CN109864993B - Application of mizoribine in preparation of drugs for preventing foot-and-mouth disease virus infection - Google Patents
Application of mizoribine in preparation of drugs for preventing foot-and-mouth disease virus infection Download PDFInfo
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- CN109864993B CN109864993B CN201910175969.5A CN201910175969A CN109864993B CN 109864993 B CN109864993 B CN 109864993B CN 201910175969 A CN201910175969 A CN 201910175969A CN 109864993 B CN109864993 B CN 109864993B
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Abstract
The invention relates to an application of mizoribine in preparing a medicament for preventing foot-and-mouth disease virus infection, belonging to the technical field of veterinary medicaments. The application of the invention can provide a high-efficiency, safe and quality-controllable anti-foot-and-mouth disease virus medicine for further controlling the spread of the foot-and-mouth disease.
Description
Technical Field
The invention relates to the technical field of veterinary medicines, and in particular relates to application of mizoribine in preparation of a medicine for preventing foot-and-mouth disease virus infection.
Background
Foot-and-mouth disease is an acute, hot and highly contact artiodactyl infectious disease caused by foot-and-mouth disease virus. The world animal health organization ranks the animal epidemic disease as a legal report animal epidemic disease, China ranks the animal epidemic disease as a type of animal epidemic disease, and ranks the foot-and-mouth disease as one of the priority disease prevention and treatment species.
FMDV genus small RNA virus family foot-and-mouth disease virus genus, there are 7 serotypes O, A, C, Asia1 and SAT I-3, there is no cross immunity phenomenon among serotypes, China is mainly epidemic type O and type A foot-and-mouth disease at present. The use of vaccines for the control of foot and mouth disease has made significant success, however vaccines can only produce complete clinical protection after 7 days of immunization of animals, and the herds will remain susceptible to infection early in the outbreak. Therefore, there is a need to develop safe and effective antiviral drugs to protect animals in the early stages of disease.
Disclosure of Invention
The invention aims to provide application of mizoribine in preparation of a medicine for preventing foot-and-mouth disease virus infection. The application can provide a high-efficiency, safe and quality-controllable anti-foot-and-mouth disease virus medicine for further controlling the spread of the foot-and-mouth disease.
The invention provides application of mizoribine in preparation of a medicine for preventing foot-and-mouth disease virus infection.
Preferably, the foot-and-mouth disease virus includes a type a foot-and-mouth disease virus and a type O foot-and-mouth disease virus.
Preferably, the mizoribine is applied at a concentration > 25 μmol/L.
The invention also provides a foot-and-mouth disease virus inhibitor which comprises mizoribine and pharmaceutically acceptable auxiliary materials.
Preferably, the concentration of mizoribine in the inhibitor is > 50 μ g/g.
Preferably, the auxiliary material comprises one or more of starch, sugar powder, dextrin, polyethylene glycol and glycerol.
The invention provides application of mizoribine in preparation of a medicine for preventing foot-and-mouth disease virus infection. The mizoribine has an inhibiting effect on cytopathic effect induced by A-type foot-and-mouth disease virus and O-type foot-and-mouth disease virus, inhibits the replication of the virus, and prolongs the survival time of infected animals. Cell tests show that mizoribine has low cytotoxicity and has an inhibiting effect on both A-type foot-and-mouth disease viruses and O-type foot-and-mouth disease viruses; further experiments confirm that mizoribine only acts early in FMDV replication; in vivo experiments show that mizoribine can delay the lethal effect of foot-and-mouth disease virus on suckling mice. Mizoribine can be used as a candidate drug for resisting FMDV infection.
Drawings
FIG. 1 is a graph of cytotoxicity of MIZ on IBRS-2 cells at various concentrations as provided in example 1 of the present invention;
FIG. 2 is a graph showing the inhibitory effect of MIZ on FMDV type O infection of IBDS-2 cells at various concentrations as provided in example 1 of the present invention;
FIG. 3 is a graph showing the inhibitory effect of MIZ on type A FMDV infection of IBDS-2 cells at various concentrations as provided in example 1 of the present invention;
FIG. 4 is a graph showing the effect of MIZ at various concentrations on FMDV mRNA inhibition in type O FMDV infected IBDS-2 cells, as provided in example 1 of the present invention;
FIG. 5 is a graph showing the effect of IFA detection provided in example 1 of the present invention on inhibition of FMDV protein expression in cells infected with type O FMDV at various concentrations of MIZ;
FIG. 6 is a graph of MIZ action on the inhibition of FMDV mRNA expression levels in virally infected cells at various time periods as provided in example 1 of the present invention;
FIG. 7 is a graph showing the inhibitory effect of MIZ on the expression level of VP1 protein in virus-infected cells at different time periods, as provided in example 1 of the present invention;
FIG. 8 is a graph showing the effect of MIZ on the death time of FMDV-infected suckling mice as provided in example 1 of the present invention.
