Disclosure of Invention
The application aims at the application of ebselen as a polymyxin synergist in inhibiting salmonella, and the research of the application discovers that the ebselen can achieve better inhibition effect in combination with a specific culture medium in an in-vitro environment. According to the application, the survival rate of salmonella is taken as a target for screening the polymyxin synergist, and the survival rate of salmonella can be obviously reduced by screening the EBS. In an in vivo test, by constructing a mouse salmonella intestinal tract infection model and combining the EBS with the colistin intraperitoneal injection, the salmonella quantity of feces, livers and spleens can be reduced, and the survival rate of animals can be obviously improved.
The technical purpose is realized by the following scheme:
use of ebselen in combination with polymyxin (CS) for the preparation of a medicament against salmonella infection.
Ebselen (EBS) is an organic selenium compound that can permeate the blood brain barrier and has a wide range of biological activities. EBS has been reported to show broad potential in the prevention and treatment of various human diseases such as stroke, neurodegenerative diseases, bipolar disorder, sensorineural deafness, cardiovascular diseases, immune system enhancement, detoxification, and antimicrobial, etc.
Preferably, in an in vitro experiment, salmonella is first incubated to log phase, then incubated with LPM broth, diluted after incubation is completed, and then ebselen is added and colistin is used in combination.
Preferably, the salmonella is salmonella standard strain ATCC14028 and polymyxin-resistant salmonella.
Preferably, the concentration of the bacterial liquid after the incubation is 1X 106～5×106 CFU/mL.
Preferably, the pH of the LPM broth is 5.5 to 6.0.
Preferably, the concentration of the ebselen is 0.0625-4 mg/L, and the concentration of the polymyxin is 0.5-32 mg/L.
A combined culture method comprises culturing Salmonella, incubating to logarithmic phase, incubating with LPM broth, diluting after incubation, and adding selenium and colistin. Preferably, the salmonella is salmonella standard strain ATCC14028 and polymyxin-resistant salmonella.
Preferably, the concentration of the bacterial liquid after the incubation is 1X 106～5×106 CFU/mL.
Preferably, the pH of the LPM broth is 5.5 to 6.0.
Preferably, the concentration of the ebselen is 0.0625-4 mg/L, and the concentration of the polymyxin is 0.5-32 mg/L.
In an in vivo test, by constructing a mouse salmonella intestinal tract infection model and combining the ebselen with polymyxin for intraperitoneal injection, the salmonella amount of feces, intestinal tracts and spleens can be reduced, and the survival rate of animals can be obviously improved. The medicine is preferably in the form of injection, tablet, pill, capsule, suspension or emulsion.
Preferably, the dosage of polymyxin in the medicament is 5 mg/kg/day, and the dosage of ebselen is 10 mg/kg/day.
Definitions of terms used in connection with the present invention, unless otherwise indicated, the initial definitions provided for terms herein apply to such groups or terms throughout the specification, and terms not specifically defined herein should be given meanings that would be given to those skilled in the art based on the disclosure and the context.
The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising the pharmaceutical dosage form, and physiologically compatible with the recipient.
The terms "salts" and "pharmaceutically acceptable salts" refer to the acid and/or base salts of the above compounds or stereoisomers thereof, with inorganic and/or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. The compound may be obtained by mixing the above compound or a stereoisomer thereof with a predetermined amount of an acid or a base as appropriate (for example, equivalent). These salts may be obtained by precipitation in solution and collected by filtration, or recovered after evaporation of the solvent, or by lyophilization after reaction in an aqueous medium. The salts of the present invention may be the hydrochloride, sulfate, hydrobromide, hydrofluoric, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salts of the compounds.
In certain embodiments, one or more compounds of the present invention may be used in combination with one another. The compounds of the invention may alternatively be used in combination with any other active agent for the preparation of a medicament or pharmaceutical composition for modulating cellular function or treating a disease of salmonella infection. If a group of compounds is used, the compounds may be administered to a subject simultaneously, separately or sequentially.
Compared with the prior art, the application has the beneficial effects that:
The invention provides treatment by combining ebselen and polymyxin, which has good synergistic effect on experiments of salmonella in vitro and in vivo. Compared with the traditional method of combining antibiotics and antibiotics to kill salmonella, the compound ebselen and polymyxin are synergistic to sterilize, so that bacterial drug resistance is not easy to induce, and the ebselen has the characteristics of wide sources, multiple effects and good treatment effect, and has good research and application significance for excavating the synergistic substitution of antibiotics and solving the bacterial drug resistance.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below in conjunction with examples of the present invention and comparative examples, and it is apparent that the described examples are only some of the examples of the present invention, but not all of the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test methods used in the examples described below are conventional methods unless otherwise specified, and the materials, reagents, etc., used are commercially available reagents and materials unless otherwise specified.
