CN111423502A - Polypeptide, derivative thereof and application thereof in preparation of anti-pulmonary fibrosis drugs - Google Patents

Abstract

The invention discloses a polypeptide specifically bound with GSK-3 β or a derivative of the polypeptide, wherein the amino acid sequence of the polypeptide is from an A20 sequence bound with GSK-3 β, the amino acid sequence of the polypeptide is shown as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 in a sequence table, and the derivative of the polypeptide comprises a chimeric peptide formed by connecting the polypeptide specifically bound with GSK-3 β and cell-penetrating peptide.

Description

Polypeptide, derivative thereof and application thereof in preparation of anti-pulmonary fibrosis drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide, a derivative thereof and application thereof in preparation of anti-pulmonary fibrosis drugs.

Background

Pulmonary fibrosis is the most serious pathological state of the lung, and its pathological changes are mostly manifested by initial lower respiratory tract inflammation, and alveolar epithelial cell and vascular endothelial cell injury, accompanied by fibroblast and type II alveolar cell proliferation, cytokine release, extracellular matrix protein and collagen deposition, ultimately leading to pulmonary changes. Pulmonary alveoli of pulmonary fibrosis patients are gradually replaced by fibrous substances, so that pulmonary tissues become hard and thick, the gas exchange capacity of the lung is gradually lost, the patients are difficult to breathe due to different degrees of hypoxia, and finally die due to exhaustion of breath. The pulmonary fibrosis is one of four major diseases of respiratory diseases, the etiology is complex, the pathogenesis is unknown, the existing medicaments and methods for treating the pulmonary fibrosis are very limited, the curative effect is poor, the prognosis is very poor, and the 5-year survival rate is only 50%.

A20 ubiquitin-modifying enzyme, also known as TNFAIP3, was first discovered in human umbilical vein endothelial cells and acts primarily by inhibiting TNF- α -mediated apoptosis, blocking NF-. kappa.B signaling pathway, etc., and is involved in the regulation of immune responses in vivo it was discovered that A20, a well-recognized anti-inflammatory protein, has been shown to play an important negative regulatory role in a variety of autoimmune diseases, such as asthma, allergy, and cystic fibrosis, etc. knockout A20 mice develop severe inflammatory responses and prematurely die.A 20 is involved in regulating macrophage differentiation by down-regulating the NF-. kappa.B signaling pathway under acute inflammatory stimuli.furthermore, increasing the expression of A20 in macrophages inhibits inflammatory responses caused by Toll-like receptors.GSK-3 β is a serine/threonine kinase that has evolved to be well conserved in the central nervous system.A polypeptide that specifically binds to GSK-3 β, such as L803-mts, is currently used in the prevention and treatment of central nervous system, and no polypeptide that specifically binds to GSK-3 β, or any related lung fibrosis, is currently reported.

Disclosure of Invention

The polypeptide, the polypeptide derivative and the pharmaceutical composition can block the interaction between GSK-3 β and A20 protein, so that the polypeptide, the polypeptide derivative and the pharmaceutical composition can be applied to the preparation of the medicine for preventing and/or treating pulmonary fibrosis.

The inventor finds that continuous and repeated lung injury promotes the expression of protein kinase GSK-3 β, high-expression GSK-3 β and A20 play interaction, induces phosphorylation of A20, inhibits activity of A20 enzyme, causes activation of alveolar macrophages, and secretes a large amount of profibrotic factors to promote proliferation, migration and activation of lung fibroblasts (main effector cells of pulmonary fibrosis), so that the conclusion that blocking of interaction between GSK-3 β and A20 is a potential way for treating pulmonary fibrosis is inferred, and the mechanism of interaction between GSK-3 β and A20 and related to pulmonary fibrosis is the first discovery of the invention, and then the inventor obtains a polypeptide targeting interaction between GSK-3 β and A20 proteins through research and development, and finds that the polypeptide can be applied to preparation of drugs for treating and/or preventing pulmonary fibrosis.

To solve the above technical problems, one of the technical solutions provided by the present invention is a polypeptide specifically binding to GSK-3 β having an amino acid sequence derived from the A20 sequence binding to GSK-3 β, as shown in SEQ ID NO:1 (the amino acid sequence thereof is β 0 eu-Val-L eu-Arg-L ys-Ala-L eu-Phe-Ser-Thr-L eu-L ys), SEQ ID NO:2 (the amino acid sequence thereof is Trp-Asn-Asp-Glu-Trp-Asp-Asn-L eu-Ile-L ys-Met-Ala) or SEQ ID NO:3 (the amino acid sequence thereof is Ile-His-Ile-Phe-Val-L eu-Asn-Ile), or a derivative of said polypeptide, wherein amino acid substitutions, deletions or additions may be appropriately introduced as long as the altered amino acid sequence is still capable of forming a polypeptide with a specific binding to GSK-3 and the activity of which is still maintained before GSK-3 β 1 is altered.

The derivative of the polypeptide comprises a chimeric peptide formed by connecting the polypeptide which specifically binds to GSK-3 β and a cell-penetrating peptide.

Preferably, the polypeptide is an α helical peptide.

Preferably, the cell-penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide, preferably to the N-terminus of the polypeptide; more preferably, the cell-penetrating peptide and the polypeptide are linked by two glutamates.

The cell-penetrating peptide of the present invention may be a cell-penetrating peptide conventional in the art as long as it can assist in introducing the polypeptide into cells to function, and in general, the cell-penetrating peptide is a short peptide molecule consisting of 10 to 30 amino acids, preferably, the cell-penetrating peptide is selected from the group consisting of a Pep2 peptide (H L YVSPW, the amino acid sequence of which is shown in SEQ ID NO:4 of the sequence Listing), a TAT peptide of an HIV-1 virus reverse transcription activator (Tat) protein (YGRKKRRQRRR, the amino acid sequence of which is shown in SEQ ID NO: 5), a transcription factor Antp peptide of a Drosophila antetype protein (RQINGFQRMKWKK, the amino acid sequence of which is shown in SEQ ID NO: 6), a Pewap-1 peptide (KETWTWWSEWPEQPKKKRKV, the amino acid sequence of which is shown in SEQ ID NO: 7), a MPG peptide (MPG 36L, the amino acid sequence of which is shown in SEQ ID NO: 6757), and a PeTWTWTWWSEWTKKKKRK 7, preferably, or more preferably, the amino acid sequence of any one or more preferably, the peptides of the amino acid sequences of.

