Anti-inflammatory effects and applications of an antimicrobial peptide from a South American frog and its modified antimicrobial peptides

Abstract

The present invention provides a South American frog antimicrobial peptide MS-PT with anti-inflammatory activity, as well as its modified antimicrobial peptides and applications, belonging to the field of biomedicine. Among them, the antimicrobial peptide MS-PT and its modified antimicrobial peptides MS-PT1 and 6A all contain 18 amino acid residues, and their molecular weights are 1810.30 Daltons, 1837.37 Daltons, and 1811.33 Daltons respectively. The antimicrobial peptide MS-PT, the modified antimicrobial peptides MS-PT1 and 6A provided by the present invention all have anti-inflammatory activity and can also alleviate the mouse sepsis model, so they can be used as potential anti-inflammatory and anti-sepsis drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an antimicrobial peptide MS-PT from the South American frog (Phyllomedusa tarsius), and modified antimicrobial peptides MS-PT1 and 6A of this peptide and their applications. Background Art

[0002] Sepsis is a common life-threatening and costly disease that affects the health of millions of patients worldwide. Clinically, to control the early inflammatory response and maintain organ function, the treatment of sepsis mainly relies on broad-spectrum antibiotics, vasopressors, and supportive therapies such as shock treatment. When treating infectious diseases with conventional antibiotics, the antibiotics destroy the structure of the bacterial cell membrane, releasing a large amount of endotoxins such as lipopolysaccharide (LPS), which activates the systemic inflammatory cascade reaction. During the occurrence of sepsis, a large number of cytokines and damage-associated molecular pattern molecules (DAMPs) produced by various cells will lead to a strong immune response, and then lead to cytokine storm and multiple organ dysfunction syndrome (MODS).

[0003] Due to the limitations of sepsis treatment, there is an urgent need to find a new treatment method to combat this problem. Antimicrobial peptides are short cationic amphiphilic peptides with antimicrobial and / or immunomodulatory activities, which are the core part of the innate immune system. They can effectively prevent and treat infections through combined antibacterial and anti-inflammatory effects. The surfaces of Gram-positive bacteria and Gram-negative bacteria are respectively covered with lipoteichoic acid and lipopolysaccharide with a large amount of negative charges, which increases the negative charge of the membrane, and cationic antimicrobial peptides can selectively act on negatively charged membrane components through electrostatic interaction. Different from the bactericidal mechanism of antibiotics, antimicrobial peptides mainly kill bacteria by directly generating physical interactions with the bacterial cell membrane or acting on intracellular targets through the membrane, so it is not easy for antimicrobial peptides to develop drug resistance. In addition to directly killing pathogens, antimicrobial peptides can also regulate immune cells directly or indirectly, affect the conduction of certain signal pathways, and control the delicate balance between pro-inflammatory and anti-inflammatory responses to exert immunomodulatory activities. Antimicrobial peptides may become an ideal substitute for traditional antibiotics. Summary of the Invention

[0004] To solve the above problems, the present invention aims to provide a new anti-inflammatory antimicrobial peptide MS-PT from the South American frog (Phyllomedusa tarsius), and modified antimicrobial peptides MS-PT1 and 6A of this peptide and their applications.

[0005] The invention of antibacterial peptides with antibacterial and anti-inflammatory effects has long been an area of concern for the inventors. The inventors have discovered that the antibacterial peptide MS-PT derived from the skin secretions of South American frogs not only has antibacterial activity but also has anti-inflammatory activity. At the same time, its modified antibacterial peptides MS-PT1 and 6A both exhibit excellent anti-inflammatory and anti-septicemia activities. Based on this discovery, the inventors have proposed the present invention:

[0006] One of the technical solutions provided by the present invention is an antibacterial peptide MS-PT from South American frogs. The inventors obtained a natural antibacterial peptide with anti-Staphylococcus aureus and anti-inflammatory activities from the skin secretions of South American frogs and named it Medusin-PT (MS-PT). This antibacterial peptide consists of 18 amino acids and has a molecular weight of 1,810.3 daltons. Jiangsu GenScript Biotech Co., Ltd. was commissioned to obtain the antibacterial peptide MS-PT using the standard Fmoc solid-phase peptide synthesis method. The amino acid sequence of this antibacterial peptide is:

[0007] NH2-Leu Leu Gly Met Ile Pro Val Ala Ile Thr Ala Ile Ser Ala LeuSerLys Leu-Amide

