Application of naringenin in preparation of medicine for preventing and treating diseases caused by pseudorabies virus

Abstract

The invention discloses application of naringenin in preparation of a medicine for preventing and treating diseases caused by pseudorabies virus, and belongs to the technical field of biological medicine. The naringenin has good anti-PRV activity in vivo and in vitro. The naringenin can inhibit virus proliferation of the pseudorabies virus in an intracellular replication stage, reduce expression of PRV gB protein, lower expression of TLR4 and P65 protein to inhibit activation of an NF-kB pathway and lower expression of an inflammatory gene, and reduce nucleation of the NF-kB / P65 protein to achieve anti-inflammatory and antiviral effects. In-vivo experiments prove that naringenin can delay the disease time of mice, reduce the virus load in organs of the mice infected by PRV and relieve pathological changes of tissues and organs of the mice, and in addition, the survival rate of the mice infected by viruses is improved by adjusting the serum cell factor level of the mice. The naringenin provides a new strategy for preventing and treating PRV infection.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of naringenin in preparing medicines for preventing and treating diseases caused by pseudorabies virus. Background Art

[0002] Pseudorabies virus (PRV) belongs to the family Herpesviridae and the genus Varicellavirus. It is the pathogen that causes pseudorabies (PR). PRV has a wide host spectrum and can infect more than 40 mammals, including pigs. The virus mainly damages the nervous system and reproductive system of infected animals and can cause death of piglets and other susceptible animals. PRV can cause cytopathic effects (CPE) in infected cells. In addition, PRV is an important model virus for studying the biology of herpes viruses and neuronal pathways. Gene-deficient vaccines have been used to prevent and control pseudorabies in China, but since 2011, a large number of mutant strains with enhanced neurotropism have appeared in the clinic, and the traditional vaccine strain Bartha-K61 can no longer provide complete protection against clinical mutant strains of PRV. In addition, recent studies have found that PRV has the risk of infecting humans and causing central nervous system diseases, fatal encephalitis and endophthalmitis. These research findings suggest that PRV has a very important public health risk. Therefore, the development of new and effective drugs is a beneficial and necessary supplement to the control of PRV.

[0003] In recent years, the antiviral effect of natural compounds has gradually emerged. Natural compounds have the advantages of low toxicity, unique chemical structure, easy access, and wide biological activity, and have gradually become a hot spot for drug development. Studies have found that flavonoids extracted from natural plants have good anti-herpes virus activity. For example, the flavonoid quercetin can effectively prevent the adsorption and entry of PRV, protect infected mice, and play an anti-PRV effect. Luteolin is a flavonoid compound widely present in honeysuckle, which can play an antiviral role by inhibiting the expression of PRV-gB gene and protein levels. Kaempferol, a flavonoid compound widely present in edible plants such as strawberries and grapes, has been found to significantly reduce the viral load of PRV in the brain, lungs, kidneys, heart and spleen of mice, inhibit the transcription of PRV early gene IE180, and reduce the inflammatory response caused by PRV, thereby playing an anti-PRV effect.

[0004] Naringenin is a dihydroflavonoid compound, mainly found in grapefruit, tomatoes, grapes and citrus fruits of the Rutaceae family. Studies have shown that naringenin has multiple pharmacological activities such as anti-inflammatory, antibacterial, antiviral, antioxidant, anti-tumor, and liver protection, and can be used in medicine, food and other fields. Naringenin also shows good antiviral effects. Studies have shown that naringenin has a significant inhibitory effect on a variety of neurotropic viruses such as dengue virus, Zika virus, Sindbis Virus and Chikungunya virus. However, there are no reports on the use of naringenin to inhibit the replication of pseudorabies virus, especially neurotropic variant strains. Summary of the invention

[0005] The purpose of the present invention is to provide a use of naringenin in the preparation of a drug for preventing and treating diseases caused by pseudorabies virus, so as to solve the problems existing in the above-mentioned prior art. The present invention evaluates the inhibitory effect of naringenin on neurotropic virus PRV in vivo and in vitro. And explores its mechanism of action against PRV, aiming to discover a new drug for preventing and treating PRV infection, and bring a new method and strategy for the control of PRV.

