Application of venenum bufonis extract combined with beta-lactam antibiotics in medicine for treating MRSA infection

Abstract

The invention provides application of a venenum bufonis extract combined with beta-lactam antibiotics in medicines for treating MRSA infection, and belongs to the technical field of biological medicines. The invention proves that the active ingredients of the venenum bufonis have strong affinity and are stably combined with target proteins such as PBP2a, BlaZ and AgrA of MRSA, so that the antibacterial effect of antibiotics is enhanced. The venenum bufonis extract and beta-lactam antibiotics are combined for use, so that a remarkable synergistic effect is achieved, and the drug resistance of drug-resistant staphylococcus aureus is overcome. Mouse model experiments prove that the venenum bufonis and penicillin G combined treatment has a remarkable treatment effect on MRSA infected mice. The combined application of the venenum bufonis and the penicillin G can effectively control the MRSA infection, and a novel and effective treatment scheme is provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of toad venom extract combined with β-lactam antibiotics in the treatment of MRSA infection. Background Technology

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a highly pathogenic bacterium with complex drug resistance, causing various infectious diseases such as pneumonia, bacteremia, and skin and soft tissue infections. Its resistance to common antibiotics is increasingly difficult to treat. With the widespread use of antibiotics, the drug resistance problem of MRSA has become increasingly serious, especially in hospital settings, where it has become one of the leading causes of nosocomial infections. Currently, the treatment of MRSA mainly relies on β-lactam antibiotics, but with the increasing drug resistance, many traditional treatment methods have become ineffective.

[0003] Toad venom (Bufonis venenum) is a traditional Chinese medicine derived from the secretions of toad skin, possessing various pharmacological effects such as clearing heat and detoxifying, anti-tumor, and anti-infective properties. In recent years, the application of toad venom in treating bacterial infections has gradually attracted attention. Studies have shown that extracts and active ingredients of toad venom can significantly inhibit the growth of various pathogenic microorganisms, including Staphylococcus aureus, Streptococcus, and Escherichia coli. The antibacterial effect of toad venom may be related to its rich chemical components, such as bufodiene lactones, bufotoxin, and polypeptides, which play an important role in anti-infective treatment. Although toad venom is widely used in traditional Chinese medicine to treat various bacterial infections, its role and molecular mechanism in combating MRSA remain unclear. Summary of the Invention

[0004] This invention provides the application of toad venom extract in combination with β-lactam antibiotics in the treatment of MRSA infection. The toad venom extract can significantly improve the therapeutic effect of anti-MRSA through synergistic effects with antibiotics.

[0005] This invention provides the application of toad venom extract in the preparation of therapeutic drugs for methicillin-resistant Staphylococcus aureus infections.

[0006] In one specific embodiment of the present invention, the toad venom extract includes toad venom injection.

[0007] This invention also provides the application of toad venom extract in the preparation of antibiotic sensitizers.

[0008] In one specific embodiment of the present invention, the β-lactam antibiotics include penicillin antibiotics and cephalosporin antibiotics.

[0009] In one specific embodiment of the present invention, the penicillin antibiotics include penicillin G and oxacillin; the cephalosporin antibiotics include ceftriaxone sodium and cefotaxime.

[0010] The present invention also provides the application of toad venom extract in combination with antibiotics in the preparation of a therapeutic drug for methicillin-resistant Staphylococcus aureus infection.

[0011] In one specific embodiment of the present invention, the toad venom extract includes toad venom injection.

[0012] In one specific embodiment of the present invention, the β-lactam antibiotics include penicillin antibiotics and cephalosporin antibiotics.

[0013] In one specific embodiment of the present invention, the penicillin antibiotics include penicillin G and oxacillin; the cephalosporin antibiotics include ceftriaxone sodium.

[0014] In one specific embodiment of the present invention, the mass ratio of the toad venom extract to penicillin G is (500~1000):(64~256).

[0015] The present invention also provides a therapeutic agent for methicillin-resistant Staphylococcus aureus infection, comprising toad venom extract and pharmaceutically acceptable excipients.

[0016] In one specific embodiment of the present invention, β-lactam antibiotics are also included.

[0017] Beneficial Effects: This invention demonstrates, through molecular docking and fluorescence quenching experiments, the strong affinity and stable binding of toad venom active ingredients to target proteins of MRSA, such as PBP2a, BlaZ, and AgrA, thereby enhancing the antibacterial efficacy of β-lactam antibiotics. Furthermore, toad venom has minimal impact on bacterial growth kinetics, with its minimum inhibitory concentration (MIC) significantly higher than its effective antibacterial concentration, effectively avoiding the antibacterial pressure common with traditional antibiotics and thus reducing the risk of drug resistance. The toad venom described in this invention is a natural traditional Chinese medicine with high safety. The combined use of toad venom extract and β-lactam antibiotics not only enhances the anti-MRSA effect but also effectively reduces the risk of adverse reactions from β-lactam antibiotics, showing promising prospects for clinical translation.

[0018] This invention combines toad venom extract with β-lactam antibiotics, exhibiting a significant synergistic effect and overcoming the drug resistance of methicillin-resistant Staphylococcus aureus (MRSA). Mouse model experiments verified that the combined treatment of toad venom and penicillin G has a significant therapeutic effect on mice infected with MRSA. Specifically, it improves mouse survival rate, reduces lung bacterial load, and improves lung tissue pathology. Furthermore, the combined application of low-dose toad venom and penicillin G significantly enhances efficacy compared to the use of the drugs alone. This indicates that the combined application of toad venom and penicillin G can effectively control MRSA infection and provides a new and effective treatment regimen. Attached Figure Description

