WO2015096756A1 - Use of polypeptide and derivatives thereof in preparing anti-pulmonary fibrosis drugs - Google Patents

Abstract

Disclosed in the present invention is the use of a polypeptide capable of specifically binding to a TRB3 protein or derivatives thereof in preparing drugs for preventing or treating pulmonary fibrosis, wherein the derivatives are chimeric peptides formed by connecting the polypeptide capable of specifically binding to a TRB3 protein to a cell-penetrating peptide. The polypeptide or derivatives thereof of the present invention are capable of specifically binding to a TRB3 protein and blocking the interaction between the TRB3 protein and a P62 protein.

Description

Application of a polypeptide and its derivative in preparing anti-pulmonary fibrosis drugs

Priority is claimed on Chinese Patent Application No. CN201310727458.2, filed on Dec. 25, 2013. This application cites the entire text of the above-mentioned Chinese patent application.

Technical field

The invention belongs to the field of biotechnology, and particularly relates to the application of a polypeptide and a derivative thereof for preparing an anti-pulmonary fibrosis medicine.

Background technique

Tissue fibrosis is a common consequence of tissue damage caused by pathogens inside and outside, and is also a fundamental pathological change in a variety of infectious and non-infectious inflammatory diseases. Tissue fibrosis is present in respiratory diseases, circulatory diseases, kidney and liver diseases, chronic pancreatitis, systemic lupus erythematosus and macular degeneration, and often increases tissue burden. Tissue fibrosis affects almost all organs and systems in the human body and is the leading cause of death in a large number of patients, posing a serious threat to human health. According to relevant statistics in the United States, nearly 45% of patients who die from various diseases worldwide can be classified as caused by various tissue fibrosis, so tissue fibrosis starts in the process of the occurrence and development of major organs in the human body. Play an important role. Pulmonary fibrosis disease is caused by hundreds of different causes such as toxic substances, spontaneous immune diseases, side effects of drugs, infections, severe trauma, lung inflammation, persistent alveolar damage, repeated destruction of extracellular matrix, repair, reconstruction and excessive deposition. A type of disease that causes normal lung tissue changes and loss of function. Currently, tissue fibrosis remains untargeted, safe and effective treatment options.

Pulmonary fibrosis is one of the most serious pathological conditions in the lungs. Most of its pathological changes are the initial lower airway inflammation, as well as alveolar epithelial cells and vascular endothelial cell damage, accompanied by fibroblasts and type II alveolar cell proliferation, cells. Factor release, extracellular matrix protein and collagen deposition eventually lead to lung changes. In the pulmonary fibrosis patients, the lung alveoli are gradually replaced by fibrous substances, resulting in the hardening and thickening of the lung tissue, the gradual loss of lung gas exchange capacity, resulting in patients with different degrees of hypoxia and breathing difficulties, and finally died of respiratory failure. Pulmonary fibrosis is one of the four major diseases of respiratory diseases. The etiology is complicated and the pathogenesis is unknown. The existing drugs and methods for treating pulmonary fibrosis are very limited, and the curative effect is unsatisfactory. The prognosis is very poor. The 5-year survival rate is only 50. %.

