Application of forsythoside A in the preparation of drugs for the prevention and / or treatment of porcine contagious pleuropneumonia

Abstract

This invention discloses the application of forsythoside A in the preparation of drugs for the prevention and / or treatment of porcine infectious pleuropneumonia, belonging to the field of biomedicine. The concentration of forsythoside A is 6.25-25 μg / mL. Cellular experiments have verified that forsythoside A can inhibit cellular inflammatory responses, reduce the expression of inflammatory factors, and effectively alleviate vascular endothelial inflammatory damage induced by *Actinobacillus pleuropneumoniae* by inhibiting the activation of CD14 / TLR4-NFκB P65. Animal experiments have verified that forsythoside A can reduce the lesions and damage to various tissues and organs after *Actinobacillus pleuropneumoniae* infection, improve weight loss caused by *Actinobacillus pleuropneumoniae* infection, alleviate inflammatory responses and metabolic dysfunction caused by *Actinobacillus pleuropneumoniae*, and exert a protective effect on the body after *Actinobacillus pleuropneumoniae* infection. This invention provides new scientific evidence for the use of natural drugs to control porcine infectious pleuropneumoniae.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of forsythoside A in the preparation of medicaments for the prevention and / or treatment of porcine infectious pleuropneumonia. Background Technology

[0002] Porcine contagious pleuropneumonia (PCP), also known as swine contact infectious pleuropneumonia, is a highly fatal and contagious respiratory disease caused by *Actinobacillus pleuropneumoniae* (APP). In recent years, the incidence and prevalence of PCP have been increasing annually. Infected pigs often exhibit acute and chronic symptoms, causing characteristic lesions such as edema, inflammation, hemorrhage, and necrosis. This bacterium can cause infection and disease in a single host, or it can cause mixed infections with pathogens such as porcine circovirus and *Pasteurella multocida*, exacerbating the disease's development and leading to high morbidity and mortality rates in pig herds. It has become one of the three major respiratory diseases in intensive poultry farming. *Actinobacillus pleuropneumoniae* has 19 different serotypes, and the dominant serotype varies significantly across different regions globally, making bacterial vaccines ineffective in controlling and preventing the disease caused by *Actinobacillus pleuropneumoniae*. In veterinary clinical practice, antibiotics such as enrofloxacin and florfenicol are mainly used to treat diseases caused by Actinobacillus pleuropneumoniae. However, long-term or excessive use of antibiotics leads to increased drug resistance and causes drug residues in animal-derived food products, resulting in significant economic losses to the pig farming industry. Therefore, protecting hosts from pathogens and finding highly effective anti-inflammatory drugs are urgent problems that the pig farming industry needs to solve.

[0003] Currently, there are two main approaches to new drug discovery: target-based drug discovery (TDD) and phenotype-based drug discovery (PDD). Target-based drug discovery involves the targeted screening and design of large or small molecule drugs for a specific target protein that is highly related to the disease mechanism. When the target is known, molecular docking software is used to visualize the active sites of these substances by utilizing the target's biological function, three-dimensional conformation, active site information, and the chemical structure characteristics of endogenous ligands or natural drugs. The ligand-drug molecule is then placed at the active site of the receptor target. By continuously optimizing parameters such as the ligand's position and conformation, the optimal conformation for binding between the ligand small molecule and the receptor target is sought, and its binding ability is predicted. Ultimately, the optimal ligand-drug molecule acting on that site is obtained. Computer-aided drug design (CADD) combined with artificial intelligence (AI), machine learning (ML), and deep learning (DL) technologies to process large amounts of biological data accelerates the drug screening and development process and reduces drug development costs. Austin Royster et al. identified a novel compound, K31, based on the SARS-CoV-2 N protein and demonstrated that K31 binds to the SARS-CoV-2 N protein and non-competitively inhibits its binding to the 5' end of the viral genomic RNA, thus exerting an antiviral effect. Sirin Theerawatanasirikul et al. designed and screened three compounds targeting the 3CL protease that could inhibit the activity of feline coronavirus (FCoV) 3CL. Through cell phenotypic assays, they demonstrated that NSC629301 and NSC71097 strongly inhibited cytopathic effects and exhibited low toxicity, while also reducing FIPV replication in CRFK cells. In target-based drug screening, although computer tools can be used to establish selection criteria and optimize drug design strategies, inconsistencies between data, parameter settings, and the models used can lead to suboptimal selection of active lead compounds. Further development of multi-parameter computer models that consider multiple factors, combined with phenotypic screening, is needed to utilize new technologies to obtain more effective therapeutic drugs and improve research efficiency.

