Antibacterial peptide P1 and medical application thereof in treating sepsis

Abstract

The invention discloses an antibacterial peptide P1 and medical application thereof in treating sepsis. The amino acid sequence of the antibacterial peptide P1 is as shown in SEQ ID NO. 1. The antibacterial peptide P1 provided by the invention has a good treatment effect on a sepsis mouse CLP model. The antibacterial peptide P1 can play a good bactericidal effect by directly destroying cell walls and cell membranes of staphylococcus aureus; the antibacterial peptide P1 also has bacterial capture ability and immunomodulatory activity, and can relieve survival rate reduction induced by sepsis, liver and lung injury caused by sepsis, liver and lung injury caused by sepsis, and organ bacterial infection caused by sepsis. Cell walls and cell membranes of staphylococcus aureus are directly destroyed, and a network structure is formed in a hydrophobic environment to capture bacteria and promote phagocytosis of immune cells. The compound is a good novel antibiotic substitute product, and can be used for preparing drugs for treating sepsis.

Description

Technical Field

[0001] The present invention belongs to the field of drug use, and in particular, relates to an antimicrobial peptide P1 and its medical use in treating sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome triggered by pathogen infection and may further develop into septic shock leading to multiple organ failure. Although significant progress has been made in the treatment of sepsis, the mortality rate of patients remains high. Global data analysis shows that approximately 48.9 million patients suffer from sepsis each year, of which an estimated 11 million die from it. A meta-analysis published in 2020 showed that the global annual incidence of sepsis exceeds 189 / 100,000 people, and the mortality rate reaches 26.7%, posing a serious threat to human health and placing a heavy burden on the global society and economy.

[0003] Bacterial infection is the main cause of sepsis, which triggers the dysregulation of the immune system. With the invasion of pathogens and the overactivation of the innate immune response, the inflammatory response is further aggravated. Although antibiotics are commonly used drugs for the treatment of sepsis, their widespread and excessive use not only puts a huge burden on organs such as the liver and kidneys, but also easily causes dysbiosis in the body, thus seriously weakening the treatment effect.

[0004] Antimicrobial peptides (AMPs) are a class of naturally occurring short-chain polypeptides (consisting of 10 to 100 amino acid residues) that can inhibit the growth of microorganisms by inducing cell lysis by destroying the integrity of the bacterial cell wall. They have the characteristics of rapid killing and broad-spectrum antibacterial activity, remain active even at micromolar concentrations, are not prone to inducing drug resistance, and have fewer side effects. Therefore, antimicrobial peptides have become the research focus of replacing traditional antibiotics in recent years, but there are still few examples of real conversion into drugs. Based on this, the development of new and highly effective antimicrobial peptides remains an important research direction in the field of sepsis treatment. Summary of the invention

[0005] The present invention aims to promote the treatment of sepsis and overcome the defects of the above-mentioned antibiotics, and provides an antimicrobial peptide P1, which is an ideal substitute for antibiotics. The amino acid sequence of the antimicrobial peptide P1 is shown in SEQ ID NO.1: KILRLLGYLLGLW.

[0006] Another object of the present invention is to provide the use of the antimicrobial peptide P1 in the preparation of a drug for treating sepsis caused by surgery. The antimicrobial peptide P1 can treat or alleviate sepsis infection and inhibit bacterial growth.

[0007] Studies have shown that the antimicrobial peptide P1 of the present invention can improve the survival rate of the sepsis CLP model and can be used to prepare drugs for treating and alleviating the reduced survival rate induced by sepsis.

[0008] The antimicrobial peptide P1 of the present invention can improve the liver and kidney function damage caused by the sepsis CLP model and can be used to prepare drugs for treating and alleviating the liver and kidney function damage induced by sepsis.

[0009] The antimicrobial peptide P1 of the present invention can reduce the pathological damage of the liver and lungs caused by the sepsis CLP model, and can be used to prepare a drug for treating and alleviating the liver and lung damage caused by sepsis.

