Use of phosphatidylcholine in the preparation of a medicine for preventing and treating porcine rotavirus infection

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of phosphatidylcholine in the preparation of drugs for the prevention and treatment of porcine rotavirus infection. This invention is the first to discover that phosphatidylcholine (PC) has significant antiviral activity in preventing and treating PoRVA infection. Experimental results show that PC can effectively inhibit PoRVA proliferation, reduce viral load, thereby alleviating diarrhea symptoms caused by PoRVA infection and reducing mortality. Furthermore, PC can also promote the repair and maintenance of intestinal barrier integrity, reduce intestinal tissue damage, and decrease the risk of secondary infections by inhibiting PoRVA infection-induced necrotizing apoptosis. These results indicate that PC has good therapeutic potential against PoRVA infection. At the same time, PC is safe to obtain and has good biocompatibility; no significant toxic side effects were observed within a reasonable dosage range, making it suitable for the prevention and / or treatment of PoRVA infection, and possessing good application prospects and promotional value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of phosphatidylcholine in the preparation of drugs for preventing and treating porcine rotavirus infection. Background Technology

[0002] Porcine rotavirus (PoRV) belongs to the genus Rotavirus in the family Reoviridae. It is a non-enveloped, double-stranded RNA virus and a major pathogen causing viral diarrhea in pigs, particularly harming newborn piglets. Infection often manifests as dehydration, watery diarrhea, growth retardation, and death, resulting in severe economic losses for large-scale pig farming. PoRV primarily attacks the epithelial cells of the jejunum and ileum in the small intestine, replicating extensively in the intestinal villi. This leads to villi blunting and atrophy, epithelial cell shedding, and impaired intestinal barrier function, thereby affecting the body's absorption of nutrients and inducing persistent diarrhea.

[0003] Based on serological and genetic differences, porcine rotavirus (PoRV) can be classified into several serotypes, including A, B, C, E, and H. Among them, serotype A (Porcine Rotavirus A, PoRVA) has the widest distribution and highest detection rate in pig herds and is considered the most pathogenic and harmful serotype, often causing highly contagious diarrhea outbreaks, especially in young piglets. The different serotypes and their genetic diversity have led to the long-term prevalence of serotype A porcine rotavirus in pig herds, increasing the difficulty of prevention and control.

[0004] Currently, prevention and control measures for PoRVA infection mainly rely on vaccination and symptomatic treatment. However, due to the complexity of PoRVA subtyping, frequent genetic variations, and interference from maternal antibodies, the protective efficacy of existing vaccines in actual production remains limited, and there is a lack of effective treatments for PoRVA infection. Therefore, developing safe, effective drugs for the prevention and treatment of PoRVA infection has significant clinical and industrial value. Summary of the Invention

[0005] The purpose of this invention is to provide the use of phosphatidylcholine (PC) in the preparation of drugs or products for the prevention and / or treatment of diseases caused by PoRVA. This invention achieves effective control of PoRVA infection through exogenous PC supplementation, providing a new, safe, and effective prevention and treatment strategy for PoRVA-related diarrheal diseases.

[0006] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: The first aspect of this invention provides the use of phosphatidylcholine in the preparation of a medicament for preventing and treating porcine rotavirus infection, wherein phosphatidylcholine has CAS NO: 97281-44-2 and a structural formula as shown in formula (I): (Formula I).

[0007] In some embodiments of the present invention, the prevention and / or treatment are defined as prevention and / or treatment.

[0008] In some embodiments of the present invention, the phosphatidylcholine is exogenous phosphatidylcholine, including eggs, soybeans or other natural or synthetic sources.

[0009] In some embodiments of the present invention, the porcine rotavirus includes group A, B, C, E and H porcine rotavirus, preferably group A porcine rotavirus.

[0010] In some embodiments of the present invention, the group A porcine rotavirus is a zoonotic disease, and its infected hosts include humans, pigs, cattle, sheep, horses, rabbits, deer, monkeys, dogs, cats, rats, chickens, and turkeys.

[0011] In some embodiments of the present invention, the drug includes human or veterinary drug preparations, preferably veterinary drug preparations.

