Ginkolide A and its combined antibacterial drugs

Abstract

The application belongs to the technical field of anti-infective biological medicine, and provides the application of ginkgolide A and its combined antibacterial drugs, wherein the application researches and finds that the ginkgolide A can inhibit the formation of Staphylococcus aureus biofilm under the condition of lower minimum inhibitory concentration, and reduce the expression level of the gene related to bacterial adhesion and biofilm formation, so as to weaken the formation ability of bacterial biofilm. Further research shows that the combined application of the ginkgolide A and the antibacterial drugs can enhance the inhibition and removal effect of the antibacterial drugs on bacteria, and the combination of the ginkgolide A and vancomycin shows more obvious antibacterial synergistic effect. In the animal model of implant-related infection, compared with the antibacterial drugs used alone, the ginkgolide A combined with vancomycin can further reduce the bacterial load at the infection site and on the surface of the implant, reduce the inflammation reaction induced by infection, and improve the bone tissue damage and abnormal bone metabolism.

Description

Technical Field

[0001] This invention belongs to the field of anti-infective biomedical technology, and particularly relates to the application of ginkgolide A and its combination with antibacterial drugs. Background Technology

[0002] Currently, implant-related bacterial infections primarily rely on surgical intervention combined with antibiotic therapy. While antibiotics such as vancomycin have good bactericidal effects, their efficacy in treating chronic and implant-related infections remains limited due to the biofilm barrier effect. Therefore, developing adjunctive therapy strategies that can interfere with the bacterial biofilm formation process and enhance the effects of traditional antibiotics is of great significance for improving the treatment outcomes of infections.

[0003] Osteomyelitis is a disease caused by pathogenic microorganisms invading bone tissue and triggering a persistent infection response. It is characterized by a prolonged course, long treatment period, and high recurrence rate. With the increasing use of orthopedic implants, implant-related infections have gradually become a significant challenge in clinical treatment. Staphylococcus aureus (S. aureus) is one of the important pathogens causing implant-related infections. This bacterium can form a stable biofilm on the surface of host tissues and implant materials by expressing adhesion-related proteins and producing extracellular polymers. After biofilm formation, bacteria are protected by the extracellular matrix, reducing the penetration efficiency of antimicrobial drugs and causing some bacteria to enter a low metabolic state, thus exhibiting strong tolerance. This is an important reason for prolonged infection, treatment failure, and recurrence.

[0004] Ginkgolide A is a naturally occurring diterpenoid lactone compound, primarily found in ginkgo leaves. Studies have shown that it possesses various biological functions, including anti-inflammatory and antioxidant effects. However, current research on the role of ginkgolide A in bacterial biofilm regulation, enhancing the anti-infective efficacy of antibacterial drugs, and treating infectious osteomyelitis remains relatively limited. Summary of the Invention

[0005] The purpose of this invention is to provide the application of ginkgolide A and its combination with antibacterial drugs, aiming to solve the problems raised in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Application of Ginkgolide A in the preparation of drugs against Staphylococcus aureus biofilm formation.

[0007] Furthermore, the ginkgolide A exerts an anti-biofilm effect at concentrations below the minimum inhibitory concentration (MIC), inhibiting the formation and maturation of Staphylococcus aureus biofilms; the MIC is 256 μg / mL.

[0008] Furthermore, the ginkgolide A reduces the expression of genes related to Staphylococcus aureus biofilm formation by regulating the expression of these genes. icaA , icaD , sarA , fnbA , fnbB and clfA This reduces the expression levels of related genes, thereby decreasing the ability of Staphylococcus aureus to form biofilms.

[0009] Another object of the present invention is to provide the use of ginkgolide A in combination with antibacterial drugs in the preparation of drugs for the prevention and treatment of Staphylococcus aureus infection.

[0010] Furthermore, the antibacterial drug includes one or more of glycopeptides, β-lactams, and aminoglycosides.

[0011] Furthermore, the antibacterial drug is vancomycin.

[0012] Furthermore, the Staphylococcus aureus infection is an implant-associated Staphylococcus aureus infection.

