CN114224891A

CN114224891A - Application of clofazimine in preparation of medicines for preventing and treating Porcine Reproductive and Respiratory Syndrome (PRRSV)

Info

Publication number: CN114224891A
Application number: CN202111646737.7A
Authority: CN
Inventors: 朱明展; 付强; 于巧萍; 陈星兆; 程云; 杨亚林; 马春全
Original assignee: Foshan University
Current assignee: Foshan University
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2022-03-25
Anticipated expiration: 2041-12-29
Also published as: CN114224891B

Abstract

The invention discloses application of clofazimine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome and resisting PRRSV. In a cell experiment, Western Blot, qRT-PCR and other technologies prove that clofazimine can obviously inhibit PRRSV infection and replication and has obvious PRRSV resistant effect in multiple layers.

Description

Application of clofazimine in preparation of medicines for preventing and treating Porcine Reproductive and Respiratory Syndrome (PRRSV)

Technical Field

The invention relates to the field of veterinary medicines, in particular to application of clofazimine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome and resisting PRRSV.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS), also known as Porcine reproductive and respiratory syndrome, is caused by PRRSV.

The blue ear disease was first developed in 1987 in the united states and subsequently spread to europe, and PRRSV was isolated in 1996 in china. Currently, PRRSV is classified into two genotypes based on genomic sequence and antigenic differences, one european type, also known as PRRSV1, represented by the Lelystad Virus (LV) strain, and the other american type, also known as PRRSV 2, represented by the ATCC VR-2332 strain. In China, PRRSV mainly takes American type as the main part, and is difficult to control because of the characteristics of immunosuppressive property, antibody dependence enhancement, persistent infection, latency time, long maintenance time and the like.

The primary target cells of PRRSV include Porcine Alveolar Macrophages (PAMs), alveolar type II pneumocytes (pneumocytes type II), peripheral monocytes, seminiferous tubule (seminiferous tubules) epithelial germ cells (epithelial germ cells) and interstitial macrophages, macrophages in ovarian follicles. Wherein the porcine alveolar macrophages are most sensitive, and the infection of the porcine alveolar macrophages by PRRSV can cause the immune system of the pig to be damaged.

Blue ear disease is also a highly contagious viral infectious disease, with endemic prevalence. For pigs of different breeds and ages, PRRSV can infect. The transmission route is wide, and the medicine can be transmitted by contact infection, air transmission and seminal fluid, and can also be transmitted vertically through placenta.

At present, the PRRS mainly comprises inactivated vaccines and attenuated vaccines, but certain defects exist, so that the prevention and control effects are limited. The inactivated vaccine has the following disadvantages: large-dose vaccination or application of concentrated antigen is required, which leads to high vaccination cost besides short immunization period and frequent need of intensive vaccination; the inactivated vaccine has weak cellular immunity, and in addition, 2-3 weeks are required for generating complete immunity, so that emergency preventive inoculation is not facilitated; there is also the possibility of incomplete inactivation and detoxification. Attenuated vaccines present the risk of virulence reversion, recombination and potential infection.

It is seen that improvements and enhancements to the prior art are needed.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide the application of clofazimine in preparing the medicines for preventing and treating the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and aims to solve the problem of poor effect of the medicines for preventing and treating the porcine reproductive and respiratory syndrome in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of clofazimine in preparing medicine for preventing blue ear disease.

Application of clofazimine in preparing medicine for treating blue ear disease.

Application of clofazimine in preparing medicine for resisting PRRSV.

Has the advantages that:

the invention discloses that clofazimine has obvious antiviral effect on PRRSV, and particularly proves that clofazimine can obviously inhibit PRRSV infection and replication through Western Blot, qRT-PCR and other technologies on multiple levels, has obvious PRRSV resistant effect, and can be applied to preparation of medicines for preventing or treating porcine reproductive and respiratory syndrome and medicines for resisting PRRSV.

Drawings

FIG. 1 shows the cytotoxicity results of clofazimine at various concentrations.

FIG. 2 shows the result of detecting the expression level of PRRSV N protein in cells by Western Blot in clofazimine and PRRSV co-treatment experiments.

FIG. 3 shows the result of detecting the PRRSV N gene expression level in cells by qRT-PCR in the experiments of clofazimine and PRRSV co-treatment.

FIG. 4 shows the result of detecting the expression level of PRRSV N protein in cells by Western Blot in the chlorine method zimine pretreatment and PRRSV post-infection experiment.

FIG. 5 shows the results of detecting the expression level of PRRSV N gene in cells by qRT-PCR in the chlorine method ziming pretreatment and PRRSV post-infection experiment.

FIG. 6 shows the result of detecting the expression level of PRRSV N protein in cells by Western Blot in the experiment of adding PRRSV N after infection and cloxacillin.

