Application of anti-inflammatory agent 35 in the preparation of drugs for preventing or treating African swine fever

Abstract

The present invention belongs to the technical field of African swine fever treatment, and specifically relates to a new use of a compound Anti-inflammatory agent 35 in the preparation of drugs for preventing or treating African swine fever. The present invention finds that Anti-inflammatory agent 35 can inhibit the expression of African swine fever virus protein p72, inhibit the number of copies of African swine fever virus genes, and inhibit the replication and infection of ASFV. Therefore, compound Anti-inflammatory agent 35 can be used to prepare drugs for preventing or treating African swine fever, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of African swine fever treatment, and specifically relates to a new use of a compound Anti-inflammatoryagent 35 in the preparation of a drug for preventing or treating African swine fever. Background Art

[0002] African swine fever (ASF) is an acute and virulent infectious disease caused by African swine fever virus (ASFV), characterized by fever and bleeding in pigs' organs. The mortality rate of domestic pigs is as high as 100%. Since there is still no effective vaccine and special treatment drug, once an African swine fever epidemic occurs, it can only be controlled by culling, but this method not only causes economic losses, but also cannot meet the needs of large-scale pig farming in my country. Therefore, how to effectively control the ASF epidemic is one of the huge challenges facing the world's pig farming industry, and it is also a major strategic issue that needs to be urgently solved in my country's ASF prevention and control.

[0003] p30 and p72 are key structural proteins in ASFV, which can neutralize the virus after it attacks susceptible cells and inhibit the virus's attachment, replication and internalization. Among them, p72 is a capsid protein and the main structural protein of African swine fever virus. It can protect viral nucleic acid from damage by nucleases or other physical and chemical factors in the environment. It also participates in the virus's infection process and has good immunogenicity. This protein is produced in the late stage of virus infection. As an important antigenic protein of ASFV, p72 is the main component of the virus's icosahedron, which is crucial for the formation of the virus capsid and participates in the virus binding to cells.

[0004] Anti-inflammatory agent 35 is an orally available curcumin analog with anti-inflammatory activity. Anti-infla mmatory agent 35 blocks mitogen-activated protein kinase (MAPK) signaling and nuclear translocation of NF-kB. Anti-infl ammatory agent 35 also inhibits neutrophil infiltration and pro-inflammatory cytokine production. Anti-inflammatory agent 35 significantly attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI) in an in vivo study.

[0005] The present invention unexpectedly discovered that Anti-inflammatory agent 35 can inhibit the expression of African swine fever virus p72, prevent the virus from invading host cells, and can be used to inhibit the early infection of ASFV; and Anti-inflammatory agent 35 can inhibit the replication of African swine fever virus and reduce the number of viral copies after African swine fever virus infection, and can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever. Summary of the invention

[0006] In view of the above technical problems, the purpose of the present invention is to provide a new use of compound Anti-inflammatoryagent 35 in the preparation of drugs for preventing or treating African swine fever. Specifically, it includes the following contents:

[0007] In a first aspect, the present invention provides the use of the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing African swine fever. The structural formula of the compound Anti-inflammatory agent 35 is shown in the following formula (I):

[0008]

[0009] Preferably, the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the expression of African swine fever virus p72 protein.

[0010] Preferably, the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the replication of African swine fever virus.

[0011] Preferably, the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier or dressing to prepare any pharmaceutically acceptable dosage form.

[0012] In a second aspect, the present invention provides the use of the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever. The structural formula of the compound Anti-inflammatory agent 35 is shown in the following formula (I):

[0013]

[0014] Preferably, the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the expression of African swine fever virus p72 protein.

[0015] Preferably, the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the replication of African swine fever virus.

[0016] Preferably, the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier or dressing to prepare any pharmaceutically acceptable dosage form.

[0017] The beneficial effects of the present invention are: the present invention unexpectedly discovered that Anti-inflammatory agent 35 can inhibit the expression of African swine fever virus p72, prevent the virus from invading host cells, and can be used to inhibit the early infection of ASFV; and Anti-inflammatory agent 35 can inhibit the replication of African swine fever virus, reduce the number of viral copies after African swine fever virus infection, and can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Cytotoxicity results of compound Anti-inflammatory agent 35;

[0019] Figure 2 The number of viral copies after African swine fever virus infection;

[0020] Figure 3 Results of RNA expression level of p72;

[0021] Figure 4 Results of p72 protein expression level. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further described below in conjunction with specific implementation methods. However, the protection scope of the present invention is not limited to the following embodiments.

