Application of resveratrol in medicine for resisting porcine reproductive and respiratory syndrome virus

Abstract

The invention discloses application of resveratrol in preparation of drugs for resisting porcine reproductive and respiratory syndrome virus, and belongs to the field of antiviral application. The resveratrol disclosed by the invention is applied to preparation of in-vitro and in-vivo drugs for resisting the porcine reproductive and respiratory syndrome virus. Western blot and RT-qPCR detection show that the resveratrol can play a role in resisting the porcine reproductive and respiratory syndrome virus in vitro and in vivo, and the infection of the porcine reproductive and respiratory syndrome virus is remarkably inhibited. Results show that the resveratrol has an antiviral effect on the porcine reproductive and respiratory syndrome virus, the resveratrol can be used for preparing the medicine for resisting the porcine reproductive and respiratory syndrome virus infection, the occurrence of the porcine reproductive and respiratory syndrome disease is prevented, and a strategy is provided for prevention and control of the porcine reproductive and respiratory syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of antiviral applications, and specifically relates to the application of resveratrol in drugs against porcine reproductive and respiratory syndrome virus (PRRSV). Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as "porcine blue ear disease", is a serious infectious disease that endangers the pig industry, characterized by reproductive disorders such as anorexia, abortion, stillbirth, mummified fetus, and weak piglets in pregnant sows and respiratory symptoms in piglets. Since the late 1980s, "mysterious pig disease" has emerged in pig farms in the United States. In the early 1990s, similar diseases were reported in Germany, and then quickly spread to countries such as the Netherlands, Belgium, and Spain. In 1992, the disease was officially named "porcine reproductive and respiratory syndrome", and its pathogen is porcine reproductive and respiratory syndrome virus (PRRSV). Currently, the disease is widespread worldwide and has had a serious impact on the global pig industry. In 1996, Guo Baoqing first isolated the PRRSV CH-1a strain from aborted fetuses in pig farms in China, confirming the existence of the virus in China. In 2006, highly pathogenic PRRS (HP-PRRS) broke out in China, causing serious economic losses to the Chinese pig industry. After 2013, the NADC-30-like strain began to prevail in China and gradually became the prevalent strain of PRRSV.

[0003] PRRSV is an enveloped single-stranded positive-strand RNA virus, belonging to the Nidovirales order together with the Coronaviridae family, and belonging to the Arteriviridae family together with Equine arteritis virus, Mouse lactate dehydrogenase-elevating virus, and Simian hemorrhagic fever virus (SHFV). The genome of PRRSV is about 15.4 kb long and consists of at least 11 open reading frames (ORFs) except for the 5′ and 3′ untranslated regions (UTR) at both ends. Among them, ORF1a and ORF1b encode two large polyproteins pp1a and pp1ab, and these two proteins are processed into at least 14 non-structural proteins and 8 structural proteins. According to the latest classification method of the International Committee on Taxonomy of Viruses (ICTV), PRRSV has been divided into PRRSV-1 type and PRRSV-2 type, and the host immune responses caused by the two show great differences. At present, the prevalent PRRSV strains in China mainly belong to PRRSV-2 type.

[0004] Currently, the main prevention strategies for PRRS include vaccination strategies such as the use of inactivated vaccines and modified live attenuated vaccines. Although vaccination undoubtedly remains the cornerstone for controlling virus outbreaks, in some cases, its single application still has limitations. Due to the immunosuppression caused by PRRSV infection, virus evolution, multiple recombination events between wild-type strains and modified live attenuated vaccines, and the limitations of currently commercially available vaccines, they cannot provide sufficient protection to prevent pigs from being infected with PRRS. Therefore, it is necessary to find a strategy for controlling the prevalence of PRRS outside of vaccination to effectively prevent and control PRRSV infection.

[0005] Chinese herbal medicines are widely used in the livestock industry due to their advantages such as pollution-free, residue-free, and low price. It has been found that many Chinese herbal medicines or their active ingredients have the effect of inhibiting virus infection. Resveratrol / trans-Resveratrol, (E)-5-[2-(4-hydroxyphenyl)-vinyl]-1,3-benzenediol, is a non-flavonoid polyphenolic compound with the chemical formula C 14 H 12 O3, with a molecular weight of 228.24. Its physical properties are odorless and white powder, soluble in organic solvents. As a natural product, it mainly exists in plants or foods such as grapes, mulberries, blueberries, peanuts, and polygonum cuspidatum. Resveratrol has pharmacological effects such as anti-inflammatory, antioxidant, anti-cancer, anti-aging, blood sugar-lowering, and blood lipid-lowering. In addition, resveratrol can also inhibit the growth of bacteria and viruses, and is a potential natural antibacterial and antiviral drug. Resveratrol has antiviral activity against a variety of viruses, including duck plague virus, herpes simplex virus type 1, pseudorabies virus, porcine epidemic diarrhea virus, etc. The application prospect of resveratrol is very broad. However, there is currently no relevant report on the effect of resveratrol on PRRSV infection. Summary of the Invention

