Antibacterial peptide and application thereof in treatment of ulcerative colitis

Abstract

The invention relates to the technical field of biological medicines, in particular to an antibacterial peptide and application thereof in treatment of ulcerative colitis. The natural antibacterial peptide aiming at the enteropathogenic bacteria is screened from an antibacterial peptide database, the existing natural antibacterial peptide is designed and modified by utilizing a bioinformatics technology and a computer-aided drug design technology to obtain the novel antibacterial peptide, and the amino acid sequence of the novel antibacterial peptide is shown as SEQ ID NO.1. The antibacterial peptide is chemically synthesized, the antibacterial activity, cytotoxicity and hemolytic activity of the antibacterial peptide are verified in experiments, meanwhile, the treatment effect of the antibacterial peptide on ulcerative colitis is verified, and it is proved that the antibacterial peptide has the application prospect for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to an antibacterial peptide and its application in the treatment of ulcerative colitis. Background Art

[0002] Ulcerative colitis is a lifelong inflammatory disease that affects the rectum and colon. Its global incidence is on the rise. This disease can highly affect the lives of patients and lead to long-term complications. Currently, the exact mechanism of this disease is still unclear, and its causes are related to lifestyle, diet, genetic factors, immune deficiencies, and intestinal flora imbalance, etc. There is currently no specific drug for treating this disease. Although treatment technologies have advanced, treatment drugs such as 5-aminosalicylic acid, antibiotics, immunosuppressants, and biological agents have been clinically used. However, these treatment methods are not universally applicable, and long-term use of these drugs may cause a series of side effects. Especially when using antibiotics to treat ulcerative colitis, drug resistance will occur.

[0003] Antibacterial peptides are a class of polypeptide substances widely present in nature with broad-spectrum antibacterial activity. Due to their unique antibacterial mechanism of action, they are regarded as new alternative drugs to traditional antibiotics. Since natural antibacterial peptides often have disadvantages such as weak activity and potential cytotoxicity of high-charge peptides, their research and application are also limited by sources, instability, toxicity, and bioavailability. Therefore, the design and optimization based on natural antibacterial peptides have become a recent research hotspot.

[0004] Due to their unique mechanism of action, antibacterial peptides are completely different from antibiotics. Among the reported mechanisms of action of antibacterial peptides, some antibacterial peptides cause bacteria to die by destroying the bacterial cell membrane structure and causing the leakage of bacterial contents; others can inhibit specific bacterial enzymes or DNA transcription and protein translation, affecting intracellular protein interactions. These mechanisms of action are not likely to cause bacterial drug resistance. Therefore, antibacterial peptides are expected to replace antibiotics to solve the problem of bacterial drug resistance and a series of problems caused by the abuse of antibiotics. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an antibacterial peptide with the advantages of high efficiency, stability, and non-toxicity, which can be used in the treatment of ulcerative colitis.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO.1.

[0008] The present invention screens natural antimicrobial peptides against intestinal pathogenic bacteria from an antimicrobial peptide database, and through the use of bioinformatics techniques and computer-aided drug design techniques, designs and modifies existing natural antimicrobial peptides to improve their activity and reduce their cytotoxicity. The present invention has modified the natural antimicrobial peptide as follows: deleting the positively charged amino acid - arginine; replacing the uncharged serine residue in the hydrophilic region with tryptophan; replacing tyrosine and threonine with proline and tryptophan, and finally designing a new antimicrobial peptide with the amino acid sequence: LWRDLICLCRNRRCNRGQLFPGWCPGWWLRCCRR. The antimicrobial peptide obtained by the present invention through N-terminal amidation modification and amino acid replacement reduces the positive charge number of the original antimicrobial peptide, reduces the cytotoxicity of the antimicrobial peptide, and at the same time improves the effect of the antimicrobial peptide by increasing hydrophobicity and amphiphilicity. The antimicrobial peptide obtained by the present invention has the advantages of high efficiency, stability and non-toxicity.

[0009] In a second aspect, the present invention provides the use of the antimicrobial peptide described above in the preparation of an antibacterial agent.