In recent years, the removal of collagen is one of the hotspots of pulmonary fibrosis treatment. Autophagy can eliminate intracellular redundant, misfolded proteins, especially the accumulation of intracellular collagen caused by tissue damage, maintain genomic stability, and inhibit the occurrence and development of fibrosis. Autophagy is also considered to be another form of programmed cell death that can induce damage to cells for clearance, thereby reducing the accumulation of cellular debris in the lung tissue. P62 is an important indicator reflecting the dynamic process of autophagy. As a "truck protein", the Ubiquitin associated domain (UBA) in the P62 protein domain binds to Ubiquitin (Ub) and recruits them to the autophagosome membrane to bind to LC3. This process mainly binds to LC3 through the LIR (LC3-Interacting Region, LIR) domain of P62 protein, mediates the degradation of ubiquitinated proteins, and the expression level of P62 also decreases. When autophagy is inhibited, the ubiquitinated protein bound to P62 cannot be degraded in time, and it is accumulated in the cytoplasm and the expression is increased. In the pulmonary fibrosis tissue, TRB3 protein (Tribbles 3) is highly expressed, and TRB3 protein can interact with P62 protein, which blocks the binding of other ubiquitinated proteins to P62 protein, resulting in inhibition of autophagy pathway. The internal collagen could not be removed, which aggravated the pathological changes of pulmonary fibrosis. Therefore, research and development of inhibitors of TRB3 protein or substances that block its binding to P62 protein have a good potential for the preparation of drugs for preventing or treating pulmonary fibrosis.

Summary of the invention

The technical problem to be solved by the present invention is to provide a polypeptide or a derivative thereof capable of specifically binding to a TRB3 protein in the preparation of a medicament for preventing or treating pulmonary fibrosis, in view of the current lack of an effective TRB3 protein inhibitor.

In order to solve the above technical problems, the present invention provides a technical solution for: a polypeptide capable of specifically binding a TRB3 protein or a derivative thereof for preparing a medicament for preventing or treating pulmonary fibrosis, which specifically binds to a TRB3 protein The polypeptide is a polypeptide as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing; the derivative of the polypeptide which specifically binds to the TRB3 protein is specifically bindable A chimeric peptide formed by the polypeptide of the TRB3 protein and the cell penetrating peptide.

The term "pulmonary fibrosis" as used in the present invention refers to pulmonary inflammation, particularly alveolar wall inflammation, due to toxic substances, spontaneous immune diseases, side effects of drugs, infection, severe trauma, and the like, and alveolar persistent damage, extracellular Repeated destruction, repair, reconstruction and excessive deposition of the matrix, that is, the formation of a large number of fibrous connective tissue and pulmonary structural disorder in the pulmonary interstitial, leading to a disorder of normal lung tissue structure and loss of function. Wherein, the pulmonary fibrosis is usually primary (specific) pulmonary fibrosis, that is, unexplained pulmonary fibrosis; or secondary pulmonary fibrosis, which is secondary to multiple causes. Pulmonary Fibrosis. The cause may be: lung infection, trauma, dust, radiation or drugs (eg bleomycin). The pulmonary fibrosis can be manifested as: pulmonary inflammation, especially interstitial pneumonia, deterioration of lung function such as chronic obstructive pulmonary disease (COPD) and lung injury (such as pulmonary interstitial formation of massive fibrous connective tissue and pulmonary structural disorder) One or more.

The polypeptide of the present invention which specifically binds to the TRB3 protein is SEQ ID NO: 1 of the sequence listing (the amino acid sequence thereof is: Ser-Leu-Ser-Gln-Met-Leu-Ser-Met), SEQ ID NO: 2 ( Its amino acid sequence is: Gly-Gly-Trp-Leu-Thr-Arg-Leu-Leu-Gln-Thr-Lys) and SEQ ID NO: 3 (the sequence of amino acids is: A polypeptide represented by any one of Ile-Gly-Ala-Ala-Leu-Asp-Thr-Ile) may be appropriately introduced with an amino acid substitution, deletion or addition as long as the altered amino acid sequence is capable of forming a specific binding to the TRB3 protein. The polypeptide and the polypeptide still retain the activity before the change.

