Application of cilnidipine in preparation of anti-transmissible gastroenteritis virus product

Abstract

The invention discloses application of cilnidipine in preparation of a product for resisting swine transmissible gastroenteritis virus, and belongs to the technical field of cytobiology and virology. According to the application disclosed by the invention, a potential anti-TGEV candidate drug, namely cilnidipine is screened from 2320 drug libraries of Discovery Probe TMFDA-applied Drug Library through high-throughput screening, the drug toxicity of the cilnidipine can be neglected, the cilnidipine shows remarkable antiviral activity on proliferation of TGEV in ST cells in vitro and presents dose dependence, and the inhibition effect can almost reach 100% when the TGEV is 30 [mu] M. The invention proves that cilnidipine has anti-TGEV virus activity for the first time, provides a new idea for the research of anti-TGEV drugs, and also provides a new perspective for the selection of TGEV antiviral drug targets.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology and virology, and in particular relates to the application of cilnidipine in the preparation of a product for resisting porcine transmissible gastroenteritis virus. Background Art

[0002] Porcine transmissible gastroenteritis virus (TGEV) is an RNA virus belonging to the Coronaviridae family and the genus Alphacoronavirus. Characterized by severe diarrhea and vomiting, as well as severe electrolyte imbalance and extreme dehydration in infected pigs, porcine transmissible gastroenteritis (TGE) is internationally considered a highly contagious swine disease, which results in a mortality rate of nearly 100% in newborn piglets. The disease was first reported in the United States by Doyle and Hutchings in 1946, and has since been reported in many countries and regions around the world (Japan, Britain, France, the Netherlands, Germany, Italy, the Soviet Union, etc.).

[0003] Antiviral drugs play an important role in the prevention and control of diseases. The research and development of antiviral drugs can not only reduce medical costs and improve treatment efficiency, but also effectively prevent infection with pathogens. Natural compounds and ingredients provide a valuable source of antiviral drugs, many of which show in vitro anti-TGEV activity, such as phloretin inhibits TGEV by reducing the expression level of proinflammatory cytokines induced by TGEV infection, curcumin has a direct inactivation effect on TGEV, mainly interfering with the adsorption stage in the TGEV life cycle, (+)-Catechin has an inhibitory effect on TGEV proliferation in vitro and participates in its antioxidant effect, eugenol protects the intestine by inhibiting NLRP3 inflammasome activation to reduce TGEV-induced pyroptosis, and bovine antimicrobial peptides show good antiviral effects on TGEV both in vitro and in vivo.

[0004] Cilnidipine molecular formula: C 27 H 28 N2O7, cilnidipine is a calcium channel blocker that can not only bind to the dihydropyridine site of the L-type calcium channel on vascular smooth muscle cells, but also inhibit Ca 2+ Through the transmembrane influx of L-type calcium channels, it relaxes and dilates vascular smooth muscle. It can also inhibit Ca 2+ Through the transmembrane influx of N-type calcium channels on the sympathetic nerve cell membrane, the release of norepinephrine from sympathetic nerve endings is reduced and the activity of sympathetic nerves is reduced.

[0005] The first step in drug development is to find drug targets. Traditional drug development strategies usually find targets by screening cellular derivatives of drug molecules. The advantage of this method is high accuracy, but it is very time-consuming and costly. In contrast, network pharmacology can use known drug targets and protein gene sequence information to establish a drug-protein-gene interaction network to predict and analyze the mechanism of drug action. Compared with traditional methods, network pharmacology can discover potential drug targets faster and more accurately.

[0006] After searching, no research has found the use of cilnidipine in the fight against porcine transmissible gastroenteritis virus. Summary of the invention

[0007] In the present invention, we combined network pharmacology with molecular docking technology to identify potential targets of cilnidipine against porcine transmissible gastroenteritis virus. Then, we verified our predictions experimentally. The candidate drugs discovered in the present invention provide a potential therapeutic approach for porcine transmissible gastroenteritis virus infection.

[0008] The invention provides application of cilnidipine in preparing a product for resisting porcine transmissible gastroenteritis virus. The CAS registration number of the cilnidipine is 132203-70-4.

[0009] Preferably, cilnidipine is the only active ingredient in the product.

[0010] Preferably, the product is a medicine.

[0011] More preferably, the medicine further contains medically acceptable excipients.

