Application of sophocarpidine in preparation of product for resisting staphylococcus aureus invasion of bovine mammary epithelial cells

Abstract

The invention discloses application of matrine in preparation of a product for resisting staphylococcus aureus invasion of bovine mammary epithelial cells. The invention provides an application of sophocarpidine in preparation of a product with at least one of the following functions: A1) preventing and treating animal infectious diseases; a2) anti-staphylococcus aureus or methicillin-resistant staphylococcus aureus; a3) reducing the adhesion of staphylococcus aureus or methicillin-resistant staphylococcus aureus to mammary gland cells or tissues; a4) the invasion of staphylococcus aureus or methicillin-resistant staphylococcus aureus to mammary gland cells or tissues is reduced; a5) retarding apoptosis of mammary gland cells or tissues induced by staphylococcus aureus or methicillin-resistant staphylococcus aureus. The matrine can be used for preparing veterinary drugs and preventing and treating animal diseases caused by staphylococcus aureus or staphylococcus aureus drug-resistant strains MRSA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of matrine in the preparation of products that resist Staphylococcus aureus invasion of bovine mammary epithelial cells. Background Technology

[0002] Staphylococcus aureus S. aureus It is an important pathogen causing infectious mammary gland infections in dairy cows. It can colonize bovine mammary epithelial cells through surface proteins and produce virulence factors such as LTA, Hla, PVL, ClfA, and FnBP to adhere to and invade mammary tissue. It can also protect itself by producing PIA, SpA, Eap, and releasing PSM toxins. S. aureus Protected from the innate and adaptive immune responses of the cow's mammary glands, thus... S. aureus Acquired methicillin-resistant Staphylococcus aureus (MRSA) strains, leading to... S. aureus Infectious bovine mammary gland disease (MMSD) is difficult to cure completely. MRSA is an intracellular parasite with higher virulence, capable of causing varying degrees of adhesion and invasion of bovine mammary epithelial cells, and inducing apoptosis. There is a need to find methods or drugs to treat MMSD in dairy cows.

[0003] Matrine is a natural quinazoline alkaloid isolated from plants such as *Sophora flavescens* and *Sophora tonkinensis*, belonging to the Fabaceae family. It is widely distributed throughout my country. Although matrine exhibits good resistance to pathogenic microorganisms, its clinical application is limited by its cytotoxicity at high concentrations. Therefore, exploring the toxicity of matrine, finding suitable concentrations, and investigating its antibacterial and anti-inflammatory activities without causing toxicity to the body will provide new insights into its clinical application. Summary of the Invention

[0004] The technical problem solved by this invention is a method or drug for treating infectious bovine mammary gland disease in dairy cows.

[0005] To address the aforementioned technical problems, the first aspect of this invention provides the application of matrine in the preparation of products having at least one of the following functionalities: A1) Prevention and control of infectious diseases in animals; A2) Anti-Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A3) Reduces the adhesion of Staphylococcus aureus or methicillin-resistant Staphylococcus aureus to breast cells or tissues; A4) Reduces the invasion of breast cells or tissues by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A5) Slows down the apoptosis of breast cells or tissues induced by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0006] In the above-described application, the pathogens causing the animal infectious diseases are Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0007] In the above application, the animal infectious disease is mastitis.

[0008] In the application described above, the mastitis referred to is infectious mastitis.

[0009] In the above-described applications, the concentration of matrine used is greater than or equal to 0.25 mg / mL.

[0010] In a second aspect, the present invention provides a composition containing matrine, comprising matrine and other drugs for the prevention and treatment of infectious diseases in animals.

[0011] Thirdly, the present invention provides a product comprising matrine or the composition described in the second aspect, and other pharmaceutically acceptable carriers.

[0012] The product described above has at least one function: A1) Prevention and control of infectious diseases in animals; A2) Anti-Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A3) Reduces the adhesion of Staphylococcus aureus or methicillin-resistant Staphylococcus aureus to breast cells or tissues; A4) Reduces the invasion of breast cells or tissues by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A5) Slows down the apoptosis of breast cells or tissues induced by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0013] The products mentioned above are veterinary drug additives, veterinary drugs, or other biological products.

[0014] The present invention also provides a method for preventing and controlling infectious diseases in animals, comprising the following steps: intervening in animals with matrine to achieve prevention and control of infectious diseases in animals.

[0015] Among them, the effective concentration of matrine in animals is greater than or equal to 0.25 mg / mL.

[0016] In some embodiments, the mammary cells are derived from mammals.

