Application of lactic acid and interferon in preparation of antiviral drugs

Abstract

The invention discloses application of lactic acid and interferon in preparation of antiviral drugs, and relates to the technical field of antivirus. Lactic acid can promote interferon-mediated antiviral ability by up-regulating expression of ISGs induced by IFN-I, and exert the antiviral function by promoting the level of IRF9-mediated lactic acid modification, thereby providing a new strategy for research and development of broad-spectrum antiviral drugs. Lactic acid 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 antiviral technology, and more specifically, to the application of lactic acid and interferon in the preparation of antiviral drugs. Background Technology

[0002] Viral infections are one of the major threats to global public health, causing millions of infections and deaths each year. Due to the diversity of viral species and their high frequency of mutations, the development of targeted drugs is extremely difficult, especially against emerging viral threats, where the available effective drugs are very limited. Therefore, the development of broad-spectrum antiviral drugs has significant clinical importance.

[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. However, the molecules that exert antiviral function are not IFN itself, but rather the downstream effector factors produced by its recognition and binding to homologous receptors, thereby activating cell signaling—namely, IFN-stimulated genes (ISGs). IFN induces the expression of hundreds of ISGs by activating the JAK-STAT signaling pathway, resulting in a broad-spectrum antiviral effect. Once interferon binds to its receptor, it induces tyrosine phosphorylation of the JAK (JAK1 / Tyk2) family, and the phosphorylated JAK family then activates STAT proteins. After activation, STATs proteins form a complex with IRF9 and enter the nucleus. The interferon-stimulated regulatory element ISRE receives the stimulation signal, inducing the transcription and expression of ISGs (IFN-stimulated genes), which ultimately perform a series of biological functions.

[0004] In antiviral immune responses, type I interferons (IFN-Is) are the most important interferons, and their broad-spectrum antiviral function has long been a focus of research. However, various factors limit the clinical antiviral efficacy of interferons, greatly restricting their use. Therefore, improving the therapeutic efficiency of IFN-Is has become a pressing clinical challenge.

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

[0006] The purpose of this invention is to provide the application of lactic acid and interferon in the preparation of antiviral drugs, thereby providing a new strategy for antiviral treatment and improving clinical antiviral activity.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides the application of lactic acid in the preparation of antiviral drugs.

[0009] Secondly, the present invention also provides the application of a lactate dehydrogenase inhibitor in the preparation of antiviral drugs.

[0010] Thirdly, the present invention also provides the application of lactate combined with interferon in the preparation of antiviral drugs.

[0011] The present invention has the following beneficial effects:

[0012] Currently, there is a lack of broad-spectrum antiviral drugs for treating viral infections. Interferon is the most commonly used broad-spectrum antiviral drug in clinical practice, but its use is limited due to its high cost and limited efficacy. This invention provides a novel drug—lactic acid. Experiments have shown that lactic acid can promote interferon-mediated antiviral activity by upregulating the expression of IFN-I-induced ISGs (IFN-stimulated genes), thereby enhancing antiviral activity. It also exerts its antiviral function by promoting IRF9-mediated lactation modification, providing a new strategy for the development of broad-spectrum antiviral drugs. After administration of lactic acid, viral infection can be significantly inhibited at the cellular level, including inhibiting the RNA levels and viral protein levels of various RNA and DNA viruses. In animal experiments, lactic acid can significantly inhibit the viral load in various organs of mice.

[0013] Lactic acid, as a major metabolite of cellular glycolysis, has a wide range of therapeutic applications. Therefore, lactic acid holds promise as a safer, broad-spectrum antiviral drug to enhance the antiviral activity of clinical interferon. Attached Figure Description

[0014] 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.

[0015] Figure 1 HT1080 cell lines were treated with different doses of lactate (0, 1, 3, 5, 10, 20, 40 mM) and 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.

[0016] Figure 2 HT1080 cells were treated with different doses of lactate (0, 1, 2, 3 mM) and infected with VSV virus. The effect of lactate on the number of VSV-infected cells was detected by viral TCID50 assay, and the viral protein VSV-G was detected by Western blotting. N = 4, mean ± SD, **p < 0.01, ***p < 0.001.

