Use of apigenin in the preparation of drugs for treating staphylococcus aureus infectious osteomyelitis

Abstract

The application provides an application of apigenin in preparation of a drug for treating staphylococcus aureus infectious osteomyelitis. The apigenin realizes the treatment of the staphylococcus aureus infectious osteomyelitis by inhibiting staphyloxanthin related virulence, reducing bacterial host adaptation ability, reducing osteogenic related cell damage and improving pathological changes of bone tissue at an infection site, provides a new active ingredient and treatment method for the treatment of drug-resistant staphylococcus aureus bone infection, and compared with an existing treatment technology for the infectious osteomyelitis which mainly depends on surgical debridement and directly inhibits or kills bacteria, has the advantages of simplicity, high efficiency, avoidance of problems such as drug resistance caused by use of a large amount of antibiotics, can be used as a replacement scheme or a combination scheme of the existing technology, and makes up for technical defects of an existing technical scheme.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a new pharmaceutical use of apigenin. Background Technology

[0002] Osteomyelitis is an infectious disease caused by pathogenic microorganisms invading bone tissue and the medullary cavity, leading to inflammation of the medullary cavity, bone necrosis, bone destruction, and local bone structural abnormalities. Due to the relatively limited blood supply to bone tissue, infection easily leads to necrotic bone, dead spaces, and local microcirculatory disturbances. Pathogens can also persistently colonize the surface of bone tissue or implants, making it difficult for antibiotics to reach and maintain effective concentrations at the site of infection. Therefore, infectious osteomyelitis typically has a long course, a high recurrence rate, and in severe cases, can cause bone defects, limb dysfunction, and even require multiple debridements or amputation.

[0003] Staphylococcus aureus ( Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens causing infectious osteomyelitis. It can adhere to bone tissue and medical implants, and can survive long-term at the site of infection through various means, including forming biofilms, evading host immune clearance, and damaging osteoblast-related cells. In recent years, methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly prevalent. Staphylococcus aureus Treatment of bone infections caused by MRSA (Metabolic Spondylitis A) is particularly challenging. Clinically, surgical debridement combined with long-term antibiotic therapy is commonly used. However, long-term antibiotic use can be limited by factors such as bacterial resistance, insufficient drug penetration into tissues, systemic adverse reactions, and infection recurrence. Therefore, developing novel treatment strategies that differ from traditional direct bacteriostatic or bactericidal modalities is of great significance for improving the treatment efficacy of Staphylococcus aureus-induced osteomyelitis.

[0004] The characteristic golden-yellow color of Staphylococcus aureus colonies primarily originates from its synthesized carotenoid pigment, staphyloxanthin. Staphyloxanthin is not merely used for colony coloration; it is an important virulence-related metabolite of Staphylococcus aureus. The staphyloxanthin molecule contains multiple conjugated double bonds, enabling it to participate in scavenging or resisting reactive oxygen species produced by host immune cells, thereby reducing oxidative stress damage to bacteria. Simultaneously, as an important component of the bacterial cell membrane, staphyloxanthin can also regulate the fluidity, stability, and physical properties of the cell membrane, enhancing the bacteria's adaptability to the host's defense environment and external stressors. Decreased staphyloxanthin levels increase the sensitivity of Staphylococcus aureus to peroxides, phagocytes, and the bactericidal environment of the blood, correspondingly weakening its survival and pathogenicity within the host.

[0005] Staphylin is synthesized through a series of enzymatic reactions, among which dehydrosqualene dehydrogenase (CrtN) is one of the key enzymes in the staphylin biosynthesis pathway, participating in the desaturation reaction that catalyzes the conversion of upstream intermediates into downstream colored carotenoid products. Inhibiting the expression or catalytic activity of CrtN can block or weaken staphylin synthesis, causing Staphylococcus aureus to lose some of its antioxidant protection and reducing its tolerance to host immune killing. Therefore, targeting the staphylin synthesis pathway, especially CrtN, does not necessarily require direct inhibition or killing of bacteria in vitro. Instead, it can enhance the host's immune system's clearance of pathogens by weakening bacterial virulence and host adaptability. This antiviral therapy strategy differs from the mode of action of traditional antibacterial drugs, theoretically reducing direct survival selection pressure and providing a new intervention pathway for drug-resistant Staphylococcus aureus infections.

[0006] Pteryxin (PTX) is a natural coumarin compound with a well-defined chemical structure and certain biological activities. According to existing reports, the medicinal uses of teryxin cover neurodegenerative diseases (Alzheimer's disease), metabolic diseases (obesity), inflammatory diseases, bone diseases (osteoporosis), and cardiovascular diseases.

[0007] Given the difficulties in treating Staphylococcus aureus-induced osteomyelitis, the continuous destruction of bone tissue at the infection site, and the lack of effective candidate drugs for drug-resistant infections in existing technologies, the purpose of this invention is to provide a drug that can effectively treat Staphylococcus aureus-induced osteomyelitis. Summary of the Invention

[0008] Based on the above objectives, the present invention first provides the application of apigenin in the preparation of a treatment for Staphylococcus aureus-infected osteomyelitis.

[0009] The apigenin described in this invention, CAS No. 13161-75-6, molecular formula C21H22O7, molecular weight 386.4 g / mol, IUPAC name [(9R,10R)-9-acetyloxy-8,8-dimethyl-2-oxo-9,10-dihydropyrano[2,3-f]chromen-10-yl] (Z)-2-methylbut-2-enoate, is mainly derived from plants such as *Peucedanum praeruptorum* Dunn (Apiaceae family), including but not limited to: *Peucedanum praeruptorum* Dunn, *Peucedanum japonicum* Thunb, *Pteryxia terebinthina*, *Seseli ericephalum*, *Libanotis condensata*, *Zizia aptera*, and *Angelica* species. furcijuga).

