Application of bexarotene in preparation of medicine for resisting PRRSV (porcine reproductive and respiratory syndrome) or treating PRRS (porcine reproductive and respiratory syndrome)

Abstract

The invention discloses an application of bexarotene in preparation of a medicine for resisting PRRSV (porcine reproductive and respiratory syndrome) or treating PRRS (porcine reproductive and respiratory syndrome). The invention provides a new application of the existing antitumor drug bexarotene, and finds that the bexarotene has an anti-PRRSV effect, can significantly inhibit the proliferation and replication of PRRSV, and reduces the expression of PRRSV-N protein and the infectivity of PRRSV; meanwhile, the compound also has a broad-spectrum anti-PRRSV effect, can inhibit various PRRSV strains with different pathogenicity, and particularly has a remarkable antiviral effect on high-pathogenicity HP-PRRSV; moreover, the compound hardly has cytotoxicity, and can be better used for resisting PRRSV infection and relieving the porcine reproductive and respiratory syndrome. The bexarotene is small in molecular weight and can easily penetrate through a cell membrane to enter cells to play an antiviral role, so that the bexarotene does not need to be delivered into the cells by a carrier, a foundation is laid for developing more novel medicines for resisting PRRSV, and more methods and means are provided for preventing and treating the PRRSV.

Description

Technical Field

[0001] The present invention belongs to the field of biological and pharmaceutical technology, and more specifically, relates to the use of bexarotene in the preparation of drugs for resisting PRRSV or treating blue ear disease in pigs. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious viral infectious disease of pigs caused by the porcine reproductive and respiratory syndrome virus (PRRSV), commonly known as "blue ear disease". PRRS was first reported in the United States in the late 1980s and is one of the pathogens that poses a great threat to the world's pig industry. The disease can cause fever, breathing difficulties, and spotty congestion or cyanosis of the skin on the limbs, and even death in pigs. It can cause abortion in sows, causing great harm to the health of the pig herd and seriously damaging the economic interests of the pig industry. In April 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) broke out in Jiangxi Province, my country. After that, the disease quickly spread to many provinces and cities across the country, with a wide epidemic area and pigs of all ages. So far, most pig farming areas in the country are still threatened by the disease, with an incidence rate generally above 50% and a mortality rate between 20% and 100%.

[0003] PRRS is an immunosuppressive disease. PRRSV can target host immune cells and weaken the host's defense ability, resulting in increased susceptibility to primary and secondary pathogens, which can affect the growth efficiency of pigs and increase morbidity and mortality. To control the prevalence of PRRS in my country, an inactivated vaccine was developed using the first PRRSV isolate in my country, CH-1a, and was put into use in pig farms after registration in 2000. In 2007, a live attenuated vaccine derived from the CH-1a isolate was also approved for registration in China. Shortly after the outbreak of atypical PRRS, China developed an inactivated vaccine using an isolated virus (JXA1). However, the effect of these vaccines in controlling PRRS was not satisfactory. In the vaccinated pig farms, clinical cases related to production failure in pregnant sows and respiratory disorders in suckling piglets occurred frequently, and they did not provide effective clinical immune protection as expected. In general, the existing vaccines cannot effectively prevent and control PRRS, so it is particularly important to find effective anti-PRRS virus drugs, which may be a new strategy for the prevention and control of PRRS.

[0004] Since the existing commercial vaccines have poor immune effects, the cross-protection ability of the vaccines against different strains is not complete, and the attenuated vaccines may revert to stronger virulence and recombinate with wild viruses into new strains during replication in pigs, there is a lack of drugs that can resist PRRSV. Therefore, it is of great significance to develop more safe, efficient and broad-spectrum anti-PRRSV drugs. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the existing PRRSV vaccines, such as poor immune effect and weak protection ability, and the insufficiency of the existing anti-PRRSV drugs, and to provide the use of bexarotene in the preparation of drugs for anti-PRRSV or for treating porcine blue ear disease.