Detailed Description
The invention provides application of Mizoribine (MIZ) in preparation of a medicine for preventing foot-and-mouth disease virus infection. In the present invention, the foot-and-mouth disease virus includes a type a foot-and-mouth disease virus and a type O foot-and-mouth disease virus. Mizoribine has inhibition effect on cell pathological changes induced by A type foot-and-mouth disease virus and O type foot-and-mouth disease virus, inhibits the replication of virus, prolongs the survival time of infected animals, and only plays a role in the early replication stage of FMDV. The medicine prepared by using mizoribine as an effective component has low cytotoxicity, has an inhibiting effect on both A type foot-and-mouth disease virus and O type foot-and-mouth disease virus, and can inhibit foot-and-mouth disease virus (FMDV) and prevent and control foot-and-mouth disease.
In the invention, the mizoribine is applied at a concentration of more than 25 mu mol/L.
The invention also provides a foot-and-mouth disease virus inhibitor which comprises mizoribine and pharmaceutically acceptable auxiliary materials.
In the present invention, the concentration of mizoribine in the inhibitor is > 50 μ g/g.
In the invention, the auxiliary material comprises one or more of starch, powdered sugar, dextrin, polyethylene glycol and glycerol. In the present invention, the glycerin is preferably glycerin for injection. The sources of the auxiliary materials in the present invention are not particularly limited, and commercially available products of conventional starch, sugar powder, dextrin, polyethylene glycol and glycerin, which are well known to those skilled in the art, may be used.
The application of mizoribine in the preparation of a medicament for preventing foot-and-mouth disease virus infection is further described in detail with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
1. Experimental Material
1.1 cells, animals, viruses and drugs
IBRS-2 cells were preserved from this group; the 3-day-old suckling mice were purchased from the laboratory animal farm of the Lanzhou veterinary research institute of the Chinese academy of agricultural sciences. FMDV (O/MY98/BY/2010 and A/GDMM/CHA/2013) is preserved and provided BY the national foot-and-mouth disease reference laboratory; MIZ was purchased from MCE and formulated in DMSO.
1.2 reagents
DMEM, fetal bovine serum FBS, trypsin medium were purchased from Gibco; MTS assay kit was purchased from Abcam corporation; TRIZOL was purchased from Invitrogen; SYBR Premix Ex TaqⅡThe kits are purchased from precious bioengineering (Dalian) Co., Ltd; RIPA lysate, BCA method protein quantification kit, SDS-PAGE gel preparation kit and ECL from Biyuntian company; BSA, PVDF membranes were purchased from BioRad; tween-20 and Tween-80 are purchased from Shanghai Bioengineering company; triton X-100, DMSO was purchased from Sigma; mouse anti-beta-actin polyclonal antibody, HRP marked anti-rabbit or anti-mouse IgG antibody are purchased from Abcam company; the rabbit anti-O type FMDV VP1 polyclonal antibody is a gift offered by Zhenghai doctor in national foot and mouth disease reference laboratory.
2. Experimental methods and results
2.1 MIZ toxicity test on IBRS-2 cells:
the cytotoxicity of MIZ on IBRS-2 cells was determined using the MTS method. After the cells are plated on a 96-well plate IBRS-2 and fully grow into a monolayer, the upper culture solution of the cells is discarded, the cells are washed for 3 times by using fresh DMEM, MIZ100 mu L which is diluted by a DMEM culture solution gradient containing 2% FBS is finally added, the corresponding DMSO concentration of the preparation solution of MIZ is used as a negative control well, and the cells are used as a cell control well without any treatment. The cells were incubated at 37 ℃ for 72 hours, the supernatant cell culture was discarded, washed three times with fresh DMEM, and 100. mu.L of fresh DMEM was added, and 20. mu.L of MTS solution was added to each well. After incubation for 4h at 37 ℃, the absorbance value at 490nm is measured on a microplate reader according toThe formula "cell activity rate ═ (OD)Medicine-ODBlank space)/(ODNegative of-ODBlank space) X 100% "toxicity of MIZ on IBRS-2 cells was calculated at various concentrations. The experiment was independently repeated three times.
The results of this experiment are shown in FIG. 1: MTS results show that the activity rate of cells is still over 90% with the continuous increase of the drug concentration, which indicates that the toxicity of mizoribine to IBRS-2 is extremely low.
