Application of misoprostol as active ingredient in preparation of products for resisting porcine epidemic diarrhea virus

Abstract

The invention discloses application of misoprostol as an active ingredient in preparation of a product for resisting porcine epidemic diarrhea virus, and belongs to the technical field of biology. The invention finds that the misoprostol has an inhibition effect on the porcine epidemic diarrhea virus for the first time, and further, cell experiments prove that the misoprostol can inhibit early infection of the porcine epidemic diarrhea virus in vitro in a dose-dependent manner, and has a very good safe concentration in Vero cells; a molecular docking model verifies that the misoprostol is combined with the integrin beta 1 protein to inhibit the infection of the porcine epidemic diarrhea virus, and the action method can be popularized to other viruses needing the integrin beta 1 protein mediated infection. The invention provides a theoretical basis for the development of the misoprostol as an antiviral drug.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to the use of misoprostol as an active ingredient in the preparation of products for resisting porcine epidemic diarrhea virus. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a re-emerging and newly emerged highly contagious enteropathogenic virus belonging to the genus Alphacoronavirus of the family Coronaviridae. It was discovered in the UK in 1971 and has since spread rapidly around the world. PEDV is highly contagious and can cause intestinal diseases characterized by diarrhea, vomiting and dehydration in pigs of all ages (especially suckling piglets), with a mortality rate of up to 100%. PEDV is a single-stranded, positive-stranded, enveloped RNA virus with a genome of approximately 28 kb, encoding 16 different nonstructural proteins and 4 structural proteins. Among them, the S protein is the most variable part of the PEDV genome. Based on the phylogenetic analysis of the S gene, PEDV is divided into G1 (G1a, G1b and G1c) and G2 (G2a, G2b, G2c and G2d) types. At present, due to the existence of various degrees of recombination in the PEDV genome, PEDV has caused the death of millions of suckling piglets and worsened the situation of the entire global pig industry, causing huge economic losses.

[0003] There is no effective vaccine or antiviral drug for the epidemic caused by PEDV. The development of effective and safe antiviral drugs for PEDV is of great significance to reduce the large-scale epidemic caused by PEDV. Misoprostol is a synthetic prostaglandin E1 analog that has the functions of enhancing gastric mucosal defense and promoting gastric mucosal repair. At present, there are few studies on the antiviral effects of misoprostol, and no studies have reported the role of misoprostol in porcine epidemic diarrhea virus infection. Summary of the invention

[0004] The purpose of the present invention is to provide the use of misoprostol as an active ingredient in the preparation of a product against porcine epidemic diarrhea virus, so as to solve the problems existing in the above-mentioned prior art. The present invention confirms through cell experiments that misoprostol can inhibit the early infection of porcine epidemic diarrhea virus in vitro in a dose-dependent manner, providing a theoretical basis for the development of misoprostol as an antiviral drug.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides use of misoprostol as an active ingredient in preparing a product for resisting porcine epidemic diarrhea virus.

[0007] Optionally, the product comprises a medicine.

[0008] Optionally, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0009] Optionally, the dosage form of the drug includes tablets, granules, mixtures and capsules.

[0010] Optionally, the medicament inhibits infection by porcine epidemic diarrhea virus.

[0011] Optionally, the infection lasts for 1-12 hours with the porcine epidemic diarrhea virus, with the drug inhibitory effect being most obvious 1-7 hours after infection.

[0012] The present invention also provides the use of misoprostol as an active ingredient in the preparation of an inhibitor targeting integrin β1 protein.

[0013] Optionally, the misoprostol inhibits the activity of the integrin β1 protein by specifically binding to the integrin β1 protein.

[0014] Optionally, the inhibitor further includes a pharmaceutically acceptable carrier or excipient.

[0015] Optionally, the dosage form of the inhibitor includes tablets, granules, mixtures and capsules.

[0016] The present invention discloses the following technical effects:

[0017] The present invention discovers for the first time that misoprostol has an inhibitory effect on porcine epidemic diarrhea virus, and further confirms through cell experiments that misoprostol can inhibit the early infection of porcine epidemic diarrhea virus in vitro in a dose-dependent manner and has a good safety concentration in Vero cells; the molecular docking model verifies that misoprostol inhibits the infection of porcine epidemic diarrhea virus by binding to integrin β1 protein, and this method of action can be generalized to other viruses that require integrin β1 protein to mediate infection.

