Use of an antimicrobial peptide in preparing a drug for resisting Staphylococcus aureus infection

Abstract

The present invention provides a use of an antimicrobial peptide in the preparation of a drug for resisting Staphylococcus aureus infection. In a pneumonia mouse model infected with Staphylococcus aureus, the therapeutic effect of GHb3K and GHbK4R is better than that of vancomycin, the inflammatory response can be significantly reduced, and the in vivo toxicity is lower than that of vancomycin. GHb3K and GHbR can significantly promote the healing of chronic infectious wounds of methicillin-resistant Staphylococcus aureus (MRSA), and the therapeutic effect is better than that of mupirocin ointment. The antimicrobial peptide provided by the present invention has the potential to be developed into a drug for treating pneumonia infected with Staphylococcus aureus and a drug for treating chronic wounds infected with MRSA.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the use of an antimicrobial peptide in preparing a drug for resisting Staphylococcus aureus infection. Background Art

[0002] As bacteria continue to develop resistance to traditional antibiotics, the demand for new antimicrobial drugs is becoming increasingly urgent. Antimicrobial peptides are an important part of the natural immune system of organisms and often have broad-spectrum antimicrobial effects. Most cationic antimicrobial peptides tend to form amphiphilic structures, selectively bind to microbial cell membranes through electrostatic adsorption, attach or insert into the cytoplasmic membrane, destroy the integrity of the membrane structure, cause leakage of contents, and effectively kill pathogenic microorganisms. Compared with traditional antibiotics, the special mechanism of action of antimicrobial peptides makes it difficult for bacteria to develop resistance to them.

[0003] Staphylococcus aureus (S. aureus), also known as "golden Staphylococcus", is widely present in the natural environment and is distributed on the skin and mucous membrane surfaces of about 60% of adults. It is one of the most common pathogens from hospitals to communities and can cause bacterial pneumonia, skin wound infections, etc. (Sakr, A.; Brégeon, F.; Mège, JL; et al. Staphylococcus aureus Nasal Colonization: An Update on Mechanisms, Epidemiology, Risk Factors, and Subsequent Infections. Frontiers inmicrobiology, 2018, 9: 2419.). Community-acquired pneumonia caused by Staphylococcus aureus is the main cause of death in children and often occurs in people over 80 years old (Self, WH; Wunderink, RG;

[0004] Williams, DJ; et al. Staphylococcus aureus Community-acquired Pneumonia: Prevalence, Clinical Characteristics, and Outcomes. Clin infect dis, 2016, 63(3): 300-309.). To make matters worse, the emergence of methicillin-resistant S. aureus (MRSA) has further increased the difficulty of effectively treating S. aureus infectious diseases. S. aureus can adhere to the surfaces of various medical devices and implanted devices, such as catheters, artificial heart valves, central venous catheters, pacemakers, etc., and can form biofilms in body tissues (Otto, M. Staphylococcal Biofilms. Microbiol Spectr 2018, 6(4): 10.1128 / microbiolspec. GPP3-0023-2018.). The bacteria in the biofilm are metabolically inactive, forming antibiotic-resistant persisters, making biofilm infections extremely difficult to treat, often leading to chronic or recurrent infections that usually require long-term antibiotic treatment or removal of implanted devices, placing a heavy burden on patients, families, and society, and making the problem of bacterial resistance more serious. In 2017, the World Health Organization (WHO) released a list of bacteria that urgently need new antibiotics, including MRSA, which is the main cause of death in patients infected with antibiotic-resistant bacteria (Tacconelli, E.; Carrara, E.; Savoldi, A.; et al. WHO Pathogens Priority List Working Group. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. The Lancet. Infectious diseases, 2018, 18(3): 318-327.).

[0005] Therefore, it is of great significance to develop new antimicrobial agents that can quickly kill bacteria and remove bacterial biofilms to address the antibiotic resistance crisis. Summary of the invention

[0006] Based on the above technical deficiencies, the present invention provides a use of an antimicrobial peptide in the preparation of a drug for resisting Staphylococcus aureus infection.

