Oligopeptide for treating neonatal bacterial meningitis and preparation method of oligopeptide

Abstract

The invention discloses an oligopeptide for treating neonatal bacterial meningitis and a preparation method of the oligopeptide, and belongs to the technical field of biological medicines. The invention successfully designs and prepares small molecular mimic peptides capable of effectively blocking escherichia coli from invading into the brain based on a lactam cyclization technology, the small molecular mimic peptides are named as EC-MP-1 and EC-MP-2, and animal level research proves that the mimic peptides disclosed by the invention can inhibit the invasion of E44 escherichia coli to HBMEC; cell and molecular level researches prove that the mimic peptide disclosed by the invention can play an effective therapeutic role. A new thought is provided for prevention and treatment of neonatal meningitis, and reliable scientific data is accumulated for subsequent development and clinical application of micromolecule polypeptide drugs.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a short peptide for treating neonatal bacterial meningitis and a preparation method thereof. Background Art

[0002] Bacterial meningitis (NBM) is an infectious disease of the central nervous system of newborns with a high mortality rate. Survivors often suffer from sequelae such as deafness, blindness, epilepsy, intellectual and motor disorders. Currently, antibiotics are the main treatment in clinical practice. However, due to the fact that pathogens are prone to drug resistance as the course of treatment progresses and antibiotics are difficult to cross the blood-brain barrier (BBB), making it difficult to maintain antibiotic concentrations in the brain, the mortality rate of neonatal bacterial meningitis has not been significantly improved. Therefore, it is necessary to develop new therapies.

[0003] The emergence of small molecule peptide drugs provides new ideas and perspectives. Their small molecular weight can penetrate the BBB; they can be administered in a non-invasive manner, which is more beneficial to patients than invasive administration methods such as lumbar puncture; compared with chemical drugs, their ingredients are safer and will not cause pathogens to develop drug resistance. They can effectively relieve brain inflammation while producing less toxic side effects, so they have a broader clinical application space. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is a short peptide for treating neonatal bacterial meningitis and a preparation method thereof. The technical solution disclosed in the present invention successfully designs and prepares small molecule analog peptides EC-MP-1 and EC-MP-2 that can effectively block Escherichia coli from invading the brain based on lactam cyclization technology, providing a new idea for the prevention and treatment of neonatal meningitis, and accumulating reliable scientific data for the subsequent development and clinical application of small molecule polypeptide drugs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The invention discloses a short peptide EC-MP-1 for treating neonatal bacterial meningitis, characterized in that the amino acid sequence of the EC-MP-1 is shown as SEQ ID No.1.

[0007] The present invention also discloses a short peptide EC-MP-2 for treating neonatal bacterial meningitis, characterized in that the amino acid sequence of the EC-MP-2 is shown as SEQ ID No.2.

[0008] The present invention also discloses the use of any of the above-mentioned short peptides for treating neonatal bacterial meningitis in the preparation of a medicine for treating neonatal bacterial meningitis.

[0009] The present invention also discloses the use of any of the above-mentioned short peptides for treating neonatal bacterial meningitis in the preparation of products for inhibiting Escherichia coli from invading human brain microvascular endothelial cells.

[0010] The present invention also discloses a method for preparing a short peptide for treating neonatal bacterial meningitis as described in any one of the above items, characterized in that the specific steps are as follows: Step 1, using the molecular docking model to screen key amino acid sequences; Step 2, dehydrating and condensing the carboxyl group on the lysine R group and the amino group on the aspartic acid R group through lactam ring formation technology to form a lactam bond to obtain the short peptide.

[0011] Furthermore, the key amino acid sequence in step 1 is shown as SEQ ID No.3.

[0012] The present invention also discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises any of the above-mentioned short peptides for treating neonatal bacterial meningitis, and a pharmaceutically acceptable carrier.

[0013] The invention also discloses the use of the above-mentioned pharmaceutical composition in preparing a medicine for treating neonatal bacterial meningitis.

[0014] The invention also discloses the use of the above-mentioned pharmaceutical composition in preparing a product for inhibiting Escherichia coli from invading human brain microvascular endothelial cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] The present invention uses the lactam cyclization technology for the first time to synthesize two cyclic analog peptides with stable natural conformations from the 331-340 peptide segment of Caspr1, which are named EC-MP-1 and EC-MP-2 respectively. The binding energy calculation results indicate that both can stably bind to IbeA and can competitively inhibit the interaction between IbeA and Caspr1. The results of CCK-8 experiments indicate that EC-MP-1 and EC-MP-2 have low toxicity to mouse hippocampal neurons and human brain microvascular endothelial cells. When the concentration reaches 512μmol / L, the relative activity of the cells is still higher than 95%.

[0017] In vitro experiments verified that the cytotoxicity of the peptides EC-MP-1 and EC-MP-2 to brain tissue cells such as hippocampal neurons and human brain microvascular endothelial cells was extremely low; bacterial adhesion experiments and bacterial invasion experiments confirmed that the main way the peptides acted was to inhibit the invasion of E. coli E44 to HBMEC, while the effect on the bacterial adhesion level was not significant; finally, Western blot experiments verified that the treatment with the peptides could effectively improve the damage of E. coli to the tight junction structure between the blood-brain barrier. In vivo experiments, we first established a newborn rat bacterial meningitis model and verified that the detection rate of E. coli in the cerebrospinal fluid of newborn rats treated with the peptides was significantly reduced, proving that the peptides can play an effective therapeutic role in vivo; then, we used H&E staining, immunofluorescence and immunohistochemistry experiments to prove that the peptides treatment could significantly alleviate the thickening of the meninges and neutrophil infiltration in newborn rats, and could inhibit the activation of glial cells in the brain and reduce the expression of inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The hotspot amino acid residues for the interaction between IbeA and Caspr1 were predicted by computational biology methods. (A) Comparison of the predicted structure of IbeA and the structure of homologs. Blue is IbeA, pink is the structure of homologs; (B) Comparison of the predicted structure of Caspr1 and the structure of homologs. Green is Caspr1, red is the structure of homologs; (C) IbeA-Caspr1 docking model diagram. Blue is IbeA, green is Caspr1; (D) Calculation results of the binding free energy of amino acids in the interaction process of the IbeA-Caspr1 docking model.