[0012] In some embodiments of the present invention, the drug is administered to pigs.

[0013] In some embodiments of the invention, the phosphatidylcholine comprises a pharmaceutically acceptable salt.

[0014] In some embodiments of the present invention, the pharmaceutically acceptable salt includes acid addition salts and base addition salts.

[0015] "Pharmaceutically acceptable acid addition salts" refer to salts that retain the biological effectiveness and properties of the free base, are not undesirable in biological or other respects, and are formed from an inorganic acid and an organic acid, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and such as, but not limited to, acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor 10 sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane 1,2 disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, fumaric acid, galactopyric acid, gentian acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, etc.

[0016] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not undesirable in biological or other respects. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazoline, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0017] Specifically, the pharmaceutically acceptable salts of phosphatidylcholine include sodium phosphatidylcholine.

[0018] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, binders, wetting agents, lubricants, disintegrants, solvents, cosolvents, solubilizers, preservatives, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

[0020] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0021] In some embodiments of the present invention, the dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0022] In some embodiments of the present invention, the route of administration of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

[0023] In some embodiments of the present invention, the drug further includes one or more other active ingredients.

[0024] In some embodiments of the present invention, the safe and effective daily dose of the phosphatidylcholine or a pharmaceutically acceptable salt thereof is 1 mg - 10 mg / kg body weight. The above dose can be administered as a single dose unit or divided into multiple dose units, and the specific dosing regimen can be adjusted according to the individual animal condition and actual application needs.

[0025] In this invention, the phosphatidylcholine or a pharmaceutically acceptable salt thereof can be used to inhibit the replication of PoRVA in in vitro cell experiments. The working concentration of the phosphatidylcholine or a pharmaceutically acceptable salt thereof is 5 μg / mL to 50 μg / mL, preferably 10 μg / mL to 15 μg / mL.

[0026] The beneficial effects of this invention are: This invention is the first to discover that PC has significant antiviral activity in preventing and treating PoRVA infection. Experimental results show that PC can effectively inhibit PoRVA proliferation, reduce viral load, thereby alleviating diarrhea symptoms caused by PoRVA infection and reducing mortality. Furthermore, PC can also promote the repair and maintenance of intestinal barrier integrity, reduce intestinal tissue damage, and decrease the risk of secondary infections by inhibiting PoRVA infection-induced necroptosis. These results indicate that PC has good therapeutic potential against PoRVA infection. At the same time, PC is safe to use and has good biocompatibility; no significant toxic side effects were observed within a reasonable dosage range, making it suitable for the prevention and / or treatment of PoRVA infection, and demonstrating good application prospects and promotional value. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a graph showing the analysis results of the metabolomics and lipidomics of clinical serum samples. A is a flowchart of sample grouping and analysis; B is a statistical graph of the distribution and quantity of identified lipid subclasses in the samples; C is a PCA analysis graph of the lipidome for each group of samples; D is a statistical graph of differentially expressed lipid molecules for each group of samples; E is a heatmap of lipid molecule abundance for each group of samples; F is a graph of predicted importance of lipid molecules for each group of samples; G is a graph of predicted importance of lipid subclasses for each group of samples; and H is a ROC curve analysis graph for PC.

[0028] Figure 2This is a graph showing the metabolic analysis and combined analysis of the cell infection model. A is a flowchart of cell sample processing and analysis; B is a statistical graph showing the distribution and quantity of lipid subclasses identified in the cell samples; C is a PCA analysis graph of the lipidome in the cell samples; D is a heatmap of lipid molecule abundance in the cell samples; E is a statistical graph of differential lipid molecules in the cell samples; F is a Venn diagram showing the analysis of clinical samples, cell samples, and reference datasets; and G is a statistical graph showing significant changes in lipid molecules across the three datasets.

[0029] Figure 3 This is a graph showing the effect of PC on PoRVA replication in IPEC-J2 cells. Specifically, A shows the cytotoxicity of PC on IPEC-J2 cells; B shows the effect of different concentrations of PC on PoRVA replication detected by RT-qPCR; C shows the effect of PC on PoRVA replication detected by IFA (scale bar: 100 μm); and D shows the effect of different concentrations of PC on PoRVA replication detected by Western blotting.