[0008] Another technical solution provided by the present invention is a modified antibacterial peptide MS-PT1 of an antibacterial peptide from South American frogs. The inventors changed the uncharged threonine at position 10 in the antibacterial peptide MS-PT in the above technical solution to a positively charged lysine to increase the net charge. This modified antibacterial peptide MS-PT1 consists of 18 amino acids and has a molecular weight of 1,837.37 daltons. Jiangsu GenScript Biotech Co., Ltd. was commissioned to obtain the modified antibacterial peptide MS-PT1 using the standard Fmoc solid-phase peptide synthesis method. The amino acid sequence of this modified antibacterial peptide is:

[0009] NH2-Leu Leu Gly Met Ile Pro Val Ala Ile Lys Ala Ile Ser Ala Leu SerLys Leu-Amide

[0010] A further technical solution provided by the present invention is a modified antibacterial peptide 6A of an antibacterial peptide from South American frogs. The inventors changed the proline at position 6 in the modified antibacterial peptide MS-PT1 in the above technical solution to alanine to increase the helicity. This modified antibacterial peptide 6A consists of 18 amino acids and has a molecular weight of 1,811.33 daltons. Jiangsu GenScript Biotech Co., Ltd. was commissioned to obtain the modified antibacterial peptide 6A using the standard Fmoc solid-phase peptide synthesis method. The amino acid sequence of this modified antibacterial peptide is:

[0011] NH2-Leu Leu Gly Met Ile Ala Val Ala Ile Lys Ala Ile Ser Ala Leu SerLys Leu-Amide

[0012] Fourth, the technical solution provided by the present invention includes the design and modification of the antibacterial peptide MS-PT from South American frogs, as well as the applications of the two modified antibacterial peptides MS-PT1 and 6A mentioned in the present invention. In addition to the proven anti-Staphylococcus aureus activity, the antibacterial peptide MS-PT also has anti-inflammatory activity. After experimental investigation, it was found that the modified antibacterial peptide MS-PT1 can inhibit the activation of the classical TLR4 inflammatory signaling pathway induced by lipopolysaccharide through its strong ability to neutralize LPS, thereby playing an anti-inflammatory role. At the same time, it has a good protective effect on the murine model of sepsis caused by LPS and can greatly improve the survival rate of mice. After experimental investigation, it was found that the modified antibacterial peptide 6A can inhibit the formation of the LPS-MD2-TLR4 complex and the activation of the classical TLR4 inflammatory signaling pathway induced by lipopolysaccharide through its physical interaction with the key target MD2, thereby playing an anti-inflammatory role. At the same time, it can relieve the inflammatory response in the sepsis model induced by cecal ligation and puncture and restore organ function. Therefore, the two modified antibacterial peptides are expected to be used as potential anti-inflammatory and anti-sepsis drugs.

[0013] In summary, the present invention has the following beneficial effects: It provides a new antibacterial peptide MS-PT from South American frogs (Phyllomedusa tarsius) with anti-inflammatory activity, as well as the modified antibacterial peptides MS-PT1 and 6A of this peptide, which are expected to be used as potential anti-inflammatory and anti-sepsis drugs. Description of the Drawings

[0014] Figure 1 Effect of antibacterial peptide MS-PT and its modified antibacterial peptide MS-PT1 on the production of TNF-α in cells;

[0015] Figure 2 Effect of antibacterial peptide MS-PT and its modified antibacterial peptide MS-PT1 on the production of IL-6 in cells;

[0016] Figure 3 Isothermal titration calorimetry for the binding of antibacterial peptide MS-PT1 to LPS;

[0017] Figure 4 Zeta potential detection of the binding of antibacterial peptide MS-PT1 to LPS;

[0018] Figure 5 Limulus reagent detection of the neutralization effect of antibacterial peptide MS-PT1 on LPS;

[0019] Figure 6 Effect of MS-PT1 on the survival rate of mice with LPS-induced sepsis

[0020] Figure 7 Effect of the modified antimicrobial peptide 6A on the production of TNF-α by cells

[0021] Figure 8 Effect of the modified antimicrobial peptide 6A on the production of IL-6 by cells

[0022] Figure 9 Effect of the modified antimicrobial peptide 6A on the NF-κB and MAPK signaling pathways downstream of TLR4

[0023] Figure 10 Interaction between the modified antimicrobial peptide 6A and the key protein MD2 on the TLR4 signaling pathway

[0024] Figure 11 Effect of the modified antimicrobial peptide 6A on the production of TNF-α in the serum of a mouse sepsis model

[0025] Figure 12 Effect of the modified antimicrobial peptide 6A on the production of IL-6 in the serum of a mouse sepsis model Detailed implementation manners

[0026] In order to make the objectives, technical solutions and advantages of this patent clearer, the following further details this patent in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not used to limit the present invention.