[0010] In the preparation of the drug provided by this invention, the therapeutic drug uses apigenin as the active ingredient to treat osteomyelitis caused by Staphylococcus aureus infection and to improve bone tissue damage caused by the infection. The Staphylococcus aureus is preferably methicillin-resistant Staphylococcus aureus, more preferably USA300 strain; the bone tissue damage includes one or more of the following: bone loss, destruction of trabecular microstructure, bone marrow cavity inflammation, cortical bone damage, and osteoclast-mediated enhanced bone resorption.

[0011] In a preferred embodiment, the Staphylococcus aureus-infected osteomyelitis refers to a disease caused by Staphylococcus aureus infection resulting in a decrease in one or more of the following indicators: bone volume, total volume, bone volume fraction, ratio of bone surface area to bone volume, connectivity density, trabecular thickness, number of trabecular bones, and bone mineral density, and / or an increase in trabecular separation.

[0012] In a preferred embodiment, the Staphylococcus aureus-infected osteomyelitis refers to a disease caused by Staphylococcus aureus infection, resulting in inflammatory cell infiltration in the bone marrow cavity, bone resorption, destruction of cortical bone structure, and an increase in multinucleated osteoclasts.

[0013] In a preferred embodiment, the treatment drug for Staphylococcus aureus-infected osteomyelitis is a drug that reduces the Staphylococcus aureus load in the bone marrow cavity after Staphylococcus aureus infection.

[0014] In a rat model of infectious osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) USA300, administration of apigenin at 20 mg / kg or 40 mg / kg improved bacterial load, bone tissue destruction, and inflammatory response in the medullary cavity to varying degrees, with the 40 mg / kg group showing the most significant overall improvement. Micro-CT scans showed that apigenin treatment restored bone volume, bone volume fraction, connectivity density, trabecular thickness, trabecular number, and bone mineral density to varying degrees, while trabecular separation decreased. Histopathological examination revealed reduced inflammatory cell infiltration in the medullary cavity, localized osteolysis, and cortical bone continuity. TRAP staining showed a decrease in TRAP-positive multinucleated osteoclasts. These results indicate that apigenin can reduce infection load, improve infection-induced bone structural damage and abnormal bone resorption, and has a therapeutic effect on infectious osteomyelitis induced by MRSA USA300.

[0015] In a preferred embodiment, the therapeutic agent for Staphylococcus aureus-induced osteomyelitis is an agent that inhibits the formation of staphylococcus aureus flavonoids.

[0016] In a preferred embodiment, the drug that inhibits the formation of glucoflavin by Staphylococcus aureus is a drug that reduces the thermal stability of Staphylococcus aureus dehydrosqualene dehydrogenase.

[0017] In a preferred embodiment, the drug that inhibits the formation of glucoflavin by Staphylococcus aureus is a drug that reduces the ability of Staphylococcus aureus to tolerate oxidative stress.

[0018] In a preferred embodiment, the drug that inhibits the formation of glucoflavin by Staphylococcus aureus is a drug that reduces the ability of Staphylococcus aureus to tolerate the bactericidal environment of whole blood.

[0019] In the technical solution provided by the present invention, the mechanism of action of apigenin against Staphylococcus aureus includes inhibiting the formation of staphylococcal flavonoids, interfering with the synthesis process of CrtN-associated staphylococcal flavonoids, and reducing the bacteria's tolerance to oxidative stress and the host's bactericidal environment, thereby weakening the virulence of Staphylococcus aureus and the host's adaptability.

[0020] More specifically, the minimum inhibitory concentration (MIC) of apigenin against USA300 was 512 μg / mL, and it inhibited glucosamine formation even at concentrations significantly lower than the MIC. Specifically, apigenin at concentrations of 32 μg / mL and 64 μg / mL did not significantly affect the growth of USA300, indicating that its effect is not primarily derived from direct bacterial inhibition. Further studies showed that apigenin can directly interact with CrtN and alter its thermal stability; surface plasmon resonance and fluorescence quenching experiments also supported the existence of this binding. After apigenin treatment, the sensitivity of USA300 to hydrogen peroxide and rabbit whole blood sterilization environments increased, and the damage to osteoblast progenitor cells was reduced. These results indicate that apigenin can reduce the pathogenicity of Staphylococcus aureus by inhibiting glucosamine synthesis, weakening bacterial antioxidant defenses, and enhancing host adaptability, providing a basis for its therapeutic effect on Staphylococcus aureus-induced osteomyelitis.

[0021] Secondly, this invention provides the application of apigenin as an inhibitor of Staphylococcus aureus dehydrosqualene dehydrogenase (CrtN) activity.

[0022] According to the findings of this invention, the equilibrium dissociation constant KD between apigenin and CrtN is 2.79 × 10⁻⁶. -5 mol / L, with a binding constant KA of 1.791 × 10⁻⁶. 4 The concentration of L / mol indicates that apigenin can directly bind to the CrtN protein. Thermal migration assays show that apigenin can alter the thermal stability of the CrtN protein, and cell thermal migration assays show that apigenin can alter the thermal stability of the CrtN protein in a cell lysis system. Molecular docking analysis shows that apigenin can enter the potential binding pocket of the CrtN protein, and the predicted binding free energy of the apigenin-CrtN complex is -6.8 kcal / mol. Docking results suggest that Ser349 and Arg330 may be key amino acid residues involved in the binding of apigenin to CrtN.

[0023] Based on the above findings, this invention provides the application of apigenin as an inhibitor of Staphylococcus aureus dehydrosqualene dehydrogenase activity. This inhibition is achieved by disrupting the thermostability of the CrtN protein.

