Application of salicylic acid-induced stress particle assembly in inhibition of African swine fever virus proliferation

Abstract

The invention discloses application of salicylic acid-induced stress particle assembly in inhibition of African swine fever virus proliferation, and belongs to the technical field of medicines. Salicylic acid is phenolic acid with a simple structure, is derived from willow barks earliest, has the effects of resisting inflammation, resisting bacteria, removing acnes and the like, and has no toxic and side effects such as obvious damage to physiological functions of organisms. The stress particles are membrane-free organelles formed in cytoplasm through liquid-liquid phase separation when eukaryotic cells cope with various environmental stresses. Experiments prove that salicylic acid shows remarkable anti-African swine fever virus activity in in-vitro experiments, and the anti-virus ability is mainly caused by inducing stress particles to form and enhancing type I interferon related natural immunity, so that salicylic acid has a good application prospect in preparation of anti-African swine fever virus drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of salicylic acid in inhibiting African swine fever virus proliferation by inducing stress granules. BACKGROUND

[0002] African swine fever (ASF) is an acute, hemorrhagic, and virulent infectious disease caused by African swine fever virus (ASFV) in pigs, which was first reported in Kenya, East Africa, in 1921. According to different clinical symptoms, it can be divided into hyperacute type, acute type, subacute type and chronic type. The main symptoms are body temperature rise, skin bleeding spots or cyanosis, vomiting and hemorrhagic diarrhea, etc. The main pathological changes are splenomegaly, lymph node enlargement and hemorrhage. Due to its 100% mortality rate, it has caused serious harm to the global pig breeding industry.

[0003] African swine fever virus is the only member of the African swine fever virus family and the only known DNA arbovirus. The virus is a large double-stranded DNA virus that mainly replicates in macrophages. Its genome is about 170-193 kb, containing 150-167 open reading frames, encoding 150-200 proteins. At present, there is no safe and effective vaccine to control the disease, so in order to reduce the economic losses caused by African swine fever, in addition to the development of reliable vaccines, the application of drugs for prevention and treatment is also an indispensable disease control approach.

[0004] Stress granules are a kind of membrane-free organelles (or biological macromolecular condensates) formed in the cytoplasm of eukaryotic cells when they respond to various environmental stresses (oxidative stress, heat shock, viral infection, nutrient deficiency, etc.). They are mainly composed of translation-stalled mRNAs, RNA-binding proteins, translation initiation factors and other related proteins. Stress granules can enhance the transduction of natural immune signaling pathways and play an antiviral role.

[0005] Salicylic acid, a natural compound extracted from willow bark, is a simple phenolic acid that can be artificially synthesized. The chemical formula of salicylic acid is C7H6O3, and the chemical structure formula is as follows:

[0006]

[0007] Salicylic acid has been widely used in the field of cosmetics due to its keratolytic, desquamatory, and anti-acne functions. In addition, salicylic acid has pharmacological activities other than skin care, such as antibacterial, anti-inflammatory, and analgesic effects. Studies have shown that salicylic acid can induce the formation of stress granules in plant cells and also in Hela cells. However, there is no report or patent on whether salicylic acid can promote the formation of stress granules in pig-derived cells and whether the use of salicylic acid can inhibit the proliferation of African swine fever virus. SUMMARY

[0008] To solve the above problems, the application provides the application of salicylic acid in inducing stress granule assembly and inhibiting the proliferation of African swine fever virus, and specifically, the application of salicylic acid in host cell immortalized porcine alveolar macrophage 3D4 / 21 in the preparation of a drug for inducing stress granule assembly to enhance type I interferon-related natural immunity and thereby inhibit the proliferation of African swine fever virus.

[0009] The application is achieved by the following technical solutions:

[0010] The application of salicylic acid in inducing stress granule assembly and inhibiting the proliferation of African swine fever virus.

[0011] The application of salicylic acid in the preparation of a drug for inhibiting the expression of B646L (p72) and CP204L (p30) genes and proteins of African swine fever virus.

[0012] The application of salicylic acid in the preparation of a drug for promoting the assembly of stress granules in 3D4 / 21 cells.

[0013] The application of salicylic acid in the preparation of a drug for promoting the expression of antiviral cytokines IFN-β, ISG15, ISG54, ISG56, and MX1.

[0014] The application of salicylic acid in the preparation of a drug for enhancing the expression levels of JAK1, TYK2, STAT1, STAT2, cGAS, and IRF3 proteins.

[0015] The application of salicylic acid in the preparation of a drug for enhancing the phosphorylation levels of JAK1, STAT2, TBK1, and IRF3.

[0016] Preferably, the effective use concentration of salicylic acid is 10-50 μM.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The present application proves that the effect of salicylic acid on inhibiting the proliferation of African swine fever virus in its host cell, immortalized porcine alveolar macrophage 3D4 / 21, is significant, the expression of African swine fever virus B646L and CP204L genes is inhibited, the expression of African swine fever virus p72 and p30 proteins is inhibited, and the titer of African swine fever virus is also inhibited.

