Preparation method and application of a fungal defensin-derived cyclic peptide and its thermosensitive preparation

Abstract

The present invention discloses a preparation method and application of a fungal defensin-derived cyclic peptide and its thermosensitive preparation, belonging to the field of biomedicine. The linear amino acid sequence of the fungal defensin-derived cyclic peptide of the present invention is ACNGVRIRQGNPKGGRKLPKDG RPGANIRACYPRKR, and the cysteines at the 2nd and 31st positions are connected by a disulfide bond to form a cyclic structure. This derived cyclic peptide first prepares a linear peptide segment through solid-phase peptide synthesis, and then synthesizes a cyclic peptide structure containing a pair of disulfide bonds in the molecule through the sulfhydryl groups on the side chains of two cysteine residues in the molecule. This derived cyclic peptide has low hemolytic activity and simultaneously has triple biological activities of antibacterial, anti-inflammatory, and promoting angiogenesis. Its artificial preparation and purification are convenient, showing the value of in-depth research and development as a candidate drug and the potential for industrialization, and having applications in preparing antibacterial drugs, drugs for inhibiting inflammation, and drugs for promoting angiogenesis or wound repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a preparation method and application of a fungal defensin-derived cyclic peptide having antibacterial, anti-inflammatory and angiogenesis-promoting functions and a thermosensitive preparation thereof. Background Art

[0002] Fungi produce a series of active substances during the infection process. On the one hand, they need to overcome the host's immune system clearance, and on the other hand, they need to absorb enough substances in a nutrient-poor environment. As a protective mechanism of the body, the inflammatory response plays an important role in immunity. Therefore, when fungi infect the host, it is necessary to suppress the inflammatory response and kill other microorganisms in the living environment.

[0003] Fungal defensins are small cysteine-rich cationic polypeptides secreted and expressed by fungi, which have antimicrobial activity, immune signaling activity, or both. They are usually composed of 38-57 amino acids, contain highly conserved intramolecular disulfide bonds, and form a specific spatial structure. The sequence of typical fungal defensins and the pairing mode of intramolecular disulfide bonds ( Figure 1 ) and spatial structure ( Figure 2 ) is shown below.

[0004] First, the antimicrobial activity of defensins makes them attractive novel therapeutic agents. Indeed, their specific lipid targeting and membrane permeability activities have the potential to address some of the key issues in current antimicrobial therapy, such as drug / antibiotic resistance and off-target effects. The relative selectivity of defensins in their killing of microorganisms is based on the composition of the microbial membrane, which, compared with host cells, lacks cholesterol and is relatively rich in negatively charged phospholipids. It is therefore widely believed that death is a consequence of microbial membrane disruption. The polar topology of defensins, with spatially separated charged and hydrophobic regions, enables them to insert into phospholipid membranes, with their hydrophobic regions buried inside the lipid membrane and their charged regions (mostly cationic) interacting with anionic phospholipid head groups and water. Subsequently, some defensins may aggregate to form “channel-like” pores; others may bind and cover the microbial membrane in a “carpet-like” manner. The end result is a disruption of membrane integrity and function, ultimately leading to the lysis of the microorganism.

[0005] Secondly, defensins also have immunomodulatory effects on the host. Defensin HNPs can enhance the phagocytosis of mouse macrophages, and defensin hBD-2 induces the activation and degranulation of mast cells, resulting in the release of histamine and prostaglandin D2 (PGD2). HNP1-3 can stimulate bronchial epithelial cells to increase interleukin-8 (IL-8) gene transcription and IL-8 production. Since the products of mast cell granules increase the influx of neutrophils, and IL-8 is a potent neutrophil chemotactic factor, defensins may indirectly promote the recruitment and accumulation of neutrophils at the site of inflammation. The degranulation of recruited neutrophils releases more defensins, leading to more IL-8 production, and both of these result in a positive feedback loop. HNP1-3 can also increase the production of tumor necrosis factor (TNF) and IL-1, while reducing the production of monocyte IL-10. At the site of microbial infection, the elevation of pro-inflammatory factors (such as IL-1, TNF, histamine, and PGD2) and the inhibition of IL-10 levels may amplify the local inflammatory response. Alpha-defensins enhance or inhibit the activation of the classical complement pathway in vitro by binding to solid-phase complement C1q or liquid-phase complement C1q, respectively. The ability of defensins to enhance phagocytosis, promote neutrophil recruitment, enhance the production of pro-inflammatory cytokines, inhibit anti-inflammatory mediators, and regulate complement activation indicates that defensins upregulate the host's innate inflammatory defense against microbial invasion.

[0006] In addition, defensins also have a relatively clear role in promoting wound healing. Generally speaking, wound healing refers to the healing process after the disconnection or defect of tissues such as the skin. This process can be divided into 4 main stages - the coagulation stage, the inflammation stage, the repair stage, and the maturation stage. 1. The coagulation stage: It refers to the time when the wound first appears, and then local hemostasis is carried out. At the same time, the body will activate the body's repair system to cause the body to play a role in blood coagulation; 2. The inflammation stage: This stage is mainly to destroy bacteria and remove debris, which is a preparatory stage for the growth of wound tissue. This stage lasts for four to six days and is often accompanied by local symptoms such as redness, swelling, heat, and pain; 3. The repair stage: This stage takes a relatively long time, about 2 to 24 days. This period can be divided into epithelial regeneration and granulation formation, mainly the proliferation and differentiation of fibroblasts, endothelial cells, etc. and the formation of new capillaries, which together form granulation tissue to fill and cover the wound and form a scar; 4. The maturation stage: It is mainly the process of scar remodeling. After the repair stage, the wound has initially healed. As time goes by, the scar tissue, scab, etc. that repair the wound are gradually adjusted, and the repair tissue adapts to the physiological function, and finally the appearance and function of the injured part are improved. The time required for scar remodeling varies according to the severity of the wound. Research in the past ten-odd years has shown that defensins have a clear role in promoting wound healing and have the effect of accelerating the formation of new blood vessels in the repair stage.