[0024] Finally, this invention provides the application of apigenin in the preparation of a protective agent against Staphylococcus aureus-induced MC3T3-E1 cell damage.

[0025] This invention found that USA300-induced MC3T3-E1 cells die due to cell membrane damage, and treatment with apigenin can significantly increase the proportion of live cells and reduce the proportion of dead cells.

[0026] Based on the above findings, this invention provides the application of apigenin in the preparation of a protective agent against Staphylococcus aureus-induced MC3T3-E1 cell damage.

[0027] This invention provides a novel use of apigenin in the preparation of drugs for treating Staphylococcus aureus-induced osteomyelitis, offering a candidate treatment regimen different from the traditional antibacterial drug mechanism for bone infections caused by Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA). In a rat model of MRSA-induced osteomyelitis, administration of apigenin at 20 mg / kg and 40 mg / kg both improved bone loss and trabecular microstructure damage to varying degrees, increasing bone volume, bone volume fraction, connectivity density, trabecular thickness, trabecular number, and bone mineral density, while reducing trabecular separation. Simultaneously, it reduced inflammatory cell infiltration in the medullary cavity, local osteolysis, and cortical bone continuity disruption, decreased TRAP-positive multinucleated osteoclasts, and reduced intramedullary bacterial load, with the 40 mg / kg group showing a more significant overall improvement. The above results indicate that apigenin can simultaneously improve infection control, inflammatory response, abnormal bone resorption, and bone tissue structural damage, and has a clear in vivo therapeutic effect on Staphylococcus aureus-induced osteomyelitis.

[0028] This invention further reveals that the minimum inhibitory concentration (MIC) of apigenin against USA300 is 512 μg / mL. Furthermore, apigenin at concentrations significantly lower than this MIC inhibits glucosamine formation. Moreover, apigenin at concentrations of 32 μg / mL and 64 μg / mL does not significantly affect bacterial growth, indicating that its therapeutic effect does not primarily rely on conventional direct antibacterial or bactericidal effects. Apigenin can interfere with the CrtN-associated glucosamine synthesis process and interact directly with CrtN, thereby weakening the tolerance of Staphylococcus aureus to oxidative stress and the host's bactericidal environment, increasing its sensitivity to hydrogen peroxide and whole blood bactericidal effects, and reducing the damage to osteoblast progenitor cells caused by bacterial infection.

[0029] Therefore, this invention achieves the treatment of Staphylococcus aureus-induced osteomyelitis by inhibiting staphylin-related virulence, reducing bacterial host adaptability, alleviating osteoblast-related cell damage, and improving pathological changes in bone tissue at the infection site. It expands the pharmaceutical applications of apigenin and provides a new active ingredient and technical approach for the treatment of drug-resistant Staphylococcus aureus bone infections. Compared with existing treatments for infectious osteomyelitis that mainly rely on surgical debridement and direct inhibition or killing of bacteria, this invention is simple, efficient, and avoids problems such as drug resistance caused by the use of large amounts of antibiotics. It can serve as an alternative or combination of existing technologies, making up for the technical deficiencies of existing solutions. Attached Figure Description

[0030] Figure 1 The effects of apigenin on flavonoid formation and bacterial growth in USA300 cells. In this study, A shows the appearance of the precipitate from USA300 cells and the pigment extracted with methanol after treatment with different concentrations of apigenin; B shows the IC50 value of apigenin inhibiting flavonoid formation. 50 Measurement results; C shows the Raman spectra of USA300 before and after apigenin treatment; D shows the MIC determination results of apigenin and vancomycin on USA300; E shows the growth curves of USA300 after treatment with different concentrations of apigenin. Figure 2 Analysis of the direct binding and interaction between apigenin and CrtN protein: A) SPR technique was used to detect the binding ability of different concentrations of apigenin to CrtN protein; B) Fluorescence quenching assay was used to analyze the interaction between apigenin and CrtN protein; C) Thermomigration assay was used to detect the effect of apigenin binding on the thermostability of CrtN protein; E) Cell thermomigration assay was used to detect the changes in the stability of CrtN protein under different temperature conditions, and the thermostability protection effect of apigenin on CrtN protein was analyzed; F) Molecular docking simulation was used to analyze the binding mode of apigenin and CrtN protein, showing its interaction with key amino acid residues SER-349 and ARG-330. Figure 3 North American apigenin reduces the tolerance of USA300 to hydrogen peroxide and rabbit whole blood sterilization environments. A shows the colony growth after no treatment and after hydrogen peroxide treatment; B shows the colony growth after no treatment and after rabbit whole blood incubation; C shows the bacterial survival rate after no treatment and after hydrogen peroxide treatment; D shows the bacterial survival rate after no treatment and after rabbit whole blood incubation. Figure 4 Apica extract alleviates USA300-induced damage to MC3T3-E1 cells. A shows the results of Calcein AM / PI staining of live and dead cells; B shows the ratio of live to dead cells; C shows the LDH release level in the cell culture supernatant. Figure 5 The therapeutic effect of apigenin on USA300 infectious osteomyelitis in rats: A is the result of femoral micro-CT scan and three-dimensional reconstruction; B is the quantitative result of bone mass and trabecular microstructure parameters; C is the result of H&E and TRAP staining of femoral tissue; D is the result of colony culture of bone marrow cavity lavage fluid; E is the relative number of colonies in the bone marrow cavity. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0032] Example 1: Apica extract inhibits the formation of flavonoids in USA300. 1.1 Preparation of materials, cells, strains and reagents Pteryxin (PTX) with a purity greater than 98% was dissolved in dimethyl sulfoxide (DMSO) and prepared into a 10 mg / mL stock solution, which was then aliquoted and stored at -20°C for later use.

