Application of metformin in preparation of antiviral drugs

Abstract

The invention discloses application of metformin in preparation of antiviral drugs, and relates to the technical field of molecular biology. Metformin can significantly inhibit the replication of a variety of RNA viruses and DNA viruses, and is verified in a variety of cell lines and mice. The invention illustrates that metformin promotes interferon-mediated antiviral ability by up-regulating expression of ISGs induced by IFN-I, and provides a new strategy for research and development of broad-spectrum antiviral drugs. Metformin is used as a clinical common drug and has low toxicity. Therefore, the metformin is expected to become a safer broad-spectrum antiviral drug for improving the antiviral activity of clinical interferon.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to the application of metformin in the preparation of antiviral drugs. Background Technology

[0002] Viruses are common pathogens in clinical practice and have been threatening human life and health, even endangering lives, in recent years. However, due to the diversity and rapid mutation of viruses, the development of traditional antiviral drugs often faces many difficulties, such as long development cycles, high costs, and the ease with which viruses develop drug resistance. Therefore, the development of broad-spectrum antiviral drugs has important clinical significance.

[0003] Currently, the broad-spectrum antiviral drugs used clinically are mainly interferons. Interferon (IFN), as one of the most important cytokines, plays a crucial role in antiviral responses. The innate immune system is the host's first line of defense against viral invasion, and its core mechanism relies on the interferon system. IFN, as a functional protein in the host, plays an important antiviral role in both innate and adaptive immunity. Among them, type I interferons (IFN-Is) play a vital role in antiviral infection. They bind to receptors on the cell surface, activate the JAK-STAT signaling pathway, and subsequently induce the expression of interferon-stimulated genes (ISGs), producing various antiviral proteins that inhibit viral replication, transcription, and translation. However, despite the important role of interferon in antiviral therapy, its clinical application still has some limitations. First, the high production cost of interferon makes it expensive, limiting its widespread use. Second, when used alone, the antiviral efficacy is sometimes unsatisfactory and may cause side effects such as flu-like symptoms and bone marrow suppression. Therefore, how to enhance the antiviral activity of interferon, reduce its dosage, and minimize side effects has become an urgent problem to be solved.

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

[0005] The purpose of this invention is to provide an application of metformin in the preparation of antiviral drugs to solve the above-mentioned technical problems.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides the application of metformin in the preparation of antiviral drugs, wherein the virus is selected from influenza virus, vesicular stomatitis virus, Sendai virus, herpes virus, dengue virus, hepatitis B virus, hepatitis C virus, or respiratory syncytial virus.

[0008] Secondly, the present invention also provides the application of metformin and type I interferon in the preparation of a combined antiviral drug, wherein the virus is selected from influenza virus, vesicular stomatitis virus, Sendai virus, herpes virus, dengue virus, hepatitis B virus, hepatitis C virus or respiratory syncytial virus.

[0009] The present invention has the following beneficial effects:

[0010] This invention, based on a drug repurposing strategy, discovers a class of hypoglycemic drugs (metformin) with significant antiviral function, capable of significantly inhibiting the replication of various RNA and DNA viruses, validated in multiple cell lines and mice. This invention elucidates that metformin enhances interferon-mediated antiviral activity by upregulating IFN-I-induced ISG expression, providing a new strategy for broad-spectrum antiviral drug development. This class of drugs (metformin) is commonly used clinically with relatively low toxicity. Metformin holds promise as a safer broad-spectrum antiviral drug to enhance the clinical antiviral activity of interferon. This invention improves the antiviral effect of IFN-I. Metformin can be administered alone as an antiviral drug or in combination with type I interferon. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 A549, HepG2, HEK293T, and RAW264.7 cell lines were pretreated with Metformin for 12 hours and then infected with VSV virus. The RNA levels of VSV virus in the low-dose lactate-treated group and the untreated group were detected by Real-time qPCR; N=4, mean±SD, ***p<0.001;