[0019] Figure 2 Design and synthesize a peptide mimetic that can compete with Caspr1 for binding to IbeA. Peptide mimetic design model diagram. Line is a straight-chain peptide, and EC-MP-1 and EC-MP-2 use lactam cyclization technology to make them cyclic peptides.

[0020] Figure 3 Liquid chromatography-mass spectrometry was used to identify the molecular weight and ion peaks of the mimetic peptides. (A) Mass spectrum of the linear peptide Line determined by LC-MS / MS; (B) Mass spectrum of EC-MP-1 determined by LC-MS / MS; (C) Mass spectrum of EC-MP-2 determined by LC-MS / MS.

[0021] Figure 4 The binding of the mimetic peptide to the IbeA docking complex is stable. (A) Binding pattern of the mimetic peptide to IbeA. Green is IbeA; blue is EC-MP-1; yellow is EC-MP-2; the yellow dotted line indicates hydrogen bonding; (B) Left: RMSD curve of EC-MP-1 binding to IbeA; right: RMSD curve of EC-MP-2 binding to IbeA.

[0022] Figure 5 The mimetic peptide did not affect the growth of hippocampal neurons and human brain microvascular endothelial cells. (A) CCK8 assay to analyze the activity of HBMEC cells after treatment with different concentrations of mimetic peptide. ns, P>0.05 (n=7); (B) CCK8 assay to analyze the activity of HT22 cells after treatment with different concentrations of mimetic peptide. ns, P>0.05 (n=7).

[0023] Figure 6 The mimetic peptide can effectively inhibit the invasion of meningococcal Escherichia coli into human brain microvascular endothelial cells. (A) The mimetic peptide blocks the invasion of bacteria; (B) The bacterial invasion experiment analyzes the changes in the number of E44 Escherichia coli colonies that invade human brain microvascular endothelial cells after the addition of the mimetic peptide. **** P<0.0001 (n=7); (C) Bacterial adhesion assay to analyze the changes in the number of E44 Escherichia coli colonies adhering to the surface of human brain microvascular endothelial cells after the addition of the mimetic peptide. ns, P>0.05 (n=7); (D) Immunofluorescence imaging to analyze the number of bacteria invading into a single HBMEC cell. VE-Cadherin is a marker for human brain microvascular endothelial cells, marked in red; LPS is a marker for Escherichia coli, marked in green; DAPI is a marker for the nucleus, marked in blue. **** P < 0.0001 (n = 7). Scale bar: 10 μm.

[0024] Figure 7 The mimetic peptides can effectively inhibit the meningitis Escherichia coli from destroying the integrity of the blood-brain barrier. Western blot analysis showed the changes in the expression levels of tight junction proteins ZO-1, Occludin and Claudin-5 in HBMEC after the addition of E44 Escherichia coli and treatment with mimetic peptides EC-MP-1, EC-MP-2 and linear peptide Line. ns, P>0.05 (n=3).

[0025] Figure 8 Peptide mimics can effectively block the invasion of meningitis Escherichia coli into the brain. (A) Neonatal rats were inoculated with E44 Escherichia coli and treated with linear peptide Line, peptide mimics EC-MP-1 and EC-MP-2, respectively. Cerebrospinal fluid was extracted 18 hours later and spread on bacterial plates after gradient dilution to count the number of colonies. Each plate was divided into four quadrants, and the concentration difference in each quadrant was 10 times. The final statistical result was the average value of the four quadrants; (B) Statistical analysis of the detection rate of E44 Escherichia coli in the blood and cerebrospinal fluid of neonatal rats in each group. ****P<0.0001 (n≥15); (C) Statistical analysis of the detection rate of E. coli E44 in the blood and cerebrospinal fluid of newborn rats in each group. Unpaired t test was used, and the data were expressed as mean ± standard error; (D) Statistical analysis of the mortality rate of newborn rats in each group. * P<0.05 (n=10).

[0026] Fig. 9 Peptide mimics can effectively alleviate brain inflammation in newborn rats with bacterial meningitis. Neonatal rats were inoculated with E. coli E44 and treated with peptide mimics EC-MP-1 and EC-MP-2 for 18 hours, and brain tissue sections were prepared for HE staining. The enlarged image shows the meninges and neutrophil infiltration (arrows). The sham group is a sham operation group treated with saline. Scale bar: 100 μm. The images shown are representative of 3 independent experiments.

[0027] Fig.10 Treatment with mimetic peptides can effectively alleviate inflammatory cell infiltration in newborn rats with bacterial meningitis. (A) Immunofluorescence staining of brain tissue was used to detect the activation of microglia in each group of newborn rats. The Sham group was a sham operation group treated with saline. IBA1 is a microglia marker, marked in red; DAPI is a cell nucleus marker, marked in blue (n=3); scale: 100μm; (B) Immunofluorescence staining of brain tissue was used to detect the activation of astrocytes in each group of newborn rats. The Sham group was a sham operation group treated with saline. GFAP is an astrocyte marker, marked in green; Lectin is a vascular marker, marked in red; DAPI is a cell nucleus marker, marked in blue, (n=3); scale: 100μm.