[0018] The invention provides a theoretical basis for the development of misoprostol as an antiviral drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a diagram for the safety validation of misoprostol in Vero cells;

[0021] Figure 2The figure shows that misoprostol has a significant inhibitory effect on PEDV; A is the detection result of mRNA expression level of different groups of viruses; B is the detection result of virus titer in different groups;

[0022] Figure 3 This is a graph showing the inhibitory effect of different concentrations of misoprostol on PEDV;

[0023] Figure 4 The mRNA (A) and TCID of misoprostol against PEDV at different stages 50 (B) Graph of the inhibitory effect;

[0024] Figure 5 Figure 2 is a graph showing the killing effect of misoprostol on PEDV; A is the detection result of mRNA expression level of viruses in different groups; B is the detection result of virus titer in different groups;

[0025] Figure 6 This is a molecular docking diagram of misoprostol directly binding to integrin β1 in cells. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The present invention includes the use of misoprostol in resisting porcine epidemic diarrhea virus, specifically as follows (A1) or (A2):

[0032] (A1) Use in the preparation of products against porcine epidemic diarrhea virus;

[0033] (A2) Application in combating porcine epidemic diarrhea virus.

[0034] In a specific embodiment, the present invention uses misoprostol as the only active ingredient for preparing a product against porcine epidemic diarrhea virus.

[0035] Optionally, the product is a medicine.

[0036] Optionally, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0037] Optionally, the pharmaceutically acceptable carrier is selected from one or more of a filler, a wetting agent, a binder, a disintegrant, a lubricant and the like.

[0038] Optionally, the drug can be further prepared into various forms such as tablets, granules, oral liquids, capsules or injections according to conventional methods in the pharmaceutical field.

[0039] Optionally, the drug also includes pharmaceutically acceptable anti-swine epidemic diarrhea virus drugs or other related active substances.

[0040] In a specific implementation case, the safe concentration range of misoprostol in Vero cells is 0-100 μM, but is not limited to 100 μM.

[0041] In a specific embodiment, misoprostol can directly and stably target integrin β1 protein and inhibit porcine epidemic diarrhea virus infection in a dose-dependent manner, and the effect is most obvious when administered within 1-7 hours after the virus infects cells.

[0042] In specific embodiments, misoprostol inhibits the early stages of viral replication.

[0043] The present invention also includes the use of misoprostol in targeting integrin β1 protein and inhibiting its activity.

[0044] In a specific embodiment, misoprostol is able to bind tightly to the integrin β1 protein, inhibiting its activity as a viral receptor.

[0045] Alternatively, misoprostol can inhibit viral infection mediated by integrin β1 protein by the above-mentioned binding method with integrin β1 protein.

[0046] Unless otherwise specified, the materials used in the embodiments of the present invention can be obtained through conventional purchase.

[0047] Example

[0048] 1. Determination of safe concentration of misoprostol for cells

[0049] Solution preparation: Dissolve misoprostol powder in DMSO solution, sterilize through a 0.22 μm sterile filter, prepare the original solution, and store it at -80°C for future use.

[0050] Vero cells were inoculated in a 96-well plate and grown to a monolayer. The cell culture medium was removed and the cells were washed three times with PBS solution. Different concentrations of misoprostol solution (5 μM, 10 μM, 30 μM, 50 μM and 100 μM) were added to the Vero cells. Six replicates were set up for each concentration of drug. A DMSO negative control group and a normal cell control were also set up. After drug treatment for 12 hours, 10 μL MTT was added to each well. After MTT incubation for 4 hours, the culture medium was replaced with 150 μL DMSO. The ultraviolet absorbance was measured at 490 nm. Figure 1 shown.

[0051] Depend on Figure 1 It can be seen that the safe concentration of misoprostol in Vero cells can reach 100μM.

[0052] 2. Inhibitory effect of misoprostol on PEDV

[0053] Vero cells were inoculated in 6-well plates. After they grew into a monolayer, the culture medium was discarded and washed three times with PBS solution. Then, cell culture medium containing 100 μM misoprostol solution and virus solution with an MOI of 0.01 were added to the cells and cultured for 12 hours. A DMSO negative control group and three repeated experiments were set up. The mRNA expression level and TCID of the virus were detected respectively. 50 , determine the virus infection, such as Figure 2 shown.

[0054] Depend on Figure 2It can be seen that compared with the DMSO negative control group, the addition of misoprostol to the culture medium can significantly reduce the viral mRNA expression level and viral titer, indicating that misoprostol has a significant inhibitory effect on PEDV.

[0055] 3. Inhibitory effect of different concentrations of misoprostol on PEDV

[0056] To analyze the inhibitory effect of misoprostol on viral infection, Vero cells were inoculated in 6-well plates. After they grew into a monolayer, the culture medium was discarded and washed three times with PBS solution. Then, cell culture medium containing 0μM, 1μM, 3μM, 5μM, 10μM, 50μM and 100μM misoprostol solution and PEDV virus solution with an MOI of 0.01 were added to the cells and cultured for 12h. A DMSO negative control group and three repeated experiments were set up. The mRNA expression level of the virus was detected to determine the infection status of the virus, such as Figure 3 shown.

[0057] Depend on Figure 3 It can be seen that when the misoprostol concentration is in the range of 0-3 μM, the viral mRNA expression level has no significant change. With the increase of misoprostol concentration, the viral mRNA expression level gradually decreases, indicating that misoprostol has a dose-dependent inhibition on PEDV.