[0027] The antimicrobial peptide MS-PT from South American frogs consists of 18 amino acids, with a molecular weight of 1810.3 daltons, and its full sequence is: NH2-Leu Leu Gly Met Ile Pro Val Ala Ile Thr Ala Ile Ser Ala Leu Ser Lys Leu-Amide

[0028] The modified antimicrobial peptide MS-PT1 consists of 18 amino acids, with a molecular weight of 1837.37 daltons, and its full sequence is: NH2-Leu Leu Gly Met Ile Pro Val Ala Ile Lys Ala Ile Ser Ala Leu Ser Lys Leu-Amide

[0029] The modified antimicrobial peptide 6A consists of 18 amino acids with a molecular weight of 1,811.33 Daltons. Its full sequence is: NH2-Leu Leu Gly Met Ile Ala Val Ala Ile Lys Ala Ile Ser Ala Leu Ser Lys Leu-Amide.

[0030] The antimicrobial peptide MS-PT and its modified peptides MS-PT1 and 6A were synthesized by Jiangsu GenScript Biotechnology Co., Ltd. through solid-phase synthesis. The purity was >90%, meeting the requirements for related activity detection experiments.

[0031] Example 1: Anti-inflammatory activity of the antimicrobial peptide MS-PT and its modified peptide MS-PT1

[0032] (1) Determination of the anti-inflammatory activity of the antimicrobial peptide MS-PT and its modified peptide MS-PT1

[0033] Primary mouse peritoneal macrophages (MPMs) were derived from male C57BL / 6 mice. The cells were cultured in an incubator at 37°C with 5% CO2. After 2 h, the medium was replaced with fresh RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) and cultured overnight. The cells were then stimulated with LPS at a final concentration of 0.5 μg / mL. The following experimental groups were set up:

[0034] Blank group: Normal culture for 2 h + 24 h;

[0035] Induction group: Incubated with LPS (0.5 μg / mL) in the incubator for 2 h and then stimulated for 24 h;

[0036] Experimental group: Incubated with LPS (0.5 μg / mL) and the sample in the incubator for 2 h and then stimulated for 24 h;

[0037] Positive control group: Incubated with LPS (0.5 μg / mL) and the positive control (20 μM TAK-242) in the incubator for 2 h and then stimulated for 24 h;

[0038] After the culture was completed, the supernatant was collected. An ELISA kit was used to determine the effects of different concentrations of the antimicrobial peptide MS-PT and its modified peptide MS-PT1 on the production of inflammatory cytokines (TNF-α, IL-6) in the induced cells. A standard curve was plotted based on the detection results of the standard products in the kit, and the concentration values of TNF-α or IL-6 were calculated. The experiment was repeated three times.

[0039] The results are as Figure 1-2As shown in the results, the antimicrobial peptide MS-PT was able to inhibit the expression of LPS-induced proinflammatory factors IL-6 and TNF-α in mouse peritoneal macrophages at a concentration of 20 μM, while the modified antimicrobial peptide MS-PT1 was able to significantly inhibit the expression of proinflammatory factors at a concentration of 10 μM, indicating that the antimicrobial peptide MS-PT1 has a strong anti-inflammatory activity.

[0040] (2) Isothermal titration calorimetry determination of the binding of antimicrobial peptide MS-PT1 to LPS

[0041] First, LPS was dissolved in PBS (10 mM) buffer at pH = 6.0 to a final concentration of 25 μM, vortexed for 10 min, ultrasonically vibrated for 10 min to remove gas, and 700 μL was added to the ITC micro-calorimeter to ensure that there were no bubbles during the addition process. The antimicrobial peptide MS-PT1 was then dissolved in the same buffer to 1 mM, ultrasonically vibrated for 10 min, and 50 μL was added to the sample syringe. The program was set to titrate 20 times, with an interval of 2 minutes, each titration amount was 2.5 μL, the titration temperature was 37°C, and the change in the heat of each titration was measured. The thermodynamic parameters such as the dissociation constant Ka, the binding constant Ka, the melting value △H and the moisture change value △S in the process of antimicrobial peptide binding to LPS were analyzed and processed by the analysis software provided by the instrument.