[0010] The present invention has detected the bactericidal activity of the obtained antimicrobial peptide, and the results show that the antimicrobial peptide has good bactericidal activity. When the concentration of the antimicrobial peptide is 7.5 μM, its bactericidal rate can reach more than 99%; when the concentration is 25 μg / mL, the bactericidal rate can reach 100%.

[0011] Preferably, the bacteria inhibited by the antibacterial agent include Gram-positive bacteria and Gram-negative bacteria.

[0012] Preferably, the Gram-positive bacteria include Staphylococcus aureus; the Gram-negative bacteria include Escherichia coli and drug-resistant Acinetobacter baumannii.

[0013] In a third aspect, the present invention provides an antibacterial agent comprising the antimicrobial peptide described above.

[0014] Preferably, the action concentration of the antimicrobial peptide is not less than 7.5 μM.

[0015] In a fourth aspect, the present invention provides the use of the antimicrobial peptide described above in the preparation of a drug or preparation for treating ulcerative colitis.

[0016] The present invention has verified through experiments the influence of the antimicrobial peptide on the animal intestinal flora and the therapeutic effect of the antimicrobial peptide on ulcerative colitis.

[0017] Preferably, the antimicrobial peptide treats ulcerative colitis by acting on the intestinal flora.

[0018] In a fifth aspect, the present invention provides a drug or preparation for treating ulcerative colitis, comprising the antimicrobial peptide described above.

[0019] The beneficial effects of the present invention are:

[0020] The present invention screens out natural antibacterial peptides against intestinal pathogenic bacteria from an antibacterial peptide database, and uses bioinformatics technology and computer-aided drug design technology to design and modify the existing natural antibacterial peptides to obtain a new antibacterial peptide, whose amino acid sequence is shown in SEQ ID NO.1. The present invention chemically synthesizes the antibacterial peptide, and verifies its antibacterial activity, cytotoxicity, and hemolytic activity in experiments. At the same time, the therapeutic effect of the antibacterial peptide on ulcerative colitis is verified, demonstrating that the antibacterial peptide has an application prospect for the treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the mass spectrometry result of the antibacterial peptide chemically synthesized by the present invention.

[0022] Figure 2 It is the predicted secondary structure diagram of the antibacterial peptide of the present invention.

[0023] Figure 3 It is the bactericidal plate diagram after treatment with antibacterial peptides at different concentrations; Figure A is the result of Staphylococcus aureus; Figure B is the result of Acinetobacter baumannii with drug resistance; Figure C is the result of Escherichia coli.

[0024] Figure 4 It is the histogram of bacterial survival rate after treatment with antibacterial peptides at different concentrations; Figure A is the result of Staphylococcus aureus; Figure B is the result of Acinetobacter baumannii with drug resistance; Figure C is the result of Escherichia coli.

[0025] Figure 5 It is the cytotoxicity result of the antibacterial peptide of the present invention.

[0026] Figure 6 It is the hemolysis result of the cells of the antibacterial peptide of the present invention.

[0027] Figure 7 It is the weight change of mice after modeling.

[0028] Figure 8 It is the weight change of mice after treatment.

[0029] Figure 9 It is the result of colon length comparison.

[0030] Figure 10 It is the result of spleen index comparison. DETAILED DESCRIPTION OF THE INVENTION

[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Example 1:

