CN117017976A - Application of thapsigargin in preparation of medicines for resisting porcine hemagglutinating encephalomyelitis virus infection - Google Patents

Application of thapsigargin in preparation of medicines for resisting porcine hemagglutinating encephalomyelitis virus infection Download PDF

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CN117017976A
CN117017976A CN202311122344.5A CN202311122344A CN117017976A CN 117017976 A CN117017976 A CN 117017976A CN 202311122344 A CN202311122344 A CN 202311122344A CN 117017976 A CN117017976 A CN 117017976A
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thapsigargin
encephalomyelitis virus
hemagglutinating encephalomyelitis
virus infection
medicines
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贺文琦
贾茜涵
石俊超
陆慧君
张竞
邱瑞召
马莹
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Jilin University
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Jilin University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention relates to application of thapsigargin in preparing medicines for resisting porcine hemagglutinating encephalomyelitis virus infection, and belongs to the technical field of veterinary medicines. Solves the technical problem that no specific therapeutic drug aiming at the pig hemagglutinating encephalomyelitis virus exists in the prior art. The invention discovers that thapsigargin can obviously inhibit proliferation of pig hemagglutinating encephalomyelitis virus for the first time. Cell models prove that thapsigargin can reduce the titer of viruses under the condition of not damaging the activity of cells and at the extremely low concentration (0.05 mu M), obviously inhibit the proliferation of the viruses on the gene, protein and cell level, has higher safety, can be used as a medicament for potentially resisting the infection of porcine hemagglutinating encephalomyelitis virus, has important significance for confirming medicament targets, and has good application prospect.

Description

Application of thapsigargin in preparation of medicines for resisting porcine hemagglutinating encephalomyelitis virus infection
Technical Field
The invention belongs to the technical field of veterinary medicines, and particularly relates to application of thapsigargin in preparation of medicines for resisting porcine hemagglutinating encephalomyelitis virus infection.
Background
Thapsigargin (Tg), a plant chemical substance that is found in the roots and fruits of the Mediterranean plant Thapsigargin, has been used in folk medicine for centuries to treat rheumatalgia, pulmonary disease and female infertility. Thereafter, tg was found to be an effective cytotoxin by inhibiting sarcoplasmic/endoplasmic reticulum Ca 2+ The atpase (SERCA) pump induces apoptosis and this biological activity facilitates the study of thapsigargin as a novel antitumor drug. In the latest studies, researchers have found that small doses of Tg elicit a highly potent broad-spectrum antiviral innate immune response.
Porcine Hemagglutinating Encephalomyelitis Virus (PHEV) belongs to a single-stranded RNA virus of the genus coronaviridae, family coronaviridae, order of monoviridae, and has a spherical shape, a diameter of 70-130 nm, a capsule membrane, and petal-shaped fibers of 20-30 nm on the surface of the capsule membrane. Coronaviruses are widely existing in the nature, the frequency of new coronavirus diseases is continuously accelerated since SARS occurs, the hazard degree is also increased, and great influence is caused on the health and life of human beings. PHEV and SARS-CoV, MERS-CoV and SARS-CoV-2 belong to members of the genus beta coronavirus, the earliest discovered coronavirus that infects pigs, and the only neurotropic coronavirus known to cause severe central nervous system infections in pigs. The swine hemagglutinating encephalomyelitis caused by PHEV infection is an acute and high-contact infectious disease, and can be classified into encephalomyelitis type, vomiting consumption type and influenza type according to clinical manifestations, and the three types can exist in the same pig group at the same time, and can also occur in different pig groups or areas. There is currently no specific therapeutic drug against this disease, so there is an urgent need to find new drugs against PHEV infection.
Disclosure of Invention
The invention aims to provide an application of thapsigargin in preparing medicines for resisting porcine hemagglutinating encephalomyelitis virus infection, and the thapsigargin is discovered to have the effect of resisting PHEV infection for the first time.
The technical scheme adopted by the invention for achieving the purpose is as follows.