[0017] In some embodiments, the mammary cells are mammary epithelial cells.

[0018] In some embodiments, the animal is a mammal.

[0019] In some embodiments, the mammal is a cow.

[0020] In some implementations, the cow is a dairy cow.

[0021] The dosage form of the drug in the products described above is an injection, a lyophilized powder for injection, an aerosol, a large-volume parenteral solution, a pill, a powder, a granule, a tablet, a capsule, an oral liquid, or an emulsion.

[0022] The pharmaceutically acceptable carriers mentioned above can be excipients, stabilizers, suspending agents, or diluents, as is well known to those skilled in the art.

[0023] Furthermore, the carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Using these materials, various dosage forms can be formulated, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added.In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0024] This invention investigates the effects of matrine on the adhesion and invasion of MRSA strains infecting MAC-T cells and the degree of apoptosis induction under low concentration (0.5 mg / mL and 0.25 mg / mL) conditions. The adhesion and invasion results show that although MRSA strains can adhere to and invade MAC-T cells to varying degrees, their ability to adhere to and invade MAC-T cells is significantly reduced under matrine intervention. This indicates that with prolonged matrine intervention in MRSA strains infecting MAC-T cells, bacterial activity decreases, and the cells' own resistance also declines, leading to a certain degree of cell death, which in turn significantly reduces bacterial adhesion and invasion rates. Therefore, with the progression of infection time, the adhesion and invasion rates of MRSA strains to MAC-T cells show a certain degree of decrease. Furthermore, immunofluorescence assays also verified that the amount of MRSA strains adhering to and invading MAC-T cells was significantly reduced under matrine intervention. This indicates that 0.25 mg / mL matrine can significantly reduce the virulence of MRSA strains against MAC-T cells, thereby significantly reducing their ability to adhere to and invade MAC-T cells.

[0025] To investigate the ability of MRSA strains to induce MAC-T cell apoptosis under matrine intervention, this study used flow cytometry to detect the apoptotic capacity of MAC-T cells in the MRSA infection group and the matrine intervention group. The results showed a significant difference in the ability of the infection group and the matrine intervention group to induce MAC-T cell apoptosis. Notably, the ability of MRSA strains to induce MAC-T cell apoptosis was significantly reduced under matrine intervention, which also demonstrates the bioactivity of matrine against MRSA infection. Furthermore, adhesin was observed during MRSA infection of MAC-T cells. clf A, clf B. Fnb A, Fnb B and CNA The mRNA expression level was also significantly reduced due to the intervention of matrine.

[0026] In summary, matrine at a concentration of 0.25 mg / mL significantly reduced the adhesion, invasion, and apoptosis-inducing ability of MRSA strains to MAC-T cells, indicating that 0.25 mg / mL matrine has a significant resistant effect against MRSA strains. Matrine at this concentration exerts its antibacterial effect by mediating several signaling pathways related to cell adhesion, invasion, apoptosis, and inflammation, thereby reducing the expression levels of inflammatory factors and apoptosis-related proteins. Matrine plays an important antibacterial role in the adhesion, invasion, and apoptosis-inducing process of MAC-T cells by drug-resistant Staphylococcus aureus (MRSA) strains and can be used in the preparation of veterinary drugs to prevent and treat animal diseases caused by Staphylococcus aureus or drug-resistant Staphylococcus aureus (MRSA) strains. Attached Figure Description

[0027] Figure 1 The image shows the recovery and adherent growth status of MAC-T cells (10X); where A: newly recovered MAC-T cells; B: MAC-T cells cultured for 48 hours.

[0028] Figure 2 To investigate the effects of matrine concentrations of 0.5 mg / mL and 0.25 mg / mL on the adhesion and invasion rates of MRSA-infected MAC-T cells, the following data were analyzed: A: Adhesion rate of MRSA-infected MAC-T cells by matrine concentrations of 0.5 mg / mL and 0.25 mg / mL; B: Influence rate of MRSA-infected MAC-T cells by matrine concentrations of 0.5 mg / mL and 0.25 mg / mL (*:). p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.001).

[0029] Figure 3 The images show cellular immunofluorescence (60X); A: Mucus map of MRSA strain on MAC-T cells; B: Invasion map of MRSA strain on MAC-T cells; C: Mucus map of MAC-T cells infected with MRSA strain after treatment with 0.25 mg / mL matrine; D: Invasion map of MAC-T cells infected with MRSA strain after treatment with 0.25 mg / mL matrine. Red represents the cell membrane stained with Dil, blue represents the cell nucleus stained with DAPI, and green represents the MRSA strain carrying the pCM29 plasmid tagged with GFP.

