Polypeptides and / or their derivatives that target and competitively inhibit the binding of CCL20 to integrin α5β1, and their applications

Abstract

The present invention belongs to the field of biomedicine, and discloses a polypeptide and / or its derivative that targets and competitively inhibits the binding of CCL20 to integrin α5β1, and its application. The amino acid sequence of the polypeptide Pep-CCL20 disclosed in the present invention is shown in SEQ ID NO: 1. The polypeptide Pep-CCL20 can specifically bind to integrin α5β1, thereby blocking the activation of lung fibroblasts by CCL20. The present invention also discloses that the polypeptide derivative is a chimeric peptide Pep-PCCL20 formed by the binding of the polypeptide Pep-CCL20 and a cell-penetrating peptide. The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO: 2. The polypeptide and / or its polypeptide derivative have significant curative effects on the treatment of pulmonary fibrosis diseases, with small toxic and side effects and safe to use.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a polypeptide and / or its derivative that can specifically bind to integrin α5β1 to target and inhibit CCL20, and their applications. Background Art

[0002] Pulmonary fibrosis is the end-stage change of a large category of lung diseases characterized by fibroblast proliferation, massive extracellular matrix aggregation, accompanied by inflammatory damage and tissue structure destruction. It is one of the four major respiratory diseases and the most severe pathological state of the lungs. Its pathological changes mostly manifest as initial lower respiratory tract inflammation, as well as damage to alveolar epithelial cells and vascular endothelial cells, accompanied by proliferation of fibroblasts and type II alveolar cells, release of cytokines, deposition of extracellular matrix proteins and collagen, ultimately leading to changes in the lungs. In patients with pulmonary fibrosis, the alveoli in the lungs are gradually replaced by fibrous substances, resulting in the hardening and thickening of lung tissue, and the gradual loss of the gas exchange ability of the lungs, leading to varying degrees of hypoxia in patients, resulting in dyspnea, and finally death due to respiratory failure. The etiology of pulmonary fibrosis is complex and the pathogenesis is unknown. Currently, the existing drugs and methods for treating pulmonary fibrosis are very limited, and the curative effect is unsatisfactory, and the prognosis is extremely poor. The 5-year survival rate is only 50%.

[0003] Chemokine ligand 20 (C-C Motif Chemokine Ligand 20, CCL20), also known as macrophage inflammatory protein 3α (MIP-3α), belongs to the CC subfamily of chemokines and generally participates in immune regulation and inflammatory processes by chemotaxing immune cells. In recent years, the role of CCL20 in the progression of tissue fibrosis diseases such as liver fibrosis and cystic fibrosis has been successively confirmed, but whether it participates in pulmonary fibrosis has not been reported. The work of the present invention found that in addition to the only known receptor CCR6, CCL20 can bind to integrin α5β1 on the surface of lung fibroblasts.

[0004] Integrins, which are composed of α-subunits and β-subunits, constitute a transmembrane receptor family. It binds to the ECM extracellularly and to the cytoskeleton intracellularly, thus "integrating" the extracellular environment with the cell interior. It can regulate the signal cascades triggered by various growth factors (including TGF-β), and thus participate in cell migration, proliferation and activation. Currently, a number of studies have shown that several integrins exacerbate TGF-β-mediated fibrotic diseases by directly activating TGF-β or affecting downstream signal transduction, suggesting that integrins are potential therapeutic targets for pulmonary fibrosis diseases.

[0005] The research results of the present invention show that continuous and repeated lung injury promotes the secretion of a large amount of CCL20 by type II alveolar epithelial cells. CCL20 in the alveolar microenvironment can specifically bind to integrin α5β1 on lung fibroblasts, thereby inducing the high expression of this receptor. The highly expressed integrin α5β1 converts extracellular inactive TGF-β into an active form, which in turn promotes the activation of lung fibroblasts, leading to the occurrence and development of pulmonary fibrosis. These results suggest that blocking the binding of CCL20 to integrin α5β1 on the surface of lung fibroblasts and inhibiting the highly activation of lung fibroblasts are potential targets for the treatment of pulmonary fibrosis. Therefore, researching and developing substances that block the action of CCL20 has good prospects for drug development in the treatment of pulmonary fibrosis. Summary of the Invention

[0006] Because the pathogenesis of pulmonary fibrosis is complex, there is currently a lack of effective therapeutic drugs. The primary objective of the present invention is to provide a polypeptide and / or its polypeptide derivative that inhibits the activation of lung fibroblasts by inhibiting the binding of CCL20 to integrin α5β1. The polypeptide is named Pep-CCL20, and its amino acid sequence is shown in SEQ ID NO:1.

