Application of lycorine in preparation of medicine for treating feline calicivirus infection

Abstract

The invention discloses an application of lycorine in preparation of a medicine for treating feline calicivirus infection, by measuring CC50 values and EC50 values in cells and healthy cells after feline calicivirus infection, the lycorine can be used as an inhibitor of feline calicivirus, the cells infected by the feline calicivirus are treated by using the lycorine, and the treatment effect of the feline on feline calicivirus infection is improved. Growth of the feline calicivirus in cells can be remarkably inhibited, the virus titer of the feline calicivirus in the cells is reduced, and the expression quantity of VP1 protein of the feline calicivirus is reduced. An indirect fluorescence immunoassay method finds that when the concentration of lycorine is 1.56-25 [mu] mol / L, the lycorine has a good consistent effect on the feline calicivirus; and when the concentration of lycorine reaches 6.25 mu mol / L, the growth of the feline calicivirus is almost completely inhibited, so that not only is a novel medicine for resisting the feline calicivirus provided, but also the feline calicivirus can be prevented and treated at low concentration.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of lycorine in preparing a medicine for treating feline calicivirus infection. Background Art

[0002] Lycorine is a natural alkaloid isolated from Amaryllidaceae plants, with the molecular formula C 16 H 17 NO4, Lycorine has strong medicinal value and has shown various biological properties, including anti-cancer, anti-plasmodial, anti-trypanosomal, anti-inflammatory, emetic, and cardiovascular protective properties. Lycorine has been reported to have broad-spectrum inhibitory activity against a variety of viruses, such as Zika virus, polio virus, novel coronavirus, herpes simplex virus, and Rift Valley fever virus.

[0003] Feline calicivirus (FCV) is a major infectious pathogen in cats worldwide. FCV, belonging to the Caliciviridae family and the Calicivirus genus, is the causative agent of respiratory disease, stomatitis, arthritis, and hemorrhagic fever in cats. The virus reproduces in the brain, sometimes forming crystals or beads. The virus is resistant to lipid solvents (ether, chloroform, and deoxycholine salts).

[0004] This highly contagious virus is widespread in cats, causing upper respiratory tract infections. Key symptoms include depression, serous and mucous rhinorrhea, conjunctivitis, stomatitis, tracheitis, and bronchitis, accompanied by biphasic fever. Other illnesses include lameness, miscarriage, oral ulcers, and chronic gastroenteritis. Feline calicivirus infection typically lasts two to three days, with the severity of symptoms varying depending on the virulence of the virus. While symptoms may resolve with clinical treatment, infected animals continue to shed the virus for extended periods, presenting a dangerous source of infection. Calicivirus transmission is common among felines, and naturally, only cats are susceptible to it, causing significant harm to their health.

[0005] Feline calicivirus infection is a common disease in cats with a high incidence rate. Due to the emergence of virulent strains in recent years, the mortality rate has gradually increased, especially for kittens under one year old. The mortality rate of FCV infection is as high as 67%.

[0006] Therefore, studying the therapeutic drugs for feline calicivirus has important scientific and practical significance. Summary of the Invention

[0007] The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide the use of lycorine in the preparation of a medicament for treating feline calicivirus infection.

[0008] The first object of the present invention is to provide the use of lycorine in preparing a medicament for reducing the activity of feline calicivirus.

[0009] The second object of the present invention is to provide the use of lycorine in preparing a medicament for treating feline calicivirus infection.

[0010] The third object of the present invention is to provide a medicine.

[0011] In order to achieve the above object, the present invention is implemented through the following scheme:

[0012] The present invention claims protection for the use of lycorine in preparing a medicine for reducing the activity of feline calicivirus.

[0013] Preferably, the reducing the activity of feline calicivirus is to inhibit the growth of feline calicivirus, reduce the viral titer of feline calicivirus and / or reduce the expression level of feline calicivirus VP1 protein.

[0014] More preferably, the feline calicivirus is the FCV-GDJM202201 strain.

