Use of forsythia suspensa essential oil in preparing traditional Chinese veterinary medicine for resisting porcine epidemic diarrhea virus

Abstract

The application provides use of forsythia suspensa essential oil in preparation of traditional Chinese veterinary medicine for resisting porcine epidemic diarrhea virus. Research results show that the forsythia suspensa essential oil can effectively inhibit the adhesion and internalization stages of the PEDV virus. Pharmacological experiments show that the forsythia suspensa essential oil can inhibit the expression of inflammatory factors such as TNF, IL-6 and CXCL8 by adjusting key target points in the IL-17 signal pathway, so as to achieve the effect of resisting the PEDV. It is shown that the forsythia suspensa essential oil has the potential of being a new type of antiviral drug, and provides a new train of thought for research and development of a new type of anti-PEDV drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of Forsythia essential oil in the preparation of traditional Chinese veterinary medicine against porcine epidemic diarrhea virus. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is an enteric disease caused by porcine epidemic diarrhea virus (PEDV) and mainly harms piglets. PEDV can be traced back to the 1970s and 1980s in the United Kingdom and China. Common symptoms of the virus include diarrhea, anorexia, and vomiting, which can reduce the immunity of piglets and eventually lead to the death of piglets, with a mortality rate of up to 100%. Since 2010, PEDV has broken out on a large scale worldwide, causing huge economic losses to the global pig industry. In China, Guangdong Province has become the main source of PEDV transmission, and the virus has been detected in multiple organs of piglets, with the highest virus content in intestinal tissue. Germany and Japan are currently the main centers of PEDV transmission in Europe and Asia, respectively. At present, vaccines are mostly used in pig farms to prevent PEDV infection, but due to the genetic diversity and variability of the virus, it brings great challenges to the treatment of PEDV, so it is urgent to develop a highly effective, low-toxicity, and minimally adverse drug.

[0003] Forsythia is the dried fruit of Forsythia suspensa (Thunb.) Vahl of Oleaceae, which has the effects of clearing heat and resolving toxins, resolving swelling and removing wind-heat. The volatile oil extracted from Forsythia is an important component for its efficacy and was first included as a quality indicator in the 2020 edition of Chinese Pharmacopoeia. Modern pharmacological studies have shown that Forsythia volatile oil has antibacterial, antioxidant, antipyretic, anti-inflammatory, and antiviral effects. Previous studies have shown that the main components of Forsythia volatile oil, α-pinene and β-pinene, have the potential to resist infectious bronchitis virus (IBV). Active ingredients in Forsythia leaves exert an anti-respiratory syncytial virus effect through the P13K / AKT signaling pathway. In addition, a recent computational study has shown that α-pinene and β-pinene have the potential to resist SARS-COV-2 and COVID-19. Essential oils and their main monoterpene compounds, such as eucalyptol, terpinen-4-ol, and α-pinene, have antiviral activity against herpes simplex virus type 1 (HSV-1), can directly inactivate virus particles, and interact with herpes virus particles in a dose-dependent manner. Currently, essential oils have been widely used in antiviral preparations. In 2019, essential oils were considered as a potential therapy for treating coronavirus, and bibliometric techniques found that only a small number of essential oils were used in the study of COVID-19. This indicates that essential oils have entered people's field of vision, which provides an opportunity for further research on the antiviral potential of essential oils.

[0004] At present, forsythia is used for the treatment of swine epidemic diarrhea, mainly in the prescription of traditional Chinese medicine, such as: patent application number: 201210596621.1, the invention name: a kind of traditional Chinese medicine for treating swine epidemic diarrhea, discloses a kind of traditional Chinese medicine for treating swine epidemic diarrhea, formula is: Xianhe grass 10g-100g, white head 10g-100g, Jianqu 10g-100g, wood 10g-100g, dried tangerine or orange peel 10g-100g, double flower 10g-100g, forsythia 10g-100g, ground plum 10g-200g, coptis 10g-50g.CN201410830022.0, the invention name: a kind of traditional Chinese medicine composition for treating swine viral diarrhea and its preparation method and use, provides a kind of traditional Chinese medicine composition for treating swine viral diarrhea, which is prepared from the following weight ratio of raw materials: gallnut 30-100 parts, coptis root 100-200 parts, honeysuckle 150-300 parts, forsythia 100-150 parts, giant knotweed 150-250 parts.Patent application number: 202010394285.7, the invention name: application of combination preparation of remdesivir and its nucleoside in resisting swine epidemic diarrhea virus, discloses that forsythoside is combined with remdesivir to resist swine epidemic diarrhea virus.

[0005] At present, there is no relevant literature report that the effective part (volatile oil) in forsythia is used alone for treating swine viral diarrhea. SUMMARY

[0006] The present application relates to the new use of forsythia essential oil.

