CN117653633A - Application of tetrandrine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome - Google Patents
Application of tetrandrine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of veterinary medicines, in particular to application of tetrandrine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome. The invention discloses the effect of tetrandrine on resisting porcine reproductive and respiratory syndrome virus for the first time, and researches on a plurality of methods prove that the tetrandrine has good antiviral effect on the porcine reproductive and respiratory syndrome virus at the cellular level. The tetrandrine provided by the invention is used as an effective component for resisting porcine reproductive and respiratory syndrome virus, has the advantages of definite action mechanism, controllable quality and lower toxicity, and has a good application prospect in the aspects of porcine reproductive and respiratory syndrome prevention and treatment.
Description
Technical Field
The invention relates to the field of veterinary medicines, in particular to application of tetrandrine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome.
Background
The porcine reproductive and respiratory syndrome is also called blue-ear disease, is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus in arteriviridae, is characterized by sow reproductive disorder, piglet and fattening pig respiratory tract symptoms and high mortality rate, is widely spread worldwide, and causes great economic loss to pig industry. Chang Jifa swine fever, eperythrozoon and other diseases, and if the treatment is not timely, the death rate of pigs is high.
The present method mainly comprises the means of vaccine control, antiviral chemical synthesis medicine, exogenous cytokine treatment and the like. The virus strain of the reproductive and respiratory syndrome virus has diversity, is extremely easy to mutate and recombine, the pathogenic and immune mechanisms of the virus are still undefined, and particularly the highly pathogenic virus strain is widely popular, so that the clinical symptoms of the virus are diversified, the protective power of the vaccine is not ideal, particularly the excessive use of attenuated live vaccine can increase the diversity of the virus strain, the clinical complexity and the control difficulty of the virus are increased, and the result is that the new virus strain is endless, the immune pig group is induced to produce immune tolerance, and the continuous infection and the secondary infection of the pig group are caused to rise.
The Chinese herbal medicine is the treasure of traditional medicine in China, has the characteristics of good curative effect, low side effect and the like, is widely accepted and favored, and is researched and developed for thousands of years, and the Chinese traditional medicine types and the use of the Chinese traditional medicine are the first in the world. Compared with western medicines, the Chinese herbal medicine has the characteristics of environmental protection, small side effect, overall regulation and the like, and has bright application prospect in preventing and treating veterinary infectious diseases. The traditional Chinese medicine has no effect faster than western medicines in treating bacterial and acute diseases, but has obvious advantages in treating viral diseases, regulating immunity and the like and is widely paid attention to. In recent years, antiviral effects of traditional Chinese medicine preparations have been increasingly emphasized.
Tetrandrine (Fangchinoline) is derived from dried root of Fangchinoline family plant powder, and has molecular formula of C 37 H 40 N 2 O 6 The molecular weight is 608.72, and the molecular structural formula I is shown as follows:
tetrandrine has various biological functions such as antiinflammatory, analgesic, antioxidant, nonspecific blocking of calcium ion channel, inhibiting histamine release, dilating coronary artery, reducing oxygen consumption of myocardium, inhibiting platelet aggregation, lowering blood pressure, and lowering blood sugar.
Tetrandrine is often used as an analgesic, antirheumatic and antihypertensive drug in traditional Chinese medicine. In recent years, their anticancer effect has been attracting attention. The literature indicates that tetrandrine can inhibit tumor cells by inducing apoptosis, blocking cell cycle progression, inhibiting tumor cell invasion and other various ways. In addition, tetrandrine is used as one of the main alkaloids contained in tetrandrine, has obvious effects of relieving pain, relieving fever, diminishing inflammation, promoting urination, dilating coronary artery, lowering blood pressure and resisting cancer, and has certain toxicity to liver and kidney when used in large dose or for a long time, wherein liver damage is the weight, and the damage degree has a certain proportional relation with the medicine dosage. Furthermore, tetrandrine is a potential drug for treating prostate cancer, which can obviously increase the expression level of pro-apoptotic protein Bax and activated Caspase-3 protein and inhibit the corresponding decrease of apoptotic protein Bcl-2. At present, the research report of the action of tetrandrine on resisting porcine reproductive and respiratory syndrome virus is not seen.
Disclosure of Invention
The invention aims to provide an application of tetrandrine in preparing a medicament for preventing and treating porcine reproductive and respiratory syndrome, so as to solve the problems in the prior art. The tetrandrine can inhibit replication of porcine reproductive and respiratory syndrome virus, and has good effect of resisting Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
In order to achieve the above object, the present invention provides the following solutions:
the invention provides an application of tetrandrine in preparing a medicament for preventing and treating porcine reproductive and respiratory syndrome.