[0007] In order to achieve the technical purpose of the present invention, the technical solution adopted by the present invention is:

[0008] A use of an antimicrobial peptide in preparing a drug for resisting Staphylococcus aureus infection, wherein the amino acid sequence of the antimicrobial peptide is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0009] Preferably, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

[0010] Preferably, the antimicrobial peptide is synthesized by N-9-fluorenylmethoxycarbonyl (Fmoc) solid phase synthesis method.

[0011] More preferably, the C-terminus of the antimicrobial peptide is amidated and purified by reverse-phase high performance liquid chromatography to a purity of ≥95%.

[0012] Preferably, the anti-Staphylococcus aureus infection drug includes a drug for treating pneumonia caused by Staphylococcus aureus infection and a drug for treating chronic wounds infected by methicillin-resistant Staphylococcus aureus.

[0013] The beneficial effects of the present invention are:

[0014] The present invention designs and synthesizes a series of antimicrobial peptides, including GHbR (amino acid sequence as shown in SEQ ID NO.1), GHb3K (amino acid sequence as shown in SEQ ID NO.2) and GHbK4R (amino acid sequence as shown in SEQ ID NO.3), and studies the anti-biofilm activity, antibacterial activity and mechanism and in vivo anti-infection effect of these antimicrobial peptides on Staphylococcus aureus. In a pneumonia mouse model infected with Staphylococcus aureus, the therapeutic effect of GHb3K and GHbK4R is better than that of vancomycin, and the inflammatory response can be significantly reduced, and the in vivo toxicity is lower than that of vancomycin. GHb3K and GHbR can significantly promote the healing of chronic infectious wounds of methicillin-resistant Staphylococcus aureus MRSA, and the therapeutic effect is better than that of mupirocin ointment. Among them, GHb3K also has the following outstanding advantages: low cytotoxicity; small molecule, easy to absorb, low allergy; high stability, long-term activity at room temperature, easy to store; simple preparation process, low production cost, etc. It has the potential to be developed into a drug for treating pneumonia caused by Staphylococcus aureus infection and a drug for treating chronic wounds infected by MRSA. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1a-Figure 1c The bactericidal kinetics of the antimicrobial peptides of the present invention against Staphylococcus aureus are shown.

[0016] Figure 2 The results show that the antimicrobial peptides of the present invention and vancomycin can induce drug resistance in Staphylococcus aureus.

[0017] Figure 3aDemonstrating the ability of the antimicrobial peptides of the present invention to inhibit biofilm formation; Figure 3b The antimicrobial peptides of the present invention are shown to have the ability to destroy mature biofilms.

[0018] Figure 4a-4c The results show that the antimicrobial peptides of the present invention affect the permeability of Staphylococcus aureus cell membrane.

[0019] Figure 5 Transmission electron micrograph showing Staphylococcus aureus treated with 12.5 μM antimicrobial peptide for 1 h.

[0020] Figure 6a-6c The cytotoxicity of the antimicrobial peptides of the present invention was demonstrated.

[0021] Figure 7a and Figure 7b The figures show the bacterial load in the organs of mice infected with Staphylococcus aureus pneumonia after treatment with the antimicrobial peptides of the present invention and vancomycin.

[0022] Figure 8 The levels of IL-1β, TNF-α, MPO and MIP cytokines in the lungs and livers of mice with pneumonia infected with Staphylococcus aureus after treatment with the antimicrobial peptides of the present invention and vancomycin were shown.

[0023] Figure 9a and Figure 9b Shown are the changes in body weight of healthy mice injected with saline, GHb3K, GHbK4R, and vancomycin.

[0024] Fig.10a and Fig.10b It was shown that GHbR and GHb3K promoted wound healing in mice infected with MRSA.

[0025] Fig.11a and Fig.11b Shown are the bacterial loads in MRSA-infected wounds of mice after GHbR and GHb3K treatment.