[0006] The purpose of the present invention is to provide a new application of the old drug bexarotene in resisting PRRSV.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a new application of the existing anti-tumor drug bexarotene in anti-PRRSV. Studies have found that bexarotene can significantly inhibit the replication and proliferation of PRRSV, and its EC50 is 2.946 μM. It can inhibit the expression of PRRSV-N protein, and also has a significant inhibitory effect on the recombinant virus PRRSV-TA-12Gluc, and can reduce the virus titer; and it has a significant anti-PRRSV effect on PRRSV strains of different pathogenicity, and has a broad spectrum, especially for HP-PRRSV. It still has a good antiviral effect, can be used to prepare drugs for relieving or treating porcine blue ear disease, and becomes a new strategy for effective prevention and control of PRRS. At the same time, since bexarotene is an oral drug that can be absorbed through the intestine, it can be premixed in feed for administration, and the administration method is extremely convenient. In addition, the pharmacokinetics of bexarotene show that the drug reaches a maximum plasma concentration about 2 hours after oral administration, and can quickly take effect, which lays a foundation for the development of more new drugs against PRRSV and provides more methods and means for the prevention and treatment of porcine blue ear disease.

[0009] Bexarotene studied in the present invention is a high-affinity selective retinoid X receptor (RXR) agonist, an anti-tumor drug, and is clinically used to treat cutaneous T-cell lymphoma. The chemical name of bexarotene is 4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)vinyl]benzoic acid, and the molecular formula is C 24 H 28 O2, molecular weight is 348.47800. Its structural formula is:

[0010]

[0011] Therefore, the present invention provides the use of bexarotene as an anti-PRRSV preparation.

[0012] The invention provides application of bexarotene in preparing an anti-PRRSV drug.

[0013] The present invention provides application of bexarotene in preparing medicine for preventing PRRSV infection.

[0014] The present invention provides application of bexarotene in preparing a medicine for alleviating or treating porcine blue ear disease.

[0015] Furthermore, the drug can inhibit the replication and proliferation of PRRSV.

[0016] Furthermore, the drug can inhibit the expression of viral PRRSV-N protein.

[0017] Furthermore, the drug can reduce viral titer.

[0018] Furthermore, the porcine blue ear disease is blue ear disease caused by HP-PRRSV or blue ear disease caused by a virus variant.

[0019] Preferably, the virus variant strain is WuH3 strain, SD16 strain, TA-12 strain, JXA1 strain or NADC30 strain.

[0020] Furthermore, the drug contains a pharmaceutically acceptable carrier or excipient.

[0021] Furthermore, the dosage form of the drug can be prepared into an injection preparation or an oral preparation, such as powder, capsule, tablet, etc., but is not limited to powder, capsule, tablet, and can be any dosage form acceptable to veterinary clinics.

[0022] Preferably, the concentration of the active ingredient of bexarotene in the drug is 1 to 20 μM.

[0023] More preferably, the concentration of the active ingredient of bexarotene in the drug is 2 to 10 μM.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides a new application of the existing anti-tumor drug bexarotene, and it is found that bexarotene has an anti-PRRSV effect, can significantly inhibit the proliferation and replication of porcine blue ear disease virus PRRSV, and reduce the infectivity of PRRSV; at the same time, it also has a broad-spectrum anti-PRRSV effect, can inhibit the expression of PRRSV-N protein of a variety of PRRSV strains with different pathogenicity, especially has a significant antiviral effect against highly pathogenic HP-PRRSV; and has almost no cytotoxicity, can be better used to resist the infection of porcine blue ear virus and alleviate porcine blue ear disease. Since bexarotene has a small molecular weight, it is very easy to pass through the cell membrane into the cell and exert an antiviral effect, so it does not need a carrier to be delivered into the cell, which lays a foundation for the development of more new anti-PRRSV drugs and provides more methods and means for the prevention and treatment of porcine blue ear disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the toxicity of bexarotene to Marc-145 cells and the drug half-effective concentration curve.

[0027] Figure 2 Fluorescence quantitative PCR (RT-PCR) was used to detect the expression level of PRRSV-N mRNA.

[0028] Figure 3 is the virus infection ratio of the recombinant virus PRRSV-TA-12Gluc.