[0028] Fig.11Peptide mimics can effectively alleviate the high expression of inflammatory factors in the brain of newborn rats with bacterial meningitis. (A) After the newborn rats were inoculated with E44 Escherichia coli and treated with peptide mimics EC-MP-1 and EC-MP-2 for 18 hours, brain tissue sections were prepared. The expression of TNF-α in newborn rats in each group was detected by IHC experiment. The Sham group was a sham operation group treated with normal saline. The dark brown staining indicates a positive reaction product (arrow). Scale bar: 100μm; (B) After the newborn rats were inoculated with E44 Escherichia coli and treated with peptide mimics EC-MP-1 and EC-MP-2 for 18 hours, brain tissue sections were prepared. The expression of IL-6 in newborn rats in each group was detected by IHC experiment. The Sham group was a sham operation group treated with normal saline. The dark brown staining indicates a positive reaction product (arrow). Scale bar: 100 μm; (C) Neonatal rats were inoculated with E. coli E44 and treated with EC-MP-1 and EC-MP-2 peptides for 18 h. Brain tissue sections were prepared and the expression of IL-2R in neonatal rats of each group was detected by IHC experiment. The sham group was a sham operation group treated with saline. The dark brown staining indicates positive reaction products (arrows). Scale bar: 100 μm. The images shown are representative of 3 independent experiments; (D) Statistical graph of positive expression area. From left to right, TNF-α, IL-6 and IL-2R. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0030] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0031] Example.

[0032] 1. Experimental materials.

[0033] 1. Experimental cells. Human brain microvascular endothelial cells (HBMEC) were donated by Professor Kwang Sik Kim of the Johns Hopkins University School of Medicine.

[0034] 2. Experimental animals. SD rats were purchased from SPIEF Biotechnology Co., Ltd. They were raised in the Experimental Animal Department of ** University in an SPF-grade environment with a 12-hour day and night cycle. All animal experimental protocols were reviewed and approved by the Animal Welfare Ethics Committee of ** University (No. ***2023016).

[0035] 3. Experimental strains. E. coli K1 strain RS218 (O18:K1:H7) was isolated from the cerebrospinal fluid of a patient with neonatal meningitis; E. coli E44 is a mutated strain with rifampicin resistance. coli K1 strain RS218 (O18:K1:H7).

[0036] 2. Experimental methods.

[0037] 1. Cell culture.

[0038] The method for preparing the culture medium of human brain microvascular endothelial cell HBMEC cell line is as follows: 10% fetal bovine serum, 10% Nu serum, 2mM glutamine, 1mM sodium pyruvate, 1×MEM vitamins and 1× non-essential amino acids are added to RPMI-1640 culture medium, and the cells are passaged every 48h.

[0039] Cell recovery: Place the EP tube containing human brain microvascular endothelial cells in a 37°C water bath and shake gently to quickly thaw the cells. Centrifuge at 800rpm for 5 minutes and discard the supernatant and add 4mL of culture medium to resuspend. Pipette the cell suspension into a cell culture dish and shake well. Place at 37°C and 5% CO 2 The cells were cultured under 4% paraformaldehyde and the culture medium was replaced the next day after the cells adhered to the wall.

[0040] Cell passaging: When the cells grow to a density of more than 90%, discard the culture medium, use 0.25% trypsin to digest for 2 minutes, and then stop the digestion. Transfer the liquid to a centrifuge tube. Centrifuge at 800 rpm for 5 minutes, discard the supernatant, add 4 mL of culture medium to resuspend, transfer to a cell culture dish, shake well, and incubate at 37°C, 5% CO 2 Cultivated under conditions.

[0041] Cell cryopreservation: The previous steps are the same as above. After discarding the supernatant, resuspend the cells with one-step freezing solution and transfer the liquid to a cell freezing tube. Place it in an ultra-low temperature refrigerator for one day and then transfer it to liquid nitrogen for storage.

[0042] 2. Bacterial culture and preparation.

[0043] Preparation of LB liquid culture medium: Use an electronic analytical balance to weigh 0.5 g yeast extract, 1 g trypsin, and 1 g sodium chloride into a conical flask, add double distilled water to make up to 100 mL, stir evenly, and place in a pressure cooker for high temperature and high pressure treatment for 20 min. The obtained LB liquid culture medium will be stored in a 4°C refrigerator.

[0044] Preparation of LB solid culture medium: Use an electronic analytical balance to weigh 0.5 g yeast extract, 1 g trypsin, 1 g sodium chloride, and 1.5 g agar powder into a conical flask, add double distilled water to make up to 100 mL, stir evenly, place in a pressure cooker for high temperature and high pressure treatment for 20 min, cool down, pour into a bacterial culture dish, and store the obtained LB solid culture medium in a 4°C refrigerator.

[0045] Preparation of BHI liquid culture medium: Use an electronic analytical balance to weigh 7.4 g of BHI powder, add double distilled water to make up to 200 mL, stir evenly and place in an autoclave for high temperature and high pressure treatment, and store the obtained BHI liquid culture medium in a 4°C refrigerator.

[0046] 3. Homology modeling and docking analysis.

[0047] Clusta-Omega software was used to align and optimize the target sequence with the homologous fragments, Modeller9.15 was used for multi-template modeling, the model was scored and screened by Discrete Optimized Protein Energy Score (DOPE Score), and the model quality was evaluated by MolProbityServer. The model was then modified using the Broyden-Fletcher-Goldfarb-Shanno (LBFGS) method.

[0048] The docking model was further simulated using ZDOCK and RosattaDock, and then the kinetic stability of the docking model was analyzed by Root Mean Squares Deviation (RMSD) using AMBER 16. The binding free energy was calculated and the hotspot amino acid residues were screened using the MM / GBSA method. Finally, the image was processed using PyMol2.5.

[0049] 4. Western Blot.

[0050] Protein extraction: Place the cells on ice, add and mix pre-cooled protease inhibitor PMSF and cell lysis buffer (RIPA Lysis Buffer) at a ratio of 1:100, wash the cells three times with 1×PBS, add the cell lysis mixture to the culture dish, collect the cells into a 1.5mL EP tube, lyse on ice for 30 minutes, then centrifuge at 4°C, 12000rpm for 15 minutes, and aspirate the supernatant into a new EP tube, which is the protein component.

[0051] BCA protein quantification: Referring to the number of samples, mix BCA solution A and BCAsolution B in a ratio of 50:1. Then add the protein standard solution to a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 μL, and add double distilled water to make up to 20 μL. Use 1×PBS to dilute the sample to be tested in an appropriate proportion and add it to the wells of the 96-well plate to be tested. Add 200 μL of working solution to each well, incubate at 37°C in the dark for 1 hour, and use an ELISA reader to measure the absorbance value of each well at 595 nm. Draw a standard curve according to the standard concentration and the corresponding absorbance value, and calculate the protein concentration of the sample to be tested based on the curve.