[0004] Forsythiaside A, with a molecular weight of 624.59 and a molecular formula of C2... 29 H 36 O 15Forsythoside A is a phenylethanoid glycoside, an active substance isolated from the dried fruit of Forsythia suspensa. Forsythoside A exhibits significant activity in treating various diseases, including inflammation, oxidative stress, neurodegeneration, and viral infections. However, there are no reports in the current technology regarding the efficacy of forsythoside A against porcine infectious pleuropneumonia caused by Actinobacillus pleuropneumoniae infection. Summary of the Invention

[0005] The purpose of this invention is to provide forsythoside A in the preparation of drugs for the prevention and / or treatment of porcine infectious pleuropneumonia, in order to solve the problems existing in the prior art. This invention discovers that forsythoside A can resist Actinobacillus pleuropneumoniae infection, providing new scientific basis for the use of natural drugs to control porcine infectious pleuropneumonia.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of forsythoside A in any of the following:

[0008] (1) Application in the preparation of antibacterial agents for Actinobacillus pleuropneumoniae;

[0009] (2) Use in the preparation of drugs for the prevention and / or treatment of porcine infectious pleuropneumonia.

[0010] Optionally, the concentration of forsythoside A is 6.25-25 μg / mL.

[0011] Optionally, the forsythoside A relieves lung and vascular endothelial damage.

[0012] Furthermore, the target of hesperidin in alleviating lung and vascular endothelial damage is CD14.

[0013] Optionally, the forsythoside A reduces the expression of inflammatory factors.

[0014] Optionally, the inflammatory factors include TNF-α, IL-1β, IL-6, and IL-18.

[0015] Furthermore, the signaling molecule by which hesperidin reduces the expression of inflammatory factors is CD14 / TLR4-NFκB P65.

[0016] The present invention also provides an antibacterial agent for Actinobacillus pleuropneumoniae, wherein the antibacterial agent comprises forsythoside A.

[0017] The present invention also provides a medicament for the prevention and / or treatment of porcine infectious pleuropneumonia, the medicament comprising forsythoside A and pharmaceutically acceptable excipients.

[0018] Optionally, the concentration of forsythoside A is 6.25-25 μg / mL.

[0019] Optionally, the excipients include at least one of diluents, fillers, excipients, binders, humectants, disintegrants, absorption promoters, surfactants, adsorbent carriers, lubricants, and flavorings.

[0020] Optionally, the medication may be taken orally or via non-gastrointestinal routes.

[0021] The present invention discloses the following technical effects:

[0022] This experiment, based on molecular docking technology, used SYBYLX-2.0 software to dock small molecule drugs in the compound library with the CD14 target, screened out drugs that interact with the CD14 target, ranked them by the scores obtained from molecular docking, and combined with the herbal origin, efficacy and meridian tropism, as well as the effect of different drug concentrations on cell viability, finally selected forsythoside A, and further verified the target-drug interaction relationship through DARTS and CETSA assays.

[0023] Cellular experiments have verified that forsythoside A can inhibit cellular inflammatory responses and reduce the expression of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-18). It can effectively alleviate vascular endothelial inflammatory damage induced by *Actinobacillus pleuropneumoniae* by inhibiting the activation of CD14 / TLR4-NFκB P65. Animal experiments have verified that forsythoside A can reduce the lesions and damage to various tissues and organs after *Actinobacillus pleuropneumoniae* infection, improve weight loss caused by *Actinobacillus pleuropneumoniae* infection, alleviate the inflammatory response and metabolic dysfunction caused by *Actinobacillus pleuropneumoniae*, and exert a protective effect on the body after *Actinobacillus pleuropneumoniae* infection.

[0024] In summary, both in vivo and in vitro test results demonstrate that forsythoside A can resist Actinobacillus pleuropneumoniae infection. This invention provides new scientific evidence for the use of natural drugs to control porcine infectious pleuropneumonia. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Visualization of the docking of CD14 with forsythoside A;

[0027] Figure 2The results of the DARTS assay for forsythoside A are shown below; A: DARTS protein banding of forsythoside A; B: Quantitative results of the DARTS protein banding of forsythoside A.