In the present invention, the polypeptide derivative is preferably a chimeric peptide formed by linking the Pep2 peptide to the N-terminus of the polypeptide represented by any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in the sequence Listing, or linking the TAT peptide to the N-terminus of the polypeptide represented by SEQ ID NO:1 in the sequence Listing, or linking the Antp peptide to the N-terminus of the polypeptide represented by SEQ ID NO:1 in the sequence Listing, and the amino acid sequence of the chimeric peptide is preferably any one of SEQ ID NO:10 (the amino acid sequence thereof is H L YVSPGG L V L RKA L FST L K), SEQ ID NO:11 (the amino acid sequence thereof is H L3 YVSPWGGWNDEWDN L MA), SEQ ID NO:12 (the amino acid sequence thereof is H L IKK YVSPWGGIHIFV L CNI), SEQ ID NO:13 (the amino acid sequence thereof is YGRKKRRQRRRGG L V L A L T L K) and FSV 39L 6 (the amino acid sequence thereof is RKA L6K).

In order to solve the technical problems, the second technical scheme provided by the invention is as follows: the active component of the medicine composition for resisting pulmonary fibrosis contains the polypeptide or the derivative of the polypeptide.

The "active ingredient" of the present invention refers to a compound having the function of preventing and/or treating pulmonary fibrosis.

Preferably, the active ingredient is a single active ingredient; or the active ingredient may also comprise other compounds having anti-pulmonary fibrosis activity.

Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

The pharmaceutical carrier in the present invention may be a conventional pharmaceutical carrier in the art, and preferably includes pharmaceutically acceptable excipients, fillers, diluents, and the like.

In the present invention, the dosage form of the pharmaceutical composition is not particularly limited, and may be a conventional dosage form in the art. The dosage form of the pharmaceutical composition is preferably solid, semi-solid or liquid. The pharmaceutical composition may also be in the form of an aqueous solution, a non-aqueous solution or a suspension. The dosage form of the pharmaceutical composition is preferably tablet, capsule, granule, injection or infusion. The route of administration of the pharmaceutical composition may be a route of administration conventional in the art, and is preferably injection or oral administration. Wherein the mode of administration by injection preferably comprises: intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous routes.

The dosage of the pharmaceutical composition of the present invention in treatment depends on the age and condition of the patient, and is preferably 0.1-15 mg/kg, more preferably 5-10 mg/kg, and preferably 5mg/kg, and the administration is preferably performed once or several times a day. The pharmaceutical composition of the present invention can be used alone or in combination with other drugs in the prevention and/or treatment of pulmonary fibrosis.

In order to solve the technical problems, the third technical scheme provided by the invention is as follows: the polypeptide or the derivative of the polypeptide and/or the pharmaceutical composition are applied to the preparation of drugs for preventing and/or treating pulmonary fibrosis diseases.

The "pulmonary fibrosis" described in the present invention may be pulmonary fibrosis which is conventional in the art. The pulmonary fibrosis is preferably pulmonary fibrosis characterized by a pathological change in idiopathic pulmonary fibrosis, resulting from a variety of factors. Preferably, the pulmonary fibrosis disease is dust, radiation and/or drug induced pulmonary fibrosis disease, the drug is, for example, bleomycin.

Preferably, the pulmonary fibrosis is preferably pulmonary fibrosis of human or animal.

Preferably, the pulmonary fibrosis disease is a primary (specific) pulmonary fibrosis (i.e., pulmonary fibrosis of unknown cause) disease, or a secondary pulmonary fibrosis disease, i.e., a pulmonary fibrosis disease secondary to the original disease; more preferably, the pulmonary fibrosis disease comprises Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis, interstitial pneumonia, sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, fibrotic pulmonary inflammation associated with collagen vascular disease, pulmonary function deterioration, or lung injury.

Preferably, the medicament for preventing and/or treating pulmonary fibrosis diseases is a medicament for reducing lung weight index, reducing the number of inflammatory cells in alveolar lavage fluid, reducing hydroxyproline content of lung tissues, reducing collagen content of lung tissues and improving lung functions; more preferably, the inflammatory cells include total leukocytes, monocytes, basophils, eosinophils and/or neutrophils.

In order to solve the technical problems, the fourth technical scheme provided by the invention is the application of the inhibitor or antagonist of GSK-3 β and/or A20 in the preparation of the drugs for preventing and treating pulmonary fibrosis.

In the present invention, anti-pulmonary fibrosis refers to prevention and/or treatment of pulmonary fibrosis.

The term "prevention" as used herein refers to the prevention or reduction of the development of pulmonary fibrosis after use in the presence of possible pulmonary fibrosis factors. The term "treatment" as used herein means to reduce the degree of pulmonary fibrosis, or to cure pulmonary fibrosis to normalize it, or to slow down the progression of pulmonary fibrosis.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The polypeptide, the polypeptide derivative and the pharmaceutical composition can block the interaction between GSK-3 β and A20 protein, so that the polypeptide, the polypeptide derivative and the pharmaceutical composition can be applied to the preparation of drugs for preventing and/or treating pulmonary fibrosis.

Drawings

FIG. 1 shows the inhibitory spectrum of A20 enzyme activity in pulmonary fibrosis mouse alveolar macrophages.

FIG. 2 is a graph showing the interaction between GSK-3 β and A20 in lung tissue of pulmonary fibroid mice, in which FIG. 2(A) shows the protein contents of GSK-3 β and A20 proteins in an initial alveolar macrophage lysate, and FIG. 2(B) shows the protein contents of GSK-3 β and A20 proteins in an alveolar macrophage lysate after IgG precipitation with an A20 antibody or a control antibody.

FIG. 3 shows the increase pattern of GSK-3 β expression in pulmonary fibrosis mouse alveolar macrophages.

FIG. 4 is a graph showing the results of the detection of mice with pulmonary fibrosis mortality caused by bleomycin reduction by Pep2-PA1, Pep2-PA2 and Pep2-PA 3.

FIG. 5 is a graph showing that Pep2-PA1, Pep2-PA2 and Pep2-PA3 reduce the interaction between GSK-3 β and A20 in the lung of mice with pulmonary fibrosis caused by bleomycin, wherein FIG. 5(A) shows the protein contents of GSK-3 β protein and A20 protein in the initial lung tissue lysate, and FIG. 5(B) shows the protein contents of GSK-3 β protein and A20 protein in the lung tissue lysate which is treated by Pep2-PA1, Pep2-PA2 and Pep2-PA3 respectively and precipitated by using an A20 antibody or a control antibody IgG.

FIG. 6 is a graph showing the results of decreasing lung weight index of mice with pulmonary fibrosis caused by bleomycin by Pep2-PA1, Pep2-PA2 and Pep2-PA 3.