The invention provides an application of thapsigargin in preparing medicines for resisting porcine hemagglutinating encephalomyelitis virus infection, wherein the application is one or two of the following (a 1) and (a 2):
(a1) The application of thapsigargin in preparing medicine for treating PHEV infection;
(a2) Application of thapsigargin in preparing medicine for preventing PHEV infection is provided.
Preferably, the PHEV infection resistant drug is a drug composition which takes thapsigargin as the only active ingredient and contains thapsigargin.
More preferably, the pharmaceutical composition is a pharmaceutical composition comprising thapsigargin and one or more pharmaceutically acceptable excipients.
Preferably, the PHEV infection resistant medicament is in the form of powder, granules, capsules or solution.
Preferably, the drug for treating PHEV infection is administered by intravenous injection, intraperitoneal injection, oral administration, aerosol inhalation or intracerebral administration.
Preferably, the concentration of the toxic carotene in the PHEV infection-resistant drug in the cells reaches 0.05 mu M to 1 mu M.
Compared with the prior art, the invention has the beneficial effects that:
the application of thapsigargin in preparing medicine for treating PHEV infection in vitro is shown by research on PHEV infected cell model, and the result shows that thapsigargin can inhibit virus proliferation in cells at very low concentration (0.05 mu M) and has expression on virus titer, gene and protein level. And thapsigargin has remarkable inhibition effect on PHEV under the condition of no cytotoxicity, and has high selection index. The thapsigargin can be used for preparing new medicines for resisting PHEV infection, and has important significance for identifying medicine targets.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the effect of different concentrations of thapsigargin solution on the viability of mouse brain neuroma mother cells (N2 a) in example 1 of the present invention. The abscissa is the logarithmic value of the concentration of thapsigargin, the left ordinate is the inhibition rate of thapsigargin to PHEV, indicated by black curve, and the right ordinate is the influence of thapsigargin to cell viability, indicated by red curve.
FIG. 2 shows the effect of different concentrations of thapsigargin on PHEV inhibition at the mRNA level in example 2 of the present invention.
FIG. 3 shows the inhibition effect of different concentrations of thapsigargin solution on the structural protein N and the non-structural proteins nsp1 and nsp8 of PHEV at the protein level in example 2 of the present invention.
FIG. 4 shows the effect of different concentrations of thapsigargin on PHEV inhibition at the cellular level in example 2 of the present invention.
Detailed Description
For a further understanding of the present invention, preferred embodiments of the invention are described below, but it is to be understood that these descriptions are merely intended to illustrate further features and advantages of the invention, and are not limiting of the claims of the invention.
The application of the thapsigargin in preparing the medicines for treating the swine hemagglutinating encephalomyelitis virus infection can be one or two of (a 1) and (a 2):
(a1) The application of thapsigargin in preparing medicine for treating PHEV infection;
(a2) Application of thapsigargin in preparing medicine for preventing PHEV infection is provided.
In the technical scheme, the PHEV infection resistant drug is a drug composition which takes thapsigargin as the only active ingredient and contains thapsigargin, wherein the drug composition is a drug composition which is formed by thapsigargin and one or more pharmaceutically acceptable auxiliary materials.
In the technical scheme, the preparation formulation of the PHEV infection resistant drug is powder, granules, capsules or solution. It should be noted that the dosage form of the PHEV infection-resistant drug is not limited thereto, and other dosage forms known to those skilled in the art are also suitable for the present invention.
In the technical scheme, the administration route of the PHEV infection resistant medicine is intravenous injection type, intraperitoneal injection type, oral administration type, aerosol inhalation type or intracerebral administration type. The administration of the drug for treating PHEV infection is not limited to this, and other administration methods known to those skilled in the art are also applicable to the present invention.
In the above technical scheme, it is preferable that the concentration of carotene in the cell, which is toxic to the PHEV infection-resistant drug, is 0.05. Mu.M to 1. Mu.M.
In the invention, thapsigargin is the prior art, and the chemical structural formula is as follows:
are commercially available.
The terms used in the present invention generally have meanings understood by those of ordinary skill in the art unless otherwise indicated.