[0015] The accession number of the FCV-GDJM202201 strain on NCBI is OQ472996.1. Its structural protein is VP1 protein. VP1 protein is a capsid protein with a size of about 70kDA. It is the main structural protein of feline calicivirus (FCV) and the main site of interaction between the virus and host cells.

[0016] The present invention also claims protection for the use of lycorine in preparing a medicine for treating feline calicivirus infection.

[0017] Preferably, the concentration of lycorine in the drug is 1.56-25 μmol / L.

[0018] More preferably, the concentration of lycorine in the drug is 6.25 μmol / L.

[0019] Preferably, the feline calicivirus is the FCV-GDJM202201 strain.

[0020] The present invention measures CC in cells infected with feline calicivirus and healthy cells. 50 and EC 50 The results indicate that Lycorine can effectively inhibit / reduce the activity of feline calicivirus without having any negative effects on cells.

[0021] The present invention also claims protection for a medicine containing 1.56-25 μmol / L of lycorine and using lycorine as the only active ingredient.

[0022] Preferably, it contains 6.25 μmol / L of lycorine.

[0023] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0024] Preferably, the dosage form of the drug is any dosage form used in pharmacy.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides the use of lycorine in the preparation of a medicine for treating feline calicivirus infection, and the CC in cells infected with feline calicivirus and healthy cells is measured. 50 Value and EC 50 The results showed that lycorine can be used as an inhibitor of feline calicivirus. When cells infected with feline calicivirus were treated with lycorine, the growth of feline calicivirus in cells was significantly inhibited, the viral titer of feline calicivirus in cells was reduced, and the expression of feline calicivirus VP1 protein was reduced. In addition, the indirect fluorescence immunoassay method showed that lycorine had a good and consistent effect on feline calicivirus when the concentration was between 1.56 and 25 μmol / L. When the concentration of lycorine reached 6.25 μmol / L, the growth of feline calicivirus was almost completely inhibited. This not only provides a new anti-feline calicivirus drug, but also achieves the prevention and treatment of feline calicivirus at low concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the chemical molecular structure of lycorine;

[0028] Figure 2 CC in Example 1 50 Regression curve graph;

[0029] Figure 3 EC in Example 1 50 Regression curve graph;

[0030] Figure 4 This is a diagram of the fluorescence observation results in Example 2;

[0031] Figure 5 This is a diagram showing the expression results of VP1 protein in Example 3. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0033] Example 1 CC of Lycorine in Cat Kidney Cells (F81 Cells) 50 and EC 50

[0034] 1. Experimental Methods

[0035] The chemical molecular structure diagram of Lycorine is as follows Figure 1 shown.

[0036] 1. Determination of the half-maximal cytotoxic concentration of lycorine in F81 cells

[0037] Lycorine was diluted in two-fold ratio using DMEM culture medium to obtain lycorine dilution solutions with concentrations of 0.78 μmol / L, 1.56 μmol / L, 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L and 100 μmol / L, respectively.

[0038] Feline kidney cells (F81 cells) were cultured at a rate of 1×10 5 The cells were plated in 96-well cell plates to obtain cell plates containing F81 cells. The half cytotoxic concentration (CC) of lycorine was detected. 50 (μM), as follows:

[0039] The experimental wells were set up as follows: different concentrations of Lycorine dilutions were added into the cell plate containing F81 cells at 100 μL / well, and each concentration of Lycorine dilution was repeated 3 times.

[0040] The blank wells were set as wells containing only F81 cells without any other substances added.

[0041] The negative wells were set as follows: 0.1% (w / w) DMEM was added to the wells containing F81 cells at 100 μL / well to culture the F81 cells.

[0042] The experimental wells, blank wells, and negative wells were cultured at 37°C for 24 hours, and the supernatant in each well was discarded. Then, 100 μL of DMEM (0.1%, w / w) and 10 μL of CCK8 reagent were added to each well and incubated in a 37°C constant temperature incubator for 1 hour. After the incubation, the OD value of each well was measured at a wavelength of 450 nm in a microplate reader, and the cell viability in each well was calculated according to Formula 1;

[0043] Formula I: Cell viability (%) = (OD value of experimental well / OD value of negative well) × 100%.

