Application of tripterine in preparation of medicine for preventing and treating porcine viral diseases

Abstract

The invention discloses an application of tripterine in preparation of a medicine for preventing and treating porcine viral diseases. According to the application disclosed by the invention, the effect of resisting PEDV, PDCoV and PRRSV activity of the tripterine is found for the first time. The tripterine has a remarkable inhibiting effect on PEDV (porcine epidemic diarrhea virus) on Vero cells, and has a remarkable inhibiting effect on virus nucleic acid level, virus protein expression and progeny virus titer of viruses on the Vero cells. The tripterine has a remarkable inhibition effect on PDCoV on a PK-15 cell, and has a remarkable inhibition effect on the viral nucleic acid level of a virus on the PK-15 cell. The tripterine has a remarkable inhibition effect on PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) on Marc-145 cells, and has a remarkable inhibition effect on the viral nucleic acid level of viruses on the Marc-145 cells. Therefore, the small molecule compound can be used as a medicine for preventing and treating infection of important porcine viruses PEDV, PDCoV and PRRSV.

Description

Technical Field

[0001] The present invention relates to the technical field of application of natural compounds, and in particular to the application of tripterygium wilfordii in the preparation of medicines for preventing and treating diseases caused by porcine epidemic diarrhea virus, porcine deltacoronavirus and porcine reproductive and respiratory syndrome virus infection. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the genus alpha-coronavirus and is the pathogen that causes porcine epidemic diarrhea (PED). PEDV infection can cause severe diarrhea, vomiting, dehydration, weight loss and other symptoms in pigs. The clinical symptoms are very similar to those of porcine transmissible gastroenteritis, which has a serious impact on the growth and development of pigs. Pigs of all ages can be infected with PEDV, but the symptoms after infection vary. Piglets are particularly serious, with a mortality rate of up to 100%, causing significant economic losses. Since PEDV is a single-stranded RNA virus, it is prone to mutation or gene recombination, resulting in new epidemic characteristics of PED, which are manifested as extremely high mortality rates (90% to 100%) in pigs, poor vaccine prevention and control effects, and immunized pigs can still be infected with PEDV. Once a large-scale outbreak of PED occurs, the incidence rate of newborn suckling piglets can reach 100%, and the mortality rate can reach 80% to 100%. A lot of research has been done on PEDV vaccine development both at home and abroad, but a vaccine with satisfactory effect has not yet been developed, and the development of antiviral drugs is imminent.

[0003] Porcine deltacoronavirus (PDCoV) is a new type of porcine enteric coronavirus that can cause diarrhea and vomiting in 5-15 day old suckling piglets, causing rapid dehydration and death from exhaustion, with a morbidity and mortality rate of 50% to 100%. A strong immune response after natural infection can prevent subsequent homologous attacks, but there is no cross-protection. Viral mutations and the reversion to virulence after immunization with attenuated vaccines remain risks faced by vaccine immunization. Therefore, it is very important to develop antiviral drugs for PDCoV.

[0004] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is a persistent virus that first appeared in the late 1980s. In the nearly 25 years since PRRSV first appeared, emerging and re-emerging strains have continued to cause global epidemics due to high-frequency mutations and recombination. PRRSV remains one of the most economically destructive diseases of pigs, and there is a trend of worsening the development of the pig farming industry. Currently, the main commercialized or developed vaccines for the prevention and control of PRRS include inactivated vaccines, modified live virus (MLV) vaccines, subunit vaccines, DNA vaccines, and viral vector vaccines, but vaccine development has been hindered due to the high variability and infectivity of PRRSV. Therefore, it is necessary to develop anti-PRRSV drugs for prevention or treatment.

[0005] Celastrol, also known as celastrol, CAS No. 34157-83-0, has a chemical structure as shown in Formula 1. Modern studies have shown that it has strong antioxidant effects, anti-cancer angiogenesis, anti-rheumatoid effects, spermicidal effects, etc. It is a natural active product worthy of attention and has outstanding efficacy in anti-cancer, tumor, and brain nerves. However, there is no relevant report on whether Celastrol can resist porcine virus infection.