The polymyxin used in the examples below is polymyxin E. Ebselen, CAS registry number 60940-34-3, molecular formula C13H9 NOSe, molecular weight 274.18, was used in the examples below.
The structural formula is as follows:
example 1 Eibusia enhances in vitro sensitivity of Salmonella to polymyxin
1. The autoclaved LPM broth at pH 5.8 was cooled for use. The standard strain ATCC 14028 of Salmonella, polymyxin-resistant Salmonella (17 ES) was stored in the laboratory and commercially available.
2. Preparation work before test:
(1) Preparing proper amount of ebselen, wherein the solvent is dimethyl sulfoxide, the final concentration of the medicine is 5120mg/L, and filtering with a filter membrane after uniformly mixing. Polymyxin was prepared as a stock solution at a concentration of 5120mg/L according to CLSI for use.
(2) The standard strain ATCC14028 of Salmonella and polymyxin-resistant Salmonella (17 ES) were inoculated onto LB agar plates and cultured to an appropriate size.
3. MIC and FICI of ebselen and polymyxin for Salmonella
(1) The standard test salmonella strains ATCC14028 and polymyxin-resistant salmonella (17 ES) were inoculated into a tube containing 4mL MH broth, placed in a 37℃shaker and incubated at 180rpm to log phase;
(2) Diluting the incubated bacteria to 100 times with LPM broth with pH of 5.8, about 106 CFU/mL for use;
(3) Taking a sterile 96-well plate, adding 180 mu L of LPM broth culture medium into a1 st well, and adding 100 mu L of LPM broth culture medium into 2 nd to 11 th wells respectively;
(4) Adding 20 mu L of medicine in column 1, blowing uniformly, sucking 100 mu L to the 2nd hole, and then analogizing, and sucking 100 mu L from the 10 th hole for discarding;
(5) 100. Mu.L of diluted bacterial liquid is added to the 1 st to 11 th wells, and 200. Mu.L of LPM broth culture broth is added to the 12 th wells;
(6) Repeating the steps (3) to (5) for three repeated parallelisms;
(7) Placing the inoculated 96-well plate into a 37 ℃ incubator for incubation for 16-18 hours, and reading the result;
(8) And according to the MIC result, performing a chessboard method. MIC results of selenium according to Table 1 show that EBS has 4/16mg/L for standard bacteria of Salmonella and polymyxin-resistant Salmonella in LPM culture medium, when MAG is combined with polymyxin, sensitivity of drug-resistant bacteria and standard bacteria to polymyxin can be remarkably reduced, chessboard method results are shown in FIG. 1, and chessboard method results show that FICI is less than 0.5, which indicates that MAG and polymyxin have good synergistic effect in treating Salmonella.
TABLE 1
4. In vitro sterilization curve of ebselen and polymyxin against salmonella
(1) The standard test salmonella strains ATCC14028 and polymyxin-resistant salmonella (17 ES) were inoculated into a tube containing 4mL MH broth, placed in a 37℃shaker and incubated at 180rpm to log phase;
(2) Diluting the incubated bacteria to 10 times with LPM broth with pH of 5.8, about 107 CFU/mL for use;
(3) Adding 1 concentration MAG or polymyxin with antibacterial concentration into an LPM broth centrifuge tube with pH value of 5.8, and then adding 0.4mL of diluted bacterial liquid obtained by diluting in the step (2) respectively, and mixing uniformly by vortex 5 s. A blank control group was set up as a quality control, and the control group was kept consistent with the test group except for the absence of drug. At this time, the concentration of the bacterial liquid is about 5X 106 CFU/mL, and the bacterial liquid is placed in a shaking table at 37 ℃ for culture at 180rpm, and the consumption of the bacterial liquid is 4mL;
(4) Taking bacterial liquid in the culture time of 0, 3, 6, 9 and 24 hours, sucking 100 mu L of bacterial liquid, adding the bacterial liquid into a 2mL centrifuge tube filled with 900 mu L of 0.85% physiological saline for 10 times of gradient dilution, sucking 25 mu L of bacterial liquid after dilution, dripping the bacterial liquid on MH agar culture medium, incubating the bacterial liquid in a 37 ℃ incubator for 18 hours, counting, and carrying out statistical analysis after experimental results are subjected to three biological repetitions. The colony count at each time point was counted and a sterilization graph was drawn.