[0010] The antimicrobial peptide P1 of the present invention can reduce the bacterial load of organs in the sepsis CLP model, and can be used to prepare drugs for treating and alleviating bacterial infection of organs caused by sepsis.

[0011] The bactericidal principle of the antimicrobial peptide P1 of the present invention is to directly destroy the cell wall and cell membrane of Staphylococcus aureus, and can be used to prepare a drug that directly destroys the cell wall and cell membrane of Staphylococcus aureus.

[0012] The antimicrobial peptide P1 of the present invention can form a network structure in a hydrophobic environment.

[0013] The antimicrobial peptide P1 of the present invention can promote the phagocytosis of RAW264.7 cells by Staphylococcus aureus, and can be used to prepare a drug that forms a network structure in a hydrophobic environment to capture bacteria and promote the phagocytosis of immune cells.

[0014] The antimicrobial peptide P1 provided by the present invention has a good therapeutic effect on the CLP model of septic mice; the antimicrobial peptide P1 has a good bactericidal effect and can exert a bactericidal effect by directly destroying the cell wall and cell membrane of Staphylococcus aureus; P1 also has bacteria capture ability and immunomodulatory activity; it is a good new antibiotic substitute product and can be used to prepare a new drug for treating sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 .Survival curve of the mouse sepsis CLP model treated with antimicrobial peptide P1.

[0016] Figure 2 . Detection results of serum biochemical indicators related to liver and kidney damage in the mouse sepsis CLP model treated with antimicrobial peptide P1.

[0017] Figure 3 .H&E staining light microscopy results of liver and lung pathology in the mouse sepsis CLP model treated with antimicrobial peptide P1 (magnification of each group: ×200).

[0018] Figure 4 . Detection of bacterial load in mouse liver, kidney and lung.

[0019] Figure 5.Electron microscopic examination results after the antimicrobial peptide P1 was co-incubated with Staphylococcus aureus for 1 hour. Figure 5 A is the transmission electron microscopy result. Figure 5 B is the scanning electron microscope detection result.

[0020] Figure 6 . Transmission electron microscopy results of antimicrobial peptide P1 in different solvents (H2O and 50% TFE).

[0021] Figure 7 .Immunofluorescence detection results after co-incubation of Staphylococcus aureus (green fluorescence) treated with antimicrobial peptide P1 and RAW264.7 cells (blue fluorescence).

[0022] Figure 8 .Flow cytometric detection results of RAW264.7 cells phagocytosing Staphylococcus aureus without antimicrobial peptide P1, after antimicrobial peptide P1 treatment for 3 hours, and after antimicrobial peptide P1 treatment for 18 hours. Specific implementation methods

[0023] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0024] Example 1 The solid phase synthesis method includes: (1) functionalization of the solid phase carrier: covalently linking the first amino acid to the solid phase carrier; (2) removal of the amino protecting group: removing the protecting group on the amino acid to expose the amino group; (3) amino acid coupling: activating the next amino acid and coupling it with the amino group on the solid phase to extend the polypeptide chain; (4) repeated cycles: sequentially removing the protecting group and coupling to gradually extend the polypeptide chain; (5) polypeptide cleavage and purification: cutting the polypeptide from the solid phase carrier, removing the protecting group, and purifying to obtain the target polypeptide, and finally synthesizing the antimicrobial peptide P1.

[0025] Table 1. Sequences of antimicrobial peptide P1 ID sequence P1 KILRLLGYLLGLW

[0026] Example 2 Antimicrobial peptide P1 effectively inhibits the growth of Gram-positive bacteria in vitro The antimicrobial activity of the peptides was evaluated using a standard broth microdilution protocol according to the MIC assay protocol of the Clinical and Laboratory Standards Institute (CLSI). After the bacteria were cultured to the logarithmic growth phase, they were washed three times with PBS and then standardized to a final bacterial cell solution (0.5-1×10 6 The antimicrobial peptide was dissolved in bovine serum albumin solution (0.2% BSA, 0.01% acetic acid solution) with an initial concentration of 512 μg / mL, and then serially diluted 2-fold in a 96-well plate, and an equal volume of bacterial suspension was added to each well to make the final bacterial concentration of 5×10 5CFU / mL. Untreated bacterial culture and medium were implemented as positive and negative control wells. Incubate at 37°C for 18-24 hours, and the MIC value is the lowest peptide concentration at which no obvious bacterial growth is observed by naked eye.