[0007] Wound healing is mainly related to three aspects: epidermal re-epithelialization, angiogenesis, and collagen deposition. The growth factor related to angiogenesis is mainly vascular endothelial growth factor A (VEGFA). Collagen deposition is one of the test criteria for the repair of the dermal part. Among them, the increased expression of Transforming Growth Factor-Beta 1 (TGF-1) will promote collagen deposition. Angiogenesis is an important physiological process in wound healing, which is mainly regulated by growth factors, especially vascular endothelial growth factor (VEGF) and angiogenin. Some studies have shown that defensins HNP1, HBD2, and HBD3 bind to cell surface receptor proteins, promote VEGF expression, and improve vascularization. In addition, the role of HBD in angiogenesis has also been determined, revealing that HBD1-4 dose-dependently increases the secretion of angiogenin.

[0008] Currently, the treatment of infectious wounds requires at least two or more drugs to achieve the functions of antibacterial, hemostasis, and wound repair. Small molecule drugs (such as antibacterial agents) have a single function, while macromolecule drugs (such as growth factor drugs) require strict storage conditions, are prone to inactivation during transportation, storage, and use, and have high logistics costs. Therefore, discovering new molecules with multiple biological functions and preparing them into suitable pharmaceutical preparations is one of the feasible methods to solve this bottleneck and pain point. Summary of the Invention

[0009] To make up for the deficiencies of the prior art and overcome the limitations of the existing clinical treatment programs for infectious wounds. The purpose of the present invention is to provide a preparation method and application of a fungal defensin-derived cyclic peptide with antibacterial, anti-inflammatory, and angiogenesis-promoting (wound repair) effects and its thermosensitive preparation.

[0010] The purpose of the present invention is achieved by the following technical solutions:

[0011] A fungal defensin-derived cyclic peptide with antibacterial, anti-inflammatory, and angiogenesis-promoting (wound repair) effects, its linear amino acid sequence is ACNGVRIRQGNPKGGRKLPKDGRPGANIRACYPRKR, and the cysteines at the 2nd and 31st positions are connected by a disulfide bond to form a cyclic structure ( Figure 3 ). The molecular formula of this fungal defensin-derived cyclic peptide is C 166 H 287 N 65 O 44 S2.

[0012] The template of the fungal defensin-derived cyclic peptide is derived from the protein Purlisin composed of 147 amino acid residues expressed by the filamentous fungus Purpureocillium lilacinum (Reference: Shen B, Cao Z, Wu Y, et al. Purlisin, a toxin-like defensin derived from clinical pathogenic fungus Purpureocillium lilacinum with both antimicrobial and potassium channel inhibitory activities. FASEB J. 2020, 34(11): 15093-15107), and its sequence is as follows:

[0013] MRFVLAALASASLASALCMPSLTCEELSPAADKACNGVCIRQGNPKGGRCLPKDGCP GANICACYPR KR SLDPVQRAEYEDGDAVLRRDLEATLDAGRELTDNVTLVKRSICCINLPGYSGLCCEAHCRKIGKPGGSCTPQNICTCN。

[0014] After the translation of protein Purlisin, through processing, the full-length polypeptide can be cleaved by protease into the underlined peptide segment, which is composed of 36 amino acid residues and has antibacterial function, but no anti-inflammatory and angiogenesis-promoting functions. The mature peptide segment ACNGVCIRQGNPKGGRCLPKDGCPGANICACYPRKR after protease processing and cleavage, the four cysteines at the 6th, 17th, 23rd, and 29th positions are respectively replaced with the basic amino acids arginine R or lysine K ( Figure 4 ). Finally, intramolecular disulfide bonds are formed by the sulfhydryl groups in the side chains of the two cysteine residues at the 2nd and 31st positions retained, thus forming a cyclic structure with the amino terminus and carboxyl terminus connected end to end.

[0015] The preparation method of the fungal defensin-derived cyclic peptide includes the following steps: preparing a linear derivative peptide sequence with the sequence of ACNGVRIRQGNPKGGRKLPKDGRPGANIRACYPRKR by solid-phase peptide synthesis technology, and forming an intramolecular pair of disulfide bonds through the sulfhydryl groups of the cysteine residues at the 2nd and 31st positions in an ionic buffer medium Tris-HCl, thereby forming a cyclic structure. By using the difference in the overall molecular polarity between the linear peptide intermediate and the derived cyclic peptide before and after the intramolecular pair of disulfide bond pairing, the final product, the fungal defensin-derived cyclic peptide, can be obtained by separation and purification through reverse phase-high performance liquid chromatography (RP-HPLC).

