CN109820852B - Application of nimodipine in preparation of drugs for preventing foot-and-mouth disease virus infection - Google Patents
Application of nimodipine in preparation of drugs for preventing foot-and-mouth disease virus infection Download PDFInfo
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- CN109820852B CN109820852B CN201910202396.0A CN201910202396A CN109820852B CN 109820852 B CN109820852 B CN 109820852B CN 201910202396 A CN201910202396 A CN 201910202396A CN 109820852 B CN109820852 B CN 109820852B
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Abstract
The invention relates to an application of nimodipine 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 drugs, in particular to application of nimodipine in preparation of drugs for preventing foot-and-mouth disease virus infection.
Background
Foot-and-mouth disease virus is a non-enveloped single-stranded positive-strand RNA virus of the picornaviridae family. It has been found that the virus comprises 7 serotypes, I, O, C, asian I, and south africa I, ii, iii, and each serotype is divided into a plurality of subtypes. Foot-and-mouth disease caused by the virus mainly infects animals with cloven hooves such as pigs, cattle and the like, and often forms blisters in the mouth, nose, hooves and other parts, accompanied by clinical symptoms such as fever, trekking and the like. Foot-and-mouth disease has multiple transmission ways, wide epidemic range and strong infectivity, is frequently outbreak in multiple countries at present, seriously threatens the development of the global animal husbandry and has great influence on the world economy and the human society. The disease is listed as the first of a type A animal epidemic disease list by the world animal health organization, China also ranks the disease at the first of a type A animal infectious disease list, and is also one of three single diseases in the China middle and long term animal epidemic disease prevention and treatment plan (2012-2020). At present, vaccine immunization is the main means for preventing and controlling foot-and-mouth disease, however, the use of the vaccine has an "immune window period", i.e. it cannot provide protection for animals within 7 days. Therefore, in order to compensate for the "immune window period", the development of novel effective antiviral drugs is urgently needed.
Disclosure of Invention
The invention aims to provide application of Nimodipine (Nimodipine) in preparation of a medicament 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 an application of nimodipine in preparing a medicament 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 concentration of the nimodipine in the medicament is 75-100 mu mol/L.
The invention also provides a foot-and-mouth disease virus inhibitor which comprises nimodipine and pharmaceutically acceptable auxiliary materials.
Preferably, the auxiliary material comprises one or more of starch, sugar powder, dextrin, polyethylene glycol and glycerol.
The invention provides an application of nimodipine in preparing a medicament for preventing foot-and-mouth disease virus infection. Nimodipine has an inhibiting effect on cytopathic effect induced by A-type foot-and-mouth disease virus and O-type foot-and-mouth disease virus, and inhibits the replication of the virus. Cell tests show that nimodipine has low cytotoxicity and has an inhibiting effect on replication of A type foot-and-mouth disease virus and O type foot-and-mouth disease virus; further experiments confirmed that nimodipine only works early in FMDV replication and does not prevent viral replication when it enters late stage of viral replication. Nimodipine can be used as an effective foot-and-mouth disease virus resisting component.
Drawings
FIG. 1 is a graph of the cytotoxicity of different concentrations of Nimodipine on IBRS-2 cells in example 1 of the present invention;
FIG. 2 is a graph showing the inhibitory effect of Nimodipine at various concentrations on the infection of type O FMDV with IBDS-2 cells in example 1 of the present invention;
FIG. 3 is a graph showing the inhibitory effect of Nimodipine at various concentrations on type A FMDV infection of IBDS-2 cells in example 1 of the present invention;
FIG. 4 is a graph showing the effect of Nimodipine at various concentrations on FMDV mRNA inhibition of type O FMDV infected cells in example 1 of the present invention;
FIG. 5 is a graph showing the effect of Nimodipine at various concentrations on the inhibition of VP1 protein expression in type O FMDV-infected cells in example 1 of the present invention;
FIG. 6 is a graph showing the effect of IFA detection of Nimodipine at different concentrations on the inhibition of FMDV protein expression in O-type FMDV infected cells in example 1 of the present invention;
FIG. 7 is a graph showing the effect of Nimodipine on FMDV mRNA inhibition of virus-infected cells at different time periods in example 1 of the present invention;
FIG. 8 is a graph showing the inhibitory effect of Nimodipine on VP1 protein expression in virus-infected cells at different time periods in example 1 of the present invention;
FIG. 9 is a graph showing the effect of Nimodipine on the death time of FMDV-infected suckling mice in example 1 of the present invention.
Detailed Description
The invention provides an application of nimodipine in preparing a medicament 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. Nimodipine has inhibitory action on cytopathic effect induced by A-type foot-and-mouth disease virus and O-type foot-and-mouth disease virus, and inhibits the replication of the virus, and nimodipine only acts in the early replication stage of FMDV, but cannot prevent the replication of the virus when entering the later replication stage of the virus. The medicine prepared by taking nimodipine as an effective component has low cytotoxicity, has an inhibiting effect on the replication of 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. The source of nimodipine in the present invention is not particularly limited, and conventional commercial products known to those skilled in the art may be used.