The cell penetrating peptide of the present invention is a cell penetrating peptide which is conventional in the art, as long as the cell penetrating peptide can assist in feeding the polypeptide into a cell to function, generally, the cell is worn. The membrane peptide is a short peptide molecule composed of 10 to 30 amino acids. The cell penetrating peptide is preferably: HLYVSPW (as shown in SEQ ID NO: 4 in the Sequence Listing, the cell penetrating peptide is named pep2 peptide, abbreviated as pep2), HIV-1 virus reverse transcription activator Trans-activator transcription (Tat) protein TAT peptide (YGRKKRRQRRR, its amino acid sequence is shown as SEQ ID NO: 5, referred to as TAT peptide), Drosophila antennae homeopathic protein transcription factor Antp peptide (RQIKIWFQNRRMKWKK, its amino acid sequence As shown in SEQ ID NO: 6, abbreviated as Antp peptide), pep-1 peptide (KETWWETWWTEWSQPKKKRKV, whose amino acid sequence is shown as SEQ ID NO: 7), MPG peptide (GALFLGFLGAAGSTMGAWSQPKSKRKV, the amino acid sequence of which is shown in SEQ ID: 8) And one or more of the RGD peptide (Arg-Gly-Asp) (the amino acid sequence of which is shown in SEQ ID NO: 9). Preferably, the polypeptide derivative is linked to the C-terminus of the cell penetrating peptide (the cell penetrating peptide, preferably a pep2 peptide, a TAT peptide or an Antp peptide) in SEQ ID NO: 1, A chimeric peptide formed after the polypeptide set forth in any one of SEQ ID NO: 2 and SEQ ID NO: 3. The cell penetrating peptide is preferably linked to the N-terminus or C-terminus of the above polypeptide, more preferably to the N-terminus of the above polypeptide. The amino acid sequence of the chimeric peptide is preferably SEQ ID NO: 10 (the amino acid sequence of which is: HLYVSPWGGSLSQMLSM), SEQ ID NO: 11 (the amino acid sequence is: HLYVSPWGGGGWLTRLLQTK), SEQ ID NO: 12 The amino acid sequence is: HLYVSPWGGIGAALDTI), SEQ ID NO: 13 (the amino acid sequence is: YGRKKRRQRRRGGGGWLTALLQTK), and SEQ ID NO: 14 (the amino acid sequence of which is: RQIKIWFQNRRMKWKKGGGGWLTRLLQTK).

The polypeptide of the present invention and derivatives thereof are used as an active ingredient for the preparation of a medicament for preventing or treating pulmonary fibrosis. Wherein the medicament for preventing or treating pulmonary fibrosis comprises a prophylactically or therapeutically effective amount of one or more of the polypeptides of the present invention and derivatives thereof, wherein "prophylactically or therapeutically effective amount" means The amount of compound that is sufficient to effectively prevent or treat the disease or condition described herein is administered to a subject in need thereof. While the amount of the compound that constitutes a "prophylactically or therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, it can be determined in a conventional manner by those skilled in the art. The dose of the medicament of the present invention is preferably 0.1 to 15 mg/kg, more preferably 5 to 10 mg/kg, and preferably 5 mg/kg, and the number of administrations is preferably once a day or several times a day. Times.

The polypeptide or a derivative thereof which specifically binds to the TRB3 protein can be prepared with a pharmaceutically acceptable carrier for preventing or treating a pulmonary fibrosis drug. The pharmaceutically acceptable carrier described therein can be conventionally selected depending on the pharmaceutical dosage form. For example, a filler, a diluent, and the like. In the medicament, the polypeptide which can specifically bind to the TRB3 protein or a derivative thereof can be used as a single active ingredient or in combination with other compounds having activity for preventing or treating pulmonary fibrosis as an active ingredient.

The pharmaceutical dosage form for preventing or treating pulmonary fibrosis according to the present invention is not particularly limited and is a conventional dosage form in the art, preferably solid, semi-solid or liquid, and may also be an aqueous solution, a non-aqueous solution or a suspension, more preferably It is a tablet, capsule, granule, injection or infusion. The route of administration of the drug is a conventional route of administration in the art, preferably injection or oral administration. The manner of administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection routes.