[0012] More preferably, the dosage form of the drug is capsule, tablet, granule, powder, solution or lozenge.

[0013] More preferably, the use is to inhibit the proliferation of porcine transmissible gastroenteritis virus in cells.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention proves for the first time that cilnidipine has a strong inhibitory effect on porcine transmissible gastroenteritis virus in ST cells, and acts in the early stage of the virus life cycle, laying the foundation for the development of new drugs against porcine transmissible gastroenteritis virus. At the same time, it is confirmed that the target protein PPARG is closely related to the anti-TGEV effect of cilnidipine. The present invention provides a new use of cilnidipine, provides a new candidate drug for the development of TGEV antiviral drugs, and provides a new research idea for the target of the interaction between antiviral drugs and TGEV. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figures A to C are schematic diagrams of high-throughput screening of the FDA drug library provided in Example 1, wherein A is a high-throughput screening analysis schedule, B is a scatter plot of the inhibitory effect of each drug, and each point represents the inhibition percentage of each drug from the drug library on TGEV (MOI = 0.05); C is an analysis flow chart for high-throughput screening. D and E are the 50% cytotoxic concentration (CC) of cilnidipine provided in Example 2, respectively. 50 ) and 50% inhibitory concentration (IC 50 )picture.

[0017] Figure 2 Schematic diagram of the anti-TGEV activity of cilnidipine in ST cells provided in Examples 3-6. AD shows ST cells infected with TGEV in the presence of different concentrations of cilnidipine (MOCK, DMSO, 15 μM, 20 μM, 25 μM and 30 μM). 24 h after infection, samples were prepared for quantitative real-time PCR (A), TCID 50 Assay (B), Western blot detection (C) and IFA (D).

[0018] Figure 3 The direct inactivation effect of cilnidipine on TGEV and the antiviral effect in the early stage of viral infection provided in Example 7. A is a virus killing test, and DMSO is used as a control group without drug. BD is the action time of cilnidipine on ST cells infected with TGEV. ST cells were infected with TGEV, and 30μM cilnidipine was introduced into the infected cells at different time intervals related to TGEV infection: -1h-24h, 1h-24h, 3h-24h, 6h-24h and 12h-24h (B). 24h after infection, samples were collected and TCID 50 The results were analyzed by quantitative real-time PCR (C) and quantitative real-time PCR (D). The control group was a group without cilnidipine.

[0019] Figure 4 Schematic diagram of the anti-TGEV target of cilnidipine provided in Example 8.

[0020] Figure 5 PPI network diagram of the anti-TGEV target provided in Example 9. A is a protein interaction network diagram of 25 potential targets. B is the PPI core network (MCC, Degree, BottleNeck from left to right, take TOP10). C is a Venn diagram of crossover genes and core genes, in which the crossover part represents the crossover gene. D is the core target pathway network of cilnidipine; blue, red and green represent cilnidipine, core targets and signaling pathways, respectively.

[0021] Figure 6 Schematic diagram of GO enrichment analysis and KEGG pathway analysis provided in Example 10. A is GO enrichment analysis, and B is KEGG signaling pathway analysis. The results of GO enrichment analysis and KEGG signaling pathway analysis are arranged in ascending order by p-value, and the top 15 objects are selected. All p-values ​​are less than 0.01.

[0022] Figure 7 Schematic diagram of the interaction between cilnidipine and target protein provided in Example 11. Molecular docking was completed by Autodock Tools (A); B is a summary of free binding energy.

[0023] Figure 8 The effects of low, medium and high doses of cilnidipine provided in Example 12 on PPARG mRNA expression (AC) and protein (DF) levels in ST cells. DETAILED DESCRIPTION

[0024] The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0025] Experimental Materials

[0026] ST cells were routinely proliferated in Dulbecco's modified Eagle's medium (Situofan) containing 10% fetal bovine serum (Beijing Quanshijin Biotechnology Co., Ltd.), and the culture environment was maintained at 37°C and 5% CO2 concentration.

[0027] The TGEV strains used were maintained in our laboratory and propagated in ST cells. To quantify the viral load, the 50% tissue culture infectious dose (TCID 50 ).