[0017] Figure 3 A549, HepG2, HEK293T, and RAW264.7 cell lines were pretreated with low-dose lactate (3 mM) for 12 hours and then infected with VSV virus. Real-time qPCR was used to detect the RNA levels of VSV virus in the low-dose lactate-treated and untreated groups. N = 4, mean ± SD, ***p < 0.001.

[0018] Figure 4 HT1080 cell lines were pretreated with low-dose lactate (3mM) 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.

[0019] Figure 5 After treating HT1080 cells with low-dose lactate or lactate dehydrogenase inhibitor, the cells were stimulated with IFNα at different time points. Real-time qPCR was used to detect the mRNA levels of representative interferon-induced genes (ISGs) in the low-dose lactate treatment group and the untreated group, or the lactate dehydrogenase inhibitor treatment group and the untreated group. N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0020] Figure 6 HT1080 cells were treated with low-dose lactate and stimulated with IFN alpha. The levels of PKR and IFIT1 proteins in the low-dose lactate-treated group and the untreated group were detected by Western blotting.

[0021] Figure 7 In the HT1080 cell line, cells were pretreated with low-dose lactate and stimulated with IFN, followed by infection with VSV, H1N1, SeV, and HSV viruses, respectively. RNA levels of different viruses in the low-dose lactate-treated and untreated groups were detected using Real-time qPCR. N = 4, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0022] Figure 8 After tissue grinding and lysis of various organs of mice, endogenous IRF9 was co-precipitated by immunoprecipitation. Exogenous IRF9 was co-precipitated in HEK293T cells overexpressing Flag-GFP-IRF9 (FG-IRF9). The protein level of L-Kla was detected by Western blotting.

[0023] Figure 9 HEK293T cells or HEK293T cells overexpressing GFP-IRF9 were treated with different doses of lactate, followed by immunoprecipitation of endogenous and exogenous IRF9, and the protein level of L-Kla was detected by Western blotting.

[0024] Figure 10 Irf9 was pretreated with low-dose lactate. + / + Or Irf9 - / - HT1080 cells were infected with VSV, H1N1, SeV, and HSV viruses, respectively. RNA levels of different viruses in the low-dose lactate-treated and untreated groups were detected using Real-time qPCR. N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 11 Irf9 was pretreated with low-dose lactate and stimulated with IFN. + / + Or Irf9 - / - In HT1080 cells, the mRNA levels of representative interferon-induced genes (ISGs) in the low-dose lactate-treated and untreated groups were detected by Real-time qPCR. N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0026] Figure 12 We overexpressed wild-type and mutant plasmids of IRF9 in HEK293T cells and used immunoprecipitation to detect the lactation level of IRF9 protein.

[0027] Figure 13 Myc-IRF9-WT and Myc-IRF9-K81R plasmids were overexpressed in HEK293T cells, and the mRNA levels of representative interferon-induced genes (ISGs) were detected by Real-time qPCR. N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0028] Figure 14Myc-IRF9-WT and Myc-IRF9-K81R plasmids were overexpressed in HEK293T cells, which were then infected with VSV, H1N1, SeV, and HSV viruses, respectively. RNA levels of different viruses were detected using real-time qPCR. N=4, mean±SD, *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 15 In Irf9 - / - FG-IRF9-WT and FG-IRF9-K81R plasmids were overexpressed in HT1080 cells. Cells were then pretreated with low-dose lactate and stimulated with IFN. The mRNA levels of representative interferon-induced genes (ISGs) were detected by Real-time qPCR. N = 4, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0030] Figure 16 Mice were injected intraperitoneally with PBS or low-dose lactate, then infected with VSV virus. Spleens were collected, homogenized, and the protein levels of PKR, IFIT1, and VSV-G were detected by Western blotting.

[0031] Figure 17 Mice were intraperitoneally injected with PBS or low-dose lactate, then infected with VSV virus. Spleens were collected, homogenized, and endogenous IRF9 was immunoprecipitated. L-Kla protein levels were detected by Western blotting.

[0032] Figure 18 Mice were infected with a virus and then injected intraperitoneally with a low dose of lactate. The mRNA levels of representative interferon-inducible genes (ISGs) in the mouse spleen were detected using Real-time qPCR. N = 5, mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0033] Figure 19 Mice were infected with a virus and then injected intraperitoneally with a low dose of lactate. The viral RNA levels 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.

