CN118001261B - Application of nordihydroguaiaretic acid in preparation of medicines for inhibiting cat infectious peritonitis virus - Google Patents

Abstract

The invention provides an application of nordihydroguaiaretic acid in preparing a medicine for inhibiting cat infectious peritonitis virus, belonging to the field of biological medicine. The nordihydroguaiaretic acid has obvious inhibition effect on cat infectious peritonitis virus, and lays a foundation for developing new antiviral drugs and treating cat infectious peritonitis. The invention also provides a medicine for inhibiting cat infectious peritonitis virus, and the active ingredients of the medicine at least comprise nordihydroguaiaretic acid.

Description

Application of nordihydroguaiaretic acid in preparation of medicines for inhibiting cat infectious peritonitis virus

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to application of nordihydroguaiaretic acid in preparation of medicines for inhibiting cat infectious peritonitis virus.

Background

Feline infectious peritonitis (Feline infectious peritonitis, FIP) is a systemic, fatal disease caused by feline infectious peritonitis virus (Feline infectious peritonitis virus, FIPV), and is also the most important infectious disease and cause of death in cats.

The main pathological characteristics of cat infectious peritonitis are suppurative granuloma and vasculitis, and along with the development of diseases, clinical manifestations can be classified into wet type (exudative type), dry type (non-exudative type) and mixed type of the two clinical forms, and common pathological areas are liver, spleen, kidney, serous, mesenteric lymph node, brain, lung, eyes and the like.

Clinically significant symptoms include dyspnea, ascites, jaundice, uveitis, other symptoms including refractory fever, anorexia, somnolence, ataxia and weight loss.

Cats at all ages are susceptible to feline infectious peritonitis, with young cats under two years of age most frequently developing.

The cat infectious peritonitis is found in the 60 th century of 20, but the symptoms of the cat infectious peritonitis are not specific, so that the diagnosis of the cat infectious peritonitis still difficult, and the results of medical history, clinical symptom observation, laboratory examination and the like are comprehensively analyzed, so that the accuracy of diagnosis is improved as much as possible.

In terms of treatment, western veterinarians are mainly treated with antiviral and symptomatic treatments.

At present, the clinical anti-FIPV medicines at home and abroad mainly comprise two major classes of protease inhibitors and nucleoside analogues, and no specific medicines and vaccines are approved for marketing at home.

Recently, the differentiation and treatment of the infectious peritonitis of cats have been advanced, for example, a scholars take the symptoms of releasing the muscles, clearing heat and drying dampness, lifting yang qi and the like as treatment rules, and select a traditional Chinese medicine compound capable of effectively treating the infectious peritonitis of cats, wherein the traditional Chinese medicine compound consists of medicines such as kudzuvine root, kuh-seng, baical skullcap root, herba lycopi, dandelion, radix bupleuri, astragalus mongholicus, pilose asiabell root and the like; the students use spleen strengthening, qi regulating, dampness eliminating, yellow removing, intestine strengthening, diarrhea stopping and organism conditioning as treatment rules, and various traditional Chinese medicine formulas are applied, and then Western medicines such as interferon, antibiotics and the like are matched for combined treatment of FIP cats.

Despite decades of research by researchers in the pathogenesis, transmission laws, and control of feline infectious peritonitis, FIP remains one of the most common fatal feline diseases today and is also considered one of the most challenging feline infectious diseases known in veterinary medicine.

At present, safer, more effective and more convenient medicines for resisting cat infectious peritonitis viruses are urgently needed in the field, so that the purpose of preventing and treating cat infectious peritonitis is achieved.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of nordihydroguaiaretic acid in preparing the medicines for inhibiting the infectious peritonitis virus of cats, and provides a new idea for treating the infectious peritonitis of cats; the invention also provides a medicine for inhibiting the infectious peritonitis virus of cats.

To achieve the above object, the first aspect of the present invention provides the use of nordihydroguaiaretic acid in the preparation of a medicament for inhibiting infectious peritonitis virus in cats.

Nordihydroguaiaretic acid (Nordihydrog μ AIARETIC ACID, NDGA), a phenolic compound extracted from evergreen carbonic acid shrubs LARREA DIVARICATA.