[0026] Fig.12 The effects of GHbR and GHb3K treatment on inflammatory cytokine production in MRSA-infected wounds of mice were shown. DETAILED DESCRIPTION

[0027] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0028] Example

[0029] The Staphylococcus aureus (ATCC25923) and methicillin-resistant Staphylococcus aureus (ATCC43300) used in the present invention were purchased from Guangdong Microbiological Culture Collection Center. Other clinically isolated methicillin-resistant Staphylococcus aureus strains (MRSA1-MRSA4) were donated by Professor Yang Ke of Guangxi University of Chinese Medicine and have been identified by biochemical methods and 16s rDNA sequencing (GenBank database, accession numbers OP824645-824648).

[0030] Temporin-GHb (GHb) is an antimicrobial peptide we cloned from the skin of frogs, but its anti-Staphylococcus aureus activity is not high. By comparing and analyzing the sequences of 110 antimicrobial peptides of the same family, it was found that the frequency of the basic amino acid lysine (Lys, K) was high, at 6.97%, while arginine (Arg, R) and histidine (His, H) were relatively low, at 1.14% and 0.9%, respectively. In the amino acid sequence of GHb, the basic amino acid is H. Different basic amino acids have different effects on the antimicrobial activity and cytotoxicity of antimicrobial peptides. Our study found that the addition of R to antimicrobial peptides often leads to increased cytotoxicity while enhancing the antimicrobial activity. Therefore, in the modification of GHb, in addition to using R to replace H, K was used to replace H, and the strategy of replacing R and K at the same time was used to obtain antimicrobial peptides with good activity and low toxicity.

[0031] Furthermore, we used GHb as a template and combined the results of big data analysis of antimicrobial peptides to design and synthesize the following three antimicrobial peptides, namely GHbR, GHb3K and GHbK4R. They are composed of 13 amino acids and carry 1-3 positive charges under physiological conditions. The amino acid sequences are:

[0032] GHbR: FIRRIIGALGRLF-NH2 (SEQ ID NO.1)

[0033] GHb3K: FIKHIIGALGHLF-NH2 (SEQ ID NO.2)

[0034] GHbK4R: FIKRIIGALGKLF-NH2 (SEQ ID NO.3)

[0035] The antimicrobial peptide was synthesized by N-9-fluorenylmethoxycarbonyl (Fmoc) solid phase synthesis method. A preparative liquid chromatograph (LC6000) was used for preparation, and a high performance liquid chromatograph (LC3000) was used for analysis. The C-terminus of the peptide was amidated and purified by reverse phase high performance liquid chromatography with a purity of ≥95%. The antimicrobial peptide was identified by electrospray mass spectrometry. No further limitation is made in the present invention.

[0036] Next, the anti-biofilm activity, antibacterial activity and mechanism, and in vivo anti-infection effects of these three antimicrobial peptides against Staphylococcus aureus were studied.

[0037] Test Case

[0038] 1. Antibacterial test

[0039] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antimicrobial peptides were tested using the two-fold broth microdilution method. A single colony was inoculated into tryptone soy broth (TSB) and cultured at 37°C for 8-12 hours. Bacteria that had grown to the logarithmic phase were diluted to 1×10 6 CFU / mL. The antimicrobial peptide was diluted two-fold in a 96-well plate to different concentrations, and an equal volume of bacterial suspension was added and incubated at 37°C for 18-24h. The wells without antimicrobial peptide were used as negative control wells, and vancomycin was used as positive control. The antimicrobial peptide concentration corresponding to the wells with no bacterial growth observed by naked eye was defined as MIC. When determining MBC, 10μL of bacterial suspension was taken from the corresponding MIC wells on the 96-well plate where MIC was measured, or from the wells above MIC, and spread on TSB agar plates (TSA) and cultured at 37°C for 24h. The antimicrobial peptide concentration at which no bacterial growth was observed was the MBC.

[0040] The results are shown in Table 1. The antibacterial activity of GHb is weak, while the other three antimicrobial peptides show extremely strong antibacterial activity, with MICs of 3.1 μM against standard strains of Staphylococcus aureus and MRSA. They also show activity against clinically isolated drug-resistant bacteria MRSA1-MRSA4, with MICs of 1.6-6.2 μM and MBCs of 3.1-25 μM.

