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

Abstract

The invention discloses application of berberine in preparation of drugs for resisting porcine reproductive and respiratory syndrome virus, and belongs to the field of antiviral application. The invention relates to an application of berberine in preparation of in-vitro and in-vivo drugs for resisting porcine reproductive and respiratory syndrome virus. According to the invention, berberine is used for pre-incubation treatment of Marc-145 cells susceptible to porcine reproductive and respiratory syndrome virus infection, Western blot and RT-qPCR detection show that berberine can significantly inhibit infection of the porcine reproductive and respiratory syndrome virus on Marc-145 in vitro, and in addition, berberine can also significantly inhibit infection of the porcine reproductive and respiratory syndrome virus on piglets in vivo. The result shows that the berberine has the effect of resisting the porcine reproductive and respiratory syndrome virus, and the berberine can be used for preparing the medicine for resisting the porcine reproductive and respiratory syndrome virus infection so as to prevent the occurrence of the porcine reproductive and respiratory syndrome disease. The invention provides a medicine source for preventing the porcine reproductive and respiratory syndrome, and has important significance for preventing and treating the porcine reproductive and respiratory syndrome virus.

Description

Technical Field

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

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV). Since its first discovery in the United States in 1987, it has spread widely throughout the world. PRRSV can infect pigs of all ages, mainly causing reproductive disorders in sows, decreased semen quality in boars, death of piglets, and respiratory symptoms in pigs of all ages, causing serious economic losses and huge disease prevention and control pressures to the pig industry worldwide.

[0003] PRRSV is a single-stranded, positive-strand, enveloped RNA virus belonging to the order Nidovirales, family Arteriviridae, genus Arterivirus. The virus particles are spherical, with a diameter of about 50 - 70 nm, containing 11 open reading frames, encoding at least 14 non-structural proteins with different functions and 8 structural proteins. PRRSV can be divided into PRRSV-1 type and PRRSV-2 type. At present, the PRRSV strains prevalent in China mainly belong to PRRSV-2 type.

[0004] Vaccination is currently considered the most effective method for preventing PRRS. Vaccination strategies including the use of attenuated live vaccines, modified live virus vaccines, genetic engineering vaccines, DNA vaccines, and immunoadjuvant vaccines are the most cost-effective means for preventing PRRS. However, due to the rapid mutation of PRRSV strains, relatively lagging vaccine research and development, frequent recombination of different strains, the existing vaccines on the market can only provide limited protection and cannot completely prevent pigs from being infected with PRRS. In addition, the continuous emergence of new strains, low cross-protection degree between strains, adaptation of attenuated vaccines to the host and recombination to even enhance virulence have complicated the selection of vaccines and the formulation of vaccination strategies, all of which have increased the difficulty of controlling PRRS, greatly hindering the prevention and control of PRRSV in the pig breeding industry. Therefore, there is an urgent need to find non-antibiotic natural compounds to effectively prevent and treat PRRS and provide new strategies for clinical control of PRRS.

[0005] Berberine (BBR) is a low-toxic natural isoquinoline alkaloid found in traditional Chinese medicines Coptis chinensis and Phellodendron amurense. The chemical formula is C 20 H 18NO4 + It has a molecular weight of 336.36 and is widely distributed in the plant kingdom. Currently, it is mostly isolated and extracted from traditional Chinese medicine plants such as Berberidaceae, Ranunculaceae, Rutaceae, Papaveraceae, Menispermaceae, Loganiaceae, and Rhamnaceae. Berberine has pharmacological effects such as anti-inflammatory, antibacterial, antiviral, antioxidant, antitumor, hypoglycemic, lipid-regulating, and immunosuppressive effects, and has been used in the clinical treatment of cardiovascular system diseases, digestive system diseases, metabolic diseases, tumors, etc. Berberine has antiviral activity against a variety of viruses, including influenza virus, herpes virus, Coxsackievirus B3, porcine rotavirus, porcine epidemic diarrhea virus, etc. However, there is currently no relevant report on the effect of berberine 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 berberine in the preparation of drugs against porcine reproductive and respiratory syndrome virus. The present invention uses berberine to pre-incubate and treat Marc-145 cells that are susceptible to porcine reproductive and respiratory syndrome virus. Through Western blot and RT-qPCR detection, it is found that berberine can significantly inhibit the infection of porcine reproductive and respiratory syndrome virus to Marc-145 cells in vitro. In addition, berberine can also significantly inhibit the infection of porcine reproductive and respiratory syndrome virus to piglets in vivo. It shows that berberine has the effect of anti-porcine reproductive and respiratory syndrome virus, and berberine can be used to prepare drugs against porcine reproductive and respiratory syndrome virus infection to prevent the occurrence of porcine reproductive and respiratory syndrome diseases. The present invention provides a new drug source for the prevention of porcine reproductive and respiratory syndrome and has important significance for the prevention of porcine reproductive and respiratory syndrome virus.