In the invention, the concentration of the nimodipine in the medicament is preferably 75-100 mu mol/L, and more preferably 100 mu mol/L.
The invention also provides a foot-and-mouth disease virus inhibitor which comprises nimodipine and pharmaceutically acceptable auxiliary materials.
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 nimodipine in preparing a medicament for preventing foot-and-mouth disease virus infection is further described in detail with reference to the following 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 FMDV and A/GDMM/CHA/2013) is preserved and provided BY the national foot-and-mouth disease reference laboratory; nimodipin 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; SYBRPremix 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; rabbit anti-O type FMDV hyperimmune serum is prepared by national foot and mouth disease reference factThe laboratory is a great green and good gift.
2. Experimental methods and results
2.1 toxicity assay of Nimodipine on IBRS-2 cells:
the cytotoxicity of Nimodipine against IBRS-2 cells was determined using the MTS method. After the cells are paved on a 96-well plate IBRS-2 and fully grow into a monolayer, the culture solution on the upper layer of the cells is discarded, the cells are washed for 3 times by using fresh DMEM, finally 100 mu L of Nimodipine diluted by the DMEM culture solution containing 2% FBS in a gradient manner is added, the corresponding DMSO concentration of the preparation solution of the Nimodipine is used as a negative control hole, and the cells are used as cell control holes 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 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% "the toxicity of Nimodipine at different concentrations on IBRS-2 cells was calculated. The experiment was independently repeated three times.
The experimental results are shown in fig. 1: MTS results show that with increasing drug concentration, the toxicity to IBRS-2 cells increases. When the concentration is 100 mu mol, the cell activity is more than 70%. When the concentration of the drug is 200. mu. mol or more, the activity of the cells decreases rapidly, and at a concentration of 600. mu. mol, the activity rate of the cells is 20%. Therefore, when the anti-FMDV is prepared using this drug, its concentration should not be higher than 100. mu. mol.
2.2 evaluation of Nimodipine activity against foot-and-mouth disease Virus 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 full monolayer, cell upper layer culture solution is discarded, the cells are washed 3 times BY fresh DMEM, and 100TCID50O/MY98/BY/2010 is inoculated. After 1h, virus liquid is removed, the cell is washed for 3 times by fresh DMEM, 100 mu L of Nimodipine which is diluted by DMEM culture solution containing 2% FBS in a gradient manner is added, the corresponding DMSO concentration of the Nimodipine preparation solution is used as a virus control hole, and Nimodipine and non-virus are used as cell control holes. Culturing at 37 deg.C for 48 hr, removing upper layer cellsThe culture was 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% "the antiviral effect of Nimodipine at different concentrations was calculated. At the same time, different groups of supernatants were collected, and q-PCR and Western Blotting were performed to detect the mRNA level of FMDV 2B gene and the FMDV VP1 protein level, respectively. RNA of the cells was extracted according to TRIZOL instructions, and fluorescent quantitative PCR was performed according to SYBR Premix Ex Taq II instructions, and β -actin was used as an internal reference gene. The primer sequence for detecting the specificity of FMDV 2B gene mRNA 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)；
beta-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
The method calculates the expression level of the sample relative to the reference gene. 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 for 5 times, 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 for 5 times by TBST for 10min, and finally detecting the FMDV VP1 protein by ECL chemiluminescence method. In order to study whether Nimodipine has an inhibitory effect on other foot-and-mouth disease virus subtypes, 100TCID50A/GDMM/CHA/2013 is used for infecting cells, and the MTS method is used for measuring the antiviral activity of the Nimodipine.
The experimental results are shown in FIGS. 2 to 5: the MTS is used for detecting whether Nimodipine has antiviral activity on FMDV, and the result shows that the Nimodipine can provide more than 85% of protection for the IBRS-2 cells (figure 2) and remarkably inhibit the expression of FMDV mRNA (figure 4) and VP1 protein level (figure 5) under the condition that drugs with different concentrations are added respectively; at a concentration of 50 μmol, Nimodipine did not provide effective protection of cells and reduced expression of FMDV mRNA and VP1 protein levels. Similarly, 100. mu. mol of Nimodipine was effective in protecting IBDS-2 cells when infected with type A foot-and-mouth disease virus (FIG. 3), suggesting that Nimodipine 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 105And (3) paving the/-hole IBRS-2 cells on a 12-hole plate, discarding culture solution on the upper layer of the cells after the IBRS-2 cells grow to be full of a monolayer, washing the cells with fresh DMEM for 3 times, and inoculating 100TCID50O/MY 98/BY/2010. After 1h, virus solution is removed, the cells are washed for 3 times by fresh DMEM, 100 mu L of Nimodipine which is diluted by a DMEM culture solution containing 2% FBS in a gradient manner is added, the corresponding DMSO concentration of a Nimodipine preparation solution is used as a virus control well, and the cells are placed at 37 ℃ for continuous culture for 12 h. Discarding the upper cell culture solution, washing with PBS for 2 times, fixing cells with 4% paraformaldehyde for 15min, discarding paraformaldehyde, adding methanol for 5min, rinsing with PBS for 3 times, 5min each time, 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 1h, rinsing with PBS for 3 times, 5min each time, adding secondary antibody (1:200) diluted with blocking solution, incubating at room temperature for 1h, rinsing with PBS for 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 experimental results are shown in fig. 6: a large amount of specific fluorescence was observed in the virus-infected IBRS-2 cells after untreated infection and in the virus-infected group treated with 50. mu. mol of Nimodipine, while a small amount of fluorescence was observed in the IBRS-2 cells of the other treated groups. This result further demonstrates that Nimodipine exhibits dose-dependent anti-foot-and-mouth disease virus activity on IBRS-2 cells.
