes, blocked with 5% skim milk powder at room temperature for 1h, and incubated overnight at 4℃ with primary antibody. After washing with PBST, secondary antibody was added and incubated at room temperature for 1h. After washing with PBST, luminescent substrate was added for exposure, development and fixation. The results are shown in Figure 7 The results showed that the protein levels of VSV and H1N1 viruses were reduced after Irisin treatment.

[0077] Figures 1 to 7 The results showed that Irisin has significant antiviral function. It can significantly inhibit the replication of a variety of RNA viruses and DNA viruses in cells, including downregulating viral RNA levels, viral protein levels and the amount of cells infected by the virus.

[0078] Example 2

[0079] This example found that the antiviral function of Irisin depends on the interferon signaling pathway.

[0080] In order to analyze whether the broad-spectrum antiviral ability of Irisin depends on the IFN signaling pathway, HT1080 cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), and treated the cells with different doses of Irisin (200, 600 μg / mL). The cells were lysed with Trizol, and then the total RNA in the cells was extracted and reverse transcribed into cDNA. The mRNA level of IFNβ was detected by fluorescence quantitative PCR (Real-time qPCR). The results are shown in Figure 8 The results showed that after HT1080 cells were treated with 200 and 600 μg / mL of Irisin, the mRNA level of IFNβ could not be significantly increased, that is, Irisin could not upregulate the production of interferon.

[0081] 293T and U3A cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), added different doses of Irisin for pretreatment for 12 hours, infected with VSV-GFP virus (with green fluorescent protein GFP label), observed the cells infected with VSV-GFP virus with a fluorescent inverted microscope, and analyzed the changes in the number of cells infected with the virus after Irisin treatment. The results are shown in Fig. 9 As shown, the results showed that Irisin could inhibit VSV virus infection in 2fTGH cells, but Irisin no longer inhibited VSV virus infection in U3A (STAT1-deficient) cells.

[0082] HT1080 cells were plated on 12-well cell culture plates (approximately 0.5×10 6 / well), treated the cells with Irisin, and then added IFNα to stimulate the cells for 12h. The cells were lysed with NP-40 lysis buffer to prepare protein samples. After SDS-PAGE electrophoresis, they were transferred to PVDF membranes, blocked with 5% skim milk powder at room temperature for 1h, added with primary antibody and incubated overnight at 4℃, washed with PBST, added with secondary antibody and incubated at room temperature for 1h, washed with PBST, and then exposed, developed and fixed with luminescent substrate. The results are shown in Fig.10 The results showed that Irisin upregulated the level of p-STAT1 induced by IFNα.

[0083] In summary, Figures 8 to 10 The results showed that Irisin could not upregulate the production of interferon. In 2fTGH cells, Irisin could inhibit the infection of VSV virus, but in U3A (STAT1-deficient) cells, Irisin no longer inhibited the infection of VSV virus. Irisin upregulated the level of p-STAT1 induced by IFNα.

[0084] Example 3

[0085] This example found that the antiviral function of Irisin depends on IFNAR1 phosphorylation.

[0086] MEF cells were plated on 12-well cell culture plates (about 0.5×10 6 / well), IFNAR1 was treated with different doses of Irisin － / － MEF cells (from IFNAR1 － / － Preparation of knockout mice, IFNAR1 － / － The knockout mice were donated by Professor Dong Chunsheng of Soochow University. The cells were lysed with NP-40 lysis buffer to prepare protein samples. After SDS-PAGE electrophoresis, the samples were transferred to PVDF membranes and blocked with 5% skim milk powder at room temperature for 1 hour. The primary antibody was added and incubated at 4°C overnight. After washing with PBST, the secondary antibody was added and incubated at room temperature for 1 hour. After washing with PBST, the luminescent substrate was added for exposure, development and fixation. The results are shown in Figure 2. Fig.11 shown.

[0087] The results showed that after IFNAR1 gene knockout cells were treated with Irisin, they were stimulated with IFNα and did not express p-STAT1 from beginning to end.

[0088] Example 4

[0089] This example found that Irisin can directly activate the IFN signaling pathway (Vero)

[0090] To further analyze whether Irisin up-regulated the IFN-I-mediated signaling pathway, cells were plated on 12-well cell culture plates (approximately 0.5×10 6 / well) and treated with different doses of Irisin (200, 600, 1000 μg / mL). The cells were lysed with Trizol, and then total RNA was extracted from the cells and reverse transcribed into cDNA. Real-time qPCR technology was used to detect the mRNA levels of representative ISGs genes. This experiment was verified in Vero cells. The results are shown in Fig.12 shown. Fig.12 The results showed that Irisin can directly activate the IFN signaling pathway and increase the expression of downstream representative interferon-stimulated genes (ISGs): Mx1, Isg15 and Rsad2, and it is dose-dependent and treatment time-dependent.

