Application of eltrombopag in preparation of medicine for preventing and treating rotavirus

Abstract

The invention belongs to the technical field of biology, and particularly relates to an application of eltrombopag in preparation of a drug for preventing and treating rotavirus, by measuring the CC50 value and IC50 value of cells infected by rotavirus and healthy cells, the eltrombopag can be used as an inhibitor of rotavirus, and an excellent antiviral effect can be achieved in the cells infected by rotavirus. Furthermore, through immunofluorescence, it is found that the eltrombopag can have a good inhibition effect on replication of the rotavirus on cells when the concentration is 2.5-10 [mu] mol / L, and a new medicinal raw material is provided for preventing and treating the rotavirus.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to application of Eltrombopag in preparing medicines for preventing and treating rotavirus. Background Art

[0002] Rotavirus (RV) is a non-enveloped double-stranded RNA virus of the Reoviridae family. It is the most important cause of severe viral gastroenteritis and may be life-threatening. Almost all children under 5 years old, the elderly and patients with chronic diseases in the world are the main populations infected by rotavirus. Rotavirus generally replicates in mature small intestinal cells and enters the blood, causing antigenemia and viremia. It causes 130 million child infections and more than 200,000 deaths each year, and causes significant losses to the livestock industry. Currently, there are two vaccines on the Chinese market: Rotavirus Live Vaccine Oral (Lanzhou Institute of Biological Products), which was launched in China in 2001, and RotaTeq TM (Merck Sharp & Dohme, USA), was launched in China in 2018. Although the introduction of rotavirus immunization programs has reduced mortality by 60%, the effectiveness of the vaccine in developing countries is low. Currently, oral and intravenous rehydration remains the main method for rotavirus treatment. However, it cannot reduce the course and severity of diarrhea because it is only symptomatic treatment. Therefore, there is an urgent need to develop specific anti-rotavirus drugs.

[0003] As a drug approved by the U.S. Food and Drug Administration, Eltrombopag has been approved for the treatment of immunocompromised patients with low platelet counts infected with hepatitis virus (HCV). Its safety and side effects have been fully evaluated and proven in clinical practice, but there are no reports of serious neurological side effects. In addition, Eltrombopag is a potent autophagy inhibitor targeting TFEB, and can also directly activate BAK and induce apoptosis. Eltrombopag also shows significant effects in anti-pathogenic infection. For example, Eltrombopag also has a strong inhibitory effect on multidrug-resistant Staphylococcus aureus and Salmonella typhi; at the same time, studies have shown that Eltrombopag has antiviral activity against a variety of viruses, such as fever with thrombocytopenia syndrome virus (SFTSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), hepatitis virus (HCV), etc.

[0004] Rotavirus is a zoonosis that infects 130 million children and kills more than 200,000 people each year, and causes significant losses to the livestock industry. However, there is no specific drug for the treatment of rotavirus in clinical practice at home and abroad. Therefore, research on therapeutic drugs for rotavirus has important scientific and practical significance. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of Eltrombopag in preparing a drug for preventing and treating rotavirus.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] One of the technical solutions of the present invention is the use of Eltrombopag as the only active ingredient in the preparation of a drug for preventing and treating rotavirus.

[0008] The second technical solution of the present invention is a drug for preventing and treating rotavirus, comprising eltrombopag and, if necessary, a pharmaceutically acceptable carrier and / or excipient.

[0009] Preferably, the dosage form of the drug is one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0010] Compared with the prior art, the present invention measures the CC of cells infected with rotavirus. 50 Value and IC 50 values, and found that Eltrombopag could be used as an inhibitor of rotavirus; further immunofluorescence revealed that Eltrombopag had a good inhibitory effect on rotavirus at a concentration of 2.5-10μmol / L, and when the concentration reached 10μmol / L, it almost completely inhibited the replication of rotavirus, providing a new candidate drug for the prevention and treatment of rotavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Eltrombopag inhibits rotavirus infection in CC 50 and IC 50 Value determination diagram: A is CC 50 Determination diagram; B is IC 50 Determination diagram.

[0012] Figure 2 Figure 3 shows the inhibitory effect of eltrombopag on the rotavirus genome copy number: A is the rotavirus genome copy number in Caco2 cells; B is the rotavirus genome copy number in MA104 cells.

[0013] Figure 3 Figure 3 shows the inhibitory effect of eltrombopag on the expression of rotavirus VP6 protein: A shows the expression of rotavirus VP6 protein in Caco2 cells; B shows the expression of rotavirus VP6 protein in MA104 cells.

