Application of Avasimibe in preparation of medicine for treating staphylococcus aureus infectious diseases

Abstract

The invention discloses an application of Avasimibe in preparation of a medicine for treating a staphylococcus aureus infectious disease. The invention reveals that Avasimibe treats staphylococcus aureus infectious diseases through dual effects of enhancing intracellular sterilization capability of macrophages and direct sterilization, and also reveals that Avasimibe has functions of inhibiting formation of a biofilm of staphylococcus aureus and inhibiting proliferation of staphylococcus aureus, and develops new indications of Avasimibe.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of Avasimibe in the preparation of a medicament for treating staphylococcus aureus infectious diseases. Background Art

[0002] Staphylococcus aureus (hereinafter referred to as S. aureus) is a Gram-positive coccus that widely exists in nature and is usually colonized in the nasal cavity, skin, and gastrointestinal tract of the human body. It is one of the common human pathogens and can cause various clinical manifestations, specifically depending on the site of infection and the virulence of the pathogen. The most common forms of infection include skin and soft tissue infections, such as abscesses, cellulitis, and erysipelas. In addition, S. aureus can also cause more severe infections, such as pneumonia, sepsis, endocarditis, and osteomyelitis. Currently, the main treatment method for S. aureus infection is still antibiotic treatment. However, on the one hand, the abuse of antibiotics has led to an increase in the drug resistance of S. aureus, and drug-resistant strains such as methicillin-resistant Staphylococcus aureus have emerged; on the other hand, S. aureus can survive within host cells, and due to poor permeability, traditional antibiotics are difficult to effectively enter host cells to kill S. aureus inside the cells. Therefore, there is still a need to explore new treatment strategies to better address this increasingly serious major challenge to public health of S. aureus infection.

[0003] Avasimibe (Chinese name: Avariceb) is a SOAT (Sterol O-acyltransferase) inhibitor that mainly acts by inhibiting the synthesis of cholesterol esters. In early preclinical studies, this drug showed significant anti-atherosclerotic effects and reduced plasma cholesterol levels, demonstrating the potential of a drug for treating hypercholesterolemia and atherosclerosis. However, subsequent clinical trial results showed that Avasimibe did not exhibit the expected clinical effects and was not superior to the existing treatment methods at that time in reducing the incidence of cardiovascular events. Therefore, the clinical development of Avasimibe for treating cardiovascular diseases and hypercholesterolemia was terminated.

[0004] However, in recent years, a number of studies have shown the treatment potential of Avasimibe in other disease fields. For example, in cancer treatment, Avasimibe causes apoptosis of cancer cells by disrupting their lipid metabolism and also shows the potential to enhance the efficacy of other chemotherapy drugs; in neuroprotection, Avasimibe reduces the formation of amyloid plaques by inhibiting SOAT, thereby slowing down the progression of Alzheimer's disease; in the treatment of metabolic diseases, since obesity is associated with dysregulated lipid metabolism and elevated cholesterol levels, which can promote insulin resistance and the development of type 2 diabetes, and by inhibiting SOAT, Avasimibe may enhance insulin sensitivity.

[0005] At present, it is still unknown whether Avasimibe can be applied to Staphylococcus aureus infectious diseases. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of Avasimibe in the preparation of drugs for treating Staphylococcus aureus infectious diseases, so as to solve the problems raised in the above background technology. To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The application of Avasimibe in the preparation of drugs for treating Staphylococcus aureus infectious diseases.

[0008] Preferably, the Avasimibe enhances the ability of macrophages to kill Staphylococcus aureus intracellularly.

[0009] Preferably, the macrophages include Raw264.7 cells, THP-1 cells, mouse primary bone marrow-derived macrophages, and other mouse primary tissue-resident macrophages.

[0010] Preferably, for the Staphylococcus aureus, the Avasimibe has bactericidal properties.

[0011] Preferably, for the Staphylococcus aureus, the minimum bactericidal concentration of the Avasimibe is 5 μM.

[0012] Preferably, the Avasimibe inhibits the biofilm formation of the Staphylococcus aureus.

[0013] Preferably, the Avasimibe inhibits the proliferation of the Staphylococcus aureus.

[0014] Preferably, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, vancomycin-intermediate-resistant Staphylococcus aureus, and vancomycin-resistant Staphylococcus aureus.

[0015] Preferably, the Staphylococcus aureus infectious diseases include but are not limited to pneumonia, systemic infection, skin abscess, endocarditis, osteomyelitis, toxic shock syndrome, sepsis, liver abscess, and meningitis.

[0016] Preferably, the osteomyelitis is implant-associated Staphylococcus aureus osteomyelitis.

[0017] The beneficial effects of the present invention are as follows: The present invention reveals that Avasimibe treats Staphylococcus aureus infectious diseases through the dual effects of enhancing the intracellular bactericidal ability of macrophages and directly killing bacteria. At the same time, it also reveals that Avasimibe has the functions of inhibiting the biofilm formation of Staphylococcus aureus and inhibiting the proliferation of Staphylococcus aureus, and develops a new indication for Avasimibe. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 The figure showing the result of Avasimibe enhancing the ability of macrophages to kill Staphylococcus aureus in the present application. Among them, A and C are representative agar plate colony growth diagrams; B and D are colony number statistical diagrams.