[0016] The theoretical molecular weight of the linear derivative peptide prepared by solid-phase synthesis is 3961.63, and the isoelectric point pI is 11.64. When forming an intramolecular pair of disulfide bonds, since 2 hydrogen atoms are removed, therefore, the theoretical molecular weight of the active conformation of the fungal defensin-derived cyclic peptide is 3961.63 - 2 = 3959.63 Da, and the theoretical isoelectric point is 11.64, which is a strongly basic cationic peptide.

[0017] The fungal defensin-derived cyclic peptide has the activity of inhibiting Gram-positive bacteria at the μM concentration level, and has good inhibitory effects on both Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) isolated clinically. At the same time, it can dose-dependently inhibit the expression of the inflammatory-related cytokine IL-1β in the human peripheral blood monocyte cell line THP-1 induced by LPS, thereby inhibiting the inflammatory response. It can also up-regulate the expression of angiogenesis-related cytokines VEGFA and TGF-β in the human umbilical vein endothelial cell line HUVEC at the mRNA level, accelerate angiogenesis, and promote wound healing. Therefore, the fungal defensin-derived cyclic peptide has the value of being developed into a drug.

[0018] The application of the fungal defensin-derived cyclic peptide or a pharmaceutically acceptable salt or hydrate or solvate thereof in the preparation of antibacterial drugs. The antibacterial includes anti-Gram-positive bacteria (including drug-resistant bacteria isolated clinically). Further, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus isolated clinically.

[0019] Use of the fungal defensin-derived cyclic peptide or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a drug for inhibiting inflammation. The inflammation inhibition is the inhibition of inflammation-related cytokines, and the inflammation-related cytokines include pro-inflammatory factors. Further, the pro-inflammatory factors include IL-1β.

[0020] Use of the fungal defensin-derived cyclic peptide or a pharmaceutically acceptable salt, hydrate or solvate thereof in the preparation of a drug for promoting angiogenesis or wound repair.

[0021] A pharmaceutical composition having antibacterial, anti-inflammatory, angiogenesis-promoting or wound-repairing effects, comprising the fungal defensin-derived cyclic peptide or a pharmaceutically acceptable salt, hydrate or solvate thereof. Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such as a filler, a pH regulator, a stabilizer, an osmotic pressure regulator, etc. The dosage form of the pharmaceutical composition can be an injection, a tablet, a powder, a granule, a capsule, an oral liquid, an ointment, a cream, a spray, a gel, etc., and can be introduced into muscle, endothelial, subcutaneous or mucosal tissues by injection, application, acupuncture, and other physically or chemically mediated methods, or can be introduced into the body after being mixed or encapsulated with other substances.

[0022] A thermosensitive preparation containing the fungal defensin-derived cyclic peptide, which is obtained by dissolving a thermosensitive material in a solution containing the fungal defensin-derived cyclic peptide. The thermosensitive material is preferably a copolymer of propylene glycol and ethylene oxide (Pluronic F-127). The temperature of the thermosensitive preparation should rise rapidly after contacting the skin, so as to form a gel protective film on the skin surface to cover the wound.

[0023] Advantages and beneficial effects of the present invention:

[0024] 1. The present invention uses the fungal defensin polypeptide as a template molecule, and through the modification of the amino acid sequence and structure, while retaining the antibacterial function, it has the functions of anti-inflammation and promoting angiogenesis. The multifunctional fungal defensin-derived cyclic peptide of the present invention has unique advantages for wound healing, especially for the anti-infection treatment and wound repair of infectious wounds.

[0025] 2. For the preparation and purification of the fungal defensin-derived cyclic peptide of the present invention, the technical route used is easy to operate, the preparation process has strong stability, and the quality of the linear polypeptide intermediate obtained by solid-phase synthesis can be detected by high-performance liquid chromatography and mass spectrometry, which is convenient for quality control. It can meet the needs of large-scale preparation and subsequent industrial mass production.

[0026] In summary, the fungal defensin-derived cyclic peptide of the present invention can provide a new choice for the development of novel active substances having antibacterial, anti-inflammatory and angiogenesis-promoting (wound-repairing) effects and their compound preparations. Description of the Drawings

[0027] Figure 1 is the sequence of a typical fungal defensin and the pairing pattern of intramolecular disulfide bonds.

[0028] Figure 2 is the spatial structure of a typical fungal defensin.

[0029] Figure 3 is the amino acid sequence of the fungal defensin-derived cyclic peptide of the present invention and the pairing sites of intramolecular disulfide bonds.

[0030] Figure 4 is a schematic diagram of the modification using the mature peptide segment of protein Purlisin as a template.

[0031] Figure 5 is the HPLC chart of the prepared linear fungal defensin-derived peptide intermediate.

[0032] Figure 6 is the HPLC chart of the prepared fungal defensin-derived cyclic peptide containing a pair of intramolecular disulfide bonds.

[0033] Figure 7 is the mass spectrometry chart of the prepared fungal defensin-derived cyclic peptide containing a pair of intramolecular disulfide bonds.

[0034] Figure 8 is the hemolytic activity result of the fungal defensin-derived cyclic peptide.

[0035] Figure 9 is the result of the fungal defensin-derived cyclic peptide inhibiting the expression of the pro-inflammatory factor IL-1β in LPS-induced THP-1 cells.

[0036] Figure 10 is the result of the fungal defensin-derived cyclic peptide upregulating the expression of the angiogenesis-related cytokine VEGFA in HUVEC cells at the mRNA level.