[0027] As shown in Table 2, the MIC of P1 against Gram-positive bacteria: Staphylococcus aureus (ATCC 25923, USA 300), Bacillus subtilis (6633), Enterococcus faecalis (29212) are all between 4-16 μg / mL; the MIC of P1 against Gram-negative bacteria: Pseudomonas aeruginosa (15442), Acinetobacter baumannii (19606), Escherichia coli (25922), Salmonella typhimurium (14028) are all greater than 128 μg / mL. P1 has a good antibacterial effect on Gram-positive bacteria and is a narrow-spectrum antimicrobial peptide.

[0028] Table 2. Antibacterial activity of P1 against 8 standard bacteria Note: a: minimum inhibitory concentration; all quality controls were within the allowable range, all growth control wells were turbid, and blank control wells were clear.

[0029] Example 3 Antimicrobial peptide P1 effectively improves the mortality rate of sepsis model mice This study used 6-8 week old male C57BL / 6J mice, randomly divided into sham operation (Sham), sepsis model (CLP) and antimicrobial peptide (CLP+P1) groups according to body weight, with 10 mice in each group. The sepsis mouse model was established as described in the literature. Before model establishment, 0.25 mL of sodium pentobarbital (40 mg / kg) was injected intraperitoneally for anesthesia. After confirming anesthesia, the mouse was fixed on a foam board in a supine position. The mouse's abdominal hair was shaved with a razor, and then the mouse's abdomen was disinfected with a 75% alcohol cotton ball. A longitudinal incision (about 1 cm in length) was made along the linea alba on the lower right side of the mouse's abdomen with ophthalmic straight scissors to fully expose the abdominal cavity. The cecum was found with blunt ophthalmic forceps, and the cecum was ligated from the root of the cecum to about 1 cm from the distal end. The cecum at the ligation site was punctured with a 19-gauge needle, and part of the intestinal contents were squeezed out from the puncture hole. The cecum was put back to its original place, and the peritoneum and skin were sutured in sequence to close the abdominal cavity. After the operation, the mouse was placed on an electric blanket for warmth, and 0.3 mL of sterile saline preheated at 37°C was subcutaneously injected for resuscitation. The CLP+P1 group was injected with P1 (10 mg / kg) intraperitoneally 1 h after model establishment. The difference between the Sham group and the CLP group is that the cecum is not ligated and punctured, and the other steps are the same. After the mice wake up, they are placed in a supine position in a cage with bedding, and food and water are added. The temperature is controlled at about 26°C. The survival rate of mice in each group is observed and counted every 1 hour for the first 24 hours, and every 2 hours for the next 12 hours.

[0030] from Figure 1 It can be seen that all mice in the CLP group died within 24 hours after modeling (percent survival 100%→0%); the survival rate of the CLP+P1 group was 50% at 24 hours after modeling, and the survival rate was 30% at 36 hours. Compared with the CLP group, the average survival rate of mice in the CLP+P1 group was significantly increased (**P<0.01), indicating that P1 has a good anti-infection effect.

[0031] Example 4 Antimicrobial peptide P1 effectively reduces organ damage and bacterial load in sepsis model mice The evaluation method of this experiment is: the mouse model of sepsis was established by cecal ligation and puncture, and the serum, liver, kidney and lung of the mouse were collected 12 hours after the model was established. The serum was sent for examination to examine the indicators related to liver and kidney damage; the liver and lung sections were embedded and H&E staining was performed to evaluate organ damage; the liver, kidney and lung homogenates were diluted and the bacterial load in the liver, kidney and lung was evaluated by the plate coating method.

[0032] This experiment used 6-8 week old male C57BL / 6J mice, randomly divided into groups according to body weight, 7 mice in each group, and housed in separate cages. They were divided into CLP group and CLP+P1 group. The mice were weighed 12 hours after modeling, and then the mice were killed and their serum, liver, kidney and lung were collected.