[0007] The amino acid sequence of the present invention also includes oligopeptide sequences with amino acid substitutions, deletions, or additions at other positions and that can specifically bind to integrin α5β1. The polypeptide and / or its derivative of the present invention is used for targeted treatment of diseases related to CCL20.

[0008] The polypeptide derivative is a chimeric peptide formed by connecting the polypeptide with a cell-penetrating peptide (a cell-penetrating peptide is a type of short peptide that can carry macromolecules into cells, and its transmembrane ability does not depend on classical endocytosis).

[0009] The cell-penetrating peptide is HLYVSPW (shown in SEQ ID NO:2 in the sequence listing), and this cell-penetrating peptide is named P.

[0010] Furthermore, the N-terminus or C-terminus of the polypeptide Pep-CCL20 is connected to the cell-penetrating peptide to obtain the chimeric peptide (Pep-PCCL20).

[0011] The second objective of the present invention is to provide the DNA sequence of the polypeptide and / or its polypeptide derivative.

[0012] The third objective of the present invention is to provide the application of a polypeptide and / or its derivative that targets and competitively inhibits the binding of CCL20 to integrin α5β1, or a polypeptide and / or its derivative that targets and competitively inhibits the binding of CCL20 to integrin α5β1 with the above specific sequence, in the preparation of drugs for the treatment of pulmonary fibrosis.

[0013] The pulmonary fibrosis includes idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

[0014] The pulmonary fibrosis includes drug-induced pulmonary fibrosis, especially drug-induced pulmonary fibrosis caused by bleomycin.

[0015] The treatment of pulmonary fibrosis is carried out by treating pulmonary inflammation, pulmonary function degradation or lung injury.

[0016] The "pulmonary fibrosis" described in the present invention is the conventional pulmonary fibrosis in the art. The pulmonary fibrosis preferably refers to the pulmonary fibrosis caused by various different factors characterized by the pathological changes of idiopathic pulmonary fibrosis. Among them, the pulmonary fibrosis preferably refers to the pulmonary fibrosis of humans or animals, and the symptoms of the pulmonary fibrosis more preferably include: pulmonary inflammation caused by pulmonary fibrosis, and pulmonary function degradation caused by pulmonary fibrosis. The etiology of the pulmonary fibrosis preferably is: pulmonary fibrosis caused by lung injury, pulmonary fibrosis caused by dust, or pulmonary fibrosis caused by drugs, and the drug preferably is bleomycin.

[0017] Among them, the pulmonary fibrosis preferably is primary (specific) pulmonary fibrosis, that is, pulmonary fibrosis of unknown cause; or secondary pulmonary fibrosis, that is, pulmonary fibrosis secondary to a previous disease. The pulmonary fibrosis more preferably is the pulmonary function degradation, pulmonary inflammation and lung injury in pulmonary fibrosis. The diseases of the pulmonary fibrosis preferably include chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis or interstitial pneumonia.

[0018] The "treatment" described in the present invention means reducing the degree of pulmonary fibrosis, or curing pulmonary fibrosis to make it normal, or slowing down the process of pulmonary fibrosis.

[0019] The fourth object of the present invention is to provide a drug for treating pulmonary fibrosis. The drug contains a polypeptide and / or its derivative that target competitively inhibits the binding of CCL20 to integrin α5β1, or the polypeptide and / or its derivative that target competitively inhibits the binding of CCL20 to integrin α5β1 with the above specific sequence, and a pharmaceutically acceptable carrier or excipient.