The invention is further illustrated below with reference to examples.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. Materials, reagents, devices, instruments, equipment and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
Half-cell toxicity concentration of thapsigargin (CC 50 ) Half maximal Inhibitory Concentration (IC) 50 ) Measurement
1. Dissolution and storage of thapsigargin
1mg thapsigargin (Meilun Bio, MB 13319) was weighed, 768.3442. Mu.l of DMSO solution was added to prepare a 2mM thapsigargin solution, and the solution was stored at-20 ℃.
2. Culture of mouse neuroma blast (N2 a cell)
Cells were removed in liquid nitrogen, rapidly lysed within 1min, resuspended in DMEM containing 10wt% fetal bovine serum and 1wt% diabody (penicillin and streptomycin), and cultured with DMEM containing 6wt% fetal bovine serum and 1wt% diabody.
3. Half-cell toxicity concentration of thapsigargin (CC 50 ) Measurement
N2a cells were seeded into 96-well plates at 37℃with 100. Mu.l of cell suspension per well and 5% CO 2 Culturing in a cell incubator, preparing the thapsigargin into solutions with different concentrations by using DMEM when the thapsigargin grows to 70% -80%, adding the solutions into 96-well plates in the form of liquid exchange, wherein the concentrations are 1 mu M, 0.5 mu M, 0.1 mu M, 0.05 mu M, 0.01 mu M, 0.005 mu M and 0.001 mu M respectively, and setting a blank control group. After 24h incubation, a medium containing 10wt% CCK-8 was prepared and added as a liquid change. The cells were developed when incubated in the incubator for 1 hour, absorbance at 450nm was measured, and viability of the cells was calculated.
The calculation formula is as follows:
cell viability = [ (experimental well-blank well)/(control well-blank well) ] ×100%
4. Half-maximal Inhibitory Concentration (IC) of thapsigargin 50 ) Measurement
N2a cells were passaged into a twelve-well plate slide at 37℃with 5% CO 2 Culturing in a cell incubator, inoculating the toxin to 8 cell holes with MOI of 1 when the cell incubator grows to 60% -70%, and inoculating the toxin at 37 ℃ and 5% CO 2 After 1h incubation in the cell incubator, 1 well was replaced with DMEM solution containing 2wt% fetal bovine serum and DMSO at the highest drug concentration as DMSO control group, and the other 7 wells were replaced with DMEM solution (2 wt% fetal bovine serum) containing 1. Mu.M, 0.5. Mu.M, 0.1. Mu.M, 0.05. Mu.M, 0.01. Mu.M, 0.005. Mu.M, 0.001. Mu.M thapsigargin as drug group, respectively, at 37℃and 5% CO 2 After incubation of the incubator for a further 24 hours. Indirect immunization with 4wt% paraformaldehyde fixationFluorescence was detected and observed using a fluorescence microscope and recorded by photographing. The number of cells (blue fluorescent cell number and red fluorescent cell number) in each well was quantified by ImageJ software, the control group was set to 100%, the other groups were compared with DMSO-treated groups, and the half Inhibition Concentration (IC) was determined by the quantified ratio of cytoprotection of the drug-treated groups 50 ) Values.
The calculation formula is as follows:
virus inhibition ratio = [1- (drug group red fluorescent cell number/corresponding well blue fluorescent cell number)/(control group red fluorescent cell number/control group blue fluorescent cell number) ]. Times.100%
As shown in FIG. 1, thapsigargin solution has no obvious toxic effect on N2a cell activity at 0.001-1. Mu.M, and half cytotoxicity concentration (CC 50 ) The thapsigargin solution has a significant inhibitory effect on PHEV at 16.12. Mu.M and a half inhibitory concentration (CC 50 ) 0.0012. Mu.M. It can be seen that the median inhibitory concentration is much less than the median cytotoxic concentration.
Example 2
Inhibition of PHEV infection by thapsigargin
7 thapsigargin solutions with different concentrations of 1 mu M, 0.5 mu M, 0.1 mu M, 0.05 mu M, 0.01 mu M, 0.005 mu M and 0.001 mu M were selected to explore the inhibition effect of the thapsigargin solutions on PHEV, and the test was verified in multiple aspects from protein level, mRNA level and cell level.