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

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

[0009]

[0010] The purpose of the present invention is to provide an application of berberine in the preparation of drugs against porcine reproductive and respiratory syndrome virus.

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

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

[0013] Further limited, 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, comprising berberine with an effective amount against PRRSV and / or a pharmaceutically acceptable excipient.

[0015] Further defined, the 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 berberine inhibits the infection of PRRSV in vitro, and the in vitro use concentration of berberine is 25 μM to 100 μM.

[0019] Further defined, after pretreating cells with berberine in vitro and then inoculating with PRRSV (MOI = 1), berberine inhibits the transcriptional level of PRRSV N mRNA.

[0020] Further defined, after pretreating cells with berberine in vitro and then inoculating with PRRSV (MOI = 1), berberine inhibits the expression level of PRRSV N protein.

[0021] The present invention also provides that berberine inhibits the infection of PRRSV in vivo, and the in vivo use concentration in weaned piglets is 50 mg / kg / d, and the administration method is by gavage.

[0022] Further defined, berberine 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] In vitro experiments show that berberine can significantly inhibit the infection of PRRSV to Marc-145; in vivo experiments prove that berberine can significantly reduce the infection of PRRSV to weaned piglets. It can be seen that berberine has a strong inhibitory effect on PRRSV and may become a candidate drug for the prevention or treatment of PRRS.

[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 1This is the result of the present invention exploring the cytotoxicity of berberine on Marc-145;

[0027] Figure 2 This is the result of the present invention verifying the inhibitory effect of berberine on PRRSV N mRNA in vitro;

[0028] Figure 3 This is the result of the present invention verifying the inhibitory effect of berberine on PRRSV N protein expression in vitro;

[0029] Figure 4 This is the result of the present invention verifying the inhibitory effect of berberine on PRRSV N mRNA in the lungs of PRRSV-infected piglets in vivo. Detailed implementation mode

[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 do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made. 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 obtained from the prior art. The reagents used are all purchased through legal commercial channels.

[0032] The berberine used in the embodiments of the present invention is purchased from Xi'an Huilin Biotechnology Co., Ltd., and its CAS number is: 2086-83-1. 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 by 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 are purchased from Seven Innovation (Beijing) Biotechnology Co., Ltd.; DMEM high-glucose medium is purchased from Gibco company, and the reverse transcription kit PrimeScript ⑨ RT reagent Kit (Perfect Real Time), and the highly specific RT-qPCR reagent TB TM Premix Ex Taq Premix Ex Taq TMII (Tli RNaseH Plus) was purchased from TaKaRa, the rabbit monoclonal antibody against PRRSV N protein was purchased from Bioss, the mouse monoclonal antibody against GAPDH protein was purchased from Proteintech, and the 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 berberine on Marc-145

[0034] 1. Experimental method:

[0035] Dissolve berberine in DMSO to a stock solution of 100 mM for subsequent use. Culture Marc-145 cells in a 37 °C, 5% CO2 incubator. When the cells grow to 80% - 90%, resuspend the cells with DMEM cell culture medium containing 10% FBS and 1% double antibody (10 kU / mL penicillin, 10 mg / mL streptomycin). Uniformly inoculate the Marc-145 cell suspension at 1×10 5 cells / mL into a 96-well cell culture plate, add 100 μL to each well, and culture in a 37 °C, 5% CO2 incubator. When the cells grow to 80% - 90%, remove the original cell culture medium and replace it with berberine diluted with DMEM cell maintenance medium containing 2% FBS and 1% double antibody (10 kU / mL penicillin, 10 mg / mL streptomycin) at concentrations of 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, 100 μL / well, and repeat three wells. At the same time, set up a cell control group and a DMSO control group. After incubating for 24 h at 37 °C and 5% CO2, discard the original solution, add 10% CCK-8 (cell maintenance medium: CCK-8 reagent = 10:1) reagent diluted with cell maintenance medium, mix well and add it to the 96-well plate, 100 μL / well, and culture in a 37 °C, 5% CO2 incubator 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 berberine is less than or equal to 100 μM, there is no significant difference in the cell survival rate after the action of berberine compared with the cell control group, indicating that berberine has no cytotoxicity to Marc-145 when the concentration is less than or equal to 100 μM. Therefore, the subsequent selected berberine action concentrations are less than or equal to 100 μM, namely 25 μM, 50 μM, and 100 μM.