[0028] Cilnidipine was purchased from Selleck Chemicals (USA). MTT reagent (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was used to evaluate cell viability. Trizol reagent (purchased from Takara) and Hiscript II FirstStrand cDNA reverse transcription kit (purchased from Novazon Biotech) were used for quantitative real-time PCR experiments. Alexa Fluor 594-conjugated goat anti-mouse antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) was used for IFA experiments. GAPDH polyclonal antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) and TGEV-N protein monoclonal antibody (purchased from Guangzhou Qianxun Biotechnology Co., Ltd.) were used for Western blot analysis.

[0029] Example 1 High-throughput screening of FDA drug library

[0030] An FDA-approved drug library containing 2320 compounds was purchased from APExBIO and stored in DMSO at a concentration of 10 mM and stored at -80 °C until use. ST cells were seeded into 96-well plates and placed in a CO2 incubator for 12 h. Figure 1 A. After the cells grow to a monolayer, treat with 0.2 μL of drug for 1 hour, then add virus infection for 1 hour. After 1 hour, discard the virus solution and replace it with drug diluent. Set up DMSO control wells and cell control wells. After 24 hours, observe the cell lesions, then add 100 μL of MTT diluted with cell maintenance solution to each well, place the cell culture plate in a 37°C incubator and incubate for 4 hours, discard MTT and add DMSO, place on a shaker and shake at low speed for 10 minutes to fully dissolve the crystals, and then use a multifunctional microplate reader to read the OD value at 490nm.

[0031] like Figure 1 As shown in B, after screening 2320 drugs, it was found that 226 compounds (9.74%) had an inhibitory effect on TGEV compared with the DMSO control group, and the inhibition rate could reach more than 50%. Then a second round of screening was conducted on the 226 compounds, of which 17 compounds (0.73%) had an inhibition rate of more than 70% on TGEV. A final round of screening was conducted on 17 compounds, of which 11 compounds (0.47%) had the strongest cell viability, and the inhibition rate on TGEV could reach more than 90%. In the end, we selected cilnidipine as a candidate antiviral drug ( Figure 1 C), because it has extremely low toxicity to ST cells, which is conducive to the safe use of drugs, and has relatively low cost, which meets market demand and is conducive to future large-scale production and application.

[0032] Example 2 Cytotoxicity test of cilnidipine on ST cells and IC 50 Determination

[0033] ST cells in the logarithmic growth phase were inoculated into 96-well plates, and 100 μL of cell suspension containing 10% DMEM FBS culture medium was added to each well, and the peripheral edge holes were filled with sterile PBS. The 96-well plates were placed in a 37°C, 5% CO2 incubator and cultured overnight. When the cells adhered to the wall and grew to 80% to 90%, the culture medium was discarded and washed with PBS. 100 μL of cilnidipine diluted to different concentrations (0mM, 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM) with 2% DMEM FBS culture medium was added to each well, and 4 replicates were set for each concentration, and a blank control group without cells and drugs and a negative control group without drugs were set at the same time. The 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours. 90 μL of fresh serum-free DMEM medium and 10 μL of MTT solution were added to each well. The culture was continued for 4 hours. The culture medium in the well was aspirated, and 100 μL of DMSO was added to each well. The well was placed on a shaker and shaken at a low speed for 10 minutes to fully dissolve the crystals. Finally, the absorbance of each well at 490 nm was measured using an ELISA reader, and the average absorbance of four replicate wells at each concentration was calculated to determine the percentage of cell viability. In addition, in order to study the antiviral potential, cells were treated with 0 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM and 100 μM of cilnidipine 1 hour after TGEV infection. The IC of cilnidipine on ST cells was determined according to the above experimental protocol. 50 value.

[0034] like Figure 1 As shown in D, compared with the negative control group, it was found that at 25 mM, the cell viability can basically reach more than 80%, with no obvious toxicity, and can be used in subsequent examples. 50 The results showed that at 25 μM, the inhibitory effect could reach more than 80% ( Figure 1 E).