In addition, nordihydroguaiaretic acid can also be isolated from the fruit of Schisandra chinensis.

Numerous studies have shown that nordihydroguaiaretic acid is useful in cancer therapy, as a hypoglycemic agent, as an anti-inflammatory and analgesic agent, as a chemoprotectant for lung cancer, in the treatment of arthritis and rheumatic diseases, as an antimicrobial agent against yeast and bacterial growth, in the treatment of gastric ulcers and dyspepsia.

As shown in formula (I), NDGA has two catechol rings that can confer very potent antioxidant activity by scavenging oxygen radicals, which may explain some of its therapeutic effects, but the rest of the medicinal mechanisms remain in the research stage.

In addition to pharmaceutical use, NDGA has also been used as a food antioxidant and nutritional supplement.

（I）

The inventor adopts a molecular butt joint method to successfully simulate the 3CLpro three-dimensional structure and the binding capacity of NDGA and cat infectious peritonitis virus FIPV, and the tight combination of NDGA and 3CLpro is confirmed for the first time.

By binding site analysis, the binding site of NDGA was found to be located near the dimerization region of 3CLpro, presumably NDGA is a potential 3CL protease inhibitor.

By using the FRET experimental method, the inhibitory effect of NDGA on 3CLpro of FIPV was first verified on the protein level, and the inhibitory effect was observed to exhibit concentration dependence.

In cytotoxicity experiments, the CCK-8 experimental method is adopted, so that the NDGA has low cytotoxicity and relatively high safety.

The effective inhibition of NDGA on FIPV at the cellular level is further qualitatively and quantitatively verified by confocal microscopy and flow cytometry experimental methods, and the inhibition effect is found to be concentration-dependent and is good.

The series of experimental results prove that the NDGA can effectively inhibit the infectious peritonitis virus FIPV of cats and has very high safety, thereby laying a foundation for developing new antiviral drugs and clinical application of the NDGA.

It was also noted that, as is clear from the above-described experimental results, nordihydroguaiaretic acid had a concentration-dependent effect on the suppression effect of FCoV viruses, and when the NDGA concentration was 2.5 μmol/L or more, the suppression effect on FCoV viruses was exhibited, and as the NDGA concentration was increased, the suppression effect on FCoV viruses was further enhanced.

Further, when the concentration of NDGA is 2.5 to 50. Mu. Mol/L, the effect of inhibiting FCoV viruses is more remarkable, and the cytotoxicity and safety are lower.

In a second aspect, the invention provides a medicament for inhibiting infectious peritonitis virus in cats, wherein the medicament comprises at least nordihydroguaiaretic acid as an active ingredient.

In other words, the active ingredient of the medicine can be nordihydroguaiaretic acid only, and other compounds can be further included on the basis.

Besides the effective components, the medicine for inhibiting the cat infectious peritonitis virus can also comprise pharmaceutically acceptable auxiliary materials.

Specifically, according to the different dosage forms of the medicines, proper auxiliary materials can be selected correspondingly.

The dosage forms of the medicine can be capsules, tablets, granules, gels, sustained release agents, oral liquid, dripping pills, emulsion, injection, nano preparations and the like.

Compared with the prior art, the invention has the following beneficial effects:

In molecular structure, NDGA is capable of tightly binding 3CLpro of FIPV; the IC50 value of half effective concentration of the NDGA to the important target 3CLpro of FIPV is 15.62 mu mol/L at the protein level, which further proves the remarkable effect of the NDGA on inhibiting the protease of the important target of FIPV; the cytotoxicity test proves that the NDGA with the experimental concentration does not show obvious cytotoxicity, which indicates that the NDGA has low cytotoxicity and higher safety; the half effective concentration EC50 value of NDGA on the FIPV is 9.958 mu mol/L on the cellular level, which shows that the NDGA has stronger inhibition capability on the cellular level, thus further proving the remarkable effect of NDGA on inhibiting the FIPV virus, thereby providing a new thought for developing medicines for preventing and treating cat infectious peritonitis.