[0020] The polypeptide or chimera described in the present invention can be used as an active ingredient for preparing an anti-pulmonary fibrosis drug. The "active ingredient" refers to a compound having the function of treating pulmonary fibrosis. The polypeptide or chimera can be prepared with one or more pharmaceutical carriers to prepare an anti-pulmonary fibrosis drug. In this drug, the polypeptide or chimera can be used alone as an active ingredient or together with other compounds having anti-pulmonary fibrosis activity as an active ingredient. The pharmaceutical carrier therein is a conventional pharmaceutical carrier in the art, and preferably includes pharmaceutically acceptable excipients, fillers, diluents, etc.

[0021] The dosage form of the drug for treating pulmonary fibrosis is not particularly limited and is a conventional dosage form in the art. Preferably, the dosage form of the drug is solid, semi-solid or liquid. The dosage form of the drug can also be aqueous solution, non-aqueous solution or suspension. More preferably, the dosage form of the drug is tablet, capsule, granule, injection or infusion. The administration route of the drug is a conventional administration route in the art, and preferably, the administration route is injection or oral administration. Among them, the injection administration method preferably includes: intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection route.

[0022] The dosage of the drug described in the present invention during treatment depends on the age and condition of the patient. The dosage is preferably 0.1 - 15 mg / kg, more preferably 5 - 10 mg / kg, and preferably 5 mg / kg. The administration frequency is preferably once a day or several times. When treating pulmonary fibrosis, the drug described in the present invention can be used alone or in combination with other drugs.

[0023] The fifth object of the present invention is to provide a polypeptide and / or its derivative that targets and competitively inhibits the binding of CCL20 to integrin α5β1, or the use of the polypeptide and / or its derivative that targets and competitively inhibits the binding of CCL20 to integrin α5β1 with the above specific sequence in the preparation of any preparation for targeting and blocking the binding of CCL20 to integrin α5β1 and reducing the activation of lung fibroblasts.

[0024] Advantageous technical effects of the present invention: The present invention provides a polypeptide that can specifically bind to integrin α5β1. This polypeptide can target and block the binding of CCL20 and integrin α5β1, reduce the activation of fibroblasts, and down-regulate the production of extracellular matrix, thereby being applied to the preparation of anti-pulmonary fibrosis drugs. The drug prepared using the above polypeptide has the advantages of significant curative effect, small toxic and side effects, and safe use in the treatment of pulmonary fibrosis diseases. Description of the Drawings

[0025] Figure 1 It is the content of CCL20 in the alveoli of normal and multiple bleomycin-induced mice determined by ELISA in Example 2.

[0026] Figure 2 It is the result of immunoprecipitation to verify the binding of CCL20 and integrin α5β1 in HEK293T cells in Example 3; among them, A represents the interaction between CCL20-EGFP protein and integrin β1-MYC protein after transfection of HEK293T cells; B represents the interaction between CCL20-EGFP protein and integrin α5-MYC protein after transfection of HEK293T cells.

[0027] Figure 3The real-time fluorescence quantitative PCR method in Example 5 was used to verify the inhibitory effect of Pep-CCL20 on the activation of fibroblasts by CCL20.

[0028] Figure 4 The cell migration experiment in Example 6 was used to verify the inhibitory effect of Pep-CCL20 on the fibroblast migration-promoting effect of CCL20. Among them, A is a typical schematic diagram; B is a statistical chart after counting the cells per field by ImageJ.

[0029] Figure 5 It is the pathological picture of Masson staining in Example 7.

[0030] Figure 6 It is the determination of hydroxyproline content in Example 7. Specific implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0033] The reagents and raw materials used in the present invention are all commercially available.

[0034] The PBS described in the embodiments refers to a phosphate buffer solution with a concentration of 0.1 M and a pH value of 7.2.

[0035] The room temperature described in the embodiments is the conventional room temperature in the art, preferably 15-30 °C.

[0036] The experimental results are expressed as mean ± standard error. After parametric or non-parametric variance test, a significant difference is considered when p < 0.05, and an extremely significant difference is considered when p < 0.01.

[0037] Example 1 Preparation of a pulmonary fibrosis animal model

[0038] 1.1 Main reagents and experimental animals

[0039] The bleomycin used in the experiment was purchased from Selleck.