[0007] The present application provides the use of forsythia essential oil in preparing traditional Chinese medicine for resisting swine epidemic diarrhea virus.

[0008] The traditional Chinese medicine is a medicine for preventing or / and treating swine epidemic diarrhea.

[0009] The forsythia essential oil contains α-pinene not less than 18% w / w, and contains β-pinene not less than 52% w / w. Preferably, the forsythia essential oil contains α-pinene 18.82% w / w, and contains β-pinene 52.74% w / w.

[0010] The pig is a piglet.

[0011] The traditional Chinese medicine is prepared into a pharmaceutically common preparation by taking forsythia essential oil as active ingredient and adding veterinary medicine acceptable adjuvant or auxiliary ingredient.

[0012] In the present application, CCK8 is used to evaluate cell viability in vivo experiment, and IFA and RT-Q-PCR are used to evaluate the antiviral activity of forsythia essential oil. In addition, the viral replication cycle is also studied to determine the stage at which forsythia essential oil plays an antiviral role.

[0013] The results showed that the Forsythia essential oil could effectively inhibit the attachment and internalization stages of PEDV virus. Other pharmacological experiments showed that Forsythia essential oil could inhibit the expression of inflammatory factors such as TNF, IL-6 and CXCL8 by regulating key targets in the IL-17 signaling pathway, thereby achieving the effect of anti-PEDV. It is shown that Forsythia essential oil has the potential to be a new type of antiviral drug, and provides a new idea for the research and development of new anti-PEDV drugs. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Forsythia essential oil could effectively inhibit PEDV infection ((A) Activity detection results of Forsythia essential oil on Vero cells (B) IFA schematic diagram after 6 hours of Forsythia essential oil, where FITC is virus infected cells and DAPI is cell nucleus. (C) IFA image processing result schematic diagram. (D) Expression of PEDV N mRNA in Vero cells under different treatments. Data are expressed as mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the model group, and ### P < 0.0001) compared with the blank group;

[0015] Figure 2 Effects of Forsythia essential oil on PEDV attachment, entry, replication and release ((A) Virus attachment test. (B) Virus internalization test. (C) Virus replication test. (D) Virus release test. All data are expressed as mean ± standard error, n represents the number of experimental groups, n = 3. The asterisk in the figure indicates that the difference is statistically significant * P < 0.05; ** P < 0.01; *** P < 0.001; ns: not significant);

[0016] Figure 3 Expression of proteins and inflammatory factors after PEDV infection of Vero cells ((A) Expression level of PEDV N protein (B) Expression level of HSP90AA1 protein (C) Expression level of MAPK14 protein (D) Expression level of CXCL8 factor (E) Expression level of TNF factor (F) Expression level of IL-6 factor (G) Expression level of COX-2 factor. Data are expressed as mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the model group, and

[0017] ### P < 0.001, #### P < 0.0001) compared with the blank group;

[0018] Figure 4Toxicity of Forsythia essential oil in vivo (A) Survival curve of mice orally administered with Forsythia essential oil at a single dose of 0, 10, 50, 250, 500, or 2500 mg / kg for 14 days. Subchronic toxicity evaluation of mice orally administered with Forsythia essential oil at a single dose of 0, 10, 50, 250, 500, or 2500 mg / kg. (B) Body weight was detected and plotted, and the mice were sacrificed on the 14th day, and (C) WBCs, (D) RBCs, (E) PLTs were detected. The results represent three independent experiments (mean ± SD), and the notes are *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the CTRL group;

[0019] Figure 5 Oral administration of Forsythia essential oil can protect piglets from PEDV infection in vivo (A) Clinical symptom score of piglets in each group. (B) Fecal score of piglets in each group. (C) Survival rate of piglets in each group. (D) Fecal virus shedding (E) Virus distribution in jejunum and ileum determined by RT-Q-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the PEDV infection group;

[0020] Figure 6 Staining results of intestinal tissue of challenged piglets after oral administration of Forsythia essential oil (100x);

[0021] Figure 7 Effect of Forsythia essential oil on the expression of related factors in PEDV pig intestinal tissue (A) Immunohistochemical results of pig intestinal tissue in each group (100x) (B) Immunohistochemical HSP90AA1 level expression of pig intestinal tissue in each group; (C) Immunohistochemical COX-2 level expression of pig intestinal tissue in each group; (D) Immunohistochemical TNF-α level expression of pig intestinal tissue in each group; Notes: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with DMEM; #### P < 0.0001; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the PEDV infection group. DETAILED DESCRIPTION

[0022] Test Example 1 Anti-porcine epidemic diarrhea virus experiment of Forsythia essential oil of the present application

[0023] 1. Method

[0024] 1.1 Reagents

[0025] Forsythia essential oil: commercially available, or refer to: Bai Yunchuan, et al. Optimization of bioenzyme-assisted extraction process, component analysis and in vitro antioxidant research of Forsythia essential oil [J]. West China Journal of Pharmacy, January 2023, Volume 38, Issue 1.