The medicine provided by the invention takes NDGA as an active ingredient or a main active ingredient, and shows obvious anti-FIPV activity, so that the purpose of treating the infectious peritonitis of cats is achieved, and a new treatment idea is provided for treating the infectious peritonitis of cats. The result of the research not only hopefully opens up a new way for the treatment of the cat infectious peritonitis, but also provides a powerful scientific basis for the treatment research of related diseases.

Drawings

FIG. 1 is a diagram showing the 3CLpro molecular docking binding patterns of NDGA and FIPV in example 1 of the present invention;

FIG. 2 shows the inhibition of FIPV by protein level NDGA in example 2 of the present invention;

FIG. 3 shows the result of an NDGA cytotoxicity test in example 3 of the present invention;

FIG. 4 shows a cell capable of emitting green fluorescence under a confocal microscope in example 4 of the present invention;

FIG. 5a is a scatter plot of FSC-SSC obtained by detection using a flow cytometer in example 4 of the present invention;

FIG. 5b is a histogram of FITC single parameters obtained by detection using a flow cytometer in example 4 of the present invention;

FIG. 5c is a histogram of FITC positivity versus Count obtained by detection using a flow cytometer in example 4 of the present invention;

FIG. 6 shows the percentage of green fluorescent cells at different NDGA concentrations detected by flow cytometry in example 4 of the present invention.

Detailed Description

Feline coronavirus (Feline coronavirus, FCoV) is a common pathogen in domestic and wild cats and is classified as enterocoronavirus (FELINE ENTERIC coronavirus, FECV) which normally causes only mild diarrhea in cats and as lethal Feline Infectious Peritonitis Virus (FIPV).

Depending on the amino acid sequence of the viral S protein and the neutralization reaction of the antibody, FCoV can be divided into two serotypes of I, II, of which type I FCoV is difficult to culture in vitro and type II FCoV is easier to grow in cell culture.

FIPV type I is the most common serotype in diseased cats, but for the few strains isolated today, most are type II.

The difficulty in separating and culturing FIPV type I presents an obstacle to the mechanism research and diagnosis of infectious peritonitis in cats.

In addition to the above factors, the research results of viruses related to feline coronaviruses, such as human frequently infected coronaviruses, are limited in reference value for feline coronaviruses, and the effects of a large number of drugs having inhibitory effects on human frequently infected coronaviruses on feline coronaviruses are often difficult to expect, which also hinders the research and diagnosis of infectious peritonitis of cats.

The main reason for this is that there are differences in biological classification, genetic structure and viral receptors between feline coronavirus and human coronavirus that are commonly infected.

Regarding biological classification: cat coronavirus FcoV is a non-segmented single-stranded RNA virus belonging to the family Coronaviridae (Coronaviridae), genus alpha coronavirus (Alphacoronavirus), type alpha coronavirus (Alphacoronavirus 1).

Whereas human frequently infected coronaviruses such as HCoV-OC43, HCoV-HKU1, MERS, SARS, SARS-CoV-2 (novel coronaviruses) belong to the genus beta coronavirus, subgenera Sarbecovirus of the family coronaviridae.

Regarding the gene structure: FCoV the virus particles are round or polymorphic, have helical symmetry, and have a diameter of about 80 to 120 a nm a.

FCoV are enveloped and have spikes on their surface of about 15 to 20 a nm a size of about 29 a kb a genome of the virus, comprising 11 Open reading frames (Open READING FRAMES, ORFs) responsible for encoding 11 viral proteins, 4 of which are structural proteins and 7 of which are non-structural proteins.

The viral genome 5' -end 2/3 region comprises two open reading frames ORF1a and ORF1b, encoding replicase polyproteins pp1a and pp1ab.

FCoV pp1a/1ab is cleaved by virally encoded papain-like protease and 3CL protease into 16 nonstructural proteins forming a replication/transcription complex that participates in the viral replication process.

FcoV of the 7 nonstructural proteins include 2 replicase proteins (1 a1 b) and 5 helper proteins (3 a, 3b, 3c, 7a and 7 b).