[0040] Unless otherwise specified, the compounds used in the experiment were all purchased from Sigma.

[0041] Specific pathogen-free (SPF) C57BL / 6 mice (male, 6 - 8 weeks old, 16 - 18 g) used in the experiment were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[0042] 1.2 Preparation of animal model of pulmonary fibrosis

[0043] Male C57BL / 6 mice (6 - 8 weeks old) were fasted overnight, anesthetized with sodium pentobarbital (45 mg / kg, i.p.), and intratracheally injected with bleomycin (1 U / kg). The administration was carried out 4 - 6 times at intervals of 14 days.

[0044] The specific protocol was as follows: After anesthetizing the mice, they were fixed in the prone position. The neck position of the mice was irradiated with a cold light source. With the right hand holding forceps, the tongue of the mice was pulled outwards, and with the left hand holding forceps, the mouth was opened as wide as possible until the glottis was exposed. Then, under the mediation of a guide wire, a 20G trocar was inserted into the trachea of the mice. After that, a microsyringe was used to inject about 1 U / kg of bleomycin into the trachea, and the mice were quickly rotated and upright for 5 minutes to enable the bleomycin to enter the left and right lung lobes evenly. The whole operation was carried out on a surgical operating table at about 60 °C. In the sham operation group, an equal amount of normal saline for injection was intratracheally injected. After an interval of 14 days, the operation was repeated, and the repetition was carried out 4 - 6 times in total.

[0045] Example 2 Detection of the expression of CCL20 in bronchoalveolar lavage fluid of mice with pulmonary fibrosis by ELISA method

[0046] 2.1 Obtaining bronchoalveolar lavage fluid from the normal group and mice with multiple modelings, the steps were as follows:

[0047] (1) The mice were anesthetized with sodium pentobarbital (45 mg / kg, i.p.) and fixed on a foam board. The skin and muscle of the neck of the mice were cut open to expose the trachea.

[0048] (2) The trachea of the mice was cut open, and a 20G trocar was inserted and fixed with surgical sutures.

[0049] (3) 1 ml of normal saline was taken with a syringe and injected into the trachea through the 20G trocar. After repeated lavage 3 times, the lavage fluid was transferred to a 1.5 ml centrifuge tube.

[0050] (4) The lavage fluid was centrifuged at 3000 rpm for 5 minutes, and the supernatant was frozen at -80 °C.

[0051] 2.2 Determination of the content of CCL20 in bronchoalveolar lavage fluid (ELISA kit was purchased from Linker Biotechnology):

[0052] (1) All reagents and samples were equilibrated to room temperature.

[0053] (2) Add 300 μL of 1× wash solution to the microplate, let it stand and soak for 30 seconds, then add 100 μL of bronchoalveolar lavage fluid or diluted standard product, and then add 50 μL of diluted detection antibody to each well. Seal the plate with a sealing film, shake at 300 revolutions per minute, and incubate at room temperature for 1.5 hours.

[0054] (3) Discard the liquid, add 300 μL of wash solution to each well to wash the plate, and wash 6 times.

[0055] (4) Add 100 μL of diluted streptavidin-horseradish peroxidase to each well, seal the plate with a sealing film, shake at 300 revolutions per minute, and incubate at room temperature for 30 minutes.

[0056] (5) Discard the liquid, add 300 μL of wash solution to each well to wash the plate, and wash 6 times.

[0057] (6) Add 100 μL of chromogenic substrate TMB to each well, protect from light, incubate at room temperature for 5 - 30 minutes, and then add 100 μL of stop solution to each well.

[0058] (7) Use an enzyme-linked immunosorbent assay reader for dual-wavelength detection, measure the maximum absorption wavelength at 450 nm and the reference wavelength at 570 nm. Subtract the measured value at 570 nm or 630 nm from the measured value at 450 nm.

[0059] 2.3 Results show that:

[0060] The level of CCL20 in the bronchoalveolar lavage fluid of the model group increased significantly with the number of times of bleomycin administration. The results are shown in Figure 1 .