[0020] Figure 2 The figure showing the bactericidal ability of Avasimibe against Staphylococcus aureus and the result of inhibiting the biofilm formation of Staphylococcus aureus in the present application. Among them, A is the OD value change diagram of the Staphylococcus aureus proliferation curve; B is the representative minimum inhibitory concentration experiment result diagram; C is the representative minimum bactericidal concentration experiment result diagram; D is the representative crystal violet staining result diagram; E is the crystal violet staining OD value statistical diagram.

[0021] Figure 3 The figure showing the inhibitory effect of Avasimibe on the proliferation of Staphylococcus aureus in the present application.

[0022] Figure 4 The figure showing that Avasimibe reduces the femoral infection degree of mice with Staphylococcus aureus osteomyelitis in the present application. Among them, A is the macroscopic view of the mouse femur; B is the mouse body weight change diagram; C is the mouse femur weight change diagram; D is the representative hematoxylin-eosin staining diagram of the mouse femur; E is the representative Gram staining diagram of the mouse femur; F is the representative scanning electron microscopy diagram of the implant; G is the statistical diagram of the histological score of the mouse femur.

[0023] Figure 5The femur bone defect in mice with Staphylococcus aureus osteomyelitis reversed by Avasimibe in the present application is shown. Among them, in Figure A, from top to bottom are the representative two-dimensional imaging results of micro-computed tomography (Micro-CT), the representative three-dimensional imaging results of Micro-CT cortical bone, and the representative three-dimensional imaging results of Micro-CT cancellous bone; Figure B is a statistical chart of bone mineral density and representative cancellous bone analysis parameters (BMD: bone mineral density; BV / TV: bone tissue volume / total tissue volume fraction; Tb.Sp: trabecular bone separation; Tb.N: trabecular bone number; Tb.Th: trabecular bone thickness; Tb.Pf: trabecular bone pattern factor); Figure C is a statistical chart of representative cortical bone analysis parameters (Tt.Ar: total cortical bone area; Ct.Ar: cortical bone area; Ct.Th: cortical bone thickness; Ct.Ar / Tt.Ar: ratio of cortical bone area to total area).

[0024] It should be noted that the accompanying drawings are not necessarily drawn to scale, but are shown only in a schematic manner that does not affect the understanding of the reader. Detailed implementation manners

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] It should also be understood that the term "and / or" used in the specification and appended claims of the present invention refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0027] Example 1:

[0028] This example is an experiment on the ability of Avasimibe to enhance the killing ability of bone marrow-derived macrophages (hereinafter referred to as BMDMs) against Staphylococcus aureus.

[0029] 1. Experimental materials

[0030] 1.1. Avasimibe, purchased from Selleck Chemicals, is a white powder and should be protected from light. It is prepared into a 20 mM stock solution using dimethyl sulfoxide (DMSO) and stored at -80°C.

[0031] 1.2. C57 mice, male, 8 weeks old, were purchased from the Experimental Animal Center of Southern Medical University and raised in a SPF-level animal experiment center.

[0032] 1.3. Tryptic Soy Broth (TSB) Medium: Dissolve 24 g of tryptone soya broth (Solarbio, LA0110) in 400 mL of distilled water. Autoclave at 121 °C for 15 - 30 min and store at 4 °C in the refrigerator for later use.

[0033] 1.4. Tryptic Soy Agar (TSA) Plates: Dissolve 20 g of tryptic soy agar medium (Huankai Microbial, 028074) in 500 mL of distilled water. Autoclave at 121 °C for 15 - 30 min. Then pour 20 mL of TSA into a sterile petri dish for bacteria to prepare agar plates. After it solidifies, seal it and store at 4 °C in the refrigerator for later use.

[0034] 1.5. Cell Lines, Reagents for Cell Extraction and Culture: L929 mouse epithelial-like fibroblasts were purchased from the cell bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai. PBS (Servicebio), RPMI 1640 medium (Servicebio), DMEM medium (Servicebio), fetal bovine serum (BI, 04 - 001 - 1ACS), 75% ethanol (Lierkang), ACK red blood cell lysate (Phygene, PH0411), penicillin / streptomycin double antibody solution (Gibco, 15140 - 122), 10 mg / mL gentamicin, 10 mg / mL lysozyme, Triton - 100 (Sigma - Aldrich, 9036 - 19 - 5).

[0035] 2. Experimental Procedures

[0036] 2.1. Preparation of L929 Supernatant: Add 5×10^5 L929 cells to 55 mL of MEMα basal medium (Gibco, C12571500BT) containing 10% FBS, inoculate into a T75 culture flask, and do not change the medium during the culture process. After 7 days, collect the supernatant and filter it through a 0.22 μm filter (Millipore, SLGVR33RB) for later use.

[0037] 2.2. Extraction and culture of BMDMs: For the extraction of BMDMs, mice were first sacrificed by cervical dislocation and immediately immersed in 75% alcohol for 5 minutes for disinfection. The lower extremities of the mice were aseptically isolated in a biosafety cabinet, and the muscle tissues around the femurs and tibias were removed. For the femurs, the femoral heads and distal growth plates were excised; for the tibias, the bones below the tibiofibular syndesmosis and proximal growth plates were excised. The processed bone tissues were placed in DMEM medium, and the medullary cavities were repeatedly rinsed with the medium until they turned white. The cell suspension was gently pipetted repeatedly, and then filtered through a 70μm tissue sieve. The filtrate was centrifuged at 1000 rpm for 5 minutes at room temperature. After discarding the supernatant, the cells were resuspended with 1 mL of ACK red blood cell lysate and placed on ice for 5 minutes to lyse red blood cells. After centrifugation, the supernatant was discarded, and the cells were washed once with PBS. The cells were resuspended with DMEM medium. The culture medium for BMDMs was DMEM containing 10% fetal bovine serum and 25% L929 supernatant. The extracted BMDMs were cultured in a constant temperature cell incubator at 37°C with a CO2 concentration of 5%. The cell growth status was observed under a microscope regularly. Half of the medium was changed on the 3rd day after extraction, and then the medium was completely changed on the 5th and 7th days. The cells were mature after 7 days and could be used for the next experiment.

