Application of Forsythoside A in the Preparation of Drugs for Infectious Bronchitis Virus in Chickens

Abstract

This invention relates to the field of biomedical technology, specifically to the application of forsythoside in the preparation of drugs for the prevention and treatment of infectious bronchitis in chickens. Immunofluorescence and Western blotting results show that forsythoside can significantly inhibit the expression of viral nucleocapsid proteins in CEK cells and reduce the viral load in CEK cells. Furthermore, forsythoside can protect against IBV infection in broiler chickens. Quantitative fluorescence results indicate a significant reduction in viral replication in the tracheal tissue of broilers. Clinical symptoms and histopathological results show that forsythoside can significantly alleviate IBV infection-induced slow weight gain, respiratory distress, and tracheal tissue damage in broilers. This invention demonstrates that forsythoside has low cytotoxicity, can effectively prevent infectious bronchitis in chickens, and has advantages such as being green and safe, leaving no residue, having few toxic side effects, and being unlikely to induce drug resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of forsythoside A in the preparation of drugs for the prevention and treatment of infectious bronchitis in chickens. Background Technology

[0002] Avian infectious bronchitis (IB) is an acute and contagious respiratory disease caused by the avian infectious bronchitis virus (IBV). IBV belongs to the genus *Gammacoronavirus*, family *Coronaviridae*, and can only infect chickens and turkeys. IBV infection causes respiratory distress, coughing, and tracheal rales in chicks; failure to lay eggs at sexual maturity; slow weight gain in adult chickens; enlarged kidneys (mottled kidneys); and blocked oviducts in laying hens, resulting in soft-shelled or deformed eggs. It is easily mixed with bacterial diseases such as *E. coli* and *Mycoplasma*, thus exacerbating clinical symptoms and posing significant challenges to disease control, seriously endangering poultry health. Currently, vaccines are the primary means of prevention; however, due to the rapid mutation rate of the virus, existing vaccines do not provide ideal immunity. Studies have shown that even in densely populated poultry farming areas in central and southern my country, outbreaks of the disease can still occur despite vaccination. Currently, there are no effective drugs for treating infectious bronchitis in chickens. Clinical studies have reported that IBV is prone to mixed infection with bacteria, which endangers the health of poultry farming. In clinical practice, antibiotics are the main treatment, leading to increased antibiotic use and increasingly serious bacterial resistance. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide an effective drug for the prevention and treatment of infectious bronchitis in chickens, which addresses the current lack of effective drugs for the prevention and treatment of infectious bronchitis in chickens, the easy mutation of the virus, and the slow speed of vaccine development.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the application of forsythoside A in the preparation of drugs against chicken infectious bronchitis virus.

[0005] The second objective of this invention is to provide the application of forsythoside A in the preparation of drugs for the prevention and treatment of infectious bronchitis in chickens.

[0006] Preferably, the infectious bronchitis of chickens includes: respiratory type, renal type, reproductive tract type, intestinal type, and muscular type.

[0007] A third object of the present invention is to provide an antiviral pharmaceutical composition comprising the aforementioned forsythoside and other antiviral drugs.

[0008] Preferably, in the pharmaceutical composition, the combination of forsythoside and other antiviral drugs is such that, without changing the existing drug dosage form, forsythoside and other antiviral drugs are combined and treated as separate drugs, or forsythoside and other antiviral drugs are formulated into the same dosage form.

[0009] Preferably, the pharmaceutical composition is formulated into an injection, aerosol, tablet, capsule, or droplet by adding a pharmaceutically acceptable carrier.

[0010] Preferably, the route of administration of the pharmaceutical composition is oral, injection, or mucosal.