[0033] Evaluation of organ damage: 1) After removing the eyeballs and taking blood, let it stand at room temperature for 20 minutes, then centrifuge it at 4°C, 3500rpm for 15 minutes, aspirate the supernatant into a new EP tube, and send the serum for testing of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (CREA), and lactate dehydrogenase (LDH). 2) H&E staining: The liver and lung tissues of mice in each group were collected and fixed with 4% paraformaldehyde solution. Ⅰ) The tissue was removed from the paraformaldehyde and placed in an embedding box. After dehydration with different concentrations of ethanol, the tissue was embedded in paraffin and sliced ​​into paraffin sections with a thickness of 5 μm. Ⅱ) Dehydration of paraffin sections: the paraffin sections were sequentially placed in xylene I for 10 minutes and xylene II for 10 minutes; the sections were sequentially placed in anhydrous alcohol, 95%, 90%, 80%, and 70% alcohol for 10 minutes each, and then washed with distilled water. Ⅲ) Hematoxylin staining: the sections were placed in hematoxylin staining solution for 3-5 minutes, rinsed with deionized water for 10 minutes, differentiated with differentiation solution, and rinsed with deionized water for 10 minutes again. IV) Eosin staining: The sections were dehydrated in alcohol solutions with a gradient of 70%, 80%, 90%, and 95% alcohol concentrations, respectively, for 2 minutes each, and then stained in eosin solution for 5 minutes; finally, they were transparentized with xylene; V) After taking out the sections, an appropriate amount of neutral gum was quickly added before the xylene dried, and finally, they were sealed with a coverslip and observed under an upright microscope.

[0034] Measurement of bacterial load: The liver, kidney and lung used to measure the bacterial load need to be cut into small pieces of similar size, rinsed in sterile PBS and placed in 1.5mL EP tubes containing a steel ball, add 1mL sterile PBS, and grind with a tissue grinder at 60Hz for 2 minutes. After grinding, put the EP tube into a centrifuge and centrifuge at 4°C, 600rpm for 3 minutes. Pipette out 100μL of supernatant into a new EP tube, add sterile PBS to gradually dilute to 100,000 times, vortex to mix, and evenly spread it on the bacterial culture dish by flat coating method. Place the culture dish in a bacterial incubator at 37°C for one day, count the number of colonies every other day, and evaluate the bacterial load in the liver, kidney and lung tissues. The results are as follows Figure 2-4 shown.

[0035] Figure 2 Serum biochemical results of septic mice without and with P1 injection. Figure 3 H&E staining results of lung and liver pathological sections of septic mice without P1 injection and with P1 injection. Figure 4 Statistical results of bacterial loads in the liver, kidney, and lung of septic mice without and with P1 injection.

[0036] according to Figure 2-4 It can be seen that P1 treatment can effectively reduce liver and kidney injury related indicators such as ALT, AST, BUN, and CREA, indicating that P1 can resist liver and kidney damage caused by CLP; the pathological H&E staining results of the liver and lungs showed that P1 can reduce the liver necrosis area and inflammatory foci caused by CLP, and improve severe lung inflammation, alveolar wall thickening and inflammatory cell infiltration; 12 hours after CLP modeling, the bacterial load in the liver, kidney and lung was as high as 10 10 CFU / g, P1 treatment can reduce the bacterial load in the liver and kidney by 0.2logs, and in particular, it can reduce the bacterial load in the lung by 0.5logs.

[0037] Example 5 Antimicrobial peptide P1 can directly destroy the cell wall and cell membrane structure of Staphylococcus aureus Staphylococcus aureus S.aureus was cultured to the logarithmic growth phase, and the bacteria were collected by low-speed centrifugation at 4000r / min for 5min. The bacteria were washed three times with PBS to remove impurities attached to the surface of the bacteria. The bacteria were treated with P1 at a concentration of 2×MIC as the experimental group, and the bacteria were treated with an equal volume of PBS as the blank group. The bacteria and the treatment solution were fully mixed, incubated at 37°C for 1h, and then centrifuged at 4000r / min for 5min, the supernatant was discarded, and the bacterial precipitate was retained.