[0030] Figure 4 for clf A, clf B. Fnb A, Fnb B and CNA relative mRNA expression levels (*: p <0.05; **: p <0.01;***: p <0.001; ****: p <0.001).

[0031] Figure 5 The apoptosis rate of MAC-T cells induced by MRSA strain and MRSA strain-induced MAC-T cells treated with 0.25 mg / mL matrine was measured by flow cytometry. A: Flow cytometry was used to detect the apoptosis rate of MAC-T cells induced by MRSA strain and MRSA strain-induced MAC-T cells treated with 0.25 mg / mL matrine. LL: Live cell region, UL: Necrotic cell region, UR: Late apoptotic cell region, LR: Early apoptotic cell region; (a): Gating strategy in the analysis of MAC-T cell apoptosis, (b): Apoptosis rate of uninfected MAC-T cells, (ch): Apoptosis rate of MAC-T cells induced by MRSA strain at different time points (2h, 4h, 6h, 8h, 10h and 12h), (in): Apoptosis rate of MAC-T cells induced by MRSA strain treated with 0.25 mg / mL matrine at different time points (2h, 4h, 6h, 8h, 10h and 12h); B: Flow cytometry was used to detect the apoptosis rate of MAC-T cells induced by MRSA strain and MRSA strain-induced MAC-T cells treated with 0.25 mg / mL matrine. matrine at a concentration of mg / mL intervened to increase the late apoptosis rate of MAC-T cells induced by MRSA strain (*: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.001). Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0035] Matrine was purchased from Sigma, product number: M5319, CAS NO.: 519-02-08.

[0036] Bovine mammary epithelial cell line (MAC-T cells) was kindly provided by Professor Peng Jie of the College of Veterinary Medicine, Gansu Agricultural University. It has already appeared in the following published articles: Yan LJ, Yang YZ, Ma XJ, et al. Effect of two different drug-resistant Staphylococcus aureus strains on the physiological properties of MAC-T Cells and their transcriptome analysis[J]. Front Vet Sci. 2022; 9:818928. Zhang Z, Yang YZ, Yan LJ, et al. Effect of matrine in MAC-T cells and their transcriptome analysis: A basic study[J]. PLoS One, 2023, 18(1): e28090 The MRSA strain is described in the following literature: Yan LJ, Yang YZ, Ma XJ, et al. Effect of twodifferent drug-resistant Staphylococcus aureus strains on the physiological properties of MAC-T Cells and their transcriptome analysis[J]. Front Vet Sci. 2022; 9: 818928, and is named Methicillin-resistant in the literature. S. aureus (MRSA). Example 1: Matrine intervention on the adhesion and invasion of MRSA strains infected with MAC-T cells I. Resuscitation and Culture of MAC-T Cells (1) Cell resuscitation MAC-T cells stored in liquid nitrogen were placed in a clean EP glove and incubated in a 37°C water bath for 3 min to rapidly thaw them. They were then transferred to 1.5 mL centrifuge tubes and centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 mL PBS and washed. The cells were then centrifuged at 1200 rpm for 5 min, and this process was repeated twice. After the third centrifugation, the supernatant was discarded, and 1 mL of 10% DMEM complete medium was added (10% cell culture medium: 445 mL DMEM + 5 mL 1% penicillin-streptomycin + 50 mL fetal bovine serum). The mixture was gently mixed and transferred to a cell culture flask. Another 4 mL of complete medium was added to bring the total volume to the desired level, and the cells were incubated at 37°C in a 5% CO2 incubator.

[0037] (2) Cell Culture Take MAC-T cells cultured in the cell culture incubator, pipette and discard the culture medium from the culture flask, add 1 mL of PBS and gently shake the culture flask horizontally to wash the cells, then discard the PBS, repeating twice. Pipette 5 mL of 10% DMEM complete culture medium into the cell culture flask and place it in a 37°C cell culture incubator to continue culturing.

[0038] (3) Cell passage After digesting the cultured cells with trypsin, 1 mL of the cell suspension was transferred to a new cell culture flask. Then, 4 mL of 10% DMEM complete culture medium was added to each flask to bring the total to 5 mL. The flasks were then incubated at 37°C. The above steps were repeated for each confluent cell flask, and each flask could be passaged into two flasks. Cells were passaged to the third generation and their condition was stable before being used for subsequent experiments. The resulting confluent monolayer of MAC-T cells was designated as MAC-T cell suspension.