[0028] Figure 3 The results of the CETSA assay for forsythoside A are shown below; A: CETSA protein band diagram of forsythoside A; B: Quantitative results of the CETSA protein band diagram of forsythoside A.

[0029] Figure 4 The MIC results for forsythoside A;

[0030] Figure 5 The effect of forsythoside A on the viability of PIEC cells;

[0031] Figure 6 The effects of Actinobacillus pleuropneumoniae on the expression of proteins (TLR4, P65, p-P65, TNF-α, CD14, IL-18, IL-6 and IL-1β) following endothelial inflammatory injury induced by Actinobacillus pleuropneumoniae;

[0032] Figure 7 for Figure 6 Quantitative results of each protein band in the sample;

[0033] Figure 8 The effect of forsythoside A on body weight in APP-infected mice;

[0034] Figure 9 The pathological changes in the lung tissue of mice in each group are shown; the scale bar is 50 μm.

[0035] Figure 10 The pathological changes in vascular tissues of mice in each group are shown; the scale bar is 20 μm.

[0036] Figure 11 Effects of forsythoside A on protein expression in mouse lung tissue; scale bar is 50 μm;

[0037] Figure 12 The effect of forsythoside A on protein expression in mouse vascular tissue; scale bar is 50 μm. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Example 1: Screening of small molecule drugs interacting with the CD14 target based on molecular docking and DARTS and CETSA assays.

[0044] 1. Test materials

[0045] 1.1 Drug Database

[0046] Taoshu Biotechnology Small Molecule Drug Database.

[0047] 1.2 Analysis Software

[0048] SYBYL-X software, DataWarrior software, Image J, GraphPad Prism.

[0049] 1.3 Strains and Cell Lines

[0050] The porcine iliac artery endothelial cells (PIEC) and Actinobacillus pleuropneumoniae strain BW39 (APP) used were donated by the National Veterinary Drug Residue Reference Laboratory of Huazhong Agricultural University and are preserved in the Hubei Key Laboratory of Animal Nutrition and Feed Science.

[0051] 1.4 Main Medicines and Reagents

[0052] The medicines and reagents required for this embodiment are shown in Table 1.

[0053] Table 1 Main Drugs and Reagents

[0054]

[0055]

[0056] 1.5 Preparation of Reagent Solutions

[0057] Forsythoside A stock solution: Dissolve 10 mg of forsythoside A in 100 μL of DMSO, mix well, filter through a membrane, and prepare a stock solution of 100 mg / mL.

[0058] 2. Test methods

[0059] 2.1 Small molecule drug molecular docking screening

[0060] Molecular docking technology was used to screen small molecule traditional Chinese medicine monomers with strong binding ability to CD14 protein. First, the CD14 protein structure was downloaded from the UniProt platform and imported into SYBYL-X software. Water molecules were removed from the CD14 receptor and hydrogen was added to create an active pocket. Drugs from the Taoshu Biotechnology small molecule drug database were imported into the software and hydrogenated. Then, molecular docking was performed sequentially between the processed small molecule ligands and the CD14 receptor. The ligands were sorted from highest to lowest docking score. The receptor and ligand were then merged, and the images were processed using PyMol software to obtain visualizations. Further screening was conducted based on subsequent experiments.

[0061] 2.2 Target stability assays for drug affinity reactions to screen small molecule drugs

[0062] (1) Cell pretreatment

[0063] PIEC cells were passaged into 60 mm cell culture dishes. After the cells grew to a suitable density, APP was added at an MOI of 100, and the cells were cultured in a transcubator for 12 h.

[0064] (2) Protein sample extraction

[0065] Discard the culture medium in the cell culture dish, wash twice with PBS, collect the cells, add 2000 μL of the prepared M-PER cell lysis buffer, lyse on ice for 20 min, then centrifuge for 15 min (4℃, 12000 rpm), and take the supernatant as the protein sample.

[0066] (3) Protein quantification

[0067] Protein sample concentrations were analyzed using the Beyotime BCA quantitative kit.