[0006] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is the use of naringenin in the preparation of drugs for preventing and treating diseases caused by pseudorabies virus.

[0007] A second technical solution of the present invention is a drug for preventing and treating diseases caused by pseudorabies virus, comprising naringenin.

[0008] The third technical solution of the present invention is a pseudorabies virus inhibitor or disinfectant, comprising naringenin.

[0009] Based on the above technical solution, the present invention has the following technical effects: The present invention has experimentally confirmed that naringenin inhibits the proliferation of PRVTJ strain on PK15 cells in a dose-dependent manner, and the 50% inhibition concentration (CC 50 ) is 0.238 mg / mL, IC 50 is 0.008 mg / mL, and the selection index (SI, CC 50 / IC 50) was 29.75, and the inhibition rate of PRVTJ strain was as high as 97.04% at the maximum non-toxic dose. Naringenin can inhibit the intracellular replication stage of the virus, but has no significant effect on virus adsorption and entry. The effect of naringenin on the TLR4 / NF-κB pathway was detected at the gene and protein levels. The results showed that PRV infection activated the NF-κB pathway, and naringenin downregulated the expression of TLR4 and P65 proteins to inhibit the activation of the NF-κB pathway, downregulated the expression of IL-18, IL-1β, TNF-α and IL-6 inflammatory genes, and reduced the nuclear expression of NF-κB / P65 proteins to exert anti-inflammatory and antiviral effects. The results of in vivo experiments in mice showed that the high-dose group (200 mg / kg) and the medium-dose group (100 mg / kg) of naringenin significantly delayed the onset of mice. All mice in the PRVTJ strain challenge group died during the experimental period, and the survival rate of mice in the medium-dose group (100 mg / kg) of naringenin was as high as 57.14%. Naringenin high (200 mg / kg), medium (100 mg / kg), and low dose groups (50 mg / kg) can significantly inhibit the replication of PRV in the brain, lungs, and kidneys of mice, and reduce the tissue pathological damage caused by PRV infection. Naringenin can exert its anti-PRV effect by regulating the levels of IL-18, IL-1β, TNF-α, and IL-6 cytokines in the serum of infected mice. These data indicate that naringenin has a good anti-PRV effect, which provides a new strategy for the prevention and control of PRV. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 The chemical structure of naringenin and the results of in vitro inhibition of PRV replication, including (A) chemical structure of naringenin, (B) cytotoxicity of naringenin to PK15 cells, (C) inhibition rate of naringenin to PRV, (D) CC of naringenin in PK15 cells 50 , (E) IC of naringenin in inhibiting the proliferation of PRV in PK15 cells 50 , (F) FQ-PCR detection of the effect of naringenin on viral DNA copy number in PRV-infected cells. Compared with the control group without naringenin treatment, ns indicates P >0.05, **** indicates P <0.0001.

[0012] Figure 2The results of the test on the inhibition of PRV replication by naringenin. PRV is the virus control group, PRV+naringenin is the naringenin experimental group, and the naringenin concentrations are 0.063 mg / mL and 0.032 mg / mL. Compared with the PRV control group, ns means P >0.05, **** indicates P < 0.0001.

[0013] Figure 3 The results of the test on the effect of naringenin on the expression of PRVgB gene and protein, including (A) relative expression of PRVgB protein, (B) gray value quantification of PRV gB protein, (C) relative expression of gB gene, (D) optimal binding result of PRV gB protein and naringenin molecule. Compared with the PRV control group, **** indicates P <0.0001.

[0014] Figure 4 The results of the test on the effect of naringenin on the expression of TLR4, P65, NF-κB and related cytokines, including (A) relative expression of TLR4 protein, (B) relative expression of P65 protein, (C) gray value quantification of TLR4 protein, (D) gray value quantification of P65 protein, (E) relative change of TLR4 gene, (F) relative change of NF-κB gene, (G) relative change of IL-1β gene, (H) relative change of IL-6 gene, (I) relative change of IL-18 gene, (J) relative change of TNF-α gene. Compared with the PRV control group, ** indicates P < 0.01, **** indicates P < 0.0001. Compared with the cell control group, # indicates P <0.05, ### indicates P < 0.001, #### indicates P < 0.0001.