[0006] (Formula 1) Summary of the invention

[0007] There is no commercialized specific therapeutic drug for PEDV, PDCoV and PRRSV, and prevention mainly relies on vaccines. However, a large number of survey reports show that the current preventive effect of vaccines is not satisfactory. The emergence of mutants has caused a sharp decline in the protective power of vaccines, and attenuated vaccines have a certain risk of relapse. Clinical prevention and treatment also use antibiotics, hormones and other drugs, but long-term use will lead to increased drug resistance of pathogenic bacteria and drug residues in pork products, which will reduce the efficacy and increase the risk of public health and safety, seriously endangering human health. Up to now, commercial drugs that can be used to resist PEDV, PDCoV and PRRSV have not yet been developed, so it is necessary to find a drug that effectively resists PEDV, PDCoV and PRRSV infection to overcome the shortcomings in current practical applications. In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide the use of tripterygium wilfordii in the preparation of drugs for preventing and treating PEDV, PDCoV and / or PRRSV infection.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: The invention discloses an application of tripterygium wilfordii in the preparation of a medicine or feed additive for preventing and / or treating a swine viral disease, wherein the viral disease comprises at least one of porcine epidemic diarrhea and blue ear disease.

[0009] Furthermore, the porcine epidemic diarrhea includes porcine epidemic diarrhea caused by porcine epidemic diarrhea virus PEDV and / or porcine deltacoronavirus PDCoV.

[0010] Furthermore, the porcine reproductive and respiratory syndrome virus (PRRSV) includes porcine reproductive and respiratory syndrome virus (PRRSV) caused by PRRSV.

[0011] The invention discloses an application of tripterygium wilfordii in the preparation of a drug for preventing and treating porcine viral infection, wherein the virus comprises at least one of porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and porcine reproductive and respiratory syndrome virus (PRRSV).

[0012] Furthermore, the anti-swine virus infection comprises at least one of inhibiting viral gene expression, inhibiting viral protein expression and inhibiting the production of progeny virus particles.

[0013] Furthermore, the viral genes include PEDV N gene, PDCoV N gene and PRRSV ORF7 gene, and the viral proteins include PEDVN protein, PDCoVN protein and PRRSV N protein.

[0014] The invention discloses an application of tripterine in the preparation of an anti-swine virus drug, wherein the virus comprises at least one of porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and porcine reproductive and respiratory syndrome virus PRRSV.

[0015] Furthermore, the porcine epidemic diarrhea virus PEDV includes the CV777 strain.

[0016] Furthermore, the porcine deltacoronavirus PDCoV includes the HKU15 strain.

[0017] Furthermore, the porcine reproductive and respiratory syndrome virus PRRSV includes the BJ-4 strain.

[0018] Furthermore, the effective concentration of tripterygium wilfordii is 0.1-0.5 μmol / L.

[0019] Furthermore, the tripterygium wilfordii includes tripterygium wilfordii and its pharmaceutically acceptable salts.

[0020] Furthermore, the medicine or feed additive comprises tripterygium wilfordii and at least one pharmaceutically acceptable excipient.

[0021] Furthermore, the dosage form of the drug is a gastrointestinal dosage form or an injection dosage form.

[0022] Furthermore, the gastrointestinal tract administration dosage form is tablets, capsules, granules, pills or pellets.

[0023] Compared with the prior art, the present invention has the following advantages and effects: The present invention discovers for the first time that Celastrol has the ability to inhibit the infection of PEDV, PDCoV and PRRSV on cells, and has the best therapeutic administration effect in PEDV infection.