[0019] The present application explores and finds that the anti-African swine fever virus effect of salicylic acid is produced by inducing stress granule assembly to enhance the transduction of type I interferon signaling pathway and promoting the expression of antiviral cytokines IFN-β, ISG15, ISG54, ISG56 and MX1, which provides a scientific and reliable theoretical basis for the clinical treatment of African swine fever. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A CCK-8 result graph of the effect of salicylic acid on the cell activity of 3D4 / 21 cells;

[0021] Figure 2 A fluorescence quantitative PCR experiment result graph of the expression level of African swine fever virus B646L and CP204L genes inhibited by salicylic acid on 3D4 / 21 cells;

[0022] Figure 3 A Western Blot experiment result graph of the expression level of African swine fever virus p72 and p30 proteins inhibited by salicylic acid on 3D4 / 21 cells;

[0023] Figure 4 A TCID50 experiment result graph of the titer of African swine fever virus inhibited by salicylic acid on 3D4 / 21 cells;

[0024] Figure 5 An experiment result graph of stress granule assembly promoted by salicylic acid on 3D4 / 21 cells;

[0025] Figure 6 An experiment result graph of the expression of antiviral cytokines IFN-β (A), ISG15 (B), ISG54 (C), ISG56 (D) and MX1 (E) of 3D4 / 21 cells promoted by salicylic acid under African swine fever virus infection;

[0026] Figure 7 An experiment result of the protein expression level of JAK1, TYK2, STAT1 and STAT2 (A) and cGAS and IRF3 (B) and the phosphorylation level of JAK1, STAT2 (A), TBK1 and IRF3 (B) of 3D4 / 21 cells promoted by salicylic acid under African swine fever virus infection. DETAILED DESCRIPTION

[0027] The present application will be further described below in combination with the drawings:

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from commercial channels unless otherwise specified. The African swine fever virus strain used is GZ201801. All tests are carried out in a biological safety protection third-level laboratory (BSL-3) according to standard operation procedures.

[0029] Example 1: Effect of salicylic acid on cell activity of 3D4 / 21 cells.

[0030] 3D4 / 21 cells were inoculated into a 96-well cell plate and cultured in a 37°C, 5% CO2 cell incubator for 24 h. Then, the cells were divided into a control group, an experimental group, and a blank group. The control group cells were added with 0.1% (v / v) dimethyl sulfoxide (DMSO) without obvious cytotoxicity, the experimental group cells were added with cell maintenance liquid containing 1, 5, 10, 20, 50, 100, and 200 μM concentrations, respectively, and the blank group was not inoculated with cells but contained maintenance liquid with a non-cytotoxic dose of dimethyl sulfoxide. Then, the cells were placed in a cell incubator for 72 h. After 72 h, 10 μL of CCK-8 reagent was added to each well, the cell plate was gently shaken to mix the reagent, and then the plate was placed in a 37°C cell incubator for 2 h. After incubation, the absorbance at 450 nm was measured using a microplate reader. The cell survival percentage was calculated by the formula [(experimental group absorbance-blank group absorbance) / (control group absorbance-blank group absorbance)] x 100%, and the cell activity at the corresponding salicylic acid concentration was calculated. As shown in Figure 1 the results showed that salicylic acid concentrations of 100 μM and below had no significant effect on the activity of 3D4 / 21 cells.

[0031] Example 2: Inhibition of African swine fever virus proliferation in 3D4 / 21 cells by salicylic acid.

[0032] 3D4 / 21 cells were divided into an experimental group and a control group. The culture medium used in the experimental group throughout the experiment was added with 10, 20, and 50 μM of salicylic acid, and the culture medium used in the control group throughout the experiment was added with 0.1% (v / v) dimethyl sulfoxide without obvious cytotoxicity. First, both groups were incubated at 37°C for 2 h. Then, both the experimental group and the control group were inoculated with 0.1 MOI of African swine fever virus and incubated at 37°C for 2 h. The virus was discarded, and the cells were washed with PBS for 3 times. After being cultured in a 37°C cell incubator for 48 h, the cells and supernatant were collected for fluorescence quantitative PCR, Western Blot, and TCID50 experiments. As shown in Figure 2As shown, the results of the qPCR experiment showed that the gene expression levels of B646L and CP204L were significantly inhibited in the experimental group with the addition of salicylic acid compared with the control group. As shown in Figure 3 As shown, the p72 and p30 protein expression levels also showed consistent results. As shown in Figure 4 As shown, the results of the TCID50 experiment showed that salicylic acid also inhibited the titer of the virus. The results showed that salicylic acid can significantly inhibit the proliferation of ASFV in 3D4 / 21 cells. The data represent the results of three independent experiments, each containing three biological replicates. The significance of the difference in data was analyzed using Student's t-test. ns indicates no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0033] Example 3: Salicylic acid promotes stress granule assembly in 3D4 / 21 cells.