[0015] Figure 5 The results of observing the expression of NF-κB / P65 protein in the nucleus of PK15 cells under a fluorescence microscope. The blue fluorescence represents the nucleus and the green fluorescence represents the NF-κB / P65 protein.

[0016] Figure 6 The results of the antiviral activity test of naringenin against PRV in mice. Among them, (A) the survival rate of mice in each group, (B) the weight change of mice in each group, (C) the viral load of tissue organs of mice in each group, and (D) the pathological sections of brain, lung, kidney, liver and heart tissues of mice. Compared with the PRV control group, ns means P >0.05, ** indicates P <0.01, *** indicatesP <0.001, **** indicates P < 0.0001.

[0017] Figure 7 The results of the test on the effect of naringenin on the expression of cytokines in mouse serum. (A) is the concentration of IL-18 cytokines in the serum of each group of mice, (B) is the concentration of IL-1β cytokines in the serum of each group of mice, (C) is the concentration of IL-6 cytokines in the serum of each group of mice, and (D) is the concentration of TNF-α cytokines in the serum of each group of mice. Compared with the PRV control group, ns indicates P >0.05,* indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001. Compared with the cell control group, # indicates P <0.05, ## indicates P <0.01, ### indicates P <0.001. DETAILED DESCRIPTION

[0018] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0019] The embodiment of the present invention provides the use of naringenin in preparing a drug for preventing and treating diseases caused by pseudorabies virus.

[0020] The embodiment of the present invention further provides a drug for preventing and treating diseases caused by pseudorabies virus, comprising naringenin.

[0021] In some specific embodiments, the dosage form of the drug is a solid dosage form, a liquid dosage form, a cream dosage form or an emulsion dosage form.

[0022] The embodiment of the present invention further provides a pseudorabies virus inhibitor or disinfectant, comprising naringenin. Example 1

[0023] 1 Materials and methods 1.1 Cells, materials, and naringenin Porcine kidney cells (PK15) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. The culture medium used for cytotoxicity and antiviral experiments contained 2% fetal bovine serum. The PRV virus strain used in the present invention was the PRVTJ (GenBank: KJ789182.1) strain, which was kindly provided by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The virus was propagated in PK15 cells, and the TCID was calculated using the Reed-Muench method. 50 For 10 -6.89 / 0.1 mL, stored at -80°C until use. Naringenin was purchased from Dalian Meilun Biotechnology Co., Ltd. with a purity of 98% and dissolved in 1% dimethyl sulfoxide (DMSO) for use.

[0024] 1.2 Cytotoxicity test of naringenin and its inhibitory activity on PRV The viability of PK15 cells was detected by CCK-8 kit at a concentration of 0.016-0.5 mg / mL of naringenin to determine the cell survival rate. 200 μL of naringenin diluted two-fold (the initial concentration of naringenin was 0.5 mg / mL, and it was diluted two-fold to 0.25 mg / mL, 0.125 mg / mL, 0.063 mg / mL, 0.032 and 0.016 mg / mL) was added to the PK15 cells that had grown to a monolayer in a 96-well plate, and incubated at 37°C and 5% CO2 for 48 h. A cell control group was also set up. After 48 h, CCK-8 solution was added according to the instructions, incubated at 37°C for 50 min, and the OD value of the well plate at a wavelength of 450 nm was measured using an enzyme reader. The cell survival rate was calculated to evaluate the cytotoxicity of naringenin. Cell survival rate (%) = (OD value of experimental group) 450 Value / cell control group OD 450 value) × 100% The inhibition rate of naringenin on PRV was detected by CCK8 method. To determine the inhibitory activity of naringenin on PRV, 100 TCID 50 The cells were incubated with PRV virus solution for 1 h, discarded, and washed 3 times with PBS. 200 μL of naringenin (0.063 mg / ml, 0.032 mg / ml, 0.016 mg / mL, 0.008 mg / ml, 0.004 mg / ml, and 0.002 mg / ml) solution was added to each well of the experimental group, and 200 μL of cell maintenance solution (2% fetal bovine serum) was added to each well of the cell control group and the virus control group for incubation. After 48 h, the OD value of each well at a wavelength of 450 nm was measured, and the virus inhibition rate was calculated to evaluate the inhibitory activity of naringenin on PRV. The half cytotoxic concentration (CC) of naringenin was calculated using GraphPad Prism 8.0 software. 50 ) and half inhibitory concentration (IC 50 ). Inhibition rate (%) = (OD 450 Value-OD of virus control group 450 value) / (OD value of cell control group 450 Value-OD of virus control group 450 value) × 100%.