[0013] Figure 2 HT1080 cell lines were pretreated with Metformin and then infected with VSV, H1N1, SeV, and HSV viruses, respectively. The RNA levels of different viruses were detected using real-time quantitative PCR (real-time qPCR); N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001;

[0014] Figure 3Mice were infected with a virus and then injected intraperitoneally with Metformin. The RNA levels of the virus in various organs of the mice were detected using Real-time qPCR. N=5, mean±SD, *p<0.05, **p<0.01, ***p<0.001;

[0015] Figure 4 Mice were injected intraperitoneally with either PBS or Metformin, and then infected with VSV virus. Survival curves were observed and plotted. N=5, mean±SD, ***p<0.001.

[0016] Figure 5 Mice were injected intraperitoneally with PBS or Metformin, then infected with VSV virus. Spleens were collected, homogenized, and the protein levels of PKR, IFIT1, and VSV-G were detected by Western Blot.

[0017] Figure 6 Mice were infected with a virus and then injected intraperitoneally with Metformin. The mRNA levels of representative interferon-inducible genes (ISGs) in the mouse spleen were detected using Realtime qPCR. N=5, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0018] Figure 7 After treating HT1080 cells with Metformin, the cells were stimulated with IFNα at different time points. The mRNA levels of representative interferon-induced genes (ISGs) in the Metformin-treated and untreated groups were detected by Real-time qPCR. N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0019] Figure 8 In the HT1080 cell line, cells were pretreated with Metformin and stimulated with IFN, and then infected with VSV, H1N1, SeV and HSV viruses, respectively. The RNA levels of different viruses in the Metformin-treated group and the untreated group were detected by Real-time qPCR; N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001;

[0020] Figure 9 Preprocessing Irf9 using Metformin + / + Or Irf9 - / -HT1080 cells were then infected with VSV, H1N1, SeV, and HSV viruses, respectively. The RNA levels of different viruses in the Metformin-treated and untreated groups were detected by Real-time qPCR. N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0021] Figure 10 Preprocessing with Metformin and stimulating Irf9 with IFN + / + Or Irf9 - / - In HT1080 cells, the mRNA levels of representative interferon-induced genes (ISGs) in the Metformin-treated and untreated groups were detected by Realtime qPCR; N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001;

[0022] Figure 11 Mice were injected intraperitoneally with either PBS or Metformin, and then infected with VSV virus. The level of L-Lactate in mouse serum was quantified by enzyme-linked immunosorbent assay (ELISA).

[0023] Figure 12 HT1080 cells, mouse primary spleen cells, or HT1080 cells overexpressing GFP-IRF9 were treated with different doses of Metformin. Then, endogenous and exogenous IRF9 were immunoprecipitated, and the protein level of L-Kla was detected by Western blotting. Detailed Implementation

[0024] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0025] Metformin is one of the most widely used oral hypoglycemic agents globally. It primarily lowers blood sugar by inhibiting hepatic gluconeogenesis and reducing insulin resistance. As a first-line antidiabetic drug, metformin has good safety and tolerability. In 1957, French diabetes expert Sterne first demonstrated the hypoglycemic efficacy of metformin through clinical trials and named it "Glucophage," meaning "sugar eater." It was launched in France that same year for the treatment of type 2 diabetes mellitus (T2DM), and has been used clinically for over sixty years. Metformin can increase insulin sensitivity, improve insulin resistance, inhibit intestinal glucose absorption, and promote its utilization. Metformin improves energy metabolism in the liver and peripheral tissues by activating the adenosine monophosphate-activated protein kinase (AMPK) pathway. This mechanism of action contributes to its good hypoglycemic effect in clinical practice. Metformin, one of the most widely used hypoglycemic drugs, is not only used to control blood sugar and treat type 2 diabetes mellitus (T2DM), but it also has potential therapeutic effects on various malignant tumors, cardiovascular diseases, thyroid diseases, obesity, aging, and polycystic ovary syndrome. Currently, research on metformin is becoming increasingly extensive, but there are few reports on whether it possesses antiviral functions.