[0058] 4. The specific stage of action of misoprostol in inhibiting viral infection

[0059] To analyze the specific stage of the inhibitory effect of misoprostol on viral infection, Vero cells were seeded in 6-well plates, and after they grew into a monolayer, the culture medium was discarded, and the cells were washed three times with PBS solution. After the cells were treated with the optimal concentration of misoprostol (100 μM) for 1 h, a virus solution containing an MOI of 0.01 was added and cultured for 12 h, which was recorded as the -1-0 infection stage. As a control, the cells were placed in a 37°C incubator for 1 h, and then the virus solution was discarded, the cells were washed three times with PBS, and the cell maintenance solution was added;

[0060] When the Vero cells inoculated in the 6-well plate grew into a monolayer, the culture medium was discarded, and the cells were washed three times with PBS solution. A virus solution containing an MOI of 0.01 was added. Misoprostol was added after 0h, 1h, 3h, 5h, and 7h of virus infection to make the drug concentration reach 100μM. After the above cells were cultured for 12h, they were recorded as 0-1, 1-12, 3-12, 5-12, and 7-12 infection stages, respectively. At the same time, a DMSO negative control group was set up to detect the mRNA expression level of the virus in each group, as shown in Figure 2. Figure 4 shown.

[0061] Depend on Figure 4A shows that compared with the control group at the -1-0 infection stage, misoprostol administration at the five infection stages of 0-1, 1-12, 3-12, 5-12 and 7-12 of viral infection had significant inhibitory effects on viral infection, especially at 1h of viral infection. The above results show that misoprostol inhibits viral infection mainly in the early stage of viral infection. Cell samples at the 1-12 infection stage were taken for TCID 50 Experiment, the results are as follows Figure 4 As shown in B, compared with the DMSO negative control group, the virus titer in the cell samples at infection stages 1-12 was significantly reduced, further confirming the inhibitory effect of misoprostol on the early infection of PEDV.

[0062] 5. Reasons for the inhibitory effect of misoprostol on viruses

[0063] (1) The killing effect of misoprostol on PEDV

[0064] To analyze the effect of misoprostol on the virus, PEDV was incubated with misoprostol (100 μM) or control DMSO in DMEM at 37°C for 1 h. The incubated mixture was diluted at a volume ratio of 1:100 (misoprostol was diluted to an inactive concentration, and the infection MOI was 0.01), and Vero cells cultured in monolayers were infected with the diluted mixture for 48 h. TCID 50 Detect the virus titer and the virus mRNA expression level.

[0065] Depend on Figure 5 It can be seen that compared with the control group DMSO, the toxicity of PEDV after mixing with misoprostol did not change significantly, indicating that misoprostol has no killing effect on PEDV.

[0066] (2) Misoprostol exerts its antiviral effect by targeting integrin β1

[0067] To analyze whether misoprostol exerts its antiviral effect by targeting integrin β1, the protein structure of integrin β1 was established and optimized by model building. Then, the surrounding water molecules, side chains and heteroatoms were removed, and hydrogen was added and energy was minimized using Discovery Studio 4.5 software. Energy minimization was also performed on misoprostol. Finally, molecular docking was used to evaluate the binding effect of misoprostol on integrin β1.

[0068] Molecular docking Figure 6As shown in the figure, through docking analysis, it was found that the docking score of misoprostol and integrin β1 can be stabilized to -22.6kcal / mol, and the two showed a stable binding effect. It can be concluded that misoprostol can target integrin β1 to inhibit the activity of integrin β1, prevent it from acting as a receptor for the virus to enter the cell, and thus exert an antiviral effect.

[0069] 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. Use of misoprostol as an active ingredient in the preparation of products against porcine epidemic diarrhea virus.

2. The use according to claim 1, characterized in that: The products include pharmaceuticals.

3. The use according to claim 2, characterized in that: The medicine also includes a pharmaceutically acceptable carrier or excipient.

4. The use according to claim 2, characterized in that: The dosage forms of the medicine include tablets, granules, mixtures and capsules.

5. The use according to claim 2, characterized in that: The drug inhibits infection of porcine epidemic diarrhea virus.

6. The use according to claim 5, characterized in that: The drug inhibits the porcine epidemic diarrhea virus infection for 1-12 hours.

7. Use of misoprostol as an active ingredient in the preparation of inhibitors targeting integrin β1 protein.

8. The use according to claim 7, characterized in that: The misoprostol inhibits the activity of the integrin β1 protein by specifically binding to the integrin β1 protein.

9. The use according to claim 7, characterized in that: The inhibitor further includes a pharmaceutically acceptable carrier or excipient.

10. The use according to claim 7, characterized in that: The dosage forms of the inhibitor include tablets, granules, mixtures and capsules.

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