[0025] PK15 cells were seeded in a 6-well plate and cultured in a 5% CO2, 37°C incubator. When the cells grew to 80%, they were taken out, washed twice with PBS, and 100 TCID 50 PRV virus solution was used for infection at 37℃ for 1 h, the virus solution was discarded, the cells were washed twice with PBS, and naringenin dilution solution (0.063mg / ml, 0.032mg / ml) was added, and the cells were cultured at 37℃ and 5% CO2. A virus control group was also set up. After 36 h, the solution was discarded, the cells were washed twice with PBS, and cell samples (cell culture supernatant and cell culture, the same below) were collected, cell DNA was extracted, and the number of viral DNA copies in infected cells was determined by FQ-PCR.

[0026] 1.3 Effect of naringenin on the PRV replication cycle Virus adsorption stage: PK15 cells were inoculated in a 6-well plate and cultured in a 5% CO2, 37°C incubator. When the cells grew to 80%, they were taken out and pre-cooled at 4°C for 1 h. They were washed twice with ice-cold PBS and 100 TCID 50 A mixture of PRV virus solution and an equal volume of naringenin (0.063 mg / ml, 0.032 mg / ml) was added to the cells and incubated at 4°C for 1 h. The virus mixture was discarded, the cells were washed twice with PBS, and the cells were added with cell maintenance solution. The cells were cultured in a 37°C, 5% CO2 incubator. A virus control group was also set up. After 36 h, the solution was discarded, and the cells were washed twice with PBS to collect the cell samples.

[0027] Virus entry stage: PK15 cells were inoculated in a 6-well plate and cultured in a 5% CO2, 37°C incubator. When the cells grew to 80%, they were taken out, washed twice with ice-cold PBS, and 100 TCID 50 PRV virus solution was added, infected at 4℃ for 1 h, the supernatant was aspirated and washed twice with PBS to remove unbound virus solution, naringenin dilution solution (0.063mg / ml, 0.032mg / ml) was added, incubated at 37℃ for 1 h, the solution was discarded, cell maintenance solution was added, and culture was continued at 37℃ and 5% CO2, and a virus control group was set up. After 36 h, the solution was discarded, and the cell samples were washed twice with PBS to collect.

[0028] Virus replication stage: PK15 cells were inoculated in a 6-well plate and cultured in a 5% CO2, 37°C incubator. When the cells grew to 80%, they were taken out, washed twice with PBS, and 100 TCID 50 PRV virus solution was added, infected at 37℃ for 1 h, virus solution was discarded, washed twice with PBS, naringenin dilution solution (0.063mg / ml, 0.032mg / ml) was added, and cultured at 37℃, 5% CO2, and a virus control group was set up. After 36 h, the solution was discarded, and cell samples were collected by washing twice with PBS.

[0029] Total cell DNA was extracted using the TIANamp Genomic DNA Kit, and the viral DNA copy number in infected cells was analyzed by real-time quantitative polymerase chain reaction (FQ-PCR). The reaction primers were 5'-GCCGAGTACGACCTCTGCC-3' (forward) and 5'-CGAGACGAACAGCAGCCG-3' (reverse); the probe was 5'-HEX-CCGCGTGCACCACGAAGCCT-BHQ1. The standard plasmid containing the gI gene was used to generate the standard curve, and the FQ-PCR reaction conditions were 94℃ pre-denaturation for 10 min; 94℃ 35 s, 60℃ 35 s, for a total of 45 cycles.