[0024] The present invention provides the use of tripterygium wilfordii in the preparation of drugs for preventing or treating porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and porcine reproductive and respiratory syndrome virus (PRRSV). The research results show that the drug tripterygium wilfordii has anti-PEDV, PDCoV and PRRSV activity. Tripterygium wilfordii has a significant inhibitory effect on PEDV on African green monkey kidney cells (Vero), and has a significant inhibitory effect on the viral nucleic acid level, viral protein expression and titer of progeny viruses on Vero cells. Tripterygium wilfordii has a significant inhibitory effect on PDCoV on porcine kidney cells (PK-15), and has a significant inhibitory effect on the viral nucleic acid level of the virus on PK-15 cells. Tripterygium wilfordii has a significant inhibitory effect on PRRSV on monkey kidney cells (Marc-145), and has a significant inhibitory effect on the viral nucleic acid level of the virus on Marc-145 cells. Therefore, the small molecule compound can be used as a drug for inhibiting infection of important porcine viruses PEDV, PDCoV and PRRSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the results of a study on the cytotoxicity of tripterygium wilfordii on Vero cells.

[0026] Figure 2 This is a graph showing the results of a study on the cytotoxicity of tripterygium wilfordii on PK-15 cells.

[0027] Figure 3 This is a graph showing the results of a study on the cytotoxicity of tripterygium wilfordii on Marc-145 cells.

[0028] Figure 4 This is a graph showing the results of a study on the effects of different concentrations of tripterygium wilfordii on the nucleic acid level of the PEDV N gene in Vero cells.

[0029] Figure 5 This is a graph showing the research results on the effects of different concentrations of tripterygium wilfordii on the expression of PEDV N protein in Vero cells.

[0030] Figure 6 This is a graph showing the research results on the effects of different concentrations of tripterygium wilfordii on the production of PEDV progeny viruses in Vero cells.

[0031] Figure 7 This is a graph showing the results of a study on the inhibitory effect of tripterygium wilfordii on PEDV infection under preventive, co-administration, and therapeutic administration.

[0032] Figure 8 This is a graph showing the research results on the effects of different concentrations of tripterygium wilfordii on the nucleic acid level of PDCoV N gene in PK-15 cells.

[0033] Fig. 9 This is a graph showing the research results on the effects of different concentrations of tripterygium wilfordii on the expression of PDCoV N protein in PK-15 cells.

[0034] Fig.10 This is a graph showing the results of a study on the effects of different concentrations of tripterygium wilfordii on the nucleic acid level of PRRSV ORF7 gene in Marc-145 cells.

[0035] Fig.11 This is a graph showing the results of a study on the effects of different concentrations of tripterygium wilfordii on the expression of PRRSV N protein in Marc-145 cells. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention. Rather than all the embodiments, all other embodiments obtained by ordinary technicians in this field without innovative work based on the embodiments of the present invention are within the scope of protection of the present invention.

[0037] The porcine epidemic diarrhea virus (PEDV) used in the following examples is the CV777 strain (GenBank: AF353511.1), which is deposited in the research group of Wang Yu'e at the College of Veterinary Medicine of Southwest University and has been published in the document "Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor ReceptorActivationImpairs the Antiviral Activity of Type I Interferon".

[0038] The porcine deltacoronavirus (PDCoV) used in the following examples is the HUK15 strain (GenBank: KM012168.1), which is deposited in the research group of Wang Yu'e at the College of Veterinary Medicine, Southwest University.

[0039] The porcine reproductive and respiratory syndrome virus (PRRSV) used in the following examples is the BJ-4 strain (Lineage 5, GenBank: AF331831.1), which is deposited in the research group of Wang Yu'e of the College of Veterinary Medicine of Southwest University and has been published in the document "Tumor Susceptibility Gene 101 (TSG101) Contributes to Virion Formation of Porcine Reproductive and Respiratory Syndrome Virus via Interaction with the Nucleocapsid (N) Protein along with the Early Secretory Pathway".

[0040] Example 1: Cytotoxicity of tripterygium wilfordii to Vero cells Vero cells (African green monkey kidney cells) were inoculated in 96-well plates at 5000 cells per well. After the cells adhered to the wall, the culture medium in the plate was discarded, and celastrol was diluted to 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75 and 2 μmol / L with 10% FBS DMEM medium (containing penicillin 100U / mL, streptomycin 50 μg / mL), 100 μL / well, and a blank cell control group was set up. Six replicates were made for each concentration and the plates were incubated at 37°C, 5% CO 2 After culturing in the incubator for 24 h, CCK-8 reagent was added at 10 μL / well, and the cells were cultured in the dark for another 2 h. The absorbance of each well was read at a reference wavelength of 450 nm using a microplate reader to calculate the cell viability.