[0011] The beneficial results achieved by this invention are that the forsythoside described in this invention can inhibit IBV replication in CEK cells, effectively protecting broilers from IBV infection. This invention demonstrates that forsythoside has low cytotoxicity, can effectively prevent infectious bronchitis in chickens, and has advantages such as being green and safe, leaving no residue, having few toxic side effects, and being unlikely to induce drug resistance. Attached Figure Description

[0012] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings;

[0013] Figure 1 Effects of Forsythoside on CEK cell activity

[0014] Figure 2 Effects of forsythoside on fluorescence expression of IBV N protein and viral load in CEK cells

[0015] Figure 3 Effects of forsythoside on body weight changes and clinical efficacy in broilers

[0016] Figure 4 Effects of Forsythosides on the Pathological and Ultrastructural Changes of Broiler Tracheal Tissue

[0017] Figure 5 Effects of Forsythoside A on Viral Load in the Respiratory Tract of Broilers Detailed Implementation

[0018] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0019]

[0020] Example 1: Virus proliferation after treatment of IBV-infected CEK cells with different concentrations of forsythoside A

[0021] To evaluate the inhibitory effect of forsythoside A on IBV replication in CEK cells, isolated primary chicken embryo kidney (CEK) cells were seeded into 6-well plates at a dose of 5 × 10⁻⁶. 5 Cells were cultured at 37°C for 15 hours, then inoculated with 200 μL of IBV (100 TCID50) virus solution and incubated in an incubator for 2 hours. After discarding the virus solution, different concentrations of forsythoside A solution were added, and the cells were cultured for another 24 hours and 36 hours. The culture was then terminated, and the replication of IBV in the cells was observed using immunofluorescence and quantitative fluorescence techniques.

[0022] To determine the safe concentration of forsythoside A in CEK cells, the CCK-8 reagent was used to detect the toxic effects of forsythoside A on CEK cells. The results are as follows: Figure 1 As shown, when the concentration of forsythoside A was greater than 250 μg / mL, the survival rate of CEK cells was greater than 67%, and when the concentration of forsythoside A was reduced to 125 μg / mL, the survival rate of CEK cells was greater than 85%, without reducing cell viability.

[0023] like Figure 2 As shown, immunofluorescence results indicated that after treatment with forsythoside A for 24 and 36 hours, the fluorescence intensity of IBVN protein in the high-concentration treatment group was significantly lower than that in the virus-infected group. Quantitative fluorescence results showed that treatment with different concentrations of forsythoside A resulted in significantly lower viral loads in IBV-infected CEK cells compared to the virus-infected group.

[0024] Example 2: Observation of the protective effect of forsythoside on tracheal tissue of broilers infected with IBV using HE staining and scanning electron microscopy.

[0025] (1) Sample collection

[0026] One hundred and forty one-day-old broiler chickens were raised in a vacuum negative pressure isolator until they reached seven days of age. They were then randomly divided into four groups (n=35 per group): a control group, an IBV infection group, a forsythoside treatment group, and a forsythoside + IBV group. The medication was added to the drinking water and administered continuously for seven days. The forsythoside dosage was 24 mg / kg BW. Then, except for the control group, the other groups were infected with IBV-M41 (10) via ocular and nasal drops at 14 days of age. 4 EID 50 Administer 0.1mL to 0.3mL daily, observing and recording the chickens' clinical symptoms and weight.

[0027] After euthanizing broilers at 19 days of age, tracheal and lung tissues were collected. Approximately 2 cm of the anterior end of the trachea was fixed in neutral formalin solution for histopathological analysis, and approximately 2 cm of the middle end of the trachea was fixed in 2.5% glutaraldehyde solution for scanning electron microscopy analysis.

[0028] (2) Preparation of HE staining samples

[0029] Tracheal and lung tissues fixed in 10% formalin solution were dehydrated, embedded in paraffin, and cut into 5μm thick sections. The sections were then sequentially immersed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min. After washing with tap water for 5 min, the sections were stained with hematoxylin for 3 min, washed with tap water for 5 min, differentiated with differentiation solution for 5 s, and then rinsed with running water after the sections returned to blue. They were then dehydrated sequentially with 85% and 95% graded alcohols for 5 min each, and stained with eosin for 5 min. The sections were then sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min. After clearing, the sections were mounted with neutral resin and observed under an optical microscope for tissue lesions.