[0033] Mouse embryonic osteoblast precursor cells (MC3T3-E1) were cultured in Ham's F-12K medium at 37°C in a 5% CO2 cell culture incubator. Staphylococcus aureus USA300 (ATCC BAA-1556) and Staphylococcus aureus USA300 crtN knockdown strain (kd- crtN (This refers to strains preserved in the laboratory.) USA300 and kd- crtN Tryptic soy broth (TSB) was cultured with shaking at 37°C and 200 r / min. The minimum inhibitory concentration (MIC) was determined using cation-adjusted Mueller-Hinton broth (CAMHB).

[0034] 1.2 Effects of apigenin on the formation of flavonoids in USA300 Single colonies of USA300 were picked and inoculated into TSB medium, and cultured overnight at 37°C with shaking at 200 rpm. The next day, the overnight bacterial culture was inoculated into fresh TSB medium at a volume ratio of 1:100, and apigenin was added to achieve final concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / mL, respectively. A control group of USA300 without apigenin was also included. All groups were co-cultured at 37°C and 200 rpm for 36 h.

[0035] After cultivation, the bacterial culture was centrifuged at 10,000 r / min for 10 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were washed twice with 0.01 mol / L phosphate-buffered saline (PBS), and then the bacterial cells were resuspended in methanol for extraction of glucoflavin. Methanol extraction was performed three times, and the supernatant was collected by centrifugation after each extraction. The three extracts were combined.

[0036] The absorbance of the combined extracts at 465 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and this absorbance was used to reflect the relative content of glucoflavin. The glucoflavin inhibition rate was calculated using the following formula: Glucoflavin inhibition rate (%) = [(control group OD) / (control group OD)] / (control group OD) ... 465 - Processing Group OD465 ) / Control group OD 465 ×100%. Dose-response curves were plotted based on the glucantin inhibition rates corresponding to different concentrations of apigenin, and the half maximal inhibitory concentration (IC50) of apigenin in inhibiting glucantin formation was calculated. 50 ).

[0037] The results are as follows Figure 1 As shown in Figures A and B, with increasing apigenin (PTX) concentration, the yellow color of the USA300 cell precipitate gradually lightened, and the yellow color of the corresponding methanol extract also gradually weakened, indicating that the content of saxanthin (STX) in USA300 decreased with increasing apigenin concentration. The dose-response curves showed that apigenin had a significant concentration-dependent inhibitory effect on saxanthin formation in USA300, with an IC50 value of [missing value]. 50 It was 4.826 μg / mL, approximately 12.49 μmol / L.

[0038] 1.3 Raman spectroscopy detection

[0039] To further verify the effect of apigenin on flavonoid formation in USA300, overnight USA300 bacterial culture was inoculated into fresh TSB medium at a volume ratio of 1:100, setting up a USA300 control group and an apigenin treatment group. In the apigenin treatment group, apigenin was added to a final concentration of 64 μg / mL. All groups were incubated at 37℃ and 200 r / min for 36 h.

[0040] After cultivation, the bacterial cells were collected by centrifugation, washed twice with sterile 0.01 mol / L PBS, and then resuspended in PBS. High-resolution Raman spectroscopy was used for detection, with an excitation wavelength of 532 nm and an objective magnification of 100x. Raman spectra of the USA300 control group and the apigenin-treated group were collected under the same detection conditions.

[0041] The results are as follows Figure 1 As shown in Figure C, the untreated USA300 measured approximately 1161.0 cm. -1 and 1535.1 cm -1 The presence of distinct Raman characteristic peaks corresponds to the carbon-carbon single and double bond vibrations in the conjugated polyene structure of apigenin. After treatment with 64 μg / mL apigenin, the intensity of these two characteristic peaks significantly decreased, consistent with the reduced yellow color of the bacterial cells, further indicating that apigenin can inhibit the formation of apigenin in USA300 apigenin.

[0042] 1.4 Determination of the minimum inhibitory concentration of apigenin against USA300 The USA300 strain, stored at -80℃, was streaked onto TSB solid medium and incubated overnight at 37℃. Single colonies were then picked and inoculated into TSB liquid medium for further overnight incubation. The following day, the overnight bacterial culture was inoculated into CAMHB medium at a 1:1000 volume ratio and incubated at 37℃ with shaking at 200 rpm until the bacterial OD reached its maximum. 600 It is approximately 0.8, then diluted with CAMHB at a ratio of 1:100 for later use.

[0043] The minimum inhibitory concentration (MIC) was determined using the microbroth dilution method. Serial dilutions of apigenin were prepared in sterile 96-well plates, with final concentrations of 1024, 512, 256, 128, 64, 32, 16, and 8 μg / mL. Vancomycin, USA300 growth, and sterile culture medium blank control groups were also included. The prepared USA300 bacterial suspension was added to the corresponding wells, with three replicates for each concentration. 2.5 μL of resveratrol solution was added to each well as an indicator of bacterial metabolic activity.

[0044] The 96-well plates were incubated at 37°C for approximately 16 hours, and bacterial growth was assessed based on the change in the azure color within the wells. When the bacteria exhibited metabolic activity, the color inside the wells changed from blue to pink; the lowest concentration of apigenin at which no significant color change was determined as the minimum inhibitory concentration (MIC).

[0045] The results are as follows Figure 1 As shown in Figure D, a significant color change occurred in the wells when the apigenin concentration was 256 μg / mL, indicating that the bacteria still possessed metabolic activity. When the apigenin concentration reached 512 μg / mL, the color in the wells remained blue, indicating that the metabolic growth of USA300 was significantly inhibited. Therefore, the MIC of apigenin against USA300 is 512 μg / mL, and the MIC of vancomycin against USA300 is 4 μg / mL.