[0026] In a first aspect, the present invention provides the application of metformin in the preparation of antiviral drugs, wherein the virus is selected from influenza virus, vesicular stomatitis virus, Sendai virus, herpes virus, dengue virus, hepatitis B virus, hepatitis C virus, or respiratory syncytial virus.

[0027] This invention, through extensive experiments, has revealed that metformin possesses significant antiviral function, effectively inhibiting the replication of various RNA and DNA viruses. This has been validated in multiple cell lines and in mice. Specifically, at the cellular level, metformin significantly inhibits viral infection, including suppressing the RNA and viral protein levels of various RNA and DNA viruses. In animal experiments, metformin significantly inhibited viral loads in various organs of mice.

[0028] Research into its antiviral mechanism revealed that metformin upregulates ISG expression by promoting lactation modification of IRF9, a key protein in the IFN signaling pathway. Specifically, metformin increases intracellular lactate production, thereby promoting IRF9 lactation and enhancing its binding affinity to the ISG promoter, thus enhancing the interferon-induced antiviral effect and achieving its antiviral function.

[0029] Experimental results show that metformin can promote the lactation modification of IRF9, thereby continuously activating the IFN signaling pathway.

[0030] Therefore, metformin holds promise as a safer, broad-spectrum antiviral drug to enhance the antiviral activity of clinical interferon. This invention avoids the high cost and limited efficacy of IFN-I drugs. Metformin can be used alone as an antiviral drug or in combination with type I interferon.

[0031] In a preferred embodiment of the present invention, the influenza virus is selected from influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

[0032] In a preferred embodiment of the present invention, the influenza A virus is selected from H1N1, H3N2, H5N1 or H7N9.

[0033] In a preferred embodiment of the present invention, the herpesvirus is selected from the herpes simplex virus genus and / or the varicella-zoster virus genus.

[0034] In a preferred embodiment of the present invention, the herpesvirus is selected from HSV-1, HSV-2, simian B virus, PRV, VZV, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, EB virus, Kaposi's sarcoma-associated herpesvirus, equine herpesvirus 1, bovine herpesvirus 1, canine herpesvirus 1, feline herpesvirus 1, simian varicella virus, phocomelia herpesvirus, or any combination thereof.

[0035] In a preferred embodiment of the present invention, the drug also includes a pharmaceutically acceptable carrier.

[0036] In one embodiment, the dosage form of the drug is a tablet, pill, powder, suspension, gel, emulsion, cream, granule, nanoparticle, capsule, suppository, injection, or spray. The dosage form of the drug is an injection or powder for injection.

[0037] In one embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain a pharmaceutically acceptable carrier, such as those selected from excipients, diluents, pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (e.g., methylcellulose), dyes, and / or flavoring agents, as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, 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 reagent selected.

[0038] In one alternative embodiment, the drug is a solid pharmaceutical preparation, such as lyophilized bacterial powder, granules, etc.

[0039] In a preferred embodiment of the present invention, the drug has at least one of the following uses:

[0040] (1) Inhibit viral replication;

[0041] (2) Downregulate viral RNA and viral protein levels;

[0042] (3) Improve the survival rate of test animals;

[0043] (4) Promote the expression of interferon-induced genes; In a preferred embodiment of the present invention, the interferon-induced genes are selected from IFIT1, Viperin, ISG15, ISG54, ISG20, Mx1, PKR or any combination thereof.

[0044] (5) Increases lactic acid production in test animals;

[0045] (6) Enhance the lactation modification of IRF9 protein;

[0046] (7) Enhance the binding of IRF9 to ISG promoters.

[0047] In a preferred embodiment of the present invention, the drug is administered by injection, gavage, or oral administration.

[0048] Secondly, the present invention also provides the application of metformin and type I interferon in the preparation of a combined antiviral drug, wherein the virus is selected from influenza virus, vesicular stomatitis virus, Sendai virus, herpes virus, dengue virus, hepatitis B virus, hepatitis C virus or respiratory syncytial virus.