[0037] Figure 11 is the result of the fungal defensin-derived cyclic peptide upregulating the expression of the angiogenesis-related cytokine TGF-β in HUVEC cells at the mRNA level.

[0038] Figure 12 is the result of the fungal defensin-derived cyclic peptide promoting the neovascularization of primary blood vessels (A) and secondary blood vessels (B) in the chicken embryo chorioallantoic membrane model. Detailed Description of the Invention

[0039] To clearly illustrate the main objectives, implementation solutions, and technical advantages of the present invention, the present invention will be clearly and completely described through the following embodiments. The following will explain the solutions of the present invention in combination with the embodiments. The described embodiments are part of the implementation of the present invention, rather than all of them, and should not be regarded as limiting the scope of the present invention. Based on the embodiments described in the present invention, all other embodiments (i.e., implementation methods) obtained by those of ordinary skill in the art without creative efforts also fall within the scope of protection of the present invention.

[0040] For those not specifying specific technologies or conditions in the embodiments, follow the technologies or conditions described in the literature in this field or according to the product specifications. Unless otherwise specified, the methods used are all conventional methods well-known in this field, and the consumables and reagents used are all commercially available unless otherwise specified. Unless otherwise stated, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in this field. In addition, any methods or materials similar or equivalent to the described content can also be applied to the present invention.

[0041]

Example 1

[0042] In the synthesis of this linear peptide segment, 9-fluorenylmethoxycarbonyl (FMOC) was used as the protecting group at the amino terminus, 4-methyl-benzhydrylamine resin·HCl (MBHA resin) was selected as the solid-phase carrier for polypeptide synthesis, and HOBt / DCC was used as the condensing agent for the reaction. The peptide chain was extended from the carboxyl terminus to the amino terminus (C-terminus → N-terminus). A mixture of 91.0% trifluoroacetic acid, 4.0% water, and 5.0% triisopropylsilane (TIA) (by mass percentage) was used to cleave the linear modified peptide containing 36 amino acid residues from the MBHA resin. After repeated precipitation with ether several times, it was purified by preparative reverse high-performance liquid chromatography RP-HPLC. Purification process: Use a C18 reversed-phase preparative column (250 mm × 20 mm, 5 μm); mobile phase: 2.5‰ trifluoroacetic acid, 0% - 65% (by volume percentage) acetonitrile as the mobile phase, and gradient elution was carried out at a flow rate of 1.5 mL / min. Detected by RP-HPLC, the retention time (RT) was 8.91 min. Calculated by the peak area normalization method, its purity > 85% ( Figure 5 ). The main peak eluate was collected, frozen in a -80°C refrigerator for 6 hours, and then placed in a freeze dryer for lyophilization.

[0043]

Example 2

[0044] Using the linear fungal defensin-derived peptide ACNGVRIRQGNPKGGRKLPKDGRPGANIRACYPRKR prepared by solid-phase synthesis, chromatographic purification, and lyophilization in Example 1 above as the raw material. Take 2 mg of the lyophilized powder in a 5 mL centrifuge tube and dissolve it with 2 mL of Tris-HCl buffer solution with a concentration of 0.20 M (pH = 10.5). After tightening the cap of the centrifuge tube, seal it with Parafilm sealing film. After confirming that there is no liquid leakage at the tube mouth, invert the centrifuge tube up and down to accelerate the dissolution of the polypeptide. Place the centrifuge tube containing the polypeptide solution in a thermostatic shaker and incubate it at 25 °C - 28 °C and 80 - 100 rpm for 18 - 24 hr. Centrifuge at high speed at 4 °C and 12000 rmp for 3 min, and take the supernatant. Purify by preparative RP-HPLC. Use a C18 reverse-phase chromatographic column (20 mm × 250 mm, 5 μm); mobile phase: 2.0‰ trifluoroacetic acid, 0% - 65% (volume percentage) acetonitrile as the mobile phase, and perform gradient elution at a flow rate of 1.5 mL / min. Detected by RP-HPLC, the RT is 10.36 min, and its purity > 95%( Figure 6 ), and store it for later use after lyophilization. Identified by ESI-Q-TOF-MS, its molecular weight is consistent with the corresponding theoretical molecular weight of the polypeptide intermediate to be prepared, m / z 1321.2[M+3H] 3+ , m / z 990.9[M+4H] 4+ , m / z792.4[M+5H] 5+ , m / z 661.6[M+6H] 6+ ( Figure 7 ).

[0045] The cyclic fungal defensin-derived peptide designed and prepared by the present invention is shown as Figure 3 .

[0046]

Example 3

[0047] After anesthesia, take blood from the orbital cavity, obtain fresh blood from healthy adult male Balb / c mice, separate mouse red blood cells and make a suspension, use sterilized normal saline as the negative control and 1% Triton X-100 as the positive control to determine the hemolytic activity of the cyclic fungal defensin-derived peptide.