2.4 evaluation of inhibition time of Nimodipine to foot-and-mouth disease virus infected IBRS-2 cells:
well-grown IBRS-2 cells on a DMEM complete medium containing 10% FBS were plated on a 12-well plate, after the IBRS-2 cells grew to a full monolayer, the cell supernatant was discarded, washed 3 times with fresh DMEM, and inoculated with 100TCID50O/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. Nimodipin was added to different wells at 0h, 2h, 4h, 8h, and 16h post-viral infection to a final concentration of 100. 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 FMDV 2B gene mRNA and FMDV VP1 protein levels, respectively.
The experimental results are shown in FIGS. 7 to 8: treatment of cells with Nimodipine at various time periods following viral infection showed significant inhibition of FMDV mRNA levels (figure 7) and VP1 protein levels (figure 8) compared to negative controls within 0-8 h of FMDV replication. The inhibition effect of Nimodipine at 16h is not obvious, which shows that Nimodipine only acts in the early stage of FMDV replication and cannot prevent virus replication when entering the later stage of virus replication.
2.5 evaluation of anti-foot-and-mouth disease virus activity of Nimodipine in vivo:
24 SPF BALB/C suckling mice of 3 days old are randomly divided into two groups, 12 mice/group, and the suckling mice and the mother mice are raised together. The experiment is divided into two groups, wherein 75 mu g of Nimodipine is injected into the neck part of 12 suckling mice in the experimental group subcutaneously, and 100 mu L of PBS containing 10% Tween-80 is inoculated into the whole neck part of 12 suckling mice in the control group subcutaneously. After 2h, all pups were injected subcutaneously in the neck with 100. mu.L PBS containing 100 LD 50O/MY 98/BY/2010. The death of the suckling mice was continuously observed and recorded. In order to ensure the safety of the experiment, the toxicity of the animals is attacked in a P3 laboratory, and the related operations conform to the related regulations of the Experimental animal management Committee of the Chinese academy of agricultural sciences.
The results of the experiment are shown in FIG. 9: the control group of suckling mice died from 30h after virus infection, all died at 54h, while the experimental group died at 60h, and the survival rate of suckling mice was 16.67% by 108 h. Therefore, Nimodipine can obviously delay the lethal effect of foot-and-mouth disease virus on 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
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
Application of nimodipine in preparation of drugs for preventing foot-and-mouth disease virus infection
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<170> SIPOSequenceListing 1.0
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Claims (1)
1. The application of nimodipine in preparing the medicament for preventing foot-and-mouth disease virus infection is characterized in that the foot-and-mouth disease virus is A type foot-and-mouth disease virus and O type foot-and-mouth disease virus, and the concentration of the nimodipine in the medicament is 75-100 mu mol/L.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1399558A (en) * | 1999-08-23 | 2003-02-26 | 免疫平衡技术有限公司 | Treatment of viral infections |
| CN1479616A (en) * | 2000-11-21 | 2004-03-03 | 霍华德・齐克 | Therapy for herpes neurological viral conditions utilizing 1,4-dihydropyridine calcium channel blockers |
| RU2281099C2 (en) * | 2000-11-21 | 2006-08-10 | Говард ЗИК | Treatment of herpetic neurological viral diseases by using 1,4-dihydropyridine blockers of calcium channels |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1399558A (en) * | 1999-08-23 | 2003-02-26 | 免疫平衡技术有限公司 | Treatment of viral infections |
| CN1479616A (en) * | 2000-11-21 | 2004-03-03 | 霍华德・齐克 | Therapy for herpes neurological viral conditions utilizing 1,4-dihydropyridine calcium channel blockers |
| RU2281099C2 (en) * | 2000-11-21 | 2006-08-10 | Говард ЗИК | Treatment of herpetic neurological viral diseases by using 1,4-dihydropyridine blockers of calcium channels |
Non-Patent Citations (2)
| Title |
|---|
| Engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (FMDV) VP1 inhibits type I interferon response in cells;Li, Xiaying等;《VETERINARY MICROBIOLOGY》;20130927;第166卷(第1-2期);第35-46页 * |
| 尼莫地平治疗小儿病毒性脑炎30例疗效观察;高淑芬;《河北医学》;20050430;第11卷(第4期);第380-381页 * |
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