[0049] Experiments showed that the combination of metformin and type I interferon significantly upregulated the levels of ISGs in mice after viral infection and in cells stimulated by IFNα, compared with type I interferon alone; and compared with type I interferon alone, the combination of metformin and type I interferon significantly inhibited the total RNA level of the virus.

[0050] In a preferred embodiment of the present invention, the influenza virus is selected from influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

[0051] In a preferred embodiment of the present invention, the influenza A virus is selected from H1N1, H3N2, H5N1 or H7N9.

[0052] In a preferred embodiment of the present invention, the herpesvirus is selected from the herpes simplex virus genus and / or the varicella-zoster virus genus.

[0053] In a preferred embodiment of the present invention, the herpesvirus is selected from HSV-1, HSV-2, simian B virus, PRV, VZV, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, EB virus, Kaposi's sarcoma-associated herpesvirus, equine herpesvirus 1, bovine herpesvirus 1, canine herpesvirus 1, feline herpesvirus 1, simian varicella virus, phocomelia herpesvirus, or any combination thereof.

[0054] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0055] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0056] Example 1

[0057] Metformin has antiviral properties.

[0058] The C57BL / 6 wild-type mice used in this embodiment were purchased from the Animal Center of Soochow University and raised in the SPF environment of Soochow University.

[0059] To investigate whether Metformin could inhibit viral infection, A549, HepG2, HEK293T, and RAW264.7 cell lines were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were pretreated with Metformin (200 μM) for 12 h and then infected with VSV (MOI=1) virus. Cells were incubated at 37°C for 24 h. Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. The RNA levels of Metformin and untreated VSV virus were detected using real-time qPCR. The results are shown below. Figure 1 As shown.

[0060] The primer and probe sequences for quantitative real-time PCR targeting VSV virus RNA are as follows:

[0061] VSV (5'-ACGGCGTACTTCCAGATGG-3' (SEQ ID NO: 1) and 5'-CTCGGTTCAAGATCCAGGT-3' (SEQ ID NO: 2)).

[0062] The reaction procedure is as follows:

[0063] Real-time PCR system: 95℃, 5 min; 95℃, 15 s, 60, 1 min (40 cycles).

[0064] HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁶ cells per well). 6 Cells were pretreated with Metformin (200 μM) for 12 h in each well, and then infected with VSV (vesicular stomatitis virus), H1N1, SeV (Sendai virus), and HSV (herpes simplex virus) (MOI=1), respectively, and incubated at 37°C for 24 h. Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. The RNA levels of different viruses were detected using real-time qPCR. The results are shown below. Figure 2 As shown.

[0065] The primer and probe sequences for quantitative real-time PCR targeting the RNA of VSV, H1N1, SeV, and HSV viruses are as follows:

[0066] SeV (5'-GATGACGATGCCGCAGCAGTAG-3' (SEQ ID NO: 3) and 5'-CCTCCGATGTCAGTTGGTTCACTC-3' (SEQ ID NO: 4));

[0067] VSV (5'-ACGGCGTACTTCCAGATGG-3' (SEQ ID NO: 5) and 5'-CTCGGTTCAAGATCCAGGT-3' (SEQ ID NO: 6));

[0068] H1N1 (5'-TTCTAACCGAGGTCGAAACG-3' (SEQ ID NO: 7) and 5'-ACAAAGCGTCTACGCTGCAG-3' (SEQ ID NO: 8));

[0069] HSV (5'-CCAACGCCAAGACGGTGTA-3' (SEQ ID NO: 9) and 5'-GGGGGTCGTGA GGAAGAAC-3' (SEQ ID NO: 10)).