[0034] Figure 20 Mice were intraperitoneally injected with either PBS or a low dose of lactate, and then infected with VSV virus. Survival curves were observed and plotted. N = 5, mean ± SD, ***p < 0.001. Detailed Implementation

[0035] 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.

[0036] Lactic acid, a byproduct of glycolysis in normal cells under hypoxic conditions, has long been considered a metabolic waste product. The discovery of the "Warburg effect" in tumor cells has led researchers to re-examine lactic acid, elucidating its crucial roles as a fuel and signaling molecule, promoting angiogenesis, and inhibiting tumor immune cells. However, the molecular mechanisms by which lactic acid regulates these biological functions remain unclear. The thousands of small metabolic molecules produced by cellular activities are not only products of protein (enzyme) catalysis but can also, in turn, influence protein function through covalent modification.

[0037] In late 2019 and early 2020, two research groups independently reported a novel post-translational modification of proteins using lactate as a substrate—protein lactation—and revealed that lactation is an important means by which lactate regulates cellular life activities. Lactate production is a key step in the glycolysis pathway. Lactate levels affect the glycolysis pathway in vivo and also influence the level of lactation modification. Lactation modification is closely related to glycolysis; glycolysis can simultaneously generate precursors for both lactation pathways and is an important pre-process of lactation modification. As a novel post-translational modification, protein lactation is crucial for understanding its function and regulatory mechanisms in physiological and pathological processes, and it also helps to deepen our understanding of the occurrence and development of many diseases, including tumors. Although existing research has elucidated the roles of lactation modification in various aspects such as tumors, Alzheimer's disease, inflammation, fibrosis, cell stemness maintenance, embryonic development, and neural regulation, the research direction of protein lactation modification as a whole is still in its early stages. Currently, research on lactic acid and lactation modification is becoming increasingly extensive, but there are few reports on whether it regulates the IFN-I signaling pathway and its antiviral function.

[0038] This invention has demonstrated through numerous experiments that lactic acid enhances the antiviral activity of interferon, and that lactic acid alone has a significant antiviral effect.

[0039] In a first aspect, the present invention provides the application of lactic acid in the preparation of antiviral drugs.

[0040] This invention reveals that lactic acid possesses broad-spectrum antiviral effects, significantly inhibiting viral infection at the cellular level, including inhibiting the RNA and viral protein levels of various RNA and DNA viruses. In animal experiments, lactic acid significantly inhibited viral loads in various organs of mice. Therefore, it shows promising potential for antiviral applications.

[0041] In a preferred embodiment of the present invention, the virus is a DNA virus or an RNA virus.

[0042] In a preferred embodiment of the present invention, the RNA virus is selected from single-stranded positive-sense RNA virus, single-stranded negative-sense RNA virus, double-stranded RNA virus, or retro-transcribed RNA virus, and the DNA virus is selected from double-stranded DNA virus or single-stranded DNA virus.

[0043] Single-stranded positive-sense RNA viruses include, for example, coronaviruses, flaviviruses, and picornaviruses.

[0044] Single-stranded negative-sense RNA viruses include, for example, influenza viruses, Ebola viruses, rabies viruses, Bunyavirales, and Orthomyxoviridae. Single-stranded negative-sense RNA viruses can also be other negative-sense segmented RNA viruses besides Bunyavirales and Orthomyxoviridae.

[0045] At least one virus from the families Phenuiviridae, Nairoviridae, and Arenaviridae within the order Bunyaviridae.

[0046] At least one virus from the genera of influenza A virus, influenza B virus, influenza C virus, and Togovirus within the family Orthomyxoviridae.

[0047] Double-stranded RNA viruses, for example, are selected from rotaviruses.

[0048] Retro-transcribed RNA viruses include, for example, human immunodeficiency virus (HIV) and human T-cell troponin virus (HTLV).

[0049] Double-stranded DNA viruses include, for example, poxviruses, herpesviruses, and adenoviruses.

[0050] Single-stranded DNA viruses, such as parvoviruses.

[0051] In a preferred embodiment of the present invention, the virus is selected from VSV, H1N1, SeV or HSV viruses.