[0030] 1.4 Effects of naringenin on gene expression PK15 cells were seeded in a 6-well plate. When the cells grew to 80%, they were taken out, washed twice with PBS, and 100 TCID 50 PRV virus solution was used for infection at 37℃ for 1 h, the virus solution was discarded, the cells were washed twice with PBS, and naringenin dilution solution (0.063mg / ml, 0.032mg / ml) was added. The cells were cultured at 37℃ and 5% CO2. A virus control group and a cell control group were set up separately. After 36 h, the solution was discarded, the cells were washed twice with PBS, and total cell RNA was extracted using the RNA Easy Fast Tissue / Cell Kit. Reverse transcription PCR (RT-PCR) was used with FastKing gDNADispelling RT SuperMix to obtain cDNA for RT-qPCR to detect relative gene expression levels. The primers used for RT-qPCR are shown in Table 1. The expression of β-actin was used to standardize the differences in total cDNA levels in the samples. −ΔΔCT Methods The mRNA expression level was calculated. The RT-qPCR reaction program was: 95℃ pre-denaturation for 5 min; 95℃ for 15 s, 55℃ for 30 s, 72℃ for 35 s, for a total of 40 cycles.

[0031] Table 1 Primer sequences , 1.5 Western Blot Experiment PK15 cells were seeded in a 6-well plate. When the cells grew to 80%, they were taken out, washed twice with PBS, and 100 TCID 50PRV virus solution was added and infected at 37℃ for 1 h. The virus solution was discarded, washed twice with PBS, and naringenin diluent (0.063mg / ml, 0.032mg / ml) was added. The cells were cultured at 37℃ and 5% CO2. A virus control group and a cell control group were set up. After 36 h, the solution was discarded, washed twice with PBS, and lysed with protein extraction buffer. The cell samples were harvested for total cell protein extraction. The proteins were separated by 10% SDS-PAGE and transferred to NC membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h, and then TLR4, P65, gB and β-actin primary antibodies were added for incubation. The membrane was incubated at 4℃ overnight. After washing 3 times with TBST, HRP-conjugated IgG secondary antibody was added and incubated on a shaker at room temperature for 1 h. Finally, the protein was visualized using enzyme chemiluminescence reagent (ECL), and the protein gray value was analyzed using ImageJ software (National Institutes of Health).

[0032] 1.6 Molecular docking prediction of naringenin and PRVgB protein target binding site The 3D structure of naringenin (PubChemCID 439246) was downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov), and the 3D structure of PRV gB protein (PDB ID: 5YS6) was downloaded from the RCSB PDB online database (https: / / www.pdbus.org / ). The protein structure was optimized using Pymol software to remove water molecules and small molecule ligands. The PRV gB protein molecular model was hydrogenated using AutoDock, and the entire protein molecule was wrapped with GridBox for molecular docking. The optimal docking result with the lowest binding energy was screened and visualized using Pymol.

[0033] 1.7 Effect of naringenin on nuclear expression of NF-κB / P65 protein PK15 cells were seeded in a 24-well plate. When the cells grew to 80%, they were taken out, washed twice with PBS, and 100 TCID 50PRV virus solution was used for infection at 37℃ for 1 h, the virus solution was discarded, the cells were washed twice with PBS, and naringenin dilution solution (0.063 mg / ml) was added, and the cells were cultured at 37℃ and 5% CO2. A virus control group and a cell control group were set up. After 36 h, the solution was discarded, the cells were washed twice with PBS, and the cells were fixed with 4% paraformaldehyde for 20 min at -20℃; after washing three times with PBS, the cells were permeabilized with 0.2% TritonX-100 for 10 min; after washing with PBS, the cells were added with P65 primary antibody (1:200) and incubated at 4℃ for 12 h; after washing with PBS, the cells were added with goat anti-rabbit secondary antibody (1:500) and incubated at room temperature for 45 min; after washing with PBS, the cells were added with Hoechst to stain the nuclei for 5 min, and the cells were placed on a shaker and washed with PBS for 3 times for 5 min each time, and finally observed under a fluorescence microscope.