[0030] (3) Preparation of scanning electron microscope samples

[0031] The fixed tracheal tissue was washed three times with phosphate buffer, 15 min each time. It was then fixed with 1% osmium tetroxide at room temperature in the dark for 2 h, followed by rinsing three times with phosphate buffer (PB), 15 min each time. The tissue was dehydrated with 30%-50%-70%-80%-90%-95%-100%-100% ethanol for 15 min each time, and then with isoamyl acetate for 15 min. The sample was dried in a critical point desiccator, then adhered tightly to a conductive carbon film double-sided adhesive and sputtered with gold for approximately 30 s on an ion sputtering stage. Finally, the sample was observed under a microscope, photographed, and analyzed.

[0032] By observing the clinical symptoms of broilers, we found that on days 3 and 5 post-infection, broilers in the virus-infected group exhibited symptoms such as lethargy, ruffled feathers, head shaking, huddling together, and drooping wings. The forsythia suspensa extract treatment group only showed mild drooping wings and head shaking on days 3 and 5 post-infection, while the control group showed no clinical symptoms. IBV infection caused slow weight gain in broilers. Forsythia suspensa extract treatment reduced the slow weight gain caused by IBV infection, but it was still slightly lower than the forsythia suspensa extract alone treatment group. Furthermore, forsythia suspensa extract alone treatment could increase weight gain in broilers, indicating that forsythia suspensa extract has a role in promoting broiler weight gain.

[0033] Histopathological analysis results as follows Figure 3As shown, only a small amount of cilia were lost from the tracheal tissue on day 1 post-infection. The tracheal lesions were most severe on day 3, mainly manifested as glandular hyperplasia in the tracheal mucosa, almost complete cilia loss, and extensive infiltration of lymphocytes and monocytes in the mucosa, accompanied by minor hemorrhage. On day 5 post-infection, a small portion of the tracheal cilia were lost, and mild hemorrhage and significant infiltration of monocytes and lymphocytes appeared in the mucosa. In the forsythia suspensa lipoprotein treatment group, IBV infection did not cause significant pathological changes in the tracheal tissue on day 1, but mild tracheal ciliary loss and mucosal hyperplasia appeared on days 3 and 5. The tracheal tissue in the control group showed no pathological changes.

[0034] Scanning electron microscopy results of the trachea are as follows Figure 4 As shown, the tracheal mucosa in the control group had a relatively normal structure, with brush cells scattered throughout without significant edema. The surface microvilli were dense, uniform in size, tightly connected, with narrow intercellular spaces and intact cell membranes. Ciliated cells were abundant, widely distributed, and had normal structure. The virus-treated group showed the most severe tracheal mucosal damage, with some brush cells exhibiting membrane rupture and disintegration, a reduced number of ciliated cells, large-area degeneration, and sparse distribution in small areas. Most ciliated cells were significantly shorter and thinner, with a small number of goblet cells present. The drug-treated group showed relatively less damage to the tracheal mucosa, with slightly shrunken and collapsed brush cells, locally sparse microvilli, a reduced number of ciliated cells, and a small number of goblet cells. This indicates that forsythoside A and baicalin protected against tracheal structural damage caused by IBV infection.

[0035] like Figure 5 As shown, the viral load in tracheal tissue gradually increased after IBV infection, reaching its peak on day 3 post-infection and then beginning to decline on day 5. The viral load in the trachea of ​​the forsythia suspensa-treated group was significantly lower than that of the virus-treated group on days 3 and 5 post-infection. The trend of viral load in the lungs after IBV infection was consistent with that in the tracheal tissue, but the viral load in the lungs of the forsythia suspensa-treated group was significantly lower than that of the virus-treated group on days 3 and 5 post-infection, and the viral load in the lungs was generally lower than that in the trachea. No viral load was detected in the control group.

[0036] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of forsythoside A in the preparation of drugs against chicken infectious bronchitis virus.

2. Application of forsythosides in the preparation of drugs for the prevention and treatment of infectious bronchitis in chickens.

3. The application as described in claim 2, characterized in that, The infectious bronchitis in chickens includes respiratory, renal, reproductive, intestinal, and muscular forms.

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