Use of lanatoside C in the preparation of a medicine for resisting porcine epidemic diarrhea virus infection

Abstract

The application discloses use of adenosine in preparation of a medicine for resisting porcine epidemic diarrhea virus (PEDV) infection. Pharmacological tests show that the adenosine can effectively inhibit proliferation of the PEDV in cells and piglets, and can be used as a pharmaceutical ingredient to be distributed with an auxiliary agent to prepare a preparation for preventing and treating diseases caused by the PEDV infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of langoside C in drugs for treating swine epidemic diarrhea virus infection. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the family Coronaviridae, genus Coronavirus. It is an enveloped, single-stranded RNA virus, approximately 28 kb in length. Two genotypes of PEDV are known: Genome I (GI) and Genome II (GII). PEDV was first isolated in Belgium in 1978. Since then, it has been reported worldwide, causing significant damage, particularly in Asian countries with developed livestock industries. PEDV primarily infects the villous epithelial cells of the small intestine, causing disruption of the tight junctions of the villous intestinal cells and a reduction in the amount of mucin. It also leads to the loss of many intestinal cells and a significant reduction in various digestive enzymes, resulting in intestinal malabsorption and indigestion.

[0003] Currently, PED prevention and control measures mainly combine biosafety control with vaccination. Clinically used PED vaccines primarily include attenuated live vaccines and inactivated vaccines. Due to the numerous genotypes of PEDV and its susceptibility to genetic mutations, leading to increased virulence, existing vaccines are insufficient to provide sustained protection. Antiviral drug therapy is considered an effective treatment for PEDV, compensating for the losses caused by ineffective vaccines. To date, there is still a lack of approved specific drugs for effective PED control; therefore, there is an urgent need to continuously explore and develop anti-PEDV drugs.

[0004] Lanatoside C (LC) is a fast-acting cardiac glycoside extracted from digitalis (Digitalis lanata Ehrh.), and is a precursor to deslanoside C and digoxin. Clinically, it is used to treat acute heart failure, exacerbations of chronic heart failure, atrial fibrillation with a rapid ventricular rate, atrial flutter, and paroxysmal supraventricular tachycardia. In addition, lanatoside C has been reported to possess anti-dengue virus, anti-tumor, and antibacterial pharmacological effects. However, no research has been reported on the anti-PEDV infection effects of lanatoside C or its application in the prevention and treatment of PEDV infection, and no related patents have been published. Summary of the Invention

[0005] This invention aims to at least partially address one of the problems in the related art. Therefore, the object of this invention is to provide the use of langoside C in the preparation of drugs for treating porcine epidemic diarrhea virus infection.

[0006] The drug in this application also contains pharmaceutically acceptable excipients; the dosage form of the drug is tablets, capsules, granules, powders, syrups, oral liquids or injections.

[0007] In this application, langoside C exhibits antiviral activity against porcine epidemic diarrhea virus.

[0008] In this application, langoside C can reduce the expression level of N protein in porcine epidemic diarrhea virus, and the concentration of langoside C used to reduce the expression level of N protein in porcine epidemic diarrhea virus is 0.125-0.5 μM.

[0009] In this application, lanolin C can inhibit the transcription of RNA in porcine epidemic diarrhea virus.

[0010] In this application, the concentration of langoside C, which inhibits RNA transcription in porcine epidemic diarrhea virus, is 0.125-0.5 μM.

[0011] In this application, langoside C can inhibit the replication of progeny viruses in porcine epidemic diarrhea virus.

[0012] The concentration of langoside C used in this application to inhibit the replication of progeny viruses in porcine epidemic diarrhea virus is 0.125-0.5 μM.

[0013] In this application, langoside C has an inhibitory effect on the replication of porcine epidemic diarrhea virus in piglets.

[0014] In this application, porcine epidemic diarrhea virus (PEDV) infection causes porcine epidemic diarrhea (PED) disease. In this application, lanolin C is used to prepare a drug for treating porcine epidemic diarrhea virus infection, but is not limited to, the use of an effective amount of the compound of the present invention for the prevention or treatment of diseases caused by porcine epidemic diarrhea virus, the alleviation of symptoms of diseases caused by porcine epidemic diarrhea virus, or the delay of the development of diseases caused by porcine epidemic diarrhea virus.

[0015] The drug described in this application also contains pharmaceutically acceptable excipients. These excipients may be carriers, excipients, diluents, mediators, etc.