[0026] 1.2 Cell lines and viruses

[0027] Vero cells (a kind of African green monkey kidney epithelial cells) were preserved in the laboratory, high-sugar DMEM cell culture medium was purchased from Hyclone Company in the United States, CO2incubator and biological safety cabinet were purchased from Thermo Company in the United States; fetal bovine serum was purchased from PAN-Biotech Company in Germany; enhanced CCK-8 kit (CT0001, Shandong Sikaijie Biotechnology Co., Ltd.) was used. The PEDV strain used in the present application is CH / HBTS / 2017 (GenBank accession number: MH581489.1). Vero cells were cultured in high-sugar DMEM medium containing 10% fetal bovine serum and 1% double-antibiotic at 37℃ in a 5% CO2incubator. When the cells were subcultured, 0.25% trypsin was used for digestion and subculture.

[0028] 1.3 Cell toxicity

[0029] The Vero cells in the cell bottle were digested, and the cell suspension after digestion was transferred to a 96-well plate, and the number of cells in each well was 1×10 4 When the cell density was 80%-90%, the Forsythia essential oil was dissolved with an ethanol solution, and then gradient dilution was performed with DMEM basic medium to make the concentration 0.04, 0.08, 0.16, 0.31, 0.63, 1.25 μL / mL, 100 μL of Forsythia essential oil diluent was added to the 96-well plate, and the same amount of DMEM was added to the cell control group and the blank control group. After 48 h, 10 μL of CCK-8 solution was added to each well and incubated for 1 h, then the OD value of each well was detected at 450 nm and the cell viability was calculated.

[0030] Cell survival rate = (OD value of the administration group - OD value of the blank group) / (OD value of the normal group - OD value of the blank group);

[0031] The OD value of the administration group represents the OD450 value of the cells incubated with Forsythia essential oil and CCK-8 solution, the OD value of the blank group represents the OD450 value of the wells without cells, and the normal group refers to the OD450 value of the cells incubated with CCK-8 solution without Forsythia essential oil.

[0032] 1.4 Virus infection

[0033] Vero cells were inoculated at 1×10 4Vero cells were seeded at a density of 1.5 x 105cells / mL into 24-well cell culture plates. Different treatment groups were added to the culture medium: control group (2% DMEM), model group (2% DMEM), low (0.16 μL / mL), medium (0.31 μL / mL), and high (0.63 μL / mL). After 24 hours of Forsythia suspensa oil treatment, the culture medium was washed three times with a PBS solution, and then different treatment groups were added: control group (2% DMEM), model group (2% DMEM + PEDV), low (0.16 μL / mL + PEDV), medium (0.31 μL / mL + PEDV), and high (0.63 μL / mL + PEDV). After 6 hours of PEDV addition, the RNA of the Vero cells in each group was collected, and the expression of β-actin and PEDV N mRNA protein was determined. The experiment was repeated, and immunofluorescence determination was performed.

[0034] 1.5 Virus Replication Cycle

[0035] 1.5.1 Adhesion Experiment

[0036] Vero cells in cell bottles were digested, and the cell suspension after digestion was transferred to 96-well plates at a cell number of 1 x 105 4 When the cell density was 80%-90%, 0.63 μL / mL of Forsythia suspensa oil culture solution was used for 1 h of treatment at 37°C and 0.5 h of pre-treatment at 4°C. The cells were washed with a PBS solution, and PEDV virus (0.01 MOI) was inoculated at 4°C for 15 min, 30 min, and 60 min, respectively. The cell culture solution was discarded, the cells were washed with PBS, and the cell lysate was collected. Real-time RT-Q-PCR was used to determine the relative content.

[0037] 1.5.2 Internalization Experiment

[0038] Vero cells in cell bottles were digested, and the cell suspension after digestion was transferred to 96-well plates at a cell number of 1 x 105 4 When the cell density was 80%-90%, PEDV (0.01 MOI) was used for 1 h of infection at 4°C, and the cells were washed with a PBS buffer for 3 times. The unabsorbed virus was discarded. One group was added with a blank culture solution, and one group was added with 0.63 μL / mL of Forsythia suspensa oil culture solution. The cells were incubated at 37°C for 30 min, 1 h, and 2 h, respectively. The cell culture solution was discarded, the cells were washed with PBS, and the cell lysate was collected. Real-time RT-Q-PCR was used to determine the relative content.