[0052] In a preferred embodiment of the present invention, the drug has at least one of the following uses:

[0053] (1) Inhibit viral replication;

[0054] (2) Downregulate viral RNA and viral protein levels;

[0055] (3) Promotes the level of IRF9-mediated lactation modification in cells, tissues or organs;

[0056] (4) Promotes the expression of interferon-induced genes.

[0057] IRF9 protein (interferon regulatory factor 9) is a key member of the interferon regulatory factor (IRF) family, playing a central role in immune responses, cell differentiation, and disease progression. It responds to type I interferons (IFN-α / β) and induces the expression of ISGs (such as OAS1, MX1, and the ADAR family), thereby inhibiting viral replication.

[0058] The above-mentioned inhibition of viral replication includes, but is not limited to: a decrease or a significant decrease in the number of viral particles after the application of lactic acid compared to before the application of lactic acid; or a decrease or a significant decrease in the number of virus-infected cells and the area of ​​infection after the application of lactic acid compared to before the application of lactic acid; or, inhibition of nucleic acid replication after the application of lactic acid compared to before the application of lactic acid.

[0059] In a preferred embodiment of the present invention, the interferon-inducible gene is selected from IFIT1, Viperin, ISG15, ISG54, or PKR. Promoting the expression of the interferon-inducible gene significantly enhances the antiviral function of interferon.

[0060] PKR (double-stranded RNA-dependent protein kinase, also known as EIF2AK2) is a core antiviral protein induced by interferon.

[0061] In a preferred embodiment of the present invention, the lactation modification is lysine L-lactylation modification.

[0062] In a preferred embodiment of the present invention, the lactic acid is L-lactic acid; in other embodiments, the lactic acid may also be D-lactic acid, or a mixture of D-lactic acid and L-lactic acid.

[0063] In a preferred embodiment of the present invention, the drug also includes a pharmaceutically acceptable carrier.

[0064] 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.

[0065] 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.

[0066] In one alternative embodiment, the drug is a solid pharmaceutical preparation, such as lyophilized bacterial powder, granules, etc.

[0067] In one alternative implementation, the drug is formulated for oral, injectable, or gavage administration.

[0068] Secondly, the present invention also provides the application of a lactate dehydrogenase inhibitor in the preparation of antiviral drugs.

[0069] The inventors discovered that lactate dehydrogenase inhibitors can also have a similar antiviral effect to lactate.

[0070] In a preferred embodiment of the present invention, the lactate dehydrogenase inhibitor is selected from sodium oxalate, pinoresinic acid, N-hydroxyindole, LDH-IN-1, or GSK2837808A.

[0071] Thirdly, this invention also provides the application of lactate combined with interferon in the preparation of antiviral drugs. After administration of lactate combined with interferon, compared with interferon alone, it can more significantly promote the expression of interferon-induced genes and inhibit viral replication.

[0072] In actual use, low doses of lactic acid and interferon can be used in combination as needed.

[0073] In a preferred embodiment of the present invention, the drug has at least one of the following uses:

[0074] (1) Inhibit viral replication;

[0075] (2) Downregulate viral RNA and viral protein levels;

[0076] (3) Promotes the level of IRF9-mediated lactation modification in cells, tissues or organs;

[0077] (4) Promotes the expression of interferon-induced genes;

[0078] In a preferred embodiment of the present invention, the interferon-inducing gene is selected from IFIT1, Viperin, ISG15, ISG54 or PKR.

[0079] In a preferred embodiment of the present invention, the lactation modification is lysine L-lactylation modification;

[0080] In a preferred embodiment of the present invention, the drug also includes a pharmaceutically acceptable carrier;

[0081] In a preferred embodiment of the present invention, the interferon is a type I interferon;

[0082] In a preferred embodiment of the present invention, type I interferon is IFNα and / or IFNb.

[0083] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0084] First, the study investigated whether low-dose lactate could inhibit viral infection. The results showed that at the cellular level, low-dose lactate significantly inhibited viral infection, including inhibiting the RNA levels and viral protein levels of various RNA and DNA viruses. In animal experiments, low-dose lactate significantly inhibited the viral load in various organs of mice.

[0085] Secondly, the study investigated whether low-dose lactate could enhance the antiviral effect induced by interferon to achieve antiviral function. The results showed that low-dose lactate increased downstream IFN signaling, thereby significantly promoting the antiviral function of interferon, indicating that low-dose lactate may exert its function by promoting the interferon signaling pathway.