FIG. 7 is a graph showing the results of Pep2-PA1, Pep2-PA2 and Pep2-PA3 reducing the number of various inflammatory cells in pulmonary alveolar lavage fluid of mice with pulmonary fibrosis caused by bleomycin. Wherein: a is the total leukocyte number, B is the monocyte number, C is the neutrophil number, D is the lymphocyte number, E is the basophil number, and F is the eosinophil number.

FIG. 8 is a pathological examination (HE) pattern of Pep2-PA1, Pep2-PA2, Pep2-PA3 for reducing pulmonary fibrosis caused by bleomycin. Wherein: a is a pseudo-surgery group, B is a bleomycin model group, C is a bleomycin and Pep2-PA1 group, D is a bleomycin and Pep2-PA2 group, E is a bleomycin and Pep2-PA3 group, and F is a bleomycin and pirfenidone group.

FIG. 9 is a graph of the results of pathological examination scores of Pep2-PA1, Pep2-PA2 and Pep2-PA3 for reducing pulmonary fibrosis caused by bleomycin.

FIG. 10 shows the pathological examination (sirius red) of Pep2-PA1, Pep2-PA2, Pep2-PA3 for reducing pulmonary fibrosis caused by bleomycin. Wherein: a is a sham operation group, B is a model group, C is a Pep2-PA1 group, D is a Pep2-PA2 group, E is a Pep2-PA3 group, and F is a positive control drug pirfenidone group.

FIG. 11 shows the effect of Pep2-PA1, Pep2-PA2 and Pep2-PA3 on the collagen content in lung tissues of mice with pulmonary fibrosis.

Fig. 12 shows the lung function test results of mice with pulmonary fibrosis. Wherein A is the amount of deep inspiration, B is the dynamic resistance, C is the dynamic elasticity, and D is the dynamic compliance.

FIG. 13 is a graph showing the results of Pep2-PA1, Pep2-PA2 and Pep2-PA3 reducing the content of hydroxyproline in lung tissues of mice with pulmonary fibrosis caused by bleomycin.

FIG. 14 is a graph showing the results of mice with pulmonary fibrosis caused by decreasing bleomycin death rate by TAT-PA1 and Antp-PA 1.

FIG. 15 is a graph showing the result of TAT-PA1 and Antp-PA1 reducing the lung weight index of mice with pulmonary fibrosis caused by bleomycin.

FIG. 16 is a graph showing the results of TAT-PA1 and Antp-PA1 reducing the number of various inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis caused by bleomycin. Wherein: a is the total leukocyte number, B is the monocyte number, C is the neutrophil number, D is the lymphocyte number, E is the basophil number, and F is the eosinophil number.

FIG. 17 is a graph showing the results of TAT-PA1 and Antp-PA1 reducing the content of hydroxyproline in lung tissues of mice with pulmonary fibrosis caused by bleomycin.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 preparation of an animal model of pulmonary fibrosis

1. Main reagent and experimental animal

Bleomycin used for the experiments was purchased from Japan Chemicals, lot X81040.

Pirfenidone, purchased from Dalian Meiren Biotech Co., Ltd., was the bulk drug with a Pirfenidone content of > 98.5%.

Other compounds used in the experiments were purchased from Sigma, unless otherwise specified.

The peptide segments of the amino acid sequences shown in the sequence tables of SEQ ID NO:1(PA1), SEQ ID NO:2(PA2) and SEQ ID NO:3(PA3) are respectively connected with cell-penetrating peptide Pep2 (the amino acid sequence of the cell-penetrating peptide Pep2 is H L YVSPW, shown in SEQ ID NO:4 in the sequence table) to form novel derivative chimeric peptides Pep2-PA1, Pep2-PA2 and Pep2-PA3, and the polypeptide or the chimeric peptide is artificially synthesized by Beijing Saibaosheng Gene technology Limited company, PA1, PA2 and PA3 are connected to the C terminal of Pep2 through two glutamic acid chains, and the sequence structure of the chimeric peptide is as follows:

the sequence of Pep2-PA1 is:

"N" -His-L eu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-L eu-Val-L eu-Arg-L ys-Ala-L eu-Phe-Ser-Thr-L eu-L ys- "C" (the sequence is shown as SEQ ID NO:10 in the sequence table).

The sequence of Pep2-PA2 is:

"N" -His-L eu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Trp-Asn-Asp-Glu-Trp-Asp-Asn-L eu-Ile-L ys-Met-Ala- "C" (the sequence is shown as SEQ ID NO:11 in the sequence table).

The sequence of Pep2-PA3 is:

n '-His-L eu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Ile-His-Ile-Phe-Val-L eu-Cys-Asn-Ile- "C' (the sequence is shown as SEQ ID NO:12 in the sequence table).

SPF grade C57B L/6 mice (male, 6-8 weeks old, 16-18 g) were purchased from institutes of Chinese medical sciences.

2. Preparation method

Male C57B L/6 (week-old 6-8 weeks) mice were fasted overnight, anesthetized with sodium pentobarbital (45mg/kg, i.p.), and injected intratracheally with bleomycin (5U/kg). The protocol was performed by incising the neck skin with as little trauma as possible, exposing the trachea with the aid of elbow forceps, puncturing the trachea with a microsyringe, injecting about 50. mu.l bleomycin into the trachea, rapidly rotating and standing for 5 minutes to allow bleomycin to uniformly enter the left and right lobes, and performing the entire procedure at about 60 ℃ surgical console.