[0041] Table 1 MIC of antimicrobial peptides against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus

[0042]

[0043]

[0044] Note: SA is Staphylococcus aureus (ATCC25923); MRSA is methicillin-resistant Staphylococcus aureus (ATCC43300); MRSA1-MRSA4 are clinically isolated methicillin-resistant Staphylococcus aureus.

[0045] 2. Bactericidal kinetics determination

[0046] The antimicrobial peptide was diluted in EP tubes by twofold gradients, and an equal volume of diluted bacterial solution was added to make the final bacterial concentration 1×10 6CFU / mL, the final concentration of peptide was 1 / 2×MIC, 1×MIC, 2×MIC, 4×MIC, and the bacterial solution not treated with peptide was used as negative control (Control). Incubate at 37°C, and take out appropriate amount of bacterial solution after 15, 30, 60, 90, 120, and 180 min, dilute to appropriate concentration with TSB liquid medium, and take 50 μL to apply on TSA plate. Incubate at 37°C for 24 h, count single colonies, and draw a bactericidal curve.

[0047] The bactericidal curve results for Staphylococcus aureus showed that ( Figure 1a-Figure 1c ), GHbR and GHbK4R had the most obvious bactericidal effects, killing bacteria within 15 min at a concentration of 2×MIC, and GHb3K killed bacteria within 60 min at a concentration of 4×MIC.

[0048] 3. In vitro drug resistance induction experiment

[0049] Take the S. aureus suspension and inoculate it in fresh TSB liquid medium containing 1 / 2MIC antimicrobial peptide, and culture it at 37℃ for 24h. Take an appropriate amount of the bacterial suspension from the previous day and re-inoculate it into fresh TSB liquid medium containing 1 / 2MIC antimicrobial peptide every day to continue culturing. The S. aureus treated with TSB liquid medium is used as a negative control, and vancomycin is used as a positive control. Repeat the operation continuously for 50 days. Determine the MIC of antimicrobial peptide against S. aureus every 5 days.

[0050] The results showed that after 50 generations of continuous passage, the MIC value of the antimicrobial peptide against S. aureus remained stable at 3.1 μM ( Figure 2 ). However, S. aureus can quickly develop resistance to vancomycin, and its MIC value increased to 32 times on the 45th day. The results showed that S. aureus is difficult to develop resistance to GHbR, GHb3K, and GHbK4R.

[0051] 4. Stability Study

[0052] The effects of different salts, temperatures, pH values ​​and storage times on the antimicrobial activity of antimicrobial peptides were evaluated. The antimicrobial peptides were dissolved to 2 mM in sterile water containing different concentrations of salt (3.0 mM, 4.5 mM, 6.0 mM KCl, 0.5 mM, 1.0 mM, 1.5 mM MgCl2, 0.05 M, 0.15 M, 0.3 M NaCl and 2.0 mM, 2.5 mM, 3.0 mM CaCl2) and incubated at 37 °C for 24 h.

[0053] The antimicrobial peptide (2 mM) solutions were treated at different temperatures (25°C, 40°C, 70°C and 100°C) for 30 min.

[0054] The antimicrobial peptide solution was incubated for 30 min in solutions with pH values ​​of 7 and 9, respectively. The antimicrobial peptide solution was stored at 25°C, 4°C and -20°C for 30 days, 60 days and 90 days, respectively. The MIC of the antimicrobial peptide after treatment was determined according to the above method.

[0055] The effects of storage temperature and time on the antimicrobial activity of antimicrobial peptides (Table 2) showed that the three antimicrobial peptides maintained good stability within 90 days and the MIC did not change when stored at 25°C, 4°C, and -20°C for 30 days, 60 days, and 90 days, respectively, compared with the untreated group. After the antimicrobial peptides were treated at 25-100°C for 30 minutes, the MIC was still 3.1 μM. The antimicrobial peptides maintained good stability in an environment of pH 7-9 (Table 3).