FIG. 7 shows the result of qRT-PCR detection of PRRSV N gene expression in cells in the experiment of adding PRRSV after infection and cloxacillin.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Clofazimine (CFZ) is mainly used as a drug for treating human body tumor type leprosy at present, and is used for treating tuberculosis by matching with other drugs, has narrow indication, and belongs to a second-line drug.

At present, the application of clofazimine to the treatment of porcine reproductive and respiratory syndrome is not reported, and the technical scheme of the invention is as follows: application of clofazimine in preparing medicines for preventing and treating blue ear disease and resisting PRRSV.

The following examples are experiments to verify the safety and efficacy of clofazimine, the results of which were statistically examined, all experiments were repeated at least 3 times independently, the results were expressed as mean and standard error, and single-factor analysis of variance and T-test analysis were used. All statistical analyses used a P <0.05 as a test standard with significant statistical differences, SPSS 16.0 and GraphPad Prism 5 as analytical software.

Example 1

Cytotoxicity test of clofazimine

1. Experimental Material

Clofazimine (CFZ) 99.23% pure, supplied by MCE.

CCK-8: the fluorescent metabolic indicator is purchased from GlpBio company and used as a living cell metabolism indicator, and can generate measurable fluorescent metabolic products under the enzymatic reduction reaction of mitochondria, and the activity of the cells can be monitored by measuring the fluorescence intensity of the fluorescent metabolic indicator.

2. Test method

Culturing Marc-145 cells by using a DMEM culture solution containing 10% fetal calf serum until the cell confluency is 60-70%, discarding the culture solution, adding a nutrient solution containing dilution of ziming fold ratio by a chlorine method for 48 hours, taking PBS as a control group, adding 10% (v/v) CCK-8, continuing culturing for 3 hours, and reading the absorbance of 450nm by using a multifunctional microplate reader.

Data processing: the cell activity of the PBS control group is taken as 100%, and the absorbance of the diluted clofazimine-treated cells is compared with the absorbance of the PBS control group, namely the relative cell activity of the clofazimine at different concentrations, and the result is shown in FIG. 1.

3. Results

As can be seen from FIG. 1, when the concentration of clofazimine is 1. mu.M or less, it is not toxic to Marc-145 cells and the activity of the cells is 100%.

Example 2

Experiment one: influence of clofazimine and PRRSV co-treatment on cell anti-PRRSV

The PRRSV 2 strain CHR6 virus is taken as an example to research the anti-PRRSV effect of clofazimine.

(1) Marc-145 cells are cultured in a 12-well plate of DMEM culture solution containing 10% fetal calf serum until the cell confluency reaches 70%, the culture solution is discarded, PBS is washed for 3 times, then the Marc-145 cells are infected by PRRSV with MOI ═ 1, and chlorine method zimine (the final concentration is 0.5 mu M and 1 mu M) is added for culture, and a control group without chlorine method zimine is set. After 24 hours of culture, the culture medium was discarded, washed 3 times with PBS, then digested with 0.25% trypsin, the cells were lysed, the cell protein concentration was determined, and the expression amount of PRRSV N protein in the cells was determined by Western Blot, as shown in fig. 2. In the figure, CFZ is clofazimine.

As can be seen from the results of FIG. 2, clofazimine can obviously inhibit the expression of PRRSV N protein, and the inhibition effect is dose-dependent. The clofazimine can obviously inhibit the expression of virus protein.

(2) Marc-145 cells are cultured in a 12-well plate of DMEM culture solution containing 10% fetal calf serum until the cell confluency reaches 70%, the culture solution is discarded, PBS is washed for 3 times, then the Marc-145 cells are infected by PRRSV with MOI ═ 1, and chlorine method zimine (the final concentration is 0.5 mu M and 1 mu M) is added for culture, and a control group without chlorine method zimine is set. After 24h of culture, the culture solution is discarded, PBS is washed for 3 times, then 0.25% pancreatin is used for digestion, the cells are cracked, the total RNA of the cells is extracted, then reverse transcription is carried out, and the relative expression quantity of PRRSV-N gene/GADPH gene in the cells is determined by qRT-PCR, and the result is shown in figure 3.

As can be seen from the results of FIG. 3, clofazimine can obviously inhibit the expression of PRRSV N gene, and the inhibition effect is dose-dependent. The clofazimine can obviously inhibit the expression of virus genes.