The invention provides a medicine for preventing and treating porcine reproductive and respiratory syndrome, and the effective components of the medicine comprise tetrandrine.
Preferably, the concentration of the active ingredient is 1-7. Mu.M.
Preferably, the concentration of the active ingredient is 7. Mu.M.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the dosage form of the medicament includes powder, granule, tablet or capsule, but is not limited to powder, granule, tablet or capsule, and can be any veterinary clinically acceptable dosage form.
The invention provides an application of tetrandrine in preparing a medicine for inhibiting porcine reproductive and respiratory syndrome virus proliferation.
The invention provides a drug for inhibiting porcine reproductive and respiratory syndrome virus proliferation, wherein the active ingredients of the drug comprise tetrandrine.
Preferably, the concentration of the active ingredient is 1-7. Mu.M.
Preferably, the concentration of the active ingredient is 7. Mu.M.
The invention discloses the following technical effects:
the invention discloses the PRRSV effect of tetrandrine for the first time, and researches on various methods prove that the tetrandrine has good antiviral effect on PRRSV at the cellular level. The tetrandrine provided by the invention is used as an effective component for resisting PRRSV, has the advantages of definite action mechanism, controllable quality and low toxicity, and has a good application prospect in the aspects of pig breeding and prevention and treatment of respiratory syndrome.
The radix stephaniae tetrandrae of the family Chinese medicine is widely applied in clinic and folk of Chinese medicine, and has high biological safety. The tetrandrine is a main medicinal component of the stephania tetrandra, has higher content in the stephania tetrandra, has mature and reliable preparation process, and can provide sufficient raw materials for preparing the anti-PRRSV medicament.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, 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 is the result of a test for determining the cytotoxicity of tetrandrine against Marc-145 by CCK-8 in example 1, wherein Concentration ofFang is the concentration of tetrandrine and Cell viability is the Cell viability;
FIG. 2 shows the inhibition of RNA proliferation of porcine reproductive and respiratory syndrome virus by tetrandrine at various concentrations in cells using real-time fluorescent quantitative PCR assay of example 2, wherein Copy number is Copy number and Fangchinoline is tetrandrine;
FIG. 3 shows the inhibition of N protein synthesis of porcine reproductive and respiratory syndrome virus in cells by Western Blot analysis of tetrandrine at different concentrations in example 3, mock as blank, con as virus control, fang as tetrandrine.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The reagents or materials used in the examples below are commercially available unless otherwise specified.
Example 1CCK-8 determination of the cytotoxicity of tetrandrine on Marc-145
Collecting Marc-145 cells in logarithmic phase, adjusting cell density to 70% -80%; plating with 96-well plates, 2X 10 per well 6 The culture medium is DEME complete culture medium, the dosage of the culture medium is 2 mL/hole, and the culture medium is prepared by mixing at 37 ℃ and 5% CO 2 After incubation in a cell incubator until cell attachment is firm, mrac-145 cells were treated with tetrandrine at final concentrations of 1, 2, 4, 8, 10, 12, 14 and 16. Mu.M, denoted as dosing group (A, treatment with cells, medium, CCK-8 solution and drug), whileBlank groups (B, no cells added, treatment with medium and CCK-8 solution) and 0 dosing group (C, no drug added, treatment with cells, medium and CCK-8 solution) were set up, each with 6 parallel wells. After the drug addition, the cells were placed in an incubator for 48 hours, 10. Mu.L of CCK-8 solution was added to each well, and incubation was continued for 1.5 hours in the incubator. After the incubation, absorbance was measured at a wavelength of 450nm using a microplate reader (A 450 ) Cell viability was calculated according to the following formula:
cell viability= (a-B)/(C-B); wherein, a (dosing group): absorbance of wells containing cells, medium, CCK-8 solution and drug solution: b (blank): absorbance of wells containing medium and CCK-8 solution without cells; c (0 dosing group): absorbance of wells containing cells, medium and CCK-8 solution without drug solution.
As can be seen from FIG. 1, the effect of different concentrations of tetrandrine on Marc-145 cells was different, and the viability of Marc-145 cells was decreased with increasing concentration of tetrandrine. The cell viability was 50% at a concentration of 12. Mu.M, thus confirming that the safe concentration of tetrandrine was 1-10. Mu.M.