[0019] Figure 1 The graph shows the MIC determination results of toad venom against MRSA USA300. Figure 2 A diagram illustrating the action of toad venom combined with β-lactam antibiotics against MRSA USA300; Figure 3 The effect of toad venom combined with β-lactam antibiotics against MSSA ATCC29213; Figure 4 The time-kill curve shows the synergistic antibacterial effect of toad venom and penicillin G. Figure 5 Graph showing the effect of different concentrations of toad venom combined with penicillin G against MRSA USA300; Figure 6 The graph shows the effect of different concentrations of toad venom on the survival rate of mice. Figure 7 Figure showing the effect of different concentrations of toad venom on the activity of HEK-293T cells; Figure 8 This is an SDS-PAGE gel electrophoresis image of BlaZ protein expression and purification. In the figure, M: protein marker; 1: bacterial culture before induction; 2: bacterial culture after induction; 3: flow broth; 4-7: imidazole elution buffer at 10mM, 20mM, 50mM, and 100mM, respectively. Figure 9 This is an SDS-PAGE gel electrophoresis image of PBP2a protein expression and purification. In the figure, M: protein marker; 1: bacterial culture before induction; 2: bacterial culture after induction; 3: flow broth; 4: binding buffer; 5-9: imidazole elution buffers of 5mM, 10mM, 20mM, 50mM, and 100mM, respectively. Figure 10The image shows an SDS-PAGE gel electrophoresis result of AgrA protein expression and purification. In the image, M: protein marker; 1: bacterial culture before induction; 2: bacterial culture after induction; 3: flow broth; 4-8: imidazole elution buffers of 10mM, 20mM, 50mM, 100mM, and 250mM, respectively. Figure 11 The image shows the fluorescence quenching analysis results of toad venom and BlaZ protein. Figure 12 The image shows the fluorescence quenching analysis results of toad venom and PBP2a protein. Figure 13 The image shows the fluorescence quenching analysis results of toad venom and AgrA protein. Figure 14 This is a schematic diagram of the molecular docking between AgrA and resibufogenin. Figure 15 This is a schematic diagram of the molecular docking between BlaZ and resibufogenin. Figure 16 This is a schematic diagram of the molecular docking mode between PBP2a and bufotaline. Figure 17 Venn diagram showing the targets of toad venom activity and targets associated with Staphylococcus aureus infection; Figure 18 KEGG enrichment analysis of target genes for toad venom in treating Staphylococcus aureus infection; Figure 19 GO enrichment analysis of target genes for toad venom therapy against Staphylococcus aureus infection; Figure 20 A protein interaction network for toad venom-related targets in Staphylococcus aureus infection treatment; Figure 21 MCODE cluster analysis diagram of toad venom targets related to Staphylococcus aureus infection treatment; Figure 22 Chemical components, targets, and pathways for the treatment of Staphylococcus aureus infection with toad venom; Figure 23 A differential expression analysis diagram of key targets in toad venom treatment for Staphylococcus aureus infection; Figure 24 This is a schematic diagram of the molecular docking mode between AKT1 and tryptanmine. Figure 25 This is a schematic diagram of the molecular docking between HIF1A and tryptanthrin. Figure 26 This is a schematic diagram of the molecular docking mode between HSP90AA1 and desacetylcinobufotalin; Figure 27 This is a schematic diagram of the molecular docking mode between MCL1 and tryptanthrin. Figure 28 This is a schematic diagram of the molecular docking of NOS2 and bufalin. Figure 29 This is a schematic diagram of the molecular docking between RAC1 and tryptanthrin. Figure 30 Figure showing the effect of high, medium, and low doses of toad venom on the survival rate of the MRSA USA300 mouse model of lung infection; Figure 31 Figure 1 shows the colony count results of lung tissue in a mouse model of MRSA USA300 lung infection treated with high, medium, and low doses of toad venom. Figure 32 Histological sections (HE staining) of lung tissue from a mouse model of MRSA USA300 lung infection treated with high, medium, and low doses of toad venom. In the figure, A: USA300 group; B: blank group; C: 5 ml / kg group; D: 10 ml / kg; E: 15 ml / kg; Figure 33 To evaluate the survival rate of a mouse model of MRSA USA300 lung infection treated with toad venom combined with penicillin G; Figure 34 Colony counts in lung tissue of a mouse model of MRSA USA300 lung infection treated with toad venom combined with penicillin G; Figure 35 The images show pathological sections (HE staining) of lung tissue from a mouse model of MRSA USA300 lung infection treated with toad venom combined with penicillin G. In the images, A: USA300 group; B: blank group; C: CSZSY group; D: penicillin G group; E: CSZSY + penicillin G group. Detailed Implementation

[0020] This invention provides the application of toad venom extract in the preparation of therapeutic drugs for methicillin-resistant Staphylococcus aureus infections.

[0021] In one embodiment, the toad venom extract described in this invention is a toad venom injection, purchased from Jiangsu Pujin Pharmaceutical Co., Ltd., and is a single-herb extract preparation of toad venom. In the embodiment, it was found that the MIC of toad venom against MRSA USA300 was 4 mg / ml, indicating significant antibacterial activity. Furthermore, high-dose injections into mice did not result in poisoning symptoms, abnormal behavior, or death. The toad venom injection also showed almost no toxicity to HEK-293T cells, indicating that toad venom has good safety.

[0022] This invention reveals that PBP2a protein (Penicillin-Binding Protein, GenBank ID: PV938972.1) and BlaZ protein (β-lactamase, GenBank ID: M62650.1) are both important drug resistance-related proteins in Staphylococcus aureus and are major causes of MRSA resistance. AgrA (GenBank ID: U85097.1) is a virulence factor of Staphylococcus aureus and a key component of the Agr (Accessory Gene Regulator) system, which plays an important role in bacterial infection and is related to bacterial virulence and hemolytic activity. In the embodiments of this invention, it was found that toad venom can bind to PBP2a protein, BlaZ protein, and AgrA and inhibit the activity of these proteins, demonstrating the good therapeutic effect of toad venom extract on MRSA infection.

[0023] This invention also provides the application of toad venom extract in the preparation of β-lactam antibiotic sensitizers.