Whereas human coronaviruses commonly infected, such as SARS-CoV, have a genome of about 29.7kb and contain 14 ORFs, ORF1a-ORF1b-ORF2-ORF4-ORF5-ORF9a encoding two polyproteins and four structural proteins, in sequence from the 5 'end to the 3' end.

In addition, 8 unique genes (ORF 3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b and ORF9 b) were arranged between ORF2 and ORF9a, encoding auxiliary proteins.

The other coronavirus, MERS-CoV, has a genome of about 30kb in length and consists of 10 ORFs, whose unique genes are ORF3, ORF4a, ORF4b and ORF5.

The genome of the novel coronavirus SARS-CoV-2 is about 29.9kb in length, has 11 ORFs encoding 12 proteins, and similar to SARS-CoV and MERS-CoV SARS-CoV-2 has typical 5 'and 3' end gene structures, encodes nonstructural and structural proteins, and also encodes the unique genes of the six accessory proteins (3 a, 6, 7a, 7b, 8 and 10).

Regarding viral receptors: the S1 region gene sequence of FCoV for both serotypes (serotype I and serotype II) is poorly homologous, indicating that types I and II FCoV enter the host cell via different receptors.

The receptor of type II FCoV is currently known as feline aminopeptidase N (fAPN), but the cellular receptor of type I FCoV is not yet known.

Whereas human commonly infected coronaviruses such as HCoV-NL63, SARS and SARS-CoV-2 bind to angiotensin converting enzyme 2 (ACE 2) mostly via the S1 subunit of the S protein, i.e.ACE 2 is the receptor of the virus into cells.

In addition, the viral receptor for HCoV-C43, HCoV-HKU1 is 9-O-acetylated neuraminic acid; the receptor of the MERS virus is DPP4; the receptor for HCoV-229E virus is human aminopeptidase N (hAPN).

In addition, the clinical symptoms of feline coronavirus and human frequently infected coronavirus are also different.

FIPV primarily infects monocytes and macrophages, with clinical symptoms of cats being generally nonspecific, with frequently occurring abnormal manifestations including anorexia, weight loss, fever, and mental depression.

Human coronaviruses mainly cause respiratory tract infections and cause gastroenteritis and neurological disorders.

Among the 7 known HCoVs, clinical symptoms are different.

HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are ubiquitous and distributed globally in the population, and commonly cause viruses causing pharyngolaryngitis and common cold in high seasons of respiratory diseases, and cause mild upper respiratory symptoms in normal population with perfect immune function, but may cause severe infection for infants and elderly with low immune function.

SARS-CoV and MERS-CoV are highly pathogenic viruses that can infect the lower respiratory tract of humans and cause severe respiratory syndrome, even death.

Given the above differences between feline coronavirus and human coronavirus that is commonly infected, no therapeutic or even therapeutic mechanism for human coronavirus is necessarily applicable to feline coronavirus treatment.

In addition, the human beings and cats have differences in physiological structure, metabolic pathways, drug reactions and the like, so that the pharmacodynamics and the pharmacokinetics characteristics of the same drugs are obviously different.

Differences in organ structure, size, proportion and tissue distribution affect the distribution, metabolism and excretion of drugs in the body, so that the efficacy and safety of the same drug may be different between humans and cats.

For example nelfinavir (nelfinavir) is a protease inhibitor that is effective against the SARS-CoV virus, but in vitro experiments the inhibition of FCoV by this drug was not ideal, probably due to the difference in 3CL protease sequences of SARS-CoV and FCoV.

And once FCoV has invaded the cell, nelfinavir loses its antiviral effect.

Nucleotide analogs are often used to treat viral diseases.

However, the adenine arabinoside analogues, uridine analogues 6-azauridine and guanosine analogues 3-deaguanosine against human herpesvirus all had poor inhibitory effect on FCoV.

All of the above reasons are important reasons for slow progress in drug development for FIP.

In addition, some drugs have a great side effect on FIP cats, and the drug development progress is also affected.

For example, ribavirin is a guanosine analogue widely used in antiviral therapies in humans, and studies have shown that it is effective in reducing the viral titre of FCoV in vitro experiments, but that clinical symptoms of cats treated with ribavirin in vivo experiments are aggravated, possibly due to ribavirin toxicity and serious side effects.