[0035] Example 3 Quantitative real-time PCR detection of the effect of cilnidipine on TGEV RNA replication

[0036] Quantitative real-time PCR was used to measure the TGEV RNA content to further evaluate the inhibitory effect of cilnidipine on the RNA content of porcine transmissible gastroenteritis virus. The specific method is as follows:

[0037] ST cells were plated on a 12-well cell culture plate, 500 μL of 10% DMEM FBS medium was added to each well, and cultured in a 37°C, 5% CO2 incubator for 24 h; new DMEM medium was replaced, and ST cells were incubated at 37°C for 1 h with TGEV at an MOI of 0.05; the culture supernatant was discarded, and the cells were washed three times with PBS buffer to wash away the virus particles that did not invade the cells; 15 μM, 20 μM, 25 μM, and 30 μM of cilnidipine were added to the wells of the cell culture plate, and a DMSO control group (0 μM of cilnidipine) was set up at the same time, and cultured at 37°C for 24 h. The cells were collected, the cell supernatant was discarded, and the cells were washed three times with PBS; the total RNA of TGEV-infected cells was extracted using the Trizol method, and quantitative real-time PCR was performed after reverse transcription.

[0038] Primers and reaction systems are shown in Tables 1 and 2.

[0039] Table 1 Primer sequences for fluorescence quantitative PCR

[0040]

[0041] Table 2 Fluorescence quantitative PCR reaction system

[0042]

[0043] The results are as follows Figure 2 As shown in A, when ST cells were infected with TGEV, compared with the DMSO control group (0 μM of cilnidipine), the TGEV mRNA content in the treatment group decreased in a dose-dependent manner with the increase of cilnidipine concentration, indicating that cilnidipine can significantly reduce the content of swine transmissible gastroenteritis virus mRNA in ST cells infected with swine transmissible gastroenteritis virus.

[0044] Example 4 Determination of cell supernatant virus titer

[0045] ST cells were inoculated into 96-well plates and cultured overnight at 37°C and 5% CO2 until the cells were fully grown; the frozen drug-treated cell supernatant was taken out and diluted in gradients and added to each well at 100 μL. Six replicate wells were set for each concentration, and a negative control group with only 2% DMEM medium was set up. The plates were cultured at 37°C and 5% CO2 until the fourth day, and the cytopathic effect was observed, the cytopathic effect wells were recorded, and the TCID 50 .

[0046] like Figure 2 B shows TCID 50 The measurement results showed that as the concentration of cilnidipine increased, the viral titer of TGEV continued to decrease.

[0047] Example 5 Western blot detection of the inhibitory effect of cilnidipine on the expression of porcine transmissible gastroenteritis virus N protein

[0048] To further determine the anti-TGEV activity of cilnidipine, the Western blot method was used to detect the inhibitory effect of cilnidipine on the expression of N protein, the main structural protein of TGEV. The specific method is as follows:

[0049] ST cells were plated on a 12-well cell culture plate, 500 μL of 10% DMEM FBS culture medium was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 h; new DMEM culture medium was replaced, and ST cells were incubated with TGEV at an MOI of 0.05 at 37°C for 1 h; the culture supernatant was discarded, and the cells were washed three times with PBS buffer to wash away the virus particles that did not invade the cells; 15 μM, 20 μM, 25 μM, and 30 μM of cilnidipine were added to the wells of the cell culture plate, and a DMSO control group (0 μM of cilnidipine) was set up at the same time, and the cells were cultured at 37°C for 24 h. The cells were washed three times with PBS buffer, 100 μL of RIPA was added to each well to lyse the cells, and the mixture was centrifuged at 12000 r / min for 3 min. The supernatant was transferred to a new 1.5 mL sterile centrifuge tube to prepare the samples of the DMSO control group and the cilnidipine treatment group. Then, the Western blot method was used, using TGEVN protein antibody and GAPDH antibody as primary antibodies, HRP-labeled goat anti-mouse antibody and HRP-labeled goat anti-rabbit as secondary antibodies to detect the expression of TGEVN protein and the intracellular reference protein GAPDH.

[0050] The results are as follows Figure 2 As shown in C, when ST cells were infected with TGEV, GAPDH was used as the cell internal reference. Compared with the DMSO control group (0 μM of cilnidipine), the expression level of TGEVN protein decreased significantly with the increase of cilnidipine concentration, indicating that cilnidipine can significantly inhibit the expression of TGEVN protein.