[0042] ITC is a calorimeter used to detect continuous changes in heat. During the titration, the interaction of the reactants triggers a binding reaction to form a macromolecule / ligand complex. The formation of the complex is often accompanied by the release and absorption of energy. However, the reaction system needs to be kept at the experimental temperature at all times. Therefore, the feedback system will provide or reduce heat to compensate for the temperature change of the reaction pool. This can directly reflect the relationship between the binding of the compound and the heat change. The binding process of MS-PT1 and LPS is accompanied by changes in thermodynamic parameters. Therefore, ITC is used to detect heat changes at 37°C to measure the degree of binding between MS-PT1 and LPS. Figure 3 As shown in the figure, a positive enthalpy change (∆H>0) is generated during the reaction between LPS and MS-PT1, which is an endothermic reaction. In addition, the reaction between MS-PT1 and LPS tends to be saturated during the last three titrations, indicating that the reaction between the two has reached a saturated state.

[0043] (3) Zeta potential detection of the binding of antimicrobial peptide MS-PT1 to LPS;

[0044] Dissolve LPS in ultrapure water to a final concentration of 50 μM, vortex for 10 min, and ultrasonically oscillate for 10 min to remove gas. At the same time, prepare antimicrobial peptide MS-PT1 at different concentrations with the same buffer, add the LPS working solution so that the molar ratio of antimicrobial peptide to LPS is 0 - 5, vortex for 10 min, and incubate at room temperature for 0.5 h. Measure the Zeta potential using a NanoZS 90 device based on the principle of phase analysis light scattering, and take the average value of three measurements for each group; use the Helmholtz-Smoluchowski equation to calculate the zeta potential from the mobility of aggregates in a driving electric field of 19.2 V·cm-1.

[0045] Due to the internal phosphate groups, LPS is overall electronegative, while MS-PT1 is overall positively charged. Therefore, there will be a change in charge during the interaction between MS-PT1 and LPS. As Figure 4 shown, in the absence of LPS, the Zeta potential value of MS-PT1 is greater than 0. In the presence of LPS, the Zeta potential value of LPS is less than 0. However, with the addition of MS-PT1, the positively charged MS-PT1 can bind to the negatively charged LPS to produce a charge compensation phenomenon, gradually neutralizing and reducing the negative charge, which shows a dose-dependent relationship with MS-PT1. As the concentration of MS-PT1 increases, the negative charge of LPS is gradually neutralized. When the molar ratio of MS-PT1 to LPS is 7.5, the negative charge is completely neutralized and the potential value is greater than 0. At this time, a charge over-compensation phenomenon may occur in the solution, and MS-PT1 may insert into the hydrophobic molecules of LPS through hydrophobic interaction after binding to LPS through electrostatic force.

[0046] (4) Detection of the neutralization effect of antimicrobial peptide MS-PT1 on LPS by Limulus reagent

[0047] The experiment of antibacterial peptide neutralizing LPS was detected using a Limulus amebocyte lysate (LAL) kit (Xiamen Limulus Reagent Biotechnology Co., Ltd., China). The experiment was carried out according to the experimental protocol provided by the seller. Stock solutions of antibacterial peptides MS-PT and MS-PT1 and the positive control drug PMB were prepared in pyrogen-free water supplied with the kit. Antibacterial peptides MS-PT, MS-PT1 or the positive control drug PMB at concentrations of 100, 50, 25, 15, 8, 4, 2, 1 and 0 μM were incubated with a 1 EU / mL LPS working solution in a non-pyrogenic flat-bottom 96-well cell culture plate at 37 °C for 30 min to allow them to bind to LPS. Then 50 μL of LAL reagent was added to an equal volume of the peptide and LPS mixture, and the mixture was incubated for another 16 min. Subsequently, 100 μL of chromogenic substrate working solution was added and incubated at 37 °C for 6 min. The reaction was terminated by adding a dilute sulfuric acid reaction termination solution, and the absorbance value was read at a wavelength of 405 nm. The time from the end of the reaction to reading the absorbance value should not exceed 5 hours.

[0048] Limulus reagents are mainly extracted from amoebocytes in the blood of marine organisms Limulus. A very small amount of LPS can activate its procoagulant enzyme and then react with specific substrates to produce a color reaction. Therefore, we can examine the change in the OD value of the color reaction to quantitatively detect the concentration of LPS, thereby indirectly reflecting the neutralizing ability of antibacterial peptides against LPS. This reaction has strong specificity and sensitivity. As Figure 5 shown, both MS-PT, MS-PT1 and the positive drug PMB can significantly inhibit the activation of LPS-mediated LAL coagulase, showing a concentration-dependent relationship. Moreover, MS-PT1 neutralized more than 80% of LPS at a concentration of 5 μM.