1. Detection method of RT-PCR (reverse transcription-polymerase chain reaction) test
N2a cells were inoculated into two six-well plates, when they grew to 70% -80%, PHEV detoxification was performed with MOI of 1, after 1h, 1 of the wells was replaced with DMEM solution containing 2wt% fetal calf serum and DMSO with the highest drug concentration as DMSO control group, the other 7 wells were replaced with 1. Mu.M, 0.5. Mu.M, 0.1. Mu.M, 0.05. Mu.M, 0.01. Mu.M, 0.005. Mu.M, 0.001. Mu.M thapsigargin and DMEM solution containing 2wt% fetal calf serum as drug group, and each well was repeated three times. After 24h incubation in the incubator, the supernatant was collected, total RNA in the adherent cells was extracted, and the mRNA expression level of PHEVN in the cells was detected.
Extracting total RNA in cellRNA extraction and reverse transcription): after incubation, the supernatant was collected and washed three times with PBS. According to the instructions of the TransZolUpPlusRNAkit of the full gold company, 1ml of TransZolUp was added per well and after complete cell lysis, the subsequent experiments were performed according to instructions. After RNA extraction, the sample concentration was determined, and reverse transcription was performed according to the sample concentration using All-In-One5XRTMastermix instructions with a total system of 20. Mu.l, wherein All-In-One5XRTMastermix 4. Mu.l, 1ng of RNA was added to the remaining nucleic-Free-H 2 And (3) supplementing O. Placing in a water bath at 37deg.C for 15min, and placing in a water bath at 60deg.C for 10min. The RNA was reverse transcribed into cDNA. Real-time fluorescence PCR detection was performed using reverse transcribed cDNA as a template. The cDNA amplification reaction system was 20. Mu.l: 10. Mu.l SYBRGreenMix, 1. Mu.l forward primer, 1. Mu.l reverse primer, 6. Mu.l water, 2. Mu.l DNA template.
PHEV forward primer: 5'-TCTGGGAATCCTGACGAG-3';
PHEV reverse primer: 5'-AGGCGCTGCAACACTTAC-3';
GAPDH forward primer: 5'-CTCAACTACATGGTCTACATGTTC-3';
GAPDH reverse primer: 5'-ATTTGATGTTAGTGGGGTCTCGCTC-3';
the detection procedure is as follows: 95 ℃ for 2min;94 ℃ for 15s; 15s at 60℃for 40 cycles; and at 72℃for 10min.
With housekeeping gene GAPDH as control, use 2 ﹣△△CT The PHEV was analyzed for changes in mRNA transcription level.
The test results are shown in fig. 2, where P <0.0001 is compared with the virus infection control group, indicating significance of the difference; ns represents no difference significance. As can be seen from fig. 2, compared with the control group, thapsigargin of the present invention has a remarkable inhibitory effect on PHEV at a non-cytotoxic concentration, and exhibits a dose-dependent relationship.
2. Detection method of Westernblot test
And (3) inoculating the N2a cells into a six-hole plate, and carrying out poison-receiving and drug-adding treatment according to 7 concentrations when the cells grow to 70-80%. After the incubation is completed, intracellular proteins are extracted, and the protein expression level of intracellular PHEV is detected.
Method for extracting intracellular proteins: after incubation, the six well plates were removed from the cell incubator and washed three times with PBS. After discarding, 1ml of PBS was added, the cells were scraped with a cell scraper, added into a 1.5ml centrifuge tube, centrifuged at 12000rpm for 10min at 4℃and after discarding the supernatant, 200. Mu.l of protein lysate (RIPALysisBuffer) and protein inhibitor (PMSF) in a volume ratio of 100:1 were added to each tube and were stirred and mixed uniformly, and after lysis on ice for 30min, the supernatant was centrifuged at 12000rpm for 10min at 4℃and 180. Mu.l of 5 Xloading buffer was added and boiled in boiling water for 10min. Protein samples were then separated using SDS-PAGE, blocked with 5wt% nonfat dry milk after transfer to PVDF membrane, incubated with primary and secondary antibodies sequentially, and finally ECL developed. The self-made antibody of PHEV is used as the antibody, and the goat anti-rabbit antibody marked by horseradish peroxidase is used as the secondary antibody, and the concentration is 1:10000.