[0086] Finally, the study analyzed how low-dose lactate promoted the IFN-I-mediated signaling pathway, further validating that low-dose lactate can enhance the antiviral activity of interferon. The results showed that the antiviral function of low-dose lactate depends on the key protein IRF9 in the IFN-I signaling pathway; low-dose lactate exerts its antiviral function by promoting the level of IRF9-mediated lactation modification.

[0087] Example 1

[0088] This embodiment tested that low-dose lactate significantly inhibited the replication of various RNA and DNA viruses in cells.

[0089] To investigate whether lactate could inhibit viral infection, HT1080 cells were seeded on 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were pretreated with different doses of lactate (0, 1, 3, 5, 10, 20, 40 mM) for 12 h, and then infected with VSV, H1N1, SeV, and HSV (MOI=1) viruses, respectively, and incubated at 37°C for 24 h. Cells were lysed using 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 1 As shown in the figure. The results showed that low doses of lactic acid could inhibit viral infection, while excessively high doses (5-40 mM) of lactic acid could promote viral infection.

[0090] HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁶ cells per well). 6 Cells were pretreated with different doses of lactate (1, 2, 3 mM) for 12 h and then infected with VSV (MOI = 1) virus. The cells were incubated at 37°C for 24 h. The effect of lactate on the amount of VSV-infected cells was detected by viral TCID50 assay, and the viral protein VSV-G was detected by Western blotting. The results are shown below. Figure 2 As shown in the figure. The results showed that after HT1080 cells were treated with 1-3 mM lactate, the viral TCID50 decreased significantly, the number of cells infected with VSV virus decreased significantly, and the protein level decreased significantly.

[0091] 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 low-dose lactate (3mM) for 12 h and then infected with VSV (MOI=1) virus. Cells were incubated at 37°C for 24 h. Cells were lysed using Trizol, and total RNA was extracted and reverse transcribed into cDNA. Real-time qPCR was used to detect the RNA levels of VSV virus in the low-dose lactate-treated and untreated groups. The results are shown below. Figure 3 As shown in the figure. The results showed that lactate treatment of A549, HepG2, HEK293T and RAW264.7 cell lines significantly inhibited the RNA level of VSV virus.

[0092] HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁶ cells per well). 6Cells were pretreated with low-dose lactate (3mM) for 12 h, and then infected with VSV, H1N1, SeV, and HSV (MOI=1) viruses, respectively, and incubated at 37°C for 24 h. Cells were lysed using 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 4 As shown in the figure. The results showed that lactate treatment of HT1080 cells significantly inhibited the RNA levels of VSV, H1N1, SeV, and HSV viruses.

[0093] In conclusion, Figures 1 to 4 The results showed that low doses of lactate could significantly inhibit the replication of various RNA and DNA viruses in cells, including downregulating viral RNA and protein levels. However, excessively high doses of lactate could promote viral infection.

[0094] Example 2

[0095] This embodiment tested the interferon signaling pathway, which is dependent on low-dose lactate to exert its antiviral function.

[0096] To further analyze whether the broad-spectrum antiviral activity of low-dose lactate depends on the interferon signaling pathway, HT1080 cells were seeded on 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were treated with low-dose lactate (5 mM) or a lactate dehydrogenase inhibitor (Oxamate, sodium oxalate) at different time points, and stimulated with IFNα (1,000 IU / ml). Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. Real-time qPCR was used to detect the mRNA levels of representative interferon-induced genes (ISGs) in the low-dose lactate treatment group and the untreated group, or the lactate dehydrogenase inhibitor treatment group and the untreated group. The results are shown below. Figure 5 As shown, low-dose lactate can increase the mRNA levels of IFN downstream signaling genes—representative interferon-induced genes (ISGs). The lactate dehydrogenase inhibitor (Oxamate) can also increase the mRNA levels of IFN downstream signaling genes—representative interferon-induced genes (ISGs).

[0097] HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁶ cells per well). 6Cells were treated with low-dose lactate (5 mM) in [number] wells, followed by stimulation with IFNα (1,000 IU / ml) at different time points. Cells were lysed using NP-40 lysis buffer, and protein samples were prepared. 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 levels of PKR and IFIT1 proteins in the low-dose lactate-treated and untreated groups were detected by Western blotting. The results are shown below. Figure 6 As shown, low-dose lactate treatment significantly increased the levels of PKR and IFIT1 proteins in cells.