Example 2 real-time fluorescent quantitative PCR assay of the inhibition of the proliferation of porcine reproductive and respiratory syndrome Virus RNA by tetrandrine in Marc-145 cells
After Marc-145 cells were grown to a monolayer in a 6-well cell culture plate, the medium was discarded, washed 2 times with PBS (phosphate buffer), and a swine reproduction and respiratory syndrome virus (highly pathogenic PRRSV WUH strain, chinese herbal extracts with antiviral activity: evaluation, mechanisms, andpotential for preventing PRV, PEDV and PRRSV infections) diluted with DMEM cell maintenance solution containing 2% fetal bovine serum, moi=0.1, 10 μl/well, incubated at 37 ℃ for 1.5 hours, the virus supernatant was discarded, washed 2 times with PBS, 1, 2, 4, and 7 μM tetrandrine, 2 mL/well, respectively, was added, and the test was run with normal control groups (no test drug, no PRRSV, only DMSO) and PRRSV control groups (no test drug). The cells were continued to be cultured at 37℃until the culture was terminated 48 hours after the infection. After observing the pathological condition, the supernatant of each well is collected. The collected cell supernatant was subjected to total RNA extraction using the procedure recommended by the total RNA extraction kit (Nanjinouzan Biotechnology Co., ltd.). And (3) immediately carrying out reverse transcription to obtain cDNA after RNA is extracted, and carrying out real-time fluorescence quantitative detection on the copy number of PRRSVNSP9 by taking the DNA as a template. Wherein, the sequence of the primer upstream and downstream of PRRSVNSP9 gene is as follows:
NSP9-F：5’-CCCTAGGACCTGTGAAC-3’，SEQ ID NO.1；
NSP9-R：5’-GGCGAGTAACTTAGGAGATG-3’，SEQ ID NO.2。
as can be seen from FIG. 2, the copy number of PRRSVNSP9 gradually decreased with increasing concentration of tetrandrine, and tetrandrine at a concentration of 1-7. Mu.M was able to effectively block replication of PRRSV. Therefore, the tetrandrine has the effect of obviously inhibiting the replication of porcine reproductive and respiratory syndrome virus through in vitro cell tests, and provides powerful support for preventing and treating porcine reproductive and respiratory syndrome through medicines.
EXAMPLE 3Western Blot analysis of the inhibition of N protein Synthesis of porcine reproductive and respiratory syndrome Virus by tetrandrine in Marc-145 cells
After Marc-145 cells were grown to a monolayer in 6 well cell culture plates, the medium was discarded, washed 2 times with PBS (phosphate buffer), swine reproduction and respiratory syndrome virus (source same as example 2) with moi=0.1 diluted with DMEM cell maintenance solution containing 2% fetal bovine serum was added, 10 μl/well incubated at 37 ℃ for 1.5h, virus supernatant was discarded, washed 2 times with PBS, 1, 4 and 7 μΜ tetrandrine respectively, 2 mL/well, cell culture was terminated 48h after infection, the supernatant was discarded, and washed 2 times with PBS. The cell culture plates were placed on ice, 100. Mu.L/well of RIPA lysate was added, the solution was aspirated into centrifuge tubes after blowing, and 12000rmp was centrifuged for 15min, the supernatant was aspirated into another clean tube for use, and the test was performed with both the Mock group (blank group, without any treatment of Marc-145 cells) and Con group (virus control group, virus only, no drug treatment). After the protein concentration of each sample was determined by BCA method, the bands of N protein and internal reference protein GAPDH of porcine reproductive and respiratory syndrome virus were detected by Western Blot. As shown in the test result in figure 3, the tetrandrine has remarkable inhibition effect on N protein synthesis of porcine reproductive and respiratory syndrome virus in Marc-145 cells in the concentration range of 1-7 mu M, and inhibits the expression of N protein of porcine reproductive and respiratory virus in a dose-dependent manner.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The application of tetrandrine in preparing medicine for preventing and treating pig reproduction and respiratory syndrome is provided.
2. A medicament for preventing and treating porcine reproductive and respiratory syndrome, which is characterized in that the active ingredients of the medicament comprise tetrandrine.
3. The medicament according to claim 2, wherein the concentration of the active ingredient is 1-7 μm.
4. A medicament according to claim 3, characterized in that the concentration of the active ingredient is 7 μm.
5. The medicament of claim 2, further comprising a pharmaceutically acceptable excipient.
6. The medicament according to claim 5, wherein the dosage form of the medicament comprises a powder, a granule, a tablet or a capsule.
7. The application of tetrandrine in preparing medicines for inhibiting porcine reproductive and respiratory syndrome virus proliferation is provided.
8. A medicament for inhibiting porcine reproductive and respiratory syndrome virus proliferation, which is characterized in that the active ingredients of the medicament comprise tetrandrine.
9. The medicament according to claim 8, wherein the concentration of the active ingredient is 1-7 μm.
10. The medicament according to claim 9, wherein the concentration of the active ingredient is 7 μm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311830331.3A CN117653633A (en) | 2023-12-28 | 2023-12-28 | Application of tetrandrine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome |
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| CN202311830331.3A CN117653633A (en) | 2023-12-28 | 2023-12-28 | Application of tetrandrine in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome |
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