[0024] In this invention, it was found that toad venom extract can bind to PBP2a and BlaZ proteins in MRSA, thereby reducing the activity of the binding proteins, thus reducing MRSA resistance and significantly improving the therapeutic effect of antibiotics. The β-lactam antibiotics described in this invention include penicillin antibiotics and cephalosporin antibiotics; the penicillin antibiotics include penicillin G and oxacillin; the cephalosporin antibiotics include ceftriaxone sodium and cefotaxime.

[0025] The toad venom extract described in this invention has a relatively weak antibacterial effect, but it exhibits a significant synergistic effect when used in combination with β-lactam antibiotics. It can sensitize the original antibiotics, restoring the antibacterial efficacy of previously ineffective antibiotics. Studies using MRSA USA300 revealed a synergistic effect between toad venom and oxacillin (FICI 0.265625); with cefepime (FICI 0.3125); with penicillin G (FICI 0.265625); with cefotaxime (FICI 0.28125); with cefoxitin (FICI 0.375); and with ceftriaxone sodium (FICI 0.315). Studies using MSSA ATCC29213 revealed a synergistic effect between toad venom and penicillin G (FICI: 0.28125); a synergistic effect between toad venom and oxacillin (FICI: 0.375); and a synergistic effect between toad venom and ceftriaxone sodium (FICI: 0.5). A significant difference between USA300 and MSSA ATCC29213 is that USA300 encodes the PBP2a protein, while MSSA ATCC29213 does not.

[0026] The present invention also provides the application of toad venom extract in combination with antibiotics in the preparation of a therapeutic drug for methicillin-resistant Staphylococcus aureus infection.

[0027] The toad venom extract of the present invention includes toad venom injection, and the β-lactam antibiotics include penicillin antibiotics and cephalosporin antibiotics. The penicillin antibiotics include penicillin G and oxacillin; the cephalosporin antibiotics include ceftriaxone sodium and cefotaxime.

[0028] In one specific embodiment of the present invention, the mass ratio of the toad venom extract and β-lactam antibiotic, especially to penicillin G, is (500~1000):(64~256).

[0029] The present invention also provides a therapeutic agent for methicillin-resistant Staphylococcus aureus infection, comprising toad venom extract and pharmaceutically acceptable excipients.

[0030] The therapeutic drugs described in this invention also include antibiotics.

[0031] To further illustrate the present invention, the application of the toad venom extract combined with antibiotics provided by the present invention in the treatment of MRSA infection is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods and materials used in the embodiments of this invention are common methods and materials in the art.

[0033] The experimental methods and materials involved in the embodiments of this invention are mainly as follows: 1. Strains: Staphylococcus aureus strain USA300 (MRSA, published in Mlynek KD, Callahan MT, Shimkevitch AV, Farmer JT, Endres JL, Marchand M, Bayles KW, Horswill AR, Kaplan JB. Effects of Low-Dose Amoxicillin on Staphylococcus aureus USA300 Biofilms. Antimicrob Agents Chemother. 2016 Apr 22;60(5):2639-51. doi:10.1128 / AAC.02070-15. PMID: 26856828; PMCID: PMC4862544.) and ATCC 29213 The strains pET28a-AgrA-BL21, pET28a-BlaZ-BL21, and pET28a-PBP2a-BL21 were obtained from the laboratory of the College of Animal Science, Jilin University. The construction method of each strain was as follows: using the genome of strain USA300 as a template, the BlaZ (GenBank ID: M62650.1), PBP2a (GenBank ID: PV938972.1), and AgrA (GenBank ID: U85097.1) genes were amplified and inserted into the BamHI and SalI spaces of the pET28a vector (purchased from Sangon Biotech Co., Ltd., catalog number B540183), respectively. The vector was then transformed into strain DH5α (purchased from Sangon Biotech Co., Ltd., catalog number B528413), and the positive plasmid was extracted and transformed into strain BL21 (purchased from Sangon Biotech Co., Ltd., catalog number B528414).

[0034] 2. Experimental animals: C57BL / 6 mice (SPF grade) were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. and raised at the Animal Experiment Center of the College of Veterinary Medicine, Jilin University. The experimental animal research protocols were all approved by the Animal Welfare and Use Committee of Jilin University.

[0035] 3. Source of the drug: Toad venom injection (CSZSY) is sourced from Jiangsu Pujin Pharmaceutical Co., Ltd. The original concentration of toad venom injection is 2mg crude drug / ml. The original concentration is defined as c. Concentrating it 2 times results in 2c, and diluting it 2 times results in 1 / 2c. The concentration can be increased or decreased in the same way.

[0036] 4. Experimental Methods 4.1 Determination of MIC The MIC was determined by broth microdilution method. The USA300 and ATCC 29213 strains were cultured in BHI medium at 37 °C overnight. After overnight culture, the strains were subcultured into CAMHB medium and continued to be cultured until the OD 600 reached 1.0 and then taken for standby. A 96-well plate was used in the experiment. All wells were filled with CAMHB medium. A concentration gradient of bufalin was set up and serially diluted by two-fold dilution, so that the bufalin concentration ranged from 2c to 1 / 128c. The antibiotic concentration used was also serially diluted by two-fold dilution. 1 μL of bacterial solution was added to the experimental wells, and no bacterial solution was added to the blank control. Three replicates were set up for each well. After sealing the 96-well plate with a sealing film, it was placed in an incubator at 37 °C and incubated for 16 h. The OD 600 value of each well was measured, which was the final result.

[0037] 4.2 In vitro combined antibacterial experiment by checkerboard method The checkerboard method was used to determine the synergistic antibacterial effect of bufalin and β-lactam antibiotics. The bacterial solution with an OD 600 of 1.0 was obtained according to the MIC determination experimental method. In the 96-well plate, the horizontal axis was the concentration gradient of the antibiotic serially diluted by two-fold dilution, and the vertical axis was the concentration gradient of bufalin serially diluted by two-fold dilution. 1 μL of bacterial solution was added to the wells used in the experiment. After sealing the 96-well plate with a sealing film, it was placed in an incubator at 37 °C and incubated for 16 h. The OD 600 value of each well was measured, which was the final result.