[0016] This application has the following beneficial effects: This application discovers a novel use of langoside C in inhibiting porcine epidemic diarrhea virus (PEDV). In Vero cells, the half-maximal effective concentration (EC50) of langoside C against PEDV strains... 50The effective value was 0.11 μM. Simultaneously, lanugin C effectively reduced the viral titer of PEDV in Vero cells, achieving a viral inhibition rate of 99% after 48 hours at the highest administered dose of 0.5 μM. Furthermore, oral administration of 1 mg / kg of lanugin C to newborn piglets significantly inhibited PEDV replication. These results indicate that lanugin C of the present invention has a significant inhibitory effect on porcine epidemic diarrhea virus (PEDV) and possesses clinical application value for the treatment or prevention of PEDV infection. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] In the attached image:

[0020] Figure 1 This is the cytotoxic effect of digoxin C on Vero cells evaluated in Example 1 (CC). 50 ) and its anti-PEDV virus activity on cells (EC 50 ) statistical chart;

[0021] Figure 2 This is an immunofluorescence image from Example 2, showing the effect of different concentrations of digoxin C on reducing the expression level of PEDV virus N protein in cells.

[0022] Figure 3 This is a Western Blot image from Example 3, showing the effect of different concentrations of lanolin C on reducing the expression level of PEDV virus N protein in cells.

[0023] Figure 4 This is a statistical graph of viral mRNA corresponding to the inhibitory effect of langoside C on PEDV replication in piglets, as analyzed by qRT-PCR in Example 6. Detailed Implementation

[0024] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the embodiments of the present invention are commercially available; unless otherwise specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0025] Example 1: Evaluation of the cytotoxic effect of digoxin C on Vero cells (CC) 50 ) and its anti-PEDV virus activity on cells (EC 50 ).

[0026] With a density of 1.5 × 10 5 Vero cells were seeded at a density of 100 μL / mL in 96-well plates. After reaching confluence and forming a monolayer, the cells were washed twice with PBS. The compound was serially diluted twice with DMEM maintenance medium containing 2% FBS. Eight concentration gradients of lanolin C (0.03-4 μM) were established, along with a solvent control group containing 4‰ DMSO and a blank control group (100 μL / well). After culturing at 37°C and 5% CO2 for 48 h, the supernatant was discarded, and 100 μL of 0.5 mg / mL MTT solution was added to each well. The cells were incubated at 37°C in the dark. After 4 h, the incubation was terminated, the supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken at low speed for 10 min to fully dissolve the formazan crystals. The OD value was measured at 570 nm using a full-wavelength microplate reader, and cell viability was calculated. The half-maximal cytotoxicity concentration (CC50) was calculated using a nonlinear regression function in GraphPadPrism 8.0 software. 50 ).

[0027] The calculation formula is as follows:

[0028]

[0029] As above, after Vero cells have grown into a monolayer, wash twice with PBS, and then dilute the virus to 100 TCID using DMEM maintenance medium containing 2% FBS. 50Viral fluid was used to prepare a blank control group of cells. The cells were incubated at 37°C with 5% CO2 for 2 hours. The compound was then serially diluted twice with DMEM maintenance medium containing 2% FBS to create eight concentration gradients of lanolin C (2-256 μM), 100 μL per well. The cells were incubated at 37°C with 5% CO2 for 48 hours before culture was terminated. Cell morphology was observed, and cells were fixed with 4% paraformaldehyde for IFA detection. The cells were observed and photographed using a fluorescence inverted microscope. The fluorescence intensity (blue and red fluorescence) of each well was quantified using ImageJ software. The DMSO-treated control group was set as 100%, and all other groups were compared with the DMSO-treated group. The half-maximal effective concentration (EC50) was determined by the quantified cell protection rate of the drug-treated groups. 50 The values ​​were calculated using a nonlinear regression function with GraphPad Prism 8.0 software.

[0030] The calculation formula is as follows:

[0031]

[0032] The test results are as follows Figure 1 As shown, Figure 1 In the middle (A), the relationship between relative cell viability and lanolin C concentration is represented. Figure 1 (B) represents the relationship between the relative viral expression rate and the concentration of lanolin C. Lanolin C, a drug of the present invention, exhibits antiviral activity against PEDV virus, and its EC50... 50 The value was 0.11 μM. After 48 h of treatment in Vero cells, the cytotoxicity index CC of lanolin C was... 50 The values ​​were all greater than 256 μM, indicating that lanolin C has low toxicity to Vero cells.

[0033] Example 2: Immunofluorescence analysis showed that different concentrations of lanolin C reduced the expression level of PEDV virus N protein in cells.