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

[0039] 1. Experimental method:

[0040] The experiment was divided into a PRRSV group, a berberine (25 μM) + PRRSV group, a berberine (50 μM) + PRRSV group, and a berberine (100 μM) + PRRSV group. The cultured cell suspension was inoculated into a 6-well cell culture plate at 5×10 6 cells / mL. When the cells grew to 80% - 90%, the berberine concentrations were diluted to 25 μM, 50 μM, and 100 μM with cell maintenance medium. The original liquid in the 6-well plate was discarded. After incubating the above three concentrations of berberine with the cells for 24 h, the medicinal liquid was discarded. After infecting the cells with PRRSV (MOI = 1) for 48 h, the total cellular RNA was extracted and reverse transcribed into cDNA. The transcriptional level of PRRSV N mRNA was detected by RT-qPCR, and the protein expression level of PRRSV N was detected by Western blot to verify the anti-PRRSV activity of berberine.

[0041] 2. Experimental results:

[0042] As Figure 2 shown, with the gradual increase in berberine concentration, the transcriptional level of PRRSV N mRNA and the expression of N protein decreased in a concentration-dependent manner, indicating that berberine has a significant inhibitory effect on PRRSV in vitro. Figure 3

[0043] Verification of the anti-PRRSV effect of berberine in vivo

[0044] 1. Experimental method:

[0045] Twelve weaned piglets with negative antigens and antibodies for PRRSV were randomly divided into 4 groups, namely a blank control group, a berberine control group, a PRRSV infection group, and a berberine + PRRSV infection group, with 3 piglets in each group. Each experimental group was fed in separate rooms to prevent cross-infection. After 3 days of adaptive feeding, the piglets were gavaged with berberine at a dose of 50 mg / kg / head for 14 days. The blank control group and the PRRSV infection group were gavaged with the corresponding volume of sterile water. On the 11th day (the 8th day of gavage), the challenge was carried out. Each pig in the PRRSV infection group and the berberine + PRRSV infection group was inoculated with 1×10 -6 TCID 50 ·mL -1 ​2 mL of the PRRSV virus solution (1 mL intramuscular injection, 1 mL intranasal drip). The challenge was conducted continuously for three days. The blank control group and the berberine control group were inoculated with 2 mL of DMEM culture medium (1 mL intramuscular injection, 1 mL intranasal drip) for three consecutive days. Berberine was continuously administered during the challenge, and the gavage administration of berberine was stopped on the seventh day after the challenge. On the 14th day after the challenge, all piglets were sacrificed, the lungs were collected, and the total tissue RNA was extracted from the lung tissue at the junction of the lesion and non-lesion areas, reverse transcribed into cDNA, and the viral load in the lung tissue was detected by RT-qPCR.

[0046] 2. Experimental results:

[0047] As Figure 4 shown, berberine at 50 mg / kg / d for 14 days in weaned piglets can significantly reduce the virus titer in the lung tissue and inhibit the infection of PRRSV in piglets, indicating that berberine can prevent the infection of PRRSV in piglets in vivo.

[0048] The above embodiments are only examples for clearly and understandably illustrating the anti-PRRSV effect of berberine, rather than limitations on the implementation modes. 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. Application of berberine in the preparation of drugs against porcine reproductive and respiratory syndrome virus.

2. The application according to claim 1, wherein Application in the preparation of drugs 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 application in the preparation of drugs against porcine reproductive and respiratory syndrome virus infection, and application in the preparation of drugs 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 application is animals and / or animal cells.

5. A drug against porcine reproductive and respiratory syndrome virus, characterized in that, Including berberine 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 forms are injection, granule, capsule, tablet.

9. The drug according to claim 5, characterized in that, It can be administered by injection, orally, or by gavage.

10. The medicament according to claim 5, characterized in that, The in vitro use concentration of berberine is 25 μM - 100 μM; the in vivo use concentration is 50 mg / kg / d.

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