An antimicrobial peptide P1 and its medical use in treating sepsis

Abstract

The present invention discloses an antimicrobial peptide P1 and its medical use for treating sepsis. The amino acid sequence of the antimicrobial peptide P1 is shown in SEQ ID NO.1. 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 can exert a good bactericidal effect by directly destroying the cell wall and cell membrane of Staphylococcus aureus; the antimicrobial peptide P1 also has bacteria-capturing ability and immunomodulatory activity, alleviating the reduced survival rate induced by sepsis, relieving liver and lung damage caused by sepsis, alleviating liver and lung damage caused by sepsis, and alleviating bacterial infection of organs caused by sepsis. It directly destroys the cell wall and cell membrane of Staphylococcus aureus, forms a network structure in a hydrophobic environment to capture bacteria and promote phagocytosis of immune cells. It is a good new antibiotic alternative product that can be used to prepare drugs for treating sepsis.

Description

Technical Field

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

[0002] Sepsis is a systemic inflammatory response syndrome triggered by pathogen infection that can progress to septic shock and multiple organ failure. Despite significant advances in sepsis treatment, the mortality rate remains high. Global data analysis shows that approximately 48.9 million patients suffer from sepsis annually, of whom an estimated 11 million die from it. A meta-analysis published in 2020 showed that the global annual incidence of sepsis exceeds 189 per 100,000 people, with a mortality rate of 26.7%, posing a serious threat to human health and placing a heavy burden on global society and the economy.

[0003] Bacterial infection is the primary cause of sepsis, triggering immune system dysfunction. With pathogen invasion and overactivation of the innate immune response, inflammatory responses are exacerbated. Although antibiotics are commonly used to treat sepsis, their widespread and excessive use not only places a significant burden on organs such as the liver and kidneys, but also easily leads to bacterial imbalance in the body, severely weakening the effectiveness of treatment.

[0004] Antimicrobial peptides (AMPs) are a class of naturally occurring short-chain polypeptides (consisting of 10 to 100 amino acid residues) that can inhibit microbial growth by inducing cell lysis by destroying the integrity of bacterial cell walls. They have the characteristics of rapid killing and broad-spectrum antibacterial activity, remain active even at micromolar concentrations, are less likely to induce drug resistance, and have fewer side effects. Therefore, antimicrobial peptides have become a research focus in recent years to replace traditional antibiotics, but there are still few examples of their actual transformation 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 shortcomings of the above-mentioned antibiotics, and provides an antimicrobial peptide P1, which is an ideal alternative to 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 alleviate the pathological damage to the liver and lungs caused by the sepsis CLP model, and can be used to prepare a drug for treating and alleviating 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 infections 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-capturing ability and immunomodulatory activity; it is a good new antibiotic alternative 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 CLP model of sepsis in mice 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 CLP model of sepsis in mice 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 microscopy results after 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 microscopy 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 treatment, antimicrobial peptide P1 treatment for 3 hours, and antimicrobial peptide P1 treatment for 18 hours. Specific implementation methods

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

[0024] Example 1

[0025] 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.

[0026] Table 1. Sequences of antimicrobial peptide P1

[0027] ID sequence P1 KILRLLGYLLGLW

[0028] Example 2 Antimicrobial peptide P1 effectively inhibits the growth of Gram-positive bacteria in vitro

[0029] 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). Bacteria were cultured to the logarithmic growth phase, washed three times with PBS, and then standardized to a final bacterial cell solution (0.5-1×10 6CFU / mL). 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. It was 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 5 CFU / mL. Untreated bacterial culture and culture medium were used as positive and negative control wells. Incubate at 37°C for 18-24 hours. The MIC value is the lowest peptide concentration at which no obvious bacterial growth is observed by the naked eye.