[0013] Another object of the present invention is to provide the use of ginkgolide A in combination with vancomycin in the preparation of a medicament for the treatment of implant-associated infectious osteomyelitis.

[0014] This invention reveals that ginkgolide A, at concentrations below the minimum inhibitory concentration (MIC), can inhibit Staphylococcus aureus biofilm formation and reduce the expression levels of genes related to Staphylococcus aureus adhesion and biofilm formation, thereby weakening the biofilm-forming ability of Staphylococcus aureus. Further research shows that the combined use of ginkgolide A and antimicrobial drugs can enhance the inhibitory and clearance effects of antimicrobial drugs against Staphylococcus aureus, with ginkgolide A and vancomycin showing a better synergistic anti-infective effect. In an animal model of implant-associated infection, compared with the use of antimicrobial drugs alone, the combination of ginkgolide A and vancomycin can further reduce the Staphylococcus aureus load at the infection site and on the implant surface, alleviate infection-induced inflammatory responses, and improve bone tissue damage and abnormal bone metabolism.

[0015] The ginkgolide A combined with an antibacterial agent provided by this invention has anti-biofilm and adjuvant anti-infective effects, and can be used to prepare drugs for the prevention and treatment of Staphylococcus aureus infection-related diseases, especially implant-related infections and infectious osteomyelitis, and has potential application value. Compared with the prior art, this invention has the following beneficial effects: 1. This invention provides a novel use of ginkgolides A in inhibiting biofilm formation, and it has been found that ginkgolides A can reduce the biofilm formation ability of Staphylococcus aureus at concentrations below the minimum inhibitory concentration.

[0016] 2. The combination of Ginkgolide A and antibacterial drugs provided by this invention can enhance the clearance effect of antibacterial drugs on Staphylococcus aureus and improve the treatment effect of infection.

[0017] 3. This invention found that ginkgolide A combined with vancomycin can reduce the Staphylococcus aureus load in implant-associated infection models and improve infection-induced inflammatory response and bone tissue damage.

[0018] 4. In this invention, ginkgolide A mainly exerts a synergistic effect by weakening the biofilm protective barrier of Staphylococcus aureus, while the antibacterial drug exerts a bactericidal effect, and the two have complementary advantages.

[0019] 5. The ginkgolide A provided by this invention has the characteristics of natural source, high safety and flexible application, and can be used as an auxiliary strategy for existing antibacterial treatment regimens, and has application value in the field of Staphylococcus aureus infection prevention and control. Attached Figure Description

[0020] Figure 1 The figures show the chemical structural formulas of ginkgolide A and vancomycin involved in this invention; in the figures, A is the chemical structural formula of ginkgolide A; and B is the chemical structural formula of vancomycin.

[0021] Figure 2 The results show the effect of ginkgolide A on the expression levels of genes related to Staphylococcus aureus biofilm formation; in the figure, A and F represent... sarA, icaA , icaD , fnbA , fnbB and clfA The relative expression level detection results.

[0022] Figure 3 The crystal violet staining results show the effect of ginkgolide A on the biofilm formation ability of Staphylococcus aureus; in the figure, A is the staining result; B is the statistical result of biofilm inhibition rate.

[0023] Figure 4 The results of laser confocal microscopy observation on the effect of ginkgolide A on the biofilm formation ability of Staphylococcus aureus are shown in the figure. In the figure, A is the laser confocal microscopy observation result of the control group; B and D are the laser confocal microscopy observation results of the ginkgolide A treatment groups of 50, 75 and 100 μg / mL, respectively.

[0024] Figure 5 The results show the synergistic effect of ginkgolide A combined with different antibacterial drugs on Staphylococcus aureus. In the figure, A is the comparison of antibacterial effects of cefazolin alone and cefazolin combined with ginkgolide A; B is the comparison of antibacterial effects of vancomycin alone and vancomycin combined with ginkgolide A; and C is the comparison of antibacterial effects of gentamicin alone and gentamicin combined with ginkgolide A.

[0025] Figure 6 The results show the changes in body weight in mice with implant-related osteomyelitis in different treatment groups.