[0049] Example 2: Antiseptic activity of antibacterial peptide MS-PT1

[0050] (1) Establishment of a murine model of septic shock

[0051] To examine the therapeutic effect of the antimicrobial peptide MS-PT1, an endotoxemia model was established by injecting LPS into mice via the tail vein. An experimental murine sepsis model was established using Salmonella typhosa LPS (Sigma-Aldrich) at a concentration of 20 mg / kg body weight. The blank control group received no drug treatment and was given an equal volume of 0.9% sterile saline by tail vein injection twice, with an interval of 1 h between the injections; in the LPS modeling treatment group, after injecting the LPS injection solution at a concentration of 20 mg / kg body weight via the tail vein, an equal volume of 0.9% sterile saline was injected 1 h later; in each of the MS-PT and MS-PT1 treatment groups, after injecting the LPS solution at a concentration of 20 mg / kg body weight, a single injection of the MS-PT solution at a concentration of 20 mg / kg body weight and the MS-PT1 solution at a concentration of 10 mg / kg body weight were respectively given via the tail vein. The survival rate of the mice within 72 h after treatment was statistically analyzed.

[0052] As Figure 6 shown, in the LPS-induced murine sepsis model, mice that did not receive antimicrobial peptide treatment within the subsequent 1 h all died within 48 h. In contrast, after treatment with MS-PT and MS-PT1, the survival rate and survival time of the mice were greatly improved. Their survival rates within seven days were as follows: more than 80% in the MS-PT (20 mg / kg) treatment group; 100% in the MS-PT1 (10 mg / kg) treatment group.

[0053] Example 3: Anti-inflammatory activity of the modified antimicrobial peptide 6A

[0054] The modified antimicrobial peptide 6A was synthesized by Jiangsu GenScript Biotechnology Co., Ltd. through solid-phase synthesis, with a purity > 90%, meeting the requirements of relevant experiments for activity detection.

[0055] (1) Determination of the anti-inflammatory activity of the modified antimicrobial peptide 6A

[0056] Primary murine peritoneal macrophages (MPMs) were derived from male C57BL / 6. The cells were cultured in an incubator at 37 °C with 5% CO2. After 2 h, the cells were replaced with fresh RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin double antibody (P / S) and cultured overnight. The cells were stimulated with LPS at a final concentration of 0.5 μg / mL. The experimental groups were set as follows: experimental group, blank group, induction group, and positive control group:

[0057] Blank group: Normal culture for 2 h + 12 h;

[0058] Induction group: Incubated with LPS (0.5 μg / mL) in the incubator for 2 h and then stimulated for 12 h;

[0059] Experimental group: Incubate LPS (0.5 μg / mL) and the sample in an incubator for 2 h, then stimulate and culture for 12 h;

[0060] Positive control group: Incubate LPS (0.5 μg / mL) and the positive control (10 μM polymyxin B (PMB)) in an incubator for 2 h, then stimulate and culture for 12 h;

[0061] After the culture is completed, collect the supernatant. Use an ELISA kit to measure the effects of different concentrations of the modified antimicrobial peptide 6A on the production of inflammatory cytokines (TNF-α, IL-6) in induced cells. Draw a standard curve according to the detection results of the standard products in the kit, calculate the concentration values of TNF-α or IL-6, and repeat the experiment three times.

[0062] The results are as Figure 7-8 shown. The modified antimicrobial peptide 6A can significantly inhibit the expression of the pro-inflammatory factors TNF-α and IL-6 induced by LPS in mouse peritoneal macrophages, indicating that the modified antimicrobial peptide 6A has strong anti-inflammatory activity.

[0063] (2) The modified antimicrobial peptide 6A inhibits the activation of the NF-κB and MAPK signaling pathways induced by LPS

[0064] Seed mouse peritoneal primary macrophages into 6-well plates, set up the experimental group, blank group, induction group, and positive control group according to the method in (1), culture for 15 min, discard the supernatant after the culture is completed, and fully lyse with a protein lysate pre-mixed with phosphatase inhibitors and protease inhibitors (inhibitor: lysate = 1:100). Measure the protein concentration by the Coomassie brilliant blue (Bradford) method, prepare samples according to the required protein loading amount, separate the samples by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis, transfer them to a polyvinylidene fluoride membrane (PVDF membrane) by wet transfer method, image with a Bio-Rad gel imaging system, and finally use Image J software to statistically analyze the gray values of each band and plot the graph.