[0038] 2.3. Culture and quantification of Staphylococcus aureus: The Staphylococcus aureus strain used in this example was isolated from a patient with chronic osteomyelitis of the tibia and identified as methicillin-sensitive. Staphylococcus aureus was stored in a -80°C refrigerator in TSB medium containing 10% DMSO. When in use, the frozen Staphylococcus aureus bacterial solution was taken out from the -80°C refrigerator and quickly thawed. 10 μL of the bacterial solution was pipetted into a shaking flask pre-added with 3 mL of TSB medium. The shaking flask was tilted at 45° to allow air to enter and placed in a bacterial shaker, and cultured at 180 rpm and 37°C until the logarithmic growth phase, which generally took 16 - 18 hours. Subsequently, 1 mL of the bacterial solution was taken, centrifuged at 2500 g for 5 minutes, and washed 3 times with PBS. Then it was resuspended with 1 mL of PBS and diluted several times. 200 μL was pipetted into a 96-well plate, and the absorbance density

[0039] ^

[0040] (optical density, OD) was measured at 600 nm using a multifunctional microplate reader (CLARIOstar, Spectra Max i3X). When OD = 0.5, the bacterial amount in the solution was approximately 1*108 CFU / mL.

[0041] 2.4. Experiment on killing Staphylococcus aureus by BMDMs: BMDMs were cultured until mature, and the medium was replaced with a medium without antibiotics. Staphylococcus aureus was used to infect the cells at a multiplicity of infection (MOI) of 10. After 30 minutes of infection, non-adherent bacteria were rinsed with PBS. BMDMs were incubated at 37°C for 1 hour in complete DMEM medium supplemented with 20 μg / mL lysozyme and 50 μg / mL gentamicin to remove the remaining extracellular bacteria. Subsequently, the cells were washed with PBS and cultured in complete BMDM medium with antibiotics and different concentrations of Avasimibe for the specified time. Then, the macrophages treated above were lysed with 0.1% Triton X-100 to release intracellular bacteria, and the cell lysate was serially diluted with PBS. 10 μL of each dilution was respectively pipetted onto agar plates and cultured in a bacterial incubator at 37°C for 12 - 16 hours, and then the number of bacterial colonies was determined.

[0042] 2.5. Statistics and analysis: All data were expressed as mean ± SD, and statistical analysis was performed using SPSS 19.0 software. Paired T-test was used between two groups; One-way ANOVA test was used among multiple groups. p < 0.05 indicated significant difference.

[0043] 3. Experimental results

[0044] 3.1. Figure 1 -A and Figure 1 -B showed that at 12 h after infection, Avasimibe at different concentrations could enhance the ability of BMDMs to kill Staphylococcus aureus intracellularly, and it changed positively with the concentration. Figure 1 -C and Figure 1 -D showed that 10 μM Avasimibe could stably enhance the ability of BMDMs to kill Staphylococcus aureus intracellularly, and it did not change with the prolongation of the killing time (Veh represents the simple infection group, and Avasimibe represents the simple infection + Avasimibe treatment group). * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0045] Example 2:

[0046] This example was an experiment on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Avasimibe against different strains of Staphylococcus aureus.

[0047] 1. Experimental materials

[0048] 1.1. Avasimibe, purchased from Selleck Chemicals, is a white powder and should be protected from light. It was dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution and stored at -80 °C.

[0049] 1.2. Tryptic Soy Broth (TSB) medium: Dissolve 24 g of tryptic soy broth (Solarbio, LA0110) in 400 mL of distilled water. Autoclave at 121 °C for 15 - 30 min and store at 4 °C in the refrigerator for later use.

[0050] 1.3. Tryptic Soy Agar (TSA) plates: Dissolve 20 g of tryptic soy agar medium (Huankai Microbial, 028074) in 500 mL of distilled water. Autoclave at 121 °C for 15 - 30 min. Then, pour 20 mL of TSA into a sterile petri dish for bacteria to prepare agar plates. After it solidifies, seal it and store it in the refrigerator at 4 °C for later use.

[0051] 2. Experimental procedures

[0052] 2.1. Staphylococcus aureus culture and quantification: The 4 Staphylococcus aureus strains used in this example were methicillin - sensitive Staphylococcus aureus strains (Primary S.A.) isolated from patients, methicillin - resistant Staphylococcus aureus strains (MRSA 37, MRSA59), and methicillin - sensitive Staphylococcus aureus strain (ATCC6538) purchased from Huankai Biotechnology. The culture and quantification steps were as described in Example 1.