[0046] 1.5 Effect of apigenin on the growth curve of USA300 USA300 was inoculated into TSB medium at a volume ratio of 1:100 and cultured overnight at 37°C and 200 r / min with shaking. The next day, the overnight bacterial culture was inoculated into fresh TSB medium at a volume ratio of 1:100 and cultured until the bacterial OD reached the specified level. 600 It is approximately 0.3.

[0047] The bacterial suspension was divided into a USA300 control group, a 32 μg / mL apigenin treatment group, and a 64 μg / mL apigenin treatment group. A blank control group containing no bacteria was also included. 100 μL of the corresponding bacterial suspension or culture medium was added to each well, and the 96-well plate was incubated at 37°C. The OD values ​​of each well were measured at 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h of incubation. 600 Plot bacterial growth curves. Each experiment should be repeated at least 3 times.

[0048] The results are as follows Figure 1 As shown in Figure E, the growth curves of the USA300 control group and the 32 μg / mL and 64 μg / mL apigenin treatment groups were basically consistent, and the OD values ​​of each treatment group at the corresponding time points were similar. 600 No significant differences were observed, indicating that 32 μg / mL and 64 μg / mL apigenin did not significantly affect the normal growth of USA300.

[0049] Example 2: Interaction between apigenin and CrtN 2.1 Surface Plasmon Resonance Experiment The interaction between apigenin and CrtN protein was detected using a Biacore 1K surface plasmon resonance system. CrtN protein was immobilized on the surface of a CM5 sensor chip, with PBS buffer as the mobile phase. After baseline stabilization, different concentrations of apigenin solution were sequentially injected, ranging from 1.56 to 100 μmol / L, at an injection rate of 30 μL / min. The binding time was 120 s, and the spontaneous dissociation time was 180 s.

[0050] The obtained response signal was fitted and analyzed using Biacore Insight software. The results are as follows: Figure 2 As shown in Figure A, the binding response signal on the chip surface gradually increases with the increase of apigenin concentration. The equilibrium dissociation constant K between apigenin and CrtN was obtained by fitting. D It is 2.79 × 10 -5 The concentration of mol / L indicates that apigenin can bind directly to the CrtN protein.

[0051] 2.2 Fluorescence quenching experiment The purified CrtN protein was diluted to 4 μmol / L with PBS and placed in black 96-well plates. Different concentrations of apigenin were added to each well to achieve a final concentration of 0–134.58 μmol / L, and the volume was brought up to the same level with PBS. The intrinsic fluorescence spectrum of the CrtN protein was acquired in the range of 280–450 nm using 280 nm as the excitation wavelength.

[0052] The results are as follows Figure 2As shown in Figure B, with increasing apigenin concentration, the intrinsic fluorescence intensity of the CrtN protein gradually decreased, exhibiting a clear concentration-dependent fluorescence quenching. The binding constant between apigenin and CrtN was calculated based on the fluorescence quenching data. K A It is 1.791×10 4 L / mol further indicates a direct interaction between apigenin and CrtN.

[0053] 2.3 Thermal migration experiment The purified CrtN protein was diluted to 2 μmol / L using thermal migration assay buffer, and two groups were set up: a CrtN control group and a apigenin treatment group. The apigenin treatment group was treated with apigenin to a final concentration of 128 μg / mL. SYPROOrange protein gel staining reagent was added to each group, and the protein melting curves were detected using a real-time quantitative PCR system within the range of 25–95℃.

[0054] The results are as follows Figure 2 As shown in Figure C, without the addition of apigenin, the melting temperature of CrtN protein was approximately 42 °C; after the addition of 128 μg / mL apigenin, the melting temperature of CrtN protein decreased to approximately 40 °C, a reduction of about 2 °C. This result indicates that apigenin can alter the thermal stability of CrtN protein, suggesting an interaction between the two.

[0055] 2.4 Cell thermal migration experiment Escherichia coli BL21(DE3) containing the pET28a-CrtN plasmid was induced to express CrtN protein with isopropyl-β-D-thiogalactoside (IPTG). The bacterial cells were collected by centrifugation and resuspended, then sonicated on ice and centrifuged again to collect the supernatant containing CrtN protein. Equal volumes of supernatant were added to either apigenin or an equal volume of DMSO to achieve a final apigenin concentration of 128 μg / mL, and incubated at room temperature for 30 min. After incubation, the samples were treated at 26.0, 40.8, 46.6, 52.4, 58.2, 64.1, and 70.0 °C for 5 min, followed immediately by an ice bath for 3 min. The soluble protein supernatant was collected again by centrifugation, and the CrtN protein bands at different temperatures were detected by SDS-polyacrylamide gel electrophoresis, followed by grayscale analysis.

[0056] The results are as follows Figure 2 As shown in Figure E, the intensity of the CrtN protein bands in both groups gradually decreased with increasing treatment temperature. This result indicates that apigenin can alter the thermal stability of CrtN protein in a cell lysis system.

[0057] 2.5 Molecular docking analysis The three-dimensional structural data of apigenin were obtained from the PubChem database (PubChem CID: 5281425). Molecular docking analysis was performed using AutoDockTools 1.5.6 software. A docking mesh was constructed using CrtN (UniProt ID: Q6GDN7) as the acceptor, with the following parameters: center_x = 10.22, center_y = 32.934, center_z = 28.349, and dimensions size_x = 64.75, size_y = 64.75, size_z = 64.75. All other docking parameters were set to the software defaults.

[0058] The results are as follows Figure 2 As shown in Figure F, apigenin can enter the potential binding pocket of the CrtN protein, and the predicted binding free energy of the apigenin-CrtN complex is -6.8 kcal / mol. The docking results suggest that Ser349 and Arg330 may be key amino acid residues involved in the binding of apigenin to CrtN.