[0006] In order to prevent pigs from being infected with PRRS, the present invention provides an application of resveratrol in the preparation of drugs for anti-porcine reproductive and respiratory syndrome virus in vitro and in vivo. Through Western blot and RT-qPCR detection, the present invention found that resveratrol can exert an anti-porcine reproductive and respiratory syndrome virus effect both in vitro and in vivo, significantly inhibiting the infection of porcine reproductive and respiratory syndrome virus. It shows that resveratrol has antiviral activity against porcine reproductive and respiratory syndrome virus, and resveratrol can be used to prepare drugs for preventing porcine reproductive and respiratory syndrome virus infection, preventing the occurrence of porcine reproductive and respiratory syndrome diseases, and providing a new strategy for the prevention and control of porcine reproductive and respiratory syndrome.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The structural formula of resveratrol is shown as follows:

[0009]

[0010] The object of the present invention is to provide an application of resveratrol in the preparation of a drug against porcine reproductive and respiratory syndrome virus.

[0011] Further defined, it is an application in the preparation of a drug for preventing PRRSV infection and / or treating porcine reproductive and respiratory syndrome caused by PRRSV.

[0012] Further defined, it includes an application in the preparation of a drug against porcine reproductive and respiratory syndrome virus infection, and an application in the preparation of a drug against the replication of porcine reproductive and respiratory syndrome virus in cells.

[0013] Further defined, the object of the said application is animals and / or animal cells.

[0014] Another object of the present invention is to provide a drug against porcine reproductive and respiratory syndrome virus, which includes resveratrol with an effective amount against PRRSV and / or a pharmaceutically acceptable excipient.

[0015] Further defined, the said pharmaceutically acceptable excipient is selected from one or more of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents and carriers.

[0016] Further defined, the dosage form of the drug is a pharmaceutically acceptable dosage form; the dosage form is injection, granule, capsule, tablet, etc.

[0017] Further defined, it can be administered by injection, orally, or by gavage.

[0018] The present invention provides that resveratrol inhibits the infection of PRRSV in vitro, and the in vitro use concentration of resveratrol is 25 - 100 μM.

[0019] Further, after resveratrol pre - treats cells in vitro and then inoculates PRRSV (MOI = 1), resveratrol inhibits the transcriptional level of PRRSV N mRNA.

[0020] Further, after resveratrol pre - treats cells in vitro and then inoculates PRRSV (MOI = 1), resveratrol inhibits the expression level of PRRSV N protein.

[0021] The present invention also provides that resveratrol inhibits the infection of PRRSV in vivo, and the in - vivo use concentration in weaned piglets is 40 mg / kg / d, and the administration method is by gavage.

[0022] Further, resveratrol inhibits the transcriptional level of PRRSV N mRNA in lung tissue in vivo.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Resveratrol can significantly inhibit the infection of Marc-145 cells by PRRSV in vitro and significantly reduce the infection of weaned piglets by PRRSV in vivo, indicating that resveratrol has a strong antiviral effect on PRRSV. The present invention is of great significance for the prevention or treatment of PRRS infection.

[0025] In order to further understand the features and technical content of the present invention, please refer to the following detailed description of the present invention and the attached drawings. However, the attached drawings are only for reference and illustration purposes and are not used to limit the present invention. Brief Description of the Drawings

[0026] Figure 1 This is the result of the present invention for identifying the cell viability of Marc-145 cells with different concentrations of resveratrol;

[0027] Figure 2 This is the result of the present invention for verifying the in vitro inhibition of PRRSV N mRNA by different concentrations of resveratrol;

[0028] Figure 3 This is the result of the present invention for verifying the in vitro inhibition of PRRSV N protein expression by different concentrations of resveratrol;

[0029] Figure 4 This is the result of the present invention for verifying the PRRSV N mRNA in the lungs of piglets infected with PRRSV inhibited by resveratrol in vivo. Detailed Embodiments

[0030] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, and at the same time do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several modifications and improvements without departing from the inventive concept of the present invention. These all belong to the protection scope of the present invention.