[0039] (4) Cell cryopreservation After digesting the cultured cells with trypsin, 10% DMEM complete medium was added to stop the digestion. The cell suspension was then transferred to a centrifuge tube and centrifuged at 8000 r / min for 5 min. The cell pellet was then resuspended in medium, and the cell density was adjusted. The cells were then transferred to 2 ml cell cryopreservation tubes, and DMEM, serum, and cell cryopreservation solution (DMSO) were added in a ratio of 5:4:1. The cell names and cryopreservation times were labeled, and the cells were stored in a cell cryopreservation box at -80℃.

[0040] MAC-T cells that had just begun to recover were observed under an inverted microscope. The results were as follows: Figure 1 As shown in Figure A, the MAC-T cells have not yet adhered to the wall and are in a round, suspended state.

[0041] Cells were cultured in 10% DMEM complete medium for 48 hours, and the results were as follows: Figure 1 As shown in B, the cells can be observed to be arranged in a cobblestone-like, tightly packed manner under a microscope.

[0042] II. Matrine's intervention on the adhesion and invasion of MAC-T cells infected by MRSA strains MRSA glycerol bacteria were inoculated into TSB (tryptone soybean broth) liquid medium, and revived and activated in a shaker at 37°C and 220 rpm to obtain MRSA bacterial suspension. Preparation of TSB medium: Add 1000 mL of distilled water to 15 g of TSB powder and stir well. Autoclave at 121°C for 20 min. For solid medium, add 7.5 g of agar and autoclave.

[0043] 1. Adhesion test (1) MAC-T cells + MRSA group (MRSA infection group): MRSA bacterial solution (2×10⁻⁶ cells / mL) was added to the MRSA culture. 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL) 5 The cells (cells / mL) were mixed in DMEM without FBS and antibiotics at an infection rate (MOI) of 100:1 and added to the cell wells. The cells were then incubated statically at 37°C and 5% CO2 for 2 h, 4 h, 6 h, 8 h, 10 h and 12 h.

[0044] MAC-T cells + MRSA + 0.5 mg / mL or 0.25 mg / mL matrine group (matrine interference group): MRSA bacterial solution (2×10⁻⁶ cells / mL) was added to the culture medium. 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL) 5 The cells were mixed with DMEM (free of FBS and antibiotics) at an infection rate of 100:1 (MOI) and added to cell wells. 10 mg / mL matrine incubation solution was added to make the matrine concentrations 0.5 mg / mL and 0.25 mg / mL, respectively. The cells were then incubated statically at 37°C and 5% CO2 for 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h.

[0045] The above-mentioned 10 mg / mL matrine incubation solution is formulated as follows: Take 100 μL of 100 mg / mL matrine aqueous solution, add 900 μL of DMEM, dispense as needed, and store at 4℃. Prepare fresh each time you use it.

[0046] The above 100mg / mL matrine aqueous solution formula is as follows: 0.1g matrine powder is mixed with 1mL ddH2O, filtered through a 0.22μM filter, and stored at 4℃ for later use.

[0047] (2) Discard the culture medium for the corresponding time period, and then wash each well of cells three times with PBS.

[0048] (3) Add 100 μL of trypsin to digest the cells. After 2 min, add 400 μL of 0.025% Triton X-100 to further lyse the cells for 2 min. Repeatedly pipette the cells to completely lyse them and obtain a bacterial suspension.

[0049] (4) Aspirate the above bacterial suspension, then dilute it 10 times with PBS, take 100 μL of the appropriate concentration of bacterial suspension and inoculate it on TSB agar plate, and incubate it overnight at 37°C.

[0050] (5) Use plate counting method to count colonies, repeating each group three times.

[0051] Adhesion rate = (colony count × dilution factor × 10 (100 μL was taken from 1 ml for plate counting)) / 2 × 10 7 (Initial bacterial count) The results are as follows Figure 2 As shown in Figure A, the adhesion ability of MRSA strain to MAC-T cells gradually increased with the increase of infection time. The adhesion rate of MRSA strain to MAC-T cells increased from 0.085% at 2h to 14.95% at 8h, and then decreased to 11.4% at 12h. Under the intervention of matrine, the ability of MRSA strain to adhere to MAC-T cells was significantly reduced. In particular, the adhesion rate of MAC-T cells incubated with 0.5 mg / mL matrine did not show a significant change with the gradual increase of MRSA infection time. Although the adhesion rate of MAC-T cells incubated with 0.25 mg / mL matrine showed a trend of first increasing and then decreasing over time, the overall adhesion rate and invasion rate were much lower than those of the MRSA infection group. Under the intervention of low concentration 0.25 mg / mL matrine, the adhesion rate of MRSA strain to MAC-T cells increased from 0.03% at 2h to 3.9% at 8h, and then decreased to 2.00% at 12h.