[0048] The specific steps are as follows: Collect whole blood with a heparin anticoagulant tube, gently invert to fully anticoagulate the blood, centrifuge at 4°C and 700 rpm for 5 min, and aspirate as much of the upper plasma as possible to retain the lower-layer red blood cells; Add 3 to 5 volumes of sterile normal saline, gently pipette with a pipette gun to suspend the red blood cells at the bottom, centrifuge at 800 rpm at room temperature for 3 min, discard the supernatant and retain the precipitated red blood cells, repeat the operation 3 times until the supernatant is colorless; Prepare a 2% (v / v) concentration of red blood cell suspension with sterile normal saline; Prepare solutions of the fungal defensin-derived cyclic peptide of the present invention at concentrations of 64 μmol / L, 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, and 2 μmol / L with sterile normal saline; Mix 75 μL of the derived cyclic peptide solution and 75 μL of the red blood cell suspension and add them to a 96-well plate, and the final concentrations of the derived cyclic peptide are 32 μmol / L, 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L, and 1 μmol / L respectively; The negative control group is red blood cells suspended with sterile normal saline, and the positive control uses red blood cells suspended with a sterile normal saline solution containing 1% Triton X-100; Place the samples in a constant temperature shaker, incubate at 36°C - 37°C at 100 rpm for 30 min; Centrifuge the samples at 4500 rpm at room temperature for 3 min, transfer 100 μL of the supernatant from each well to another 96-well plate; Measure the absorbance at a wavelength of 490 nm with a full-wavelength microplate reader, and calculate the hemolysis percentage through the following formula, where H is the absorbance at a wavelength of 490 nm: Hemolysis rate (%) = (H sample - H negative ) / (H positive - H negative ) × 100%, as shown in Figure 8 .

[0049] The results show that the fungal defensin-derived cyclic peptide of the present invention still does not show obvious hemolytic effect (hemolysis rate % < 10%) when the final concentration is as high as 32 μmol / L.

[0050]

Example 4

[0051] The Gram-positive bacteria to be tested include standard reference strains (Staphylococcus aureus, S. epidermidis) and clinically isolated methicillin-resistant Staphylococcus aureus (MRSA). The standard strains are from the China Center of Type Culture Collection (CCTCC), and the clinically isolated drug-resistant pathogenic bacteria are provided by the Department of Laboratory Medicine, Renmin Hospital of Wuhan University. The minimum inhibitory concentration (MIC) was determined by the serial dilution method, and the antibacterial activity of the derived cyclic peptide was characterized by the MIC value.

[0052] The specific method is as follows:

[0053] The bacteria to be tested were inoculated onto sterilized Luria-Bertani (LB) solid medium plates by the three-zone streaking method and incubated overnight at 37 °C in an inverted position in a constant temperature incubator (incubation time > 8 hr). A single colony was picked with an inoculation loop and transferred to a newly prepared sterilized liquid LB medium, and incubated at 37 °C and 160 rpm on a shaker until the logarithmic growth phase. The optical density (OD 600 ) value of the bacterial solution at a wavelength of 600 nm was measured using an ultraviolet spectrophotometer. According to the conversion relationship of 1 OD ≈ 1×10 9 CFU / mL, the bacterial solutions of the S. aureus standard reference strain and the clinically drug-resistant strain MRSA were diluted with sterilized liquid medium to a concentration of approximately 2×10 6 CFU / mL.

[0054] First, 75 μL of sterilized LB medium was added to a sterile 96-well plate; then, 75 μL of the polypeptide solution with a concentration of 128 μmol / L dissolved in sterilized LB medium was added to the first well. After mixing evenly, 75 μL was taken and added to the second well, and the operation was carried out in sequence. Finally, 75 μL was aspirated from the sixth well and discarded, and serial dilutions were performed in sequence; finally, 75 μL of the diluted bacterial solution with a concentration of ~2×10 6 CFU / mL was added to each well and mixed evenly. The wells without added polypeptide were used as negative controls.

[0055] The above groups were cultured at 37 °C and 180 rpm for 18 - 24 hours, and the absorbance value at a wavelength of 600 nm was measured. The minimum inhibitory concentration (MIC) was taken as the concentration at which no bacterial growth was detected (OD 600The lowest value of the final concentration of the fungal defensin-derived cyclic peptide contained in the wells (≤0.05). The bacterial suspensions in the negative control wells were all turbid, and the absorbance OD at 600 nm 600 > 2.0. The antibacterial activity results of the fungal defensin-modified peptide of the present invention are shown in Table 1.

[0056] Table 1 Antibacterial activity of the fungal defensin-modified peptide of the present invention

[0057]

[0058] From the results in Table 1, it can be seen that the designed and prepared fungal defensin-derived cyclic peptide of the present invention has good antibacterial activity against Gram-positive bacterium S. aureus (MIC value = 8 μM), and at the same time also has a certain inhibitory effect on the clinically isolated drug-resistant pathogenic bacterium MRSA (MIC value = 32 μM).

[0059]

Example 5

[0060] Take THP-1 cells cultured to a confluence rate of 80% - 90%, aspirate and discard the culture medium, and wash twice with sterile PBS buffer; add an appropriate amount of trypsin and digest at 37 °C for 20 seconds, gently tap to make the cells detach from the bottom of the culture container, and add RPMI 1640 medium containing 10% fetal bovine serum, 0.05 mM β-mercaptoethanol and 1% P / S double antibody to gently pipette the cells to make a single-cell suspension; dilute the cells with the medium and adjust the cell concentration to 2.5×10 5 cells / mL, inoculate into 24-well plates, 0.5 mL per well. Incubate the culture plates at 37 °C, 5% CO2 for 8 - 12 hours, aspirate and discard the supernatant. Add 0.5 mL of RPMI 1640 complete medium to the blank group; add 0.5 mL of RPMI1640 complete medium containing LPS (the final concentration of LPS is 15 μg / mL) to the LPS model group and the experimental group. Incubate the culture plates at 37 °C, 5% CO2 for 2 hours. Add 0.5 mL of RPMI 1640 complete medium to the blank group and the LPS model group; add 0.5 mL of DMEM medium containing 10% FBS of the fungal defensin-derived cyclic peptide of the present invention to the experimental group, with the final concentration of the derived cyclic peptide being 30 μmol / L (high-dose group), the final concentration of 15 μmol / L (medium-dose group), and the final concentration of 7.5 μmol / L (low-dose group). Incubate the culture plates at 37 °C, 5% CO2 overnight, centrifuge at 4 °C, 1500 rmp for 15 min. Take the supernatant and dilute it by an appropriate multiple, and use enzyme-linked immunosorbent assay (ELISA) to measure the content of cytokine IL-1β in the supernatant.