[0038] Calculation formula for the synergistic antibacterial index: FICI = FIC A + FIC B; where FIC A is the newly measured MIC of drug A / the MIC of drug A used alone, and FIC B is the newly measured MIC of drug B / the MIC of drug B used alone; when FICI ≤ 0.5, it is considered that the two drugs show a synergistic effect; when 0.5 < FICI ≤ 1, it is considered that the two drugs show an additive effect; when 1 < FICI ≤ 2, it is considered that the two drugs show an irrelevant effect; when FICI > 2, it is considered that the two drugs show an antagonistic effect.

[0039] 4.3 Determination of Time-kill curve The bactericidal curve experiment was carried out using CAMHB medium. The initial concentration of the USA300 bacterial solution was adjusted to 1×10 6 CFU / mL, and sub-inhibitory concentration of bufalin or antibiotic was added thereto. Incubated at 37 °C, samples were taken and measured at 0 h, 4 h, 8 h, 12 h and 24 h. After obtaining the samples, the sample concentration was serially diluted by two-fold dilution. The diluted sample liquid was dropped on the LB solid plate, sealed with a sealing film, and then the plate was placed in an incubator at 37 °C and cultured overnight. The next day, the colony growth was observed and the colony count was performed.

[0040] 4.4 Antibacterial zone experiment First, 6mm filter paper discs were made using a punch, followed by autoclaving. Then, different concentrations of toad venom (0°C, 1 / 2°C, 1 / 4°C) LB solid culture medium were prepared. Penicillin G solution was prepared in a laminar flow hood, and the paper discs were immersed in the solution, dried, and set aside. The USA300 culture medium was then incubated to OD. 600 When the concentration is 0.1, 100 μL of bacterial suspension is spread on a plate, and then the dried paper is attached to the solid culture medium. The plate is sealed with sealing film and placed in an incubator at 37°C. After incubation for 12 hours, the plate is removed and the diameter of the inhibition zone is observed.

[0041] 4.5 Acute toxicity test Five female C57BL / 6 mice (6-8 weeks old, weighing 18-20 g) in each group were injected intraperitoneally with toad venom at doses of 5 ml / kg, 10 ml / kg, and 15 ml / kg, respectively. The control group mice were injected with the corresponding volume of physiological saline. Abnormal behavior, poisoning symptoms, and survival of the mice in different dose groups were observed for 72 hours.

[0042] 4.6 Cytotoxicity assay The effect of different concentrations of toad venom on the viability of HEK-293T cells was detected using a CCK-8 assay kit. HEK-293T cells are human embryonic kidney cells. In culture, HEK-293T cells were proliferated in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotic solution, and incubated at 37°C and 5% CO2. The HEK-293T cell suspension was aliquoted into 96-well plates for cell experiments (1 × 10⁶ cells per well). 4 (3 cells per well) Three replicates per well. After 24 h of incubation, these cell cultures were treated with different concentrations of toad venom (0, 1 / 8 c, 1 / 4 c, 1 / 2 c, c), and then incubated for another 24 h. After incubation at 37 °C for 1 h, the cell viability was assessed. Finally, the absorbance at 450 nm was measured using an ELISA reader, and the data were recorded.

[0043] 4.7 Expression and purification of PBP2a, Blaz, and AgrA proteins 4.7.1 Expression of the target protein PBP2a-pET28a, BlaZ-pET28a, and AgrA-pET28a strains previously preserved in the laboratory were transformed into *E. coli* BL21. Subsequently, PBP2a-pET28a-BL21, BlaZ-pET28a-BL21, and AgrA-pET28a-BL21 bacterial suspensions were streaked on kanamycin-resistant LB agar plates. Single colonies that grew after streaking were transferred to LB liquid medium and cultured. Induction culture was performed at low temperature (37°C) for 16 h with 1 mmol / L IPTG. After incubation, bacterial cells were collected, centrifuged (10000 rpm, 10 min), and the supernatant was discarded. The bacterial cells were resuspended and lysed by sonication (190 W, 30 min), centrifuged, and the supernatant was collected for later use.

[0044] 4.7.2 Purification of the target protein PBP2a protein Blaz and AgrA protein were purified using a Ni-NTA His Bind Resin nickel column. The supernatant was incubated with the nickel column, and the filtered liquid was the runoff. Subsequently, different concentrations of imidazole were used for elution. The eluted sample solution was then analyzed by SDS-PAGE. Coomassie Brilliant Blue was used to stain the gel, and after destaining, the bands on the gel were observed and photographed. Finally, the purified protein sample solution was collected, concentrated, and frozen at -80°C for further experiments.

[0045] 4.8 Fluorescence Quenching Experiment The target protein sample solution was placed in a cuvette, and then toad venom was continuously added to prepare sample solutions with different concentration gradients of toad venom. The sample solutions of different concentrations were measured using a fluorescence spectrophotometer. The excitation wavelength was set to 280 nm, and the fluorescence emission spectrum data in the wavelength range of 280-400 nm were collected.

[0046] 4.9 Molecular docking 3D structures of the active ingredients in toad venom were downloaded from the PubChem database, and these small molecule structures were optimized using ChemBio3D Ultra 14.0. Protein structures used in the study were collected from the RCSB database (https: / / www.rcsb.org / ), dehydrated and hydrogenated the proteins, and imported both the proteins and ligands into AutoDocktools (v1.5.6) to determine the binding pockets. The structures were exported as "pdbqt" format and molecular docking was performed using AutoDock Vina 1.1.2.