[0068] (4) Co-incubation of protein and drug

[0069] The protein sample was divided into two equal parts, each 500 μL. One part was added with the drug to achieve a final concentration of 100 μg / mL, and the other part was added with the corresponding volume of solvent control. The mixture was then placed on a rotating shaker and incubated at room temperature for 1 h.

[0070] (6) Co-incubation of protein with streptomycin protease

[0071] The drug group and the solvent control group were each divided into 4 equal parts, each containing 100 μL. One blank control group was set up, and the other 3 groups were added with streptomycin protease, so that the ratio of streptomycin protease to protein was 1:2000, 1:4000 and 1:8000, respectively. The mixtures were incubated at room temperature for 30 min.

[0072] (7) Protein denaturation

[0073] After lysis, loading buffer was added to the protein sample mixture, and the mixture was mixed well. The mixture was then heated at 98°C for 10 min for high-temperature denaturation before proceeding with the subsequent Western blot experiment.

[0074] 2.3 Cell thermal transfer assay for drug screening

[0075] (1) Preparation and extraction of cell protein-like substances

[0076] The same method as 2.2 for screening small molecule drugs by target stability test of drug affinity reaction (1) and (2).

[0077] (2) Incubation of molecules and proteins

[0078] Protein samples were aliquoted into two tubes, each containing 600 μL, and divided into an experimental group and a control group. The experimental group was given a final concentration of 100 μg / mL of drug, while the control group was given an equal volume of DMSO with the same solvent concentration. The tubes were placed on a rotating shaker and incubated at room temperature for 1 h.

[0079] (3) Gradient temperature incubation

[0080] Each protein group was divided into 200 μL eight-row tubes, with each tube containing 75 μL. The control group and drug protein samples were heated in a Bio-Rad T100 gradient PCR instrument at 40.6, 43.5, 48.1, 53.5, 57.9 and 61°C for 3 min, respectively, and then cooled to room temperature.

[0081] (4) Protein denaturation

[0082] After incubating the protein at a gradient temperature, add 25 μL of loading buffer, mix well, heat at 98 °C for 10 min for high-temperature denaturation, and then perform subsequent Western blot experiments.

[0083] 2.4 Statistical Analysis

[0084] One-way differential analysis of the data was performed using SPSS software, and data visualization was performed using Graphpad Prism 8 software ("*" indicates p<0.05, indicating a significant difference, and "**" indicates p<0.01, indicating an extremely significant difference).

[0085] 3. Test Results

[0086] 3.1 Results of molecular docking drug screening

[0087] Drug screening was conducted using molecular docking technology. Small molecule drugs from the molecular library were docked one by one with the CD14 target. The drugs were then sorted from highest to lowest based on their total docking score, resulting in a number of drugs with a score greater than 8. To further identify highly effective anti-inflammatory drugs and determine their efficacy, further screening was conducted through literature review. Based on the drug's origin, efficacy, meridian tropism, and the impact of individual drugs on cell viability, and considering economic factors, forsythoside A was found to be associated with the lung meridian (Table 2), closely related to inflammation, and possessing good anti-inflammatory effects. Molecular docking showed that forsythoside A can bind to CD14 via hydrogen bonds between His-257, Asp-202, Asp-254, Gly-358, Arg149, Lys-175, and Glu-56. Figure 1 Furthermore, preliminary experiments revealed that forsythoside A has a strong intervention effect on the inflammatory response induced by Actinobacillus pleuropneumoniae. After molecular docking and phenotype-based drug screening, forsythoside A was finally selected as the drug for the next step of research.

[0088] Table 2. Meridian tropism and efficacy of forsythoside A

[0089]

[0090] 3.2 DARTS assay to verify drug-protein binding

[0091] Target stability assays based on drug affinity reactions (DARTS) were used to further validate the binding of the drug to the CD14 protein. As the concentration of streptomycin protease increases, the protein is enzymatically cleaved. When the target protein binds to the drug molecule, it typically becomes stable; that is, compared to the solvent control group, the amount of undegraded protein is increased at the same concentration of streptomycin protease. Results are as follows: Figure 2As shown, at streptomycin protease to protein ratios of 1:2000 and 1:4000, the CD14 protein level in the forsythoside A group was significantly higher than that in the DMSO solvent control group (p<0.01). At a ratio of 1:8000, the CD14 protein level in the forsythoside A group was significantly higher than that in the DMSO solvent control group (p<0.05). These results indicate that forsythoside A can improve CD14 stability to a certain extent and can bind to CD14 protein.