[0026] Figure 7 The results show the bacterial load detection results of femoral tissue and implant surface in mice with implant-associated osteomyelitis in different treatment groups; in the figure, A is the statistical result of bacterial load in bone tissue of different treatment groups; B is the statistical result of bacterial load in implant of different treatment groups.

[0027] Figure 8 The results of hematoxylin-eosin (H&E) staining of femoral tissue from mice with implant-related osteomyelitis in different treatment groups are shown. In the figure, AE are H&E staining images of femoral tissue from mice in the control group, Staphylococcus aureus group, Ginkgolide A group, vancomycin group, and vancomycin + Ginkgolide A group; F is the quantitative analysis result of histopathological damage score.

[0028] Figure 9 The results of serum inflammatory factors in mice with implant-associated osteomyelitis in different treatment groups are shown in the figure. In the figure, AC represent the detection results of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), respectively.

[0029] Figure 10 The results of tartrate-resistant acid phosphatase (TRAP) staining in femoral tissue of mice with implant-related osteomyelitis model in different treatment groups are shown. In the figure, AE represents the TRAP staining results of femoral tissue of mice in the control group, Staphylococcus aureus group, Ginkgolide A group, vancomycin group, and vancomycin + Ginkgolide A group; F represents the number of osteoclasts as a statistical result.

[0030] Figure 11 The results of serum bone metabolism-related indicators in mice with implant-related osteomyelitis in different treatment groups are shown in the figure. In the figure, A is the detection result of osteopontin (OPN); B is the detection result of osteocalcin (OCN); C is the detection result of type I procollagen amino-terminal propeptide (PINP); D is the detection result of type I collagen cross-linked carboxyl-terminal peptide (CTX); and E is the detection result of tartrate-resistant acid phosphatase 5b (TRAP-5b).

[0031] Figure 12The figures show the results of Micro-CT scans and bone microstructure parameter analysis of the femur in mice with implant-related osteomyelitis in different treatment groups. In the figures, AE represents the Micro-CT scan results of the femur in the control group, Staphylococcus aureus group, Ginkgolide A group, vancomycin group, and vancomycin + Ginkgolide A group, respectively. FI represents the quantitative analysis results of bone mineral density (BMD), bone volume / total volume ratio (BV / TV), trabecular bone number (Tb.N), and trabecular bone pattern factor (Tb.Pf), respectively. The significance markers in the figures are as follows: NS: no significant difference; *: P≤0.05; **: P≤0.01; ***: P≤0.001. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] In one embodiment of the present invention, targeting Staphylococcus aureus ( Staphylococcus aureus To address the challenges of biofilm formation during infections caused by bacteria such as Staphylococcus aureus (S. aureus), which leads to decreased antibiotic sensitivity, persistent bacterial colonization, and difficulty in effectively controlling implant-related infections, a solution containing ginkgolide A (structural formula as shown) is provided. Figure 1 A composition of vancomycin (shown) and an antibacterial agent; the antibacterial agent includes one or more of glycopeptides, β-lactams, and aminoglycosides; preferably, the antibacterial agent is vancomycin (structural formula shown). Figure 1 (As shown).

[0034] Ginkgolide A is used to inhibit biofilm formation by bacteria such as Staphylococcus aureus. Ginkgolide A exerts its anti-biofilm effect below the minimum inhibitory concentration (MIC) (256 μg / mL), inhibiting the formation of mature bacterial biofilms and related pathogenic processes, and enhancing the clearance effect of antibacterial drugs against Staphylococcus aureus and other bacterial infections. It is particularly suitable for the prevention and treatment of implant-related infectious osteomyelitis. Specifically, Ginkgolide A reduces, but is not limited to, bacterial biofilm formation-related genes by regulating their expression. icaA , icaD , sarA , fnbA , fnbB and clfA This reduces the expression levels of related genes, thereby decreasing the bacteria's ability to form biofilms.