[0052] 2. Invasion test (1) MAC-T cells + MRSA group: MRSA bacterial culture (2×10⁻⁶ cells / mL) was added to the culture. 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL) 5 The cells were mixed with DMEM (free of FBS and antibiotics) at an infection rate of 100:1 (MOI, the ratio of Staphylococcus aureus to cells) and added to cell wells. The cells were then incubated statically at 37°C and 5% CO2.

[0053] MAC-T cells + MRSA + (0.5 mg / mL or 0.25 mg / mL) matrine group: MRSA bacterial solution (2×10⁻⁶) was added to the culture medium. 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL)5 The cells (cells / mL) were mixed in DMEM without FBS and antibiotics at an infection rate (MOI) of 100:1 and added to the cell wells. Matrine incubation solution was added to make the matrine concentrations 0.5 mg / mL and 0.25 mg / mL, respectively. The cells were then incubated statically in an incubator at 37°C and 5% CO2.

[0054] (2) Discard the culture medium for the corresponding time period, and then wash each well of cells three times with pre-cooled PBS.

[0055] (3) Add 1 mL of gentamicin culture medium with a final concentration of 50 mg / mL and incubate at 37°C for 2 h to eliminate extracellular bacteria.

[0056] (4) After removing the cell culture medium, wash the cells three times with PBS.

[0057] (5) Add trypsin to digest the cells, then add 0.025% Triton X-100 to further lyse the cells, and repeatedly pipette the cells to completely lyse them.

[0058] (6) Aspirate the above suspension, then serially dilute it with PBS, and inoculate the bacterial suspension of appropriate concentration onto TSB agar plates and incubate overnight at 37°C.

[0059] (7) Use plate counting method to count colonies, repeat three times for each group.

[0060] Invasion rate = (colony count × dilution factor × 10 (100 μL was taken from 1 mL for plate counting)) / 2 × 10 7 (Initial bacterial count) The results are as follows Figure 2 As shown in Figure B, the invasion rate of MAC-T cells by MRSA strain increased from 0.045% at 2 h to 5.465% at 8 h, and then decreased to 2.350% at 12 h. No significant change was observed in the invasion rate of MAC-T cells incubated with 0.5 mg / mL matrine as the infection time of MRSA strain increased. Under the intervention of 0.25 mg / mL matrine, the invasion rate of MAC-T cells by MRSA strain increased from 0.01% at 2 h to 0.155% at 8 h, and then decreased to 0.05% at 12 h, showing no significant trend. However, the line graph comparing the MRSA infection group and the matrine intervention group showed that the invasion rate of MAC-T cells by MRSA strain was significantly reduced under the intervention of 0.25 mg / mL matrine.

[0061] In conclusion, a low concentration of 0.25 mg / mL matrine can significantly reduce the adhesion and invasiveness of MRSA strains to MAC-T cells.

[0062] 3. Cell immunofluorescence detection 1) Preparation of competent Staphylococcus aureus cells (1) Streak MRSA glycerol bacteria stored at -80℃ onto TSB solid plates and incubate at 37℃ for 24h; (2) Select a single clone from the TSB plate and inoculate it into liquid culture medium, and incubate it in a shaker at 37°C for 24 h; (3) Take the overnight culture and transfer it back to TSB liquid medium for expansion culture. Shake at 37℃ until OD600 is 0.6, let stand on ice for 10 min, centrifuge at 4℃ and 5000 rpm for 20 min, and collect the bacteria. (4) Resuspend the bacterial cells in an equal volume of pre-cooled 0.5M sucrose aqueous solution, let stand on ice for 20 min, and centrifuge at 5000 rpm for 20 min at 4℃. (5) Resuspend the bacterial cells in 1 / 2 volume of pre-cooled 0.5M sucrose aqueous solution, let stand on ice for 40 min, centrifuge at 5000 rpm for 20 min at 4℃, and collect the bacteria; (6) Resuspend the bacterial cells in 5 ml of pre-cooled 0.5 M sucrose aqueous solution, aliquot into 100 μL portions, and store at -80 °C. Obtain MRSA competent cells.