[0061] Example 3 Use the method of co-immunoprecipitation to verify the binding of CCL20 and integrin α5β1 in HEK293T cells

[0062] 3.1 Co-immunoprecipitation reagents

[0063] Co-immunoprecipitation lysis buffer: 0.6057 g of Tris base, 1.7532 g of NaCl, 0.1017 g of MgCl2·6H2O, 0.0742 g of EDTA, 10 mL of glycerol, 10 mL of 10% NP40, add deionized water to 150 mL, adjust the pH value to 7.6 with HCl, make up the volume to 191 mL, mix well, filter through a 0.45 μm filter membrane, and store at 4°C.

[0064] Anti-Myc beads are purchased from Bimake.

[0065] 3.2 Specific operation steps

[0066] (1) HEK293T cells were transfected with plasmids: plasmid integrin β1-MYC, plasmid integrin β1-MYC and plasmid CCL20-GFP, plasmid integrin α5-MYC, plasmid integrin α5-MYC and plasmid CCL20-GFP. Plasmid CCL20-GFP and plasmid integrin α5-MYC were purchased from Ubest Biotechnology, and plasmid integrin β1-MYC was purchased from Sino Biological.

[0067] (2) Lyse the cells with immunoprecipitation lysis buffer, harvest about 4-10 mg of total cell protein, and adjust each group of proteins to the same concentration. Take 200 μg of each group of proteins, take 200 μg of each group of proteins as the input group of cell lysate, and use the resulting input group of cell lysate as a control. (3) Add 20 μL of Anti-Myc beats to the remaining protein, resuspend it thoroughly, and shake it slowly at 4°C overnight.

[0068] (4) Place the sample on a magnetic rack and wait for the magnetic beads to adsorb to the wall, then discard the liquid. Add 0.5 mL of immunoprecipitation lysis buffer, invert to mix, adsorb the magnetic beads again, and discard the liquid. Repeat the wash five times, add 50 μL of 2× SDS gel loading buffer, mix, denature at 95°C for 10 min, and quickly transfer to an ice bath to cool. Take part or all of it for SDS-polyacrylamide gel electrophoresis.

[0069] 3.3 Results

[0070] The results are as follows Figure 2 As shown (A shows HEK293T cells transfected with integrin β1-MYC and CCL20-GFP; B shows HEK293T cells transfected with integrin α5-MYC and CCL20-GFP), Figure 2 The results show that intracellular CCL20 protein binds to integrin α5 and integrin β1. The preparation method of the input cell lysate is as shown above. The input represents the protein content of integrin α5, integrin β1 and CCL20 protein contained in the initial HEK293T cell lysate, that is, the protein stock solution before precipitation by MYC antibody (since the transfected integrin α5 and integrin β1 proteins are both MYC-tagged, MYC antibody magnetic beads are selected). Cells transfected with integrin α5 and integrin β1 only were used as the control group, and cells transfected with CCL20 were used as the experimental group. The results showed that the results after transfection of the input group were consistent with expectations, and the control group and the experimental group cells were transfected with equal amounts of integrin protein.

[0071] The output represents the integrin α5, integrin β1, and CCL20 contained in the HEK293T cell lysate after adsorption by the MYC antibody magnetic beads. Since both integrin β1 and integrin α5 carry MYC, the tags can adsorb integrin β1 and integrin α5 in Figures A and B respectively. And CCL20 can bind to integrin β1 and integrin α5, so it can be adsorbed together by the MYC antibody magnetic beads. Therefore, the protein blot lanes incubated with the GFP antibody in the experimental groups of Figures A and B show black. The control group was not transfected with CCL20 containing GFP, so the protein blot lanes incubated with the GFP antibody in Figures A and B show blank. The above experimental results fully prove that the CCL20 protein can bind to both integrin α5 and integrin β1 proteins.

[0072] Example 4 Detection of the binding ability between polypeptide Pep-CCL20 and integrin α5 protein by surface plasmon resonance

[0073] The amino acid sequence of the Pep-CCL20 polypeptide is TWVKYIVRLLSKKVK (SEQ ID NO: 1).