[0044] Based on the survival rate of each well in the blank well and the experimental well and the concentration of the Lycorine dilution solution, CC 50 Regression curve and CC 50Regression equation; combined with CC 50 The regression equation was used to calculate the concentration of Lycorine dilution in the corresponding experimental well when the cell survival rate was 50%, which was the half cytotoxic concentration (CC) of Lycorine. 50 (μM).

[0045] 2. The effective concentration of Lycorine to inhibit half of the virus in F81 cells (EC 50 )

[0046] Taking the cell plate containing F81 cells obtained in step 1 as an example, the EC of lycorine in F81 cells was detected. 50 , as follows:

[0047] Feline calicivirus FCV (FCV-GDJM202201 strain, accession number OQ472996.1) was used to infect F81 cells in a cell plate at an MOI of 0.1 to obtain FCV-infected F81 cell wells, and blank cell wells not inoculated with the virus were set.

[0048] Lycorine was diluted with DMEM medium to obtain lycorine dilutions with concentrations of 1.56 μmol / L, 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L and 25 μmol / L. Then, different concentrations of lycorine dilutions (1.56 μmol / L, 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L and 25 μmol / L) were added to different FCV-infected F81 cell wells at 1 mL / well. After culturing at 37°C for 24 h, the supernatant and cultured cells were separated and analyzed by TCID 50 The inhibitory effect of the technology on feline calicivirus in cultured cells was detected by plotting the EC with virus titer as the ordinate and lycorine concentration as the abscissa. 50 Regression curve and calculation of EC 50 The regression equation was to substitute the viral titer at half inhibition of feline calicivirus into the EC 50 In the regression equation, the drug effect concentration of Lycorine that inhibits half of the feline calicivirus in cells is obtained, that is, EC 50 (μM).

[0049] 2. Experimental Results

[0050] CC 50 The regression curve is as follows Figure 2 As shown, EC 50 The regression curve is as follows Figure 3 shown.

[0051] CC of Lycorine in F81 Cells 50 and EC 50 The measurement results are shown in Table 1.

[0052] Table 1CC 50 and EC 50 Measurement results

[0053] cell <![CDATA[EC 50 (μmol / L)]]> <![CDATA[CC 50 (μmol / L)]]> F81 0.5398 73.79

[0054] The results showed that the CC of Lycorine in F81 cells 50 It reached 73.79 μmol / L, indicating that Lycorine has less toxicity to cells; and Lycorine has an EC of 1.577 μmol / L against feline calicivirus in F81 cells. 50 It is 0.5398 μmol / L, indicating that low concentrations of lycorine have the effect of resisting feline calicivirus, which is far below the half cytotoxic concentration.

[0055] Example 2 Determination of the effective concentration of lycorine against feline calicivirus in F81 cells

[0056] 1. Experimental Methods

[0057] Feline kidney cells (F81 cells) were cultured at a rate of 1×10 5 Cells were inoculated into 12-well plates and cultured in DMEM at 37°C for 12 hours. Feline calicivirus (FCV) (FCV-GDJM202201 strain) was then inoculated. After 1 hour, the cell culture medium was replaced and cells were treated with different concentrations of lycorine. The cells were collected and assayed as follows:

[0058] F81 cells in good growth condition were cultured at a rate of 1×10 5 The cells were inoculated into a 12-well plate with 100 cells / well, cultured in DMEM at 37°C for 12 h, and then inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) at an MOI of 1.0. After 1 h, the cell culture medium was replaced to obtain a cell plate of F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain); 7 cell plates of F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) were prepared in parallel.

[0059] A cell plate of F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) was used as a blank group, and the blank group was not treated.

[0060] A cell plate of F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) was used as a control group, and 0.1% (w / w) DMEM solution was added at 1 mL / well.

[0061] Then, 1.56 μmol / L, 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L and 25 μmol / L of lycorine solution were added to the remaining 5 cell plates inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) at 1 mL / well as experimental groups.