EXAMPLE 2Western-Blot detection of enzymatic Activity of pulmonary fibrosis mouse alveolar macrophage A20A proper amount of alveolar macrophages from the animal model prepared in example 1 were added with lysis buffer [ containing 0.1mM EDTA (ethylenediaminetetraacetic acid), 0.1mM EGTA (ethyleneglycoldiethylenediamine tetraacetic acid), 10mM KCl (potassium chloride) and 10mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), and 50mM NaF (sodium fluoride), 0.1M Na₃VO₄(sodium vanadate), 0.1M Na₃PO₄(sodium pyrophosphate), 1. mu. mol/L protease inhibitors Aprotinin (Aprotinin), Trypsin inhibitor (Trypsin inhibitor), PMSF (phenylmethylsulfonyl fluoride), L eupeptins (leupeptins) and DTT (dithiothreitol)]HomogenateThen, the mixture is placed on ice for 15min, and is shaken occasionally, 10% NP-40 is added rapidly and mixed evenly, the mixture is centrifuged for 5min at 4 ℃, 12000rpm, the supernatant is taken, the Protein concentration is measured by a Coomassie brilliant blue method, the Protein concentration of samples of a sham operation group and a bleomycin group is adjusted to be the same, 2 mu G A20 antibody (purchased from CST and numbered 5630) or common IgG antibody (purchased from cell signaling and numbered 2729) which is the same as the A20 antibody is added, 10 mu L Protein A/G Plus-Agarose is added for full resuspension, 4 ℃ slow rotation and shaking overnight is carried out, 3000rpm is added for centrifugation for 5min, the supernatant is carefully sucked, 0.5m L immune coprecipitation lysate is added, the mixture is mixed evenly, the mixture is placed on ice for 1min, 4 ℃, 3000rpm is centrifuged for 30sec, the supernatant is carefully sucked, the washing is repeated for 5 times, the mixture is placed for 5min before the final centrifugation, the supernatant is carefully sucked, 50 mu L eluent (obtained from Pice immune coprecipitation kit), the result is obtained, the supernatant is obtained, the result is used for detecting the activity of Marse Protein A, the mouse lung fibrosis, the supernatant is detected by centrifugation, the supernatant A, the supernatant is extracted by a supernatant, the supernatant is extracted at room temperature, the supernatant is extracted, the centrifugation of the supernatant of the mouse.

Example 3 demonstration of binding of GSK-3 β to protein A20 in alveolar macrophages by co-immunoprecipitation

The co-immunoprecipitation reagents were as follows:

lysate A solution comprising 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl 2.6H2O, 0.0742g EDTA, 10m L glycerol, 10m L10% NP40, deionized water to 150m L, adjusting pH to 7.6 with HCl, fixing volume to 191m L, mixing well, filtering with 0.45 μm filter membrane, and storing at 4 ℃.

Lysate B solution 200. mu. L2, 2M β -phosphoglycerol, 4M L2.5, 2.5M NaF, 2M L100 mM NaVO₃2M L100 mMPMSF, 200 mu L1M DTT, L eu, Pep and Apr of 1mg/M L of 200 mu L respectively, and the total volume is 9M L. before use, the mother liquor of each component in the B liquor is unfrozen, and is respectively added into the A liquor according to the composition proportion and mixed evenly.

Protein A/G Plus-Agarose is available from Santa cruz, USA.

The specific operation steps are as follows:

(1) and taking alveolar macrophages in the alveolar lavage fluid of the mice.

(2) The cells were lysed with a co-immunoprecipitation lysate, and about 4-10mg of total cell protein was harvested and each histone was adjusted to the same concentration. Each group of proteins was collected at 200. mu.g, 200. mu.g of each group of proteins was collected as an input group of cell lysate, and the cell lysate of the input group was used as a control.

(3) Adding 2 mu g A20 antibody (purchased from CST, numbered 5630) or common IgG antibody (purchased from cell signaling, numbered 2729) with the same species of A20 antibody into the residual Protein, adding 10 mu L Protein A/GPlus-Agarose, fully resuspending, slowly rotating and shaking overnight at 4 ℃, centrifuging at 3000rpm for 5min, carefully sucking off the supernatant, if a small amount of supernatant can not be sucked into the Agarose, adding 0.5m L immune coprecipitation lysate, uniformly mixing, standing in an ice bath for 1min, centrifuging at 4 ℃, 3000rpm for 30sec, carefully sucking off the supernatant, repeatedly washing for 5 times, carefully sucking off the supernatant 5min before the final centrifugation, adding 50 mu L2 × gel loading buffer, uniformly mixing, denaturing at 95 ℃ for 5min, rapidly transferring to the ice bath for cooling, centrifuging at 12000rpm for 2min, wherein the supernatant is a precipitated Protein sample, and performing SDS-polyacrylamide gel electrophoresis on part or all of the Protein sample.

The results are shown in FIG. 2 (wherein FIG. 2(A) shows the protein contents of GSK-3 β protein and A20 protein in the starting alveolar macrophage lysate; wherein FIG. 2(B) shows the protein amounts of GSK-3 β protein and A20 protein in the alveolar macrophage lysate after precipitation by A20 antibody or control antibody IgG), and the results of FIG. 2 show the phenomenon that GSK-3 β protein and A20 protein in alveolar macrophage lysate bind to each other. the preparation method of the input cell lysate is as described above, and the input represents the protein contents of GSK-3 β and A20 in the starting alveolar macrophage lysate, that is, the contents of GSK-3 β and A20 protein in the protein stock before precipitation by A20 antibody or control antibody IgG (since A20 antibody is IgG type antibody, IgG antibody was selected as control), and the results show that the contents of GSK-3 β protein and A20 protein in the cell lysate of the group are identical.

The output represents the protein content of the protein stock solution after the precipitation of the A20 antibody or the IgG of the control antibody, and the IgG antibody is used as the control antibody of the A20 antibody and cannot precipitate the A20 protein, so the A20 Western blot lane in the cell lysate treated by the IgG antibody is blank, while the A20 antibody is used as the experimental group antibody and can be combined with the A20 protein and precipitate the A20 protein, so the result of the cell lysate treated by the A20 antibody is that the A20 Western blot lane is black, just because the interaction exists between the A20 protein and the GSK-3 β protein, the GSK-3 β protein can be precipitated when the A20 protein is precipitated by using the A20 antibody, so the GSK-3 β Western blot lane of the cell lysate treated by the A20 antibody is black, the A20 protein cannot be precipitated, and the result of the GSK-3 Western blot of the cell lysate treated by the IgG antibody is sufficient for the experiment of the IgG β protein and the GSK β protein.

Example 4 the expression of GSK-3 β protein in pulmonary fibrosis mouse alveolar macrophages was detected by the Western blot method.

An appropriate amount of alveolar macrophage from the animal model prepared in example 1 was added with lysis buffer [ containing 0.1mM EDTA (ethylenediaminetetraacetic acid), 0.1mM EGTA (ethyleneglycol diethylenediaminetetraacetic acid), 10mM KCl (potassium chloride) and 10mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), and 50mM NaF (sodium fluoride), 0.1M Na₃VO₄(sodium vanadate), 0.1M Na₃PO₄(sodium pyrophosphate), 1. mu. mol/L protease inhibitors Aprotinin (Aprotinin), Trypsin inhibitor (Trypsin inhibitor), PMSF (phenylmethylsulfonyl fluoride), L eupeptins (leupeptins) and DTT (dithiothreitol)]After homogenizing, placing on ice for 15min, oscillating at intervals, quickly adding 10% NP-40, mixing uniformly, centrifuging for 5min at 4 ℃ at 12000rpm, taking supernatant, measuring protein concentration by a Coomassie brilliant blue method, adjusting the protein concentration to be the same for Western Blot analysis, and detecting GSK-3 β, developing color by using Amersham color developing solution (NBT/BCIP staining kit IK5030 of Huamei company), measuring the light density value analysis of each strip by Western Blot analysis software (gelPro32), and obtaining a result shown in figure 3, wherein the result in figure 3 shows that the GSK-3 β protein in the lung tissues of the pulmonary fibrosis mice is in a high expression state.