[0074] 4.1 Surface plasmon resonance experiment was carried out on a surface plasmon resonance instrument Biacore T200, and the operation steps were carried out according to the instructions of the plasmon resonance instrument Biacore T200. The specific steps are as follows:

[0075] (1) The purified integrin α5β1 protein (purchased from Sino Biological Inc.) was coupled to a CM5 chip (purchased from GE Healthcare) through amino coupling, and the unbound protein was eluted at a flow rate of 10 μL / min, and the surface of the chip was equilibrated for 2 hours. Among them, the specific steps of amino coupling, elution, and equilibration refer to the relevant instructions of the CM5 chip of GE Healthcare.

[0076] (2) 250 μL of Pep-CCL20 polypeptide fragments with different concentrations (5000, 2500, 1250, 625, 312.5, 156.25, 9.76 nM) were automatically injected. The whole surface plasmon resonance experiment was carried out at 25 °C. The buffer used was HBS-EP buffer [0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% (w / w) surfactant]. The binding curves of polypeptides with different concentrations and integrin α5β1 protein were simulated using the built-in analysis software of Biacore T200, and the affinity between the polypeptide and integrin α5β1 protein was calculated.

[0077] 4.2 Results show that:

[0078] As shown in Table 1, Table 1 shows that the peptide Pep-CCL20 has a high affinity for integrin α5β1 protein.

[0079] Table 1 Affinity Test of Polypeptide Pep-CCL20 and Integrin α5β1 Protein

[0080] Polypeptide Name Affinity Constant (KD) with Integrin α5β1 Protein Pep-CCL20 <![CDATA[1.603×10 -6 M]]>

[0081] Example 5 Verification of the Effect of Chimeric Peptide Pep-CCL20 on Inhibiting CCL20-induced Fibroblast Activation by Real-Time Fluorescent Quantitative PCR Method

[0082] The specific operation steps are as follows:

[0083] 5.1 Cell Collection

[0084] (1) MRC5 cells (human embryonic lung fibroblasts) were evenly seeded in a six-well plate and divided into a control group (PBS), an experimental group (only adding human recombinant CCL20 factor), and a treatment group (adding human recombinant CCL20 factor and Pep-CCL20). They were pre-starved for 24 hours.

[0085] (2) For the treatment group, Pep-CCL20 (final concentration 5 μM) was added and incubated for 30 minutes.

[0086] (3) Human recombinant CCL20 factor (purchased from Peprotech) was added to the experimental group and the treatment group.

[0087] (4) After 48 hours, the cells were washed 3 times with PBS and then the cells of the three groups were collected.

[0088] 5.2 RNA Extraction. The extraction method referred to the Promega RNA Extraction Kit.

[0089] (1) The cells were respectively added with 300 μL of RNA lysis buffer and 300 μL of RNA dilution buffer. After thorough mixing, they were left at room temperature for 7 minutes.

[0090] (2) After centrifuging at the maximum speed of the centrifuge for 5 minutes, the supernatant was taken and 300 μL of absolute ethanol was added. After thorough mixing, it was transferred to a centrifugal column.

[0091] (3) After centrifuging at 12000×g for 1 minute, the liquid in the tube was discarded.

[0092] (4) 600 μL of RNA washing buffer was added and centrifuged at 12000×g for 1 minute.

[0093] (5) 50 μL of diluted DNase was added and left at room temperature for 15 minutes. Then 600 μL of RNA washing buffer was added and centrifuged at 12000×g for 1 minute, and the liquid in the tube was discarded.

[0094] (6) After repeating the washing twice, the centrifugal column was centrifuged at 12000×g for 2 minutes.

[0095] (7) Transfer the centrifugal column to a collection tube, add 50 μL of enzyme-free water to the center of the column, wait for 5 minutes at room temperature, centrifuge at 12,000×g for 2 minutes, and store the collected liquid at -80 °C.

[0096] 5.3 Then reverse transcribe the extracted RNA into cDNA using a rapid reverse transcription kit (Beijing Quanshijin Biotechnology). The steps are as follows:

[0097] (1) Take 100 ng - 5 μg of total RNA, make up the volume to 7 μL with enzyme-free water, add 10 μL of 2×ES Reaction Mix, 1 μL of RT / RI Enzyme mix, 1 μL of gDNA Remover, and 1 μL of Oligo(dT) 18 Primer, and mix well.