[0047] 4.10 Network Pharmacological Analysis 4.10.1 Data Collection Toad venom is a single-herb extract of toad venom. Therefore, when collecting the active ingredients of toad venom, only toad venom was collected. The main components of toad venom were collected through the herb database. The QSAR-TargetNet (Probability>0.9, http: / / targetnet.scbdd.com / home / index / ), SwissTargetPrediction (Probability>0.4, http: / / swisstargetprediction.ch / ), and SEA (P-Value<1.0e-15, https: / / sea.bkslab.org / ) databases were used for target fishing of the main active ingredients of toad venom. The genecards (Relevancescore>10, https: / / www.genecards.org / ) and CTD (Inference Score≥15, https: / / ctdbase.org / ) databases were used to collect Staphylococcus aureus infection-related targets.

[0048] 4.10.2 Enrichment analysis of target genes in toad venom therapy for Staphylococcus aureus infection The Venn package in R was used to construct a Venn diagram of the targets of toad venom active ingredients and targets related to Staphylococcus aureus infection, thus identifying genes involved in toad venom treatment of Staphylococcus aureus infection. These target genes were then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses. P Significant enrichment terms with values ​​≤0.05 were used to analyze the molecular mechanism of action of toad venom in treating Staphylococcus aureus infection.

[0049] 4.10.3 Construction of the Target Protein Interaction Network (PPI) Protein-protein interaction predictions for the target genes of toad venom in treating Staphylococcus aureus infection were performed using the String 11.0 database (https: / / cn.string-db.org / ). The results were then imported into Cytoscape 3.8.2 (http: / / metascape.org / ) for visualization. In Cytoscape, nodes with higher connectivity to target proteins in the network were assigned larger sizes and darker colors. The target genes for toad venom in treating Staphylococcus aureus infection were submitted to Metascape, and the MCODE algorithm was used to identify protein subnetworks. The protein networks were clustered, and enrichment analysis was performed on each cluster.

[0050] 4.10.4 Construction of Toad Venom Compound-Target-Pathway Network The main active ingredients screened from toad venom and their targets related to Staphylococcus aureus infection were imported into Cytoscape 3.8.2 to construct a compound-target network. Furthermore, some key pathways enriched by KEGG in the previous stage were imported into Cytoscape to construct a target-pathway network, ultimately yielding the toad venom compound-target-pathway network.

[0051] 4.10.5 Differential Expression Analysis of Key Targets The expression of key target genes in blood samples from 32 patients with Staphylococcus aureus bloodstream infection and 43 healthy individuals was analyzed using GEO gene chip expression data GSE33341.

[0052] 4.11 Experiment on the therapeutic effect of toad venom on mouse pneumonia 4.11.1 Preparation of bacterial culture The USA300 strain was inoculated into LB liquid medium and cultured overnight at 37°C, 200 rpm. It was then subcultured into 500 ml TSB liquid medium (1:100) and cultured until OD200 was reached. 600 The bacterial culture used in the experiment was 1.0. After centrifugation, the bacterial cells were collected, washed several times, and then resuspended in 1.5 ml of PBS for further experimental use.

[0053] 4.11.2 Survival rate experiment in mice with pneumonia Fifty male C57 mice, weighing approximately 20g and 8 weeks old, were selected and, after acclimatization, were randomly divided into five groups of 10 mice each: a control group (saline injection), a USA300 infection group, a low-dose toad venom treatment group (5ml / kg), a medium-dose toad venom treatment group (10ml / kg), and a high-dose toad venom treatment group (15ml / kg). Mice were anesthetized with ether, held upright by the neck, and 30μL of the bacterial solution obtained in the previous step was dripped into the left nostril, allowing the mice to inhale it completely. After the mice regained consciousness, they were returned to their cages. Two hours later, the mice were administered the same intraperitoneal injection according to the grouping and dosage guidelines. Administering the medication every 12 hours, and the mice's survival status was recorded for a total of 96 hours.

[0054] 4.11.3 Colony Count of Bacterial Load in the Lungs of Pneumonia-Infected Mice The experimental groupings in this section were consistent with those in 4.12.2, with each group consisting of 5 C57 mice. The total bacterial dose administered was half of the dose given in 4.12.2. Mice were sacrificed 24 hours after administration according to the different groups. Lung tissue was removed, rinsed with physiological saline, weighed, and then ground evenly in sterile physiological saline. The tissue was then diluted and inoculated onto TSB solid culture plates. The culture plates were sealed with sealing film and placed in a constant temperature incubator for 12 hours. Colony counting was performed when colonies became clear.

[0055] 4.11.4 Pathological study of lungs in mice with pneumonia The experimental groups and bacterial dosages for this part were the same as in 4.12.3, with 5 male mice in each group. After 24 hours of drug administration, the mice were sacrificed, and the left lungs of the mice were removed. The lung tissue morphology was observed and images were recorded. The mouse lungs were then fixed in 10% formalin. The fixed lung tissue was dehydrated, embedded in paraffin, and sectioned. After dewaxing in xylene solution, the tissue was stained with hematoxylin and eosin. The stained pathological sections were observed under an optical microscope.

[0056] 4.12 Experiment on the treatment of mouse pneumonia by toad venom combined with penicillin G 4.12.1 Survival rate experiment of mice with pneumonia Fifty male C57 mice weighing approximately 20g were randomly divided into 5 groups of 10 mice each: a control group (saline injection), a USA300 infection group, a low-dose toad venom treatment group (5ml / kg), and a penicillin G treatment group (6.5×10⁻⁶). 5 The treatment group received low-dose toad venom (U / kg) combined with penicillin G (5ml / kg). Toad venom was administered via intraperitoneal injection, while penicillin G was administered via subcutaneous injection.

[0057] 4.13 Statistical Analysis The statistical analysis in this invention was performed using GraphPad Prism 9 software. The experiment was repeated multiple times to obtain three sets of valid data. Results are expressed as mean ± standard error. For two or more groups, t-tests or analysis of variance (ANOVA) were used respectively. P A value <0.05 is considered statistically significant. P <0.05, P <0.01, P <0.001, the difference in ns was not significant.