[0030] Figure 4 This is a diagram showing the experimental design and clinical symptom results of a treatment experiment on piglets. A is the experimental flowchart; B is the survival curve of the piglets; C is the trend of weight change in piglets after challenge; D is the statistical chart of clinical score data for piglets; E is the result of viral load detection in swab samples; and F is the result of viral load detection in the intestinal tissue of piglets in the challenge and treatment groups.

[0031] Figure 5 These are anatomical diagrams and histopathological section results from a piglet prevention and treatment experiment. A is the anatomical diagram; B is the HE and IHC staining analysis diagram of the jejunum; C is the HE and IHC staining analysis diagram of the ileum; the scale bar for B and C is 100 μm.

[0032] Figure 6 These are scanning electron microscope (SEM) images of small intestinal villi from a piglet preventive treatment experiment. Image A shows the jejunum; image B shows the ileum; the images are displayed at scale bars of 10.0 μm, 200 μm, and 100 μm, respectively.

[0033] Figure 7 These are images showing the detection of intestinal necrosis and apoptosis, as well as intestinal integrity, in piglets. Image A shows the colocalization IFA detection of E-cadherin and p-MLKL in piglet intestinal tissue; image B shows the colocalization IFA detection of ZO-1 and p-MLKL in piglet intestinal tissue; the scale bar is 100 μm. Detailed Implementation

[0034] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0035] The experimental procedure in this embodiment is summarized as follows: First, clinical samples collected from pig farms were tested for antigens and antibodies. Based on the results, the samples were divided into three groups: a negative control group (N), where both antigen and antibody tests were negative; a positive group (P), where both antigen and antibody tests were positive, indicating the pigs were infected; and a recovery group (R), where antigens were negative but antibodies were positive, indicating the pigs were in the recovery phase after infection. Non-targeted metabolomics and lipidomics were performed using the LC-MS platform to further elucidate the host metabolic evolution characteristics during PoRV infection and recovery, and a differentially expressed molecule, PC, with a strong correlation to PoRVA infection was identified, suggesting it may have a significant impact on PoRVA infection. Therefore, this invention verified whether PC has an inhibitory effect on PoRVA replication in the passaged porcine small intestinal epithelial cell line (IPEC-J2), and conducted animal experiments, administering PC as a preventative treatment to infected PoRVA-infected piglets to test its clinical efficacy.

[0036] The materials used in this experiment are as follows: (1) Cells and viruses The PoRVA strain and IPEC-J2 cells were preserved in our laboratory. Both the strain and cells were conventional strains and cells that are publicly available in the current technology. The 3-day-old antigen-antibody double-negative suckling piglets were purchased from the Experimental Animal Center of Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd.

[0037] (2) Main reagents PCs used in both in vivo and in vitro experiments were purchased from MCE; the enhanced CCK8 kit was purchased from Yisheng Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Sigma; the EZBioscience® EZ-press RNA Purification Kit was purchased from EZBioscience; the Hifair® V C58P2 Multiplex One Step RT-qPCR Probe Kit (UDGPlus) was purchased from Yisheng Biotechnology; and other reagents were prepared using conventional methods in the field.

[0038] Example 1: Metabolic and lipid changes of PoRVA in vivo from infection to recovery and in vitro cell infection at different times. Clinical serum samples were divided into a control group (N group), a positive group (P group), and a recovery group (R group). Non-targeted polar metabolomics and lipidomics were performed using the LC-MS platform to further elucidate the host metabolic evolution characteristics during PoRV infection and recovery. Based on the analysis of differentially expressed metabolites, key lipid subclasses were further analyzed in depth. It was found that several PC lipid molecules were significantly downregulated in pigs after PoRV infection (compared to the N group in the P group), and these PC lipid molecules were significantly upregulated in pigs after recovery (compared to the P group in the R group). Furthermore, differential analysis of the PC lipid subclass also revealed that the abundance of the PC lipid subclass was significantly upregulated in pigs after recovery (compared to the P group in the R group).