[0035] Furthermore, the combined use of ginkgolide A and antibacterial drugs produces a synergistic antibacterial effect, enhancing the ability of antibacterial drugs to clear Staphylococcus aureus planktonic bacteria and biofilm-associated bacteria. This composition can reduce the bacterial load in implant-related infections, including reducing the amount of biofilm-associated bacteria on the implant surface and the number of bacteria in infected bone tissue. Therefore, it can be used to prevent and treat implant-related infections caused by Staphylococcus aureus, especially implant-related infectious osteomyelitis. Specifically, this composition enhances the antibacterial effect of antibacterial drugs by inhibiting Staphylococcus aureus biofilm formation, reduces the bacterial load at the site of infection, and alleviates infection-induced inflammatory responses and tissue damage, thereby improving abnormal bone structure.

[0036] It should be noted that the experimental methods used in the embodiments of the present invention are all conventional experimental methods in the art, and the reagents, consumables and experimental materials used can all be obtained through commercial channels.

[0037] Example 1: Evaluation of the direct antibacterial activity of ginkgolide A against Staphylococcus aureus, as detailed below: 1. Staphylococcus aureus was inoculated into Mueller-Hinton broth (MHB) medium and cultured at 37°C and 180 rpm until the logarithmic growth phase. Subsequently, the bacterial culture was diluted to a final concentration of approximately 5 × 10^6. 5 CFU / mL. The minimum inhibitory concentration (MIC) of ginkgolide A against Staphylococcus aureus was determined using the microbroth dilution method. Different concentrations of ginkgolide A were added to 96-well plates and mixed with an equal volume of bacterial culture for incubation. Final concentrations of ginkgolide A were set using a two-fold serial dilution method: 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 μg / mL. Bacterial culture without ginkgolide A was used as the growth control group, and sterile PBS was used as the blank control group. Each experimental group was configured with three replicates. After incubation at 37℃ for 18–22 h, bacterial growth was observed, and the lowest drug concentration at which no obvious turbidity was observed was taken as the MIC value.

[0038] Experimental Results: The results showed that ginkgolide A had a certain inhibitory effect on Staphylococcus aureus, with a MIC value of 256 μg / mL. These results indicate that, below the MIC concentration, ginkgolide A did not significantly affect the growth of Staphylococcus aureus, providing experimental evidence for further research on its regulation of Staphylococcus aureus biofilm formation and enhancement of antibacterial drug effects under sub-inhibitory concentrations.

[0039] Example 2: The effect of ginkgolide A on the expression of genes related to Staphylococcus aureus biofilm formation, as detailed below: Staphylococcus aureus strains were inoculated and cultured at 37℃ and 180 rpm until the logarithmic growth phase. A control group and a ginkgolide A treatment group were included, with the ginkgolide A treatment group receiving final concentrations of 50, 75, and 100 μg / mL, respectively. All these concentrations were below the minimum inhibitory concentration (MIC) of ginkgolide A against Staphylococcus aureus. After culturing, bacterial cells were collected by centrifugation and washed with pre-cooled PBS to remove residual culture medium components. Total RNA was then extracted using a bacterial RNA extraction kit, and cDNA was obtained by reverse transcription. Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of genes related to Staphylococcus aureus biofilm formation, including... sarA, icaA, icaD, fnbA, fnbB and clfA Using 16S rRNA as an internal reference gene, 2 -ΔΔCT The method calculates the relative expression levels of each target gene.

[0040] Experimental results: such as Figure 2 As shown, compared with the control group, treatment with 50, 75, and 100 μg / mL ginkgolide A significantly reduced the levels of Staphylococcus aureus. sarA, icaA, icaD, fnbA, fnbB and clfA The expression levels of genes related to biofilm formation were all decreased, exhibiting a concentration-dependent change. These results indicate that ginkgolide A can regulate the expression of genes related to Staphylococcus aureus biofilm formation and participate in the regulation of the biofilm formation process.

[0041] Example 3: Effect of Ginkgolide A on the biofilm formation ability of Staphylococcus aureus. Details are as follows: Crystal violet staining detection: The experiment was divided into a control group and treatment groups with ginkgolide A at concentrations of 50, 75, and 100 μg / mL. Staphylococcus aureus was inoculated into 96-well plates and cultured statically at 37°C for 24 hours under different concentrations of ginkgolide A to form a biofilm. After incubation, the culture medium was discarded, and air-dried gently with sterile PBS to remove airborne bacteria. Methanol was then added to each well for fixation for 15 minutes, followed by air drying. Then, 0.1% crystal violet staining solution was added, and the plates were stained at room temperature for 15 minutes. After staining, the plates were thoroughly rinsed with distilled water, and the bound crystal violet was dissolved in 33% glacial acetic acid. The absorbance at 590 nm was measured using a microplate reader to evaluate the amount of biofilm formed under different treatment conditions.