[0092] 3.3 CETSA test to verify the effect of drugs on the thermal stability of proteins

[0093] To better verify the binding of the drug to the CD14 protein, a cell thermal migration assay (CETSA) was performed. This showed that as temperature increases, the protein degrades. When the target protein binds to the drug molecule, it typically becomes stable; that is, compared to the solvent control group, the amount of undegraded protein increases at the same temperature. Figure 3 As shown, under treatment conditions of 48.1℃, the protein content of CD14 in the forsythoside A treatment group was significantly higher than that in the solvent control group (p<0.05); under treatment conditions of 43.5℃, 53.5℃, 57.9℃, and 61℃, the protein content of CD14 in the forsythoside A treatment group was extremely significantly higher than that in the solvent control group (p<0.01). The results indicate that forsythoside A can improve the thermal stability of the CD14 target protein to a certain extent, and that forsythoside A has a certain binding capacity with CD14.

[0094] Example 2: Forsythoside A against Actinobacillus pleuropneumoniae infection

[0095] To further investigate the role of forsythoside A in Actinobacillus pleuropneumoniae infection, this study determined the cytotoxicity of forsythoside A against PIEC cells and its minimum inhibitory concentration against Actinobacillus pleuropneumoniae, selected the optimal drug concentration of forsythoside A for treating PIEC, and studied the intervention effect of forsythoside A through a PIEC cell inflammatory damage model induced by Actinobacillus pleuropneumoniae and a CD14 overexpression assay. The aim was to explore the molecular mechanism by which the drug alleviates vascular endothelial inflammatory damage induced by Actinobacillus pleuropneumoniae.

[0096] 1. Strains and cell lines

[0097] The porcine hip artery endothelial cells (PIEC) and Actinobacillus pleuropneumoniae strain BW39 used were donated by the National Veterinary Drug Residue Reference Laboratory of Huazhong Agricultural University and are preserved by the Hubei Key Laboratory of Animal Nutrition and Feed Science.

[0098] The ATCC 25922 used was provided by the Hubei Key Laboratory of Animal Nutrition and Feed Science.

[0099] 2. Determination of the minimum inhibitory concentration of forsythoside A

[0100] The minimum inhibitory concentration (MIC) of hesperidin was determined using the broth dilution method. Final drug concentrations were set at 2048, 1024, 512, 256, 128, 64, 32, 16, and 8 μg / mL. TSB medium without bacterial culture was used as a negative control, bacterial culture without drug was used as a positive control, and DMSO was used as a drug solvent control. ATCC 25922 was used as the quality control bacteria, and florfenicol was used as the quality control drug.

[0101] 3. Determination of cell viability by forsythoside A

[0102] The effect of hesperidin on PIEC cell viability was determined using the CCK8 assay. Drug concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.6525, and 0.87625 μg / mL were used, with a culture medium control group and a blank cell control group included. A bar chart showing the effect of hesperidin on cell viability was plotted based on absorbance data.

[0103] 4. Effects of forsythoside A on the expression of inflammatory factors in cells infected with APP

[0104] PIEC cells were divided into a blank cell group, an APP-infected group, and APP + forsythoside A low, medium, and high concentration groups (6.25, 12.5, and 25 μg / mL). After overnight culture, forsythoside A was added for pretreatment for 2 h. Actinobacillus pleuropneumoniae with an MOI of 100 was added to the APP-infected group and the low, medium, and high concentration groups, and the cells were mixed and co-cultured for 12 h. Western blot analysis was used to detect the expression levels of related proteins.

[0105] 5. Statistical Analysis

[0106] One-way differential analysis of the data was performed using SPSS software, and graphs were plotted using Graphpad Prism 8 ("*" or "#" indicates p<0.05, indicating a significant difference; "**" or "##" indicates p<0.01, indicating an extremely significant difference).