[0014] Figure 4 The results of eltrombopag inhibiting rotavirus immunofluorescence observation: A is Caco2 cell rotavirus immunofluorescence; B is MA104 cell rotavirus immunofluorescence. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0016] The Caco2 cells used in the following examples are preserved by the Guangdong Provincial Key Laboratory of Animal Molecular Design and Precision Breeding. Caco2 cells are a cell line derived from human colon adenocarcinoma. The cell line can spontaneously differentiate into polar monolayer cells similar to small intestinal epithelial cells and is the best in vitro model for studying anti-rotavirus drugs. Other related reagents used, if not otherwise specified, are all commercially available agents, and the methods involved, if not otherwise specified, are all known methods.

[0017] The compound Eltrombopag used was purchased from Selleck, and its chemical molecular structure is shown below:

[0018]

[0019] Rotavirus strains used: Rotavirus vaccine strain LLR (G10P

[15] ), maintained by the Viral Diarrhea Laboratory of the Chinese Center for Disease Control and Prevention, subcultured on MA104 cells; MA104 cells (African green monkey embryonic kidney cells), maintained by the Viral Diarrhea Laboratory of the Chinese Center for Disease Control and Prevention.

[0020] In order to verify whether Eltrombopag can be used as the only active ingredient to prepare drugs for the prevention and treatment of rotavirus, the following tests were conducted:

[0021] 1. Toxicity of Eltrombopag in Caco2 cells

[0022] Caco2 cells were plated in 96-well plates (the culture medium was DMEM culture medium containing 10% FBS, 1% amphotericin, and 1% gentamicin, and 0.1 mL of culture medium was added to each well) with 100 μL of cell suspension per well, and cultured to a cell density of about 70%; Eltrombopag was diluted with culture medium to obtain Eltrombopag solutions with concentrations of 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, respectively, which were recorded as experimental group drugs, and a blank control group was set up at the same time; 10 μL of experimental group drugs of different concentrations were added to each well, and cultured at 37 ° C, 5% CO2 for 24 hours. 10 μL of CCK-8 solution was added to each well under light-proof conditions, and placed in a 37 ° C incubator for light-proof culture for 4 hours. The absorbance at 450 nm was measured using an enzyme marker. The cell survival rate was calculated by the formula to determine the optimal drug concentration. Cell survival rate = [(As-Ab] / (Ac-Ab)] × 100%, inhibition rate = [(Ac-As] / (Ac-Ab)] × 100%.

[0023] The results are as follows Figure 1As shown in A and B in Figure 1, eltrombopag solution has no significant toxicity to Caco2 cells within a concentration of 10 μM. The IC50 value of eltrombopag in Caco2 cells is 4.35 μM, indicating that venetola can exert a good anti-rotavirus effect at a low concentration. The results suggest that venetola is a potential rotavirus inhibitor.

[0024] 2. Effect of the compound Eltrombopag on rotavirus replication

[0025] (1) Effect on the copy number of rotavirus in cells

[0026] Caco2 and MA104 cells (2×10 6 / well), the experimental group was inoculated with rotavirus (MOI = 0.01) and adsorbed for 2 hours at 37°C and 5% CO2; the cells were treated with different concentrations (1, 2.5, 5, 10 μM) of Eltrombopag, and the infection control group was treated with rotavirus (MOI = 0.01) mixed with DMSO. The blank control group was not treated. The cell plate was incubated at 37°C and 5% CO2 for 24 hours, and then the total cellular RNA was extracted using the Omega Virus RNA Extraction Kit (R6874-02), and reverse transcribed into cDNA.

[0027] Finally, the viral genome copy number was detected by fluorescence quantitative PCR targeting the rotavirus NSP3 gene. The total volume of the fluorescence quantitative PCR reaction system was 25 μL, including 12.5 μL Premix Ex Taq (2×); 0.5 μL upstream and downstream primers, 0.5 μL probe, 2 μL template, and sterile deionized water to 25 μL; the upstream primer sequence was: 5'-ACCATCTWCACRTRACCCTCTATGAG-3'; the downstream primer sequence was: 5'-GGTCACATAACGCCCCTATAGC-3'; the probe sequence was: 5'-FAM-AGTTAAAAGCTAACACTGTCAAA-MGB3'.