[0061] The results showed that the fungal defensin-derived cyclic peptide of the present invention could significantly inhibit the expression of the pro-inflammatory cytokine IL-1β in the human monocytic leukemia cell line THP-1 induced by LPS, as Figure 9 shown.

[0062]

Example 6

[0063] I. Cell culture:

[0064] Heat the constant temperature water bath and maintain it at (36 ± 1) °C. Quickly take out the HUVECs cell line from the liquid nitrogen tank, quickly place it in the water bath and gently shake it. After dissolving in the water bath within 1 min, transfer it to a clean workbench. Use a pipette to aspirate all the cell cryopreservation solution into a centrifuge tube, centrifuge at a speed of 1000 rpm for 5 min, and discard the supernatant. Use Gibico human large vessel endothelial cell basal medium (M200500) and add 10 mL of 50× low serum growth supplement (LSGS, S00310). Add 1 mL of fresh culture medium to a sterilized centrifuge tube and pipette the cell pellet to resuspend it into a uniformly dispersed cell suspension, and transfer it to a culture flask. Then add 5 mL of fresh culture medium, place it in a constant temperature incubator containing 5% CO2 (37 °C), and change the medium every other day.

[0065] Take out the cells and observe the cell growth status and density under the inverted microscope in bright field. When the cells grow to more than 85% of the culture flask and the cell status is good without bacterial contamination, passage the cells. Discard the old culture medium, wash the bottle wall with 3 mL of preheated sterilized PBS 3 times, discard the PBS, and then add 800 μL of trypsin to digest the cells. Observe the cell digestion and detachment under the microscope. When a large number of cells become round and some cells detach from the bottle wall, immediately add 2 mL of complete culture medium to terminate the digestion. Use a pipette to pipette the cells from the bottle wall, collect the cell solution in the culture flask and transfer it to a 15 mL centrifuge tube, centrifuge at a speed of 1000 rpm for 5 min. Discard the supernatant, add 1 mL of fresh culture medium and pipette it into a uniformly dispersed cell suspension, and transfer it to a culture flask. Then add 2 mL of fresh culture medium and place it in the incubator for culture. After the cells are passaged 3 times, carry out subsequent cell experiments.

[0066] Cells were cultured in 6-well plates. When the cell confluence reached approximately 80%, the culture medium containing different components was replaced. The experiment was divided into four groups: ① control group, replacing with complete medium; ② low-dose experimental group, replacing with complete medium containing the fungal defensin-derived cyclic peptide of the present invention at a final concentration of 7.5 μM; ③ medium-dose experimental group, replacing with complete medium containing the fungal defensin-derived cyclic peptide of the present invention at a final concentration of 15 μM; ④ high-dose experimental group, replacing with complete medium containing the fungal defensin-derived cyclic peptide of the present invention at a final concentration of 30 μM. After culturing for 24 hours, the culture solution was discarded and the cells were washed once with pre-warmed sterilized 1×PBS.

[0067] II. Total RNA Extraction

[0068] Discard the culture solution and wash once with 1×PBS. Add 1 ml of RNA isolater to each well of the 6-well plate to fully cover the cell surface, and then pipette to blow down the cells. If the cells adhere firmly, a cell scraper can be used to detach the cells. Transfer the lysate to a 1.5 mL centrifuge tube and pipette repeatedly until fully lysed, and let it stand on ice for 5 minutes. Add 1 / 5 volume of chloroform to the above lysate. Vigorously shake for 15 - 20 seconds to form an emulsion, let it stand at 4°C for 5 minutes, and centrifuge at 4°C and 12,000 rpm for 15 minutes. Carefully take out the centrifuge tube. At this time, the solution is divided into three layers: a colorless aqueous phase (upper layer), a white middle layer, and a red organic layer (lower layer). Carefully aspirate 400 - 500 μL of the upper aqueous phase into a new centrifuge tube. Add an equal volume of pre-cooled isopropanol, invert and mix well, and let it stand at 4°C for 10 min. Centrifuge at 4°C and 12,000 rpm for 10 minutes, and a white precipitate can be seen. Carefully discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water. Flick the bottom of the tube to suspend the precipitate and invert several times, and let it stand at room temperature for 3 - 5 minutes. Centrifuge at 4°C and 12,000 rpm for 5 minutes and discard the supernatant. Dry the precipitate in a clean environment at room temperature with the opening uncovered for 3 minutes, add an appropriate amount of DEPC water to dissolve the precipitate, and pipette gently to completely dissolve it. Take a small amount for detection, and store the rest in a -80°C refrigerator. The purity of the product was detected by a NanoDrop spectrophotometer. The OD 260 / OD 280 The ratio between 1.8 and 2.2 indicates a relatively high RNA purity. The calculation formula for the product concentration is RNA concentration (ng / μL) = OD 260 × dilution factor × 40.