[0052] Electrophoresis: Prepare the upper gel solution and the lower gel solution according to the proportions in the instructions, insert a 10-hole comb, and let it stand for 20 minutes. After the gel is completely solidified, assemble the gel plate and electrophoresis equipment, add an appropriate amount of protein sample to each channel, adjust the voltage to 100V to the boundary between the concentrated gel and the separation gel, and then adjust the voltage to 120V. Turn off the power after electrophoresis to the bottom of the glass plate.

[0053] Transfer: Cut the filter paper and PVDF membrane of appropriate size according to the size of the gel after electrophoresis. After activating the membrane with methanol for 5 minutes, transfer it to the pre-cooled transfer solution together with the gel and filter paper to slowly balance. After 20-30 minutes, put the transfer clip in the order of sponge, filter paper, PVDF membrane, gel, filter paper, and sponge from the transparent surface. Be careful that there should be no bubbles between the gel and the membrane. Assemble the transfer clip and wet transfer tank, fill it with pre-cooled transfer solution, place the entire device in a foam box filled with ice, and set the transfer voltage and time according to the molecular weight of the target protein.

[0054] Blocking: After the transfer is completed, take out the PVDF membrane, soak it completely with 5% skim milk powder TBST diluent, and incubate it slowly on a shaker at room temperature for 1 hour.

[0055] Hybridization: Wash the blocked PVDF membrane with 1×TBST three times and place it in the primary antibody diluent diluted at a ratio of 1:800, and incubate slowly overnight at 4°C on a shaker. On the second day, wash the PVDF membrane with 1×TBST three times, 10 minutes each time, add the secondary antibody diluent diluted at an appropriate ratio, incubate slowly at room temperature on a shaker for 1 hour, and then wash it with 1×TBST three times, 10 minutes each time.

[0056] ECL luminescence: Prepare an appropriate amount of ECL colorimetric solution according to the proportion in the instruction manual, absorb excess water on the PVDF membrane with absorbent paper, place it on an exposure board, evenly add an appropriate amount of luminescent solution to cover the entire membrane, and place it in an exposure instrument for exposure.

[0057] 5. GST pulldown.

[0058] After mixing the two target proteins evenly, add the treated GST tag affinity beads, put them into an EP tube, and turn the drum overnight at 4°C. Take it out the next day, centrifuge it at 12000g for 10min, discard the supernatant, and add 1mL wash buffer to wash. Use the drum to mix at 4°C for 15min, centrifuge it at 1000g for 30s, discard the supernatant, and repeat 3 times. Finally, add 25μL of 2×RSB to the sample, cook the sample at 95°C for 5min, centrifuge it instantly, and store it at -20°C for later use.

[0059] Collect the GST fusion protein gel after electrophoresis, cut off the concentrated gel and put it in a container, add 50mL of double distilled water to wash away SDS and other substances on the gel and reduce the staining background, repeat 3 times, the last time the gel is not covered, and the liquid level is at least 3 gel thicknesses higher. After rinsing, suck out the residual liquid, add about 20mL of Coomassie Brilliant Blue Rapid Staining Solution to the container for Coomassie Brilliant Blue staining, and stain on a horizontal shaker at room temperature for 30 minutes until the protein bands are clear. Discard the staining solution and add an appropriate amount of double distilled water to wash away the residual staining solution. Finally, add 100mL of double distilled water and wash on a horizontal shaker at room temperature for 15 minutes until a gel staining effect with very low background is obtained.

[0060] 6. Bacterial invasion assay.

[0061] The formula of the culture medium for bacterial invasion experiment is RPMI-1640 medium with 5% fetal bovine serum and 1 mmol / L sodium pyruvate added.

[0062] HBMEC were plated in a 24-well plate. After full growth, the experimental culture medium was replaced. After 30 minutes, E44 Escherichia coli (MOI = 100) was added and placed in a bacterial incubator for 1.5 hours. After washing 3 times with 1×PBS, gentamicin was added and cultured for 1 hour. The liquid in the well was discarded, and after washing 3 times, 200μL of double distilled water was added and allowed to stand for 20 minutes to allow the cells to absorb water, swell, and rupture. Then, a 1mL pipette tip was used to scrape the cells, collect the supernatant and cell debris in the well, and 20μL was taken after gradient dilution in multiples of 10 and applied to each of the four quadrants and counted separately.

[0063] 7. Perfusion of neonatal rats.

[0064] Weigh 32 g of paraformaldehyde powder and dissolve it in double distilled water. Stir evenly at 60°C and add NaOH until the liquid becomes transparent. Make the volume to 400 mL to obtain 8% paraformaldehyde. Add 400 mL of 0.2 M PBS to 400 mL of 8% paraformaldehyde, adjust the pH value to 7.2-7.4 to obtain 4% paraformaldehyde, and store it at 4°C.

[0065] After anesthetizing the newborn mice, fix them on the perfusion table. After disinfecting the ventral skin with alcohol cotton balls, cut the chest and abdominal skin, cut the chest diaphragm and the ribs on both sides, insert the perfusion needle into the newborn mouse 2mm from the left ventricle, cut the right atrial appendage with scissors, inject the normal saline in the perfusion bottle into the newborn mouse, and discharge the blood in the body along the systemic circulation. After the liver turns white, switch the three-way tube; continue to perfuse 4% paraformaldehyde into the newborn mouse. The body of the newborn mouse gradually stiffens, and the tail is slightly lifted. After perfusing about 10mL until the body is completely stiff, cut off the head of the newborn mouse and carefully peel off the skull. After cutting the connecting nerves, remove the newborn mouse brain completely and immerse it in 4% paraformaldehyde for fixation overnight.

[0066] 8. Establishment of a newborn rat bacterial meningitis model and cerebrospinal fluid sampling.