[0030] As shown in Table 2, the MICs of P1 against Gram-positive bacteria, including Staphylococcus aureus (ATCC 25923, USA 300), Bacillus subtilis (6633), and Enterococcus faecalis (29212), were all between 4 and 16 μg / mL. The MICs of P1 against Gram-negative bacteria, including Pseudomonas aeruginosa (15442), Acinetobacter baumannii (19606), Escherichia coli (25922), and Salmonella typhimurium (14028), were all greater than 128 μg / mL. P1 exhibits good antimicrobial activity against Gram-positive bacteria and is a narrow-spectrum antimicrobial peptide.

[0031] Table 2. Antibacterial activity of P1 against 8 standard bacteria

[0032]

[0033] Note: a: minimum inhibitory concentration; all quality controls were within the allowable range, all growth control wells were turbid, and all blank control wells were clear.

[0034] Example 3 Antimicrobial peptide P1 effectively improves the mortality rate of sepsis model mice

[0035] This study used 6-8 week old male C57BL / 6J rats, randomly divided into 10 groups according to body weight and housed in separate cages. The rats were divided into sham operation (Sham), sepsis model (CLP) and antimicrobial peptide (CLP+P1) groups. Sepsis mice were induced as described previously. Before induction, 0.25 mL of sodium pentobarbital (40 mg / kg) was injected intraperitoneally for anesthesia. After confirmation of anesthesia, the mice were fixed in the supine position on a foam board. The abdominal hair was shaved with a razor, and the abdomen was disinfected with 75% alcohol cotton balls. A longitudinal incision (approximately 1 cm in length) was made along the linea alba on the lower right side of the abdomen using ophthalmic straight scissors to fully expose the abdominal cavity. Blunt ophthalmic forceps were used to locate the cecum. The cecum was ligated approximately 1 cm distal to the root of the cecum. A 19-gauge needle was used to puncture the cecum at the ligation site, and intestinal contents were squeezed out of the puncture. The cecum was replaced, and the peritoneum and then the skin were sutured sequentially to close the abdominal cavity. After surgery, the mice were placed on an electric blanket for warmth and resuscitation with 0.3 mL of sterile saline preheated at 37°C. The CLP+P1 group was intraperitoneally injected with P1 (10 mg / kg) 1 h after induction of the model. The sham group differed from the CLP group in that cecal ligation and puncture were not performed; all other procedures were the same. After the mice regained consciousness, they were placed supine in a cage with bedding, provided with food and water, and the temperature was maintained at approximately 26°C. The survival rate of mice in each group was observed and calculated every hour for the first 24 hours and every two hours for the next 12 hours.

[0036] from Figure 1 As shown, all mice in the CLP group died within 24 hours after modeling (percent survival 100% → 0%). The survival rate in the CLP+P1 group was 50% at 24 hours and 30% at 36 hours. Compared with the CLP group, the mean survival rate of mice in the CLP+P1 group was significantly increased (**P < 0.01), indicating that P1 has a strong anti-infection effect.

[0037] Example 4 Antimicrobial peptide P1 effectively reduces organ damage and bacterial load in sepsis model mice

[0038] This study evaluated the effects of cecal ligation and puncture on the establishment of a sepsis mouse model. Twelve hours after initiation, serum, liver, kidney, and lung samples were collected. Serum was analyzed for liver and kidney injury markers. Liver and lung sections were embedded and stained with H&E to assess organ damage. Liver, kidney, and lung homogenates were diluted and plated to assess bacterial loads in the liver, kidney, and lung.

[0039] This study used 6- to 8-week-old male C57BL / 6J mice. They were randomly divided into groups of 7 according to body weight and housed separately. They were divided into a CLP group and a CLP+P1 group. Mice were weighed 12 hours after model establishment and sacrificed. Serum, liver, kidney, and lung samples were collected.