[0056] Table 2 Effects of storage time and temperature on the antimicrobial activity of antimicrobial peptides

[0057]

[0058] Table 3 Effects of temperature and pH on the antimicrobial activity of antimicrobial peptides

[0059]

[0060] Table 4 Effect of cations on the antimicrobial activity of antimicrobial peptides

[0061]

[0062] Moreover, in the presence of cations, there was no adverse effect on the antibacterial activity of antimicrobial peptides and it could even promote the antibacterial activity of GHbR (Table 4).

[0063] The above results show that the three antimicrobial peptides have excellent stability and can remain stable under different storage conditions and body fluid environments. 5. Inhibitory effect of antimicrobial peptides on the formation of Staphylococcus aureus biofilm and the effect of destroying mature biofilm

[0064] The antimicrobial peptide anti-Staphylococcus aureus biofilm effect was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The antimicrobial peptide was serially diluted two-fold (final concentration 0.4-3.1 μM) to 100 μL in a 96-well plate, and an equal volume of bacterial solution (final concentration 1×10 6CFU / mL), and culture at 37°C for 24h. Discard the supernatant, wash three times with phosphate buffered saline (PBS), air-dry at room temperature, add 95μL PBS and 5μL MTT (5mg / mL) to each well, incubate at 37°C in the dark for 2-4h, and discard the supernatant. Add 150μL dimethyl sulfoxide (DMSO) to dissolve the blue-purple crystalline formazan, incubate at room temperature in the dark for 20min, and measure the absorbance at 570nm (MultiskanSpectrum, BioTek, Winooski, VT, USA). When determining the clearance effect of antimicrobial peptides on mature biofilm of Staphylococcus aureus, 200μL of bacteria were inoculated into a 96-well plate (1×10 6 CFU / mL), cultured at 37°C for 24 h, washed 3 times with PBS, air-dried at room temperature, added with 200 μL of 3.1-25 μM antimicrobial peptides, cultured at 37°C for 24 h, and the rest of the steps were the same as above. The bacterial solution not treated with antimicrobial peptides was used as a negative control (Control).

[0065] The results showed that the antimicrobial peptides have the ability to inhibit the formation of biofilms by Staphylococcus aureus and remove mature biofilms, and the activity is concentration-dependent. GHbR has the ability to inhibit biofilm formation, inhibiting 87% of biofilms at 3.1 μM, and GHb3K and GHbK4R can inhibit about 65% of biofilm formation at the same concentration ( Figure 3a ). Antimicrobial peptides also have the activity of clearing mature biofilms. GHbR and GHbK4R have the ability to clear mature biofilms. At 25 μM, they can clear 80% of biofilms. GHb3K can clear 70% of biofilms at 25 μM ( Figure 3b ). However, at 25 μM, the inhibition rate and clearance rate of GHb were only about 20%.

[0066] Therefore, compared with GHb, the other three antimicrobial peptides showed stronger ability to inhibit the formation of S. aureus biofilm and clear mature biofilm.

[0067] 6. PI permeation test to detect the effect of antimicrobial peptides on bacterial cell membrane

[0068] The propidium iodide (PI) uptake experiment was used to evaluate the membrane permeability of antimicrobial peptides against Staphylococcus aureus. When the cell membrane is damaged and PI penetrates into the cell and binds to DNA, PI emits strong fluorescence, indicating that the integrity of the bacterial cell membrane is destroyed and the permeability increases.

[0069] The antimicrobial peptide was diluted twice in a 96-well plate (100 μL, 3.2-25 μM), the S. aureus was washed 3 times with PBS and diluted, and 92 μL of bacterial solution (final concentration 1×10 8 CFU / mL) and 8 μL PI (final concentration 20 μM) were mixed. The determination was performed using a multifunctional microplate reader at an excitation wavelength of 560 nm and an emission wavelength of 620 nm (Spark, Tecan, Switzerland). The program was set at 37°C, and the measurement was performed every 5 minutes for a total of 2 hours. The bacterial solution not treated with antimicrobial peptides was used as a negative control (Control).