[0034] 1.8 Determination of the inhibitory effect of naringenin on PRV in vivo 46 female Kunming mice weighing 20 ± 2g were purchased from Changsheng Experimental Animal Center. After 1 week of adaptation and feeding in the experimental animal laboratory of Northeast Agricultural University, they were randomly divided into 5 groups, namely: blank control group (6 mice), PRV challenge control group (10 mice), naringenin high-dose group (H) (10 mice), naringenin medium-dose group (M) (10 mice) and naringenin low-dose group (L) (10 mice). The mice in the high-, medium- and low-dose groups of naringenin were gavaged with 200 mg / kg, 100 mg / kg and 50 mg / kg of naringenin, respectively, once a day for three days. On the 4th day, the mice in the PRV challenge control group and the naringenin experimental group were intramuscularly injected with 1000 TCID 50 The blank control group mice were intramuscularly injected with an equal amount of DMEM with 100 μL of PRV virus solution. 2 h after the mice were challenged with the virus, the naringenin experimental group continued to be gavaged at the above dose, once a day, for 4 days, and the weight and food intake of the mice were recorded every day. On the third day after the mice were challenged with the virus (Note: the day of the challenge was recorded as the 0th day after the challenge), 3 mice were randomly selected from each group for blood collection and dissection, and the organ lesions of the mice were observed. The heart, liver, brain, lung and kidney tissues of the mice were collected and weighed, and the remaining mice were observed to determine the survival rate.

[0035] The survival rate of each group was calculated according to the following formula: Survival rate = number of surviving mice / total number of remaining mice.

[0036] Part of the collected organ tissues was homogenized in physiological saline to extract DNA to determine the viral load in the tissues. On the 4th day after PRV infection, the mouse brain, lung, kidney, liver and heart tissues were collected, viral DNA was extracted, and the viral copy number in each organ tissue was detected by FQ-PCR.

[0037] The other part was fixed in 4% paraformaldehyde, and then tissue sections were prepared and histopathological examination was performed. On the third day after PRV infection, three mice were randomly selected from each group for blood collection and used to determine the four cytokines in mouse serum. The collected blood samples were coagulated at 4°C for 30 minutes and centrifuged at 5000r / min for 10 minutes to obtain serum, and the ELISA kit was used to determine the content of TNF-α, IL-18, IL-1β and IL-6 cytokines in mouse serum.

[0038] 1.9 Statistical methods All cell experiments were repeated three times and the results are presented as mean ± SD. The statistical significance of the data was assessed by two-tailed student's t-test using GraphPad Prism 8.0 software (La Jolla, CA, USA). p Values ​​< 0.05 were considered statistically significant. (ns: p >0.05,* p <0.05,** p <0.01,*** p <0.001 and **** p <0.0001, # p <0.05,## p <0.01,### p <0.001 and #### p <0.0001).

[0039] 2 Results 2.1 Determination of cytotoxicity of naringenin to PK15 cells and its inhibition of PRV proliferation in PK15 cells Naringenin was diluted with DMEM to a final concentration of DMSO less than 1%, which had no effect on viruses and cells. The PK15 cell viability was evaluated by CPE observation and CCK8 methods to determine the cytotoxicity of naringenin. The results showed that as the concentration of naringenin increased, the cell viability gradually decreased; when the concentration of naringenin was less than 0.063 mg / mL, the viability of PK15 cells was not significantly different from that of the cell control group ( Figure 1 Therefore, this concentration was selected as the safe concentration for subsequent cell experiments. The half cytotoxic concentration (CC 50 ) was 0.238mg / mL. The inhibitory activity of naringenin on PRV in PK15 cells was evaluated by CPE observation, CCK8 and FQ-PCR. CCK8 results showed that naringenin had a significant inhibitory effect on cell death induced by PRV infection and was dose-dependent; when the concentration of naringenin was 0.063mg / mL, the inhibition rate was as high as 97.04% ( Figure 1(C) Calculate the IC of naringenin 50 is 0.008 mg / mL, and the selectivity index (SI, CC 50 / IC 50 ) was 29.75. In the viral copy number determination experiment, the viral copy number decreased in a dose-dependent manner in the presence of naringenin. When the concentration of naringenin was 0.063 mg / mL, the viral copy number decreased by 98.84% ( Figure 1 (F). The above results indicate that naringenin can significantly inhibit the proliferation of PRV in PK15 cells.