[0039] 1.5.3 Replication Experiment

[0040] Vero cells in cell bottles were digested, and the cell suspension after digestion was transferred to 96-well plates at a cell number of 1 x 105 4When the cell density was 80%-90%, the cells were infected with 0.63 μL / mL of Forsythia suspensa essential oil and PEDV (0.01 MOI) at 37°C for 1 h, the cell culture solution was removed, then a group was added with blank culture solution, and a group was added with 0.63 μL / mL of Forsythia suspensa essential oil culture solution, and they were incubated at 37°C for 2 h, 4 h, 6 h, respectively, the cells were washed with PBS, and the cell lysate was collected, and the relative content was determined by real-time RT-Q-PCR.

[0041] 1.5.4 Release experiment:

[0042] The Vero cells in the cell bottle were digested, and the cell suspension after digestion was transferred to a 96-well plate, and the number of cells in each well was 1×10 4 When the cell density was 80%-90%, the cells were infected with PEDV (0.01 MOI) at 37°C for 1 h, the virus adsorption and penetration were ensured, the cell supernatant was removed, and the cells were washed with PBS three times, 10 h after infection, a group was added with blank culture solution, and a group was added with 0.63 μL / mL of Forsythia suspensa essential oil culture solution, and they were treated at 37°C for 0.5 h, 1 h, 2 h, and the cell supernatant was taken, and the virus copy number was determined by real-time RT-Q-PCR.

[0043] 1.6 Verification of IL-17 signaling pathway related targets by RT-Q-PCR

[0044] On the basis of the above study that Forsythia suspensa essential oil can effectively inhibit the adhesion and internalization stages of PEDV, the mechanism of Forsythia suspensa essential oil in inhibiting the adhesion stage of PEDV was further explored. The experimental model was established according to 1.4, and the experimental method was implemented according to 1.5.1. The related indexes such as PEDV N, HSP90AA1, MAPK14, TNF, CXCL8, IL-6, COX-2, etc. were detected by RT-Q-PCR method, and the mechanism of Forsythia suspensa essential oil in resisting PEDV was explored.

[0045] 1.7 In vivo experiment

[0046] The toxicity and anti-PEDV activity of Forsythia suspensa essential oil in vivo were measured as described above, 36 BALB / c mice were purchased from Shaanxi University of Chinese Medicine, and randomly divided into 6 groups (6 mice / group), and fed in separate rooms, on the 0th day, the first group of mice was gavaged with 0.4 mL of DMEM as a control group. The 2nd-6th group of mice were gavaged with 0.4 mL of DMEM containing 10, 50, 250, 500 and 2500 mg / kg of Forsythia suspensa essential oil. In the acute toxicity study, the death of mice was observed and recorded within 14 days. For sub-chronic toxicity assessment, the body weight of each mouse was measured every day after administration of Forsythia suspensa essential oil, and the mice were autopsied after 14 days of inoculation, and blood samples of all mice were collected for blood cell detection.

[0047] In the protection test of PEDV, 30 hybrid conventional newborn piglets of 4 days old were divided into 5 groups and fed in 5 independent rooms, and the 30 newborn piglets were confirmed to be negative for PEDV, TGEV and porcine rotavirus by using colloidal gold rapid detection kit. The low-dose group of forsythia suspense essential oil was 20 mg / kg, the medium-dose group was 40 mg / kg, and the high-dose group was 80 mg / kg, according to 10xTCID 50 The virus amount was given to the piglets orally, and the DMEM group was orally given the same volume of cell culture medium. The death of piglets in the PEDV group was recorded, and autopsy and sampling were immediately performed. The body temperature and body weight of piglets were recorded 12 hours before inoculation, and recorded every 12 hours until death. The clinical symptoms of lethargy and diarrhea of all piglets were evaluated every day as described above. After the death of all piglets, the jejunum tissue of piglets was collected, and histopathology and immunohistochemical staining were used for examination.

[0048] 1.8 Immunofluorescence detection of PEDV infected Vero cells (IFA)

[0049] The PEDV infected cells were washed with PBS solution three times, and then fixed with methanol: acetone (4:1) solution at 4℃ for 30 minutes. Then the cells were washed with PBS solution three times, 5 minutes each time. The primary antibody was added to the sample, and incubated at 4℃ overnight. The primary antibody was removed, and the sample was washed with PBS solution three times, 5 minutes each time. Then the secondary antibody was added, and incubated at 37℃ for one hour in the dark. Then, the sample was washed with PBS solution three times, 5 minutes each time. Then the cells were stained with DAPI nuclear staining solution for 5-8 minutes, washed with PBS solution three times, and finally observed and photographed with a fluorescence inverted microscope.