[0070] like Figure 4a-4c As shown, when S. aureus was incubated with antimicrobial peptides, the fluorescence values ​​of GHbR, GHb3K and GHbK4R increased in a concentration-dependent manner as the peptide concentration increased. When S. aureus was treated with 12.5 μM GHbR, GHb3K and GHbK4R, the maximum fluorescence value was reached at 5 min. The results showed that GHbR, GHb3K and GHbK4R exerted their antibacterial effects by increasing the permeability of bacterial cell membranes and destroying bacterial cell membranes.

[0071] VII. Transmission electron microscopy observation of bacterial morphology treated with antimicrobial peptides

[0072] The S. aureus in the logarithmic growth phase was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, the cells were washed with PBS and resuspended, and the antimicrobial peptide (final concentration 12.5 μM) was mixed with an equal volume of bacterial solution (final concentration 1×10 9 CFU / mL), with no peptide added as a blank control (Control), incubated at 37°C for 1 hour. Centrifuged at 8000rpm for 5 minutes, discarded the supernatant, washed with PBS, fixed with 2.5% glutaraldehyde for 4 hours, centrifuged, washed with PBS three times, dehydrated with 30%, 50%, 70%, and 100% ethanol in sequence for 10 minutes, resuspended the bacteria in 80μL anhydrous ethanol, and dropped 10μL of the bacterial solution onto a copper grid, stained with 2% phosphomolybdic acid, and observed under a transmission electron microscope.

[0073] like Figure 5 As shown in the figure, the bacterial cell structure of the blank control group (Control) was intact and the cells were regular and round. The shape of Staphylococcus aureus treated with 12.5 μM GHbR, GHb3K and GHbK4R changed significantly, the cells shrank, the cell membrane ruptured, and the contents leaked. This further confirmed that the antimicrobial peptides targeted the bacterial cell membrane, causing the cell membrane to rupture and the contents to leak out, resulting in death.

[0074] 8. Cytotoxicity

[0075] Human alveolar epithelial cells (A549), human breast cancer cells (MCF-7), and human keratinocytes (HaCaT) were cultured in high-glucose DMEM (containing 1% penicillin and streptomycin) containing 10% fetal bovine serum (FBS) in a 37°C 5% CO2 incubator. When the cell coverage reached 80%-90%, the cells were digested with trypsin and diluted to 1×10 5cell / mL, add 100 μL to each well of a 96-well plate and culture for 24 hours. Discard the original culture medium, add 3.1-200 μM peptide diluted with DMEM containing 2% FBS to each well, cells not treated with peptides are used as control wells (Control), and blank wells without peptides and cells are used. Continue to culture for 24 hours, discard the supernatant, wash 3 times with PBS, add 100 μL culture medium, 10 μL CCK8 solution, and incubate for 2-4 hours. Measure the absorbance at 450 nm.

[0076] Cytotoxicity of antimicrobial peptides Figure 6a-6c As shown. At a concentration of 200 μM, GHb3K had no cytotoxicity to HaCaT, MCF-7 and A549 cells. GHbR and GHbK4R had certain toxicity to cells, with half inhibitory concentrations (IC50) of 15.4 and 18.5 for HaCaT cells and 13.6 and 30.4 μM for A549, respectively. The results showed that GHb3K had low toxicity among the three antimicrobial peptides.

[0077] IX. Construction and treatment of pneumonia model in mice infected with Staphylococcus aureus

[0078] Seventy-two healthy male BALB / cJ mice (20-24 g) aged 6-8 weeks were randomly divided into 9 groups, with 8 mice in each group, of which 1 group of healthy mice served as the blank group (Control). The remaining 8 groups of mice were anesthetized with pentobarbital and inoculated intranasally with 30 μL of Staphylococcus aureus (5×10 9 CFU / mL, keep the mice vertically inverted for 30 seconds, and inject GHb3K or GHbK4R (5, 10, 15 mg / Kg) intraperitoneally 2 hours after infection, and vancomycin is used as a positive control (10 mg / Kg). The model group (Model) and the blank group (Control) mice were intraperitoneally injected with 100 μL of normal saline. The drug was administered once every 8 hours. After 3 doses, the mice were anesthetized, blood was collected from the eyeballs, the mice were killed, and the organs (lungs, heart, liver, kidneys, spleen) were collected, and the bacterial load of the organs was recorded.