[0063] 2) Electric transfer (1) Remove MRSA competent cells at -80℃ and let them thaw naturally on ice. Add 10μL of pCM29 plasmid with GFP tag to 100μL of MRSA competent cells (agr-Dependent interactions of Staphylococcus aureusUSA300 with human polymorphonuclear neutrophils; Yun Yun Pang 1, Jamie Schwartz, Matthew Thoendel, Laynez W Ackermann, Alexander R Horswill, William M Nauseef Affiliations Expand; PMID: 20829608; PMCID: PMC2982852; DOI: 10.1159 / 000319855, the name in the literature is pCM29 plasmid), and let stand on ice for 30min. (2) Transfer the competent cells with added plasmid to a pre-cooled electrode cup, electrolyze once or twice at 2.5 kV and 2.5 ms, then transfer to an EP tube and quickly add 400 μL TSB medium, and incubate on ice for 5 min. (3) Place it in a float and revive it in a shaker at 37°C and 220 rpm for 1 hour; (4) Remove the EP tube, centrifuge at 5000 rpm for 2 min, discard the supernatant, and leave 50 μL of liquid at the bottom of the tube; (5) Mix the remaining liquid and precipitate in the tube by blowing and stirring, then transfer it to TSB solid medium, spread it evenly with an L stick, and place it in an incubator at 37°C for 12-16 hours until individual colonies grow. (6) Pick individual colonies into TSB liquid medium, and culture them in a shaker at 37°C and 220 rpm for 12 h. Mix the bacterial solution with glycerol at a ratio of 1:1 and dispense into EP tubes. Store at -80°C for a long time to obtain MRSA strains with pCM29 plasmid.

[0064] The MRSA strain carrying the pCM29 plasmid was cultured in TSB liquid medium to obtain MRSA bacterial culture containing the pCM29 plasmid.

[0065] 3) Preparation of cell slides (1) After the cultured MAC-T cells were digested with trypsin, they were resuspended in complete culture medium; (2) Place the required cell spread sheets (18 mm) in a sterile culture dish; (3) Take a certain number of cell suspensions and drop them onto the climbing slides, and incubate them in a 37℃, 5% CO2 incubator for about 24 hours. (4) Perform the following: MAC-T cells + MRSA group: MRSA bacterial culture carrying pCM29 plasmid (2×10⁻⁶ cells) 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL) 5 The cells (cells / mL) were mixed in DMEM without FBS and antibiotics at an infection rate (MOI) of 100:1 and added to the cell wells. The cells were then incubated statically at 37°C and 5% CO2 for 2 h, 4 h, 6 h, 8 h, 10 h and 12 h.

[0066] MAC-T cells + MRSA + (0.25 mg / mL) matrine group: MRSA bacterial culture carrying PCM29 plasmid (2×10⁻⁶) was added to the culture. 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL) 5 The cells (cells / mL) were mixed in DMEM without FBS and antibiotics at an infection rate (MOI) of 100:1 and added to cell wells. Matrine incubation solution was added to the wells to a concentration of 0.25 mg / mL. The cells were then incubated statically at 37°C and 5% CO2 for 2 h, 4 h, 6 h, 8 h, 10 h and 12 h.

[0067] 4) Immunofluorescence staining (1) Discard the incubation solution from the static culture product obtained in 3) above, and wash with PBS 3 times, 2 min each time; (2) After treating with 1 mL of 4% paraformaldehyde for 20 min, discard the solution and rinse with PBS 3 times for 5 min each time. (3) Add DAPI (0.5-10 μg / mL) and incubate for 20 min, then discard the solution. Wash with PBS 4 times, 5 min each time. (4) Add Dil (10 μM) and incubate for 20 min, then discard the solution. Wash with PBS 4 times, 5 min each time. (5) Mark the glass slide, drop an appropriate amount of glycerin, take out the slide and cover it with the glycerin drop, being careful not to have air bubbles; (6) The sealed slides are stored at 4°C, observed and photographed at a live cell workstation, and the results are analyzed.

[0068] Immunofluorescence assays were used to detect the ability of MRSA-infected groups and 0.25 mg / mL matrine-treated groups to adhere to and invade MAC-T cells.

[0069] The results are as follows Figure 3 As shown, the amount of MRSA strains with green fluorescence entering the cell membrane and nucleus changes continuously with different degrees of adhesion and invasion. With the increase of infection time, the amount of MRSA strains entering the cell nucleus and cell membrane gradually increases. In the 0.25 mg / mL matrine intervention group, the ability to adhere to and invade MAC-T cells is reduced, which is consistent with the results measured by plate counting method.