[0039] Furthermore, the importance of lipid molecules was scored using a Random Forest (RF) model. It was found that multiple PC lipid molecules ranked high in importance both after PoRV infection (comparison between P and N groups) and after recovery (comparison between R and P groups). At the lipid subclass level, RF was used to predict the importance of lipid subclasses, and PC was also found to be among the most important. Figure 1 (F). Furthermore, the reliability of PC as a biomarker of viral infection was analyzed using classic receiver operating characteristic (ROC) curves (F). Figure 1 The results also showed that PC exhibited the best diagnostic efficacy, with an area under the curve (AUC) as high as 0.914 (95% CI: 0.876-0.949). This result confirms a strong correlation between PC and PoRV infection and also suggests the key role of PC in maintaining the body's normal physiological state.

[0040] Subsequently, virus-infected cell samples were prepared, with six replicates per group. Cells were infected with PoRVA and collected at 12, 24, 36, and 48 hours post-infection for LC-MS analysis to verify the reliability of the clinical sample results. The results are as follows: Figure 2 As shown, the clinical serum and cell models showed high consistency in the analysis of differential metabolites and lipids. The results revealed that the cell model data contained multiple PC lipid molecules shared with the clinical serum model data, as well as others, which showed a downregulation trend after PoRV infection (comparing the P group to the N group). Since multiple PC lipid molecules underwent significant changes, and it is known that PC lipid molecules with different carbon chains can be transformed through cyclic processes such as deacylation and reacylation, commercially available high-purity PC lipid molecules (CAS NO: 97281-44-2) were used in subsequent experiments for further verification.

[0041] Example 2: Effect of PC on PoRVA replication in IPEC-J2 cells 1. PC toxicity test on IPEC-J2 cells The cytotoxicity of PC to IPEC-J2 cells was determined using the CCK8 cytotoxicity assay, as detailed below: IPEC-J2 cells were seeded into 96-well plates, leaving one column unseeded. Cells were cultured at 37°C and 5% CO2 until complete confluence. The culture medium was aspirated, and the cells were washed three times with PBS. PC was dissolved in DMSO to prepare a stock solution, which was then diluted with DMEM to different concentrations (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL), with five replicates per concentration, 100 µL / well. After incubation at 37°C and 5% CO2 for 24 h, the solution was aspirated, and the cells were washed three times with PBS. A 10% (v / v) CCK8 solution was prepared in DMEM and added to the 96-well plates (100 μL / well). The plates were incubated at 37°C and 5% CO2 for 1 h. The absorbance (λ = 450 nm) was measured using a microplate reader, and cell viability was calculated after 24 h of treatment with different concentrations of the solution.

[0042] The results are as follows Figure 3 As shown in Figure A, the maximum safe concentration of PC for IPEC-J2 is 50 μg / mL.

[0043] 2. Effect of PC on PoRVA replication in IPEC-J2 cells - RT-qPCR IPEC-J2 cells were seeded in 24-well plates and cultured at 37°C and 5% CO2 until the cell confluence reached 80-90%. The culture medium was then discarded, and the cells were washed three times with PBS. PoRVA was used to infect the cells with an MOI of 0.1. The cells were incubated at 37°C and 5% CO2 for 1 hour. The culture medium was then replaced with one containing PC (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) and another without PC. Samples were collected 24 hours after infection.

[0044] RNA was extracted using the EZBioscience® EZ-press RNA Purification Kit according to the manufacturer's instructions, and then reverse transcribed into cDNA using the TaKaRa kit. The reaction mixture was prepared according to the SYBR GreenReal-time PCR Master Mix kit. RT-qPCR was performed using an Applied Biosystems® 7500 system. The specific program was: 50℃ for 2 min; 95℃ for 10 min; 95℃ for 15 s; 60℃ for 15 s; 72℃ for 30 s; amplification cycle number of 40. The relative expression level of PoRVA-VP6 mRNA in cells was detected. Primer sequences are as follows: PoRVA-VP6-F: 5'-GGCTTTAAACGAAGTCTTC-3' (SEQ ID NO: 1); PoRVA-VP6-R: 5'-GGTCACATCCTCTCACT-3' (SEQ ID NO: 2); GAPDH-F: 5'-CCTTCCGTGTCCCTACTGCCAAC-3' (SEQ ID NO: 3); GAPDH-R: 5'-GACGCCTGCTTCACCACCTTCT-3' (SEQ ID NO: 4).