[0070] To analyze the antiviral function of Metformin in animals, 6-8 week old mice were infected with VSV (1x10⁻¹⁰). 8VSV virus was lysed (PFU) for 72 hours, followed by intraperitoneal injection of Metformin (50 mg / kg, n=5) or PBS (Ctrl, n=5), respectively. Twelve hours later, heart, liver, spleen, lung, and kidney tissues were collected and homogenized. Trizol was used for lysis, and total RNA was extracted from each tissue and reverse transcribed into cDNA. Real-time qPCR was used to detect the VSV virus RNA levels in each mouse organ. The results are shown below. Figure 3 As shown.

[0071] Eight-week-old mice were intraperitoneally injected with either Metformin (50 mg / kg, n = 10) or PBS (Ctrl, n = 10), and 12 hours later were treated with VSV (1 x 10⁻⁶). 8 Mice were infected with PFU virus, and their survival curves were observed and plotted. The results are as follows: Figure 4 As shown.

[0072] Figures 1 to 4 The results showed that Metformin has significant antiviral function. It can significantly inhibit the replication of various RNA and DNA viruses in cells. At the same time, compared with the control group, the RNA levels of various viruses in mice injected with Metformin were significantly downregulated, indicating that Metformin can effectively reduce viral infection in mice.

[0073] Example 2

[0074] Metformin promotes IFN-I-mediated antiviral activity.

[0075] The C57BL / 6 wild-type mice used in this embodiment were purchased from the Animal Center of Soochow University and raised in the SPF environment of Soochow University.

[0076] To analyze whether the broad-spectrum antiviral activity of metformin is mediated by regulating IFN-I-mediated antiviral signaling, mice aged 6-8 weeks were infected with VSV (1x10⁻¹). 8 PFU virus was administered for 72 hours, followed by intraperitoneal injection of Metformin (50 mg / kg, n=5) or PBS (Ctrl, n=5). Mouse spleen tissue was collected, homogenized, and cells were lysed with NP-40 lysis buffer to prepare protein samples. After SDS-PAGE electrophoresis, the samples were transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 1 hour, incubated overnight at 4°C with primary antibody, washed with PBST, incubated with secondary antibody at room temperature for 1 hour, washed with PBST, and then exposed, developed, and fixed with luminescent substrate. The protein levels of PKR, IFIT1, VSV-G, and tubulin in mouse spleen tissue were detected by Western blotting. The results are as follows: Figure 5 As shown.

[0077] Infect 6-8 week old mice with VSV (1x10⁻¹) 8 PFU virus was administered to mice 72 hours later, followed by intraperitoneal injection of Metformin (50 mg / kg, n=5) or PBS (Ctrl, n=5). Spleen tissue was collected from mice and homogenized. Lysis was performed using Trizol, followed by extraction of total RNA from the spleen tissue and reverse transcription into cDNA. Real-time qPCR was used to detect the mRNA levels of representative interferon-induced genes (ISGs) in the mouse spleen tissue. The results are shown below. Figure 6 As shown.

[0078] The primer and probe sequences for detecting the mRNA levels of IFIT1, ISG15, ISG54, ISG20, and Mx1 are as follows:

[0079] mouse Ifit1(5'-GCCTATCGCCAAGATTTAGATGA-3' and 5'-TTCTGGATTTAACCGGACAGCA-3');

[0080] mouse Isg15 (5'-GGTGTCCGTGACTAACTCCAT-3' and 5'-CTGTACCACTA GCATCACTGTG-3');

[0081] mouse Isg54 (5'-GGAGAGCAATCTGCGACAG-3' and 5'-GCTGCCTCATTT AGACCTCTG-3');

[0082] mouse Isg20 (5'-GCCATTTGGTGAAGCCAGGCTA-3' and 5'-AGCCTGTCTGT GGACGTGTCAT-3');

[0083] mouse Mx1(5'-GACCATAGGGGTCTTGACCAA-3' and 5'-AGACTTGCTCTTT CTGAAAAGCC-3')

[0084] HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁶ cells per well). 6Cells were treated with Metformin (200 μM) in / wells and stimulated with IFNα (1,000 IU / ml) at different time points. Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. The mRNA levels of representative interferon-induced genes (ISGs) in the Metformin-treated and untreated groups were detected using real-time qPCR. The results are shown in the figure. Figure 7 As shown.