The inventors found in the study that nordihydroguaiaretic acid NDGA exhibited significant anti-FIPV activity, and accordingly conducted a series of studies and experiments, as will be described below by way of specific examples.

Example 1 molecular docking mimics the 3CLpro binding level of NDGA and FIPV

Immunohistochemical staining of feline coronavirus antigen in diseased tissue is considered to be a gold standard for diagnosis of FIP in clinical practice for diagnosis and treatment of feline infectious peritonitis.

The current view suggests that internal mutations in FIPV are caused by a single core/macrophage eosinophil transition.

The 3C-like protease (3C-likeprotease, 3 CLpro) is also called main protease (Mpro) and consists of 306 amino acids, and can further cleave the multimeric protein of coronaviruses, thereby generating helicase, RNA-dependent RNA polymerase and other relevant replication elements, and has important roles in virus proliferation and assembly.

The substrate binding site of the protease may serve as a starting point for antiviral drug design.

The active site of 3CLpro represents an important target for anti-coronavirus drug development due to its highly conserved sequence and the necessity for viral replication.

In order to study the structural basis of NDGA for inhibiting 3CLpro of FIPV, this example predicts the three-dimensional structure and binding capacity of small-molecule NDGA to target protein by using a virtual molecule docking method.

In the simulation, the stability of the binding of the two was evaluated by Autodock calculation of the intermolecular potential.

Because the scoring value of receptor-ligand docking integrates parameters such as ligand energy, receptor energy, binding energy between the two and the like, the larger the absolute value of the score value is, the stronger the affinity of the receptor is, and the stronger the binding between the receptor and the ligand is, therefore, the scoring value is smaller than that of the original ligand molecule as a primary screening condition.

The crystal structure of the FIPV 3CLpro complex is downloaded from the protein database (PDB ID:7 SNA).

The downloaded crystal structure is a structure that binds to the small molecule drug GC 376.

The crystal structure of the downloaded FIPV 3CLpro complex is processed as follows: the small molecule drug GC376 and the redundant H ₂ O molecules are deleted, so that preparation is made for molecular docking research.

The central coordinates of the active pocket of the 3CLpro complex of the feline infectious peritonitis virus are determined to be-46.4155, 16.5741 and-2.19207 according to the binding site of the 3CLpro protein in the small molecule drug GC376, and the size of the active pocket is 25 nm ×25 nm ×25 nm.

And running molecular docking software in a windows system by utilizing Autodock vina software, simulating the combination process of NDGA and 3CLpro, and obtaining a docking score.

After obtaining the results of the molecular docking experiments, a visual analysis was performed to generate a binding pattern map to observe and analyze the manner in which NDGA binds to 3CLpro, and the relative positions of the binding sites.

As shown in fig. 1, the binding pattern analysis showed that NDGA binds tightly to 3CLpro with binding sites near the dimerization region of 3 CLpro.

This means that NDGA may influence the dimerization state of 3CLpro by interacting with this region.

Since dimerization in viral replication is often a critical biological process, NDGA is presumed to exert an inhibitory effect by interfering with this process.

The docking fraction of NDGA with 3CLpro is-11.59 kcal/mol.

The more negative the docking score and the more absolute (i.e., the less the value of the docking score), the more generally indicates that the intermolecular bonds are tight, as it represents that the bonding process is an energetically favorable process.

In this example, a docking fraction of-11.59 kcal/mol indicates that the binding between NDGA and 3CLpro is relatively stable.

The degree of tightness of the binding pattern and the choice of binding site suggests that NDGA may be a potential FIPV inhibitor.

The potential inhibitor property provides a powerful theoretical basis for subsequent experiments.

Example 2 protein level verification of the Effect of NDGA on 3CLpro of FIPV

The fluorescence resonance energy transfer (flμ orescence resonance ENERGY TRANSFER, FRET) assay is a biomolecular interaction detection method based on optical phenomena.

In FRET experiments, after one fluorescent molecule absorbs energy, the energy can be transferred to another adjacent molecule by resonance and cause it to excite and fluoresce.