[0029] Figure 4 Western-blot was used to detect the expression level of PRRSV-N protein.

[0030] Figure 5 This is a fluorescent image of the PRRSV-N protein expression level detected by immunofluorescence IFA (DAPI in the image indicates the cell nucleus, and Merge indicates the combination of PRRSV-N protein and cell nucleus).

[0031] Figure 6 This is a graph showing the results of the plaque assay to detect virus titer.

[0032] Figure 7 is the expression level of PRRSV-N protein of different PRRSV strains. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The cells and PRRSV strains used in the following examples were all derived from and stored in the laboratory of the research group of the present invention; bexarotene was provided by MedChemExpress (MCE), CAS No. 153559-49-0.

[0036] For the statistical analysis of the following examples, all experiments were repeated at least 3 times independently, and the results were expressed as mean values ​​and standard errors, and analyzed using one-way analysis of variance and T test; all statistical analyses used P<0.05 as the test standard for significant statistical differences, and the analysis software was SPSS16.0 and GraphPad Prism5.

[0037] Example 1 Cytotoxicity and drug half effective concentration of bexarotene

[0038] 1. Cytotoxicity testing of drugs

[0039] CCK-8 kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was used for drug cytotoxicity detection. Marc-145 cells (or PAMs cells) were cultured with DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 60-70%, the culture medium was discarded, and nutrient solution containing bexarotene dilution solution (diluted with DMEM culture medium, the dilution concentrations were: 2.5, 5.0, 10.0, 20.0 μM) was added for 36 hours, and a negative control group and a blank control group were set at the same time, and then CCK-8 solution was added to continue culturing for 2 hours; the absorbance value at 450nm was detected using a multifunctional microplate reader, and a bexarotene cytotoxicity graph was drawn, with the cell activity of the negative control group as 100%, and the cell survival percentage = [(absorbance value of cells treated with bexarotene in multiple dilutions - absorbance value of the blank control group) / (absorbance value of the negative control group - absorbance value of the blank control group)] × 100%, which is the relative cell activity of bexarotene at different concentrations.

[0040] The cytotoxicity of bexarotene Figure 1 As shown, the CC50 curve of the cells in the figure shows that when the concentration of bexarotene reaches 20.0 μM, it still does not show cytotoxicity, indicating that bexarotene has little toxicity to cells.

[0041] 2. Medication half effective concentration

[0042] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 70-75%, the culture medium was discarded, and replaced with DMEM culture medium containing 2% fetal bovine serum containing bexarotene multiple dilution solution (diluted with DMEM culture medium, the dilution concentrations were: 2.5, 5.0, 10.0, 20.0 μM), HP-PRRSV was inoculated at a multiplicity of infection MOI = 0.5, and a control group without bexarotene and a negative control group were set up, and then cultured in a 37°C, 5% CO2 incubator, washed 3 times with PBS after 36 hours, and the cells were fixed and permeabilized with ice methanol. Subsequently, immunofluorescence analysis (IFA) was used to detect the expression level of PRRSV-N protein. The PRRSV-N fluorescence intensity of the control group without bexarotene was taken as 100%, and the formula was used to calculate: effective concentration = (PRRSV-N fluorescence intensity of the experimental groups with different concentrations of bexarotene / PRRSV-N fluorescence intensity of the control group without bexarotene). The drug effective concentration curve was drawn and the drug half effective concentration was calculated.

[0043] The results of the median effective concentration of bexarotene are as follows Figure 1 As shown, according to the drug half effective concentration curve EC50, the half effective concentration of bexarotene is 2.946 μM, indicating that bexarotene has a good anti-PRRSV effect.

[0044] Example 2 Antiviral Test of Bexarotene

[0045] 1. Fluorescence quantitative PCR (RT-PCR) detection

[0046] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 70-75%, the culture medium was discarded, and replaced with DMEM culture medium containing 2% fetal bovine serum containing bexarotene multiple dilution solution (diluted with DMEM culture medium, the dilution concentrations were: 5.0, 7.5, 10.0 μM), HP-PRRSV was inoculated at a multiplicity of infection MOI=0.5, and a control group without bexarotene was set up, and then cultured in a 37°C, 5% CO2 incubator, washed 3 times with PBS after 36 hours, and then the cells were digested with 0.25% trypsin, RNA in the cells was extracted, and quantitative reverse transcription was performed after measuring the RNA concentration. Finally, the PRRSV-N mRNA expression level was detected by fluorescent quantitative PCR.