[0098] (2) Incubate at 42 °C for 15 minutes and at 85 °C for 5 seconds. Collect the cDNA as a template for qPCR.

[0099] qPCR primers:

[0100] GAPDH

[0101] Forward: 5'-GTCTCCTCTGACTTCAACAGCG-3'; SEQ ID NO: 3;

[0102] Reverse: 5'-ACCACCCTGTTGCTGTAGCCAA-3; SEQ ID NO: 4;

[0103] α-SMA

[0104] Forward: 5'-CTATGCCTCTGGACGCACAACT-3'; SEQ ID NO: 5;

[0105] Reverse: 5'-CAGATCCAGACGCATGATGGCA-3; SEQ ID NO: 6.

[0106] Design mouse GAPDH and α-SMA primers, and use the reverse transcribed cDNA as a template for qPCR. The qPCR system is: 10 μL of KAPA enzyme, 200 nM of primer, 50 ng of template, and 8 μL of water.

[0107] 5.4 Results show that:

[0108] CCL20 can upregulate the expression of α-smooth muscle actin (α-SMA) in MRC5 cells, but treatment with the polypeptide Pep-CCL20 can reverse this upregulation. The results are shown in Figure 3 .

[0109] Example 6 Cell Migration Experiment Verifies that Pep-CCL20 Inhibits the Fibroblast Migration-promoting Effect of CCL20

[0110] 6.1 The specific operation steps are as follows:

[0111] (1) MRC5 cells were pre-starved for 24 hours.

[0112] (2) Take a Transwell chamber (purchased from Corning) and invert it in a large dish. Uniformly spread 60 μL of fibronectin (10 μg / mL) on the bottom of the chamber and let it dry in the cell culture hood.

[0113] (3) Place the dried Transwell chamber in a 24-well plate. Add the following to the outer chambers respectively: MEM medium, MEM medium + recombinant CCL20 factor (25 nM), MEM medium + recombinant CCL20 factor (25 nM) + Pep-CCL20 (final concentration 5 μM), MEM medium + recombinant CCL20 factor (25 nM) + Pep-CCL20 (final concentration 10 μM). The volume of liquid in each outer chamber is 500 μL.

[0114] (4) Digest MRC5 cells, resuspend them to 1×10 6 cells / mL, and then add 100 μL to each chamber. Place it in an incubator at 37 °C for 12 hours.

[0115] (5) Take out the chamber, discard the liquid inside the chamber, rinse it with PBS, and then place it in 4% paraformaldehyde for 30 minutes.

[0116] (6) After rinsing again with PBS, place it in crystal violet staining solution overnight.

[0117] (7) After rinsing the chamber with PBS, gently wipe the residual liquid and cells inside the chamber with a cotton swab, and then let it dry.

[0118] (8) Take a photo of the bottom of the chamber under a microscope and perform cell counting using ImageJ software.

[0119] 6.2 Results:

[0120] CCL20 promotes the migration of MRC5 cells. After adding the polypeptide Pep-CCL20, as the concentration of the polypeptide increases, the ability to promote migration weakens. The results are shown in Figure 4 .

[0121] Example 7 Verification of the Anti-pulmonary Fibrosis Effect of Chimeric Peptide Pep-PCCL20 Using a Pulmonary Fibrosis Animal Model

[0122] 7.1 The animal models prepared in Example 1 were grouped and administered drugs 10 days after modeling. The grouping and drug administration are shown in Table 2 (i.p. indicates intraperitoneal injection):

[0123] Table 2 Grouping and drug administration of the pulmonary fibrosis animal models after modeling

[0124]

[0125] 7.2 Masson staining pathological imaging analysis

[0126] Masson staining is a classic and authoritative method for collagen fiber staining. After staining, muscle fibers are red, while collagen fibers are blue, mainly differentiating muscle fibers and collagen fibers.