[0069] III. RNA Reverse Transcription

[0070] Prepare the RNA reverse transcription system according to a 10 μL reaction system, including 2 μL of 5×Prime Script RT Master Mix (Perfect Real Time), 3 μL of the extracted total RNA (RNA content is approximately 400 ng), and supplement with 5 μL of RNAse Free water. After mixing the prepared reaction solution, perform the reverse transcription reaction in the following program: 37 °C for 15 min (reverse transcription reaction); 85 °C for 5 sec (inactivation reaction of reverse transcription); 4 °C for 1 hr (temporary storage). After the reverse transcription is completed, aliquot the cDNA and store it at -20 °C.

[0071] Ⅳ. RT-qPCR determination of relative expression

[0072] Design primers using Primer 5.0 software. The primers for the internal reference gene GAPDH, target genes VEGFA, and TGF-β are as follows:

[0073] GAPDH Forward: 5’-TGTGGGCATCAATGGATTTGG-3’,

[0074] GAPDH Reverse: 5’-ACACCATGTATTCCGGGTCAAT-3’;

[0075] VEGFA Forward: 5’-AGGGCAGAATCATCACGAAGT-3’,

[0076] VEGFA Reverse: 5’-AGGGTCTCGATTGGATGGCA-3’;

[0077] TGF-β Forward: 5’-CTAATGGTGGAAACCCACAACG-3’,

[0078] TGF-β Reverse: 5’-TATCGCCAGGAATTGTTGCTG-3’.

[0079] Prepare the reaction system according to a volume of 20 μL: 10 μL of AceQ qPCR SYBR Green Master Mix (2×); 0.5 μL of Forward Primer (10 μM); 0.5 μL of Reverse Primer (10 μM); 2 μL of cNDA template; 7 μL of sterilized water. The RT-qPCR reaction procedure is as follows: pre-denaturation at 95°C for 3 minutes (1 cycle); denaturation at 95°C for 10 seconds (40 cycles); annealing and extension at 60°C (40 cycles); 15 seconds at 95°C (melting curve, 1 cycle); 30 seconds at 60°C (melting curve, 1 cycle); 15 seconds at 95°C (melting curve, 1 cycle).

[0080] After the reaction, judge the specificity of the primer according to its melting curve and obtain the CT values of each target gene and internal reference gene in each group through the amplification curve, and use the 2 -ΔΔCT method for data analysis and statistical analysis. The results show that the fungal defensin-derived cyclic peptide described in the present invention can significantly up-regulate the genes VEGFA ( Figure 10 ) and TGF-β ( Figure 11 ) related to angiogenesis at the mRNA level.

[0081]

Example 7

[0082] Using the completely randomized experimental design method, 24 hatching eggs were randomly divided into: normal saline control group, low-dose group (7.5 μM derived cyclic peptide), medium-dose group (15 μM derived cyclic peptide), and high-dose group (30 μM derived cyclic peptide). First, wipe the stains on the eggshell surface with 1‰ bromogeramine solution, and then disinfect with 75% alcohol. The large end (air chamber end) of the hatching egg is facing up, at a 45° inclination angle with the egg tray, and incubated in an incubator at 37.5°C. A water tray is placed in the incubator to maintain the relative humidity at 40% - 70%, and ventilation holes are left to ensure sufficient oxygen supply. During the incubation process, turn the eggs 4 - 5 times a day to prevent embryo adhesion and promote amniotic movement. On the 7th day of incubation, disinfect the surface of the egg air chamber (large end) area of about 2 cm × 3 cm with a sterile cotton swab dipped in 75% alcohol, and then grind and cut the eggshell with a dental grinding wheel. After grinding out a dent, use an ophthalmic curved forceps to clamp off the eggshell and shell membrane. After the window is exposed, first drop 1 small drop of normal saline on the air chamber membrane with a disposable syringe. After the normal saline slowly spreads, the blood vessels of the chicken embryo chorioallantoic membrane under the air chamber membrane will appear. And the chorioallantoic membrane of the chicken embryo under the dropped normal saline is separated from the upper air chamber membrane and sinks downward. Use the syringe needle to carefully push aside and lift the air chamber membrane at this place, and hold an ophthalmic curved forceps in the other hand to clamp and tighten the lifted air chamber membrane. At the same time, with the assistance of the syringe needle, gently peel off the air chamber membrane bit by bit to completely expose the chicken embryo chorioallantoic membrane.

[0083] After exposing the chicken embryo chorioallantoic membrane, the carrier prepared in advance is placed in its central blood vessel scarce area so that the fungal defensin derived cyclic peptide to be tested can fully play its role. According to the grouping, 15 μ L of corresponding medicine is dripped with a pipette and then the pseudo air chamber is sealed with transparent tape to form a transparent observation window to observe the survival of the chicken embryo. After the window is sealed, continue to incubate and ensure that the whole operation process should be aseptic. After 3 days of drug incubation, cut the transparent tape paper with surgical scissors, drip about 8 drops of methanol: acetone=1: 1 fixative from the observation window, pre-fix for 20 minutes, wait for the blood vessels of the chicken embryo chorioallantoic membrane to coagulate completely, carefully remove the eggshell and egg shell membrane above the chicken embryo chorioallantoic membrane plane with ophthalmic curved forceps, and cut the chicken embryo chorioallantoic membrane completely with ophthalmic scissors with the object to be tested as the center, place it in a plate filled with physiological saline and spread it on a carrier sheet with tweezers after shaking, and store it in the shade. New capillaries were counted under a stereo microscope at 1×10 magnification. The first-class vessels were within 1 mm of the experimental site edge, and the second-class vessels were within 5 mm of the experimental site edge. The first-class and second-class vessels were counted separately and statistically analyzed.