As shown in FIG. 3, it can be seen from FIG. 3 that thapsigargin has a very good inhibitory effect on PHEV when the concentration is more than 0.05. Mu.M, as shown in FIG. 3, both the structural protein N and the non-structural proteins nsp1 and nsp8 of PHEV.
3. Detection method for indirect immunofluorescence test
And (3) inoculating N2a cells into the twelve-hole plate cell climbing sheet, and carrying out poison-receiving and drug-adding treatment according to 7 concentrations when the cells grow to 60% -70%. After 24h incubation, fixation was performed with 4% paraformaldehyde solution and the cellular level of PHEV content was detected.
Indirect immunofluorescence assay procedure: the nutrient solution in the twelve-well plate was discarded, fixed with 4wt% paraformaldehyde preheated at 37℃for 10min at room temperature, and then discarded, and fixed with-40℃precooled methanol in a refrigerator at 4℃for 10min. After being discarded, PBS was used for three times, the membrane was permeabilized with 0.5wt% Triton X-100 at room temperature for 5min, PBS was used for three times, 5wt% skimmed milk powder was used for sealing at 37 ℃ for 1h, PBS was used for three times, the slide glass was taken out, marked on a glass slide, placed in a cassette, and the self-made PHEV-N protein antibody was incubated at 4 ℃ overnight. After washing with PBS for 15min, goat anti-rabbit red fluorescent antibody was incubated away from light, after incubation at 37℃for 1h, washing with PBS for 15min, sealing with anti-fluorescence quenching sealing tablet, observing with fluorescence microscope and recording with photographs.
As shown in FIG. 4, it can be seen from FIG. 4 that virus-infected cells were first present in the 0.01. Mu.M drug concentration as the thapsigargin concentration was decreased and the number of virus-infected cells was gradually increased as the drug concentration was further decreased. The results indicate a dose-dependent inhibition of PHEV infection by thapsigargin at the cellular level.
It is apparent that the above embodiments are merely examples for clarity of illustration and are not limiting examples. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (6)

1. Use of thapsigargin in the manufacture of a medicament for combating hemagglutinating encephalomyelitis virus infection in a pig, characterized in that said use is one or both of the following (a 1), (a 2):
(a1) Application of thapsigargin in preparing medicines for treating pig hemagglutinating encephalomyelitis virus infection;
(a2) Application of thapsigargin in preparing medicine for preventing pig hemagglutinating encephalomyelitis virus infection is provided.
2. The use of thapsigargin according to claim 1 for preparing medicines for resisting porcine hemagglutinating encephalomyelitis virus infection, wherein the medicines for resisting porcine hemagglutinating encephalomyelitis virus infection are pharmaceutical compositions containing thapsigargin as the only active ingredient.
3. The use of thapsigargin according to claim 1 for preparing a medicament for treating swine hemagglutinating encephalomyelitis virus infection, wherein the pharmaceutical composition is a pharmaceutical composition comprising thapsigargin and one or more pharmaceutically acceptable excipients.
4. The use of thapsigargin according to claim 1 for preparing medicines for resisting porcine hemagglutinating encephalomyelitis virus infection, wherein the medicines for resisting porcine hemagglutinating encephalomyelitis virus infection are in the forms of powder, granules, capsules or solutions.
5. The use of thapsigargin according to claim 1 for preparing a medicament for treating porcine hemagglutinating encephalomyelitis virus infection, wherein the administration route of the medicament for treating porcine hemagglutinating encephalomyelitis virus infection is intravenous injection, intraperitoneal injection, oral administration, aerosol inhalation or intracerebral administration.
6. The use of thapsigargin according to claim 1 for preparing a medicament against porcine hemagglutinating encephalomyelitis virus infection, wherein the concentration of thapsigargin in cells is 0.05 μm-1 μm.
CN202311122344.5A 2023-09-01 2023-09-01 Application of thapsigargin in preparation of medicines for resisting porcine hemagglutinating encephalomyelitis virus infection Pending CN117017976A (en)

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