[0038] Subsequently, glutaraldehyde electron microscopy fixative (2.5%, w v-1) was added to fix at room temperature for 2 hours, and then placed at 4°C for overnight fixation. For transmission electron microscopy (TEM) observation, after overnight fixation, the samples were dehydrated in gradient ethanol solutions (30%, 50%, 70% and 80%) for 15 minutes each, and then dehydrated with gradient acetone solutions (90%, 95%) for 15 minutes each. Subsequently, the samples were dehydrated twice with anhydrous acetone for 20 minutes each. The sample was placed in a 1:1 mixture of anhydrous acetone and Spurr resin for 1 hour at room temperature, then transferred to a 1:3 mixture of anhydrous acetone and resin for 3 hours, and finally transferred to pure Spurr resin overnight. The sample was placed in an EP tube containing Spurr resin and heated at 70°C for 10 hours.

[0039] The samples were sliced ​​using a LEICA EMUC7 ultramicrotome, stained with uranyl acetate and alkaline lead citrate for 10 minutes, and observed using a Hitachi Model H-7650TEM. For scanning electron microscopy (SEM), the samples were washed three times in phosphate buffer (0.1M, pH 7.0) for 15 minutes each after overnight fixation, then post-fixed in 1% osmium tetroxide (OsO4) phosphate buffer for 1 hour, and then washed three times in PBS for 15 minutes each. The samples were dehydrated in gradient ethanol solutions (30%, 50%, 70%, 80%, 90% and 95%) for 15 minutes each, and then dehydrated twice with anhydrous ethanol for 20 minutes each. Finally, the samples were fully dried in a Hitachi Model HCP-2 critical point dryer, and the bacterial cells were sprayed with gold for 4-5 minutes on a HitachiModel E-1010 ion gold plater, and observed using a Hitachi Model SU-8010SEM.

[0040] The results are as follows Figure 5 As shown in A and B, the normal S. aureus cells that were not treated with antimicrobial peptides were plump and spherical. The S. aureus cells treated with P1 had blurred edges and adhesion between cells, showing signs of cell wall dissolution, membrane damage, and extensive cytoplasm leakage. It is speculated that P1 can achieve a bactericidal effect by destroying the cell wall and cell membrane structure of S. aureus, causing leakage of cell contents.

[0041] Example 6 Antimicrobial peptide P1 can capture Staphylococcus aureus through self-assembly 25 mg of antimicrobial peptide P1 sample was dissolved in 1 mL of pure water and 1 ml of 50% TFE, and the sample was dropped on the carbon film on the TEM grid, followed by glutaraldehyde electron microscopy fixative (2.5%, w v-1) for 2 h at room temperature, and then placed at 4 ° C overnight for fixation. For transmission electron microscopy (TEM) observation, after overnight fixation, the sample was dehydrated with gradient ethanol solutions (30%, 50%, 70% and 80%) for 15 minutes each, and then dehydrated with gradient acetone solutions (90%, 95%) for 15 minutes each. Subsequently, the sample was dehydrated twice with anhydrous acetone for 20 minutes each. The sample was placed in a 1:1 mixture of anhydrous acetone and Spurr resin for 1 hour at room temperature, then transferred to a 1:3 mixture of anhydrous acetone and resin for 3 hours, and finally transferred to pure Spurr resin overnight. The sample was placed in an EP tube containing Spurr resin and heated at 70 ° C for 10 hours.

[0042] The samples were sliced ​​using a LEICA EMUC7 ultramicrotome, stained with uranyl acetate and alkaline lead citrate for 10 minutes respectively, and observed using a Hitachi Model H-7650TEM. The results are shown in Figure 6 The antimicrobial peptide P1 shown in the figure presents a uniform vesicle-like nanostructure in aqueous solution. However, in the hydrophobic environment of 50% TFE, the vesicle-like nanostructure will continue to aggregate as a basic unit to form a larger vesicle structure, and even further aggregate into agglomerates. Based on this, it is speculated that the vesicle-like nanostructured P1 may begin to aggregate into a larger nanostructure after contacting the hydrophobic bacterial cell wall, thereby capturing bacteria.