[0042] 2. Laser confocal microscopy: The experiment was divided into a control group and treatment groups with ginkgolide A at concentrations of 50, 75, and 100 μg / mL. The changes in the biofilm structure of *Staphylococcus aureus* were observed using SYTO 9 / PI fluorescence staining combined with laser confocal microscopy. *Staphylococcus aureus* was inoculated into confocal culture dishes, treated with different concentrations of ginkgolide A, and cultured for 24 hours to form a biofilm. The bacteria were gently washed with PBS to remove airborne bacteria, then incubated with SYTO 9 / PI staining solution in the dark for 30 minutes. After washing to remove unbound dye, the bacteria were observed using a laser confocal microscope.

[0043] Experimental results: such as Figure 3 and Figure 4 As shown, compared with the control group, treatments with 50, 75, and 100 μg / mL ginkgolide A all reduced the amount of Staphylococcus aureus biofilm formation in a dose-dependent manner. These results indicate that ginkgolide A can inhibit Staphylococcus aureus biofilm formation below the minimum inhibitory concentration (MIC).

[0044] Example 4: Evaluation of the synergistic antibacterial effect of ginkgolide A combined with antibacterial drugs. Details are as follows: After overnight incubation, Staphylococcus aureus was diluted, and the bacterial concentration was adjusted to approximately 1 × 10⁻⁶ by counting colony forming units (CFU). 6 CFU / mL. Different antibacterial drugs, including vancomycin, cefazolin, and gentamicin, were added to achieve concentrations of 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC, respectively. The experiment included antibacterial drug-only treatment groups and groups treated with antibacterial drugs in combination with ginkgolide A, with a final concentration of ginkgolide A of 100 μg / mL. After incubation at 37℃ for 16–18 h, bacterial activity was assessed to evaluate the synergistic antibacterial effect between ginkgolide A and the antibacterial drugs.

[0045] Experimental results: such as Figure 5 The results showed that, compared with the use of antibiotics alone, the inhibitory effect of each antibiotic against Staphylococcus aureus was enhanced after the addition of Ginkgolide A. In particular, the combination of Ginkgolide A and vancomycin exhibited a more significant inhibitory effect, further reducing bacterial activity even at lower concentrations of vancomycin. These results indicate that Ginkgolide A can enhance the clearance effect of antibiotics, especially vancomycin, against Staphylococcus aureus, thereby improving the efficacy of anti-infective treatment.

[0046] Example 5: The intervention effect of ginkgolide A combined with vancomycin on an implant-related osteomyelitis model is as follows: Establishment of a mouse implant-related infection model and drug intervention: Male C57BL / 6 mice (8 weeks old, weighing 20±2g) were selected as the research subjects. All animals were randomly divided into 5 groups, including: control group (not infected with Staphylococcus aureus, only implanted and treated with an equal volume of physiological saline); Staphylococcus aureus group (i.e., infection model group); Ginkgolide A group (treated with Ginkgolide A after the infection model was established); Vancomycin group (treated with Vancomycin after the infection model was established); Vancomycin + Ginkgolide A group (treated with both Ginkgolide A and Vancomycin after the infection model was established). The infection model was constructed as follows: Before the experiment, mice were anesthetized with tribromoethanol (250 mg / kg, intraperitoneal injection). After adequate anesthesia, the hair in the right femoral region was shaved, and local disinfection was performed using povidone-iodine and isopropanol. Under aseptic conditions, the lateral region of the right femur was exposed, and a bone hole was established at the midline of the femur using a 27G needle, and a sterile stainless steel needle of approximately 2 mm in length was implanted. Then, 2 μL of Staphylococcus aureus bacterial solution (1×10⁻⁶) was slowly injected into the bone hole. 5 An infection model was established by injecting an equal volume of sterile saline solution (CFU / mL). After inoculation, the bone pores were sealed with bone wax, and the skin was sutured layer by layer. Drug treatment groups received medication starting on the first postoperative day. The Ginkgolide A group received intraperitoneal injection of Ginkgolide A (dissolved in saline containing 1% DMSO and 5% Tween-80) at a dose of 20 mg / kg once daily; the Vancomycin group received intraperitoneal injection at a dose of 20 mg / kg once daily; the combined treatment group received both the above doses of Ginkgolide A and Vancomycin. All animals were housed in a constant temperature (22±3℃) environment with free access to food and water. Animal condition and weight changes were observed and recorded daily during the experiment. Animals were sacrificed on the 14th postoperative day, and femoral tissue, serum, and implants were collected for subsequent testing.