[0045] The results are as follows Figure 3 As shown in B.

[0046] 3. Effects of PC on PoRVA replication in IPEC-J2 cells - IFA IPEC-J2 cells were seeded in 24-well plates and cultured at 37°C and 5% CO2 until the cell confluence reached 80-90%. The culture medium was discarded, and the cells were washed three times with PBS. PoRVA was used to infect the cells at an MOI of 0.1, and the cells were incubated at 37°C and 5% CO2 for 1 hour. The medium was then replaced with PC-containing and PC-free medium, respectively. After 24 hours of infection, the culture medium was discarded, and the cells were washed three times with PBS for 5 minutes each time. 300 μL of pre-chilled 4% paraformaldehyde was added to each well, and the cells were incubated at room temperature for 15 minutes. The cells were then washed three times with PBST for 5 minutes each time, and 0.4% Triton X was added. 100 solution, incubate at room temperature for 10 days 20 min. Wash 3 times with PBST, 5 min each time, then add 3% BSA and block at room temperature for 1 h. Wash 3 times with PBST, 5 min each time, then incubate the mouse anti-PoRVA-VP6 protein primary antibody at 37°C for 1 h. Wash 3 times with PBST, 5 min each time, then dilute the FITC fluorescent secondary antibody with PBST at a ratio of 1:500 and incubate at 37°C in the dark for 1 h. Under dark conditions, wash 3 times with PBST, 5 min each time, then dilute the DAPI fluorescent dye with PBST at a ratio of 1:1000 and incubate at 37°C in the dark for 10 min. Under dark conditions, wash 5 times with PBST. Repeat 7 times, observe the fluorescence intensity using a fluorescence microscope and take pictures.

[0047] The results are as follows Figure 3 As shown in Figure C, the proliferation of PoRVA after PC treatment was detected by indirect immunofluorescence assay (IFA). The specific fluorescence of PoRVA-VP6 protein in the drug-treated group was significantly reduced compared with that in the positive control group.

[0048] 4. Effect of PC on PoRVA replication in IPEC-J2 cells - Western blot IPEC-J2 cells were seeded in 6-well plates and cultured at 37°C and 5% CO2 until the cell confluence reached 80-90%. The culture medium was discarded, and the cells were washed three times with PBS. PoRVA was used to infect the cells with an MOI of 0.1. The cells were incubated at 37°C and 5% CO2 for 1 h. The culture medium was then replaced with PC-containing and PC-free medium, respectively. After 24 h of infection, the culture medium was discarded, and the cells were washed three times with PBS. 200 μL of IRA lysis buffer was added to each well, and the cells were lysed on ice for 10 min. The cell pellet was mixed by pipetting and transferred to 1.5 mL sterile EP tubes. The cells were centrifuged at 12,000 r / min for 5 min at 4°C. 180 μL of the supernatant was aspirated and mixed with 36 μL of 5× loading buffer. The mixture was then incubated at 100°C for 10 min to obtain Western blot protein samples.

[0049] Electrophoresis was performed using a 10-well, 10% precast gel. The sample loading order was: Marker, 0 μg / mL (negative control), 0 μg / mL (positive control), 5 μg / mL, 10 μg / mL, and 15 μg / mL PC-treated sample.

[0050] The electrophoresis apparatus was programmed to perform electrophoresis at 80V for 30 min and at 120V for 1 h. After electrophoresis, a semi-dry transfer system was used. The transfer system was assembled in the following order: black side (negative electrode) - sponge - filter paper - protein gel - PVDF membrane - filter paper - sponge - white side (positive electrode). After assembly, air bubbles were removed using a clean test tube or glass rod. The transfer apparatus was programmed to perform transfer at 15V for 40 min. After transfer, the PVDF membrane was removed, washed with TBST for 5 min, and then sealed in 5% skim milk powder at room temperature for 1 h.