[0041] The results are as follows Figure 1 As shown in Figure 2, the cell cytotoxic concentration (CC) of tripterygium wilfordii on Vero cells 50 ) was 1.583 μmol / L. The results showed that tripterygium wilfordii had low toxicity to Vero cells.

[0042] Example 2: Cytotoxicity of tripterygium wilfordii to PK-15 cells PK-15 cells (pig kidney cells) were inoculated in 96-well plates at 5000 cells per well. After the cells adhered to the wall, the culture medium in the plate was discarded, and celastrol was diluted to 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5 and 1.75 μmol / L with 10% FBS DMEM medium (containing penicillin 100 U / mL, streptomycin 50 μg / mL), 100 μL / well, and a blank cell control group was set up. Six replicates were made for each concentration and the plates were incubated at 37°C and 5% CO2 After culturing in the incubator for 24 h, CCK-8 reagent was added at 10 μL / well, and the cells were cultured in the dark for another 2 h. The absorbance of each well was read at a reference wavelength of 450 nm using a microplate reader to calculate the cell viability.

[0043] The results are as follows Figure 2 As shown, the cell median cytotoxic concentration (CC 50 ) was 1.695 μmol / L. The results showed that tripterygium wilfordii had little toxicity to PK-15 cells.

[0044] Example 3: Cytotoxicity of tripterygium wilfordii to Marc-145 cells Marc-145 cells (African green monkey embryonic kidney cells) were inoculated in 96-well plates at 5000 cells per well. After the cells adhered to the wall, the culture medium in the plate was discarded, and celastrol was diluted to 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75 and 2 μmol / L with 10% FBS DMEM medium (containing penicillin 100 U / mL, streptomycin 50 μg / mL), 100 μL / well, and a blank cell control group was set up. Six replicates were made for each concentration and the plates were incubated at 37°C, 5% CO 2 After culturing in the incubator for 24 h, CCK-8 reagent was added at 10 μL / well, and the cells were cultured in the dark for another 2 h. The absorbance of each well was read at a reference wavelength of 450 nm using a microplate reader to calculate the cell viability.

[0045] The results are as follows Figure 3 As shown, tripterygium wilfordii had very little cytotoxicity to Marc-145 cells, and cell viability did not decrease significantly when treated with the highest tripterygium wilfordii concentration of 2 μM.

[0046] Example 4: Effects of different concentrations of tripterygium wilfordii on the level of PEDV N gene nucleic acid in Vero cells Vero cells were cultured at 3 × 10 5 Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO. 2After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with the PEDV CV777 strain at an MOI of 0.01. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for further incubation for 24 h. After 24 h, the total RNA was extracted using a total RNA extraction kit according to the instructions. After reverse transcription, the PEDV N gene primers and SYBR Green I were used as fluorescent dyes to perform fluorescence quantitative PCR to detect the relative expression of the PEDV N gene. The reaction conditions were: 95°C for 5 min to activate the hot start enzyme; 95°C for 10 s to denature, 60°C for 30 s to anneal, and 45 cycles of reaction; the fluorescence signal was collected at this step.

[0047] The results are as follows Figure 4 As shown in the figure, compared with the control group, triptolide at 0.1 μmol / L significantly inhibited the transcription level of PEDV N gene ( p -value=0.0027), and the inhibition of PEDV N gene was concentration-dependent at 0.1-0.5 μmol / L concentration. This indicates that tripterygium wilfordii has the effect of inhibiting the nucleic acid level of PEDV N gene.

[0048] Example 5: Effects of different concentrations of tripterygium wilfordii on the expression of PEDV N protein in Vero cells Vero cells were cultured at 3 × 10 6 Each well was inoculated in a 6-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO 2 After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with the PEDV CV777 strain at an MOI of 0.01. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for another 24 h. After 24 h, the cell supernatant was discarded, the cell culture was collected, the total protein was extracted, and the difference in PEDV N protein expression was detected by Western blot.