[0033] The present invention searches for natural antimicrobial peptides that can act on intestinal pathogenic bacteria in the Antimicrobial Peptide Database, and screens out a natural antimicrobial peptide with the amino acid sequence: LSRDLICLCRNRRCNRGELFYGTCAGPFLRCCRRRR. According to the database information, this antimicrobial peptide was isolated from the small intestine of C57BL / 6 mice, with a hydrophobicity of 31% and a net charge of +10. This natural antimicrobial peptide is active against Salmonella enterica, Salmonella typhimurium phoP strain, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. However, highly charged antimicrobial peptides have strong cytotoxicity, so the present invention modified this natural antimicrobial peptide as follows: deleting the positively charged amino acid - arginine; the amphiphilicity of antimicrobial peptides also has a great impact on antimicrobial activity and antimicrobial spectrum selectivity, replacing the uncharged serine residue in the hydrophilic region with tryptophan to enhance amphiphilicity; replacing tyrosine and threonine with proline and tryptophan to increase hydrophobicity, and finally designing a new antimicrobial peptide with the amino acid sequence: LWRDLICLCRNRRCNRGQLFPGWCPGWWLRCCRR. After modification, prediction was carried out using an algorithm, and the prediction results showed that the accuracy of it being an antimicrobial peptide was 99.9%, its net charge was +7, and its hydrophobicity remained at 50%. The present invention chemically synthesized the modified and screened antimicrobial peptide sequence, and then carried out mass spectrometry analysis. The mass spectrometry results showed ( Figure 1 ), the antimicrobial peptides designed by the present invention and the chemically synthesized antimicrobial peptides had almost the same molecular weight, which were 4293.13 g / mol and 4294.11 g / mol respectively, and the purity was higher than 95%. The secondary structure was predicted using Alphafold3, and the result showed an α-helix structure ( Figure 2 ). The present invention used this chemically synthesized antimicrobial peptide to carry out subsequent experiments.

[0034] Example 2:

[0035] In this example, the bactericidal activity of the antimicrobial peptide chemically synthesized in Example 1 was detected. The bacteria detected included: the typical Gram-positive bacterium - Staphylococcus aureus, the typical Gram-negative bacterium - Escherichia coli, and multi-drug resistant Acinetobacter baumannii. The minimum bactericidal concentration (MBC) is the lowest drug concentration that can kill bacteria, and it was determined by the colony counting method.

[0036] Process of the bactericidal experiment:

[0037] The bacterial suspensions of Staphylococcus aureus, Escherichia coli, and multi-drug resistant Acinetobacter baumannii were diluted with PBS to a viable bacteria count of 10 5 cfu / mL. The antimicrobial peptide was diluted with PBS to different concentrations: 400 μg / mL, 200 μg / mL, 100 μg / mL, and 50 μg / mL.

[0038] The diluted bacterial suspension and the antimicrobial peptide were mixed at a volume ratio of 1:1 and cultured together in an incubator at 37°C for 2 hours, and then inoculated onto a solid LB medium and cultured at 37°C for 12 h. At the same time, PBS was used as a control treatment. MBC is determined by the number of colonies formed on the solid medium. By the colony counting method, according to the number of colonies on each plate, the bactericidal rate of the antimicrobial peptide was calculated according to formula (1).

[0039] Bactericidal rate (%) = (number of colonies on the experimental group plate / number of colonies on the control group plate) × 100% —— Formula (1)

[0040] The bactericidal results are as Figure 3-4 shown. From the results of the bactericidal plates, it can be seen that the antimicrobial peptide described in the present invention has significant bactericidal activity against three representative bacteria. When the concentration is 25 μg / mL, the bactericidal rate can reach 100% ( Figure 3 ).

[0041] Using GraphPad Prism 8 to construct a survival rate histogram, the results show that when the concentration of the antimicrobial peptide is 7.5 μM, its bactericidal rate can reach more than 99% ( Figure 4 ).

[0042] Example 3:

[0043] In this example, a cytotoxicity experiment was carried out on the antimicrobial peptide (AMPs) chemically synthesized in Example 1.

[0044] Mouse macrophages RAW264.7 were cultured to the logarithmic growth phase and inoculated into 96-well plates at a density of 10 5 cells per well, and placed in a cell culture incubator (5% carbon dioxide, 37°C) for 24 hours. The supernatant was discarded, and AMPs were added to DMEM medium to make the final concentrations of AMPs 12.5, 25, 50, 100, and 200 μg / mL respectively. Samples without AMP were used as blank controls and cultured for 12 h. Then 10 μL of CCK-8 was added to each well and incubated for another 2 h. Finally, the absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. The cell survival rate was calculated according to formula (1), and GraphPad Prism 8 was used for plotting.

[0045] Cell survival rate (%) = (OD of experimental well - OD of blank well) / (OD of control well - OD of blank well) × 100% —— Formula (2)

[0046] The toxicity results of macrophages RAW264.7 showed that at concentrations of 0 - 200 μg / mL, the antimicrobial peptide had no toxic effect on macrophages ( Figure 5 ).