[0067] Newborn 5-day-old SD rats were injected subcutaneously with 10 5 E44 Escherichia coli, and then the rats in the peptide-mimicking group were injected with 5 mg / kg of the peptide-mimicking EC-MP-1 or peptide-mimicking EC-MP-2, and the linear peptide Line-treated group was injected with 5 mg / kg of Line; the rats in the control group were injected with an equal amount of normal saline, recorded as the Sham group.

[0068] Use surgical scissors to cut the scalp of newborn rats, insert a microsyringe into the intersection of the sagittal suture and the coronal suture, slowly extract about 3 μL of cerebrospinal fluid and store it in a 1.5 mL EP tube. Use physiological saline to dilute the sample in multiples of 10, divide the bacterial plate evenly into four quadrants, apply 20 μL of sample dilution in each quadrant and place it in a bacterial incubator, and count the number of colonies after 15 hours.

[0069] 9. Frozen sections.

[0070] The neonatal rat brain fixed overnight was transferred to 20% sucrose for dehydration for one day, and then transferred to 30% sucrose for dehydration for two days. After dehydration, the brain was placed upright in a cubic tin foil box and injected with OCT to completely cover the brain. Small bubbles were emptied at 4°C, and the brain was frozen at -20°C for 4 hours and then transferred to an ultra-low temperature refrigerator for storage and finally sliced ​​using a freezing microtome.

[0071] 10. Immunofluorescence staining.

[0072] Cell immunofluorescence: Fix the cells with pre-cooled 4% paraformaldehyde for 30 minutes, then use 60μL blocking solution to block at room temperature for 30 minutes. After blocking, add 50μL primary antibody working solution at 4℃ overnight, incubate 50μL secondary antibody working solution at room temperature in the dark for 1 hour, then add 50μL DAPI to stain for 5 minutes, and finally add anti-fluorescence quenching sealing agent to seal the slices and observe and photograph using a confocal microscope.

[0073] Immunofluorescence staining of brain tissue: Take out the frozen sections from the ultra-low temperature refrigerator and restore them to room temperature. Immerse the sections in 0.4% Triton-X100 at 37°C for 20 minutes, then immerse the sections in citrate for 25 minutes of antigen repair at 98°C. After cooling naturally at room temperature, wash the slides three times with 1×PBS. Use 5% donkey serum to block at 37°C for 2 hours, then add the primary antibody working solution and place it in a 4°C refrigerator overnight. Then incubate the secondary antibody working solution at room temperature in the dark for 2 hours, then add DAPI staining for 5 minutes, and finally add anti-fluorescence quenching sealing agent to seal the slides and observe and photograph using a confocal microscope.

[0074] 11. Hematoxylin-eosin staining (HE).

[0075] Tissue embedding: The tissue blocks fixed with 4% paraformaldehyde were dehydrated with different concentrations of ethanol, then placed in xylene for 30 min, and then immersed in xylene and paraffin at 60°C for 2 h. The tissue blocks were embedded and sectioned.

[0076] Place the slices in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 85% ethanol, 75% ethanol, 1×PBS, hematoxylin stain, distilled water, 1% hydrochloric acid alcohol, distilled water, and eosin stain in order. Wipe the liquid around the slide, add a drop of neutral gum, cover with a cover glass, and dry at room temperature. Finally, use an imaging system to scan the entire slide after drying.

[0077] 12. Immunohistochemistry.

[0078] The tissue embedding and dehydration steps are the same as 11.

[0079] After adding antigen repair solution and heating for 9 minutes, use an immunohistochemistry pen to draw a circle and then add donkey serum. Incubate at room temperature for 30 minutes for blocking. After blocking, place the slices in a 4°C refrigerator to incubate the primary antibody working solution overnight. The next day, add the secondary antibody working solution and incubate for 1 hour. Then add neutral gum to the tissue for sealing. Finally, use an imaging system to scan the entire slide after drying.

[0080] 13. LC-MS / MS chromatographic mass spectrometry conditions.

[0081] ESI source positive ion mode; mobile phase is acetonitrile (chromatographic grade); flow rate is 0.3mL / min; chromatographic column is ACQUITYUPLC BEH C18 (2.1×50mm), particle size is 1.7μm; column oven temperature is set to 40℃; total analysis time is 10min; injection volume is 10µL.

[0082] 14. Statistical analysis.

[0083] Image J was used to analyze the results. GraphPad Prism statistical software was used for data analysis. The results were expressed as mean ± standard error. Unpaired t-test and two-way ANOVA were used to analyze the significance of the differences between the groups of data. When the P value was less than 0.05, it was considered to be statistically significant, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ns indicates that the data were not statistically significant.

[0084] 3. Experimental results.

[0085] 1. Predict the site of interaction between IbeA and Caspr1: Modeller 9.15 was used to model the IbeA and Caspr peptides based on the above homologous structures. The comparison between the three-dimensional structure and the template is shown in the figure ( Figure 1 AB). ZDOCK and RosattaDock software were then used to obtain the docking structure, and Amber16 was used to analyze the kinetic stability of the complex, and the IbeA-Caspr1 docking model with the smallest binding energy was selected ( Figure 1 C). The binding free energy of all amino acids was calculated by MM / GBSA method ( Figure 1 D) to screen out the key amino acid sites in the interaction process of the docking model.

[0086] 2. The analog peptides EC-MP-1 and EC-MP-2 were designed and prepared based on the key amino acid sites.

[0087] After confirming the key amino acids for the interaction between IbeA and Caspr1, the 331-340 peptide segment of Caspr1 was obtained. This amino acid sequence not only has a minimal secondary structure but also can stably interact with the 322-329 peptide segment of IbeA. It was named Line: RVNIADLAVR (SEQ ID No. 3). The 4th isoleucine (I), 5th alanine (A), 8th alanine (A), and 9th valine (V) at the N-terminus of Line have little effect on the change in binding free energy. According to the mutation principle of simple unbranched non-aromatic amino acids, they were replaced with lysine (K), lysine (K), aspartic acid (D), and aspartic acid (D), respectively. Since the natural conformation of Line is an unstable straight chain, we further used the lactam cyclization technology to dehydrate and condense the carboxyl group on the lysine R group and the amino group on the aspartic acid R group to form a lactam bond ( Figure 2) as the skeleton of the mimetic peptide to make it more stable, and successfully synthesized the mimetic peptides EC-MP-1: RVNIk*DLAd*R (SEQ ID No.1) and EC-MP-2: RVNk*ADLd*VR (SEQ ID No.2). Note: The * position is the lactam bond ring position.