[0098] HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁶ cells per well). 6 Cells were pretreated with low-dose lactate (3mM) for 12 h and stimulated with IFN. They were 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. The RNA levels of different viruses in the low-dose lactate-treated and untreated groups were detected by real-time qPCR. The results are shown below. Figure 7 As shown, low-dose lactate can significantly enhance the antiviral function of interferon.

[0099] Figures 5 to 7 The results showed that low-dose lactate could increase downstream IFN signaling, thereby significantly promoting the antiviral function of interferon, indicating that low-dose lactate may function by promoting the interferon signaling pathway.

[0100] Example 3

[0101] This embodiment tested the antiviral function of low-dose lactate, which is dependent on IRF9 lactation.

[0102] To further investigate the specific mechanism by which low-dose lactate exerts its antiviral function, we obtained heart, liver, spleen, lung, and kidney tissues from 6-8 week old mice, followed by tissue homogenization and lysis. Cells were lysed using NP-40 lysis buffer to prepare protein samples, and endogenous IRF9 protein was immunoprecipitated separately. 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 to luminescent substrate for exposure, development, and fixing. The L-Kla modification level of endogenous IRF9 protein was detected by Western blotting. The results are as follows: Figure 8As shown in the left figure, the results indicate that the endogenous IRF9 protein exhibits a higher level of L-Kla modification.

[0103] HEK293T cells were seeded onto 6-well cell culture plates (approximately 1.0 × 10⁶ cells per well). 6 Cells were divided into several wells ( / well), and then overexpressed with the FG-IRF9 plasmid and incubated at 37°C for 24 h. Cells were lysed with NP-40 lysis buffer to prepare protein samples. Exogenous IRF9 protein was co-immunoprecipitated using HEK293T whole-cell lysis buffer. 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 to luminescent substrate for exposure, development, and fixing. The L-Kla modification level of exogenous IRF9 protein was detected by Western blotting. The results are shown below. Figure 8 As shown in the right figure, the results indicate a high level of L-Kla modification in the exogenous IRF9 protein.

[0104] To further verify that low-dose lactate exerts its antiviral function by regulating the lactation modification of IRF9, we seeded HEK293T cells on 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 lactate (1 mM, 3 mM, 5 mM) and incubated at 37°C for 24 h. Cells were lysed with NP-40 lysis buffer to prepare protein samples. Endogenous and exogenous IRF9 proteins were co-immunoprecipitated using HEK293T whole-cell lysis buffer. 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 (left) and exogenous (right) IRF9 proteins were detected by Western blotting. The results are shown below. Figure 9 As shown in the figure. The results showed that L-Kla modification levels of endogenous and exogenous IRF9 proteins were significantly increased after treatment with different doses of lactate.

[0105] Irf9 + / + Or Irf9 - / - HT1080 cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6Cells were pretreated with low-dose lactate (3mM) for 12 h, followed by infection 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. Irf9 was detected by real-time PCR. + / + Or Irf9 - / - RNA levels of different viruses in cells were observed in the low-dose lactate-treated group and the untreated group. The results are as follows: Figure 10 As shown, after Irf9 gene knockout, viral RNA levels significantly increased compared to before knockout, without L-lactate treatment. After Irf9 gene knockout, viral RNA levels significantly increased compared to before knockout, with L-lactate treatment. Before Irf9 gene knockout, viral RNA levels significantly decreased after L-lactate treatment compared to before L-lactate treatment. This indicates that L-lactate function is closely related to the Irf9 gene, and adding L-lactate after Irf9 gene knockout has no significant effect on viral RNA levels. Adding L-lactate before Irf9 gene knockout has a significant effect on viral RNA levels.

[0106] 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 low-dose lactate (5 mM) 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 cells from low-dose lactate-treated and untreated groups were compared. The results are as follows: Figure 11 As shown in the figure. The results showed that after Irf9 gene knockout, the mRNA levels of representative interferon-inducible genes (ISGs) decreased significantly compared with before knockout. Before Irf9 gene knockout, low-dose lactate treatment could significantly increase the mRNA levels of representative interferon-inducible genes (ISGs).