[0031] The methods and equipment adopted in the present invention are conventional methods and equipment in the technical field. The methods not specifically described in detail in the present invention are all conventional laboratory methods and can be understood from the prior art. The reagents used in the present invention are all purchased through legal commercial channels.

[0032] The resveratrol used in the embodiments of the present invention was purchased from Shanghai Macklin Biochemical Co., Ltd., and its CAS number is: 501-36-0. The PRRSV strain used in the embodiments of the present invention is HP-PRRSV, which is preserved by this experiment. The PRRSV-susceptible cell Marc-45 (African green monkey embryonic kidney cells) is preserved in this laboratory. The reagents required for the present invention, Cell Counting Kit (CCK-8 kit), Total RNA Extraction Kit for Cells, and the total RNA extraction reagent Trizol were purchased from Seven Innovation (Beijing) Biotechnology Co., Ltd.; DMEM high-glucose medium was purchased from Gibco; Reverse Transcription Kit PrimeScript TM RT reagent Kit (Perfect Real Time), highly specific RT-qPCR reagent TB Premix Ex Taq TM II (Tli RNaseH Plus) was purchased from TaKaRa; Rabbit monoclonal antibody against PRRSV N protein was purchased from Bioss; Mouse monoclonal antibody against GAPDH protein was purchased from Proteintech; Goat anti-mouse IgG / horseradish peroxidase-labeled and goat anti-rabbit IgG / horseradish peroxidase-labeled antibodies were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0033] Cytotoxicity of resveratrol on Marc-145 cells

[0034] 1. Experimental method:

[0035] The resveratrol was dissolved in DMSO to a stock solution of 200 mM for subsequent use. The Marc-145 cells were cultured in a 37°C, 5% CO2 incubator. When the cells grew to 80% - 90%, the cells were resuspended with DMEM cell culture medium containing 10% FBS and 1% double antibody (10 kU / mL penicillin, 10 mg / mL streptomycin), and the Marc-145 cell suspension was adjusted to 1×10 5Inoculate a 96-well cell culture plate evenly at 5×10 cells / mL, add 100 μL to each well, and culture it in an incubator at 37 °C and 5% CO₂. When the cells grow to 80% - 90%, remove the original cell culture medium and replace it with resveratrol diluted with DMEM cell maintenance medium containing 2% FBS and 1% double antibiotics (10 kU / mL penicillin, 10 mg / mL streptomycin) at concentrations of 0 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM, and 800 μM, 100 μL per well, repeat three wells, and set up a cell control group and a DMSO control group at the same time. After incubating for 24 h at 37 °C and 5% CO₂, discard the original solution, add 10% CCK-8 reagent diluted with cell maintenance medium (cell maintenance medium: CCK-8 reagent = 10:1), mix well and add it to the 96-well plate, 100 μL per well, and culture it in an incubator at 37 °C and 5% CO₂ for 30 - 45 min; measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Record the data and calculate the cell survival rate.

[0036] 2. Experimental results:

[0037] As Figure 1 shown, when the concentration of resveratrol is less than or equal to 100 μM, there is no significant difference in the cell survival rate after the action of resveratrol compared with the cell control group, indicating that resveratrol has no cytotoxicity to Marc-145 cells when the concentration is less than or equal to 100 μM. Therefore, the subsequent selected resveratrol concentration is less than or equal to 100 μM, namely 25 μM, 50 μM, and 100 μM.

[0038] Verify the in vitro anti-PRRSV effect of resveratrol at different concentrations

[0039] 1. Experimental method:

[0040] The experiment is divided into four groups: PRRSV group, resveratrol (25 μM) + PRRSV group, resveratrol (50 μM) + PRRSV group, and resveratrol (100 μM) + PRRSV group. Dilute the resveratrol concentration to 25 μM, 50 μM, and 100 μM with cell maintenance medium when the cultured cell suspension is inoculated into a 6-well cell culture plate at 5×10 6 cells / mL. When the cells grow to 80% - 90%, discard the original solution in the 6-well plate. After incubating the above three concentrations of resveratrol with the cells for 24 h, discard the liquid medicine. After infecting the cells with PRRSV (MOI = 1) for 48 h, extract the total cellular RNA and reverse transcribe it into cDNA, and detect the transcriptional level of PRRSV N mRNA by RT-qPCR.