[0034] Vero cells were seeded into 96-well plates and allowed to grow into a confluent monolayer. The cells were washed twice with PBS, and the virus was diluted to 100 TCID using DMEM maintenance medium containing 2% FBS. 50Viral fluid was prepared and a blank control group was set up. The cells were incubated at 37℃ in a 5% CO2 incubator for 2 hours. Afterwards, the cells were washed twice with PBS to remove unbound viral particles. A blank control group, a solvent control group, and groups containing different concentrations of lanolin C were also prepared, with 100 μL per well. After 48 hours of incubation, the supernatant was discarded, the culture was terminated, and the cells were fixed with 4% paraformaldehyde at room temperature, 150 μL per well. After 15 minutes, the cells were washed three times with PBS, and 50 μL of 0.3% Triton-X100 solution was added to each well, and the cells were incubated at room temperature. After 10 minutes, the cells were washed three times with PBS, and 100 μL of 2% BSA solution was added to each well, and the cells were incubated at 37℃. After 1 hour, the cells were washed three times with PBS, and 50 μL of Anti-PEDV-NAntibody (1:800 dilution) was added to each well, and the cells were incubated overnight at 4℃. The cells were then washed three times with PBS for 5 minutes each time, and 1g HCl (Anti-Mouse) was added to each well in the dark. 50 μL of DAPI (300 nM) solution (1:1000 dilution) was added to each well and incubated at 37 °C. After 1 h, the wells were washed three times with PBS for 5 min each time. 50 μL of DAPI (300 nM) solution was added to each well in the dark and the wells were incubated at room temperature. After 10 min, the wells were washed three times with PBS for 5 min each time. The samples were observed and photographed using an inverted fluorescence microscope.

[0035] The test results are as follows Figure 2 As shown, the drug lanolin C of the present invention significantly reduced the expression level of PEDVN protein in Vero cells within a concentration range of 0.125-0.5 μM, and exhibited a good dose-response relationship.

[0036] Example 3: Western blot analysis showed that different concentrations of lanolin C reduced the expression level of PEDV virus N protein in cells.

[0037] Vero cells were seeded in 6-well plates at 2 mL per well. After the cells grew into a monolayer, the following experiments were performed. The steps for infecting Vero cells with PEDV and adding the drug were the same as in Example 1. After incubation in an incubator for 48 h, the culture was terminated, the supernatant was discarded, and the cells were washed twice with PBS. The cell culture plate was placed on ice, and 120 μL of RIPA lysis buffer was added per well. After repeated pipetting, the liquid was transferred to a centrifuge tube and centrifuged at 13,000 rpm for 15 min. The supernatant was transferred to another clean tube for later use. After determining the protein concentration of each sample using the BCA method, the bands of PEDV protein and the internal reference protein GAPDH were detected by Western blotting.

[0038] The test results are as follows Figure 3 As shown, the drug lanolin C of the present invention significantly reduced the expression level of PEDVN protein in Vero cells within a concentration range of 0.125-0.5 μM, and exhibited a good dose-response relationship.

[0039] Example 4: qRT-PCR analysis of the inhibitory effect of different concentrations of lanolin C on PEDV viral RNA proliferation in cells.

[0040] The steps for infecting Vero cells with PEDV and adding the drug were the same as in Example 1. After incubation in an incubator for 48 hours, the culture was terminated. After observing cell morphology, the cell plate was repeatedly frozen and thawed three times at -80℃ and 4℃ to ensure complete cell lysis, resulting in the release of all the virus into the cell supernatant. The supernatant from each well was then collected. Total RNA was extracted from the collected cell supernatant according to the recommended procedure of the total RNA rapid extraction kit. Reverse transcription was performed immediately after RNA extraction. Using cDNA as a template and β-Actin as an internal reference gene, Real-Time PCR was used to detect the copy number of the PEDV gene. The changes in PEDV-N mRNA were evaluated using the virus control group as a reference.

[0041] PEDV-N gene upstream and downstream primer sequences:

[0042] PEDV-NF: 5'-CGCAAAGACTGAACCCACTAATTT-3'

[0043] PEDV-NR: 5'-TTGCCTCTGTTGTTACTTGGAGAT-3'

[0044] β-Actin gene upstream and downstream primer sequences:

[0045] β-Actin-F: 5'-GGACTTCGAGCAGGAGATGG-3'

[0046] β-Actin-R: 5'-AGGAAGGAGGGCTGGAAGAG-3'

[0047] The drug lanoside C of this invention significantly inhibits PEDV RNA transcription in Vero cells within a concentration range of 0.125-0.5 μM, exhibiting a favorable dose-response relationship. At the highest administered dose of 0.5 μM, the viral inhibition rate reached 99.92% after 48 hours of administration.

[0048] Example 5: Endpoint dilution method was used to analyze the inhibitory effect of different concentrations of lanolin C on PEDV progeny viruses in cells.