[0053] 2.2. Minimum inhibitory concentration (MIC) detection: The Staphylococcus aureus cultured to the logarithmic growth phase was serially diluted to 5*10 ^ 5 CFU / mL. Then, Avasimibe was serially diluted in a 96 - well plate. Subsequently, the diluted bacterial suspension was added to each well containing Avasimibe so that the final bacterial concentration in the well was approximately 2.5*10 ^ 5 CFU / mL. The final concentration gradient of Avasimibe was from 1.25 μM to 20 μM, and the total volume of the liquid in the well was 200 μL. Negative and positive controls were set up. After sealing the 96 - well plate, it was placed in an incubator at 37 °C and incubated for 16 - 18 hours. Finally, after the incubation, the growth of bacteria in each well was detected by visual inspection and using an enzyme - linked immunosorbent assay (ELISA) reader to determine the OD value, and the results were recorded for data analysis.

[0054] 2.3. Minimum bactericidal concentration (MBC) detection: After the minimum inhibitory concentration detection was completed, samples were taken incrementally in concentration from the wells with a concentration one level lower than the concentration without visible bacterial growth. 10 μL of the suspension was taken from each well and inoculated onto an agar plate. Then, the agar plate was placed in a bacterial incubator at 37 °C and incubated for 12 - 16 hours to observe the growth of bacteria.

[0055] 3. Experimental Results

[0056] 3.1. Figure 2 -A and Figure 2 -B showed that for 4 Staphylococcus aureus strains, the minimum inhibitory concentration of Avasimibe was 5 μM for all of them. Figure 2 -C showed that for 4 Staphylococcus aureus strains, when Avasimibe was at 5 μM, the bacteria in the wells at this concentration could not form colonies on the agar plate, that is, the minimum bactericidal concentration of Avasimibe was 5 μM. Therefore, the minimum inhibitory concentration of Avasimibe was equal to the minimum bactericidal concentration, so Avasimibe had bactericidal activity against Staphylococcus aureus.

[0057] Example 3:

[0058] This example was an experiment on the inhibition of biofilm formation of Avasimibe against different Staphylococcus aureus strains.

[0059] 1. Experimental Materials

[0060] 1.1. Avasimibe, purchased from Selleck Chemicals, was a white powder and stored away from light. It was prepared into a 20 mM stock solution using dimethyl sulfoxide (DMSO) and stored at -80 °C.

[0061] 1.2. Tryptic Soy Broth (TSB) medium: 24 g of tryptic soy broth (Solarbio, LA0110) was dissolved in 400 mL of distilled water. It was autoclaved at 121 °C for 15 - 30 min and stored at 4 °C in the refrigerator for later use. 1.3 Other reagents: 95% ethanol, PBS (Servicebio).

[0062] 1.3. Crystal violet staining solution, purchased from Biosharp.

[0063] 2. Experimental Procedures

[0064] 2.1. Pre - culture of bacterial solution: Staphylococcus aureus was inoculated into TSB medium and cultured overnight at 37 °C (about 16 - 18 hours).

[0065] 2.2. Dilution of bacterial solution: The overnight - cultured bacterial solution was diluted to 5*10 ^ 5 CFU / mL with fresh TSB medium.

[0066] 2.3. Gradient dilution of avasimibe: According to the required concentration range (the final concentration gradient of avasimibe is 2.5 μM to 20 μM), serial dilutions of avasimibe are performed in a 96-well plate. Subsequently, the diluted bacterial suspension is added to each well containing avasimibe, so that the final concentration of bacteria in the well is approximately

[0067] ^

[0068] 2.5×105 CFU / mL, the total volume of the liquid in the well is 200 μL, and negative and positive controls are established. After sealing the 96-well plate, it is placed in an incubator at 37 °C and incubated statically for 24 hours.

[0069] 2.4. Fixation: Carefully pour out the culture medium in the well, and each well is gently washed 3 times with 200 μL of sterile PBS buffer to remove the non-adherent bacteria. Subsequently, the microplate is dried in an oven at 60 °C for 1 hour to fix the adhered biofilm.

[0070] 2.5. Staining: Add 200 μL of 0.1% crystal violet staining solution to each well and stain at room temperature for 15 minutes. Pour out the staining solution. Gently wash each well 3 times with distilled water to remove the excess dye. Invert the microplate on absorbent paper and gently pat dry.

[0071] 2.6. Measurement of absorbance: Add 200 μL of 95% ethanol to each well and gently shake or oscillate at room temperature for 15 minutes to dissolve the stained biofilm. The absorbance of each well is measured at a wavelength of 570 nm. The reading of the negative control well is used as the background value for deduction, and the values are recorded.

[0072] 2.7. Statistics and analysis: All data are expressed as mean ± SD, and statistical analysis is performed using SPSS 19.0 software. One-way ANOVA test is used among multiple groups. P < 0.05 indicates significant difference.

[0073] 3. Experimental results

[0074] 3.1. Figure 2 -D and Figure 2 -E show that for 4 strains of S. aureus, when the concentration of avasimibe is 10 μM, compared with the 0 μM group, the purple staining in the well is significantly lightened, even approaching colorless, indicating that the formation of biofilm is strongly inhibited. The OD value also decreases significantly, indicating that avasimibe has the ability to inhibit the formation of S. aureus biofilm.

[0075] Example 4:

[0076] This example is the inhibitory experiment of avasimibe on the proliferation of S. aureus.

[0077] 1. Experimental materials

[0078] 1.1. Avasimibe, purchased from Selleck Chemicals, is a white powder and should be protected from light. It was prepared into a 20 mM stock solution with dimethyl sulfoxide (DMSO) and stored at -80 °C.