[0088] 3. The molecular weight of the mimetic peptide is smaller and the molecular ion peak is stronger.

[0089] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to identify and analyze the peptide sequences of the analog peptides EC-MP-1 and EC-MP-2. The results showed that the molecular weights of the three peptides were consistent with expectations ( Figure 3 AC): The molecular weight of Line is about 1126Da, the molecular weight of the analog peptide EC-MP-1 is about 1181Da, and the molecular weight of the analog peptide EC-MP-2 is about 1167Da. In addition, the abundance of the doubly charged quasi-molecular ion peaks [M+2H]+ (m / z 563.8, m / z 591.3, m / z 584.3) of Line, EC-MP-1 and EC-MP-2 is significantly stronger than that of other quasi-ion peaks, and there is no interference from other peaks around them, so they can be used as parent ions for subsequent further analysis.

[0090] 4. The mimetic peptide can stably bind to the IbeA docking complex.

[0091] To verify the binding level of the mimetic peptides EC-MP-1 and EC-MP-2 with IbeA (225-343), we performed molecular dynamics simulations using PyMOL2.5 software and selected the final conformation of md for mapping analysis ( Figure 4A), the results show that the two short peptides basically maintain the helical binding in the original position, and the binding is mainly maintained by hydrogen bonds and hydrophobic interactions. Among them, ALA-326, ILE-334 on the analog peptide EC-MP-1 and VAL-326 of IbeA; ALA-338, LYS-335 and PHE-322, LEU-319 of IbeA; LEU-337, ALA-332 and VAL-326 of IbeA have hydrophobic interactions. And ARG-331 on EC-MP-1 and ASP-282, ASP-254 on IbeA have hydrogen bonds and ionic interactions (salt bridges). In addition, LEU-337, ASP-336 on the analog peptide EC-MP-2 and PHE-322 on IbeA; VAL-332 and LEU-319 on IbeA have hydrophobic interactions. ARG-340 on EC-MP-2 forms a hydrogen bond with THR-325 on IbeA; VAL-332, ARG-331 and GLU-266 form hydrogen bonds and ionic interactions (salt bridges).

[0092] The docking complex of the mimetic peptide EC-MP-1 and the mimetic peptide EC-MP-2 with IbeA (225-343) was simulated by conventional molecular dynamics simulation for 20 ns using the Desmond software, and the binding energy of the mimetic peptide with IbeA was calculated using the MM / GBSA method. It can be seen that the blue curve of EC-MP-1 has converged at the beginning of the simulation, and the root mean square deviation (RMSD) continues to remain at around 3 angstroms ( Figure 4 B left); The blue curve of EC-MP-2 decreases and remains stable in the later stage of the simulation, and the RMSD also remains at about 3 angstroms ( Figure 4 B right). Overall, the RMSD curve of EC-MP-1 is more stable, and the interaction with IbeA fluctuates less. Therefore, based on conventional molecular dynamics simulations, we predict that both EC-MP-1 and EC-MP-2 can stably bind to IbeA, and EC-MP-1 can compete with Caspr1 (203-355) for IbeA.

[0093] 5. The mimetic peptide has low cytotoxicity to brain tissue cells.

[0094] CCK8 was used to test the effects of EC-MP-1 and EC-MP-2 at different concentrations (0-512 μmol / L) on hippocampal neuronal cells (HT22) ( Figure 5 A) and human brain microvascular endothelial cells (HBMEC) ( Figure 5B) Effect on viability. The cells were inoculated in a 96-well plate, and 10 μL of CCK8 reagent was added to each well after 24 hours. The absorbance at 450 nm was measured using a microplate reader after 1 hour. The experimental results showed that the mimetic peptide had low toxicity to different types of brain tissue cells, as shown by the fact that when the concentration was 512 μmol / L, the viability of HT22 and HBMEC was still unaffected, indicating that the mimetic peptide had low cytotoxicity.

[0095] 6. Cytological experiments were used to analyze the effect of peptide mimics on blocking the invasion of E44 Escherichia coli into HBMEC.

[0096] 6.1 The mimetic peptide can effectively inhibit the invasion of E. coli E44 into HBMEC.

[0097] EC-MP-1 and EC-MP-2 were added to HBMEC to a final concentration of 256 μmol / L, and then 5 μL of E44 Escherichia coli (E. coli K1 strain with rifampicin resistance) with an MOI of 100 was added to each well. After co-culture in an incubator for 1.5 h, the number of colonies adhering to the surface of HBMEC and invading into HBMEC was analyzed by cell adhesion and cell invasion assays ( Figure 6 A).

[0098] The results of bacterial adhesion experiments showed that the three peptides did not affect the adhesion of E. coli E44 to human brain microvascular endothelial cells; the results of bacterial invasion experiments showed that the number of E. coli E44 colonies invading human brain microvascular endothelial cells was significantly reduced by about 64.7% after treatment with the two mimetic peptides ( Figure 6 B), this result shows that both EC-MP-1 and EC-MP-2 can effectively reduce the invasion of E. coli E44 into human brain microvascular endothelial cells at the cellular level. However, the linear peptide Line, which does not use the lactam cyclization technology, cannot inhibit the invasion of E. coli E44 into human brain microvascular endothelial cells, which shows that lactam cyclization can effectively enhance the effect of the mimetic peptide, and the blocking ability of EC-MP-2 is stronger than that of EC-MP-1 ( Figure 6 C).