[0051] Discard the blocking solution and wash three times with TBST, 5 min each time. Then, remove the membrane and place it in the primary antibody against mouse anti-PoRVA-VP6 protein, incubate overnight at 4°C, then wash three times with TBST, 5 min each time. Place the membrane in a secondary antibody solution of HRP-labeled goat anti-mouse IgG diluted with TBST (1:10000 dilution), and incubate at room temperature for 1 h. After incubation, wash the membrane three times with TBST, 5 min each time, immerse the membrane in TBST, prepare ECL colorimetric solution under light-protected conditions, and develop and photograph using a gel scanning system. Next, the membrane was eluted with membrane regeneration buffer at room temperature for 10 min, blocked again, and then placed in mouse anti-GAPDH protein primary antibody and incubated at room temperature for 1 h. Then, it was washed three times with TBST for 5 min each time. The membrane was then placed in HRP-labeled goat anti-mouse IgG secondary antibody solution diluted with TBST (dilution ratio 1:10000) and incubated at room temperature for 1 h. After that, the membrane was washed three times with TBST for 5 min each time, and then immersed in TBST. ECL colorimetric solution was prepared under light-protected conditions, and the membrane was developed and photographed using a gel scanning system.

[0052] The results are as follows Figure 3 As shown in Figure D, the proliferation of PoRVA virus after PC treatment was detected by Western blotting (WB): After treatment with different concentrations of PC, the gray value of the corresponding PoRVA-VP6 protein band gradually decreased with the increase of drug concentration, showing a dose-dependent effect, indicating that PC has an inhibitory effect on PoRVA proliferation.

[0053] Example 3: The alleviating effect of PC on PoRVA-infected piglets 1. Experimental Design Twenty-four 3-day-old commercial piglets (purchased from Wens Foodstuff Group Co., Ltd.) were tested negative for PoRVA, PEDV, PDCoV, and TGEV antibodies and antigens by ELISA and PCR, respectively. They were randomly divided into 4 groups (6 piglets per group): The first group was the blank control group (blank group). Each pig was given an oral dose of DMEM equal to the challenge dose, and the oral administration group was given an oral dose of empty solvent (corn oil) equal to the challenge dose. The second group was the control group (PC group). Each pig was orally administered the same volume of DMEM as the challenge dose, and the PC solution at a concentration of 2.5 mg / mL (dose 10 mg / kg body weight). The third group was the challenge group (PoRVA, RV group). Each pig was orally administered 2 mL of a 1×10⁻⁶ titer. 6 TCID 50 / mL of PoRVA virus solution, and the same volume of empty solvent as the oral administration group; The fourth group was the challenge treatment group (PC+PoRVA, PC+RV group), with each pig orally administered 2 mL of a 1×10⁻⁶ titer. 6 TCID 50 PoRVA virus solution / mL, oral concentration is 2.5 mg / mL, PC solution dosage is 10 mg / kg body weight).

[0054] PC solution: Taking 1 mL of working solution as an example, add 100 μL of 25.0 mg / mL clear EtOH stock solution to 900 μL of corn oil and mix thoroughly.

[0055] Experiment and sampling arrangements: After being transported to the sterilized experimental site, piglets were randomly divided into four groups and placed in independent feeding units. Groups three and four were orally challenged with the virus, while groups one and two were injected with an equal volume of DMEM. Groups two and four began receiving the drug after challenge and continued until the end of the experiment. Clinical symptoms of the piglets in each group were monitored and recorded daily, including weight, diarrhea, feed intake, and mental state.

[0056] Pigs that died after infection were immediately dissected, and the remaining pigs were all dissected on the seventh day after infection, as detailed below: 1) Take photos of the dissected pigs in each group to record and observe and check whether there are pathological changes in the intestines, such as thinning of the intestinal wall, transparency, and watery contents; 2) Collect small intestinal tissue (duodenum, jejunum, ileum) from each group of dissected pigs, and process it into two portions: One sample was fixed with 10% formaldehyde solution for HE staining and immunohistochemical detection. The pathological changes in the intestinal tissue were observed under a microscope, including villus morphology, villus loss, villus height and crypt depth, and PoRVA-VP6 protein expression.