[0023] The experiments described in the following examples were performed with biosafety permits and African swine fever laboratory activity permits:

[0024] In accordance with the relevant requirements of biosafety level 3 laboratories (BSL-3) and African swine fever-related biosafety, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences has obtained the permission from the Ministry of Agriculture and Rural Affairs of the People's Republic of China to conduct research on highly pathogenic ASFV pathogens and animals through the Biosafety Committee of the Lanzhou Veterinary Research Institute, the Laboratory Animal Ethics Committee, the Biosafety Committee of the Chinese Academy of Agricultural Sciences, the Laboratory Animal Ethics Committee of the Lanzhou Veterinary Research Institute, and the Biosafety Committee of the Lanzhou Veterinary Research Institute. The research has been filed with the Ministry of Agriculture and Rural Affairs of the People's Republic of China and meets the national biosafety level requirements.

[0025] The experimental cells and virus sources described in the following examples are:

[0026] Primary porcine alveolar macrophages (PAM) and primary bone marrow macrophages (BMDM) were obtained from 2-4 month old healthy SPF Bama miniature pigs. After aseptic collection of cells, red blood cells were removed using red blood cell lysis buffer (purchased from Biosharp). After low-speed centrifugation, the supernatant was discarded and the cell pellet was resuspended in RPMI 1640 complete medium (purchased from Gibco) containing 10% FBS (purchased from PAN) and cultured in a 37°C, 5% CO2 incubator.

[0027] The ASFV CN / GS / 2018 isolate was from the National African Swine Fever Regional Laboratory (Lanzhou), belonging to genotype II, with a virus titer of 5×10 7 TCID 50 / mL, which is the 4th generation seed virus after PAM cell expansion.

[0028] The compound Anti-inflammatory agent 35 was purchased from MedChemExpress.

[0029] Unless otherwise specified, other reagents in the experiment are common commercially available reagents; unless otherwise specified, the operations in the experiment are all methods known in the art.

[0030] Example 1 Cytotoxicity of Anti-inflammatory Agent 35

[0031] The stable in vitro cell screening system was constructed and the cytotoxicity of the small molecule compound Anti-inflammatory age nt 35 was detected by CCK-8 method. Porcine alveolar macrophages (PAM, 2×10 5 / well), overnight culture, add different concentrations of compound Anti-inflammatory agent 35 (100nM, 200nM, 300nM, 500nM, 1000nM, 5000nM) to the wells, set up blank wells (containing only culture medium) and control wells (containing cells and culture medium), incubate the culture plate in the incubator for 16h, add 10μL of CCK-8 solution to each well of the plate, incubate the culture plate in the incubator for 1-4h, and mix gently on a shaker before reading the plate. Then read the absorbance at 450nm with an ELISA reader to calculate the cell survival rate.

[0032] The results are as follows Figure 1 As shown, when the concentration of compound Anti-inflammatory agent 35 is less than or equal to 1000nM, it has almost no toxicity to cells and has good safety.

[0033] Example 2 Effect of Anti-inflammatory Agent 35 on African Swine Fever Virus Infection, Replication and Gene Transcription Expression

[0034] 1. Gene copy number

[0035] Porcine alveolar macrophages (PAM, 1×10 6 / well), the experimental group was treated with compound Anti-inflammatory agent 35 (500nM) and Anti-inflammatory agent 35 (1000nM) for 16h, and the control group was treated with DMSO (1%) for 16h. The treated experimental and control groups were infected with ASFV CN / GS / 2018 strain (0.1MOI) for 24h. The cells were collected, and the cell culture was collected after repeated freezing and thawing at -80℃, centrifuged, and the supernatant was taken. The effect of compound Anti-inflammatory agent 35 on ASFV virus replication was detected by Q-PCR method, and the ASFV virus gene copy number was calculated by formula.

[0036] The total volume of the Q-PCR reaction system is 25 μL, including 12.5 μL of 2×Pro Taq HS Probe Premix, 1 μL of upstream and downstream primers, 1 μL of ASFV probe primer, 3 μL of template, and sterile deionized water is added to the total volume of 25 μL.

[0037] The reaction program was: 95°C, 2 min; 95°C, 7 s, 60°C, 12 s, 3 cycles; 95°C, 6 s, 58°C, 11 s, 40 cycles.

[0038] Upstream primer: 5'-CTGCTCATGGTATCAATCTTATCGA-3'; downstream primer: 5'-GATACCACAAGATCAGCCGT-3'.