[0047] Test results such as Figure 2 As shown, bexarotene has a significant inhibitory effect on the expression of PRRSV-N mRNA, and as the concentration of bexarotene increases, the inhibitory effect becomes more obvious and presents a dose-dependency, indicating that bexarotene can inhibit the expression of PRRSV-N mRNA.

[0048] 2. Detection of recombinant virus luciferase protein expression level

[0049] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 70-75%, the culture medium was discarded, and replaced with DMEM culture medium containing 2% fetal bovine serum containing bexarotene multiple dilution (dilution concentrations were: 5.0, 7.5, 10.0 μM), and PRRSV-TA-12Gluc recombinant virus (the strain was donated by Professor Li Yanhua's research group of Yangzhou University) was inoculated at a multiplicity of infection MOI=0.5, and a control group without bexarotene was set up, and then cultured in a 37°C, 5% CO2 incubator. After 36 hours, the cell supernatant was collected in an ELISA plate, and after adding the coelenterazine reaction substrate for reaction, the absorbance at 480 nm was detected using a multifunctional ELISA instrument, and the absorbance of the control group without bexarotene was taken as 100%. After treating MARC-145 cells infected with TA-12 recombinant PRRSV expressing Gaussia luciferase (Gluc) gene with different concentrations of bexarotene, the expression level of Gluc protein was detected, and the expression amount of Gaussia luciferase protein was calculated and converted into virus infection ratio.

[0050] The results are as follows Figure 3 As shown, different concentrations of bexarotene can significantly inhibit the expression of the luciferase protein of the recombinant virus PRRSV-TA-12Gluc, indicating that bexarotene has a significant inhibitory effect on the recombinant virus PRRSV-TA-12Gluc.

[0051] 3. Western blot detection

[0052] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 70-75%, the culture medium was discarded, and replaced with DMEM culture medium containing 2% fetal bovine serum containing bexarotene multiple dilution solution (diluted with DMEM culture medium, the dilution concentrations were: 5.0, 7.5, 10.0 μM), HP-PRRSV was inoculated at a multiplicity of infection MOI=0.5, and a control group without bexarotene and a blank control group were set up, and then cultured in a 37°C, 5% CO2 incubator, washed 3 times with PBS after 36 hours, and then the cells were digested with 0.25% trypsin, the protein concentration was determined after the cells were lysed, and the expression level of PRRSV-N protein was detected by Western blot.

[0053] Test results such as Figure 4As shown, bexarotene inhibited the expression of PRRSV-N protein, and when the concentration of bexarotene reached 7.5 μM, PRRSV-N protein was almost undetectable, indicating that bexarotene has excellent anti-PRRSV ability.

[0054] 4. Immunofluorescence (IFA) detection

[0055] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 70-75%, the culture medium was discarded, and replaced with DMEM culture medium containing 2% fetal bovine serum containing 10.0 μM bexarotene, HP-PRRSV was inoculated at a multiplicity of infection MOI=0.5, and a control group without bexarotene and a blank control group were set up, and then cultured in a 37°C, 5% CO2 incubator, washed three times with PBS after 36 hours, and then fixed and permeabilized with ice methanol, and the expression level of PRRSV-N protein was detected by immunofluorescence (IFA).

[0056] Test results such as Figure 5 As shown, compared with the control group without bexarotene, the cells treated with bexarotene were almost not infected with PRRSV and the proliferation of PRRSV was inhibited, indicating that bexarotene has anti-PRRSV infection activity.