[0047] 2. Femoral bone and implant bacterial load detection: To evaluate the impact of different treatment regimens on the bacterial load at the infection site, femoral tissue and implants from each group of mice were collected for bacterial load detection. Ex vivo femoral tissue was collected, cryogenically ground in liquid nitrogen, and then fully resuspended in sterile PBS. After serial dilution, the samples were plated on bacterial culture plates and incubated under suitable conditions. The bacterial load was calculated based on the number of colonies. Results are expressed as log10 CFU / g bone tissue. Simultaneously, the removed implants were placed in sterile PBS and shaken thoroughly to release bacteria adhering to the implant surface. After serial dilution and incubation, the implant-related bacterial load was calculated.

[0048] 3. Femoral tissue pathological examination: After collecting femoral tissue from each group, it was fixed in 4% paraformaldehyde for 48 h. After decalcification, it was embedded in paraffin and tissue sections were prepared. Hematoxylin-eosin (H&E) staining was used to observe the pathological changes in bone tissue. According to the osteomyelitis histopathological scoring criteria, the acute inflammation, chronic inflammation, periosteal reaction, and osteonecrosis were comprehensively evaluated.

[0049] 4. Serum inflammatory factor detection: Blood samples were collected from mice in each group 14 days after infection, and serum was separated. The levels of serum inflammatory factors IL-1β, IL-6, and TNF-α were detected using ELISA. The concentrations of each indicator were calculated based on a standard curve.

[0050] 5. Osteoclast activation detection: Femurs from each group of mice were collected, fixed in 4% paraformaldehyde for 48 hours, decalcified, and then embedded in paraffin. Coronal sections of the embedded femurs were prepared, and TRAP staining was used to detect osteoclast formation. Changes in the number of TRAP-positive cells in the bone tissue of different treatment groups were observed and analyzed under a microscope.

[0051] 6. Detection of bone metabolism-related indicators: Blood samples were collected from mice in each group on day 14 post-infection, and serum was separated. Bone metabolism-related indicators, including osteogenic indicators OCN, OPN, and PINP, and bone resorption-related indicators TRAP-5b and CTX, were detected using ELISA. The levels of each indicator were calculated based on the standard curve of the kit.

[0052] 7. Micro-CT Detection of Bone Structure Changes: Femurs of mice from each group were collected, fixed with 4% paraformaldehyde, and then examined using a high-resolution micro-CT scanning system (SkyScan1276). Scanning parameters were set as follows: scanning voltage 55 kV, current 145 μA, integration time 400 ms. Two-dimensional and three-dimensional bone structure images were obtained after reconstructing the scan data using NRecon software. A region of interest (ROI) was selected from 0.444 mm below the distal femoral growth plate to 1.2 mm proximally. Bone microstructural parameters, including bone mineral density (BMD), bone volume / total volume ratio (BV / TV), trabecular bone number (Tb.N), and trabecular bone pattern factor (Tb.Pf), were detected.