[0058] Example 1: In vitro activity of toad venom combined with β-lactam antibiotics against MRSA 1.1 Determination of the MIC of toad venom The minimum inhibitory concentration (MIC) of toad venom was determined using the broth microdilution method according to the guidelines of the American Clinical and Laboratory Standards Institute (ACSI). The results are as follows: Figure 1 As shown, it can significantly inhibit bacteria at 4 mg / ml, i.e., the MIC is 4 mg / ml (2c).

[0059] 1.2 Inhibitory effect of toad venom combined with antibiotics on Staphylococcus aureus The results of the experiment on the effects of toad venom combined with β-lactam antibiotics against MRSA USA300 are shown in Table 1. Toad venom showed a synergistic effect with oxacillin (FICI 0.265625); with cefepime (FICI 0.3125); with penicillin G (FICI 0.265625); with cefotaxime (FICI 0.28125); with cefoxitin (FICI 0.375); and with ceftriaxone sodium (FICI 0.315). Figure 2 As shown, toad venom combined with these six β-lactam antibiotics exhibits good synergy against MRSA USA300. Among them, toad venom combined with penicillin G and oxacillin has the best synergistic effect, indicating that toad venom combined with β-lactam antibiotics is superior to cephalosporin antibiotics against USA300 and can significantly improve the sensitivity of penicillin G and oxacillin.

[0060] The results of the experiment comparing toad venom combined with β-lactam antibiotics against MSSA ATCC29213 are shown in Table 2. Toad venom combined with cefoxitin showed no synergistic effect (FICI > 1); toad venom combined with penicillin G showed a synergistic effect (FICI 0.28125); toad venom combined with cefepime showed no synergistic effect (FICI > 1); toad venom combined with cefotaxime showed an additive effect (FICI 0.75); toad venom combined with oxacillin showed a combined effect (FICI 0.375); and toad venom combined with ceftriaxone sodium showed a synergistic effect (FICI 0.5). Figure 3 As shown, toad venom combined with β-lactam antibiotics has a significant synergistic effect against MSSA ATCC29213 and penicillin antibiotics, but no synergistic effect or only a weak synergistic effect with cephalosporin antibiotics.

[0061] Table 1 Synergistic Index of Toad Venom Combined with β-Lactam Antibiotics Against MRSA USA300

[0062] Table 2 Synergistic Index of Toad Venom Combined with β-Lactam Antibiotics Against MSSA ATCC29213

[0063] 1.3 Bactericidal curves of toad venom combined with penicillin G The initial concentration of the USA300 bacterial culture was adjusted to 10. 6 CFU / ml was used to divide the samples into a USA300 control group, a 64 μg / ml penicillin G group, a 1 / 2 c toad venom group, and a combined 64 μg / ml penicillin G and 1 / 2 c toad venom group to verify the synergistic antibacterial effect of the bactericidal curve. Figure 4As shown, the USA300 control group grew rapidly from 0 to 8 hours, entering the logarithmic growth phase, and then entered a plateau phase after 12 hours, essentially ceasing growth. Both the 1 / 2 c toad venom group and the 64 μg / ml penicillin G group showed weak inhibitory effects on USA300 within 4 hours, followed by another rapid growth phase before reaching a plateau. The combination of the 1 / 4 MIC toad venom group and the 1 / 8 MIC penicillin G group resulted in a significant decrease in USA300 levels from 0 to 8 hours, with a plateau reached after 8 hours, indicating a good synergistic antibacterial effect between the two drugs.

[0064] 1.4 Antibacterial zone The results of the inhibition zone experiment are as follows Figure 5 As shown, when the concentration of toad venom is 0, penicillin G does not form a significant inhibition zone, but the diameter of the inhibition zone increases with the addition of toad venom.

[0065] 1.5 Acute toxicity test like Figure 6 As shown, no poisoning symptoms or abnormal behavior were observed in any of the mice within 72 hours, and no deaths occurred, indicating that toad venom has good safety.

[0066] 1.6 Cytotoxicity assay The CCK8 assay was used to determine the killing effect of toad venom on human HEK-293T cells. The results were as follows: Figure 7 As shown, when cells were treated with toad venom at concentration c, the survival rate of HEK-293T cells was almost unchanged compared with the control group, with a survival rate of nearly 100%, indicating that the original concentration of toad venom has almost no toxicity to HEK-293T cells and has good safety.

[0067] Example 2: Confirmation of the effect of toad venom active ingredients on MRSA bacterial target sites BlaZ and PBP2a 2.1 Expression and purification of PBP2a, AgrA, and BlaZ proteins Based on the results of the synergistic antibacterial experiment, it was speculated that toad venom may bind to PBP2a and BlaZ proteins, thereby inhibiting the activity of these two proteins and thus sensitizing β-lactam antibiotics. Therefore, PBP2a and BlaZ proteins were expressed and purified to prepare for subsequent binding verification experiments. Figure 8 and Figure 9 As shown, BlaZ and PBP2a proteins have been successfully expressed and purified, with molecular weights of 29.7 kDa and 73 kDa, respectively.

[0068] Toad venom has a good therapeutic effect on MRSA infection, which is speculated to be related to its regulation of Staphylococcus aureus virulence. Therefore, AgrA was expressed and purified for further experimental verification. Figure 10 As shown, AgrA protein has been successfully expressed and purified, with a molecular weight of 15 kDa.

[0069] 2.2 Fluorescence Quenching Experiment Fluorescence quenching experiments were performed on the obtained high-purity PBP2a, AgrA, and BlaZ proteins, and the results are as follows: Figure 11 , 12 As shown in Figure 13, the fluorescence intensity of the three proteins gradually decreased as the concentration of toad venom increased, indicating that the active ingredients of toad venom have a strong binding effect with the three proteins.