[0107] 6. Test Results

[0108] 6.1 Minimum inhibitory concentration of forsythoside A

[0109] The results are as follows Figure 4Forsythoside A caused turbidity in all wells at concentrations ranging from 8 to 2048 μg / mL. After spreading the turbid bacterial suspensions onto TSA plates, bacteria grew in all wells. The TSB medium control wells remained clear with no bacterial growth, while the solvent control wells and the bacterial suspension positive control wells showed turbidity. This indicates that the minimum inhibitory concentration (MIC) of forsythoside A is ≥2048 μg / mL. Florfenicol's MIC against the quality control bacterium ATCC 25922 was 4 μg / mL, meeting the quality control requirements.

[0110] 6.2 Effects of forsythoside A on cell viability

[0111] The results of the CCK8 test showed that ( Figure 5 PIEC cells maintained a viability of over 90% when co-incubated with forsythoside A at concentrations of 25, 12.5, 6.25, 3.125, 1.6525, and 0.87625 μg / mL. Subsequent experiments involved treating PIEC cells with low, medium, and high concentrations of forsythoside A at concentrations of 6.25, 12.5, and 25 μg / mL.

[0112] 6.3 Forsythoside A inhibits the expression of APP-induced PIEC inflammatory factors.

[0113] Following infection with *Actinomyces pleuropneumoniae*, PIEC cells showed upregulated expression of CD14, TLR4, p-P65, TNF-α, IL-1β, IL-6, and IL-18 proteins (p<0.01). Furthermore, treatment with forsythoside A at concentrations of 6.25, 12.5, and 25 μg / ml inhibited, in a concentration-dependent manner, the upregulation of these proteins in PIEC cells induced by *Actinomyces pleuropneumoniae*. Figures 6-7 The above results indicate that Actinobacillus pleuropneumoniae infection of vascular endothelial cells can lead to an abnormal increase in its related inflammatory proteins, while forsythoside A can effectively alleviate Actinobacillus pleuropneumoniae-induced vascular endothelial inflammatory damage by inhibiting the activation of CD14 / TLR4-NFκB P65.

[0114] Example 3: Alleviating effect of forsythoside A on Actinobacillus pleuropneumoniae-induced inflammatory response in mice.

[0115] 1. Preparation of drugs and solutions

[0116] Forsythoside A solution: Accurately weigh 600 mg of forsythoside A, dissolve it in 60 mL of sterile water, mix well, filter through a membrane, and prepare a stock solution of 60 mg / mL.

[0117] 2. Experimental animals and grouping

[0118] The experimental animals were 4-5 week old female BALB / c mice, purchased from the Hubei Provincial Experimental Animal Research Center, approval number: WPU 202411003.

[0119] (1) Management of the breeding environment

[0120] Before raising mice, the animal room was fumigated and disinfected. Before the formal experiment, the mice were pre-raised for 3 days to de-stress and adapt to the living environment. During the rearing period, the air conditioning temperature was set at 22-24℃, and they were given feed and drinking water.

[0121] (2) Grouping of experimental animals

[0122] Sixty BALB / c mice were randomly divided into six groups of ten each: Actinobacillus pleuropneumoniae infection group, Actinobacillus pleuropneumoniae infection + 15 mg / kg bw forsythoside A treatment group (15 mg / kg forsythoside A group), Actinobacillus pleuropneumoniae infection + 30 mg / kg bw forsythoside A treatment group (30 mg / kg forsythoside A group), Actinobacillus pleuropneumoniae infection + 60 mg / kg bw forsythoside A treatment group (60 mg / kg forsythoside A group), Actinobacillus pleuropneumoniae infection + 10 mg / kg bw flunixin meglumine treatment group (10 mg / kg flunixin meglumine group), and a blank control group. The mice were numbered.

[0123] (3) Mouse treatment

[0124] Bacterial infection method: A mouse infection model was established by intraperitoneal injection of Actinobacillus pleuropneumoniae, with a challenge dose of 10. 6 CFU / each.

[0125] Administration: Different concentrations of forsythoside A (15, 30, and 60 mg / kg bw) and flunixin meglumine (10 mg / kg bw) were administered intramuscularly. The blank control group and the Actinobacillus pleuropneumoniae infection group were injected with the same volume of PBS. The first administration was 2 hours before the intraperitoneal injection of Actinobacillus pleuropneumoniae, followed by once daily for 5 consecutive days, after which samples were collected.