[0059] Example 3: Celeryin reduces the tolerance of USA300 to the host's bactericidal environment. 3.1 Hydrogen peroxide sensitivity test Staphylococcus aureus USA300 was inoculated into TSB medium and cultured overnight with shaking at 37°C and 220 r / min. The next day, the overnight bacterial suspension was inoculated into fresh TSB medium at a volume ratio of 1:100, with separate USA300 control and apigenin treatment groups. Apigenin was added to the apigenin treatment group to a final concentration of 64 μg / mL. All groups were cultured for another 36 h at 37°C and 200 r / min. After culture, 500 μL of bacterial suspension from each group was centrifuged at 12,000 r / min for 2 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were washed twice with 0.01 mol / L sterile phosphate buffer, and then resuspended in 500 μL of sterile phosphate buffer. The OD of the bacterial suspension was then measured. 600 Adjust to approximately 0.1. Take 250 μL of bacterial suspension from each tube, add 30% hydrogen peroxide solution to bring the final hydrogen peroxide concentration to 1.5%, seal, and incubate at 37°C for 1 h. After incubation, add catalase to each tube to a final concentration of 1000 U / mL, and continue incubation at 37°C and 200 r / min for 30 min to decompose residual hydrogen peroxide and terminate the reaction. A corresponding control group without added hydrogen peroxide was also set up. After the reaction, the bacterial suspension was serially diluted, and an appropriate amount of the diluted solution was spread onto TSB agar plates and incubated overnight at 37°C. Colony counting was then performed, and bacterial survival rate was calculated based on the colony counts of the hydrogen peroxide-treated groups and the corresponding untreated groups.

[0060] The results are as follows Figure 3 A and Figure 3 As shown in Figure C, under conditions without hydrogen peroxide, there was no significant difference in colony count and survival rate between the USA300 control group and the apigenin pretreated group, indicating that 64 μg / mL apigenin pretreatment did not significantly affect the baseline survival of USA300. After treatment with 1.5% hydrogen peroxide, the bacterial survival rate of both groups decreased significantly, with the colony count and survival rate of the apigenin pretreated group further decreasing, indicating that apigenin pretreatment enhanced the sensitivity of USA300 to hydrogen peroxide damage and reduced its tolerance to oxidative stress.

[0061] 3.2 Rabbit whole blood sterilization experiment USA300 was inoculated into TSB medium and cultured overnight at 37°C and 200 rpm. The next day, the overnight bacterial culture was inoculated into fresh TSB medium at a volume ratio of 1:100, with separate USA300 control and apigenin treatment groups. Apigenin was added to the apigenin treatment group to a final concentration of 64 μg / mL. All groups were cultured for another 36 h at 37°C and 200 rpm. After culture, 500 μL of bacterial culture from each group was centrifuged at 12,000 rpm for 2 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were washed twice with 0.01 mol / L sterile phosphate (PBS) buffer, then resuspended in 500 μL of phosphate buffer, and the OD of the bacterial suspension was determined. 600 Adjust to approximately 0.1. Take 150 μL of the above OD 600 Add approximately 0.1 g of bacterial suspension to 850 μL of phosphate buffer to adjust the OD of the bacterial suspension. 600 The concentration was further adjusted to approximately 0.015. 40 μL of the diluted bacterial suspension was added to 360 μL of fresh anticoagulated rabbit whole blood and incubated at 37°C and 200 r / min for 6 h. A corresponding control group without rabbit whole blood was also included. After incubation, the bacterial suspensions were serially diluted, and appropriate amounts of the diluted solutions were spread onto TSB agar plates and incubated overnight at 37°C. Colony counts were then performed, and bacterial viability was calculated based on the colony counts in the whole blood incubation group and the corresponding untreated group.

[0062] The results are as follows Figure 3 B and Figure 3 As shown in Figure D, without the addition of rabbit whole blood, there was no significant difference in colony count and survival rate between the USA300 control group and the apigenin pretreated group. After incubation with fresh rabbit anticoagulated whole blood for 6 hours, the colony count and survival rate of both groups decreased significantly. The colony count and survival rate of the apigenin pretreated group were significantly lower than those of the USA300 control group, indicating that apigenin pretreatment weakened the tolerance of USA300 to the sterilization environment of whole blood.

[0063] Example 4: Protective effect of apigenin on MC3T3-E1 cells 4.1 Protective effect of apigenin against USA300-induced MC3T3-E1 cell damage MC3T3-E1 cells (mouse embryonic osteoblast precursor cells, Zishan Biotechnology; catalog number: STCC20062P) were added at a rate of 1×10⁻⁶. 5 Inoculate 100 cells / well into 24-well plates using Ham's F-12K medium containing 10% fetal bovine serum and incubate at 37°C and 5% CO2 for 24 h. Resuscitate Staphylococcus aureus USA300 and inoculate into TSB medium at a 1:100 volume ratio, incubating until the bacterial OD reaches 100%. 600 Approximately 0.5. The bacterial cells were collected by centrifugation, washed twice with PBS buffer, and then resuspended in Ham's F-12K medium. The experiment included a normal cell control group, a USA300 infection group, a 32 μg / mL apigenin treatment group, a 64 μg / mL apigenin treatment group, and a CrtN knockdown strain infection group. The CrtN knockdown strain was prepared as follows: using Staphylococcus aureus USA300 genomic DNA as a template, a specific targeting sgRNA was designed based on the crtN gene sequence and cloned into a CRISPRi vector containing the dCas9 regulatory element to construct a recombinant CRISPRi plasmid. Subsequently, the recombinant plasmid was introduced into Staphylococcus aureus USA300 competent cells, and positive strains carrying the CRISPRi regulatory system were obtained through screening, thus obtaining the USA300-crtN knockdown strain. In the apigenin treatment group, apigenin was added to the USA300 bacterial suspension to a final concentration of 32 μg / mL or 64 μg / mL; kd- crtN The infection group used the same bacterial suspension preparation and infection conditions as the USA300 group. Subsequently, the corresponding bacterial suspensions were added to the MC3T3-E1 cell culture system and incubated for 5 h. After incubation, staining was performed according to the Calcein AM / PI live / dead cell detection kit instructions. Images were observed and acquired using a fluorescence microscope. Calcein AM-positive cells showed green fluorescence, representing live cells; PI-positive cells showed red fluorescence, representing dead cells with damaged cell membranes. The ratio of live to dead cells was quantitatively analyzed using ImageJ software. Simultaneously, cell culture supernatants from each group were collected, and lactate dehydrogenase (LDH) release levels were measured according to the lactate dehydrogenase detection kit instructions to evaluate the degree of cell membrane damage.