[0091] The cells were plated on 12-well cell culture plates (approximately 0.5 × 10 6 / well), and treated the cells with different doses of Irisin (0, 100, 200 μg / mL). The cells were lysed with NP-40 lysis buffer, and the protein samples were prepared. After SDS-PAGE electrophoresis, they were transferred to PVDF membranes, blocked with 5% skim milk powder at room temperature for 1 hour, and incubated overnight at 4°C with the addition of primary antibody. After washing with PBST, the secondary antibody was added and incubated at room temperature for 1 hour. After washing with PBST, the luminescent substrate was added for exposure, development and fixation. This experiment was verified in Raw264.7, 2fTGH, and HT1080. The results are shown in Fig.13 The results showed that Irisin treatment could activate the IFN signaling pathway and increase the expression of STAT1, p-STAT1, and p-Tyk2.

[0092] Example 5

[0093] This example found that the effect intensity of Irisin and IFN was compared

[0094] HT1080 cells were plated in 12-well cell culture plates (approximately 0.5 × 10 6 / well), added different doses of Irisin (200, 600, 1000ng / L) for pretreatment for 12h, infected with VSV-GFP virus (with green fluorescent protein GFP label), observed the cells infected with VSV-GFP virus with a fluorescent inverted microscope, and analyzed the changes in the number of cells infected with the virus after Irisin treatment. The results are shown in Fig.14 The results showed that Irisin treatment could significantly reduce the number of cells infected with the virus in a dose-dependent manner.

[0095] Example 6

[0096] In this example, Irisin was found to be a new ISG.

[0097] HT1080 cells were plated on 12-well cell culture plates (approximately 0.5×10 6 / well), and stimulated the cells with IFNα. The cells were lysed with Trizol, and then total RNA was extracted from the cells and reverse transcribed into cDNA. Real-time qPCR technology was used to detect the mRNA level of Irisin. The results are shown in Fig.15 The results showed that IFNα could increase the level of FNDC5 mRNA.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of irisin in the preparation of antiviral drugs.

2. The use according to claim 1, characterized in that: The virus is selected from RNA viruses and / or DNA viruses.

3. The use according to claim 2, characterized in that: The RNA virus is selected from a positive-strand RNA virus, a negative-strand RNA virus, a double-strand RNA virus or a retrovirus; the DNA virus is selected from a double-strand DNA virus or a single-strand DNA virus.

4. The use according to claim 3, characterized in that: The negative-strand RNA virus is selected from at least one virus of the family Rhabdoviridae, Paramyxoviridae, Bunyavirales and Orthomyxoviridae; Preferably, the virus in the Rhabdoviridae family is VSV virus; the virus in the Paramyxoviridae family is Sendai virus (SeV); The positive-strand RNA virus is selected from the family Picornaviridae; Preferably, the virus of the Picornaviridae family is a Coxsackievirus; Preferably, the double-stranded DNA virus is selected from the family Herpesviridae; Preferably, the negative-strand RNA virus is selected from at least one virus of the families Phenuiviridae, Nairoviridae and Arenaviridae under the order Bunyavirales; Preferably, the negative-strand RNA virus is selected from at least one virus of Severefever with thrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV) and Guertu virus (GTV) under the family Leukoviridae; Preferably, the negative-strand RNA virus is selected from at least one of Songlingvirus (SGLV) and Crimean Congo hemorrhagic fever virus (CCHFV) under the Nairoviridae family; Preferably, the negative-strand RNA virus is selected from at least one virus of the family Arenaviridae: lymphocytic choriomeningitis virus LCMV, Machupo virus (MACV) and Lassa virus; Preferably, the negative-strand RNA virus is selected from at least one virus of the genus Influenza A virus, genus Influenza B virus, genus Influenza C virus and genus Togotovirus under the family Orthomyxoviridae.

5. The use according to any one of claims 1 to 4, characterized in that: The drug has at least one of the following uses: (1) Inhibit viral replication; (2) Down-regulate viral RNA levels, viral protein levels, and the number of cells infected by the virus; (3) Promote the expression of multiple interferon-induced genes (ISGs); Preferably, the interferon-induced gene is selected from IFIT1, Viperin, ISG15 or ISG54; (4) Activating the IFN signaling pathway; preferably, activating the tyrosine phosphorylation of the JAK (JAK1 / Tyk2) family; preferably, relying on IFNAR1 phosphorylation to exert antiviral effects; (5) Upregulate the levels of STATs proteins.

6. The use according to any one of claims 1 to 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the drug is injection or oral preparation.

8. Use of irisin and interferon in the preparation of combined antiviral drugs.

9. The use according to claim 8, characterized in that: The virus is selected from RNA virus and / or DNA virus, and the interferon is IFN-I subtype.

10. The use according to claim 9, characterized in that: The RNA virus is selected from a positive-strand RNA virus, a negative-strand RNA virus, a double-strand RNA virus or a retrovirus; the DNA virus is selected from a double-strand DNA virus or a single-strand DNA virus.

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