[0053] Experimental results: such as Figure 6 As shown, compared with the Staphylococcus aureus-infected group, the weight loss of mice in the Ginkgolide A group, vancomycin group, and vancomycin + Ginkgolide A group was improved, with the improvement in weight loss being more significant in the combined treatment group of vancomycin + Ginkgolide A. Figure 7As shown, compared with the group infected with Staphylococcus aureus, both the Ginkgolide A group and the vancomycin group were able to reduce the bacterial load on the femoral tissue and implant surface, while the vancomycin + Ginkgolide A group showed a more significant reduction effect, suggesting that Ginkgolide A can enhance the control effect of vancomycin on Staphylococcus aureus infection and enhance the antibacterial therapeutic effect. Figure 8 As shown, H&E staining results indicate that Staphylococcus aureus infection leads to increased inflammatory infiltration of the bone marrow cavity, destruction of trabecular bone structure, and tissue damage. Treatment with ginkgolide A combined with vancomycin significantly improves these pathological changes and reduces the degree of tissue damage. Figure 9 As shown, compared with the control group, the serum inflammatory factor levels of mice infected with Staphylococcus aureus were significantly increased. Treatment with ginkgolide A and vancomycin alone could reduce inflammation levels, while the combined treatment of vancomycin and ginkgolide A showed a more significant reducing effect. These results suggest that ginkgolide A combined with vancomycin can effectively alleviate the inflammatory response induced by Staphylococcus aureus infection. Figure 10 As shown, TRAP staining results revealed that Staphylococcus aureus infection significantly promoted osteoclast formation, while treatment with ginkgolide A combined with vancomycin significantly reduced the number of TRAP-positive cells. This suggests that the combination can inhibit infection-induced abnormal bone resorption. Figure 11 As shown, compared with the Staphylococcus aureus-infected group, both ginkgolide A and vancomycin alone increased serum OCN, OPN, and PINP levels, and decreased TRAP-5b and CTX levels. The combined treatment of vancomycin and ginkgolide A showed even more significant effects. This suggests that ginkgolide A combined with vancomycin can improve the bone metabolic imbalance caused by decreased bone formation and increased bone resorption under infectious conditions. Figure 12 As shown, Micro-CT analysis revealed that Staphylococcus aureus infection led to decreased femoral bone mineral density and destruction of trabecular bone structure. Ginkgolide A combined with vancomycin treatment significantly improved bone microstructure, increased BMD, BV / TV and Tb.N levels, and decreased Tb.Pf levels.

[0054] In summary, the results of this embodiment demonstrate that ginkgolide A combined with vancomycin can reduce the bacterial load at the infection site and on the implant surface in a Staphylococcus aureus implant-associated osteomyelitis model, reduce inflammatory responses, decrease abnormal osteoclast activation, improve increased bone resorption, and alleviate infection-related bone tissue damage and bone metabolic disorders. Therefore, the composition of ginkgolide A combined with antibacterial drugs provided by this invention can serve as an adjunctive treatment strategy with both antibacterial synergistic effects and infection-related bone protection, and has potential application value in the prevention and treatment of bacterial infections such as Staphylococcus aureus, especially implant-associated osteomyelitis.

[0055] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. Application of Ginkgolide A in the preparation of drugs against Staphylococcus aureus biofilm formation.

2. The application according to claim 1, characterized in that, Ginkgolide A exerts its anti-biofilm effect at concentrations below the minimum inhibitory concentration (MIC), inhibiting the formation and maturation of Staphylococcus aureus biofilm; the MIC is 256 μg / mL.

3. The application according to claim 2, characterized in that, Ginkgolide A regulates the expression of genes related to Staphylococcus aureus biofilm formation, reducing [various factors]. icaA , icaD , sarA , fnbA , fnbB and clfA This reduces the expression levels of related genes, thereby decreasing the ability of Staphylococcus aureus to form biofilms.

4. Application of Ginkgolide A in combination with antibacterial drugs in the preparation of drugs for the prevention and treatment of Staphylococcus aureus infection.

5. The application according to claim 4, characterized in that, The antibacterial drugs include one or more of glycopeptides, β-lactams, and aminoglycosides.

6. The application according to claim 5, characterized in that, The antibacterial drug is vancomycin.

7. The application according to claim 5, characterized in that, The Staphylococcus aureus infection mentioned is an implant-associated Staphylococcus aureus infection.

8. Application of Ginkgolide A in combination with vancomycin in the preparation of a drug for the treatment of implant-related infectious osteomyelitis.

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