[0050] 1.9 RNA extraction and real-time quantitative PCR

[0051] The total RNA was extracted from Vero cells according to the instructions of Trizol, and then the cDNA was synthesized using Prime Script TM RT kit and gDNA Eraser. To establish the RT-Q-PCR reaction, the cDNA was synthesized using SYBR Green Low ROX qPCR MasterMix. To generate standard curves, a plasmid containing the PEDV N gene was serially diluted, and the mRNA expression of β-actin was used as a relative reference for each experiment. The specific primers are shown in Table 1. The reaction conditions of the fluorescence quantitative PCR instrument were: 94℃ for 3 min, 94℃ for 10 sec, 60℃ for 30 sec, 40 cycles.

[0052] Table 1 Specific primers

[0053]

[0054]

[0055] 1.10 HE staining to observe pathological changes

[0056] The pig farm tissue was flushed with physiological saline, soaked in 4% paraformaldehyde for 12 h, dehydrated, paraffin-embedded, sliced, coated, heated, stained with hematoxylin and eosin, dehydrated and sealed. The slice was imaged using a microscopic imaging system, and the specific lesions were observed.

[0057] 1.11 Immunohistochemical analysis

[0058] The pig intestinal tissue sections were incubated overnight at 4°C and compared with specific primary antibodies of HSP90AA1, COX-2, and TNF. The sections were washed with phosphate buffered saline, and then poly-HRP anti-rabbit IgG secondary antibody was added. The sections were treated with diaminobenzidine (DAB) after washing with phosphate buffered saline. Hematoxylin was used for counterstaining, and observed under an optical microscope. Image analysis software Image-Pro Plus 6 was used to calculate the average integral positive area from 3 randomly selected areas.

[0059] 1.12 Statistical analysis

[0060] The statistical software GraphPad Prism 9.5.0 was used to perform t-test and one-way ANOVA on all results to determine significant differences between groups. The data is expressed as the mean and standard deviation (SD) of one group in three independent experiments.

[0061] 2. Results

[0062] 2.1 Forsythia essential oil can effectively inhibit PEDV infection

[0063] Figure 1 As can be seen in (A), there was a significant difference (P < 0.001) at a concentration of 1.25 μL / mL of Forsythia essential oil compared to the control group. This indicates that at this concentration, Forsythia essential oil will cause damage to the cells. Figure 1 (B) shows the results of cell fusion and separation after PEDV infection. FITC staining shows green fluorescence, indicating viral infection, and DAPI staining is used to observe the cell nucleus. The Merge image shows that Forsythia essential oil has a certain inhibitory effect on PEDV. Visualization analysis of IFA data by Image j shows that Figure 1 (C), Forsythia essential oil has a significant therapeutic effect on PEDV (P < 0.001). As Figure 1(D) shows that the expression of PEDV N protein in the model group was significantly higher than that in the blank group (P < 0.0001). However, in the high, medium and low concentration forsythia essential oil groups, the expression of PEDV N protein was significantly lower than that in the model group. In addition, the expression of PEDV N protein decreased with the increase of concentration. This shows that forsythia essential oil can inhibit the replication of PEDV, and has a dose-dependent effect.

[0064] Table 2 Effect of forsythia essential oil on Vero cell activity n = 3

[0065]

[0066]

[0067] Table 3 Analysis of IFA visualization results n = 3

[0068]

[0069] Table 4 Effect of forsythia essential oil on the expression of PEDV N protein n = 3

[0070]

[0071] 2.2 Effect of forsythia essential oil on PEDV adhesion, internalization, replication and release

[0072] To further explore the mechanism of forsythia essential oil in inhibiting PEDV infection, RT-Q-PCR was used to detect the relative content of PEDV N mRNA and β-actin to determine the effect of forsythia essential oil on PEDV adhesion, internalization and replication. As shown in Figure 2 (A), the adhesion of virus on Vero cells showed that with the extension of treatment time, forsythia essential oil could effectively inhibit the adhesion of PEDV (P < 0.001). When evaluating the effect of forsythia essential oil on PEDV internalization, as shown in Figure 2 (B), the treatment with forsythia essential oil for 30 minutes after PEDV infection for 1 hour showed that forsythia essential oil could effectively inhibit the internalization of virus (P < 0.01). Subsequently, forsythia essential oil was added during the virus replication stage to detect the effect of forsythia essential oil on PEDV replication, as shown in Figure 2 (C), compared with the blank group, there was no significant difference in PEDV N mRNA level treated with forsythia essential oil. In addition, the virus release test (as shown in Figure 2 (D)) showed that there was no significant difference in the copy number of virus particles between the blank group and the essential oil group, indicating that forsythia essential oil did not affect the release of PEDV virus. In summary, forsythia essential oil mainly inhibits PEDV infection by affecting the adhesion and internalization of virus.