[0040] Assessment of Organ Damage: 1) After removing the eyeballs and collecting blood, the blood was allowed to stand at room temperature for 20 minutes. The blood was then centrifuged at 4°C, 3500 rpm for 15 minutes. The supernatant was transferred to a new EP tube and the serum was analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (CREA), and lactate dehydrogenase (LDH). 2) H&E Staining: Liver and lung tissues were collected from each group and fixed with 4% paraformaldehyde. I) Tissue was removed from paraformaldehyde and placed in an embedding cassette. After dehydration with various concentrations of ethanol, the tissue was embedded in paraffin and serially sectioned into 5 μm thick paraffin sections. II) Dehydration of Paraffin Sections: Paraffin sections were sequentially placed in xylene I for 10 minutes, xylene II for 10 minutes, and then placed in an alcohol concentration gradient of absolute, 95%, 90%, 80%, and 70% alcohol for 10 minutes each, followed by washing with distilled water. III) Hematoxylin Staining: Sections were stained in hematoxylin solution for 3-5 minutes, rinsed with deionized water for 10 minutes, differentiated with differentiation solution, and rinsed again with deionized water for 10 minutes. IV) Eosin staining: Dehydrate the sections in a gradient of 70%, 80%, 90%, and 95% alcohol solutions, each for 2 minutes. Stain in eosin solution for 5 minutes. Finally, clear the sections with xylene. V) After removing the sections, quickly add an appropriate amount of neutral gum before the xylene dries. Finally, seal the sections with a coverslip and observe under an upright microscope.

[0041] 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 a 1.5mL EP tube containing a steel ball, 1mL sterile PBS is added, and the tube is ground at 60Hz for 2 minutes using a tissue grinder. After grinding, the EP tube is placed in a centrifuge and centrifuged at 4°C and 600rpm for 3 minutes. Aspirate 100μL of supernatant into a new EP tube, add sterile PBS and gradually dilute to 100,000 times. After vortex mixing, evenly spread it on the bacterial culture dish by the plate 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.

[0042] Figure 2 Serum biochemical results of septic mice without and with P1 injection. Figure 3 The 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.

[0043] according to Figure 2-4It can be seen that P1 treatment can effectively reduce liver and kidney damage-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 lungs was as high as 10 10 CFU / g, P1 treatment can reduce the bacterial load in the liver and kidney by 0.2 logs, and in particular, it can reduce the bacterial load in the lung by 0.5 logs.

[0044] Example 5 Antimicrobial peptide P1 can directly destroy the cell wall and cell membrane structure of Staphylococcus aureus

[0045] Staphylococcus aureus was cultured to the logarithmic growth phase and harvested by low-speed centrifugation at 4000 rpm for 5 minutes. The bacteria were then washed three times with PBS to remove surface impurities. The experimental group was treated with P1 at a concentration of 2× the MIC, while the blank group was treated with an equal volume of PBS. The bacteria and treatment solution were thoroughly mixed, incubated at 37°C for 1 hour, and then centrifuged at 4000 rpm for 5 minutes. The supernatant was aspirated and the bacterial pellet was retained.

[0046] Subsequently, glutaraldehyde electron microscopy fixative (2.5%, w v-1) was added and fixed 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 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 samples were dehydrated twice with anhydrous acetone for 20 minutes each. The samples were placed in a 1:1 mixture of anhydrous acetone and Spurr resin at room temperature for 1 hour, then transferred to a 1:3 mixture of anhydrous acetone and resin for 3 hours, and finally transferred to pure Spurr resin overnight. The samples were placed in an EP tube containing Spurr resin and heated at 70°C for 10 hours.

[0047] Samples were sectioned using a LEICA EMUC7 ultramicrotome, stained with uranyl acetate and alkaline lead citrate for 10 minutes each, and observed using a Hitachi Model H-7650TEM. For scanning electron microscopy (SEM), after overnight fixation, samples were washed three times for 15 minutes each in phosphate buffer (0.1 M, pH 7.0), then postfixed in 1% osmium tetroxide (OsO4) in phosphate buffer for 1 hour and washed three times in PBS for 15 minutes each. Samples were dehydrated using a graded ethanol solution (30%, 50%, 70%, 80%, 90%, and 95%) for 15 minutes each, followed by two dehydrations in absolute ethanol for 20 minutes each. Finally, samples were thoroughly dried in a Hitachi Model HCP-2 critical point dryer. Bacterial cells were gold-sprayed on a Hitachi Model E-1010 ion gold plater for 4-5 minutes and observed using a Hitachi Model SU-8010 SEM.