[0040] 2.2 Effects of naringenin on specific stages of the PRV life cycle In order to further explore the effect of naringenin on the PRV replication cycle, the number of viral copies of infected cells during the PRV adsorption, entry, and replication stages was determined using probe-based real-time fluorescence quantitative PCR. In the adsorption experiment, naringenin and PRV were added to cells at the same time at 4°C. The results showed that there was no significant difference between the naringenin experimental group and the PRV control group ( P > 0.05). In the entry experiment, the virus infected cells at 4°C, and then a maintenance solution containing naringenin was added. The results showed that the viral load of cells infected with naringenin in the experimental group was not significantly different from that in the virus control group ( P > 0.05), indicating that naringenin has no significant effect on the process of virus entry. After the virus enters the cell, it begins to use the energy and raw materials provided by the cell to replicate itself. In the replication experiment, the naringenin experimental group was able to significantly inhibit the number of viral copies in infected cells in a dose-dependent manner ( P <0.0001).

[0041] 2.3 Naringenin inhibits the expression of PRV gB protein gB protein is one of the most conserved envelope glycoproteins in the herpesvirus family and is an indispensable key protein in the fusion process of virus entry and intercellular transmission. In order to further study the effect of naringenin on the expression of PRV gB protein. First, RT-qPCR was used to detect the expression level of gB gene, and then Western Blot was used to detect the expression level of gB in PRV-infected cells and Image J software was used to analyze the gray value of the protein. Finally, molecular docking technology was used to predict the target site of naringenin binding to gB protein. The results showed that naringenin can significantly downregulate the PRV gB gene ( Figure 3 C) and protein ( Figure 3 In addition, naringenin can form hydrogen bond interactions with PRV gB protein at residues LEU-414, GLN-413, and ASP-415 ( Figure 3The binding energy is -3.26 kcal / mol, indicating that naringenin can form a stable binding with PRV gB protein.

[0042] 2.4 Naringenin inhibits the expression of TLR4 and P65 proteins and the release of inflammatory factors In order to further study the effect of naringenin on the NF-κB pathway after PRV infection of PK15 cells, RT-qPCR was first used to detect the expression levels of TLR4, P65, IL-1β, IL-6, IL-18 and TNF-α genes. Secondly, Western Blot was used to detect the expression levels of TLR4 and P65 key proteins, and Image J software was used to analyze the grayscale value of the protein. The results showed that naringenin could significantly downregulate the gene expression of TLR4, P65, IL-1β, IL-6, IL-18 and TNF-α. And naringenin can significantly downregulate the expression levels of TLR4 and P65 proteins ( Figure 4 ).

[0043] 2.5 Naringenin reduces NF-κB / P65 nuclear expression The nuclear translocation of NF-κB / P65 protein indicates the activation of NF-κB signaling pathway. Immunofluorescence was used to detect the effect of naringenin on the nuclear expression of NF-κB / P65 protein. Figure 5 As shown, the nuclear entry of NF-κB / P65 in the PRV control group was more than that in the cell control group, and the nuclear entry of NFκB / P65 in the naringenin experimental group was significantly reduced compared with the PRV control group.

[0044] 2.6 Naringenin inhibits PRV infection in mice In order to evaluate the inhibitory effect of naringenin on PRV in mice, in vivo animal experiments were performed as described above. On the third day after the infection, mice in the PRV infection group showed restlessness, itching, convulsions, moist skin at the infection site, and scratching and bleeding. They died within a few hours from the onset of typical neurological symptoms; on the sixth day, all mice in the PRV infection group died, with a survival rate of 0%; mice in the medium and high dose groups of naringenin showed clinical symptoms and died on the fifth and fourth days after the infection, respectively. On the tenth day after the infection, 2, 4 and 1 mice survived in the high, medium and low dose groups of naringenin, respectively, with mouse survival rates of 28.57% (2 / 7), 57.14% (4 / 7) and 14.28% (1 / 7), respectively (Note: There were 10 mice in each group of the naringenin experimental group and the virus control group, and 7 mice remained after 3 mice were randomly selected for blood sampling and dissection.), and all mice in the blank control group survived ( Figure 6Middle A); The weight change curve of mice showed that compared with the blank control group mice, the weight of the PRV-challenged mice continued to decrease after the onset of symptoms on the 3rd day; the average weight growth rate of the mice in the naringenin experimental group slowed down on the 5th day, but the weight of the mice showed a significant upward trend after the 6th day, and the weight of the mice showed a downward trend on the 9th day. ( Figure 6 Middle B).