[0126] (4) Observation of mouse signs

[0127] Mice were weighed daily, and changes in body weight were recorded. Clinical symptoms of mice after viral challenge and changes after drug administration were observed.

[0128] (5) Mouse dissection and tissue collection

[0129] Six days after the viral challenge, the mice were dissected, blood was collected from the eyeballs, the mice were euthanized by dislocation of the neck, the abdominal and thoracic cavities were opened, and lung and aortic vascular tissues were collected. After the autopsy, the carcasses were disposed of in a harmless manner.

[0130] (6) Blood biochemistry and routine blood tests

[0131] The collected blood samples were processed. An appropriate amount of anticoagulated blood was used for blood cell count testing, a portion was centrifuged, and the supernatant plasma was used for various blood biochemical tests.

[0132] (7) Histopathological examination

[0133] After collecting blood vessel and lung tissue, a suitable amount of tissue blocks were cut off, fixed in 4% paraformaldehyde, and then prepared into sections through processes such as embedding, dewaxing, staining, dehydration and mounting. The sections were then examined under a microscope, and images were collected and analyzed.

[0134] (8) Immunohistochemistry

[0135] The prepared slides were dewaxed, hydrated, antigen retrievaled, inactivated, blocked with 5% BSA, incubated with primary antibody overnight at 4°C, incubated with genus-specific secondary antibody, developed with DAB, counterstained, dehydrated and mounted to prepare slides. The slides were then examined under a microscope, and images were acquired and analyzed.

[0136] 3. Statistical Analysis

[0137] One-way ANOVA was performed using SPSS software to analyze the data between groups. The results are expressed as mean ± standard deviation (mean ± SD). p < 0.05 indicates a significant difference, and p < 0.01 indicates a highly significant difference.

[0138] 4. Test Results

[0139] 4.1 Changes in physical signs in mice

[0140] (1) Clinical manifestations and necropsy of mice

[0141] In the blank control group, normal mice were in good spirits, energetic and active, with smooth and shiny fur, normal water and food intake, and stable weight. In the infected group, mice infected with Actinobacillus pleuropneumoniae showed lethargy, huddled together, disheveled fur, increased eyelid discharge, significantly decreased water intake, anorexia, sticky feces, and in some cases, bloody stools, and weight loss. Post-mortem examination revealed pulmonary hemorrhage, with some lungs adhering to the pleura, and enlarged liver and spleen, exhibiting fibrinous pleuropneumonia. Compared to the challenge group, the drug treatment groups showed significant improvement in vital signs, with food and water intake returning to normal levels, significant weight recovery, and significantly reduced lesions in various organs and tissues after mortem examination.

[0142] (2) Changes in mouse body weight

[0143] Changes in mouse body weight under different treatments are shown in the figure. Figure 8 As shown, mice in the blank control group grew normally. After infection with Actinobacillus pleuropneumoniae, mice in the challenge group and the groups treated with different doses of drugs showed significant changes in body weight in the initial stage, with a downward trend in both groups. As the number of administrations increased, the food intake of mice in the groups treated with different doses of drugs tended to normalize and their body weight steadily recovered, approaching the level of the normal group. This indicates that treatment with different doses of forsythoside A has a certain ameliorative effect on the weight loss caused by Actinobacillus pleuropneumoniae infection.

[0144] 4.2 Effects of forsythoside A on hematological and plasma biochemical parameters in mice infected with APP

[0145] To investigate the changes in various indicators in the blood of mice infected with Actinobacillus pleuropneumoniae using forsythoside A, blood samples were collected from mice for blood cell differential counting. As shown in Table 3, compared with the blank control group, the number and percentage of neutrophils (Neu), white blood cells (WBC), and monocytes (Mon) were increased in the Actinobacillus pleuropneumoniae infection group, while the number and percentage of lymphocytes (Lym) and eosinophils (Eos) were decreased. This indicates that the body undergoes a severe inflammatory response after Actinobacillus pleuropneumoniae infection. Compared with the infection group, the treatment groups with forsythoside A (15, 30, and 60 mg / kg bw) and flunixin meglumine (10 mg / kg bw) all alleviated the above conditions. As shown in Table 4, compared with the blank control group, the infection group mice had increased levels of creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and creatinine (CREA), and decreased levels of albumin (ALB) and glucose (GLU). Compared with the infection group, the abnormal changes in the above biochemical indicators were alleviated to some extent after treatment with 15, 30 and 60 mg / kg bw forsythoside A and 10 mg / kg bw flunixin meglumine. The results indicate that Actinobacillus pleuropneumoniae infection in mice leads to a series of inflammatory responses and metabolic disorders such as liver and kidney dysfunction. Forsythoside A can alleviate the inflammatory response and metabolic dysfunction caused by Actinobacillus pleuropneumoniae.