[0065] Since the NF-κB and MAPKs signaling pathways are classical downstream signaling pathways of LPS / TLR4, we studied the effects of the modified antimicrobial peptide 6A on the NF-κB and MAPKs signaling pathways. Based on the results of the previous experiment, we selected concentrations of 5 and 10 μM for the mechanism study. As Figure 9 shown, compared with the control group, LPS significantly increased the phosphorylation of ikkα / β, JNK, ERK, P38 and the degradation of IκBα.

[0066] (3) Interaction between the modified antimicrobial peptide 6A and the key protein MD2 on the TLR4 signaling pathway

[0067] Primary peritoneal macrophages of mice were seeded into medium dishes, and protein samples were obtained according to method (1). First, non-specific binding was removed using protein A+G agarose magnetic beads. After 2 h, the supernatant was aspirated, 2 μg of IgG / TLR4 antibody was added, and the mixture was placed on a mixer at 4 °C and incubated overnight. The next day, 20 μL of fresh protein A+G magnetic beads was added to the above supernatant, and the mixture was placed on a mixer at 4 °C and incubated for 2 h. Subsequently, the supernatant was discarded, protein samples were prepared, and immunoblot analysis was performed according to the method in (2).

[0068] The modified antimicrobial peptide 6A can exert anti-inflammatory activity by inhibiting the activation of key proteins in the TLR4 downstream pathway. We predicted that the anti-inflammatory effect of the modified antimicrobial peptide 6A is mediated by its action on the upstream molecule MD2, as Figure 10 shown, treatment of primary peritoneal macrophages of mice with the modified antimicrobial peptide 6A (10 μM) can inhibit the formation of the MD2-TLR4 complex induced by LPS.

[0069] Example 4: Anti-sepsis activity of antimicrobial peptide 6A

[0070] (1) Construction of a mouse sepsis model

[0071] Male C57BL / 6 mice were randomly divided into four groups, including a control group (CON), a CLP model group (CLP), a CLP model + high-dose administration group of the modified antimicrobial peptide 6A (CLP+6A 10 mg / kg), and a CLP model + low-dose administration group of the modified antimicrobial peptide 6A (CLP+6A 5 mg / kg). A mouse sepsis model was constructed by cecal ligation and puncture (CLP): Each mouse was anesthetized by intraperitoneal injection of 0.2 mL of pentobarbital sodium (100 mg / kg), the hair was shaved and disinfected, laparotomy was performed in a sterile area, the cecum was exposed, ligated at a position 1 cm from the end, and punctured twice with a 16-gauge needle. Slight extrusion was performed to release a small amount of intestinal contents, then the cecum was returned to the abdominal cavity, the abdominal skin was sutured, and epidermal disinfection was performed. At the same time, mice in the sham operation group underwent the same procedure without ligation or puncture. All mice were immediately injected subcutaneously with normal saline and kept warm after the operation until the mice regained consciousness, and then they were returned to the cage for observation. At 30 min after CLP, the modified antimicrobial peptide 6A or normal saline was injected respectively, and serum was collected 6 h after CLP and the levels of inflammatory factors were measured.

[0072] (2) Determination of TNF-α and IL-6 in the serum of the mouse sepsis model

[0073] It was the same as the determination method in Example 1 (1). As Figure 11-12As shown, compared with the control group, the levels of serum IL-6 and TNF-α in CLP model mice were significantly increased. After treatment with the modified antimicrobial peptide 6A, the increase in these indicators was significantly alleviated, indicating that the modified antimicrobial peptide 6A effectively alleviated the large-scale systemic inflammation caused by sepsis.

[0074] The above-described embodiments only express several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this patent, several deformations, combinations, and improvements can be made to the above-mentioned embodiments, and these all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be subject to the claims.

Claims

1. A modified antimicrobial peptide 6A of a South American frog antimicrobial peptide, characterized in that: The modified antimicrobial peptide 6A contains 18 amino acid residues, with a molecular weight of 1811.33 Daltons, and its amino acid sequence is shown in SEQ ID NO.

3.

2. Use of the modified antimicrobial peptide of the South American frog antimicrobial peptide according to claim 1, characterized in that: The application of the modified antimicrobial peptide 6A for the preparation of potential anti-inflammatory and anti-sepsis drugs.

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