[0051] Example 6 IFA detection of anti-TGEV activity of cilnidipine

[0052] ST cells were plated on a 12-well cell culture plate, 500 μL of 10% DMEM FBS medium was added to each well, and cultured in a 37°C, 5% CO2 incubator for 24 hours; new DMEM medium was replaced, and ST cells were incubated at 37°C for 1 hour with TGEV at a virus infection multiplicity (MOI) of 0.05; the culture supernatant was discarded, and the cells were washed three times with PBS buffer to wash away the virus particles that did not invade the cells; 15 μM, 20 μM, 25 μM, and 30 μM of cilnidipine were added to the wells of the cell culture plate, and a DMSO control group (0 μM of cilnidipine) was set up at the same time, and cultured at 37°C for 24 hours. Then the culture medium was discarded, rinsed three times with PBS, 250 μL of 4% paraformaldehyde was added to each well, and placed at room temperature for about 20 minutes, and then fixed with 5% skim milk powder. Rinse with PBS three times, add monoclonal antibody to TGEVN protein and incubate at 37°C for 1 hour, rinse with PBS three times, and add Alexa The cells were incubated with 594 fluorescently labeled goat anti-mouse antibody at 37°C for 1 h, rinsed with PBS three times, stained with DAPI at room temperature for 10 min, and observed under a fluorescence microscope. The red fluorescence intensity indicates the proliferation of TGEV.

[0053] The results are as follows Figure 2 As shown in D, compared with the DMSO control group (0 μM of cilnidipine), as the concentration of cilnidipine increased, TGEV-specific immunofluorescence gradually decreased in infected ST cells, which means that cilnidipine has good anti-transmissible gastroenteritis virus activity under this concentration condition.

[0054] Example 7 Observation of the effect of adding drugs at different time points on virus proliferation

[0055] To understand the mechanism by which cilnidipine inhibits TGEV infection, we investigated whether cilnidipine has a direct virucidal effect on TGEV. After cilnidipine was mixed with TGEV, they were incubated at 37 °C for 1 h, and then the TCID 50 Determine the virus titer. Figure 3 As shown in A, our results showed that cilnidipine could directly inactivate TGEV, which was significantly different from the DMSO control group.

[0056] Then, to explore the antiviral effect of cilnidipine, we used two different drug treatment regimens. Figure 3B. First, ST cells were seeded into 12-well plates in the treatment regimen before virus infection of cells. Once the cells reached 80%–90%, the original culture medium was discarded, the cells were gently rinsed with PBS, and then 500 μL of 30 μM cilnidipine was added. Positive and negative control groups were set up at the same time, and each treatment group included three replicate wells. After that, the 12-well plates were incubated in a 37°C, 5% CO2 incubator for 1 h. Subsequently, ST cells were infected with TGEV at an MOI of 0.05 and further incubated under the same conditions for 1 h. After infection, the cells were washed twice with PBS to remove unbound viral particles. Next, 500 μL of DMEM medium containing 2% FBS and double antibodies was added to each well, and the cells were incubated again in a 37°C, 5% CO2 incubator for 24 h.

[0057] Secondly, in the treatment protocol after virus infection of cells, once ST cells in the 12-well plate reached 80%–90%, TGEV virus solution with an MOI of 0.05 was added, and then incubated in a 37°C, 5% CO2 incubator for 1 h, and then cilnidipine at a concentration of 30 μM was added at different time points (1h-24h, 3h-24h, 6h-24h, 12h-24h). After drug treatment, the cells were further incubated in a 37°C, 5% CO2 incubator for 24 h.

[0058] Finally, RNA was extracted from the cells and the viral supernatant was collected to analyze the effects of cilnidipine on TGEV mRNA expression and viral titer at different time points.

[0059] The results are as follows Figure 3 As shown in C, cilnidipine significantly reduced the TGEV virus titer 1 h after TGEV infection. Figure 3 D shows the quantitative real-time PCR results and TCID 50 The results were consistent, indicating that TGEV NmRNA expression was significantly downregulated 1 h after infection. This downregulation emphasized the reduction in viral nucleic acid replication. Based on these combined findings, it was suggested that cilnidipine exerted an effective inhibitory effect in the early stages of TGEV infection, possibly by interfering with the process of viral adsorption and invasion of host cells.

[0060] Example 8 Target of Cilnidipine against TGEV

[0061] The SMILES chemical structure of cilnidipine was searched in the PubChem database. Based on this SMILES structure, a total of 117 genes were obtained after merging the drug target gene data and removing duplicates in the SuperPRED, Genecard, and CTD databases. 622 disease targets related to TGE were collected by comparing CTD. Finally, the intersection of the above drug targets and disease targets was taken by VennDiagram, and a total of 25 potential targets related to the anti-TGEV effect of cilnidipine were obtained ( Figure 4 ).