[0079] 1.2. Tryptic Soy Broth (TSB) medium: Dissolve 24 g of tryptic soy broth (Solarbio, LA0110) in 400 mL of distilled water. Autoclave at 121 °C for 15 - 30 min and store at 4 °C for later use.

[0080] 1.3. Bacterial staining kit was purchased from Pythonbio

[0081] 1.4. Other reagents and consumables: glass slides, PBS (Servicebio).

[0082] 2. Experimental procedures

[0083] 2.1. Pre - culture of bacterial solution: Inoculate Staphylococcus aureus in TSB medium and incubate overnight at 37 °C (about 16 - 18 hours).

[0084] 2.2. Dilution of bacterial solution: Dilute the overnight - cultured bacterial solution with fresh TSB medium to 10^8 CFU / mL.

[0085] 2.3. Gradient dilution of Avasimibe: Transfer the diluted bacterial solution to EP tubes, 1 mL per tube. According to the required concentration range (the final concentration gradient of Avasimibe is MIC, 1 / 2 MIC, 1 / 4 MIC), dilute Avasimibe and add it to the EP tubes, and set up a positive control group.

[0086] 2.4. Staining: After adding the reagents, incubate in an incubator at 37 °C for half an hour. Then centrifuge at 2500 g for 3 minutes to obtain bacterial pellets, and resuspend with 2 mL of PBS. Then pipette 1 mL of the bacterial suspension, add 1.5 μL of SYTO9 and 1.5 μL of PI, and incubate at room temperature in the dark for 10 minutes.

[0087] 2.5. Observation and photography: Pipette 5 μL of liquid from each tube onto a glass slide, cover it with a square coverslip, transfer it to a fluorescence microscope for observation. Bacteria showing green fluorescence are live bacteria, and those showing red fluorescence are dead bacteria. Select representative areas for photography.

[0088] 3. Experimental results

[0089] 3.1. SYTO-9 is a green fluorescent nucleic acid dye that can penetrate cell membranes and can be used for staining live and dead bacteria. PI is a nuclear stain that can stain DNA and emits red fluorescence after embedding in double-stranded DNA. PI cannot penetrate intact cell membranes, but can penetrate the cell membranes of dead bacteria and stain the nuclei red. Figure 3 It shows that when the concentration of Avasimibe gradually increases, the green fluorescence intensity gradually weakens and the red fluorescence intensity gradually increases, indicating that when Avasimibe is at MIC, it has the ability to kill Staphylococcus aureus.

[0090] Example 5:

[0091] This example is an experimental treatment of Staphylococcus aureus osteomyelitis in mice with Avasimibe.

[0092] 1. Experimental materials

[0093] 1.1. Instruments and consumables required for establishing an implant-related Staphylococcus aureus osteomyelitis model and femoral bone sampling: small animal operating table, ophthalmic scissors, ophthalmic forceps, microsurgical forceps, constant temperature incubator, 50 mL centrifuge tube, 12 mL plastic shaking tube, isopropanol, absolute ethanol, M1*2 stainless steel self-tapping screw, handheld small electric drill, small cross screwdriver, sterile gauze, sterile cotton swab, needle holder, 5-0 silk thread.

[0094] 1.2. Tissue fixative: Weigh 20 g of paraformaldehyde, add it to 500 mL of PBS solution, and keep it in an oven at 60 °C overnight. Take it out after complete dissolution.

[0095] 1.3. Bone tissue decalcifying solution: Dissolve 93 g of EDTA and 11.5 g of NaOH in 500 mL of double-distilled water, dissolve it thoroughly on a shaker, and adjust the pH to 8 with 1 M sodium hydroxide solution.

[0096] 1.4. Hematoxylin staining solution: Solution A: Weigh 2 g of hematoxylin and dissolve it in 40 mL of absolute ethanol; Solution B: Weigh 100 g of potassium alum and dissolve it in 600 mL of double-distilled water. Mix the two solutions and boil for 2 minutes. Make up to 600 mL with double-distilled water, add 400 mg of sodium iodate and mix well.

[0097] 1.5. Eosin staining solution: First dissolve 0.5 - 1 g of eosin in 100 mL of 90% alcohol, and add 20 μL of glacial acetic acid after dissolution.

[0098] 1.6. 1% hydrochloric acid alcohol differentiation solution: Dilute 1 mL of concentrated hydrochloric acid in 99 mL of 75% alcohol.

[0099] 1.7. Gram staining reagents: neutral resin, absolute ethanol, Gram staining (Modified Brown&Breen) kit (ScyTek, BBS-2-IFU).

[0100] 1.8. Electron microscope fixative: Glutaraldehyde fixative, purchased from Biosharp.

[0101] 2. Experimental procedures

[0102] 2.1. Establishment of the Staphylococcus aureus osteomyelitis model related to implants and the treatment regimen of Avasimibe

[0103] 2.1.1. Pretreatment of stainless steel self-tapping screws: Put M1*2 stainless steel self-tapping screws into a 1.5 mL EP tube, add 1 mL of isopropanol, place it on a shaker, and shake it at low speed for 5 minutes. Then discard the isopropanol and add absolute ethanol, and place it on the shaker again to shake at low speed for 5 minutes. Then discard the absolute ethanol and replace it with PBS, and wash 3 times. The above steps are to clean the machine oil on the stainless steel self-tapping screws to prevent the subsequent bacterial solution from not adhering to the surface of the self-tapping screws. Subsequently, put 20 - 30 washed self-tapping screws into each 1.5 mL EP tube. After stuffing a cotton ball at the opening of the EP tube, transfer it to a high-pressure autoclave for high-temperature and high-pressure sterilization.

