Application of Nomilin in the preparation of drugs for improving the body's antiviral immunity

Abstract

The present invention belongs to the technical field of chemical drugs, and specifically relates to the application of Nomilin in the preparation of drugs that improve the body's antiviral immunity. The purpose of the present invention is to develop more antiviral drugs. The technical solution of the present invention is the application of Nomilin in the preparation of drugs that improve the body's antiviral immunity. The present invention discloses that Nomilin promotes the expression of interferon-stimulated genes (ISGs) by promoting the production of virus-induced IFN-I, thereby promoting the antiviral ability mediated by IFN-I, thereby significantly inhibiting the infection of various RNA viruses and DNA viruses. The present invention provides a new option for treating infections of various viruses such as HBV, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical medicines, and particularly relates to the application of nomilin in preparing medicines for improving the antiviral immunity of an organism. Background Art

[0002] Virus particles are tiny and have no complete cell structure. They are ubiquitous in our daily lives. They usually enter the body through air, insects, contact and other transmission methods, thus causing many inflammations and diseases. In the past few decades, humans around the world have repeatedly suffered from the threats of influenza virus, severe acute respiratory syndrome (SARS), dengue virus (DENV), Zika virus, Ebola virus and other viruses. Due to the diversity of virus species and the high frequency of virus mutation, the development of targeted drugs is difficult, especially for the threat of sudden viruses. The specific drugs that can be selected are very limited. Therefore, it is of great clinical significance to improve the efficacy of existing broad-spectrum antiviral drugs.

[0003] The broad-spectrum antiviral drugs currently used in clinical practice are mainly type I interferons (IFN-I). In 1957, Isaacs, Lindenmann and others used chicken embryo chorionic follicles to study the phenomenon of influenza interference and found that cells produced a protein that could make uninfected cells resistant to subsequent invading viruses. This protein interfered with the replication of the infected virus and was therefore named interferon. Based on differences in sequence homology, interferon (IFN) is divided into three families, namely type I, type II and type III. In humans and mice, the type I interferon family includes multiple IFN-α subtypes (13 in humans and 14 in mice), as well as single IFN-β, IFN-ε, IFN-κ, IFN-ω (human) and IFN-ζ (mouse) subtypes. The type II interferon family has only one member, IFNγ, whose proinflammatory and immunomodulatory functions are different from those of type I and type III interferons; type III interferons include four subtypes in humans: IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), and IFN-λ4. Although type III interferons are induced by similar pathogen perception pathways as type I interferons and activate related antiviral, antiproliferative, and immunomodulatory gene expression programs, type I interferons are clearly more effective in antiviral protection.

[0004] As the first line of defense for the host against viral invasion and replication, after the innate immune system is activated, pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs) and DNA receptor cGAMP enzyme (cyclic-GMP-AMP synthase, cGAS), can recognize specific viral components and then trigger a series of signal cascade reactions, inducing the production of IFN-I in cells. Once IFN-I binds to its receptor, it will induce JAK (JAK1 / Tyk2) The phosphorylated JAK family then activates STATs proteins. After activation, STATs proteins form a complex with IRF9 and enter the nucleus, binding to the regulatory element ISRE, inducing the transcription and expression of ISGs (IFN-stimulated genes), and ultimately these ISGs perform a series of biological functions. IFN-I plays an important role in the body's innate immune process, especially its broad-spectrum antiviral function, which has always been valued by researchers. However, due to its high cost and poor clinical antiviral efficacy, its use is greatly limited. Therefore, how to improve the therapeutic efficiency of IFN-I by promoting the production of IFN-I under viral infection has become a clinical problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of the present invention is to develop more antiviral drugs.

[0006] The technical solution of the present invention is the application of Nomilin in the preparation of medicines for improving the antiviral immunity of the body.

[0007] The invention also provides the use of Nomilin in preparing antiviral drugs.

[0008] Specifically, the virus is an RNA virus or a DNA virus.

[0009] Furthermore, the RNA virus is VSV, SeV, RSV or H1N1.

[0010] Furthermore, the DNA virus is HSV-1.

[0011] The present invention also provides the use of Nomilin in preparing a medicine for promoting the body to produce interferon.

[0012] Furthermore, the interferon is IFN-I.

[0013] Furthermore, the IFN-I is IFNβ.

[0014] The invention also provides a medicine for improving the anti-viral immunity of an organism, comprising Nomilin.

[0015] Furthermore, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0016] The invention also provides an antiviral drug, comprising Nomilin.

[0017] Furthermore, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0018] The invention also provides a medicine for promoting the body to produce interferon, including Nomilin.

[0019] Furthermore, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0020] Beneficial effects of the present invention: The present invention discloses that the limonoid compound Nomilin can be used to improve the body's antiviral immunity. The present invention verifies through cytological experiments that Nomilin promotes the expression of interferon-stimulated genes (ISGs) by promoting the production of virus-induced IFN-I, thereby promoting the antiviral ability mediated by IFN-I, thereby significantly inhibiting the infection of multiple RNA viruses and DNA viruses. The present invention provides a new option for treating infections of multiple viruses such as HBV, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :Raw264.7 cell line was pretreated with different concentrations of Nomilin for 24 h, 48 h and 72 h, CCK-8 reagent was added, and then the OD450 of cells at different time points was detected by a multifunctional microplate reader to reflect the cell proliferation. N=3, mean ± SD, ns, p>0.05.