[0043] RAW264.7 cells were cultured to a density of 70-80% in DMEM high-glucose medium containing 10% FBS. FITC fluorescently labeled Staphylococcus aureus S.aureus was cultured to the logarithmic growth phase, and the bacteria were collected by low-speed centrifugation at 4000r / min for 5min. The bacteria were washed three times with PBS and suspended in PBS. The bacterial suspension was added to the RAW264.7 cell culture dish. At the same time, 1×MIC concentration of P1 was added to co-incubate with bacteria and RAW cells as the experimental group, and an equal volume of PBS was used as the blank group for incubation at 37°C for 1h. After the co-incubation, the supernatant was discarded, and PBS was added to wash three times for 5 minutes each time. 1μl / ml Hoechst staining solution was used for nucleus staining for 10min. After the staining was completed, the supernatant was discarded, and PBS was added to wash three times for 5 minutes each time. The stained samples were observed under a fluorescence microscope.

[0044] The results are as follows Figure 7As shown, the proportion of bacteria phagocytosed by RAW264.7 cells treated with antimicrobial peptide P1 was significantly higher than that of bacteria phagocytized by RAW264.7 cells not treated with antimicrobial peptide P1. It is speculated that P1 may begin to aggregate to form larger nanostructures after contacting the hydrophobic bacterial cell wall, which is easier to capture bacteria and more likely to activate the phagocytosis of bacteria by macrophages.

[0045] Example 7 Antimicrobial peptide P1 has an immunomodulatory effect on macrophages RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS to a density of 70-80%. Staphylococcus aureus S.aureus labeled with PI fluorescence was cultured to the logarithmic growth phase, and the bacteria were collected by low-speed centrifugation at 4000r / min for 5min, and the bacteria were washed three times with PBS and suspended in PBS. The bacterial suspension was added to the RAW264.7 cell culture dish. At the same time, 1×MIC concentration of P1 was added to co-incubate with bacteria and RAW264.7 cells as the experimental group, and an equal volume of PBS was used as the blank group, and incubated at 37°C for 20min. After the co-incubation, the supernatant was discarded, and PBS was added for washing three times, each time for 5 minutes. The cells were carefully scraped with a cell scraper, and the cells were collected by low-speed centrifugation at 1500rpm / min. 200μl PBS was added to resuspend the cells, and 0.5μl of FITC fluorescent-labeled F4 / 80 antibody was added, and incubated in the dark for 15min. The cells were collected by low-speed centrifugation at 1500rpm / min, and then PBS was added for washing three times, each time for 5 minutes. 300μl PBS was added to resuspend the cells and filter the membrane to make a single cell suspension, and then flow cytometry was performed. The results are shown in Figure 2. Figure 8 As shown, the proportion of bacteria phagocytosed by RAW264.7 cells treated with antimicrobial peptide P1 was significantly higher than that of bacteria phagocytized by RAW264.7 cells not treated with antimicrobial peptide P1. It is speculated that in addition to directly killing bacteria, P1 is also more likely to activate the phagocytosis of bacteria by macrophages.

Claims

1. An antimicrobial peptide P1, characterized in that: The amino acid sequence of the antimicrobial peptide P1 is shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide P1 according to claim 1 in the preparation of a drug for treating sepsis.

3. The use according to claim 2, characterized in that: The sepsis is sepsis caused by surgery.

4. The use according to claim 2, characterized in that: The antimicrobial peptide P1 can treat and alleviate the decreased survival rate induced by sepsis, can treat and alleviate the liver and lung damage caused by sepsis, can treat and alleviate the bacterial infection of organs caused by sepsis, directly destroy the cell wall and cell membrane of Staphylococcus aureus, form a network structure in a hydrophobic environment to capture bacteria and promote phagocytosis of immune cells.

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