Example 5 Pep2-PA1, Pep2-PA2, Pep2-PA3 were tested against pulmonary fibrosis using an animal model of pulmonary fibrosis.

The animal model prepared in example 1 was administered in groups 10 days after molding, and the groups and administration conditions are shown in table 1 (i.p. intraperitoneal administration, i.g. intragastric administration):

TABLE 1 grouping administration after model building of pulmonary fibrosis animal model

1. Determination of mouse mortality

The death conditions of the experimental animals in each group are counted and calculated every day from the 10 th day after the model building, the survival rate of the animals in a certain group without death is calculated to be 100%, the survival rate of the animals in a certain group without death is 0%, and the result can be seen from fig. 4. Compared with the sham operation group, the survival rate of the model group is obviously reduced. After drug treatment, the administration groups of Pep2-PA1, Pep2-PA2 and Pep2-PA3 can obviously improve the survival rate of the fibrosis mice, and the administration group of the positive control drug pirfenidone can reduce the death of experimental animals, but has no statistical difference. # is p <0.01 compared to sham, p <0.05 compared to model, p <0.01 compared to model. The experimental results show that the polypeptide and the derivative thereof can effectively reduce the death rate of the pulmonary fibrosis model mouse, and have the advantages of less toxic and side effects and safer use.

2. Method for detecting interaction of pulmonary fibrosis mouse lung GSK-3 β protein and A20 protein caused by bleomycin by using co-immunoprecipitation

The specific operation method is the same as that in example 3. the result is shown in fig. 5 (wherein a shows the protein content of GSK-3 β protein and a20 protein contained in the initial lung tissue alveolar macrophage lysate; wherein B shows the protein content of GSK-3 β protein and a20 protein obtained by precipitating alveolar macrophage lysate respectively by Pep2-PA1, Pep2-PA2, and Pep2-PA 3), it can be seen from fig. 5 that the GSK-3 β 0 protein and a20 protein in the pulmonary fibrosis tissue are bound to each other by using an a20 antibody or a control antibody IgG, after drug treatment, Pep2-PA2, and Pep2-PA2 can all reduce the mutual binding between GSK-3 2 protein and a2 protein, that the protein content of GSK-3 protein and a2 protein in the initial alveolar macrophage lysate and the initial alveolar macrophage lysate can not be bound to the a2 protein by using a2 antibody or the control antibody, and the protein of the aforementioned PA2, the aforementioned PA 2-2 protein precipitate can not be considered as a2 antibody, and the aforementioned three control antibody, the aforementioned antibodies can not be considered as a2, the aforementioned antibodies, and the aforementioned antibodies can be considered as a2, the control antibody precipitation of the aforementioned antibodies, and the aforementioned protein of the aforementioned protein.

3. Detection of pulmonary weight index in mice

The lung weight index was obtained by finely dissecting the mouse lung and weighing the wet weight, and dividing the lung weight (mg) by the mouse body weight (g), and the results are shown in FIG. 6. As can be seen from fig. 6, the lung weight index of mice was significantly increased after administration of bleomycin compared to the sham group. The lung weight index of the fibrotic mice can be obviously reduced after administration of Pep2-PA1, Pep2-PA2 and Pep2-PA3, and the lung weight index of experimental animals can be slightly reduced by a positive control drug of pirfenidone, but the statistical difference is not generated. Where # # is p <0.01 compared to sham, p <0.05 compared to model, and p <0.01 compared to model.

4. Detecting the number of various inflammatory cells in mouse alveolar lavage fluid

The mouse was dissected at the neck, the exposed organs were cannulated, the volume of PBS lavage was 0.8ml, the number of lavages was 3-5, the lavage fluid recovered was centrifuged at 4 ℃ and 1500rpm for 10 minutes, the supernatant was recovered and placed at-20 ℃ for cytokine detection, 1m L PBS containing 1% BSA was used to resuspend the cells, 10. mu. L of the resuspension solution was used for cell counting, and the results were analyzed using a hematology analyzer, as shown in FIG. 7, the total leukocyte count, monocyte count, basophil count, eosinophil count, and neutrophil count were significantly increased in the mouse alveolar lavage fluid after bleomycin administration compared to the sham group, Pep2-PA1, Pep2-PA2, Pep2-PA3 administration, and the positive control pirfenidone were able to reduce the above-mentioned inflammatory cell count in the pulmonary fibrosis lavage fluid to different extents compared to the sham group, p is 0.01. the model p # is 0.01 compared to the sham group.

Example 6 pathological evaluation of pulmonary fibrosis

1. Pathologic morphology analysis of pulmonary fibrosis caused by bleomycin

HE staining method, i.e. hematoxylin-eosin staining method. Hematoxylin staining solution is alkaline staining solution, which is important to make chromatin in cell nucleus and ribosome in cytoplasm bluish; eosin is an acid dye that primarily reddens components in the cytoplasm and extracellular matrix. General tissue changes and tissue products can be shown by this staining method, which is the most common staining method for morphology.

The lung tissue of the right lower lobe of the experimental animal is taken and fixed by 4 percent paraformaldehyde and then embedded by paraffin. In the largest cross-sectional section of the wax block embedded in lung tissue, HE staining was used to observe the basic pathological changes, and the results are shown in fig. 8. Wherein, A is a pseudo-operation group, B is a model group, C is a Pep2-PA1 group, D is a Pep2-PA2 group, E is a Pep2-PA3 group, and F is a positive control drug pirfenidone group. As can be seen from FIG. 8, HE stained tissues in the lung of the sham operated mice were clearly seen, alveolar structures were intact, and no changes in inflammatory and fibrotic pathology were observed. After being administrated, the Pep2-PA1, the Pep2-PA2 and the Pep2-PA3 can relieve the lung inflammation caused by bleomycin, effectively improve the lung injury and restore the normal structure of the lung. Especially Pep2-PA1 group, the lung inflammation basically disappears, and the lung tissue structure is clearer.