[0104] 2.1.2. Preparation of bacteria-coated screws: After resuscitating and quantifying Staphylococcus aureus, dilute the bacterial solution to 1*10 ^ 4 CFU / mL. Subsequently, place the sterile self-tapping screws in the bacterial solution and shake them at low speed on a shaker for 15 minutes. Then pour the bacteria-coated screws into a sterile Petri dish, suck out the residual liquid, place them in a 37°C incubator for 5 - 10 minutes, and then place them in a sterile Petri dish and put them on ice for later use.

[0105] 2.1.3. Model establishment: Male C57BL / 6 mice at 8 - 9 weeks of age, fasted and water-deprived for more than 6 hours before surgery.

[0106] 2.1.3.1. Anesthesia: Inject 500 μL of tribromoethanol working solution intraperitoneally into the mice. After the anesthetic takes effect, fix the mice in the supine position on a foam pad. The foam pad can have a height of 10° - 15°, making the mice present a head-high and foot-low position, with both lower limbs spread and fixed to expose the surgical area of the right femur and hip.

[0107] 2.1.3.2. Skin preparation: Prepare the skin of the mice's thighs, and the trimming range is from the knee joint to the groin, and try to trim it to the outside as much as possible. Fix the lower abdomen with transparent tape. Disinfect the surgical area three times with 75% alcohol, and cover it with a sterile drape.

[0108] 2.1.3.3. Incision: Locate the surface landmark of the greater trochanter of the right femur and make an incision about 0.5 cm parallel to the longitudinal axis of the right femur.

[0109] 2.1.3.4. Expose the surgical area: Find the femur in the muscle space, bluntly separate the muscle with a curved forceps, and use a hemostat to expand and expose the field of view at the distal end of the third trochanter of the femur.

[0110] 2.1.3.5. Insertion of the bacteria-containing nail: At the distal horizontal plane of the third trochanter of the femur, at the midpoint of the long axis of the femur, drill a vertical hole with an electric drill (only drill out the nail position to avoid excessive bleeding affecting the vision). Hold a screwdriver and rotate and fix the bacteria-containing nail or the sterile self-tapping nail (note that do not over-rotate the self-tapping nail to avoid drilling through the femur or causing a fracture of the femoral shaft).

[0111] 2.1.4. Suture and postoperative observation: Stop bleeding with a sterile cotton swab. After the muscles are aligned, suture the skin of the incision with 5-0 silk thread for 1-2 stitches after aligning the incision skin. Disinfect the surgical area with 75% alcohol, and the operation is completed.

[0112] 2.1.5. Grouping and treatment regimen of Avasimibe: Randomly divide the established Staphylococcus aureus osteomyelitis mouse model into 3 groups, namely the simple infection group (vehicle), the low-concentration treatment group (Avasimibe (15 mg / kg)), and the high-concentration treatment group (Avasimibe (30 mg / kg)). The treatment groups were given the corresponding concentration of Avasimibe after the operation, and then administered the drug every other day. The simple infection group was given corn oil containing DMSO. The experimental endpoint was the 8th day after infection.

[0113] 2.2. Tissue fixation, decalcification, dehydration, embedding and sectioning.

[0114] 2.2.1. Fixation: After removing the right femur of the mouse in vitro, take out the self-tapping nail, and then place it in a 4% paraformaldehyde solution for fixation for 24 hours.

[0115] 2.2.2. Decalcification: After the specimen is fixed, wash it once with PBS, and then put it into the EDTA decalcifying solution. Replace the decalcifying solution every 3 days, and decalcify for a total of 10 days.

[0116] 2.2.3. Dehydration and embedding: After checking that the softness of the bone tissue is appropriate, place it in a tissue embedding cassette, continuously rinse with running water for 30 minutes, and then immerse it in the following liquids in sequence for dehydration: 70% ethanol I for 1 hour → 70% ethanol II for 1 hour → 80% ethanol I for 1 hour → 80% ethanol II for 1 hour → 95% ethanol I for 1 hour → 95% ethanol II for 1 hour → absolute ethanol I for 1 hour → absolute ethanol II for 1 hour → xylene I for 1 hour → xylene II for 1 hour. Preheat the paraffin tissue embedding machine. After the wax block in the machine is completely dissolved, immerse the specimen in wax for 2 hours, and then perform embedding. Add wax liquid to the metal mold, put in the tissue specimen, place the metal mold on the cooling table, quickly align the position of the specimen. At this time, the bottom wax liquid solidifies to fix the position of the specimen, and then continue to add wax liquid until the mold is filled. Press a plastic embedding cassette on the top and place it on the cooling table to continue cooling. After the wax block solidifies, store it at room temperature or transfer it to a -20°C refrigerator.

[0117] 2.2.4. Sectioning: Take out the tissue wax block and place it on the cooling table. Use a microtome to make continuous sections along the coronal plane of the femur. The section thickness is 4 μm. Gently flatten the sections on the water surface at 42 °C, carefully pick them up with an adhesive glass slide, and put them into a drying oven to dry for 2 hours to ensure that the sections are completely attached to the glass slide. Place the specimens at room temperature and use them as soon as possible.

[0118] 2.3. Gram (Gram) staining.