[0146] Table 3. Results of routine blood tests

[0147]

[0148] Table 4. Results of Blood Biochemistry Tests

[0149]

[0150]

[0151] Note: * indicates p < 0.05 compared to the control group; # indicates p < 0.05 compared to the infected group; ## indicates p < 0.01 compared to the infected group.

[0152] 4.3 Histopathological changes of forsythoside A in APP-infected mice

[0153] Histopathological examination results of mouse lung tissue are as follows Figure 9 As shown, in the lung pathological sections, the lung structures of the blank control group were normal under microscopic examination. In contrast, the lung tissue of mice infected with Actinobacillus pleuropneumoniae showed inflammatory cell infiltration, alveolar wall thickening, alveolar collapse and atrophy, and disordered arrangement with blurred boundaries between alveoli. The results indicate that Actinobacillus pleuropneumoniae infection caused lung damage and inflammatory response in mice. After intervention with forsythoside A, the inflammatory cell infiltration in the lung tissue of mice decreased, and the abnormal changes such as alveolar thickening, collapse, and atrophy were alleviated. The pathological microscopic examination results of vascular tissue are shown below. Figure 10 As shown, the vascular endothelium of infected mice was locally ruptured, and the fibrous structure was locally broken and disordered; the vascular structure of the blank control group was basically normal. Forsythoside A intervention reduced the vascular endothelial damage induced by Actinobacillus pleuropneumoniae infection in mice. The results indicate that Actinobacillus pleuropneumoniae infection induces an inflammatory response in mice, leading to damage and dysfunction of the lungs and vascular tissues. Forsythoside A can alleviate lung and vascular endothelial damage induced by Actinobacillus pleuropneumoniae.

[0154] 4.4 Effects of forsythoside A on the expression of inflammatory proteins in the tissues of APP-infected mice

[0155] Immunohistochemical assays were used to investigate the expression of inflammatory proteins in the lungs and vascular tissues of mice infected with Actinobacillus pleuropneumoniae, and the intervention effect of forsythoside A. Figure 11 and Figure 12As shown, immunohistochemical results of mouse lungs and blood vessels revealed increased expression of CD14, TNF-α, IL-1β, and IL-18 proteins in the lungs and blood vessels of mice after infection with *Actinobacillus pleuropneumoniae*. This suggests that *Actinobacillus pleuropneumoniae* infection induced an inflammatory response in both the lungs and blood vessels of mice, leading to varying degrees of inflammatory damage. After treatment with forsythoside A, compared with the infection group, the expression of CD14 and inflammatory factors TNF-α, IL-1β, and IL-18 in the lungs and blood vessels of mice was reduced to varying degrees. These results indicate that *Actinobacillus pleuropneumoniae* infection leads to a certain degree of inflammatory response in the lungs and blood vessels of mice, triggering upregulation of CD14 and inflammatory factors TNF-α, IL-1β, and IL-18. Forsythoside A treatment inhibited the expression of pro-inflammatory cytokines in the lungs and blood vessels, suggesting that forsythoside A has a protective effect in the *Actinobacillus pleuropneumoniae*-induced inflammatory injury model in mice.

[0156] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of forsythoside A as the only active ingredient in the preparation of a medicament for the prevention and / or treatment of porcine contagious pleuropneumonia.

2. Use according to claim 1, wherein The concentration of forsythoside A is 6.25-25 μg / mL.

3. The use according to claim 1, wherein The forsythoside A alleviates lung and vascular endothelial injury.

4. The use according to claim 1, wherein The forsythoside A reduces the expression of inflammatory factors.

5. The use according to claim 4, wherein the compound is ###0002### The inflammatory factors include TNF-α, IL-1β, IL-6 and IL-18.

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