[0048] The results are as follows Figure 5 As shown in A and B, normal S. aureus cells, untreated with antimicrobial peptides, exhibit plump, spherical cells. However, S. aureus cells treated with P1 exhibit blurred cell edges and adhesions, showing signs of cell wall dissolution, membrane disruption, and extensive cytoplasmic leakage. It is hypothesized that P1 achieves its bactericidal effect by disrupting the cell wall and membrane structure of S. aureus, leading to leakage of cellular contents.

[0049] Example 6 Antimicrobial peptide P1 can capture Staphylococcus aureus through self-assembly

[0050] A 25 mg sample of the antimicrobial peptide P1 was dissolved in 1 mL of purified water and 1 mL of 50% TFE. The sample was then dropped onto a carbon film on a TEM grid and fixed with 2.5% glutaraldehyde (w / v) for 2 hours at room temperature before being placed at 4°C overnight. For transmission electron microscopy (TEM) observation, after overnight fixation, the sample was dehydrated using a gradient ethanol solution (30%, 50%, 70%, and 80%) for 15 minutes each, followed by a gradient acetone solution (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's resin at room temperature for 1 hour, then transferred to a 1:3 mixture of anhydrous acetone and resin for 3 hours, and finally transferred to pure Spurr's resin overnight. The sample was placed in an EP tube containing Spurr's resin and heated at 70°C for 10 hours.

[0051] The samples were sliced ​​using a LEICA EMUC7 ultramicrotome, stained with uranyl acetate and alkaline lead citrate for 10 minutes each, and observed using a Hitachi Model H-7650TEM. Figure 6 The antimicrobial peptide P1 exhibits uniform vesicular nanostructures in aqueous solution. However, in the hydrophobic environment of 50% TFE, the vesicular nanostructures aggregate as building blocks, forming larger vesicles and even further clustering. This suggests that the vesicular nanostructures of P1 may aggregate into larger nanostructures upon contact with the hydrophobic bacterial cell wall, thereby capturing the bacteria.

[0052] 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 5 minutes. 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 to treat the blank group and incubated 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. The cell nucleus was stained with 1μl / ml Hoechst staining solution 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.

[0053] The results are as follows Figure 7 As shown in the figure, 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 phagocytic effect of macrophages on bacteria.

[0054] Example 7 Antimicrobial peptide P1 has an immunomodulatory effect on macrophages

[0055] RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS to a density of 70-80%. PI fluorescently labeled Staphylococcus aureus (S. aureus) was cultured to the logarithmic growth phase, and the bacteria were collected by low-speed centrifugation at 4000 rpm for 5 minutes. 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 the bacteria and RAW264.7 cells for co-incubation as the experimental group. An equal volume of PBS was used as the blank group and incubated at 37°C for 20 minutes. After the co-incubation, the supernatant was discarded and the cells were washed three times with PBS for 5 minutes each time. The cells were carefully scraped with a cell scraper and collected by centrifugation at 1500 rpm / min. The cells were resuspended in 200 μl PBS and 0.5 μl of FITC fluorescent-labeled F4 / 80 antibody was added and incubated in the dark for 15 minutes. The cells were collected by centrifugation at 1500 rpm / min and washed three times with PBS for 5 minutes each time. The cells were resuspended in 300 μl PBS and filtered to form a single cell suspension. The results were as follows. Figure 8 As shown in the figure, 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 phagocytic effect of macrophages on bacteria.

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 caused by Gram-positive bacteria, characterized in that: The sepsis is sepsis caused by surgery.

3. The use according to claim 2, characterized in that The antimicrobial peptide P1 can treat and alleviate the reduced survival rate induced by sepsis caused by Gram-positive bacteria, treat and alleviate liver and lung damage caused by sepsis, treat and alleviate bacterial infection of organs caused by sepsis caused by Gram-positive bacteria, 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.

Images