[0070] 2.3 Molecular docking of PBP2a, AgrA, BlaZ proteins with active ingredients in toad venom Bufalin, bufotaline, cinobufagin, gamabufalin, and resibufogenin are the five active ingredients of toad venom specified for testing in the Chinese Pharmacopoeia. Molecular docking was used to further verify the interactions of these active ingredients with AgrA, BlaZ, and PBP2a (allosteric sites). Table 3 shows the binding energies of these active ingredients with AgrA, BlaZ, and PBP2a. Among them, resibufogenin showed the best binding with AgrA, with a binding energy of -7 kcal / mol; bufotaline showed the best binding with PBP2a, with a binding energy of -7.8 kcal / mol; and resibufogenin showed the best binding with BlaZ, with a binding energy of -8.2 kcal / mol. Figure 14 , Figure 15 and Figure 16 As shown, resibufogenin can form hydrogen bonds with LYS167 residues when it binds to AgrA, and it can form hydrogen bonds with SER70 and SER130 when it binds to BlaZ. Bufotaline can form hydrogen bonds with ASP295 and ASN146 when it binds to PBP2a.

[0071] Table 3. Scoring of the binding energy of the main active components of toad venom to PBP2a, AgrA, and BlaZ protein molecules.

[0072] Example 3: Exploring the in vivo mechanism of toad venom in treating MRSA infection 3.1 Acquisition of toad venom components and target fishing The mechanism of toad venom in treating MRSA infection was explored using network pharmacology. Eighty-one major chemical components of toad venom were collected from the Herb database. The study showed that the main active components of toad venom are bufotenoids and bufotenines. After ADMET property filtering, eight bufotenine compounds and 30 bufotenolone compounds were finally obtained, totaling 38 major active components, as shown in Table 4. The first column, Ingredient id, represents the compound ID from the Herb database; the second column categorizes bufotenine compounds into category A and bufotenolone compounds into category B, numbered sequentially; the third column lists the compound name; and the fourth column lists the canonical smiles of the compound structures.

[0073] Target fishing was performed on 38 active ingredients using QSAR-TargetNet, SwissTargetPrediction, and SEA databases, yielding a total of 220 targets. Genecards and CTD databases were used to collect Staphylococcus aureus infection-related targets, resulting in 702 Staphylococcus aureus infection-related targets. A Venn diagram was constructed showing the relationship between toad venom target genes and Staphylococcus aureus disease-related genes, ultimately identifying 33 targets related to toad venom in the treatment of Staphylococcus aureus infection. Figure 17 ).

[0074] Table 4. List of active compounds of bufotoxin, bufotoxin, stryptamines, and bufo lactones.

[0075] 3.2 GO and KEGG enrichment analysis The 33 target sites of toad venom for treating Staphylococcus aureus infection were identified, and GO and KEGG enrichment analyses of these target genes were performed using R scripts, such as... Figure 18 As shown, KEGG analysis revealed that these genes were significantly enriched in Fc gamma R-mediated phagocytosis, C-type lectin receptor signaling pathway, Tuberculosis, Prostate cancer, Relaxin signaling pathway, B cell receptor signaling pathway, PD-L1 expression and PD-1 checkpoint pathway in cancer, Salmonella infection, IL-17 signaling pathway, etc. These pathways include bacterial infection, cancer, and immune-related pathways.

[0076] like Figure 19 As shown, GO analysis revealed that these key targets are enriched in response to tolipopolysaccharide, response to molecule of bacterial origin, ficolin-1-rich granule, heme binding, tetrapyrrole binding, histone deacetylase binding, etc.

[0077] 3.3 Protein-protein interaction network and analysis of chemical components, targets, and pathways in toad venom Figure 20 Using the string database, protein interaction network analysis was performed on 33 key targets of toad venom in the treatment of Staphylococcus aureus infection. It was found that HIF1A, MMP9, AKT1, HSP90AA1, PTG2 and other targets have high connectivity and are located in the core of the network, which may play more important functions. Figure 21 MCODE cluster analysis of 33 key targets of toad venom in the treatment of Staphylococcus aureus infection revealed that these 6 target proteins form a subnetwork, which is associated with and enriched in this network of interleukin-4 and interleukin-13 signaling.

[0078] Figure 22 The diagram shows the components, targets, and pathways of the toad venom compounds, further illustrating their targets and pathways.

[0079] 3.4 Differential expression analysis of key target genes Figure 23 To investigate the differential expression of six targets in the key sub-network of toad venom-related targets for Staphylococcus aureus infection in the gene chip dataset GSE33341, gene chip expression profiles of blood samples from healthy individuals and Staphylococcus aureus-infected individuals in the GEO dataset GSE33341 were extracted. The results showed that, except for MCL1, the remaining genes AKT1, HIF1A, HSP90AA1, NOS2, and RAC1 were significantly differentially expressed in both healthy individuals and Staphylococcus aureus-infected individuals.

[0080] 3.5 Molecular docking of key targets and corresponding compounds in toad venom therapy for Staphylococcus aureus infection To further verify the interaction between the active ingredients of toad venom and the target sites, molecular docking was performed between the key targets and their corresponding compounds. The binding energies of these active ingredients with AKT1, HIF1A, HSP90AA1, NOS2, RAC1, and MCL1 are shown in Table 5. Figures 24 to 29As shown, AKT1 can form hydrogen bonds with THR211 and SER205 residues when binding to tryptamine; HIF1A can form hydrogen bonds with GLN239 and ARG238 when binding to tryptanthrin; HSP90AA1 can form hydrogen bonds with LEU107 and TYR139 when binding to desacetylcinobufotalin; MCL1 can form hydrogen bonds with TARG187 residues when binding to tryptanthrin; NOS2 can form hydrogen bonds with ARG199 and TRP372 when binding to bufalin; and RAC1 can form hydrogen bonds with ASP11 and SER86 when binding to tryptanthrin.

[0081] Table 5. Molecular docking binding energies of the main active components of toad venom to their corresponding target proteins.