[0022] Figure 2 :Raw264.7 and THP-1 cell lines were pretreated with different doses of nomilin for 24 h, infected with VSV virus, and the RNA levels of different viruses were detected by real time qPCR technology. N=3, mean ± SD, *p<0.05, **p<0.01, ***p<0.001.

[0023] Figure 3 : Raw264.7 cells were pretreated with different concentrations of nomilin and then infected with VSV-GFP virus. The viral protein VSV-G was detected by Western Blot technology and the changes in the number of cells infected with VSV-GFP virus after nomilin treatment were observed under a fluorescent inverted microscope.

[0024] Figure 4 : Raw264.7 cells were pretreated with 10 μM nomilin for 0, 3, 6, 12 and 24 h and then infected with VSV virus. The viral protein VSV-G was detected by Western Blot technology.

[0025] Figure 5 :Raw264.7 cells were pretreated with 10 μM nomilin and infected with RSV, H1N1 and HSV viruses, respectively. Real time qPCR was used to detect RNA or gDNA levels of different viruses. N=3, mean ± SD, ***p<0.001.

[0026] Figure 6 :A549 cells were pretreated with 10 μM nomilin and infected with RSV, H1N1 and HSV viruses, respectively. Realtime qPCR was used to detect RNA or gDNA levels of different viruses. N=3, mean ± SD, ***p<0.001.

[0027] Figure 7 : Raw264.7 and VERO cells were pretreated with different concentrations of nomilin for 24 h and then infected with VSV virus, respectively. The viral protein VSV-G was detected by Western Blot technology.

[0028] Figure 8 : Raw264.7 and THP-1 cells were pretreated with different concentrations of nomilin for 24 h, and then the cells were infected with SeV or VSV virus to promote the expression of IFNβ. Real time qPCR technology was used to detect the changes in IFNβ mRNA levels in the nomilin-treated group and the untreated group. N=3, mean ± SD, *p<0.05, **p<0.01, ***p<0.001.

[0029] Fig. 9 :Raw264.7, THP-1 and A549 cells were pretreated with 10 μM nomiline, and the cells were infected with SeV for 0, 6 and 12 h, respectively. Real time qPCR was used to detect the changes in IFNβ mRNA levels in the nomiline-treated and untreated groups. N=3, mean ± SD, *p<0.05, **p<0.01, ***p<0.001.

[0030] Fig.10:Raw264.7 cells were pretreated with 10 μM nomilin for 24 h, and then infected with SeV for 0, 6, and 12 h, respectively. Real time qPCR was used to detect the changes in mRNA levels of representative ISGs genes Cxcl10 and Ccl5 between the nomilin-treated and untreated groups. N=3, mean ± SD, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0032] Unless otherwise specified, the Raw264.7 and THP-1 cells involved in the embodiments of the present invention are from ATCC, and the viruses are model viruses used by various scientific research institutions and stored in this laboratory. The reagents involved in the embodiments of the present invention are all commercially available products, and CCK-8 and reverse transcription kits (Novagen Biotech) can be purchased through commercial channels.

[0033] Example 1 Nomilin does not affect cell proliferation

[0034] In order to explore whether nomilin affects cell proliferation, Raw264.7 cells (macrophages) were plated on 96-well cell culture plates (about 0.5×10 5 / well), different concentrations of nomilin (0.1, 1.0, 10, 50 μM) were added for pretreatment for 24 h, CCK-8 reagent was added at different time points (24, 48 and 72 h), incubated in a 37°C incubator for 2 h, and then OD450 was detected using a multifunctional microplate reader to reflect cell proliferation. The results are shown in Figure 1 shown.

[0035] Figure 1 The results showed that treatment of Raw264.7 cells with different concentrations of nomilin (0.1, 1.0, 10, 50 μM) and different time periods (24 h, 48 h and 72 h) did not affect the proliferation of Raw264.7 cells.

[0036] Example 2 Nomilin significantly inhibits viral infection

[0037] To explore whether nomilin can inhibit viral infection, Raw264.7 and THP-1 cells (human monocytic leukemia cells) were plated on 12-well cell culture plates (about 0.5 × 10 6 / well), pretreated with different concentrations of nomilin (0.1, 1.0, 10, 50 μM) for 24 h, infected with VSV (vesicular stomatitis virus) (MOI = 0.1) virus, and incubated in a 37°C incubator for 20 h. The cells were lysed with Trizol, and then total RNA was extracted from the cells and reversely transcribed into cDNA. Real-time qPCR technology was used to detect the RNA levels of different viruses. The results are shown in the figure. Figure 2 shown.