[0119] 2.3.1. Deparaffinization and hydration: Place the sections on a slide rack and put them into the following staining jars one by one. Xylene I for 15 minutes → Xylene II for 15 minutes → Absolute ethanol I for 5 minutes → Absolute ethanol II for 5 minutes → 95% ethanol I for 5 minutes → 95% ethanol II for 5 minutes → 80% ethanol for 5 minutes → 70% ethanol for 5 minutes → Immerse in distilled water for 1 minute. Gently wash the slides on a shaker with PBS three times.

[0120] 2.3.2. Cover the tissue sections with gentian violet solution and incubate for 2 minutes.

[0121] 2.3.3. Rinse the slides with distilled water to remove the excess gentian violet solution.

[0122] 2.3.4. Cover the tissue sections with Lugol's iodine solution and incubate for 1 minute.

[0123] 2.3.5. Wash the slides in running deionized water to remove the excess iodine solution, and repeat this step once.

[0124] 2.3.6. Drop the Gram decolorizer on the slides until the color no longer exudes. (Note the action time of the decolorizer. Dropping for more than 5 seconds may remove the gentian violet staining).

[0125] 2.3.7. Quickly rinse the slides in deionized water, then cover the tissue sections with safranin O solution and incubate for 4 minutes.

[0126] 2.3.8. Quickly rinse the sections in deionized water to remove the excess staining, then immerse the tissue sections in absolute alcohol once and blot the excess alcohol.

[0127] 2.3.9. Cover the tissue sections with picric acid - acetone solution and gently shake and incubate for 2 - 10 seconds, then immediately quickly rinse the sections in absolute alcohol. If the tissue is still dark red, repeat this step until most of it appears yellow.

[0128] 2.3.10. Let the sections air dry naturally, put them into xylene for 5 minutes for dehydration and clearing, and repeat this step once. Finally, mount the sections with neutral resin.

[0129] 2.4. Hematoxylin-Eosin (H&E) staining.

[0130] 2.4.1. Deparaffinization and hydration: As described above.

[0131] 2.4.2. Hematoxylin staining, differentiation, and blueing: Place the hydrated slides in hematoxylin staining solution and let stand for 3 minutes, rinse with running water for 2 minutes, differentiate with 1% hydrochloric acid alcohol for 2 - 3 seconds, and continue to rinse with running water for 10 minutes.

[0132] 2.4.3. Eosin staining, dehydration, and clearing: Place the sections in 70% alcohol for 5 minutes → 90% alcohol for 5 minutes, then stain with eosin staining solution for 8 seconds, and continue to dehydrate and clear in the order of 95% ethanol I for 2 minutes, 95% ethanol II for 2 minutes, absolute ethanol for 2 minutes, and xylene for 2 minutes, and seal with neutral resin.

[0133] 2.5. Histopathological scoring

[0134] The histological Smeltzer method was used to score each specimen section, which is the sum of four aspects: intramedullary acute inflammation (0 to 4 points), intramedullary chronic inflammation (0 to 4 points), periosteal inflammation (0 to 4 points), and bone necrosis (0 to 4 points). The scoring criteria are shown in Table 1.

[0135] Table 1 Histopathological score criteria by Smeltzer’s

[0136] Table 1 Histopathological score criteria by Smeltzer’s

[0137]

[0138]

[0139] 2.6. MicroCT scanning

[0140] After removing the right femur of the mouse, the muscle tissue around the femur was removed and soaked in 4% paraformaldehyde for 24 hours. First, the self-tapping screws were removed from the fixed femur. After wetting, the specimens were wrapped with sealing film to prevent drying. According to the usage specifications, the specimens were placed and fixed on the Micro-CT scanning table. The scanning parameters were set as follows: voltage 55 kV, current 145 mA, integration time 400 ms, and isotropic pixel size 12 μm. After the scanning was completed, the scanning data was converted into an image format. The software CT vox (Bruker, version 3.0.0r1122) was used to perform three-dimensional reconstruction of the ROI and export the three-dimensional image. The software CT Analyser (Bruker, version 1.15.4.0+) was used to draw the regions of interest (Range of Interest, ROI) of cortical bone and cancellous bone respectively for relevant parameter analysis. To ensure the reliability and uniformity of the analysis data, we customized the ROI analysis program in CT Analyser. According to this program, the analysis results (as shown in Table 2) could be obtained quickly and accurately. The cancellous bone analysis parameters included: bone volume / tissue volume (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular space (Tb.Sp), bone mineral density (BMD), and trabecular bone pattern factor. The cortical bone analysis parameters included: cortical area (Ct.Ar), cortical bone thickness (Ct.Th), and total cross-sectional area of cortical bone (Transection area, Tt.Ar).

[0141] Table 2 ROI analysis program

[0142]

[0143]

[0144] 2.7. Scanning electron microscopy imaging

[0145] 2.7.1. Primary fixation: The self-tapping screw samples were placed in 2.5% glutaraldehyde solution and fixed at room temperature for 2 hours. Usually, 0.1 M phosphate buffer solution (PBS) was used as the solvent of the fixing solution, and the pH was controlled at 7.2 - 7.4.

[0146] 2.7.2. Washing: Wash the samples 3 times with PBS buffer for 10 minutes each time to remove residual fixative.

[0147] 2.7.3. Gradient dehydration: Dehydrate the samples successively in ethanol with different concentration gradients (30%, 50%, 70%, 90%, 100%), soaking in each concentration for 10 minutes.

[0148] 2.7.4. Drying: Use the critical point drying method with CO2 as the drying medium.