[0082] Example 4: The therapeutic effects of toad venom and its combination with penicillin G on a mouse model of MRSA lung infection. 4.1 Therapeutic effect of toad venom on MRSA pulmonary infection model mice 4.1.1 Toad venom improved the survival rate of mice with MRSA lung infection. A mouse pneumonia model was established by intranasal administration of MRSA USA300 bacterial suspension. Mice were then treated with high, medium, and low doses of toad venom, and survival rates were observed. Figure 30 As shown, all mice in the USA300 treatment group died after 48 hours. The survival rate of mice in the 5 ml / kg toad venom treatment group was 30% after 48 hours, the survival rate in the 10 ml / kg toad venom group was 50% after 48 hours, and the survival rate in the 15 ml / kg toad venom group was 70% after 36 hours, with no further mortality. The survival rate of mice in the control group was 100%. This indicates that toad venom has a good therapeutic effect on MRSA infection in the lungs of mice, and the survival rate of mice continuously increases with increasing dosage.

[0083] 4.1.2 Toad venom reduced the bacterial load in the lungs of MRSA pneumonia model mice. The bacterial load in mouse lungs is a good indicator of infection status and the effectiveness of drug treatment. By reducing the bacterial dose, mice were sacrificed after 48 hours, their lungs were removed, ground, and colony counting was performed on the lung tissue. Figure 31 As shown, the bacterial load in the lung tissue of the USA300 mouse infection group was as high as 9.63 log 10 The bacterial load in the lung tissue of the toad venom treatment group (CFU / g, 5ml / kg toad venom) was 7.2 log. 10 CFU / g, bacterial load at 10ml / kg is 6.56 log.10 CFU / g, bacterial load at 15 ml / kg is 5.97 log. 10 The results showed that toad venom treatment could reduce the bacterial load in the lungs of MRSA-infected mice. The effect of reducing the bacterial load in the lungs of mice was more obvious with the increase of toad venom dosage, indicating that toad venom has a good therapeutic effect on MRSA-infected mice.

[0084] 4.1.3 Effects of toad venom on lung tissue pathological changes in MRSA pneumonia model mice Mice in different experimental groups were sacrificed, their lungs were harvested, and the lungs were washed with physiological saline, photographed, and subjected to HE staining to observe the pathological state of the mouse lung tissue. Figure 32 As shown, the lungs of mice in the USA300-infected group were darker in color, more congested, and showed increased inflammatory cell infiltration compared to the normal control group. In the toad venom treatment group, the lung color gradually recovered, and inflammatory cell infiltration was significantly reduced, with the effect becoming more pronounced with increasing dosage.

[0085] 4.2 Treatment of MRSA pulmonary infection model mice with toad venom combined with penicillin G 4.2.1 Toad venom combined with penicillin G improved the survival rate of mice with MRSA lung infection. Mice with MRSA lung infection were treated with a low dose of 5 ml / kg toad venom combined with penicillin G. An in vivo anti-infection experiment was conducted to observe whether it had a synergistic antibacterial effect in vivo. Figure 33 As shown, the survival rate of mice treated with USA300 was only 10% after 48 hours, while the survival rate of mice treated with toad venom (5 ml / kg) was 30% after 48 hours, and the survival rate of mice treated with penicillin G was 10% after 48 hours. In the toad venom combined with penicillin G treatment group, mice no longer died after 24 hours, with a final survival rate of 90%, while the survival rate of mice in the control group was 100%. This indicates that the combined use of toad venom and penicillin G has a good synergistic therapeutic effect on MRSA infection in the lungs of mice, making previously resistant to penicillin G resensitive.

[0086] 4.2.2 Toad venom combined with penicillin G reduced the bacterial load in the lungs of MRSA pneumonia model mice. like Figure 34 As shown, the bacterial load in the lung tissue of the USA300 mouse infection group was as high as 9.21 log 10 The bacterial load in the lung tissue of the toad venom treatment group (CFU / g, 5ml / kg toad venom) was 7.00 log. 10 CFU / g, the bacterial load in the penicillin G treatment group was 8.23 ​​log. 10 The bacterial load in the toad venom combined with penicillin G treatment group was 4.97 log CFU / g. 10The results showed that toad venom combined with penicillin G could significantly reduce the bacterial load in the lungs of MRSA-infected mice, and had a good synergistic effect on MRSA-infected mice.

[0087] 4.2.3 Effects of toad venom combined with penicillin G on lung tissue pathological changes in MRSA pneumonia model mice like Figure 35 As shown, the lungs of mice in the USA300-infected group became darker in color, with severe congestion and increased inflammatory cell infiltration. The penicillin G-treated group also showed darker lung color and increased inflammatory cell infiltration. The lung pathology in the toad venom-treated group showed slight improvement. The lung tissue in the toad venom combined with penicillin G-treated group showed significant improvement in color and a significant reduction in inflammatory cell infiltration. These results indicate that the combined use of the two drugs has a good therapeutic effect.

[0088] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of toad venom extract in the preparation of therapeutic drugs for methicillin-resistant Staphylococcus aureus infections.

2. The application according to claim 1, characterized in that, The toad venom extract includes toad venom injection.

3. Application of toad venom extract in the preparation of β-lactam antibiotic sensitizers.

4. The application according to claim 3, characterized in that, The β-lactam antibiotics include penicillin antibiotics and cephalosporin antibiotics.

5. Application of toad venom extract in combination with antibiotics in the preparation of drugs for the treatment of methicillin-resistant Staphylococcus aureus infection.

6. The application according to claim 5, characterized in that, The toad venom extract includes toad venom injection.

7. The application according to claim 5, characterized in that, The β-lactam antibiotics include penicillin antibiotics and cephalosporin antibiotics.

8. The application according to claim 7, characterized in that, The penicillin antibiotics include penicillin G and oxacillin; the cephalosporin antibiotics include ceftriaxone sodium.

9. A treatment drug for methicillin-resistant Staphylococcus aureus (MRSA) infections, characterized in that, This includes toad venom extract and pharmaceutically acceptable excipients.

10. The therapeutic drug according to claim 9, characterized in that, It also includes β-lactam antibiotics.

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