[0079] Two hours after intranasal instillation of bacteria, the lung tissue of the model group (Model) became larger, while the lung tissue of the blank group (Control) did not change. HE staining results showed that the alveolar interstitial congestion and edema, pulmonary vascular dilation, and a large number of inflammatory cells infiltrated the lung tissue of the model group mice. The number of bacteria in the lung tissue of the model group mice counted on the mannitol sodium chloride agar plate was greater than 10 6 CFU / g, indicating that the mouse pneumonia model was successfully established.

[0080] The number of bacteria in the lung tissue of mice was counted after treatment ( Figure 7a and Figure 7b), compared with the model group, the bacterial load in the lung tissue of the treatment group was reduced. Compared with the vancomycin treatment group, the low-dose antimicrobial peptide group was equivalent to vancomycin, and the bacterial load in the medium and high-dose groups was significantly lower than that in the vancomycin group. After vancomycin treatment, the number of Staphylococcus aureus in the heart, liver, spleen, lung, and kidney decreased by 16%, 21%, 32.6%, 23%, and 15%, respectively. The number of Staphylococcus aureus in tissues decreased in a concentration-dependent manner after GHb3K and GHbK4R treatment. In the high-dose GHb3K group, the number of Staphylococcus aureus decreased by 48%, 20%, 50%, 49%, and 15%, respectively; in the high-dose GHbK4R group, the number of Staphylococcus aureus decreased by 31%, 20%, 43%, 46.5%, and 46%, respectively. In the high-dose vancomycin, GHb3K, and GHbK4R groups, the bacterial load in the lungs of mice decreased to 7.8×10 7 , 5.3×10 5 and 5.4×10 5 CFU / g. Therefore, the antimicrobial peptides GHb3K and GHbK4R were more effective than vancomycin in treating bacterial pneumonia caused by S. aureus in mice.

[0081] Lung and liver tissues were taken and the effects on cytokines IL-1β, TNF-α, MIP-2, and MPO were determined using ELISA kits. Figure 8 The results showed that after infection with Staphylococcus aureus, the levels of IL-1β, TNF-α, MIP-2 and MPO in lung and liver tissues increased significantly, and the levels of these inflammatory factors were significantly reduced after antimicrobial peptide treatment.

[0082] 10. In vivo toxicity test of antimicrobial peptides

[0083] To evaluate the in vivo toxicity of GHb3K and GHbK4R, healthy BALB / cJ mice were divided into 4 groups and injected with saline (Control), 15mg / kg GHb3K, 15mg / kg GHbK4R, and 10mg / kg vancomycin, respectively. The drugs were injected intraperitoneally for 8 days, and the weight of the mice was recorded every day. It was found that there was no significant difference in the weight of the mice among the 4 groups ( Figure 9a and Figure 9b). Eight days after intraperitoneal injection of the drug, the blood indicators of the mice, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), were measured, and there were no significant differences between the different groups. This shows that GHb3K and GHbK4R have no obvious hepatotoxicity. The pathological sections of the mice were evaluated, and it was found that after vancomycin treatment, histopathological observations showed that the liver and kidneys were toxic, the hepatic cord structure in the liver was disordered, and there was inflammatory cell infiltration. The number of inflammatory cells in the renal corpuscles in the kidneys increased, and the renal capsule cavity became narrowed. No necrosis, fibrosis, and inflammatory lesions were observed in the heart, liver, spleen, lungs, and kidneys of mice treated with GHb3K and GHbK4R, indicating that GHb3K and GHbK4R are not toxic in vivo at the doses used.