[0049] Vero cells were seeded into 12-well plates and allowed to grow into a confluent monolayer. The cells were washed twice with PBS. A blank control group, a solvent control group, and groups containing different concentrations of LC drug were set up. Except for the blank control group, which received maintenance medium containing 2% FBS, all other wells contained 100 TCID50 diluted with maintenance medium. 50100 μL of PEDV virus solution was collected per well and incubated in a 37°C, 5% CO2 incubator. After 2 hours, the cells were washed twice with PBS to remove unbound virus particles. For the drug-treated groups, the corresponding compound was added at a 2-fold serial dilution in DMEM maintenance medium with 2% FBS. For the blank control and solvent control groups, 1 mL of fresh DMEM maintenance medium was added to each well, and the cells were incubated for further growth. After 48 hours, the culture was terminated, and the cell plate was subjected to three freeze-thaw cycles at -80°C and 4°C to ensure complete release of the virus into the supernatant. 100 μL of the freeze-thawed solution was collected per tube, and 3-4 replicates were collected for subsequent experiments.

[0050] With a density of 1.5 × 10 5 / mLVero cells were seeded in 96-well plates at 100 μL per well. After the monolayer had grown to confluence, the cells were washed twice with PBS. The original sample solution was then serially diluted 10-fold with DMEM maintenance medium, and 100 μL of the diluted solution was added to each well of the cell plate, with 4 replicates. After incubation for 48 h, the viral titer of different samples was detected using an IFA assay, and the results were recorded and analyzed.

[0051] The romaine C of the present invention has a significant inhibitory effect on the replication of PEDV progeny virus in Vero cells in the concentration range of 0.125-0.5 μM, and exhibits a good dose-response relationship.

[0052] Example 6: qRT-PCR analysis of the inhibitory effect of langoside C on PEDV replication in piglets.

[0053] Nine 3-day-old piglets were randomly divided into three groups: a PEDV challenge group, a digoxin C treatment group, and a negative control group. Each piglet was housed separately and allowed to acclimatize for one day before the experiment. The PEDV challenge group and the digoxin C treatment group were orally challenged, while the negative control group was orally administered PBS. When piglets developed diarrhea, they were treated with 1 mg / kg digoxin C, administered orally twice daily for 4 consecutive days. After challenge, fecal samples were collected from piglets in the experimental groups at fixed times daily and stored at -80℃. 0.1 g of fecal sample was weighed, mixed thoroughly with 1 mL of PBS, centrifuged (12000 rpm, 10 min, 4℃), and the fecal supernatant was collected. Viral mRNA levels were detected using qRT PCR.

[0054] The test results are as follows Figure 4 As shown, the vertical axis represents the logarithm of the initial RNA copy number in PEDV, i.e., PEDV RNA (log 10The graph shows the number of copies / reaction, with the vertical axis representing the number of days post-infection. The PBS group represents the negative control group, the DMSO group represents the virus-infected control group, and the LC group represents the langoside C treatment group. In the figure, compared with the virus-infected control group, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001, indicating significant difference. The experimental results are as follows... Figure 4 As shown, compared with the DMSO group, oral administration of digoxin C significantly reduced the viral load of PEDV in the feces of infected piglets, suggesting that digoxin C has an inhibitory effect on PEDV replication in piglets.

[0055] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. Application of langoside C in the preparation of drugs against porcine epidemic diarrhea virus infection.

2. The application of langoside C according to claim 1 in the preparation of a drug for treating porcine epidemic diarrhea virus infection, characterized in that, The lanolin C has antiviral activity against porcine epidemic diarrhea virus.

3. The application of langoside C according to claim 1 in the preparation of a drug for treating porcine epidemic diarrhea virus infection, characterized in that, The lanolin C can inhibit the replication of progeny viruses in porcine epidemic diarrhea virus.

4. The application of langoside C according to claim 3 in the preparation of a drug for treating porcine epidemic diarrhea virus infection, characterized in that, Alphazoline C at concentrations of 0.125–0.5 μM can inhibit the replication of progeny viruses in porcine epidemic diarrhea virus.

5. The application of langoside C according to claim 1 in the preparation of a drug for treating porcine epidemic diarrhea virus infection, characterized in that, The lanolin C has an inhibitory effect on the replication of porcine epidemic diarrhea virus in piglets.

6. The application of langoside C according to claim 1 in the preparation of a drug for treating porcine epidemic diarrhea virus infection, characterized in that, The drug also contains pharmaceutically acceptable excipients; the dosage form of the drug is tablets, capsules, granules, powders, syrups, oral liquids, or injections.

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