[0062] The experimental group, blank group and control group were cultured in a 37°C constant temperature incubator for 24 hours, and then indirect immunofluorescence (FITC and DAPI were used as fluorescent dyes, respectively) was performed, and the fluorescence results were observed using a fluorescence microscope.

[0063] 2. Experimental Results

[0064] Fluorescence observation results are shown in Figure Figure 4 As shown, the first row is the fluorescence observation result graph of each group when FITC is used as the fluorescent dye, the second row is the fluorescence observation result graph of each group when DAPI is used as the fluorescent dye, and the third row is the overlay graph of the fluorescence observation result graph in the first row and the fluorescence observation result graph in the second row.

[0065] The results showed that compared with the control group, the addition of lycorine to F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) could significantly inhibit the feline calicivirus FCV (FCV-GDJM202201 strain) in the cells; compared with the blank group, in cells added with 6.25 μmol / L lycorine, the fluorescence of feline calicivirus FCV (FCV-GDJM202201 strain) was almost completely inhibited.

[0066] The results show that the effective anti-feline calicivirus FCV (FCV-GDJM202201 strain) concentration of lycorine in F81 cells is 6.25 μmol / L.

[0067] Example 3 Effect of Lycorine on Viruses in F81 Cells Infected with Feline Calicivirus

[0068] 1. Experimental Methods

[0069] Cat kidney cells (F81 cells) in good growth condition were cultured at a rate of 1×10 5 Cells were inoculated into 12-well plates at 100 μg / well, cultured in DMEM at 37°C for 12 h, and then inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) at an MOI of 1.0. After 1 h, the cell culture medium was replaced to obtain F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain).

[0070] 1.56 μmol / L, 3.125 μmol / L, 6.25 μmol / L, 12.5 μmol / L and 25 μmol / L of lycorine solution were added to different F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) at 1 mL / well to obtain experimental group 1. 0.1% (w / w) DMEM solution was added to F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain) at 1 ml / well to obtain control group 1.

[0071] The F81 cells without any treatment were used as blank group 1.

[0072] Each group of cells was cultured in a 37°C constant temperature incubator for 24 hours, and then RIPA lysis buffer (ST507-10 mL, Beyotime) containing PMSF was added to each group of cells at 200 μL / well. After lysis on ice for 30 minutes, each group of cells was pipetted and transferred to a sterilized 1.5 mL centrifuge tube using a pipette. The expression of VP1 protein of feline calicivirus FCV (FCV-GDJM202201 strain) was detected by Western Blot to obtain the VP1 protein expression result diagram.

[0073] 2. Experimental Results

[0074] VP1 protein expression results are shown in the figure Figure 5 As shown, the results showed that compared with the blank group and the control group, when 6.25 μmol / L lycorine was added to the F81 cells inoculated with feline calicivirus FCV (FCV-GDJM202201 strain), the expression of VP1 protein of feline calicivirus was almost completely inhibited; and when 12.5 μmol / L lycorine was added, the VP1 protein of feline calicivirus in the cells was completely inhibited.

[0075] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. The application of lycorine in the preparation of a medicine for reducing the activity of feline calicivirus.

2. The use according to claim 1, characterized in that: The reducing the activity of feline calicivirus is to inhibit the growth of feline calicivirus, reduce the virus titer of feline calicivirus and / or reduce the expression level of feline calicivirus VP1 protein.

3. The use according to claim 2, characterized in that: The feline calicivirus is the FCV-GDJM202201 strain.

4. Application of lycorine in the preparation of medicines for treating feline calicivirus infection.

5. The use according to claim 4, characterized in that: The concentration of lycorine in the medicine is 1.56-25 μmol / L.

6. The use according to claim 5, characterized in that: The concentration of lycorine in the drug is 6.25 μmol / L.

7. The use according to claim 6, characterized in that: The concentration of lycorine in the drug is 12.5 μmol / L.

8. The use according to claim 4, characterized in that: The feline calicivirus is the FCV-GDJM202201 strain.

9. A drug, characterized in that It contains 1.56-25 μmol / L of lycorine and has lycorine as the only active ingredient.

10. The drug according to claim 9, characterized in that Contains 6.25μmol / L of lycorine.

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