[0084] The results showed that compared with the drug group, the vascular density of the saline control group was significantly lower than that of the fungal defensin-derived cyclic peptide group, which was dose-dependent, that is, the high-dose group had the largest number of new blood vessels. The statistical data of the primary and secondary blood vessel numbers are shown in Figure 2. Figure 12 A and Figure 12 As shown in B, the difference was statistically significant (P<0.05).

[0085] [Example 8] Gel (thermosensitive gel spray)

[0086] Take 5mL of distilled water and 5mL of PBS solution containing 60μM derivative cyclic peptide, sprinkle 2.5g of thermosensitive polymer material Pluronic F-127, a polymer of polypropylene glycol and ethylene oxide, evenly on the liquid surface, place it in a 4℃ refrigerator overnight to dissolve it, and prepare a solution with a mass concentration of 25% of Pluronic F-127 and a final concentration of 30μM derivative cyclic peptide. The solution containing the thermosensitive material and the derivative cyclic peptide is in a liquid state at 4℃, and begins to change phase at about 26℃ as the temperature rises, the viscosity increases, and when the temperature reaches body temperature 36℃~37℃, a gel with certain strength and toughness can be formed. Therefore, the thermosensitive solution containing the derivative cyclic peptide is stored in a low temperature environment of 2℃~8℃, and after being sprayed on the skin surface by a spray device for seconds, a uniform gel coating will be immediately produced on the wound due to the increase in local temperature, thereby exerting its effect.

[0087] [Example 9] Tablets

[0088] Take 0.2 g of the fungal defensin-derived cyclic peptide of the present invention, 3.5 g of starch, and 3.5 g of dextrin, mix them, and use a pharmaceutical-grade polyvinylpyrrolidone (PVP) with a mass concentration of 35% - 40% as a binder to granulate, size the granules, and compress into tablets to obtain the tablets.

[0089]

Example 10

[0090] Take 1 g of the fungal defensin-derived cyclic peptide of the present invention, 1.4 g of glyceryl monostearate, 4.8 g of stearic acid, 0.4 g of white petrolatum, 2.4 g of liquid paraffin, 2.0 g of lanolin, 0.16 g of triethanolamine, and 30 mL of distilled water, mix well to obtain.

[0091]

Example 11

[0092] Take 2 g of the fungal defensin-derived cyclic peptide of the present invention and 20 g of mannitol, place them in a container, dissolve with an appropriate amount of PBS buffer (0.15 M, pH 7.4), add water for injection to 250 mL, shake well, add 6 - 10 g of activated carbon for injection, stir at room temperature for about 45 minutes, filter roughly, filter and sterilize with a 0.22 μm sterilizing filter membrane, dispense into vials of 2 mL each, and use the quick-freezing method to cool at a rate of 5°C - 10°C per minute until the temperature reaches -45°C - -50°C, maintain for 2 - 3 hours, evacuate, slowly raise the temperature under vacuum at a rate of 3°C - 5°C per hour, stop raising the temperature when the temperature reaches room temperature (about 25°C), take out after the temperature approaches room temperature, cover and seal to obtain the freeze-dried injection.

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A fungal defensin-derived cyclic peptide, characterized in that: The linear amino acid sequence of the fungal defensin-derived cyclic peptide is ACNGVRIRQGNPKGGRKLPKDGRPGANIRACYPRKR, and the cysteines at positions 2 and 31 are linked by a disulfide bond to form a cyclic structure.

2. A method for preparing the fungal defensin-derived cyclic peptide according to claim 1, characterized in that: It includes the following steps: preparing a linear derivative peptide sequence with the sequence of ACNGVRIRQGNPKGGRKLPKDGRPGANIRACYPRKR by using solid-phase polypeptide synthesis technology, and forming a pair of intramolecular disulfide bonds through the sulfhydryl groups of the side chains of the cysteine residues at positions 2 and 31 in an ionic buffer medium of Tris-HCl, thereby obtaining the fungal defensin-derived cyclic peptide.

3. Use of the fungal defensin-derived cyclic peptide according to claim 1 or a pharmaceutically acceptable salt or hydrate thereof in the preparation of a drug, characterized in that: The drug described above is an antibacterial drug; the antibacterial activity is against Gram-positive bacteria, and the Gram-positive bacteria include Staphylococcus aureus and Staphylococcus epidermidis.

4. An antibacterial pharmaceutical composition, characterized in that: It contains the fungal defensin-derived cyclic peptide described in claim 1 or a pharmaceutically acceptable salt or hydrate thereof; the antibacterial activity is against Gram-positive bacteria, and the Gram-positive bacteria include Staphylococcus aureus and Staphylococcus epidermidis.

5. The pharmaceutical composition according to claim 4, characterized in that: It also contains a pharmaceutically acceptable carrier; its dosage forms include injections, tablets, powders, granules, capsules, oral liquids, ointments, creams, sprays, and gels.

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