[0085] The primer and probe sequences for detecting the mRNA levels of IFIT1, Viperin, ISG54, and Mx1 are as follows:

[0086] human Ifit1(5'-CACAAGCCATTTTCTTTGCT-3' and 5'-ACTTGGCTGCATATC GAAAG-3');

[0087] human Isg54 (5'-GGAGAGCAATCTGCGACAG-3' and 5'-GCTGCCTCATTTAGACCTCTG-3');

[0088] human Isg15 (5'-GGGACCTGACGGTGAAGATG-3' and 5'-CGCCGATCTTCTGGGTGAT-3');

[0089] human Viperin (5'-TGCTTAAGGAAGCTGGTATGGAG-3' and 5'-TCACCAACTTGCCCAGGTAT-3');

[0090] HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁶ cells per well). 6 Cells were pretreated with Metformin (200 μM) for 12 h and stimulated with IFNα. They were then infected with VSV, H1N1, SeV, and HSV (MOI=1) viruses and incubated at 37°C for 24 h. Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. The RNA levels of different viruses in the Metformin-treated and untreated groups were detected by real-time qPCR. The primers, system, and conditions used for amplification were the same as in Example 1, and the results are as follows: Figure 8 As shown.

[0091] Figures 5 to 8The results showed that Metformin significantly promoted interferon-mediated antiviral function and upregulated ISG levels in mice after viral infection and in IFNα-stimulated cells, indicating that Metformin may function by promoting the interferon signaling pathway. The combination of metformin and type I interferon significantly upregulated ISG levels in mice after viral infection and in IFNα-stimulated cells compared to type I interferon stimulation alone. Compared to type I interferon stimulation alone, the combination of metformin and type I interferon significantly inhibited total viral RNA levels.

[0092] Example 3

[0093] This embodiment found that Metformin's antiviral function depends on the key protein IRF9 in the IFN signaling pathway.

[0094] To further analyze how Metformin exerts its broad-spectrum antiviral ability by relying on the interferon signaling pathway, Irf9 was used. + / + Or Irf9 - / - HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were pretreated with Metformin (200 μM) for 12 h, then infected with VSV, H1N1, SeV, and HSV (MOI=1) viruses, respectively, and incubated at 37°C for 24 h. Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. Irf9 was detected by real-time PCR. + / + Or Irf9 - / - RNA levels of different viruses in Metformin-treated and untreated cells were measured. The primers, system, and conditions used for amplification were the same as in Example 1, and the results are as follows: Figure 9 As shown.

[0095] Irf9 + / + Or Irf9 - / - HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were pretreated with Metformin (200 μM) for 12 h and stimulated with IFNα (1,000 IU / ml). Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. Irf9 was detected by real-time qPCR. + / + Or Irf9 - / -The mRNA levels of representative interferon-induced genes (ISGs) in Metformin-treated and untreated cells were measured. The primers, system, and conditions used for amplification were the same as in Example 2, and the results are as follows: Figure 10 As shown.

[0096] Figures 9 to 10 The results showed that Metformin's antiviral function depends on the key protein IRF9 in the interferon signaling pathway. Compared to cell stimulation by type I interferon alone, the combination of metformin and type I interferon significantly increased the mRNA levels of interferon-induced genes (ISGs).

[0097] Example 4

[0098] This embodiment found that Metformin affects the level of IRF9 lactation modification by promoting lactic acid production.

[0099] To analyze the specific mechanism by which Metformin exerts its antiviral function in animals, 6-8 week old mice were infected with VSV (1x10⁻¹⁰). 8 After 72 hours of PFU virus administration, mice were injected intraperitoneally with either Metformin (50 mg / kg, n=5) or PBS (Ctrl, n=5). The concentration of L-Lactate in mouse serum was then detected using enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 11 As shown.