FRET assay is a gold standard for protease activity and is commonly used as the primary assay for screening 3CLpro inhibitors (see literature "Ma C, Tan H, Choza J, Wang Y, Wang J. Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays. Acta Pharm Sin B. 2022; 12(4): 1636-1651."）.

This example demonstrates the inhibitory effect of NDGA on 3CLpro of FIPV at the protein level using FRET technology.

The specific experimental operation steps are as follows:

(1) The 10mmol/L storage concentration of NDGA (manufactured by MedChemExpress Biotechnology Co., ltd. In U.S.A.) was adjusted to 50. Mu. Mol/L, 40. Mu. Mol/L, 30. Mu. Mol/L, 20. Mu. Mol/L, 12.5. Mu. Mol/L, 10. Mu. Mol/L, 6.25. Mu. Mol/L, respectively, using a buffer.

(2) The above test compounds of different concentrations were added to a black 96-well elisa plate at 25 μl per well.

At least 3 duplicate wells were made for each concentration; subsequently 3CLpro at a concentration of 3. Mu. Mol/L was added to each well, 25. Mu.L per well and incubated at 37℃for 30 min.

A blank control group and a negative control group were set up at the same time as the above-described experimental group, wherein 50 μl of buffer was used in each well of the blank control group, and 25 μl of buffer was used in the negative control group in place of the test compound.

(3) After the sample was prepared, substrate Dabcyl-KTSAVLQSGFRKME-Edans (Souzhou offshore protein technologies Co., ltd.) was rapidly added to each well at a concentration of 12.5. Mu. Mol/L, and 50. Mu.L per well.

(4) Immediately putting the black 96-hole ELISA plate into an ELISA apparatus (TECAN INFINITE M1000 PRO), and setting parameters as follows: excitation wavelength 340 nm, emission wavelength 535 nm, and RFU (RELATIVE FL μ orescence μnit, relative fluorescence units) values within 15 min were determined.

(5) According to the experimental result, a linear region is plotted by taking time (min) as an X axis and RFU as a Y axis, and a curve of the change of a fluorescence signal with time reflecting the change of the enzyme activity is drawn.

And taking the RFU value after the reaction is stable to carry out subsequent calculation.

Enzyme activity and IC50 values were calculated using GRAPHPAD PRISM. Percent (%) inhibition=100× (RFU of 1-experimental group ≡rfu of control group).

This calculation was used to compare the inhibitory effect of different concentrations of NDGA on 3CLpro and compared to the control group.

The experimental results are shown in FIG. 2.

As can be seen from fig. 2, NDGA exhibits a concentration-dependent inhibition at the protein level.

Specifically, NDGA exhibits an inhibitory effect on 3CLpro in the concentration range of 6.25 to 50 μmol/L, and the inhibition rate of 3CLpro increases with increasing NDGA concentration, and the final inhibition rate is maintained at about 90%.

The IC50 value was calculated to be 15.62. Mu. Mol/L.

The IC50 value indicates that a half maximum inhibitory concentration can be achieved at relatively low drug concentrations, further corroborating the effectiveness of NDGA as a potential inhibitor, showing the potential of NDGA in inhibiting 3CLpro activity of FIPV.

EXAMPLE 3 cytotoxicity assay of NDGA

CCK-8 assays are used to rapidly and sensitively detect cell proliferation and cytotoxicity.

The basic principle of the experiment is as follows: in the presence of an electron coupling reagent, 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-8) can be reduced by intramitochondrial dehydrogenase to generate a highly water-soluble orange-yellow formazan product (Formazan), the color depth of which is directly proportional to cell proliferation and inversely proportional to cytotoxicity, and the color depth and the cell number are in a linear relationship for the same cells, so that the OD value is measured at the wavelength of 450 nm by using an enzyme-labeled instrument, and the number of living cells can be indirectly reflected.

This example tested the toxic effects of NDGA at different concentrations on CRFK cells (the excellent center of innovation in molecular science, academy of sciences of china), and the specific procedures included:

(1) CRFK cells (feline kidney cells) in the logarithmic growth phase were taken and prepared as cell suspensions. CRFK cells were seeded in 96-well plates, 1×10 ⁴ cells per well, and incubated in a 5% carbon dioxide incubator at 37 ℃ for 12 hours.