[0073] Table 5 Influence of Forsythia essential oil on the adhesion stage of PEDV n = 3

[0074]

[0075] Table 6 Influence of Forsythia essential oil on the internalization stage of PEDV n = 3

[0076]

[0077] Table 7 Influence of Forsythia essential oil on the replication stage of PEDV n = 3

[0078]

[0079] Table 8 Influence of Forsythia essential oil on the release stage of PEDV n = 3

[0080]

[0081] 2.3 Verification of relevant target points by RT-Q-PCR

[0082] In order to study the mechanism of action of Forsythia essential oil in inhibiting PEDV virus, the present application uses the RT-Q-PCR method to study the relevant target points. As shown in Figure 3 (A-C) shows that the expression amount of PEDV N and MAPK14 protein in the model group is higher than that in the control group (P < 0.01), and after adding Forsythia essential oil, the expression amount of PEDV N protein and MAPK14 in the high, medium and low groups is significantly reduced (P < 0.001), and with the increase of the dose, the expression amount of PEDV N protein and MAPK14 protein will decrease, indicating that Forsythia essential oil has a dose-dependent effect, indicating that Forsythia essential oil can inhibit PEDV infection by regulating MAPK14. As shown in Figure 3 (B) shows that the expression amount of HSP90AA1 protein in the model group is significantly less than that in the blank group (P < 0.001), and the expression amount in the high, medium and low groups is higher than that in the model group; it is shown that the expression amount of HSP90AA1 is reduced after PEDV infects Vero cells, and the addition of Forsythia essential oil increases the expression amount of HSP90AA1, indicating that the overexpression of HSP90AA1 can inhibit the infection of PEDV. The above studies show that HSP90AA1 and MAPK14 are related to the anti-PEDV effect of Forsythia essential oil on Vero cells.

[0083] After PEDV infects Vero cells, the cell RNA is extracted and reverse transcribed into cDNA, and the transcription level of IL-6, TNF, COX-2 and CXCL8 is detected by RT-qPCR. As shown in Figure 3(D) -(G) showed that PEDV infection up-regulated the transcriptional levels of IL-6, TNF, COX-2 and CXCL8, compared with the treatment of forsythia oil, the expression of IL-6, TNF, COX-2 and CXCL8 was reduced by forsythia oil treatment, and forsythia oil at high concentration group could significantly reduce the expression of IL-6, TNF, COX-2 and CXCL8 (P < 0.001). This study showed that PEDV infection of Vero cells could promote the expression of cell inflammatory factors, and forsythia oil could inhibit the expression of inflammatory factors, so as to achieve the effect of anti-PEDV.

[0084] Table 9 Expression of proteins and inflammatory factors after PEDV infection of Vero cells n = 3

[0085]

[0086] 2.4 Acute toxicity study of forsythia oil on mice

[0087] In an acute toxicity study, mice were orally administered with forsythia oil at 0, 10, 50, 250, 500 and 2500 mg / kg, and no mice death was observed after 14 days in each dose group Figure 4 A). For subchronic toxicity evaluation, mice were orally administered with different concentrations of forsythia oil for 14 days. In this experiment, there was no significant difference in body weight between the forsythia oil treatment group and the control group of mice (CTRL) at 14 days Figure 4 B), oral administration of forsythia oil did not affect the changes of white blood cells and platelets Figure 4 C), however, the change of red blood cells increased (P < 0.05) when treated with high dose (500 mg / kg), but the number of red blood cells was within the normal range (6.5-11.5 x 106 / μL). Therefore, the oral dose was set to 250 mg / kg to investigate whether forsythia oil could protect piglets from PEDV attack.

[0088] Table 9 Effect of forsythia oil on the number of blood cells in mice after gavage for 14 days n = 6

[0089]

[0090]

[0091] 2.5 Protection of forsythia oil against PEDV infection in piglets in vivo

[0092] Animal experiments showed that the degree of clinical symptoms was lighter in piglets treated with forsythia oil after PEDV infection than in piglets only infected with PEDV Figure 5A). To clarify the effect of Forsythia suspensa essential oil on the fecal characteristics of PEDV-infected piglets, after 12 hours, the PEDV-infected group showed varying degrees of diarrhea; after 36 hours, severe watery diarrhea appeared; and after 48 hours, severe watery diarrhea persisted until death. In contrast, only a few piglets in the Forsythia suspensa essential oil group showed watery diarrhea. The fecal characteristic score of piglets in the Forsythia suspensa essential oil group was higher than that of the healthy control group but lower than that of the PEDV-infected control group. Figure 5 B). At 48 hpi, 4 piglets died in the PEDV-infected group treated with Forsythia suspensa essential oil (6 piglets / group), all piglets in the PEDV-infected group died (6 piglets / group), and no piglets died in the DMEM group (6 piglets / group). Figure 5 C).