[0107] Figures 8 to 11 The results showed that the antiviral function of low-dose lactate depends on lactation modification of IRF9, a key protein in the IFN-I signaling pathway. Low-dose lactate exerts its antiviral function by promoting the level of IRF9-mediated lactation modification.

[0108] Example 4

[0109] This embodiment identifies sites in IRF9 that have undergone lactation modification.

[0110] To further investigate the key sites of lactation modification in IRF9, HEK293T cells were seeded on 6-well cell culture plates (approximately 1.0 × 10⁶ cells per well). 6 Cells were overexpressed in both wild-type and mutant IRF9 plasmids ( / well), and incubated at 37°C for 24 h. Cells were lysed with NP-40 lysis buffer to prepare protein samples. Exogenous IRF9 protein was then co-immunoprecipitated, followed by SDS-PAGE electrophoresis, transferred to a PVDF membrane, 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 level of exogenous IRF9 protein was detected by Western blotting. The results are shown below. Figure 12 As shown in the results, in cells overexpressing the IRF9 mutant plasmid, and in cells overexpressing the IRF9 mutant K81R, the L-Kla modification level of exogenous IRF9 protein was significantly decreased. This indicates that the L-Kla modification site of IRF9 protein may be related to K81R.

[0111] HEK293T cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were incubated at 37°C for 24 hours after overexpressing Myc-IRF9-WT and Myc-IRF9-K81R plasmids ( / well). Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. The mRNA levels of representative interferon-induced genes (ISGs) were detected using real-time qPCR. The results are shown below. Figure 13 As shown, Figure 13 The "-" in the text refers to the control group with the empty vector. The results showed that in cells overexpressing the Myc-IRF9-K81R plasmid, the mRNA levels of its representative interferon-inducible genes (ISGs) were significantly reduced.

[0112] HEK293T cells were seeded onto 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were overexpressed with Myc-IRF9-WT and Myc-IRF9-K81R plasmids ( / well), and infected with VSV, H1N1, SeV, and HSV (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 14 As shown in the figure, the results indicate that cells overexpressing the Myc-IRF9-K81R plasmid showed a significant increase in viral RNA levels after viral infection.

[0113] HT1080 cells with conditional knockout of the IRF9 gene were seeded on 12-well cell culture plates (approximately 0.5 × 10⁻⁶ cells per well). 6 Cells were overexpressing FG-IRF9-WT or IFG-IRF9-K81R mutant plasmids ( / well), pretreated with low-dose lactate (5mM) for 12h, and stimulated with IFNα (1,000 IU / ml) for 8h. Cells were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. The mRNA levels of representative interferon-induced genes (ISGs) were detected using real-time qPCR. The results are shown below. Figure 15 As shown in the results, HT1080 cells with conditional knockout of the IRF9 gene overexpressing the IFG-IRF9-K81R mutant plasmid maintained extremely low mRNA levels of representative interferon-induced genes (ISGs) even after treatment with low doses of lactate and / or IFNα. This means that after the IRF9 gene is knocked out, low doses of lactate and IFNα cannot promote IRF9-mediated lactation modification.

[0114] The results showed that low-dose lactic acid can exert its antiviral function by promoting the level of lactation modification of IRF9.

[0115] Example 5

[0116] This embodiment demonstrates that low-dose lactic acid can effectively reduce viral infection in mice.

[0117] 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.

[0118] To analyze the antiviral function of low-dose lactate in animals, 6-8 week old mice were infected with VSV (1x10⁻¹⁰). 8PFU virus was administered for 72 hours, followed by intraperitoneal injection of low-dose L-lactic acid (10 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, and VSV-G in mouse spleen tissue were detected by Western blotting. The results are as follows: Figure 16 As shown in the figure. The results indicate that low-dose lactate can downregulate the level of VSV-G protein in mouse spleen tissue and increase the levels of PKR and IFIT1 proteins in mouse spleen tissue.