2.2 evaluation of the anti-foot-and-mouth disease Virus Activity of 2.2 MIZ on IBRS-2 cells:
well-grown IBRS-2 cells on a DMEM complete medium containing 10% FBS are paved on a 96-well plate, after the IBRS-2 cells grow to a monolayer, the upper layer culture solution of the cells is discarded, the cells are washed for 3 times by using fresh DMEM, and 100TCID is inoculated50O/MY 98/BY/2010. After 1h, the virus solution was removed, washed 3 times with fresh DMEM, MIZ100 μ L diluted with a gradient of 2% FBS in DMEM was added, MIZ was used as virus control wells for the corresponding DMSO concentration, and MIZ-free, virus-free cells were used as cell control wells. The cells were incubated at 37 ℃ for 48h, the supernatant cell culture was discarded, washed three times with fresh DMEM, and 100. mu.L of fresh DMEM was added, and 20. mu.L of MTS solution was added to each well. After incubation at 37 ℃ for 4h, the absorbance at 490nm was measured on a microplate reader, according to the formula "cell activity rate ═ ODMedicine-ODBlank space)/(ODNegative of-ODBlank space) X 100% "antiviral effect was calculated at various concentrations of MIZ. At the same time, different groups of supernatants were collected and the mRNA level of FMDV2B gene was determined by q-PCR. RNA from cells was extracted according to TRIZOL instructions, according to SYBR Premix Ex TaqⅡThe operating instruction carries out fluorescence quantitative PCR, and beta-actin is used as an internal reference gene. The primer sequence for detecting the mRNA specificity of the FMDV2B gene is as follows:
FMDV-for,5’-CAACAAAACACGGACCCGAC-3’(SEQ ID NO.1)；
FMDV-rev,5’-TTGTACCAGGGTTTGGCCTC-3’(SEQ ID NO.2)；
the primer sequence of beta-actin is as follows:
β-actin for,5’-GACCACCTTCAACTCGATCA-3’(SEQ ID NO.3)；
β-actin-rev,5’-GTGTTGGCGTAGAGGTCCTT-3’(SEQ ID NO.4)。
the reaction system is as follows: SYBR Premix ExTaq: 12.5. mu.L, upstream primer: 1 μ L, downstream primer: 1 μ L, cDNA: 1 μ L, sterilized water: 9.5. mu.L, the reaction program is: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 56 for 30s, and extension at 72 ℃ for 30s, for 40 cycles. According to 2-△△CTThe method calculates the expression level of the sample relative to the reference gene. To investigate MIZ whether it has antiviral effects on other foot and mouth disease virus subtypes, 100TCID was used50A/GDMM/CHA/2013 infected cells, as described above.
The experimental results are shown in FIGS. 2 to 4: the MTS test MIZ shows that MIZ can provide more than 60% of protection of IBDS-2 cells (FIG. 2) and significantly inhibit the expression level of FMDV mRNA (FIG. 4) when different concentrations of drugs are added respectively to the FMDV to determine whether FMDV has anti-FMDV virus activity. While MIZ does not provide effective protection of cells at concentrations below 25. mu. mol. Similarly, MIZ at concentrations of 12. mu. mol and above was effective in protecting IBDS-2 cells when infected with type A foot-and-mouth disease virus (FIG. 3), indicating that MIZ also has antiviral activity against type A FMDV.
2.3 Indirect immunofluorescence assay for FMDV protein expression in infected cell groups
The density is 3 x 105Spreading/well IBRS-2 cells on 12-well plate, removing upper culture solution after IBRS-2 cells grow to monolayer, washing with fresh DMEM for 3 times, and inoculating 100TCID50O/MY 98/BY/2010. After 1h, the virus solution was removed, washed 3 times with fresh DMEM, MIZ100 μ L diluted with a gradient of 2% FBS in DMEM medium was added, and the virus control wells were incubated at 37 ℃ for 12h with the corresponding DMSO concentration of the MMF preparation solution. Removing upper cell culture fluid, washing with PBS for 2 times, fixing cells with 4% paraformaldehyde for 15min, removing paraformaldehyde, adding methanol for 5min, rinsing with PBS for 3 times, each time for 5min, adding blocking solution (10% FBS, 0.3% Triton X-100, 89.7% PBS) for blocking for 10min, adding primary antibody (1:100) diluted with blocking solution, incubating at room temperature for 1 hr, rinsing with PBS for 3 times, each time for 5min, adding secondary antibody (1:200) diluted with blocking solution, incubating at room temperature for 1 hr, and incubating PB for 1 hrS rinse 5 times, 5min each time. Finally, 300. mu.L of DAPI was added to each well for staining, the staining was performed for 5min, PBS was rinsed 2 times for 5min each time, and the results were observed with a fluorescence microscope.