[0034] 3D4 / 21 cells were cultured on cell slides and divided into an experimental group, a negative control group, and a positive control group. The experimental group cells were added with 50 μM salicylic acid in the culture medium; the negative control group cells were added with 0.1% (v / v) dimethyl sulfoxide in the culture medium; and the positive control group cells were added with sodium arsenite (20 μM) in the culture medium. The three groups of cells were incubated at 37°C for 2 h, and then the cells were fixed with 4% paraformaldehyde and broken with 0.25% Triton X-100. After blocking with 1% BSA for 1 h, the cells were incubated with diluted G3BP1 polyclonal antibody at 37°C for 1 h. After incubation, the cells were incubated with diluted goat anti-rabbit secondary antibody at 37°C for 45 min; after incubation, the cells were stained with nuclei, and finally the slides were sealed; the formation of stress granules was observed using a laser confocal microscope. As shown in Figure 5 The results showed that the addition of salicylic acid can significantly promote the formation of stress granules in 3D4 / 21 cells.

[0035] Example 4: Salicylic acid promotes the expression of antiviral cytokines under African swine fever virus infection.

[0036] 3D4 / 21 cells were divided into an experimental group and a control group. The culture medium used in the experimental group throughout the experiment was added with 10, 20, and 50 μM salicylic acid, and the culture medium used in the control group throughout the experiment was added with 0.1% (v / v) dimethyl sulfoxide without obvious cytotoxicity. First, the two groups of cells were incubated at 37°C for 2 h. Then, both the experimental group and the control group were inoculated with 0.1 MOI African swine fever virus and incubated at 37°C for 2 h, and the virus was discarded and the cells were washed with PBS 3 times. After incubation at 37°C in a cell incubator for 48 h, the cells were collected. The cells were used for qPCR and Western Blot experiments. As shown in Figure 6As shown, the results of the quantitative PCR experiment show that the gene expression levels of IFN-β, ISG15, ISG54, ISG56 and MX1 in the experimental group with the addition of salicylic acid are significantly increased compared with the control group.

[0037] As shown, the results of the quantitative PCR experiment show that the gene expression levels of IFN-β, ISG15, ISG54, ISG56 and MX1 in the experimental group with the addition of salicylic acid are significantly increased compared with the control group. Figure 7 As shown, the results of the Western Blot experiment show that the protein expression levels of JAK1, TYK2, STAT1, STAT2, cGAS and IRF3 and the phosphorylation levels of JAK1, STAT2, TBK1 and IRF3 in the experimental group with the addition of salicylic acid are significantly enhanced and dose-dependent compared with the control group.

[0038] The results show that salicylic acid can positively regulate IFN-β and ISGs and enhance antiviral innate immunity. The data represent the results of three independent experiments, each containing three biological replicates. The significance of the differences in the data was analyzed using Student's t-test. ns represents no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0039] From the above examples, it is concluded that the present application proves that salicylic acid has a significant effect on inhibiting the proliferation of African swine fever virus in its host cell, immortalized porcine alveolar macrophage 3D4 / 21, inhibits the expression of African swine fever virus B646L and CP204L genes, inhibits the expression of African swine fever virus p72 and p30 proteins, and also inhibits the titer of African swine fever virus.

[0040] The present application explores that the anti-African swine fever virus effect of salicylic acid is produced by inducing stress granule assembly to enhance the transduction of type I interferon signaling pathway and promoting the expression of antiviral cytokines IFN-β, ISG15, ISG54, ISG56 and MX1, which provides a scientific and reliable theoretical basis for the clinical treatment of African swine fever.

[0041] The above is merely a specific implementation of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Application of salicylic acid-induced stress particle assembly in inhibiting African swine fever virus proliferation.

2. Application of salicylic acid in the preparation of drugs that inhibit the gene expression and protein expression of African swine fever virus B646L (p72) and CP204L (p30).

3. Application of salicylic acid in the preparation of drugs that promote the assembly of stress granules in 3D4 / 21 cells.

4. Application of salicylic acid in the preparation of drugs that promote the expression of antiviral cytokines IFN-β, ISG15, ISG54, ISG56, and MX1.

5. Application of salicylic acid in the preparation of drugs that enhance the expression levels of JAK1, TYK2, STAT1, STAT2, cGAS, and IRF3 proteins.

6. Application of salicylic acid in the preparation of drugs that enhance the phosphorylation levels of JAK1, STAT2, TBK1, and IRF3.

7. The application according to any one of claims 1 to 6, characterized in that, The effective concentration of the salicylic acid is 10-50 μM.

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