The results show that the in vitro sterilization curve of FIG. 1 shows that the bacterial load of the EBS and the polymyxin after 24 hours of combination is reduced by more than 100 times compared with that of single medicine, and the synergy is remarkable, and the EBS and the polymyxin combination have excellent effect of killing salmonella in vitro.
Example 2 Eibuten enhances the killing effect of polymyxin on Salmonella in vivo
1. Test materials 80C 57BL/6J female mice, mice stomach-filling needle, sterile syringe were purchased at university of medical science in south China, guangdong province.
2. Preparation before the test, 5mg/kg of polymyxin, 10mg/kg of EBS stock solution and 100000mg/L of streptomycin were prepared. Salmonella ATCC14028 strain was inoculated onto LB agar plates and cultured to an appropriate size. Each group had 9 mice, for a total of 4 groups (control group, polymyxin treated group, EBS treated group, combination treated group, respectively).
3. Establishment of salmonella infection mouse model
(1) Each mouse was orally treated with 20mg of streptomycin and water cut run out of grain was performed 4 hours before treatment.
(2) After one day of streptomycin treatment, 100. Mu.L of ATCC14028 bacteria solution of about 108 was lavaged. The ATCC14028 strain to be tested was inoculated into a tube containing 4mL of LB broth, and incubated in a shaker at 37℃at 180rpm until the tube was removed from the logarithmic phase (about 108 cells).
4. Target organ colony colonization number detection after mouse infection
(1) After 1 day of infection, EBS (10 mg/kg, intraperitoneal injection, once a day) was administered in single and combined doses with polymyxin (5 mg/kg, intraperitoneal injection, once a day).
(2) After continuous treatment with the above doses for 3 days, mice in the treatment group and quality control group were sacrificed by cervical dislocation, various organs were collected, and colony counts were performed.
As a result, the target organs (spleen, liver, feces) of mice were significantly reduced after EBS and polymyxin combination treatment compared to the best single-drug treatment group, and the bacterial load was reduced by more than 100-fold compared to single-drug treatment (see FIG. 2).
5. Treatment survival test after mice infection
(1) After 1 day of infection, EBS (10 mg/kg, intraperitoneal injection, once a day) was administered in single and combined doses with polymyxin (5 mg/kg, intraperitoneal injection, once a day).
(2) After a sustained treatment with the drug dose of (1) for 7 days, mice survival curves of the treatment group and the quality control group were recorded and plotted.
The effects of significantly reducing the bacterial load in the liver of animals treated by the combination of EBS and polymyxin compared with the bacterial load treated by the single drugs EBS and polymyxin in a mouse salmonella infection model are shown in the figure 2a, the effects of significantly reducing the bacterial load in the spleen of animals treated by the combination of EBS and polymyxin compared with the bacterial load treated by the single drugs EBS and polymyxin are shown in the figure 2b, the effects of significantly reducing the bacterial load in the feces of animals treated by the combination of EBS and polymyxin compared with the bacterial load treated by the single drugs EBS and polymyxin are shown in the figure 2c, and the effects of significantly increasing the survival rate of animals treated by the combination of EBS and polymyxin compared with the survival rate treated by the single drugs EBS and polymyxin are shown in the figure 2 d.
Conclusion the control group survival rate was 0%, the EBS treated group survival rate was 0%, the polymyxin treated group survival rate was 10%, the EBS combined polymyxin survival rate was 80% and the EBS combined polymyxin protection rate was significantly higher than that of the polymyxin treated group after treatment day 7 (see figure 2). This shows that the combination of EBS and polymyxin also has good synergistic effect in the treatment of Salmonella infection in vivo.
In conclusion, the invention takes the survival rate of salmonella as a target, and the combination of the compound EBS and polymyxin is screened out to have good in vitro killing effect on the salmonella. Finally, a salmonella infection mouse model is successfully established by oral infection of salmonella, and further, it is proved that the combination of EBS and polymyxin can reduce colonial colonization of organs with different tissues and reduce death rate of mice infected with salmonella, and the researches provide ideas and foundation for research and development of new medicines of polymyxin synergists.
It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and are not intended to limit the present invention to the specific embodiments thereof. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.