[0028] The viral copy number calculation results of Caco2 and MA104 cells after rotavirus infection and treatment with different concentrations of Eltrombopag are as follows: Figure 2 As shown in A and B. The results showed that in Caco2 and MA104 cells, compared with the control group, with the increase of Eltrombopag concentration, the rotavirus genome copy number gradually decreased, indicating that Eltrombopag can inhibit rotavirus replication in a dose-dependent manner.

[0029] (2) Effects on the expression of rotavirus VP6 protein in cells

[0030] Caco2 and MA104 cells (2×10 6 / well), the experimental group was inoculated with rotavirus (MOI = 0.01) and cells were adsorbed for 2 hours under 5% CO2. Then, the cells were treated with different concentrations (1, 2.5, 5, 10 μM) of Eltrombopag. The infection control group was treated with rotavirus (MOI = 0.01) mixed with DMSO. The blank control group was not treated. The cell plate was incubated at 37°C and 5% CO2 for 24 hours, the cell culture supernatant was discarded, and the cells were rinsed three times with PBS. After adding 1× SDS-PAGE loading buffer, the proteins were denatured (100°C, 10 min), and centrifuged for 10 min (12 000r / min), after homogenizing the protein sample, add it to the sample tank of the gel, perform electrophoresis (120V, 30min), after the bromophenol blue dye drops to the edge of the gel, transfer it to a 0.2μm nitrocellulose membrane (NC membrane) (100V, 1h), block it with 5% skim milk powder (1h), discard the skim milk powder and wash the NC membrane with 1×TBST, add the primary antibody diluent (mouse rotavirus VP6 monoclonal antibody, 1:1000 dilution) and place it in a 4℃ refrigerator overnight, wash the NC membrane with 1×TBST (5 times / 5min), add the corresponding secondary antibody dilution (incubate for 1h), wash it with 1×TBST (5 times / 5min), finally expose it with ECL luminescent liquid to detect the expression of rotavirus VP6 protein.

[0031] The results are as follows Figure 3 As shown in A and B in Figure 3, in Caco2 and MA104 cells, compared with the control group, as the concentration of Eltrombopag solution increased, the expression of rotavirus VP6 viral protein decreased, indicating that Eltrombopag can inhibit rotavirus replication in a dose-dependent manner.

[0032] (3) Effects on rotavirus immunofluorescence in cells

[0033] Caco2 cells (2×10 6 / well), the experimental group was inoculated with rotavirus (MOI = 0.01), and after adsorption for 2 hours under 5% CO2, the cells were treated with different concentrations (1, 2.5, 5, 10 μM) of Eltrombopag; the infection control group was treated with rotavirus (MOI = 0.01) mixed with DMSO, which was recorded as the DMSO group; the blank control was not treated, which was recorded as the Mock group. The cell plate was placed at 37°C and 5% CO2 for 24 hours, the cell culture supernatant was discarded, and after rinsing with PBS three times, it was fixed with 4% paraformaldehyde for 30 minutes. Then it was permeabilized by treating with 0.3% Triton for 10 minutes. Blocking: Block with 5% BSA at room temperature for 1 hour. Then discard the blocking solution, directly add the primary antibody dilution solution (mouse rotavirus VP6 monoclonal antibody, 1:200 dilution) prepared with 5% BSA, and incubate at 4°C overnight (protected from light). Then use the fluorescent secondary antibody labeled with fluorescein isothiocyanate (FITC) and incubate at 37°C for 1 hour. After incubation, the antibody was discarded, and the cells were washed three times with PBST before observation under an inverted fluorescence microscope.

[0034] The results are as follows Figure 4 As shown in A and B in the figure, in Caco2 and MA104 cells, compared with the control group, as the concentration of Eltrombopag solution increased, the rotavirus fluorescence decreased significantly, indicating that Eltrombopag can inhibit rotavirus replication in a dose-dependent manner.

[0035] In summary, Eltrombopag can be used as the sole active ingredient to prepare drugs for the prevention and treatment of rotavirus. Of course, the prepared drugs contain Eltrombopag and also contain pharmaceutically acceptable carriers and / or excipients; the dosage form of the drug is one of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

[0036] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of eltrombopag as the sole active ingredient in the preparation of drugs for the prevention and treatment of rotavirus.

2. A drug for preventing and treating rotavirus, characterized in that: Contains eltrombopag.

3. The drug for preventing and treating rotavirus according to claim 2, characterized in that: It also contains pharmaceutically acceptable carriers and / or excipients.

4. The drug for preventing and treating rotavirus according to claim 2 or 3, characterized in that: The dosage form of the medicine is one of tablets, sprays, granules, capsules, oral liquids, injections and suspensions.

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