The results of the experiment are shown in FIG. 5 (scale 100 μm): a large amount of specific fluorescence was observed in the virus-infected IBRS-2 cells after untreated infection and virus-infected groups treated with 6, 12. mu. mol MIZ, while a small amount of fluorescence was observed in IBRS-2 cells of other treated groups. This result further confirmed MIZ that anti-foot-and-mouth disease virus activity on IBRS-2 cells is dose dependent.
2.4MIZ evaluation of inhibition time of foot-and-mouth disease virus infection of IBRS-2 cells:
well-grown IBRS-2 cells on a DMEM complete medium containing 10% FBS are spread on a 12-well plate, after the IBRS-2 cells grow to a monolayer, the upper layer culture solution of the cells is discarded, the cells are washed 3 times with fresh DMEM, and 100TCID is inoculated50O/MY 98/BY/2010. After 1h, the virus solution was removed, washed 3 times with fresh DMEM, and DMEM with 2% FBS was added as 0 h. MIZ was added to different wells at 0h, 2h, 4h, 8h, and 16h after virus infection to give a final concentration of 75. mu. mol. And meanwhile, a negative control without adding the medicine is set. CO at 37 deg.C2Culturing in a constant-temperature cell culture box for 48 h. Different groups of supernatants were collected and q-PCR and Western Blot were used to detect FMDV2B gene mRNA and FMDV VP1 protein levels, respectively. Extracting protein with protein lysate, and determining the concentration of the extracted protein by using BCA method. Preparing 12% separation gel to carry out protein SDS-PAGE denaturing electrophoresis, and after 2 hours of electrophoresis, electrically transferring the protein to a PVDF membrane. After the membrane is transferred for 2 hours, the membrane is put into 5 percent of freshly prepared skim milk powder for sealing for 1 hour. After blocking, the membrane was placed in rabbit anti-O FMDV VP1 polyclonal antibody (1:3000) and mouse anti-beta-actin polyclonal antibody (1:4000) and incubated overnight in a refrigerator at 4 ℃. Washing the membrane with TBST for 10min 5 times, then placing the membrane into corresponding secondary antibody HRP-labeled goat anti-rabbit IgG, HRP-labeled goat anti-mouse IgG (1:3000), incubating at room temperature for 1h, washing the membrane with TBST for 10min 5 times, and finally, developing and detecting FMDV VP1 protein by ECL chemiluminescence method.
The experimental results are shown in FIGS. 6 to 7: cells were treated with MIZ at different time periods after viral infection and the results showed significant inhibition of FMDV mRNA levels (figure 6) and VP1 protein levels (figure 7) compared to the negative control within 0-8 h of FMDV replication. The inhibition of MIZ was not evident at 16h, indicating that MIZ only acted early in FMDV replication and did not prevent viral replication when late in virus replication.
2.5 evaluation of anti-foot-and-mouth disease virus Activity of 2.5 MIZ in vivo:
24 SPF BALB/C suckling mice 3-4 days old are randomly divided into two groups, 12 mice/group, and the suckling mice and the mother mice are raised together. The type-O FMDV virus is derived from cell adaptive viruses of foot-and-mouth disease reference laboratories of China. The experiment was divided into two groups, in which 12 mice in the experimental group were injected with 50. mu.g of MIZ subcutaneously into the neck, and 12 mice in the control group were all inoculated with 100. mu.L of PBS containing 10% DMSO and 5% Tween-80 subcutaneously into the neck. After 2h, all suckling mice were injected subcutaneously in the neck with 100LD50PBS of type O FMDV at 100. mu.L. The death of the suckling mice was continuously observed and recorded. In order to ensure the safety of the experiment, the animal toxicity attack is carried out in a P3 laboratory, and the related operation conforms to the relevant regulations of the animal management committee of experiments.
The experimental results are shown in fig. 8: the control group of suckling mice died after 36h of virus infection, and all died at 60h, while the experimental group died after 48h and all died at 108 h. The total death time of the experimental group is delayed by 48 hours compared with that of the control group of suckling mice, so that MIZ can delay the lethal effect of the foot-and-mouth disease virus on the suckling mice.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Application of mizoribine in preparation of drugs for preventing foot-and-mouth disease virus infection
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Claims (2)
1. The application of mizoribine in preparing medicaments for preventing foot-and-mouth disease virus infection; the foot-and-mouth disease virus is A type foot-and-mouth disease virus and O type foot-and-mouth disease virus.
2. The use according to claim 1, wherein the mizoribine is present at a concentration of > 25 μmol/L.
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