Inflammatory staging was performed based on the results of HE staining, with the following criteria (grades 0-5): level 0: normal tissue. Level 1: minimal inflammatory changes. And 2, stage: mild to moderate inflammatory changes without significant destruction of lung tissue structure. And 3, level: moderate inflammatory injury (thickening of the alveolar diaphragm). 4, level: moderately severe inflammatory lesions, the formation of tissue masses, or areas of localized pneumonia destroy the normal structure of lung tissue. 5: severe inflammation injury, severe damage of local lung tissue structure to cause lumen closure, etc. Fig. 9 is the result of case analysis inflammatory grading, and it can be seen from fig. 9 that significant inflammation of the lungs occurred in mice after administration of bleomycin, compared to the sham group. The administration of Pep2-PA1, Pep2-PA2, Pep2-PA3 and the positive control drug pirfenidone can significantly reduce the pulmonary inflammation caused by bleomycin. Where # # is p <0.01 compared to sham, p <0.05 compared to model, and p <0.01 compared to model.

2. Canadella red special staining pathological imaging analysis

The sirius red can specifically stain the fibrosis collagen, and is a common special staining method for tissue sections.

The lung tissue of the right lower lobe of the animal was taken and fixed with 4% paraformaldehyde and then embedded in paraffin. The fibrosis condition is observed by sirius red staining on the section of the maximum cross section of the wax block embedding lung tissue. A high-definition pathological picture (200 times) of the sirius red special staining is obtained by applying a high-definition element color pathological graph and text analysis system Spotadvanced 3.0. The stained area of collagen and the area of lung tissue under each visual field after sirius red staining were measured using Image-Pro plus 5.1. The relative amount of collagen is expressed as the ratio of the stained area to the area of lung tissue in the visual field. 10 samples are analyzed in each group in the bleomycin-induced pulmonary fibrosis experiment, 6 fields are randomly selected from each sample, and the average value represents the relative content and expression intensity of an animal collagen tissue in a lung tissue. Groups "absolute area of collagen under field of view" were compared by parametric analysis of variance. The results are shown in FIG. 10 and FIG. 11, where A is the sham group, B is the model group, C is the Pep2-PA1 group, D is the Pep2-PA2 group, E is the Pep2-PA3 group, and F is the positive control drug pirfenidone group. As can be seen from fig. 10 and 11, no significant collagen staining was seen in the sirius red staining of the lung of sham operated mice. After the bleomycin administration, the lung of the mice has obvious collagen accumulation and a large amount of fibrotic tissues are formed. The administration of Pep2-PA1, Pep2-PA2 and Pep2-PA3 can relieve accumulation of collagen in lung caused by bleomycin and improve pulmonary fibrosis. # is p <0.01 compared to sham, p <0.05 compared to model, p <0.01 compared to model.

Example 7 evaluation of pulmonary function in pulmonary fibrosis mice

Pulmonary function is a gold index for clinical detection of pulmonary fibrosis in patients. A decrease in lung function is often accompanied by an increase in fibrosis, while an improvement in lung function is often also indicative of a restoration of lung tissue structure.

The Pulmonary Fibrosis model mice obtained in example 1 were anesthetized with pentobarbital sodium (45mg/kg, i.p.), and tested for Pulmonary function by a Flexitent small animal Pulmonary function apparatus, T L C, SnaPshlots (for testing methods, see L v X, Wang X, L i K, et al. Rupatadine Protections against Pulmonary Fibrosis, PAF-medial Senesence in Rodents [ J ]. Plcon, 2013,8(7): e 68631.).

The detection results are shown in fig. 12, wherein a is the deep inspiratory capacity, B is the dynamic resistance, C is the dynamic elasticity, and D is the dynamic compliance. As can be seen from fig. 12, compared with the sham-operated group, the bleomycin-induced pulmonary fibrosis mice significantly decreased deep inspiratory capacity, increased dynamic resistance and dynamic elasticity of the lung, and significantly decreased compliance. The lung function is remarkably recovered after the treatment of the medicines of Pep2-PA1, Pep2-PA2 and Pep2-PA 3. Where # # is p <0.01 compared to sham, p <0.05 compared to model, and p <0.01 compared to model.

Example 8 determination of hydroxyproline content in pulmonary fibrosis mice

Hydroxyproline accounts for 13.4% of collagen, accounts for a very small amount of elastin, and is absent in other proteins, so that the content of collagen is detected by hydroxyproline. Detecting the content of the hydroxyproline in the left lung of the animal, and evaluating the condition of the pulmonary fibrosis. The specific method comprises the following steps: taking all lung lobes on the left side of the model animal prepared in example 1, recording wet weight, ultrasonically homogenizing with physiological saline to prepare 10% tissue homogenate, taking about 150 mu l of homogenate supernatant, adding 500 mu l of alkali hydrolysis liquid, uniformly mixing by vortex, treating for 40min by alkali hydrolysis under the condition of 120 ℃ and 0.1Kpa (the method refers to the instruction of kit of Nanjing Biotechnology Limited company, slightly modified), adjusting pH value, fixing volume, treating with active carbon, and taking supernatant. Hydroxyproline assay was performed according to the instructions (chloramine-T method). The results are shown in FIG. 13, and it can be seen from FIG. 13 that the hydroxyproline content in the model group and its significance are increased compared with those in the sham operation group, indicating that the fibrosis pathology is seriously changed. The content of hydroxyproline in the lung of the fibrotic mouse can be obviously reduced after the administration of the Pep2-PA1, the Pep2-PA2 and the Pep2-PA 3. Where # # is p <0.01 compared to sham, p <0.05 compared to model, and p <0.01 compared to model.

In the experiment, the results of pathological examination, pathological imaging and other means are analyzed, and the Pep2-PA1, Pep2-PA2 and Pep2-PA3 can obviously inhibit pulmonary fibrosis caused by bleomycin; the death rate of the mice with pulmonary fibrosis is obviously reduced; the lung weight index of a pulmonary fibrosis mouse is obviously reduced; obviously reducing the number of various inflammatory cells in pulmonary fibrosis mouse alveolar lavage fluid; the contents of hydroxyproline and collagen in pulmonary tissues of the mice with pulmonary fibrosis are obviously reduced; obviously improve the lung function of the pulmonary fibrosis mouse. The experimental results prove that the Pep2-PA1, the Pep2-PA2 and the Pep2-PA3 have excellent treatment prospects in the aspect of pulmonary fibrosis.

EXAMPLE 9 preparation of TAT-PA1, Antp-PA1 pharmacodynamics evaluation of pulmonary fibrosis animal models

1. Main reagent and experimental animal

Bleomycin used for the experiments was purchased from Japan Chemicals, lot No. X90147.