[0093] To clarify the effect of Forsythia suspensa essential oil on the excretion of PEDV in challenged piglets, the copy number of the PEDV N gene in collected anal swab samples was detected using RT-Q-PCR, such as ( Figure 5 D) At 12, 24, and 36 hours after infection, the viral copy number in the feces of the PEDV-infected group was 10. 8.5 copies / mL, 10 8.5 copies / mL and 10 8.23 The viral copy number in the high-dose Forsythia essential oil group was 10 copies / mL. 4.5 copies / mL, 10 5.56 copies / mL and 10 4.43 copies / mL. This indicates that Forsythia suspensa essential oil can significantly reduce the viral load in anal swabs from PEDV-infected piglets (p<0.01).

[0094] To clarify the effect of Forsythia suspensa essential oil on enteroviruses challenged in piglets, the copy number of the PEDV N gene in collected jejunal and ileal samples was detected using RT-Q-PCR, such as ( Figure 5 E) At 48 hours post-challenge, the viral copy numbers in the jejunum and ileum of the PEDV-infected group were 10. 9.5 copies / mL, 10 9.5 The viral copy number in the high-dose Forsythia essential oil group was 10 copies / mL. 4.36 copies / mL, 10 3.80 copies / mL. This indicates that Forsythia suspensa essential oil can significantly reduce the viral load in anal swabs from PEDV-infected piglets (p<0.001).

[0095] Table 10 Clinical symptom scores of PEDV-infected piglets treated with Forsythia suspensa essential oil ( n=6)

[0096]

[0097] Table 11 Feces symptom score of PEDV infected piglets treated with Forsythia essential oil n = 6

[0098]

[0099] Table 12 Effect of Forsythia essential oil on survival rate of PEDV infected piglets n = 6

[0100]

[0101] Table 13 Virus load in anal swabs of virus challenged piglets treated with Forsythia essential oil n = 3

[0102]

[0103] Table 14 Virus load in jejunum and ileum of virus challenged piglets treated with Forsythia essential oil n = 3

[0104]

[0105] 2.6 Effect of Forsythia essential oil on histopathology of virus challenged piglets

[0106] To further clarify the effect of Forsythia essential oil on the pathological changes of the intestines of virus challenged piglets, H.E staining was used to analyze the changes in the intestines of the piglets in the experiment. The results, as shown in Table 11, indicated that the intestinal villi of the PEDV infected group were significantly atrophied, the villi proper layer was thinned, the crypts were shallow, and the capillary vessels in the mucosa were congested; the small intestinal villi of the piglets in the Forsythia essential oil group were complete in structure and clear in outline, and no obvious histological lesions were observed. The piglets in the DMEM group had no obvious histological lesions. Figure 6

[0107] 2.7 Effect of Forsythia essential oil on expression of related factors in PEDV pig intestinal tissue

[0108] ​Heat shock protein 90(HSP90) includes HSP90α and HSP90β, which is an important molecular chaperone highly conserved in evolution. It can be involved in various biological processes, such as viral replication, immune regulation, and signal transduction. COX-2 and TNF-α are important members of the cytokine network and play an important role. The expression of HSP90AA1, COX-2 and TNF-α in pig intestinal tissue was observed and detected by immunohistochemical method. The results showed that compared with the DMEM group, the expression levels of COX-2 and TNF-α in pig intestinal tissue after PEDV infection increased significantly(P<0.0001), and the expression level of HSP90AA1 decreased significantly(P<0.0001). Compared with the PEDV infection group, the use of different doses of forsythia essential oil made the expression of TNF-α and COX-2 decrease significantly, and the expression level of HSP90AA1 increased significantly. Among them, the secretion level of TNF-α and COX-2 in the high dose group(80mg / kg) of forsythia essential oil decreased significantly(P<0.0001), and the expression level of HSP90AA1 increased significantly(P<0.001) as Figure 7 (B), (C), (D).

[0109] Table 15 Statistical table of HSP90AA1, COX-2 and TNF-α expression levels in piglets infected with PEDV in different groups n=3

[0110]

[0111] 3. Conclusion and discussion

[0112] Porcine epidemic diarrhea virus (PEDV) is an acute enteric infectious disease with high infectivity. At present, there is no effective antiviral drug for PEDV, and it is important to develop new and effective anti-PEDV drugs. Studies have shown that forsythia oil has inhibitory effect on many viruses, but the antiviral target of forsythia oil and its mechanism are still unclear. The present application focuses on the relationship between PEDV and HSP90AA1 and MAPK14. HSP is a kind of conserved and indispensable chaperone protein in eukaryotes, which can be divided into five families according to its relative molecular mass: HSP110, HSP90, HSP70, HSP60 and HSP. HSP90 exists in multiple isoforms, mainly HSP90α and HSP90β in cytoplasm. The interaction between nsP3 and nsP4 of chikungunya virus (CHIKV) and HSP90 can block its in vitro and in vivo replication. HSP is one of the regulators of apoptosis, which can regulate antigen presenting cells (APC), leading to autoimmune and antiviral effects of the body. HSP90 promotes protein folding and transport in non-stressed cells, and regulates cell homeostasis in stress response. Overexpression of HSP90AA1 can inhibit CSFV replication, and knocking down the gene of HSP90AA1 can promote the replication of CSFV. The above studies show that HSP90AA1 is involved in different stages of the life cycle of various viruses. This reflects the great potential of HSP as an antiviral target.