[0099] In addition, we verified the blocking effect of the mimetic peptide again through immunofluorescence experiments. We used anti-E. coli lipopolysaccharide (LPS) antibodies to label E. coli, which showed green fluorescence; and used anti-vascular endothelial cadherin (VE-Cadherin) antibodies to label HBMEC, which showed red fluorescence. The extracellular green fluorescence was quenched with 0.4% trypan blue. The results showed that after treatment with the mimetic peptide, the number of E. coli E44 invading into the cells was significantly reduced by about 74.3%, which is consistent with the results of the invasion experiment ( Figure 6 D).

[0100] 6.2. Peptide simulations can effectively improve the damage of E. coli E44 to the integrity of the blood-brain barrier.

[0101] Tight junction (TJ) proteins are an adhesion complex between epithelial cells and endothelial cells, which maintain tissue integrity by controlling paracellular permeability and cell polarity. Therefore, this study used Western blot to detect the expression of tight junction proteins in brain microvascular endothelial cells ( Figure 7 ), verify the effect of peptide mimic treatment on the integrity of the blood-brain barrier. Peptides were added to the monolayer HBMEC to a final concentration of 256 μmol / L, and 20 μL of wild-type E44 Escherichia coli cultured in BHI medium for 17 h was added to each well. Protein was extracted after culturing for 1.5 h. The results of Western blot experiments showed that compared with the control group, the expression level of ZO-1 in HBMEC was slightly decreased after treatment with the two peptide mimics, while the expression levels of Occludin and Claudin-5 did not change significantly; however, if the linear peptide Line without lactam cyclization was treated and only E44 Escherichia coli was treated, the expression levels of ZO-1 and Occludin were significantly reduced, and the expression of Claudin-5 was not affected. The above results show that the cyclic peptide mimics with a stable biological structure obtained by us can effectively improve the damage of Escherichia coli to the integrity of the blood-brain barrier.

[0102] 7. A newborn rat bacterial meningitis model was constructed to investigate the blocking effect of the mimetic peptide.

[0103] Based on the above cell-level experimental results, we constructed a newborn rat model of bacterial meningitis: 5-day-old SD rats were selected and the newborn rats in the simulated peptide treatment group were injected subcutaneously with 10 μg of insulin in their heads using an insulin syringe. 5 coli and 5 mg / kg EC-MP-1 or EC-MP-2; the newborn rats in the Line treatment group were injected subcutaneously with an insulin syringe on their heads for 10 5 coli and 5 mg / kg of Line without lactam ring formation; the newborn rats in the control group were injected subcutaneously with 10 5 E44 Escherichia coli and an equal amount of normal saline.

[0104] 7.1 The mimetic peptide can effectively block E. coli E44 from invading the blood-brain barrier.

[0105] The newborn rats in each group were injected according to the above modeling method. After 18 hours, the cerebrospinal fluid and blood of each group of rats were extracted and spread on rifampicin-resistant LB solid medium after concentration gradient dilution. The colony forming units (CFUs) were counted at 37°C overnight and the logarithm was calculated ( Figure 8 A). The statistical results showed that there was no significant difference in the detection rate of E44 Escherichia coli in the blood of newborn rats in each group, indicating that the mimetic peptide did not inhibit bacteremia. The detection rate of E44 Escherichia coli in the cerebrospinal fluid of newborn rats in the mimetic peptide treatment group was significantly reduced by about 64% compared with the control group ( Figure 8 B); however, the detection rate of E. coli E44 in the cerebrospinal fluid of newborn rats in the Line treatment group without lactam ring formation was not significantly different from that in the control group. The statistical results showed that the cyclic peptide analogue could effectively inhibit E. coli E44 from crossing the blood-brain barrier and entering the brain of newborn rats ( Figure 8 C).

[0106] After the model was successfully constructed, the mortality of rats in each group was recorded every 12 hours, and the mortality rate of newborn rats within 96 hours was calculated ( Figure 8 D), the results showed that only 10 5 50% of the newborn rats infected with E44 E. coli died within 24 hours after infection. However, more than 60% of the newborn rats in the peptide-treated group survived within 96 hours after infection. This shows that peptide-treated newborn rats can effectively reduce the mortality rate of newborn SD rats with bacterial meningitis and significantly prolong the survival time.

[0107] In the above detection of the number of E. coli E44 in the cerebrospinal fluid of newborn rats, we found that subcutaneous injection of Line into newborn rats with bacterial meningitis had no significant inhibitory effect on the invasion of E. coli E44 into the brain. Therefore, out of consideration for the ethics of experimental animals, this article did not set up a Line treatment group in the statistical mortality rate and the following in vivo experiments to reduce the number of experimental animals.

[0108] 7.2 Peptide mimics can effectively alleviate brain inflammation in newborn rats with bacterial meningitis.

[0109] In order to more intuitively observe the inflammation of the meninges in rats with meningitis in the peptide-mimicking group, brain slices of newborn rats in each group were prepared by HE staining.

[0110] The results showed that 10 5 The meninges of newborn rats inoculated with E. coli E44 showed thickening and damage and neutrophil infiltration (arrows). 5 coli E44 and the mimetic peptide EC-MP-1, as well as newborn rats inoculated with 10 5The meninges of newborn rats infected with E. coli E44 and the EC-MP-2 mimetic peptide were intact and thin, and there were no obvious histopathological changes such as neutrophil infiltration ( Fig. 9 ). The above results indicate that the mimetic peptide can effectively alleviate the inflammatory response in the brain.

[0111] 7.3 Peptide mimics can effectively alleviate inflammatory cell infiltration in the brain of newborn rats with bacterial meningitis.

[0112] After bacterial infection, inflammatory cells in the brain, such as microglia and astrocytes, are activated to produce inflammatory factors. Therefore, we used immunofluorescence to observe the activation of microglia and astrocytes in each group of newborn rats.