[0045] In order to determine the viral load in mouse tissues and organs, 3 mice were randomly selected from each group, and the brain, lung, kidney, heart and liver tissues of the mice were collected and weighed. Then the total DNA in the tissues and organs was extracted, and the changes in the viral DNA copy number were determined by FQ-PCR. The results showed that PRV was detected in the brain, lung, kidney, heart and liver tissues of mice in the PRV-challenged control group, among which the viral load in the brain tissue was the highest, followed by the lung, kidney, liver and heart; compared with the PRV-challenged control group, the naringenin experimental group was able to significantly reduce the viral load in the brain, lung and kidney of mice. Especially in the brain and lung tissues, the medium-dose group of naringenin (100 mg / kg) had the best effect. However, there was no significant effect on the viral load in the liver and heart tissues ( Figure 6 Middle C).

[0046] To further explore whether naringenin has a protective effect on tissue damage caused by PRV, histopathological examinations were performed on the brain, lung, kidney, heart, and liver tissues of each group of mice. Figure 6 D in the middle shows that compared with the blank control group mice, the PRV-challenged control group mice had more severe cerebral vascular congestion and congestion; inflammatory cells infiltrated around the cerebral blood vessels, forming a typical "vascular cuff" phenomenon. The lungs showed congestion, hemorrhage, and edema. The alveolar epithelium proliferated, the alveolar wall thickened, and the alveolar septum widened. The renal tubules were congested and congested, the glomeruli were swollen, and the renal capsule cavity was narrowed. The central veins of the liver were congested, and the hepatocytes were extensively edematous. The heart showed myocardial cell hemorrhage, myocardial fiber rupture, and dissolution. The naringenin experimental group was able to alleviate tissue-related pathological changes.

[0047] 2.7 Changes in serum cytokines in mice To further study the effect of naringenin on cytokines in the serum of PRV-infected mice, blood was collected from each group of mice to separate the serum, and the changes of cytokines IL-18, IL-1β, IL-6 and TNF-α in the serum of mice were detected using ELISA kits. Figure 7It was shown that compared with the blank control group mice, PRV infection caused a significant increase in the levels of cytokines TNF-α, IL-6, IL-18 and IL-1β in the serum of mice. Compared with the PRV challenge control group, the levels of IL-18, IL-1β and TNF-α cytokines in the serum of mice in the high-dose naringenin group were significantly reduced, and the level of IL-6 cytokine did not change significantly. The levels of IL-18, IL-1β, IL-6 and TNF-α cytokines in the medium-dose naringenin group were significantly reduced. The serum factor IL-18 in the serum of mice in the low-dose naringenin group was significantly reduced, and there was no significant change in IL-1β, IL-6 and TNF-α.

[0048] In summary, naringenin has good anti-PRV activity both in vivo and in vitro. Naringenin can inhibit viral proliferation in the intracellular replication stage, reduce the expression of PRV gB protein, downregulate the expression of TLR4 and P65 proteins to inhibit the activation of the NF-κB pathway and downregulate the expression of inflammatory genes, and reduce the nuclear entry of NF-κB / P65 protein to exert anti-inflammatory and antiviral effects. In vivo experiments showed that naringenin can delay the onset of mice, reduce the viral load in mouse organs, and reduce the pathological changes in mouse tissues and organs to improve the survival rate of mice. PRV-infected mice can also be protected by regulating the level of mouse serum cytokines. In general, naringenin has the potential to become a new strategy for the prevention and treatment of PRV infection in the future.

[0049] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of naringenin in the preparation of drugs for preventing and treating diseases caused by pseudorabies virus.

2. A drug for preventing and treating diseases caused by pseudorabies virus, characterized in that: Including naringenin.

3. The drug according to claim 2, characterized in that The dosage form of the drug is a solid dosage form, a liquid dosage form, a paste dosage form or an emulsion dosage form.

4. A pseudorabies virus inhibitor or disinfectant, characterized in that: Including naringenin.

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