[0064] The results are attached. Figure 4As shown, the normal cell control group was dominated by green fluorescent cells with fewer dead cells; after infection with USA300, the number of green fluorescent cells decreased significantly and the number of red fluorescent cells increased significantly. At the same time, the LDH release level in the culture supernatant increased significantly, indicating that USA300 can cause significant damage to MC3T3-E1 cells.

[0065] Compared with the USA300 infection group, treatment with 32 μg / mL and 64 μg / mL apigenin increased the proportion of green fluorescent live cells and decreased the proportion of red fluorescent dead cells, as well as reduced LDH release levels in the culture supernatant. The protective effect was more pronounced in the 64 μg / mL apigenin group. The cell death rate and LDH release levels in the CrtN knockdown strain infection group were also lower than those in the USA300 infection group. Fluorescence imaging and quantitative analysis showed that both apigenin treatment and CrtN knockdown could alleviate USA300-induced MC3T3-E1 cell damage.

[0066] Example 5: The therapeutic effect of apigenin on Staphylococcus aureus-induced osteomyelitis in rats. 5.1 Establishment, grouping, and administration of a rat model of infectious osteomyelitis Male SD rats aged 6–8 weeks, weighing 280–300 g, were selected. After acclimatization, the experimental animals were randomly divided into four groups: a normal control group, a USA300 infection group, a apigenin 20 mg / kg group, an apigenin 40 mg / kg group, and a vancomycin 40 mg / kg group, with 6 rats in each group.

[0067] Staphylococcus aureus USA300 was streaked onto TSB agar plates and incubated overnight at 37°C. Single colonies were picked and inoculated into TSB liquid medium, and incubated overnight with shaking at 37°C and 200 rpm. The next day, the bacterial suspension was inoculated into fresh TSB medium at a 1:100 volume ratio and incubated until OD500 reached. 600 The value was approximately 1.0. The cells were then centrifuged at 5000 r / min for 6 min, the supernatant was discarded, and the cells were resuspended in sterile phosphate buffer to prepare a USA300 bacterial suspension for later use.

[0068] Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. Under strict aseptic conditions, the surgical area was prepared and disinfected. An incision was made in the midline of the knee joint, and the knee was fully exposed by dissection towards the medial parapatellar region. A burr was drilled into the distal femur to access the medullary cavity, an 18G metal needle was inserted, and a solution containing 1×10⁻⁶ sodium pentobarbital was injected into the medullary cavity. 8An infectious osteomyelitis model was established using a bacterial suspension of CFU USA300. The normal control group underwent the same surgical procedure, with a metal needle inserted and an equal volume of sterile saline injected into the bone marrow cavity. Drug administration began 2 hours post-surgery. The apigenin 20 mg / kg and 40 mg / kg groups received subcutaneous injections of the corresponding doses of apigenin; the vancomycin group received a subcutaneous injection of 40 mg / kg vancomycin; the normal control group and the USA300 infection group received no drug intervention. All treatment groups received medication every 24 hours for 14 consecutive days. The general condition of the rats was observed during the experiment.

[0069] 5.2 Micro-CT Detection Fourteen days after drug administration, rats were sacrificed, and the infected femur was aseptically isolated. Femoral tissue for Micro-CT examination was fixed in 10% neutral buffered formalin for 5 days, then transferred to 70% ethanol and stored at 4°C. Micro-CT was used to scan the distal femur at a voltage of 80 kV, a current of 0.06 mA, and a scan time of approximately 200 s. After scanning, three-dimensional reconstruction was performed, and quantitative analysis of bone mass and trabecular microstructure parameters was conducted within the defined region of interest. The detection indicators include bone volume (BV), total volume (TV, also known as tissue volume, refers to the total spatial volume contained within the defined volume of interest (VOI), including mineralized bone tissue and the volume occupied by non-bone tissues such as trabecular gaps and medullary cavity), bone volume fraction (BV / TV, representing the proportion of mineralized bone tissue volume to the total VOI volume, an important parameter for evaluating bone mass), bone surface area to bone volume ratio (BS / BV), connectivity density (Conn.D, referring to the connectivity density of the trabecular network, i.e., the degree to which trabeculae connect, branch, and form a continuous three-dimensional network structure within the VOI, obtained after standardization of the total volume TV, commonly measured in mm⁻³. It is usually calculated based on the Euler characteristic of the three-dimensional structure and does not refer to the connection between osteocytes, bone and implants, or bone and surrounding tissues), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), trabecular number (Tb.N), and bone mineral density. density, BMD).