Example 3

Experiment two: influence of chlorine method zimine pretreatment and PRRSV after infection on cell anti-PRRSV

(1) Culturing Marc-145 cells in a 12-well plate of a DMEM medium containing 10% fetal calf serum until the cell confluency is 70%, discarding the culture solution, washing with PBS 3 times, adding clofazimine (0 mu M, 0.5 mu M and 1 mu M) with different concentrations into different groups respectively, culturing for 4h, then infecting the Marc-145 cells with PRRSV with MOI of 1, continuing culturing for 24h at 37 ℃ in the DMEM medium containing 2% fetal calf serum, discarding the culture solution after finishing the culture, washing with PBS 3 times, then digesting with 0.25% pancreatin, cracking the cells, determining the cell protein concentration, and determining the expression amount of PRRSV N protein in the cells by Western Blot, wherein the result is shown in FIG. 4.

As can be seen from the results in FIG. 4, clofazimine can obviously inhibit the expression of PRRSV N protein, and the inhibition effect is dose-dependent. The results show that the virus protein expression can be obviously inhibited by adding clofazimine and then inoculating the virus.

(2) Culturing Marc-145 cells in a 12-well plate of a DMEM medium containing 10% fetal calf serum until the cell confluency is 70%, discarding the culture solution, washing with PBS 3 times, adding clofazimine (0 mu M, 0.5 mu M and 1 mu M) with different concentrations into different groups respectively, culturing for 4h, then infecting the Marc-145 cells with PRRSV with MOI of 1, continuing culturing for 24h at 37 ℃ in the DMEM medium containing 2% fetal calf serum, discarding the culture solution after the culture is finished, washing with PBS 3 times, then digesting with 0.25% pancreatin, cracking the cells, extracting total RNA of the cells, then carrying out reverse transcription, and determining the relative expression quantity of the PRRSV-N gene/GADPH gene in the cells by qRT-PCR, wherein the result is shown in FIG. 5.

As can be seen from the results of FIG. 5, clofazimine can obviously inhibit the expression of PRRSV N gene, and the inhibition effect is dose-dependent. The results show that the virus gene expression can be obviously inhibited by adding the clofazimine and then inoculating the virus.

Example 4

Experiment three: influence of PRRSV infection and clofazimine addition on cell anti-PRRSV

(1) Culturing Marc-145 cells in a 12-well plate of a DMEM medium containing 10% fetal calf serum until the cell confluency is 70%, discarding the culture solution, washing with PBS 3 times, culturing for 4h by inoculating toxin PRRSV with MOI 1, adding different concentrations of clofazimine (0 mu M, 0.5 mu M and 1 mu M) into different groups, continuously culturing for 24h at 37 ℃ in the DMEM culture solution containing 2% fetal calf serum, discarding the culture solution after the culture is finished, washing with PBS 3 times, digesting with 0.25% pancreatic enzyme, cracking the cells, determining the cell protein concentration, and determining the expression level of PRRSV N protein in the cells by Western Blot, wherein the result is shown in figure 6.

As can be seen from the results of FIG. 6, clofazimine can obviously inhibit the expression of PRRSV N protein, and the inhibition effect is dose-dependent. The results show that the virus inoculation is firstly carried out and then the clofazimine is added, so that the virus protein expression can be obviously inhibited.

(2) Culturing Marc-145 cells in a 12-well plate of a DMEM medium containing 10% fetal calf serum until the cell confluency is 70%, discarding the culture solution, washing with PBS 3 times, culturing for 4h by inoculating toxin PRRSV with MOI 1, adding different concentrations of clofazimine (0 mu M, 0.5 mu M and 1 mu M) into different groups, continuously culturing for 24h at 37 ℃ in the DMEM culture solution containing 2% fetal calf serum, discarding the culture solution after the culture is finished, washing with PBS 3 times, digesting with 0.25% pancreatin, cracking the cells, extracting total RNA of the cells, carrying out reverse transcription, and determining the relative expression quantity of the PRRSV-N gene/GADPH gene in the cells by qRT-PCR (quantitative reverse transcription-polymerase chain reaction), wherein the results are shown in FIG. 7.

As can be seen from the results of FIG. 7, clofazimine can obviously inhibit the expression of PRRSV N gene, and the inhibition effect is dose-dependent. The result shows that the clofazimine can obviously inhibit the expression of virus genes after the virus inoculation.

The invention proves that clofazimine has the capacity of inhibiting the expression of virus genes and proteins when added at different periods through the embodiment on the level of cell experiments, and can be applied to the preparation of medicines for preventing or treating the blue-ear disease and resisting the PRRSV.

It should be understood that equivalents and modifications of the technical solution and inventive concept thereof may occur to those skilled in the art, and all such modifications and alterations should fall within the scope of the appended claims.

Claims

1. Application of clofazimine in preparing medicine for preventing blue ear disease.

2. Application of clofazimine in preparing medicine for treating blue ear disease.

3. Application of clofazimine in preparing medicine for resisting PRRSV.