[0041] The experiment was divided into six groups: blank control group, resveratrol (100 μM) control group, PRRSV group, resveratrol (25 μM) + PRRSV group, resveratrol (50 μM) + PRRSV group, and resveratrol (100 μM) + PRRSV group. The cultured cell suspension was divided into 5×10 6 / mL inoculated 6-well cell culture plates, when the cells grew to 80% to 90%, the resveratrol concentration was diluted to 25μM, 50μM, and 100μM with cell maintenance solution, and the original solution in the 6-well plate was discarded. The above three concentrations of resveratrol were incubated with cells for 24 hours and then the solution was discarded. After the cells were infected with PRRSV (MOI = 1) for 48 hours, the cell protein samples were extracted and the expression level of PRRSVN protein was detected by Western blot to verify the in vitro anti-PRRSV activity of resveratrol.

[0042] 2. Experimental results:

[0043] like Figure 2 and Figure 3 As shown, as the concentration of resveratrol gradually increased, the PRRSVN mRNA transcription level and N protein expression decreased in a concentration-dose dependent manner, indicating that resveratrol has a significant inhibitory effect on PRRSV infection in vitro.

[0044] Verification of the anti-PRRSV effect of resveratrol in vivo

[0045] 1. Experimental methods:

[0046] Twelve weaned piglets with negative antigens and antibodies for PRRSV were randomly divided into 4 groups, including a blank control group, a resveratrol control group, a PRRSV infection group, and a resveratrol + PRRSV infection group, with 3 piglets in each group. Each experimental group was fed in a separate room to prevent cross infection. After 3 days of adaptive feeding, the piglets were gavaged with 40 mg / kg / head of resveratrol for 14 days. The blank control group and the PRRSV infection group were gavaged with the corresponding volume of sterile water, and the virus was challenged on the 11th day (the 8th day of gavage). Each pig in the PRRSV infection group and the resveratrol + PRRSV infection group was inoculated with 1×10 -6 TCID 50 mL -1 2mL of PRRSV virus solution (1mL intramuscular injection, 1mL nasal drops). The virus was challenged for three consecutive days, and the blank control group and the resveratrol control group were inoculated with 2mL DMEM culture medium (1mL intramuscular injection, 1mL nasal drops) for three consecutive days. Resveratrol was continuously administered during the virus challenge, and resveratrol gavage was stopped on the seventh day after the virus challenge. On the 14th day after the virus challenge, all piglets were killed, lung swelling was collected, and lung tissue at the junction of lesions and non-lesions was taken to extract total tissue RNA, which was reverse transcribed into cDNA, and the viral load in the lung tissue was detected by RT-qPCR.

[0047] 2. Experimental results:

[0048] As Figure 4 shown, resveratrol at a dose of 40 mg / kg / d for 14 days in weaned piglets can significantly reduce the virus titer in lung tissues and inhibit PRRSV infection in piglets, indicating that resveratrol can prevent PRRSV infection in piglets in vivo.

[0049] The above embodiments are only examples for clearly and understandably illustrating the anti-PRRSV effect of resveratrol, rather than limitations on the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of resveratrol in the preparation of a drug against porcine reproductive and respiratory syndrome virus.

2. The application according to claim 1, wherein Use in the preparation of a drug for preventing PRRSV infection and / or treating anti-porcine reproductive and respiratory syndrome caused by PRRSV.

3. The application according to claim 1, characterized in that, Including use in the preparation of a drug against porcine reproductive and respiratory syndrome virus infection, and use in the preparation of a drug against the replication of porcine reproductive and respiratory syndrome virus in cells.

4. The application according to claim 1, wherein The object of the said use is animals and / or animal cells.

5. A drug against porcine reproductive and respiratory syndrome virus, characterized in that, Including resveratrol with an effective amount against PRRSV and / or pharmaceutically acceptable excipients.

6. The drug according to claim 5, characterized in that, The said pharmaceutically acceptable excipients are selected from one or more of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents and carriers.

7. The drug according to claim 5, characterized in that, The dosage form of the drug is a pharmaceutically acceptable dosage form.

8. The drug according to claim 5, characterized in that, The dosage form is injection, granule, capsule, tablet.

9. The medicament according to claim 5, wherein It can be administered by injection, orally, or by gavage.

10. The drug according to claim 5, characterized in that, The in vitro use concentration of resveratrol is 25 - 100 μM; the in vivo use concentration is 40 mg / kg / d.

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