Pirfenidone, purchased from Dalian Meiren Biotech Co., Ltd., was the bulk drug with a Pirfenidone content of > 98.5%.

The compounds used in the experiments were purchased from Sigma, unless otherwise specified.

The peptide segments of the amino acid sequence shown in the sequence table SEQ ID NO:1(PA1) are respectively connected with TAT peptide (YGRKKRRQRRR of cell-penetrating peptide HIV-1 virus reverse transcription activator (Tat) protein, the amino acid sequence of which is shown in SEQ ID NO: 5) and transcription factor Antp peptide (RQIKIWFQNRRMKWKK of drosophila antennal homeoprotein, the amino acid sequence of which is shown in SEQ ID NO: 6) to form new derivatives TAT-PA1 and Antp-PA1, and the polypeptides or chimeric peptides are artificially synthesized by Baisheng Gene technology Limited Beijing. PA1 is linked to the C-terminus of TAT and Antp by two chains of glutamate. The sequence structure of the chimeric peptide is as follows:

the sequence of TAT-PA1 is:

"N" -Tyr-Gly-Arg-L ys-L ys-Arg-Arg-Gln-Arg-Arg-Gly-Gly-L eu-Val-L eu-Arg-L ys-Ala-L eu-Phe-Ser-Thr-L eu-L ys- "C" (the sequence is shown as SEQ ID NO:13 in the sequence table);

the sequence of Antp-PA1 is:

"N" -Arg-Gln-Ile-L ys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-L ys-Trp-L ys-L ys-Gly-Gly-L eu-Val-L eu-Arg-L ys-Ala-L eu-Phe-Ser-Thr-L eu-L ys- "C" (the sequence is shown as SEQ ID NO:14 in the sequence table).

SPF grade C57B L/6 mice (male, 6-8 weeks old, 16-18 g) were purchased from institutes of Chinese medical sciences.

2. The preparation method is the same as that of example 1

The animal model prepared in example 1 was administered in groups 10 days after molding, and the groups and administration conditions are shown in table 2 (i.p. intraperitoneal administration, i.g. intragastric administration):

TABLE 2 TAT-PA1, Antp-PA1 pharmacodynamic test pulmonary fibrosis animal model grouped administration after model building

1. Determination of mouse mortality

The death conditions of the experimental animals of each group were counted and calculated every day from the 10 th day after the model building, the survival rate of the animals of a certain group without death was calculated to be 100%, the survival rate of all the animals of a certain group was calculated to be 0%, and the result can be seen from fig. 14. Compared with the sham operation group, the survival rate of the model group is obviously reduced. After drug treatment, the TAT-PA1 and Antp-PA1 administration groups can obviously improve the survival rate of the fibrosis mice. # is p <0.01 compared to sham and p <0.05 compared to model. The experimental result shows that the polypeptide PA1 can effectively reduce the death rate of the pulmonary fibrosis model mouse by being connected with the known cell-penetrating peptide.

2. Detection of pulmonary weight index in mice

The lung weight index was obtained by finely dissecting the mouse lung and weighing the wet weight, and dividing the lung weight (mg) by the mouse body weight (g), and the results are shown in FIG. 15. As can be seen from fig. 15, the lung weight index of mice was significantly increased after administration of bleomycin compared to the sham group. TAT-PA1 and Antp-PA1 can obviously reduce the lung weight index of the fibrotic mouse after being administrated. Where # # is p <0.01 compared to sham and # is p <0.01 compared to model.

3. Detecting the number of various inflammatory cells in mouse alveolar lavage fluid

The mice were subjected to cervical dissection and organs were exposed for intubation. The lavage amount of PBS is 0.8ml, and the lavage times are 3-5 times. Centrifuging the recovered lavage liquid at 4 ℃ and 1500rpm for 10 minutes, recovering the supernatant, and placing at-20 ℃ for cell factor detection; the cell pellet was resuspended in 1ml PBS containing 1% BSA, and 10. mu.l of the resuspension was taken for cell counting. The analysis was performed using a blood cell analyzer. The results are shown in FIG. 16. As seen in FIG. 16, the total leukocyte count, monocyte count, basophil count, eosinophil count, and neutrophil count in the alveolar lavage fluid of mice administered with bleomycin were significantly increased as compared with those of the sham group. TAT-PA1 and Antp-PA1 can reduce the number of the inflammatory cells in pulmonary fibrosis mouse alveolar lavage fluid to different degrees after being administrated. Where # # is p <0.01 compared to sham, p <0.05 compared to model, and p <0.01 compared to model.

4. Mouse lung tissue hydroxyproline detection

Taking all lung lobes on the left side of an animal, recording wet weight, ultrasonically homogenizing with physiological saline to prepare 10% tissue homogenate, taking about 150 mu l of homogenate supernatant, adding 500 mu l of alkali hydrolysis liquid, uniformly mixing by vortex, carrying out alkali hydrolysis treatment for 40min under the condition of 120 ℃ and 0.1Kpa (the method refers to kit instructions of Nanjing to build bioengineering technology Co., Ltd, and is slightly changed), adjusting pH value, fixing volume, and taking supernatant after active carbon treatment. Hydroxyproline assay was performed according to the instructions (chloramine-T method). The results are shown in FIG. 17, and it can be seen from FIG. 17 that the hydroxyproline content in the model group and its significance are increased compared with those in the sham operation group, indicating that the fibrosis pathology is seriously changed. TAT-PA1 and Antp-PA1 can obviously reduce the content of hydroxyproline in the lung of a fibrotic mouse after being administrated. Where # # is p <0.01 compared to sham, p <0.05 compared to model, and p <0.01 compared to model.

In the experiment, the experimental analysis result shows that TAT-PA1 and Antp-PA1 can obviously inhibit pulmonary fibrosis caused by bleomycin; the death rate of the mice with pulmonary fibrosis is obviously reduced; the lung weight index of a pulmonary fibrosis mouse is obviously reduced; obviously reducing the number of various inflammatory cells in pulmonary fibrosis mouse alveolar lavage fluid; the hydroxyproline content of lung tissues of the mice with pulmonary fibrosis is obviously reduced. The experimental results prove that TAT-PA1 and Antp-PA1 have excellent treatment prospect in the aspect of pulmonary fibrosis.

The experimental results are expressed by mean value plus or minus standard error, and the significant difference is considered to be existed by comparing that p is less than 0.05 and p is less than 0.01 through parameter or nonparametric variance test. Statistics of pathology grading data significant differences were considered using the chi-square test, comparing p <0.05 and extremely significant differences were considered p < 0.01.