[0127] Take the right lower lobe lung tissue of the animal, fix it with 4% paraformaldehyde and embed it in paraffin. Cut sections on the largest cross-section of the wax block embedding the lung tissue, and observe the fibrosis status by Masson staining. Use the high-definition digital color pathological image analysis system SpotAdvanced 3.0 to obtain high-definition pathological pictures of Masson special staining (100 times). The results are shown in Figure 5 . The results showed that after treatment with the polypeptide Pep-PCCL20, the area of blue collagen fibers in the lung tissue of mice decreased significantly.

[0128] 7.3 Determination of hydroxyproline content in pulmonary fibrosis mice

[0129] Hydroxyproline accounts for 13.4% in collagen, a very small amount in elastin, and does not exist in other proteins. Therefore, the content of collagen is detected by hydroxyproline. Detect the content of hydroxyproline in the whole left lung of the animal to evaluate the situation of pulmonary fibrosis. The specific method is as follows: Take the same lung lobe of the model group and treatment group animals prepared in Example 7, record the wet weight, add 1 mL of alkaline hydrolysis solution for homogenization and vortex mixing, and then treat it by alkaline hydrolysis method at 95 °C and 0.1 Kpa for 20 min (the method refers to the kit instruction manual of Nanjing Jiancheng Bioengineering Technology Co., Ltd., with slight modifications). Adjust the pH value and make up the volume, and take the supernatant after activated carbon treatment. Perform the determination of hydroxyproline according to the instruction manual (chloramine T method). The results are shown in Figure 6 , from Figure 6 it can be seen that after treatment with Pep-PCCL20, the content of hydroxyproline in the lungs of fibrotic mice can be significantly reduced.

[0130] In this experiment, through pathological examination, pathological imaging and other means of analyzing the results, it was found that Pep-PCCL20 can significantly inhibit bleomycin-induced pulmonary fibrosis; significantly reduce the hydroxyproline and collagen content in the lung tissue of pulmonary fibrosis mice. The above experimental results prove that Pep-PCCL20 has excellent therapeutic prospects in pulmonary fibrosis.

[0131] The results of the above embodiments show that the polypeptide of the present invention has a significant effect against pulmonary fibrosis diseases and can be used as an active ingredient for preparing anti-pulmonary fibrosis drugs.

[0132] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide and / or its derivative that competitively inhibits the binding of CCL20 to integrin α5β1 in a targeted manner, characterized in that, The amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO: 1; the polypeptide derivative is a chimeric peptide formed by the connection of the polypeptide and a cell-penetrating peptide.

2. The polypeptide and / or its derivative that targets and competitively inhibits the binding of CCL20 to integrin α5β1 according to claim 1, characterized in that, The cell-penetrating peptide has the amino acid sequence shown in SEQ ID NO:

2.

3. The polypeptide and / or its derivative according to claim 2 that targets and competitively inhibits the binding of CCL20 to integrin α5β1, characterized in that, The N-terminus or C-terminus of the polypeptide is connected to the cell-penetrating peptide to obtain the chimeric peptide.

4. Use of the polypeptide and / or its derivative according to any one of claims 1-3 for competitively inhibiting the binding of CCL20 to integrin α5β1 in the preparation of a medicament for treating pulmonary fibrosis.

5. The application according to claim 4, wherein The pulmonary fibrosis is idiopathic pulmonary fibrosis or secondary pulmonary fibrosis.

6. The application according to claim 4, characterized in that The pulmonary fibrosis is drug-induced pulmonary fibrosis.

7. The application according to claim 6, wherein The drug-induced pulmonary fibrosis is drug-induced pulmonary fibrosis caused by bleomycin.

8. The application according to any one of claims 4-7, characterized in that, The treatment of pulmonary fibrosis is carried out by treating pulmonary inflammation, pulmonary function degradation or lung injury.

9. A drug for treating pulmonary fibrosis, characterized in that, The medicament contains the polypeptide and / or its derivative according to any one of claims 1-3 for competitively inhibiting the binding of CCL20 to integrin α5β1, and a pharmaceutically acceptable carrier or excipient.

10. Use of the polypeptide and / or its derivative according to any one of claims 1-3 for competitively inhibiting the binding of CCL20 to integrin α5β1 in the preparation of any preparation for targetedly blocking the binding of CCL20 to integrin α5β1 and reducing the activation of lung fibroblasts.

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