[0038] Raw264.7 cells were plated on 12-well cell culture plates (approximately 0.5 × 10 6 / well), added different concentrations of nomilin (0.1, 1.0, 10, 50 μM) for pretreatment for 24 h, infected with VSV (MOI=0.1) virus, and incubated in a 37°C incubator for 20 h. First, the cells infected with VSV-GFP virus were observed under a fluorescent inverted microscope to analyze the changes in the number of cells infected with the virus after nomilin treatment. Secondly, the cells can be lysed with NP-40 lysis buffer to prepare protein samples, which are transferred to PVDF membranes after SDS-PAGE electrophoresis and blocked with 5% skim milk powder at room temperature for 1 h. The primary antibody is added and incubated overnight at 4°C. After washing with PBST, the secondary antibody is added and incubated at room temperature for 1 h. After washing with PBST, the luminescent substrate is added for exposure, development and fixation. The results are shown in the figure. Figure 3 shown.

[0039] Raw264.7 cells were plated on 12-well cell culture plates (approximately 0.5 × 10 6 / well), pre-treated with nomilin (10 μM) for 0, 3, 6, 12 and 24 h, infected with VSV (MOI = 0.1) virus, and incubated in a 37°C incubator for 20 h. 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 1 h, and incubated overnight at 4°C with primary antibody. After washing with PBST, secondary antibody was added and incubated at room temperature for 1 h. After washing with PBST, luminescent substrate was added for exposure, development and fixation to detect changes in VSV-G protein levels. The results are shown in the figure. Figure 4 shown.

[0040] Raw264.7 cells were plated on 12-well cell culture plates (approximately 0.5 × 10 6 / well), pre-treated the cells with nomilin (10 μM) for 24 h, infected with RSV, H1N1 and HSV-1 (MOI=1.0) viruses, and incubated in a 37°C incubator for 20 h. 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 or gDNA levels of different viruses. The results are shown in the figure. Figure 5 shown.

[0041] A549 cells (lung cancer human alveolar basal epithelial cells) were plated in 12-well cell culture plates (about 0.5 × 10 6 / well), pre-treated the cells with nomilin (10 μM) for 24 h, infected with RSV, H1N1 and HSV-1 (MOI=1.0) viruses, and incubated in a 37°C incubator for 20 h. 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 or gDNA levels of different viruses. The results are shown in the figure. Figure 6 shown.

[0042] Figures 2 to 6 The results showed that Nomilin 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.

[0043] Example 3 Nomilin significantly promotes the production of IFN-I under viral infection

[0044] In order to analyze whether the broad-spectrum antiviral ability of nomilin is through regulating IFN-I-mediated antiviral signals, Raw264.7 and VERO cells (African green monkey kidney cells (Vero cells) are a kind of aneuploid cells) were plated on 12-well cell culture plates (about 0.5×10 6 / well), pretreated with different concentrations of nomilin (0.1, 1.0, 10, 50 μM) for 24 h, infected with VSV (MOI=0.1) virus, and incubated in a 37°C incubator for 20 h. 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 1 h, and incubated overnight at 4°C with primary antibody. After washing with PBST, secondary antibody was added and incubated at room temperature for 1 h. After washing with PBST, luminescent substrate was added for exposure, development and fixation to detect VSV-G protein levels. The results are shown in Figure 7 shown.

[0045] Raw264.7 and THP-1 cells were plated on 12-well cell culture plates (approximately 0.5 × 106 / well), pretreated with different concentrations of nomilin (0.1, 1.0, 10 μM) for 24 h, infected with VSV and SeV viruses, and incubated at 37°C for 12 h. 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 mRNA level of IFNβ. The results are shown in the figure. Figure 8 shown.

[0046] Raw264.7 cells were plated on 12-well cell culture plates (approximately 0.5 × 10 6 / well), pre-treated the cells with nomilin (10 μM) for 24 h, and then infected with SeV virus for 0, 6, and 12 h. 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 mRNA level of IFNβ. The results are shown in Fig. 9 shown.

[0047] Figures 6 to 9 The results showed that nomilin could significantly promote the production of IFN-I under viral infection, which indicated that nomilin may function by promoting the interferon signaling pathway.

[0048] Example 4 Nomilin increases the expression of interferon-induced genes (ISGs)

[0049] To further analyze whether nomilin upregulated the IFN-I-mediated signaling pathway, Raw264.7 cells were plated on 12-well cell culture plates (approximately 0.5 × 10 6 / well), pre-treated with nomilin (10 μM) for 24 h, and then infected with SeV virus for 0, 6, and 12 h. The cells were lysed with Trizol, and then total RNA was extracted from the cells and reversely transcribed into cDNA. The mRNA levels of representative ISGs (Cxcl10 / Ccl5) ​​genes were detected by fluorescence quantitative PCR (Real-time qPCR). The results are shown in Fig.10 shown.

[0050] Fig.10 The results showed that nomilin could significantly promote the production of antiviral ISGs (Cxcl10 / Ccl5) ​​under viral infection, indicating that nomilin may function by promoting the interferon signaling pathway.

[0051] Obviously, the above embodiments are merely examples for the purpose of clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. The use of Nomilin in the preparation of a drug for improving the body's antiviral immunity, characterized in that: The virus is VSV.

2. The use of Nomilin in the preparation of antiviral drugs, characterized in that: The virus is RSV, H1N1 or HSV-1.

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