(2) Test compounds (6.25. Mu. Mol/L, 12.5. Mu. Mol/L, 20. Mu. Mol/L, 25. Mu. Mol/L, 40. Mu. Mol/L, 50. Mu. Mol/L) were added to the culture wells at various concentrations.

A negative control group was set and only the maintenance medium, i.e. DMEM solution containing 2% fetal bovine serum, was added. After allowing the test compound and DMEM solution containing 2% fetal bovine serum to act on the cells for 24 hours, the effect on cell proliferation was evaluated.

(3) Mu.L of CCK8 reagent (Beijing Nuo Bayer Biotechnology Co., ltd.) was added to each well, and incubated at 37℃in a 5% carbon dioxide incubator for 1 hour.

(4) Absorbance at 450 nm was measured using a microplate reader.

Statistical analysis and data visualization were performed on the data with GRAPHPAD PRISM and the survival of CRFK cells was calculated to assess the effect of NDGA on cells and to infer the cytotoxicity level of the test compounds, the experimental results are shown in figure 4.

As shown in FIG. 4, the cell viability was higher than 80% at the concentration of NDGA ranging from 0 to 50. Mu. Mol/L, indicating that the cytotoxicity of NDGA was low and the safety was high.

Example 4. Determination of the effectiveness of NDGA at the cellular level;

4.1 confocal microscopy;

(1) CRFK cells in logarithmic growth phase were taken and prepared into cell suspensions.

CRFK cells were seeded in 6-well cell culture plates, 5×10 ⁵ cells per well, and incubated at 37 ℃ in a 5% carbon dioxide incubator for 12 hours.

(2) The culture medium in the original 6-well plate was discarded, and 2 mL complete medium per well, i.e., a DMEM solution of 10% fetal bovine serum, was added. CRFK cells were infected with AY-FCoV-GFP virus with green fluorescent label at MOI=0.1, i.e.150. Mu.L of virus stock was added per well.

Incubate at 37℃and 5% carbon dioxide incubator for 1 hour.

(3) Discarding virus liquid in a 6-hole plate, adding compounds to be tested (2.5 mu mol/L,5 mu mol/L,10 mu mol/L,20 mu mol/L and 40 mu mol/L) with different concentrations into a culture hole, and simultaneously setting a negative control group and a blank control group, wherein the negative control group is not added with the compounds to be tested and is only a DMEM solution for maintaining a culture medium, namely 2% fetal bovine serum; the blank group was CRKF cells not infected with virus, only the maintenance medium.

(4) After incubation at 37 ℃ and 5% carbon dioxide incubator for 24 hours, i.e. after the test compound acts on the cells for 24 hours, the effect of the test compound on CRKF cells is observed under confocal microscope, the FITC channel with wavelength 488 nm is selected, the parameters are kept unchanged, and the image is taken.

The location and intensity of fluorescence observed by confocal microscopy is a critical source of information.

The existence position and intensity of fluorescence reflect the distribution and quantity of virus in the cell, and the existence position of virus in the cell can be judged, and the virus quantity is estimated preliminarily.

The decrease in fluorescence intensity is a biological indicator that NDGA inhibits AY-FCoV-GFP virus at the cellular level.

Fig. 4 is a captured image.

The results showed that the blank group did not fluoresce, indicating that CRKF cells not infected with virus did not fluoresce green.

The results of this blank group provide a benchmark for subsequent drug inhibition.

The fluorescence intensity after addition of NDGA was significantly reduced and concentration-dependent compared to the negative control group.

Specifically, the fluorescence intensity decreased with increasing NDGA concentration, indicating that NDGA inhibition of FCoV viruses increased.

These results demonstrate that NDGA has the ability to effectively inhibit FCoV viruses at the cellular level.

4.2 Flow cytometry detection;

(1) CRFK cells in logarithmic growth phase were taken and prepared into cell suspensions.

CRFK cells were seeded in 6-well cell culture plates, 5×10 ⁵ cells per well, and incubated at 37 ℃ in a 5% carbon dioxide incubator for 12 hours.