[0047] Example 4:

[0048] In this example, a cell hemolysis experiment was conducted on the antibacterial peptides (AMPs) chemically synthesized in Example 1.

[0049] Fresh blood was taken, centrifuged, and the supernatant was discarded. After washing 4 - 6 times with PBS, red blood cells were collected until the supernatant. A 2% V / V red blood cell suspension dispersed in PBS was mixed with AMPs such that the final concentrations of AMPs were 12.5, 25, 50, 100, and 200 μg / mL respectively. A PBS sample was used as the negative control, and a 2% Triton X - 100 sample was used as the positive control. After gentle incubation at 37 °C for 2 h, the supernatant was collected by centrifugation, and the absorbance of the sample at 570 nm was measured using an enzyme - linked immunosorbent assay (ELISA) reader. The hemolysis rate was calculated according to Equation (3), and a hemolysis rate histogram was plotted using GraphPad Prism 8.

[0050] Hemolysis rate (%) = [OD (experimental group) - OD (PBS group)] / [OD (positive control group) - OD (PBS group)] —— Equation (3)

[0051] The results of cell hemolysis are as Figure 6 shown. Comparing the antibacterial peptide (LR) of the present invention with other antibacterial peptides (KC, amino acid sequence: KWWIKKVFKWIKGIGKEVVIRTGIEIAACKIKGEC), it can be seen that at a concentration of 100 μg / mL, LR hardly causes hemolysis, while the hemolysis rate of KC is much higher than that of LR.

[0052] Example 5:

[0053] This example verifies the application of the antibacterial peptides (AMPs) chemically synthesized in Example 1 in ulcerative colitis.

[0054] Experimental procedure:

[0055] Healthy female C57 mice at 6 - 8 weeks of age (body weight 18 - 20 g) were randomly grouped. The mice in the experimental group were given 2.5% DSS water for free drinking for 7 days, and the mice in the control group were given normal water. By weighing their body weights daily and observing the occult blood in the feces of the mice, the establishment of the UC model was judged. The spleen index and colon length are important indicators of the UC disease phenotype. The successfully modeled mice in the experimental group were intragastrically treated with AMPs at a dose of 5 mg / kg for 7 days, and the control group was intragastrically treated with PBS at the same dose for 7 days. As the treatment time of the experimental group of mice with AMPs increased, the body weight of the mice gradually increased. After the end of the treatment cycle, the colon length of the mice was significantly increased compared with the PBS - treated group, and the spleen index also decreased significantly after the end of the AMPs treatment cycle.

[0056] Determination of spleen index: First, measure the body weight (grams) of the mice: weigh the mice and record their body weights; then measure the spleen weight (grams) of the mice: weigh with a balance and record the spleen weight. Calculate the spleen index according to Equation (4).

[0057] Spleen index = Spleen weight (grams) / Body weight of mice (grams) — Equation (4)

[0058] The results are as Figure 7-10 shown. Treatment with DSS water caused a significant decrease in the body weight of the mice. The changes in body weight, colon length, and spleen index of the mice indicated that after treatment with AMPs, the symptoms of ulcerative colitis were all restored.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

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

1.

2. Use of the antimicrobial peptide as claimed in claim 1 in the preparation of an antimicrobial agent.

3. The use according to claim 2, characterized in that The bacteria inhibited by the antibacterial agent include Gram-positive bacteria and Gram-negative bacteria.

4. The use according to claim 3, characterized in that The Gram-positive bacteria include Staphylococcus aureus; the Gram-negative bacteria include Escherichia coli and drug-resistant Acinetobacter baumannii.

5. An antibacterial agent, characterized in that The invention comprises the antimicrobial peptide according to claim 1.

6. The antibacterial agent according to claim 5, characterized in that The effective concentration of the antimicrobial peptide is not less than 7.5 μM.

7. Use of the antimicrobial peptide according to claim 1 in the preparation of a medicine or preparation for treating ulcerative colitis.

8. The use according to claim 7, characterized in that The antimicrobial peptide treats ulcerative colitis by acting on intestinal flora.

9. A medicine or preparation for treating ulcerative colitis, characterized in that: The invention comprises the antimicrobial peptide according to claim 1.

Images