[0100] To further verify that Metformin exerts its antiviral function by regulating the lactation modification of IRF9, we seeded HT1080 cells, primary mouse spleen cells, or HT1080 cells overexpressing GFP-IRF9 onto 6-well cell culture plates (approximately 1.0 × 10⁻⁶ cells per well). 6 Cells were divided into several wells and treated with different doses of Metformin (50, 100, 200 μM) and incubated at 37°C for 24 h. Cells were lysed with NP-40 lysis buffer to prepare protein samples. Endogenous (i.e., IRF9 secreted by HT1080 cells and mouse primary spleen cells) and exogenous (i.e., overexpressing GFP-IRF9) IRF9 proteins were co-immunoprecipitated. After SDS-PAGE electrophoresis, the samples were transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 1 h, incubated overnight at 4°C with primary antibody, washed with PBST, incubated with secondary antibody at room temperature for 1 h, washed with PBST, and then exposed, developed, and fixed with luminescent substrate. The L-Kla modification levels of endogenous and exogenous IRF9 proteins were detected by Western blotting. The results are shown below. Figure 12 As shown.

[0101] Figures 11 to 12The results showed that Metformin can increase lactic acid production, thereby affecting the lactation modification of IRF9.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of metformin in the preparation of antiviral drugs, characterized in that, The virus is selected from influenza virus, vesicular stomatitis virus, Sendai virus, herpes virus, dengue virus, hepatitis B virus, hepatitis C virus, or respiratory syncytial virus.

2. The application according to claim 1, characterized in that, The influenza virus is selected from influenza A, influenza B, influenza C, or influenza D viruses.

3. The application according to claim 2, characterized in that, The influenza A virus is selected from H1N1, H3N2, H5N1 or H7N9.

4. The application according to claim 1, characterized in that, The herpesviruses are selected from the genus Herpes simplex virus and / or the genus Varicella herpesvirus; Preferably, the herpesvirus is selected from HSV-1, HSV-2, simian B virus, PRV, VZV, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, EB virus, Kaposi's sarcoma-associated herpesvirus, equine herpesvirus 1, bovine herpesvirus 1, canine herpesvirus 1, feline herpesvirus 1, simian varicella virus, phocomelia herpesvirus, or any combination thereof.

5. The application according to any one of claims 1-4, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

6. The application according to any one of claims 1-4, characterized in that, The drug has at least one of the following uses: (1) Inhibit viral replication; (2) Downregulate viral RNA and viral protein levels; (3) Improve the survival rate of test animals; (4) Promote the expression of interferon-induced genes; preferably, the interferon-induced genes are selected from IFIT1, Viperin, ISG15, ISG54, ISG20, Mx1, PKR or any combination thereof. (5) Increases lactic acid production in test animals; (6) Enhance the lactation modification of IRF9 protein; (7) Enhance the binding of IRF9 to ISG promoters.

7. The application according to any one of claims 1-4, characterized in that, The drug can be administered by injection, gavage, or oral administration.

8. The application of metformin and type I interferon in the preparation of a combined antiviral drug, characterized in that, The virus is selected from influenza virus, vesicular stomatitis virus, Sendai virus, herpes virus, dengue virus, hepatitis B virus, hepatitis C virus, or respiratory syncytial virus.

9. The application according to claim 8, characterized in that, The influenza virus is selected from influenza A virus, influenza B virus, influenza C virus, or influenza D virus; Preferably, the influenza A virus is selected from H1N1, H3N2, H5N1 or H7N9.

10. The application according to claim 9, characterized in that, The herpesviruses are selected from the genus Herpes simplex virus and / or the genus Varicella herpesvirus; Preferably, the herpesvirus is selected from HSV-1, HSV-2, simian B virus, PRV, VZV, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, EB virus, Kaposi's sarcoma-associated herpesvirus, equine herpesvirus 1, bovine herpesvirus 1, canine herpesvirus 1, feline herpesvirus 1, simian varicella virus, phocomelia herpesvirus, or any combination thereof.

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