The PBS used in the examples of the present invention, phosphate buffered saline, was 0.1M in concentration and 7.2 in pH.

The results of the above examples show that the polypeptide of the present invention or the derivative of the polypeptide has a significant effect of resisting pulmonary fibrosis diseases, and can be used as an active ingredient for preparing a medicament for resisting pulmonary fibrosis.

It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Beijing Weifeng Yimin technology Limited

<120> polypeptide, derivatives thereof and application thereof in preparation of anti-pulmonary fibrosis drugs

<130>P180115945C

<160>14

<170>PatentIn version 3.5

<210>1

<211>12

<212>PRT

<213>Artificial Sequence

<220>

<223>PA1

<400>1

Leu Val Leu Arg Lys Ala Leu Phe Ser Thr Leu Lys

1 5 10

<210>2

<211>12

<212>PRT

<213>Artificial Sequence

<220>

<223>PA2

<400>2

Trp Asn Asp Glu Trp Asp Asn Leu Ile Lys Met Ala

1 5 10

<210>3

<211>9

<212>PRT

<213>Artificial Sequence

<220>

<223>PA3

<400>3

Ile His Ile Phe Val Leu Cys Asn Ile

1 5

<210>4

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223> Pep2 peptide

<400>4

His Leu Tyr Val Ser Pro Trp

1 5

<210>5

<211>11

<212>PRT

<213>Artificial Sequence

<220>

<223> TAT peptide of reverse transcription activator protein of HIV-1 virus

<400>5

Tyr Gly ArgLys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210>6

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223> transcription factor Antp peptide of drosophila antennal homeoprotein

<400>6

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>7

<211>21

<212>PRT

<213>Artificial Sequence

<220>

<223> Pep-1 peptide

<400>7

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val

20

<210>8

<211>27

<212>PRT

<213>Artificial Sequence

<220>

<223> MPG peptide

<400>8

Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly

1 5 10 15

Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val

20 25

<210>9

<211>3

<212>PRT

<213>Artificial Sequence

<220>

<223> RGD peptide

<400>9

Arg Gly Asp

1

<210>10

<211>21

<212>PRT

<213>Artificial Sequence

<220>

<223> Pep2-PA1 sequence

<400>10

His Leu Tyr Val Ser Pro Trp Gly Gly Leu Val Leu Arg Lys Ala Leu

1 5 10 15

Phe Ser Thr Leu Lys

20

<210>11

<211>21

<212>PRT

<213>Artificial Sequence

<220>

<223> Pep2-PA2 sequence

<400>11

His Leu Tyr Val Ser Pro Trp Gly Gly Trp Asn Asp GluTrp Asp Asn

1 5 10 15

Leu Ile Lys Met Ala

20

<210>12

<211>18

<212>PRT

<213>Artificial Sequence

<220>

<223> Pep2-PA3 sequence

<400>12

His Leu Tyr Val Ser Pro Trp Gly Gly Ile His Ile Phe Val Leu Cys

1 5 10 15

Asn Ile

<210>13

<211>25

<212>PRT

<213>Artificial Sequence

<220>

<223> TAT-PA1 sequence

<400>13

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Leu Val Leu

1 5 10 15

Arg Lys Ala Leu Phe Ser Thr Leu Lys

20 25

<210>14

<211>30

<212>PRT

<213>Artificial Sequence

<220>

<223> Antp-PA1 sequence

<400>14

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

Gly Gly Leu Val Leu Arg Lys Ala Leu Phe Ser Thr Leu Lys

20 25 30

Claims (10)

1. A polypeptide specifically binding to GSK-3 β or a derivative of the polypeptide is characterized in that the amino acid sequence of the polypeptide is from an A20 sequence combined with GSK-3 β, the amino acid sequence of the polypeptide is shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3 in a sequence table, and the derivative of the polypeptide comprises a chimeric peptide formed by connecting the polypeptide specifically binding to GSK-3 β with cell penetrating peptide;

preferably, the polypeptide is an α helical peptide.

2. The polypeptide or derivative of the polypeptide of claim 1, wherein the cell-penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide; preferably, the cell-penetrating peptide and the polypeptide are linked by two glutamates.

3. The polypeptide or the derivative of the polypeptide according to claim 1 or 2, wherein the cell-penetrating peptide is selected from one or more of TAT peptide of HIV-1 virus reverse transcriptase activator protein, Antp peptide of transcription factor of drosophila antennapedia homeoprotein, Pep-1 peptide, Pep-2 peptide, MPG peptide and RGD peptide, preferably Pep-2 peptide.

4. The polypeptide or derivative of the polypeptide of claim 3, wherein the amino acid sequence of the chimeric peptide is as set forth in SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO:13 or SEQ ID NO: as shown at 14.

5. A pharmaceutical composition for treating pulmonary fibrosis, wherein the active ingredient comprises the polypeptide or the derivative of the polypeptide as claimed in any one of claims 1 to 4; preferably:

the active ingredient is a single active ingredient; or, the active ingredient also comprises other compounds with anti-pulmonary fibrosis activity.

6. The pharmaceutical composition of claim 5, further comprising one or more pharmaceutically acceptable carriers;

and/or, the pharmaceutical composition is a pharmaceutical composition of which the administration route is injection administration or oral administration.

7. Use of a polypeptide or a derivative of said polypeptide according to any one of claims 1 to 4 and/or a pharmaceutical composition according to claim 5 or 6 for the preparation of a medicament for the prevention and/or treatment of a pulmonary fibrotic disease; preferably, the pulmonary fibrosis disease is dust, radiation and/or drug induced pulmonary fibrosis disease, the drug is, for example, bleomycin.

8. The use of claim 7, wherein the pulmonary fibrotic disease is a primary pulmonary fibrotic disease or a secondary pulmonary fibrotic disease; preferably, the pulmonary fibrotic disease comprises chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, interstitial pneumonia, sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, collagenous vascular disease-related fibrosis alveolitis or pulmonary function degeneration.

9. The use of claim 7, wherein the agent for preventing and/or treating pulmonary fibrotic disease is an agent that reduces lung weight index, reduces the number of inflammatory cells in alveolar lavage fluid, reduces hydroxyproline content of lung tissue, reduces collagen content of lung tissue, improves lung function; preferably, the inflammatory cells include total leukocytes, monocytes, basophils, eosinophils and/or neutrophils.

The use of an inhibitor or antagonist of GSK-3 β and/or a20 in the manufacture of a medicament for the prevention or treatment of pulmonary fibrosis.

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