[0070] 4. Detection of Staphylococcus aureus adhesin gene expression level 1) Primer design and synthesis According to the NCBI β-actin and adhesin genes clfA, clfB, FnbA, FnbB and CNA The complete coding region sequence of the cDNA was obtained, primers were designed and their specificity was verified (Table 1), and β-actin was used as an internal reference gene.

[0071] Table 1 shows the RT-qPCR primer design.

[0072] 2) Extraction of total RNA from cells (1) MAC-T cells will be cultured at an appropriate density in 6-well plates.

[0073] (2) MAC-T cells + MRSA group: MRSA bacterial culture (2×10⁻⁶ cells / mL) was added to the culture. 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL) 5The cells (cells / mL) were mixed in DMEM without FBS and antibiotics at an infection rate (MOI) of 100:1 and added to the cell wells. The cells were then incubated statically at 37°C and 5% CO2 for 2 h, 4 h, 6 h, 8 h, 10 h and 12 h.

[0074] MAC-T cells + MRSA + (0.25 mg / mL) matrine group: MRSA bacterial solution (2×10⁻⁶) was added to the culture medium. 7 CFU / mL) and MAC-T cell lysate (2×10⁻⁶ CFU / mL) 5 The cells (cells / mL) were mixed in DMEM without FBS and antibiotics at an infection rate (MOI) of 100:1 and added to cell wells. Matrine incubation solution was added to the wells to a concentration of 0.25 mg / mL. The cells were then incubated statically at 37°C and 5% CO2 for 2 h, 4 h, 6 h, 8 h, 10 h and 12 h.

[0075] (3) Discard the incubation solution at the corresponding time and wash the cells three times with pre-cooled PBS.

[0076] (4) Use TRIzon Reagent to extract RNA solution and store at -80℃ to prevent degradation.

[0077] 3) cDNA synthesis Follow the reverse transcription reagent instructions: (1 μg) Total RNA + (1 μL) Random Primer (N9) (0.1 μg / μL) + (10 μL) 2×TS Reaction Mix + (1 μL) TransScript® RT / RI EnzymeMix. Finally, add RNasc-free water to bring the total volume to 20 μL, mix well, incubate at 25°C for 10 min, at 42°C for 15 min, and heat at 85°C for 5 s to obtain cDNA. Store at -20°C.

[0078] 4) Real-time quantitative PCR detection of the relative expression levels of clfA, clfB, FnbA, FnbB and CNA Take 1 μg of total RNA, 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL each of forward and reverse primers, and finally add RNasc-free water to bring the total volume to 20 μL. After mixing, incubate at 95℃ for 300 s, 94℃ for 10 s, 57℃ for 30 s, and 72℃ for 20 s, for 42 cycles. β-actin was used as an internal control gene, with each group repeated 3 times, and the relative gene expression level was used for calculation.

[0079] The relative expression levels of these five genes were measured at 2h, 4h, 6h, 8h, 10h and 12h in the MRSA infection group and the 0.25 mg / mL matrine intervention group.

[0080] The results are as follows Figure 4 As shown, the expression levels of the five genes in both groups gradually increased with the increase of MRSA infection time, reaching the highest at 8 hours, and then decreased. However, it was clearly observed that the mRNA expression levels of the five adhesin genes in the MRSA infection group were significantly higher than those in the matrine intervention group. cIf A showed the highest expression level at 8 hours in the MRSA infection group. cIf A and CNA The expression level was highest at 8 hours in the matrine intervention group. Fnb B was expressed at the lowest levels in both the infection group and the matrine intervention group.

[0081] In conclusion, this demonstrates that matrine at a concentration of 0.25 mg / mL can indeed significantly reduce the adhesion of MRSA strains to MAC-T cells.

[0082] III. Assay of Matrine Intervention in MRSA Strains to Induce Apoptosis in MAC-T Cells Using the green fluorescent probe YF ® Apoptosis was detected in MAC-T cells induced by MRSA strains at different time points using 488-labeled Annexin V and red fluorescent propidium iodide (PI) and in MAC-T cells induced by MRSA strains treated with 0.25 mg / mL matrine.

[0083] (1) After the MAC-T cells have filled the cell flask, they are digested with trypsin and then plated in a 6-well plate.