[0049] The results are as follows Figure 5As shown in the figure, compared with the uninfected group, the expression of PEDV N protein in the infected control group (0 μmol / L group) and the tripterygium wilfordii treated group was significantly increased. However, compared with the infected control group, tripterygium wilfordii inhibited the expression of PEDV N protein in a concentration-dependent manner at a concentration of 0.1-0.5 μmol / L. Grayscale quantitative analysis showed that the expression levels of PEDV N protein under the treatment of tripterygium wilfordii at a concentration of 0.1-0.5 μmol / L were 0.97 times, 0.64 times, 0.39 times, 0.20 times and 0.11 times that of the infected control group, respectively, indicating that tripterygium wilfordii has the effect of inhibiting the expression of PEDV N protein.

[0050] Example 6: Effects of different concentrations of tripterygium wilfordii on the production of PEDV progeny viruses in Vero cells Vero cells were cultured at 3 × 10 5 Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO. 2 After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with the PEDV CV777 strain at an MOI of 0.01. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for another 24 h. After 3 days of infection, the positive rate of cytopathic effect in each well was observed and recorded. The virus titer (TCID 50 )value.

[0051] The results are as follows Figure 6 As shown, tripterygium wilfordii inhibited the production of PEDV infectious virus particles in a concentration-dependent manner at a concentration of 0-0.5 μmol / L, indicating that tripterygium wilfordii has the effect of inhibiting the production of PEDV infectious progeny virus particles.

[0052] Example 7: Inhibitory effect of tripterygium wilfordii on PEDV infection under preventive administration, co-administration and therapeutic administration Vero cells were cultured at 3 × 10 5Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 0.5 μmol / L tripterygium wilfordii to incubate the cells for 4 h, then the medium was discarded, and the cells were rinsed with PBS three times and infected with PEDV CV777 strain with MOI=0.01. The virus solution was discarded 1 h after infection, and 10% FBS DMEM was replaced for continued incubation as the prevention group (Pre-treatment); after the cells adhered to the wall, they were infected with PEDV CV777 strain with MOI=0.01 containing 0.5 μmol / L tripterygium wilfordii. The virus solution was discarded 1 h after infection, and 10% FBS DMEM was replaced for continued incubation as the co-administration group (During-time); after the cells adhered to the wall, they were infected with PEDV CV777 strain with MOI=0.01. The virus solution was discarded 1 h after infection, and 10% FBS DMEM was replaced for continued incubation as the co-administration group (During-time). The cells were incubated in DMEM for 24 hours before infection, which was the treatment group (Post-time); the cells were treated with 0.5 μmol / L tripterygium wilfordii from 4 hours before infection to 24 hours after infection, which was the full-time administration group (Full-time); the cells were not added with tripterygium wilfordii, which was the control group (Control); and the cells were not infected with the virus, which was the blank group (Mock). After 24 hours of virus infection, the cell supernatant was discarded, the cell culture was collected, the total protein was extracted, and the difference in the expression of PEDV N protein was detected by Western blot.

[0053] The results are as follows Figure 7 As shown, compared with the control group, the full-course administration group, the co-administration group, the treatment group and the prevention group all significantly inhibited the expression level of PEDV N protein. Grayscale quantitative analysis showed that the expression level of PEDV N protein in the full-course administration group was 0.08 times that of the control group, the expression level of PEDV N protein in the prevention group was 0.67 times that of the control group, the expression level of PEDV N protein in the co-administration group was 0.87 times that of the control group, and the expression level of PEDV N protein in the treatment group was 0.28 times that of the control group. The full-course administration group had the most significant effect, indicating that tripterygium wilfordii is the most promising in preventing and treating PEDV infection.

[0054] Example 8: Effects of different concentrations of tripterygium wilfordii on PDCoV N gene nucleic acid levels in PK-15 cells Vero cells were cultured at 3 × 10 5 Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO. 2After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with the PDCoV HKU15 strain at an MOI of 0.1. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for further incubation for 24 h. After 24 h, the total RNA was extracted using a total RNA extraction kit according to the instructions. After reverse transcription, the PEDV N gene primers and SYBR Green I were used as fluorescent dyes to perform fluorescence quantitative PCR to detect the relative expression of the PDCoV N gene. The reaction conditions were: 95°C for 5 min to activate the hot start enzyme; 95°C for 10 s to denature, 60°C for 30 s to anneal, and 45 cycles of reaction; the fluorescence signal was collected at this step.