[0149] 2.7.5. Sample pasting: Fix the dried samples on the aluminum stage with conductive tape to ensure the stability of the samples.

[0150] 2.7.6. Gold plating: Deposit gold in an ion sputtering instrument, usually with a thickness of 10 - 20 nm, to improve the conductivity of the samples and reduce the charge accumulation during imaging.

[0151] 2.7.7. Sample installation and vacuum treatment: Place the prepared samples on the sample stage, install them in the sample chamber of the scanning electron microscope, and start the vacuum pumping system to the required vacuum level.

[0152] 2.7.8. Image acquisition: Select an appropriate acceleration voltage according to the conductivity of the samples. By adjusting the working distance and objective lens current, make the image in the field of view clear, and adjust the contrast and brightness to optimize the image quality. Determine the appropriate magnification, capture and save the image. Different regions or different magnifications of the image can be captured multiple times as needed to obtain more comprehensive information.

[0153] 3. Experimental results

[0154] 3.1. Figure 4 A shows that macroscopically, compared with the femurs of the Vehicle group, which were pale due to Staphylococcus aureus osteomyelitis, the intervention of Avasimibe made the femurs more blood - colored. Figure 4 B shows that the use of Avasimibe caused a decreasing trend in the body weight of mice, but at the observation endpoint, there were no statistical differences among the groups. Figure 4 C shows that the use of Avasimibe did not cause a statistical difference in the mass of the infected femurs.

[0155] 3.2. Figure 4 D shows that compared with the extensive abscesses in the medullary cavity of the femurs and the large - scale bone destruction in the cancellous bone of the distal femur in the Vehicle group, the use of Avasimibe reversed the bone destruction caused by osteomyelitis and reduced the spread range of the abscesses. Figure 4Group G showed a significantly decreased histological score at two concentrations of Avasimibe, suggesting that Avasimibe significantly improved femoral inflammation and bone destruction (compared with the Vehicle group, *** for p < 0.001).

[0156] 3.3. Figure 4 The Gram staining results of E showed that there was an obvious positive staining area in the Vehicle group, while the positive staining areas in the two Avasimibe groups were significantly reduced and the staining degree was lighter. It is suggested that the use of Avasimibe significantly reduced the bacterial load in the infected femur (the yellow arrow indicates Staphylococcus aureus).

[0157] 3.4. Figure 4 The scanning electron microscopy results of F showed that there was a large connected biofilm on the surface of the self-tapping screw in the Vehicle group, and Staphylococcus aureus showed a round and smooth normal morphology. On the surface of the self-tapping screw in the two Avasimibe groups, the integrity of the biofilm was damaged, showing filamentous connections, and the cell bodies of Staphylococcus aureus were deformed and shrank. It is suggested that the use of Avasimibe significantly disrupted the biofilm formation of Staphylococcus aureus and affected the normal physiological state of Staphylococcus aureus.

[0158] 3.5. Figure 5 A showed that the cortical bone thickness in the Vehicle group was thinned, and the cancellous bone showed a sparse arrangement, while there was no bone destruction in the cortical bone in the two Avasimibe groups, and the cancellous bone also showed a dense structure arrangement similar to the normal state. Figure 5 B and Figure 5 C showed that the intervention of Avasimibe increased the representative parameters of bone density, cancellous bone and cortical bone with statistical differences, suggesting that the use of Avasimibe significantly improved the femoral bone destruction caused by Staphylococcus aureus osteomyelitis (BMD: bone mineral density; BV / TV: bone tissue volume / total tissue volume fraction; Tb.Sp: trabecular separation; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Pf: trabecular pattern factor; Tt.Ar: total cross-sectional area of cortical bone; Ct.Ar: cortical bone area; Ct.Th: cortical bone thickness; Ct.Ar / Tt.Ar: ratio of cortical bone area to total area).

[0159] For the embodiments of the present invention, it should also be noted that, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other to obtain new embodiments.

[0160] As described above, it is only the preferred embodiment of the present invention, and there is no limitation to the present invention in any form. The protection scope of the present invention shall be subject to the protection scope of the claims. Although the present invention has been disclosed above with the preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Use of Avasimibe in the preparation of medicines for treating Staphylococcus aureus infectious diseases.

2. The use according to claim 1, characterized in that: Avasimibe enhances the intracellular killing ability of macrophages against Staphylococcus aureus.

3. The use according to claim 2, characterized in that: The macrophages include Raw264.7 cells, THP-1 cells, mouse primary bone marrow-derived macrophages, and other mouse primary tissue-resident macrophages.

4. The use according to claim 1, characterized in that: Avasimibe is bactericidal against Staphylococcus aureus.

5. The use according to claim 4, characterized in that: For the Staphylococcus aureus, the minimum bactericidal concentration of Avasimibe is 5 μM.

6. The use according to claim 1, characterized in that: Avasimibe inhibits biofilm formation of the Staphylococcus aureus.

7. The use according to claim 1, characterized in that: The Avasimibe inhibits the proliferation of the Staphylococcus aureus.

8. The use according to any one of claims 1 to 7, characterized in that: The Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, vancomycin-intermediate-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus.

9. The use according to claim 1, characterized in that: The Staphylococcus aureus infectious diseases include but are not limited to pneumonia, systemic infection, skin abscess, endomyocarditis, osteomyelitis, toxic shock syndrome, sepsis, liver abscess and meningitis.

10. The use according to claim 9, characterized in that: The osteomyelitis is implant-related Staphylococcus aureus osteomyelitis.

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