[0084] (2) After the cells have grown well (2×10) 5 CFU / mL), one group added a concentration of 2×10 7 MRSA bacterial suspension at CFU / mL (infected group), and another group was treated with a concentration of 2×10⁶ CFU / mL. 7 MRSA bacterial suspension at CFU / mL and matrine incubation solution were prepared at a concentration of 0.25 mg / mL (intervention group) and incubated for 2h, 4h, 6h, 8h, 10h, and 12h, with three replicates for each incubation time. A control group was set up at 0h incubation, and three wells were used for untreated cells. One well served as a negative control, and the other two wells were used for single staining to adjust for compensation. Heat-killed cells were used as a positive control.

[0085] (3) After incubating the cells for 0h, 2h, 4h, 6h, 8h, 10h and 12h respectively, the supernatant of all groups of cells was discarded, and the cells were washed 3 times with pre-cooled PBS and then discarded.

[0086] (4) Digest each well of cells with 500 mL of EDTA-free trypsin for 4 min, add 500 mL of 10% culture medium to stop digestion, and place in a 37℃ CO2 incubator for 20 min to recover and avoid false positives.

[0087] (5) Take 300g of cell suspension and centrifuge at 4℃ for 5min. Discard the supernatant and collect the cells in a centrifuge tube.

[0088] (6) Wash twice with pre-cooled PBS, centrifuge at 300g, 4℃ for 5min. Collect the cells in a new centrifuge tube and resuspend the cells in 500μL of 1× binding buffer.

[0089] (7) Add 5 μL of YF488-Annexin V and 5 μL of PI working solution to each tube (YF®488-Anneix V / PI Apoptosis Kit - Bioscientific and Technological Corporation - Cat:Y6002L, Lot:331D0101). For single-staining cells in two wells, add only one dye (YF488-Annexin V and PI) to each tube for compensation and regulation of flow cytometry staining.

[0090] (8) The cells were kept in a dark greenhouse and incubated on ice for 15 min. The cells were filtered through a 70 μm cell filter and the apoptosis was detected by flow cytometer.

[0091] The results are as follows Figure 5 As shown, MRSA strains can induce apoptosis in MAC-T cells to varying degrees, and the apoptosis rate in both the infection and intervention groups increased with the duration of MRSA infection. However, the apoptosis rate in the MRSA infection group was significantly higher than that in the matrine intervention group. The apoptosis rate of MAC-T cells induced by MRSA strains increased from 0.89% to 12.7%, while the apoptosis rate of MAC-T cells induced by MRSA strains increased from 0.89% to 5.12% under the intervention of 0.25 mg / mL matrine. Notably, the apoptosis rate in the MRSA infection group was significantly higher than that in the matrine intervention group.

[0092] This trend indicates that after MRSA strains invade MAC-T cells under the intervention of matrine at a concentration of 0.25 mg / mL, their virulence and activity are reduced, thus slowing down the apoptosis induced by MRSA strains in MAC-T cells.

[0093] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of matrine in the preparation of products with at least one of the following functions: A1) Prevention and control of infectious diseases in animals; A2) Anti-Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A3) Reduces the adhesion of Staphylococcus aureus or methicillin-resistant Staphylococcus aureus to breast cells or tissues; A4) Reduces the invasion of breast cells or tissues by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A5) Slows down the apoptosis of breast cells or tissues induced by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

2. The application according to claim 1, characterized in that: The pathogens causing the animal infectious diseases are Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

3. The application according to claim 1 or 2, characterized in that: The infectious disease in the animal is mastitis.

4. The application according to claim 3, characterized in that: The mastitis mentioned is infectious mastitis.

5. The application according to any one of claims 1-4, characterized in that: In the application described, the concentration of matrine used is greater than or equal to 0.25 mg / mL.

6. A composition containing matrine, including matrine and other drugs used to prevent and treat infectious diseases in animals.

7. A product comprising matrine or the composition of claim 6, and other pharmaceutically acceptable carriers.

8. The product according to claim 7, characterized in that: The product has at least one function: A1) Prevention and control of infectious diseases in animals; A2) Anti-Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A3) Reduces the adhesion of Staphylococcus aureus or methicillin-resistant Staphylococcus aureus to breast cells or tissues; A4) Reduces the invasion of breast cells or tissues by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; A5) Slows down the apoptosis of breast cells or tissues induced by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

9. The application according to any one of claims 1-5 or the product according to claim 7 or 8, characterized in that: The product is a veterinary drug additive, veterinary drug, or other biological product.

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