[0113] Microglia were labeled with anti-IBA1 antibody, showing red fluorescence; astrocytes were labeled with anti-GFAP antibody, showing green fluorescence; and blood vessels were labeled with anti-Lectin antibody, showing red fluorescence. 5 Compared with newborn rats inoculated with 10 E44 E. coli 5 E. coli E44 and EC-MP-1 mimetic peptide in rat brain microglia ( Fig.10 A) The activation was reduced by about 61.15% (P < 0.01), and the 5 The activation of microglia in the brain tissue of newborn rats inoculated with 10 E. coli and the mimetic peptide EC-MP-2 was reduced by about 64.72% (P < 0.001). 5 coli and the EC-MP-1 peptide mimetic in the brain tissue of newborn rats ( Fig.10 B) activation decreased by about 45.14% (P < 0.01), and 10 5 The activation of astrocytes in the brain tissue of newborn rats treated with E. coli E44 and EC-MP-2 mimetic peptides was reduced by about 37.59% (P < 0.01). However, there was no significant change in the two mimetic peptide-treated rats compared with the sham-operated rats injected with saline (P > 0.05). The above results indicate that mimetic peptides can effectively inhibit the activation of glial cells in the brain, thereby alleviating the symptoms of brain inflammation.

[0114] 7.4 Peptide mimics can effectively alleviate the high expression of inflammatory factors in the brain of newborn rats with bacterial meningitis.

[0115] Inflammatory factors are molecules involved in regulating the immune and inflammatory processes. Tumor necrosis factor-α (TNF-α) is a potent activator of neutrophils and can mediate adhesion and chemotaxis; interleukin-6 (IL-6) can activate T lymphocytes and fibroblasts to produce lymphokines; interleukin-2 receptor (IL-2R) is not easy to pass through the blood-brain barrier and has a low expression level in the cerebrospinal fluid of normal people. The abnormal increase in the expression levels of TNF-α, IL-6, and IL-2R is closely related to the pathogenesis of meningitis. Therefore, we performed immunohistochemical staining to detect the expression of TNF-α, IL-6, and IL-2R in the brains of newborn rats in each group.

[0116] The statistical results showed that compared with the sham-operated group of newborn rats injected with saline, the rats inoculated with 10 5 coli and the mimetic peptides EC-MP-1 or EC-MP-2 in the brain tissue of newborn rats. Fig.11 A) IL-6 ( Fig.11 B) IL-2R ( Fig.11 C) expression levels did not change significantly (P>0.05); however, compared with the newborn rats in the sham operation group, the expression of TNF-α in the brain tissue of the newborn rats inoculated with E. coli E44 increased significantly (P<0.0001), IL-6 increased significantly (P<0.0001), and IL-2R increased significantly (P<0.001). In tissue sections, the above molecules were strongly positive. In summary, the mimetic peptide can effectively improve the inflammatory symptoms in the brain of newborn rats with bacterial meningitis and inhibit the release of inflammatory factors TNF-α, IL-6 and IL-2R in the brain.

[0117] The present invention verifies the function of the mimetic peptide from both the cellular level and the animal level. In vitro experiments first verify that the mimetic peptides EC-MP-1 and EC-MP-2 have extremely low cytotoxicity to brain tissue cells such as hippocampal neurons and human brain microvascular endothelial cells, indicating that the mimetic peptides have good biosafety; then bacterial adhesion experiments and bacterial invasion experiments are used to confirm that the main way in which the mimetic peptides work is to inhibit the invasion of E44 Escherichia coli on HBMEC, while the effect on the bacterial adhesion level is not significant; finally, Western blot experiments are used to verify that the treatment with mimetic peptides can effectively improve the destruction of the tight junction structure between the blood-brain barrier by Escherichia coli. In vivo experiments, we first established a newborn rat bacterial meningitis model and verified that the detection rate of Escherichia coli in the cerebrospinal fluid of newborn rats treated with mimetic peptides was significantly reduced, proving that the mimetic peptides can play an effective therapeutic role in vivo; then we used H&E staining, immunofluorescence and immunohistochemistry experiments to prove that the treatment with mimetic peptides can significantly alleviate the thickening of the meninges and neutrophil infiltration in newborn rats, and can inhibit the activation of glial cells in the brain and reduce the expression of inflammatory factors. The above experimental results can prove the effectiveness of peptide mimics in blocking neonatal bacterial meningitis.

[0118] In summary, the present invention successfully designed and prepared small molecule analog peptides EC-MP-1 and EC-MP-2 based on lactam cyclization technology, which can effectively block the invasion of Escherichia coli into the brain, providing new ideas for the prevention and treatment of neonatal meningitis, and accumulating reliable scientific data for the subsequent development and clinical application of small molecule polypeptide drugs.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A short peptide EC-MP-1 for treating neonatal bacterial meningitis, characterized in that: The amino acid sequence of EC-MP-1 is shown in SEQ ID No.

1.

2. A short peptide EC-MP-2 for treating neonatal bacterial meningitis, characterized in that: The amino acid sequence of EC-MP-2 is shown in SEQ ID No.

2.

3. Use of the short peptide having the effect of treating neonatal bacterial meningitis according to any one of claims 1 to 2 in the preparation of a medicament for treating neonatal bacterial meningitis.

4. Use of the short peptide for treating neonatal bacterial meningitis according to any one of claims 1 to 2 in the preparation of a product for inhibiting Escherichia coli from invading human brain microvascular endothelial cells.

5. A method for preparing a short peptide for treating neonatal bacterial meningitis as claimed in any one of claims 1 to 2, characterized in that: The specific steps are as follows: Step 1, using the molecular docking model to screen key amino acid sequences; Step 2, dehydrating and condensing the carboxyl group on the lysine R group and the amino group on the aspartic acid R group through lactam ring formation technology to form a lactam bond to obtain the short peptide.

6. The preparation method according to claim 3, characterized in that: The key amino acid sequence in step 1 is shown as SEQ ID No.

3.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the short peptide for treating neonatal bacterial meningitis according to any one of claims 1 to 2, and a pharmaceutically acceptable carrier.

8. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating neonatal bacterial meningitis.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of a product for inhibiting Escherichia coli from invading human brain microvascular endothelial cells.

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