[0070] The results are as follows Figure 5 A and Figure 5As shown in Figure B, compared with the normal control group, the USA300 infection group showed a significant reduction in distal femoral bone mass and disruption of trabecular microstructure, manifested as varying degrees of decrease in bone volume (BV), BV / TV, BS / BV, Conn.D, Tb.Th, Tb.N, and BMD, and an increase in Tb.Sp, while TV remained unchanged. Compared with the USA300 infection group, treatment with apigenin at 20 mg / kg and 40 mg / kg resulted in varying degrees of increase in BV, BV / TV, Conn.D, Tb.N, and BMD, a decrease in Tb.Sp, and varying degrees of improvement in other trabecular parameters. The overall improvement was more significant in the 40 mg / kg apigenin group than in the 20 mg / kg group, with a trend largely consistent with that of the vancomycin 40 mg / kg group. These results indicate that apigenin can alleviate bone loss and trabecular microstructure damage caused by USA300 infection.

[0071] 5.3 Histopathological Examination Femoral tissue samples were collected and fixed in 4% paraformaldehyde, followed by decalcification in 12% EDTA solution. After decalcification, the tissues were routinely dehydrated, cleared, and embedded in paraffin to prepare serial sections with a thickness of 4 μm. Hematoxylin-eosin staining and tartrate-resistant acid phosphatase staining were then performed. Tissue images were observed and acquired using an optical microscope. Hematoxylin-eosin staining was used to evaluate inflammatory cell infiltration in the bone marrow cavity, localized bone resorption, and cortical bone structure destruction; tartrate-resistant acid phosphatase staining was used to observe changes in TRAP-positive multinucleated osteoclasts.

[0072] The results are as follows Figure 5 As shown in Figure C, the femoral structure in the normal control group remained intact, and no significant inflammatory cell accumulation was observed in the bone marrow cavity. In the USA300 infection group, a large number of inflammatory cells infiltrated the bone marrow cavity, accompanied by local osteolysis and disruption of cortical bone continuity. TRAP staining showed a significant increase in TRAP-positive multinucleated osteoclasts in the infection group. Treatment with apigenin at 20 mg / kg and 40 mg / kg reduced inflammatory cell infiltration in the bone marrow cavity, alleviated local osteolysis and cortical bone structural destruction, and decreased TRAP-positive multinucleated osteoclasts. The improvement was more pronounced in the 40 mg / kg group, and the overall trend was consistent with that of the vancomycin group. These results indicate that apigenin can reduce the inflammatory response at the infection site and inhibit infection-related osteoclast activation and bone resorption.

[0073] 5.4 Detection of bacterial load in the bone marrow cavity Fourteen days after drug administration, rat femurs were isolated under aseptic conditions. The medullary cavity of the femur was flushed with sterile phosphate-buffered saline (PBS) using a Hamilton microsyringe, and the flushing fluid was collected and transferred to sterile centrifuge tubes. The flushing fluid was serially diluted, and appropriate amounts of samples at different dilutions were plated onto TSB agar plates containing defibrinated rabbit blood and incubated at 37°C for 24 h before colony counting. The relative colony counts for each group were calculated using the USA300 infection group as 100%.

[0074] The results are as follows Figure 5 D and Figure 5 As shown in Figure E, almost no bacterial colonies were detected in the normal control group, while a large number of colonies grew in the USA300 infection group. Treatment with apigenin at 20 mg / kg and 40 mg / kg significantly reduced the number of colonies in the bone marrow cavity lavage fluid. The relative colony count in the 40 mg / kg apigenin group decreased to 8.29 ± 0.61%, showing a better overall reduction than the 20 mg / kg group, but still less than the vancomycin 40 mg / kg group. These results indicate that continuous administration of apigenin for 14 days can reduce the USA300 bacterial load in infected bone marrow cavities.

Claims

1. Application of apigenin in the preparation of drugs for the treatment of Staphylococcus aureus-induced osteomyelitis.

2. The application according to claim 1, characterized in that, Staphylococcal osteomyelitis refers to a disease caused by Staphylococcus aureus infection that results in a decrease in one or more of the following indicators: bone volume, total volume, bone volume fraction, ratio of bone surface area to bone volume, connectivity density, trabecular thickness, number of trabecular bones, and bone mineral density, and / or an increase in trabecular separation.

3. The application according to claim 1, characterized in that, Staphylococcal osteomyelitis refers to a disease caused by Staphylococcus aureus infection, resulting in inflammatory cell infiltration in the bone marrow cavity, bone resorption, destruction of cortical bone structure, and an increase in multinucleated osteoclasts.

4. The application according to claim 1, characterized in that, The aforementioned drug for treating Staphylococcus aureus-infected osteomyelitis is a drug that reduces the Staphylococcus aureus load in the bone marrow cavity after Staphylococcus aureus infection.

5. The application according to claim 1, characterized in that, The drug for treating Staphylococcus aureus-induced osteomyelitis is a drug that inhibits the formation of staphylococcus aureus flavonoids.

6. The application according to claim 5, characterized in that, The drug that inhibits the formation of glucoflavin by Staphylococcus aureus is a drug that reduces the thermal stability of Staphylococcus aureus dehydrosqualene dehydrogenase.

7. The application according to claim 5, characterized in that, The drug that inhibits the formation of glucoflavin by Staphylococcus aureus is a drug that reduces the ability of Staphylococcus aureus to tolerate oxidative stress.

8. The application according to claim 5, characterized in that, The drug that inhibits the formation of glucoflavin by Staphylococcus aureus is a drug that reduces the ability of Staphylococcus aureus to tolerate the bactericidal environment of whole blood.

9. Application of apigenin as an inhibitor of Staphylococcus aureus dehydrosqualene dehydrogenase activity.

10. Application of apigenin in the preparation of a protective agent against Staphylococcus aureus-induced MC3T3-E1 cell damage.

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