(2) The culture medium in the original 6-well plate was discarded, and 2 mL complete medium per well, i.e., a DMEM solution of 10% fetal bovine serum, was added.

CRFK cells were infected with AY-FCoV-GFP virus with a green fluorescent label at an MOI of 0.1, i.e.150. Mu.L of virus stock was added to each well.

Incubate at 37℃and 5% carbon dioxide incubator for 1 hour.

(3) The virus solution in the 6-well cell culture plate was discarded.

Test compounds (2.5. Mu. Mol/L, 5. Mu. Mol/L, 10. Mu. Mol/L, 12. Mu. Mol/L, 15. Mu. Mol/L, 20. Mu. Mol/L) were added to the culture wells at various concentrations.

Simultaneously setting a negative control group and a blank control group, wherein the negative control group is not added with a compound to be detected and is only a maintenance culture medium, namely a DMEM solution of 2% fetal bovine serum; the blank group was CRKF cells not infected with virus, and only maintenance medium was added.

(4) After incubation for 24 hours at 37 ℃ in a 5% carbon dioxide incubator, i.e. after the test compound acts on the cells for 24 hours, the cells are digested with 0.25% trypsin solution, the cells are discretized into individual cells with PBS, the cell suspension is taken out, passed through a 40 μm cell sieve, and added to a flow cell dedicated tube for preparation.

(5) The proportion of fluorescent cells in the cells was determined in the FITC channel of the flow cytometer.

According to the positions of cells in the graph, the voltage of FSC and SSC is regulated in a parameter label in Cytometer FACScanto columns, so that the cells of the main group are in a central position in the graph, the positions of the cells are determined, and a proper loop gate is looped, so that data are recorded.

10,000 Cells were grasped by flow cytometry and the proportion of cells with green fluorescence was recorded.

Analysis was performed using FlowJo software and statistical analysis and data visualization were performed using GRAPHPAD PRISM and experimental results are shown in figures 5-6.

FIG. 5a is a FSC-SSC scatter plot; FIG. 5b is a FITC single parameter histogram; FIG. 5c is a FITC-positive-Count histogram.

As shown in fig. 5, the flow cytometry results for the negative control group are plotted showing the quantitative analysis of the percentage of fluorescent cells by flow cytometry.

The method can accurately evaluate the quantity of the virus in the cells and provides a reliable index for evaluating the effectiveness of the drug at the cellular level.

The percentage of fluorescent cells reflects the viral content in the cells, and therefore, the fewer fluorescent cells, the less virus is present in the cells, which further demonstrates the better inhibitory effect of the drug.

The inhibition effect of NDGA on FIPV can be accurately estimated through quantitative analysis of the percentage of fluorescent cells of a flow cytometer.

In fig. 6, NDGA concentrations of 12 μmol/L, 15 μmol/L, and 20 μmol/L were significantly different from the negative group (P < 0.0001), NDGA concentration of 10 μmol/L was significantly different from the negative group (P < 0.001), and NDGA concentration of 5 μmol/L was significantly different from the negative group (P < 0.05).

As is clear from FIG. 6, NDGA shows a concentration-dependent inhibitory effect at the cellular level, specifically, the fluorescence intensity gradually decreases as the concentration of NDGA increases, so that the inhibition rate of NDGA against FCoV virus is positively correlated with the EC50 value of 9.956. Mu. Mol/L.

This result clearly indicates that NDGA is able to effectively inhibit FIPV virus in cells.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the experimental scheme described in the previous embodiments can be modified, or some or all experimental conditions can be adjusted; such modifications and adaptations do not depart from the essence of the corresponding technical solutions in the scope of the technical solutions of the embodiments of the present invention.

Claims (3)

1. Use of nordihydroguaiaretic acid in the manufacture of a medicament for the treatment of infectious peritonitis in cats.

2. The use according to claim 1, wherein the concentration of nordihydroguaiaretic acid is not less than 2.5 μmol/L.

3. The use according to claim 2, wherein the concentration of nordihydroguaiaretic acid is 2.5 to 50 μmol/L.