[0057] Another sample was used for viral load detection: 100 mg of intestinal tissue sample was weighed, 1 mL of lysis buffer and 2 grinding beads were added, and the sample was ground thoroughly in a grinder. After grinding, the sample was centrifuged at 4°C and 12000 r / min for 10 min, and the supernatant was collected for subsequent RNA extraction and RT-qPCR detection.

[0058] 2. Clinical symptoms During the experiment, one piglet died in the challenge group on the third, fourth, and sixth days after challenge; the only piglet that died in the treatment group was on the fifth day after challenge. The survival curves are shown below. Figure 4 As shown in Figure B. Weight changes are as follows. Figure 4 As shown in Figure C, piglets in the challenge group experienced severe weight loss due to diarrhea, while piglets in the drug-treated group showed significant improvement in weight. Clinical scoring results are as follows: Figure 4 As shown in Figure D, the results indicate that the PC administration group effectively alleviated diarrhea symptoms in piglets, and the piglets' overall condition was better than that of the challenge group. The swab viral load test results are as follows: Figure 4 As shown in Figure E, the results indicate that the PC administration group significantly reduced the PoRVA load in swabs.

[0059] 3. Viral load detection in duodenal, jejunal, and ileal tissue samples The relative expression level of PoRVA-VP6 mRNA in intestinal tissue was determined by RT-qPCR, and the experimental steps were the same as those in Example 2.

[0060] The results are as follows Figure 4 As shown in Figure F, compared with the challenge group, the content of PoRVA-VP6 mRNA in the duodenum, jejunum and ileum tissues of piglets was significantly reduced after PC treatment, indicating that PC has the effect of inhibiting PoRVA replication in vivo.

[0061] 4. Histopathological changes Intestinal tissue sections were observed under a microscope using hematoxylin and eosin (HE) staining to analyze intestinal lesions. Results were as follows: Figure 5 As shown: Compared with the blank control group, the duodenal, jejunal, and ileal tissue sections of the treatment group showed no obvious lesions, indicating that PC had no adverse effects on the piglet intestines. Compared with the blank control group, the intestinal tissue sections of the challenge group showed obvious lesions such as shortened and shed intestinal villi, which are characteristic pathological manifestations after PoRVA infection. In contrast, the challenge treatment group maintained normal intestinal wall thickness and did not show obvious shortening or shed intestinal villi. These results indicate that PC treatment can effectively alleviate the tissue damage to the piglet intestines caused by PoRVA infection.

[0062] 5. Intestinal villi damage status The ultrastructure of the intestinal mucosa was observed using scanning electron microscopy (SEM), and the results are as follows: Figure 6As shown, the intestinal mucosa of piglets in both the control group and the PC group exhibited normal physiological morphology at different magnifications. Under low magnification, the intestinal mucosal folds were intact, regularly arranged, and the surface was smooth and without defects. Under medium and high magnification, the intestinal villi were arranged in a uniform finger-like or leaf-like pattern, dense and neat, with rounded and full villi tips, long and slender shapes, and clear boundaries between villi, without adhesion or breakage. The normal intestinal villi in the PC group also indicates that PC treatment does not have an abnormal effect on the ultrastructure of the intestinal villi in piglets. The ultrastructure of the intestinal villi in RV group piglets showed severe damage. Under low magnification, the integrity of the intestinal mucosa surface was disrupted, with obvious depressions and defects in local areas. Under medium magnification, the intestinal villi were disordered, with a large number of villi falling off. The remaining villi were severely atrophied, shortened, and thickened, and they adhered and fused together to form irregular clump-like structures. Under high magnification, the villi tips ulcerated and collapsed, and the surface texture disappeared, presenting a rough and damaged pathological state. This phenomenon is highly consistent with the ultrastructural lesions of the intestinal mucosa in piglets reported in previous literature due to PoRV infection. This phenomenon provides direct ultrastructural evidence of intestinal malabsorption caused by viral infection.