[0113] Mitogen-activated protein kinase (MAPK) is an important mediator of signal transmission from the cell surface to the nucleus, which regulates important physiological and pathological processes such as cell growth, differentiation and inflammation. Studies have shown that p38MAPK specific inhibitors can inhibit the replication of respiratory syncytial virus and influenza A virus. Duck Tembusu virus (DTMUV), influenza A virus, blue tongue disease virus, rabies virus and chikungunya virus can activate the MAPK signaling pathway and participate in viral replication. The results showed that the expression of MAPK8 protein was significantly reduced after treatment with 0.3mg / mL strychnine before, during and after PEDV infection. In summary, MAPK may be a key pathway for the treatment of PEDV.

[0114] In summary, HSP90AA1 and MAPK14 are involved in different stages of the viral life cycle alone or together. In this study, the changes in the mRNA of HSP90AA1, MAPK14 and pro-inflammatory factors were detected by RT-Q-PCR. The results showed that the mRNA expression of HSP90AA1 was significantly reduced, and the mRNA expression of MAPK14 and pro-inflammatory factors was significantly increased after viral infection compared with the control group. However, the expression of HSP90AA1 was significantly increased, and the expression of MAPK14 and pro-inflammatory factors was significantly decreased after treatment with Forsythia essential oil. The up-regulation of HSP90AA1 expression may affect the expression of TRAF6, which can inhibit the expression of MAPK14, thereby inhibiting the expression of inflammatory factors such as IL-6, CXCL8, TNF and COX-2. In vivo experiments showed that Forsythia essential oil had no effect on mice at a dose of 500mg / kg by gavage. After conversion with the body surface area of pigs, the dosages of Forsythia essential oil were 20mg / kg, 40mg / kg and 80mg / kg. The results showed that the Forsythia essential oil group could effectively improve the clinical symptoms and fecal symptoms of piglets, while the PEDV infection group showed water-like jet diarrhea. For the survival rate of piglets, the survival rate of the DMEM group was 100%, and all piglets in the PEDV infection group eventually died. The survival rate of piglets in the Forsythia essential oil group was 33.3%. With the extension of time, it was found that the Forsythia essential oil group could effectively improve the virus load in the feces, jejunum and ileum of the PEDV infection group, indicating that Forsythia essential oil could effectively inhibit the infection of PEDV. Immunohistochemical experiments showed that HSP90AA1 was significantly reduced, and TNF and COX-2 were significantly increased after viral infection. However, after the addition of Forsythia essential oil, HSP90AA1 was significantly increased, and TNF and COX-2 were significantly decreased after viral infection. It was indicated that Forsythia essential oil could be used as one of the target drugs for anti-virus.

[0115] 5. Summary

[0116] The present application discusses the treatment potential of Forsythia essential oil for PEDV, predicts the possible mechanism of action of Forsythia essential oil in treating PEDV by network pharmacology technology, and further clarifies the mechanism of action of Forsythia essential oil in treating PEDV through pharmacodynamics experiments. The results showed that terpinen-4-ol and alpha-terpinene in Forsythia essential oil could regulate the key target points HSP90AA1 and MAPK14 in the IL-17 signaling pathway, thereby inhibiting the expression of inflammatory factors. Given the widespread use of Forsythia in daily life, the development of Forsythia as a targeted preparation for treating PEDV can provide a new direction for the treatment of PEDV with Forsythia.

Claims

1. The use of Forsythia suspensa essential oil in the preparation of traditional Chinese veterinary medicine for treating swine epidemic diarrhea virus; wherein the Forsythia suspensa essential oil contains not less than 18% w / w of α-pinene and not less than 52% w / w of β-pinene.

2. The use of Forsythia essential oil in the preparation of a traditional Chinese veterinary medicine for treating porcine epidemic diarrhea; wherein the Forsythia essential oil contains not less than 18% w / w of α-pinene and not less than 52% w / w of β-pinene.

3. The use according to claim 1 or 2, characterized in that: The forsythia essential oil contains 18.82% w / w α-pinene and 52.74% w / w β-pinene.

4. The use according to claim 1 or 2, characterized in that: The pig mentioned is a piglet.

5. The use according to claim 1 or 2, characterized in that: The aforementioned traditional Chinese veterinary medicine is prepared into a pharmaceutically commonly used formulation by adding Forsythia essential oil as the active ingredient and adding excipients or auxiliary ingredients acceptable to veterinary medicine.

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