[0084] 11. Construction and treatment of MRSA-infected skin wound mouse model

[0085] Adult healthy female Kunming mice (18-22 g) were raised normally for 7 days, and the hair on the back of the mice was shaved with an electric shaver, washed with saline, dried, and disinfected with 75% alcohol. After intraperitoneal injection of pentobarbital anesthesia, a circular full-thickness skin wound with a diameter of 10 mm was cut in the epidermis of the mice with a skin punch, and disinfected with iodine. 0.5 mL of MRSA suspension (1×10 8 CFU / mL), and after the bacterial solution was absorbed, the mice were fed separately in cages. 64 mice were randomly selected each time, with 8 mice in each group, and divided into 8 groups, namely, negative group (Model), positive group (Mupirocin), GHbR high, medium and low dose (12.5μM, 25μM, 50μM) treatment group, GHb3K high, medium and low dose (12.5μM, 25μM, 50μM) treatment group. A blank group (Control) was also set up, that is, a full-thickness skin wound mouse group that was not infected with bacteria. 24h after infection, treatment began, once a day, the negative group (Model) was smeared with 50μL of normal saline, the positive group was smeared with mupirocin ointment (according to the instructions), the treatment group was smeared with 50μL of antimicrobial peptide solution of different concentrations, and the blank group was smeared with 50μL of normal saline. Take pictures once on the 0th day, the 3rd day, the 6th day, and the 10th day. After the treatment, the mice were killed and the skin was disinfected with alcohol cotton balls. 0.05-0.1 g of skin from the wound of each group was taken and cut into pieces. 1 mL of sterile saline was added to an EP tube for every 0.1 g of pathological tissue and homogenized. The homogenate was diluted to an appropriate concentration and spread on a blood agar plate. The agar plate was cultured in a 37°C incubator for 24 h and the number of colonies was recorded.

[0086] The high, medium and low dose groups of GHbR and GHb3K treatment could significantly accelerate the repair of chronic infectious wounds in mice ( Fig.10a , Fig.10bThe wound repair degree of the high-dose antimicrobial peptide group was better than that of the positive control drug mupirocin ointment. After 10 days of treatment, the number of bacteria in the wound was significantly reduced, and the low-dose antimicrobial peptide group was equivalent to the positive control (Mupirocin) ( Fig.11a and Fig.11b On the third day of treatment, TNF-α, IL-1β and IL-6 in the high-dose antimicrobial peptide group were significantly reduced, TNF-α basically returned to the blank control (Control) level, and IL-1β and IL-6 levels were even lower than the blank control ( Fig.12 ). The reduction of the three inflammatory factors in the positive control group was lower than that in the antimicrobial peptide treatment group. After 10 days of treatment, the inflammatory factors in the antimicrobial peptide and drug treatment groups basically returned to the blank control level. The results showed that the therapeutic effect of GHbR and GHb3K on MRSA-infected wounds was better than that of the positive control drug mupirocin ointment.

[0087] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Use of an antimicrobial peptide in the preparation of a drug for resisting Staphylococcus aureus infection, wherein the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

2. Use of an antimicrobial peptide according to claim 1 in the preparation of a drug for resisting Staphylococcus aureus infection, characterized in that: The Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

3. Use of an antimicrobial peptide according to claim 1 in the preparation of a drug for resisting Staphylococcus aureus infection, characterized in that: The antimicrobial peptide is synthesized by N-9-fluorenylmethoxycarbonyl solid phase synthesis method.

4. Use of an antimicrobial peptide according to claim 1 or 3 in the preparation of a drug for resisting Staphylococcus aureus infection, characterized in that: The C-terminus of the antimicrobial peptide is amidated and purified by reverse phase high performance liquid chromatography with a purity of ≥95%.

5. Use of an antimicrobial peptide according to claim 1 or 2 in the preparation of a drug for resisting Staphylococcus aureus infection, characterized in that: The anti-Staphylococcus aureus infection medicine includes a medicine for treating pneumonia infected by Staphylococcus aureus and a medicine for treating chronic wounds infected by methicillin-resistant Staphylococcus aureus.

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