[0119] Infect 6-8 week old mice with VSV (1x10⁻¹) 8 Mice were infected with PFU (polyurethane) virus for 72 hours, followed by intraperitoneal injection of low-dose L-lactic acid (10 mg / kg, n=5) or PBS (Ctrl, n=5). Spleen tissue was collected from mice, homogenized, and lysed with NP-40 lysis buffer to prepare protein samples. Endogenous IRF9 protein was immunoprecipitated, and after SDS-PAGE electrophoresis, the samples were transferred to a PVDF membrane, 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 again, and then exposed, developed, and fixed with a luminescent substrate. The L-Kla modification level of endogenous IRF9 in mouse spleen tissue was detected by Western blotting. The results are as follows: Figure 17 As shown in the figure, low-dose lactate can increase the L-Kla modification level of endogenous IRF9 in mouse spleen tissue.

[0120] Infect 6-8 week old mice with VSV (1x10⁻¹) 8 PFU virus was administered to mice for 72 hours, followed by intraperitoneal injection of low-dose L-lactic acid (10 mg / kg, n=5) or PBS (Ctrl, n=5), respectively. Spleen tissue was collected from mice and homogenized. The tissue was lysed using Trizol, and total RNA was extracted and reverse transcribed into cDNA. Real-time qPCR was used to detect the mRNA levels of representative interferon-induced genes (ISGs) in mouse spleen tissue. The results are shown below. Figure 18 As shown in the figure. The results showed that L-lactic acid could increase the mRNA level of representative interferon-inducible genes (ISGs) in mouse spleen tissue.

[0121] Infect 6-8 week old mice with VSV (1x10⁻¹)8 After 72 hours of inoculation with PFU (phenylephrine-lactate-full-urine), mice were intraperitoneally injected with low doses of L-lactic acid (10 mg / kg, n=5) or PBS (Ctrl, n=5), respectively. Twelve hours later, heart, liver, spleen, lung, and kidney tissues were collected and homogenized. The tissues were lysed with Trizol, and total RNA was extracted and reverse transcribed into cDNA. Real-time qPCR was used to detect the RNA levels of VSV virus in each organ. The results are shown below. Figure 19 As shown in the figure. The results indicate that L-lactic acid can reduce the RNA levels of VSV virus in various organs.

[0122] Eight-week-old mice were intraperitoneally injected with low doses of L-lactic acid (10 mg / kg, n = 10) or PBS (Ctrl, n = 10), respectively. Twelve hours later, they were administered 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 20 As shown in the figure. The results showed that low-dose L-lactic acid could improve the survival rate of mice infected with the virus.

[0123] 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. Application of lactic acid in the preparation of antiviral drugs.

2. The application according to claim 1, characterized in that, The virus is either a DNA virus or an RNA virus.

3. The application according to claim 2, characterized in that, The RNA virus is selected from single-stranded positive-sense RNA virus, single-stranded negative-sense RNA virus, double-stranded RNA virus, or retro-transcribed RNA virus, and the DNA virus is selected from double-stranded DNA virus or single-stranded DNA virus.

4. The application according to claim 3, characterized in that, The virus is selected from VSV, H1N1, SeV or HSV viruses.

5. The application according to claim 1, 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) Promotes the level of IRF9-mediated lactation modification in cells, tissues or organs; (4) Promotes the expression of interferon-induced genes; Preferably, the interferon-inducible gene is selected from IFIT1, Viperin, ISG15, ISG54 or PKR; Preferably, the lactation modification is lysine L-lactylation modification.

6. The application according to claim 1, characterized in that, The lactic acid is L-lactic acid; Preferably, the drug further includes a pharmaceutically acceptable carrier.

7. Application of lactate dehydrogenase inhibitors in the preparation of antiviral drugs.

8. The application according to claim 7, characterized in that, The lactate dehydrogenase inhibitor is selected from sodium oxalate, pinoresinic acid, N-hydroxyindole, LDH-IN-1, or GSK2837808A.

9. Application of lactate combined with interferon in the preparation of antiviral drugs.

10. The application according to claim 9, 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) Promotes the level of IRF9-mediated lactation modification in cells, tissues or organs; (4) Promotes the expression of interferon-induced genes; Preferably, the interferon-inducible gene is selected from IFIT1, Viperin, ISG15, ISG54 or PKR; Preferably, the lactation modification is lysine L-lactylation modification; Preferably, the drug further includes a pharmaceutically acceptable carrier; Preferably, the interferon is a type I interferon; Preferably, the type I interferon is IFNα and / or IFNb.

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