[0055] The results are as follows Figure 8 As shown in the figure, compared with the control group, triptolide at 0.1 μmol / L significantly inhibited the transcription level of PDCoV N gene ( p -value<0.0001), and the inhibition of PDCoV N gene was concentration-dependent at a concentration of 0.1-0.5 μmol / L. This indicates that tripterygium wilfordii has the effect of inhibiting the nucleic acid level of PDCoV N gene.

[0056] Example 9: Effects of different concentrations of tripterygium wilfordii on PDCoV N protein expression in PK-15 cells PK-15 cells were cultured at 3×10 6 Each well was inoculated in a 6-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO 2 After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with PDCoV HKU15 strain at MOI = 0.1. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for another 24 h. After 24 h, the cell supernatant was discarded, the cell culture was collected, the total protein was extracted, and the difference in PDCoV N protein expression was detected by Western blot.

[0057] The results are as follows Fig. 9As shown in the figure, compared with the uninfected group, the expression of PEDV N protein in the infected control group (0 μmol / L group) and the tripterygium wilfordii treated group was significantly increased. However, compared with the infected control group, tripterygium wilfordii inhibited the expression of PDCoV N protein in a concentration-dependent manner at a concentration of 0.1-0.5 μmol / L. Grayscale quantitative analysis showed that the expression levels of PDCoV N protein under the treatment of 0.1-0.5 μmol / L tripterygium wilfordii were 0.77 times, 0.64 times, 0.56 times, 0.52 times and 0.28 times that of the infected control group, respectively, indicating that tripterygium wilfordii has the effect of inhibiting the expression of PDCoV N protein.

[0058] Example 10: Effects of different concentrations of tripterygium wilfordii on the production of PDCoV progeny viruses in PK-15 cells PK-15 cells were cultured at 3×10 5 Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO. 2 After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with PDCoV HKU15 strain at MOI = 0.1. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for another 24 h. After 3 days of infection, the positive rate of cytopathic effect in each well was observed and recorded. The virus titer (TCID 50 )value.

[0059] Example 11: Effects of different concentrations of tripterygium wilfordii on the nucleic acid level of PRRSV ORF7 gene in Marc-145 cells Marc-145 cells were cultured at 3 × 10 5 Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO. 2After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with the PRRSV BJ-4 strain at an MOI of 0.1. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for further incubation for 24 h. After 24 h, the total RNA was extracted using a total RNA extraction kit according to the instructions. After reverse transcription, the PRRSV ORF7 gene primers and SYBR Green I were used as fluorescent dyes to perform fluorescence quantitative PCR to detect the relative expression of the PRRSVORF7 gene. The reaction conditions were: 95°C for 5 min to activate the hot start enzyme; 95°C for 10 s to denature, 60°C for 30 s to anneal, and 45 cycles of reaction; the fluorescence signal was collected at this step.

[0060] The results are as follows Fig.10 As shown in the figure, compared with the control group, triptolide at 0.3 μmol / L significantly inhibited the transcription level of PRRSVORF7 gene ( p -value=0.0192), and the inhibition of PRRSV ORF7 gene was concentration-dependent at a concentration of 0.1-0.5 μmol / L. This indicates that tripterygium wilfordii has the effect of inhibiting the nucleic acid level of PRRSV ORF7 gene.

[0061] Example 12: Effects of different concentrations of tripterygium wilfordii on the expression of PRRSV N protein in Marc-145 cells Marc-145 cells were cultured at 3 × 10 6 Each well was inoculated in a 6-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO 2 After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with the PRRSV BJ-4 strain at an MOI of 0.1. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for another 24 h. After 24 h, the cell supernatant was discarded, the cell culture was collected, the total protein was extracted, and the difference in the expression of PRRSV N protein was detected by Western blot.