[0063] In summary, in the piglet treatment experiment, PC, as a metabolite, demonstrated the advantages of being non-toxic and having no side effects in the control group. For PoRVA-challenged piglets, PC treatment significantly alleviated clinical symptoms such as diarrhea. Furthermore, PC treatment significantly reduced viral load in the duodenum, jejunum, and ileum of infected piglets, alleviating small intestinal tissue damage. Therefore, PC has the potential to be used in the preparation of drugs for the prevention and treatment of PoRVA infection.

[0064] Example 4: Exploring the mechanism by which PC exerts its anti-PoRVA effect To investigate the mechanism by which PC exerts its anti-PoRVA effect, the jejunal tissue sections of piglets obtained in Example 3 were further analyzed. In this example, E-cad (red fluorescence) and p-MLKL (green fluorescence) dual immunofluorescence staining were used to detect the intestinal tissue of piglets.

[0065] The results are as follows Figure 7 As shown in Figure A: In the blank group, E-cadherin (E-cadherin) showed a continuous and clear linear distribution on the intestinal epithelial cell membrane, while p-MLKL showed only a weak fluorescent signal. In the RV group, the continuity of E-cadherin was significantly impaired, exhibiting breakage and disordered distribution, while the fluorescence signal of p-MLKL was significantly enhanced and mainly distributed in the villous epithelial region. Compared with the RV group, the fluorescence distribution of E-cadherin was significantly restored and its continuity was enhanced in the PC+RV group, while the fluorescence signal of p-MLKL was significantly reduced. These results indicate that PoRVA infection can lead to damage to intestinal epithelial adhesion junctions and activate necrotizing apoptotic signals, while PC treatment can alleviate the above damage, mitigate the disruption of adhesion junctions by viral infection, and thus effectively maintain the integrity of the intestinal mucosal barrier.

[0066] Tight junctions (TJs) are important structures constituting the intestinal physical barrier. Among them, ZO-1, as a key skeletal protein of the TJ family, plays a central role in maintaining intestinal epithelial cell polarity, regulating paracellular pathway permeability, and ensuring the integrity of the intestinal mucosal barrier. In this embodiment, dual immunofluorescence staining with ZO-1 (red fluorescence) and p-MLKL (green fluorescence) was used to detect intestinal tissue in piglets.

[0067] The results are as follows Figure 7 As shown in Figure B: In the blank group, the ZO-1 fluorescence signal showed a continuous band distribution with intact structure, while the p-MLKL signal was extremely weak. In the RV group, the continuity of ZO-1 fluorescence was significantly decreased, showing a broken and scattered punctate distribution, indicating that PoRVA infection severely damaged the tight junction structure of intestinal epithelial cells, while the p-MLKL signal was significantly enhanced. Compared with the RV group, the continuity of ZO-1 fluorescence was significantly improved in the PC+RV group, the distribution tended to be more regular, and the p-MLKL signal was significantly decreased. These results further indicate that PC treatment can improve PoRVA infection-induced damage to intestinal tight junctions and maintain the physical integrity of the intestinal epithelial barrier.

[0068] This embodiment demonstrates that PC can significantly inhibit the activation of p-MLKL-related necroptosis signals in the intestine of PoRVA-infected piglets and improve the continuity distribution of E-cad and ZO-1, suggesting that PC can maintain the integrity of the intestinal barrier by protecting the intestinal epithelial adhesion junctions and tight junction structures, thereby exerting an anti-PoRVA infection effect.

[0069] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of phosphatidylcholine as the sole active ingredient in the preparation of drugs for the prevention and treatment of group A rotavirus infection in pigs; The structural formula of the phosphatidylcholine is shown in formula (I): (Equation I).

2. The application according to claim 1, characterized in that: The drugs include veterinary drug preparations.

3. The application according to claim 2, characterized in that: The drug is administered to pigs.

4. The application according to claim 3, characterized in that: The phosphatidylcholine includes pharmaceutically acceptable salts.

5. The application according to claim 4, characterized in that: The drug includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that: The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, cosolvents, solubilizers, preservatives, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

7. The application according to claim 5, characterized in that: The dosage form of the drug includes at least one of the following: suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

8. The application according to claim 5, characterized in that: The route of administration of the drug includes at least one of the following: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

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