[0062] Example 9 PPI network of target

[0063] Enter 25 potential targets into the String database, ( Figure 5 A) Predict their interactions and their interacting proteins, and obtain a total of 93 pairs of interactions. For the obtained interaction data, the PPI network was constructed in the cytoscape software, and the core node subnetwork in the PPI network was calculated using the cytoHubba plug-in in cytoscape. The core genes identified by the MCC, Degree and BottleNeck algorithms were intersected with the 25 intersection genes, and finally 6 hub-genes were identified: ESR1, BCL2, PPARG, TGFB1, REN, FGF2 ( Figure 5 B, 5C). Then, a drug-gene-pathway network diagram was constructed based on the six key genes ( Figure 5 D).

[0064] Example 10GO and KEGG analysis

[0065] The 25 potential targets were input into ClusterProfiler, and a total of 1085 GO results were enriched. The enrichment results were sorted according to the p-value, and the top 5 results were selected for plotting ( Figure 6A). The biological processes mainly involve "cellular response to external stimulus" and "response to peptide hormone". For cellular components, proteins related to "cytoplasmic vesicle lumen" and "secretory granule lumen" were enriched. In terms of molecular function, the target proteins were mainly closely related to "RNA polymerase ll-specific DNA-bindingtranscription factor binding" and "DNA-binding transcription factor binding". Through KEGG pathway analysis, a total of 71 pathways were obtained. After sorting according to the p value, the top 15 signal pathways were selected for mapping ( Figure 6 B).

[0066] Example 11 Molecular docking

[0067] Molecular docking was performed using AutoDockTools-4.2.6 software, which can explain the interaction and binding mode between the target protein and cilnidipine at the molecular level. Cilnidipine was connected to the active pocket of the protein, and its theoretical binding mode was displayed using PyMOL software ( Figure 7 A). The binding free energy of cilnidipine to PPARG is -8.6 kcal / mol ( Figure 7 B). The results showed that cilnidipine tightly binds to PPARG.

[0068] Example 12 Determination of PPARG and TGEVN mRNA / protein expression

[0069] like Figure 8 As shown in A, B, and C, after virus infection, the expression of PPARG mRNA levels in the low-dose (25 μM), medium-dose (50 μM), and high-dose (100 μM) cilnidipine-treated groups was reduced compared with the DMSO group. As the culture time of cilnidipine and TGEV increased, the mRNA expression of PPARG in the virus control group (DMSO) gradually increased compared with the cell control group (MOCK). However, compared with the DMSO group, the mRNA expression of PPARG in the low-dose, medium-dose, and high-dose groups was significantly reduced.

[0070] At 12h, 24h and 36h of treatment, the protein expression of PPARG in the low-dose group, medium-dose group and high-dose group was significantly lower than that in the DMSO group. Figure 8DF. These results indicate that TGEV infection of ST cells can promote the expression of PPARG. After cilnidipine treatment, the expression of PPARG protein was decreased, and the results confirmed the correlation between PPARG and the anti-TGEV effect of cilnidipine on ST cells.

[0071] In summary, the present invention proves for the first time that cilnidipine has anti-TGEV viral activity and acts in the early stage of the virus, providing a valuable reference for the development of anti-TGEV drugs. Then, network pharmacology was used to discover the target protein PPARG for cilnidipine and TGEV, providing a new perspective for the target of the interaction between antiviral drugs and TGEV, and emphasizing the potential of network pharmacology in accelerating and optimizing drug target discovery.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of cilnidipine in the preparation of anti-porcine transmissible gastroenteritis virus products, characterized in that: The CAS registration number of the cilnidipine is 132203-70-4.

2. The use according to claim 1, characterized in that: Cilnidipine is the only active ingredient in the product.

3. The use according to claim 1, characterized in that: The product described is a drug.

4. The use according to claim 3, characterized in that: The medicine also contains medically acceptable auxiliary materials.

5. The use according to claim 4, characterized in that: The dosage form of the drug is capsule, tablet, granule, powder, solution or lozenge.

6. The use according to any one of claims 1 to 5, characterized in that: The application is to inhibit the proliferation of porcine transmissible gastroenteritis virus in cells.

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