[0057] 5. Plaque assay (PFU) detection

[0058] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 70-75%, the culture medium was discarded, and replaced with DMEM culture medium containing 2% fetal bovine serum containing 10.0 μM bexarotene, and HP-PRRSV was inoculated at a multiplicity of infection MOI = 0.5, and a control group without bexarotene and a blank control group were set up, and then cultured in a 37°C, 5% CO2 incubator. After 36 hours, the cell supernatant was collected for plaque assay to detect the virus titer.

[0059] Test results such as Figure 6 As shown, the number of plaques was significantly reduced compared with the control group without bexarotene, indicating that bexarotene can significantly inhibit PRRSV replication. The HP-PRRSV titer was significantly reduced after bexarotene treatment, indicating that bexarotene has significant antiviral activity.

[0060] Example 3 Broad-spectrum anti-PRRSV effect of bexarotene

[0061] Marc-145 cells (or PAMs cells) were cultured in DMEM culture medium containing 10% fetal bovine serum for about 18 hours until the cell density reached 70-75%, the culture medium was discarded, and replaced with DMEM culture medium containing 2% fetal bovine serum containing bexarotene multiple dilution solution (diluted with DMEM culture medium, the dilution concentrations were: 5.0, 7.5, 10.0 μM), and PRRSV strains of different pathogenicity (WuH3 strain, SD16 strain, TA-12 strain, JXA1 strain, NADC30 strain) were inoculated at a multiplicity of infection MOI=0.5, and a control group without bexarotene and a blank control group were set up, and then cultured in a 37°C, 5% CO2 incubator, washed 3 times with PBS after 36 hours, and then digested with 0.25% trypsin, the protein concentration was measured after the cells were lysed, and the expression level of PRRSV-N protein was detected by Western blot.

[0062] Test results such as Figure 7 As shown, bexarotene has a significant anti-PRRSV effect on PRRSV strains of different pathogenicity and is dose-dependent. PRRSV-N protein expression is almost undetectable at 10 μM. The above results indicate that bexarotene has a broad-spectrum anti-PRRSV effect.

[0063] In summary, the present invention provides a new application of bexarotene, an old drug for treating skin T-cell lymphoma, and found that bexarotene has an anti-PRRSV effect, can significantly inhibit the proliferation and replication of porcine blue ear disease virus PRRSV, and reduce the infectivity of PRRSV; at the same time, it also has a broad-spectrum anti-PRRSV effect, can inhibit the expression of PRRSV-N protein of a variety of different pathogenic PRRSV strains, especially has a significant antiviral effect against highly pathogenic HP-PRRSV; and has almost no cytotoxicity, can be better used to resist the infection of porcine blue ear virus and alleviate porcine blue ear disease. Since bexarotene has a small molecular weight, it is very easy to pass through the cell membrane into the cell and exert an antiviral effect, so it does not need a carrier to be delivered into the cell, which lays a foundation for the development of more new anti-PRRSV drugs and provides more methods and means for the prevention and treatment of porcine blue ear disease.

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The use of bexarotene in an anti-PRRSV preparation, characterized in that: The structural formula of bexarotene is:

2. The use of bexarotene in the preparation of an anti-PRRSV drug, characterized in that: The structural formula of bexarotene is:

3. The use of bexarotene in the preparation of a medicament for preventing PRRSV infection, characterized in that: The structural formula of bexarotene is:

4. Use of bexarotene in the preparation of a drug for alleviating or treating porcine blue ear disease, characterized in that: The structural formula of bexarotene is:

5. The use according to any one of claims 1 to 4, characterized in that: The drug can inhibit the replication and proliferation of PRRSV.

6. The use according to any one of claims 1 to 4, characterized in that: The drug can inhibit the expression of viral PRRSV-N protein.

7. The use according to any one of claims 1 to 4, characterized in that: The drug is able to reduce viral titers.

8. The use according to claim 4, characterized in that: The porcine blue ear disease is blue ear disease caused by HP-PRRSV or blue ear disease caused by virus variants.

9. The use according to claim 8, characterized in that: The virus variants are WuH3, SD16, TA-12, JXA1 or NADC30.

10. The use according to any one of claims 1 to 4, characterized in that: The medicine contains a pharmaceutically acceptable carrier or excipient.

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