[0062] The results are as follows Fig.11As shown in the figure, compared with the uninfected group, the expression of PRRSV N protein in the infected control group (0 μmol / L group) and the tripterygium wilfordii treated group was significantly increased. However, compared with the infected control group, tripterygium wilfordii inhibited the expression of PRRSV N protein in a concentration-dependent manner at a concentration of 0.1-0.5 μmol / L. Grayscale quantitative analysis showed that the expression levels of PRRSV N protein under the treatment of tripterygium wilfordii at a concentration of 0.1-0.5 μmol / L were 0.88 times, 0.84 times, 0.77 times, 0.76 times and 0.61 times that of the infected control group, respectively, indicating that tripterygium wilfordii has the effect of inhibiting the expression of PRRSV N protein.

[0063] Example 13: Effects of different concentrations of tripterygium wilfordii on the production of PRRSV progeny viruses in Marc-145 cells Marc-145 cells were cultured at 3 × 10 5 Each well was inoculated in a 24-well plate. After the cells adhered to the wall, the culture medium in the plate was discarded and replaced with 10% FBS DMEM containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 μmol / L tripterygium wilfordii. The cells were incubated at 37°C and 5% CO. 2 After 4 h of incubation in the incubator, the supernatant was discarded and the cells were infected with PRRSV BJ-4 strain at an MOI of 0.1. The virus solution was discarded 1 h after infection and replaced with 10% FBS DMEM containing the corresponding concentration of tripterygium wilfordii for another 24 h. After 3 days of infection, the positive rate of cytopathic effect in each well was observed and recorded. The virus titer (TCID 50 )value.

[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 described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of tripterygium wilfordii in the preparation of a drug or feed additive for preventing and / or treating porcine viral diseases, characterized in that: The viral disease includes at least one of porcine epidemic diarrhea and blue ear disease.

2. The use according to claim 1, characterized in that: The porcine epidemic diarrhea includes porcine epidemic diarrhea caused by porcine epidemic diarrhea virus PEDV and / or porcine delta coronavirus PDCoV; The porcine reproductive and respiratory syndrome virus (PRRSV) is included in the porcine reproductive and respiratory syndrome virus (PRRSV) caused by the porcine reproductive and respiratory syndrome virus (PRRSV).

3. The use of tripterygium wilfordii in the preparation of drugs for anti-swine viral infection, characterized in that: The virus includes at least one of porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and porcine reproductive and respiratory syndrome virus PRRSV.

4. The use according to claim 3, characterized in that: The anti-swine virus infection comprises at least one of inhibiting viral gene expression, inhibiting viral protein expression and inhibiting the production of progeny virus particles.

5. The use according to claim 4, characterized in that: The viral genes include PEDV N gene, PDCoV N gene and PRRSV ORF7 gene, and the viral proteins include PEDVN protein, PDCoVN protein and PRRSV N protein.

6. The use of tripterygium wilfordii in the preparation of anti-swine virus drugs, characterized in that: The virus includes at least one of porcine epidemic diarrhea virus PEDV, porcine deltacoronavirus PDCoV and porcine reproductive and respiratory syndrome virus PRRSV.

7. The use according to any one of claims 3 to 6, characterized in that: The porcine epidemic diarrhea virus PEDV includes the CV777 strain; The porcine deltacoronavirus PDCoV includes the HKU15 strain; The porcine reproductive and respiratory syndrome virus PRRSV includes the BJ-4 strain.

8. The use according to any one of claims 3 to 6, characterized in that: The effective concentration of the tripterygium wilfordii is 0.1-0.5 μmol / L.

9. The use according to any one of claims 3 to 6, characterized in that: The tripterygium wilfordii includes tripterygium wilfordii and its pharmaceutically acceptable salts; The medicine or feed additive comprises tripterygium wilfordii and at least one pharmaceutically acceptable excipient; The dosage form of the medicine is a gastrointestinal administration dosage form or an injection dosage form.

10. The use according to claim 9, characterized in that: The gastrointestinal tract administration dosage form is tablets, capsules, granules, pills or dripping pills.

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