[0039] The results of gene copy number detection are as follows Figure 2 As shown, the virus replication ability was found to be high 24 hours after ASFV infection; after adding Anti-inflammatory agent 35 (500nM) and Anti-inflammatory agent 35 (1000nM), the compound Anti-inflammatory agent 35 was able to inhibit the ASFV virus gene copy number compared with the Mock control group; and as the concentration of Anti-inflammatory agent 35 increased, the inhibitory effect on the ASFV replication ability became more obvious. The above results show that the compound Anti-inflammatory agent 35 can significantly inhibit the African swine fever virus gene copy number and inhibit ASFV virus replication while ensuring safety (at a concentration of less than or equal to 1000nM).

[0040] 2. p72 RNA transcription level

[0041] Porcine alveolar macrophages (PAM, 1×10 6 / well), the experimental group was treated with compound Anti-inflammatory agent 35 (500nM) and Anti-inflammatory agent 35 (1000nM) for 16h, and the control group was treated with DMSO (1%) for 16h. The treated experimental group and control group were infected with ASFV CN / GS / 2018 strain (MOI=0.1), cultured at 37°C, 5% CO2 for 24h, and the cell culture was collected. The cells were washed once with PBS, centrifuged, and the supernatant was discarded. Total RNA was extracted by Trizol method, and cDNA was synthesized by iScriptTM Reverse Transcription Supermix for RT-qPCR (Bio-Rad) kit, and the RNA expression difference of p72 was detected by Q-PCR method.

[0042] The total volume of the Q-PCR reaction system was 20 μL, including 10 μM upstream and downstream primers, 90 ng cDNA, 10 μL SYBR Green supermix (2×) (Bio-Rad), and sterile deionized water was added to 20 μL. The reaction conditions were: 95°C, 3 min; 95°C, 5 s, 60°C, 30 s, 40 cycles, where the p72 primer sequences were: upstream primer 5'-CTGCTCATGGTATCAATCTTATCGA-3'; downstream primer 5'-GATACCACAAGATCRGCCGT-3'.

[0043] The results of the p72 RNA expression experiment are as follows Figure 3 As shown, the compound Anti-inflammatory agent 35 can inhibit the RNA expression level of p72 in the African swine fever virus gene, and when the dose of the compound Anti-inflammatory agent 35 is 1000nM, the inhibition rate of the RNA expression level of p72 is nearly 70%.

[0044] 3. p72 protein expression level

[0045] Porcine alveolar macrophages (PAM, 1×10 6 / well), the experimental group was treated with compound Anti-inflammatory agent 35 (500nM) and Anti-inflammatory agent 35 (1000nM) for 16h, and the control group was treated with DMSO (1%) for 16h. The treated experimental group and control group were infected with ASFVCN / GS / 2018 strain (MOI=0.1), cultured at 37°C, 5% CO2 for 24h, and the cell culture was collected. The cells were washed once with PBS, centrifuged, and the supernatant was discarded. Total protein was extracted, and the difference in p72 protein expression was detected by western-blotting method.

[0046] The experimental results of p72 protein expression level are as follows Figure 4 As shown in the figure, compared with the Mock group, the expression level of p72 protein was higher after ASFV virus infection; after treatment with the compound Anti-inflammatory agent 35, the expression of p72 protein decreased; and as the concentration of Anti-inflammatory agent 35 increased, the expression of p72 protein decreased more significantly. This shows that the use of the compound Anti-inflammatory agent 35 can significantly inhibit the expression of p72 and inhibit the replication of ASFV virus.

[0047] In summary, the compound Anti-inflammatory agent 35 of the present invention can significantly inhibit the expression of African swine fever virus protein p72, prevent the virus from invading host cells, and can be used to inhibit the late infection of ASFV. In addition, the compound Anti-inflammatory agent 35 can significantly inhibit the copy number of ASFV virus genes and can be used as an inhibitor of African swine fever virus. The combined use of the inhibitor is beneficial to the prevention or treatment of African swine fever.

[0048] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made based on the structure, features and principles of the patent scope of the present invention should be included in the patent application scope of the present invention.

Claims

1. Use of the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing African swine fever, wherein the structural formula of the compound Anti-inflammatory agent 35 is shown in the following formula (I):

2. The use according to claim 1, characterized in that The compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the expression of African swine fever virus p72 protein.

3. The use according to claim 1, characterized in that The compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the replication of African swine fever virus.

4. The use according to claim 1, characterized in that The compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier to prepare any pharmaceutically acceptable dosage form.

5. Use of the compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating African swine fever, wherein the structural formula of the compound Anti-inflammatory agent 35 is shown in the following formula (I):

6. The use according to claim 5, characterized in that The compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the expression of African swine fever virus p72 protein.

7. The use according to claim 5, characterized in that The compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof inhibits the replication of African swine fever virus.

8. The use according to claim 1, characterized in that The compound Anti-inflammatory agent 35 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier to prepare any pharmaceutically acceptable dosage form.

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