"Prophylaxis" as used herein means preventing or reducing the production of pulmonary fibrosis after use in the presence of possible pulmonary fibrosis factors. "Treatment" as used herein means reducing the extent of pulmonary fibrosis, or healing pulmonary fibrosis to normalize it, or slowing the progression of pulmonary fibrosis.

"Required subject" refers to a warm-blooded animal that may have the disease or condition described herein in the presence of a possible pulmonary fibrotic factor; or a warm-blooded animal, such as a mammal, having the diseases and conditions described herein. The present invention is preferably human or mouse.

Based on the common knowledge in the art, the above various preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

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

A positive progressive effect of the present invention is that the present invention provides a polypeptide which specifically binds to a TRB3 protein (the sequence of which is as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing) and The use of the derivative in the preparation of a medicament for preventing or treating pulmonary fibrosis can effectively improve the lung function of a patient with pulmonary fibrosis, and the curative effect is remarkable, and the medicament has the advantages of less toxic side effects and safe use.

DRAWINGS

Figure 1 is a graph showing high expression of TRB3 protein in lung tissue of mice with pulmonary fibrosis.

Fig. 2(A) and Fig. 2(B) are diagrams showing the interaction between TRB3 protein and p62 in the lung tissue of lung fiber mice. Fig. 2(A) is the protein content of TRB3 protein and P62 protein in the initial lung tissue lysate. . Fig. 2(B) is a graph showing the protein amount of the TRB3 protein and the P62 protein after the lung tissue lysate was precipitated by the P62 antibody or the control antibody IgG.

Fig. 3(A) and Fig. 3(B) are diagrams showing the phagocytosis of collagen by lung epithelial cells by flow cytometry. Fig. 3(A) is a flow chart of cells, and Fig. 3(B) is flow cytometry. Results statistics.

Figure 4 shows that pep2-A1, pep2-A2, and pep2-A3 reduce the mortality test of bleomycin-induced pulmonary fibrosis in mice. Fruit map.

Fig. 5(A) and Fig. 5(B) are diagrams showing the interaction of peb2-A1, pep2-A2 and pep2-A3 in the lung TRB3 protein and p62 protein in bleomycin-induced pulmonary fibrosis mice, Fig. 5 (A) shows the protein content of TRB3 protein and P62 protein contained in the initial lung tissue lysate. Fig. 5(B) is a diagram showing the protein amount of the TRB3 protein and the P62 protein precipitated by the P62 antibody or the control antibody IgG after the lung tissue lysate was treated with pep2-A1, pep2-A2, and pep2-A3, respectively.

Figure 6 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing lung weight index in mice with pulmonary fibrosis induced by bleomycin.

Figure 7 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing the number of various inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin. Among them: A is the total number of white blood cells, B is the number of monocytes, C is the number of neutrophils, D is the number of lymphocytes, E is the number of basophils, and F is the number of eosinophils.

Figure 8 is a pathological examination (HE) diagram of pep2-A1, pep2-A2, and pep2-A3 to reduce bleomycin-induced pulmonary fibrosis. Among them: A is the sham operation group, B is the model group, C is the pep2-A1 group, D is the pep2-A2 group, E is the pep2-A3 group, and F is the positive control drug pirfenidone group.

Figure 9 is a graph showing the results of pathological examination scores of pep2-A1, pep2-A2, and pep2-A3 for reducing bleomycin-induced pulmonary fibrosis.

Figure 10 is a graph showing the pathological examination of bleomycin-induced pulmonary fibrosis (Sirius Red) by pep2-A1, pep2-A2, and pep2-A3. Among them: A is the sham operation group, B is the model group, C is the pep2-A1 group, D is the pep2-A2 group, E is the pep2-A3 group, and F is the positive control drug pirfenidone group.

Figure 11 is a graph showing the effect of pep2-A1, pep2-A2, and pep2-A3 on collagen content in lung tissue of mice with pulmonary fibrosis.

Figure 12 is a graph showing the results of lung function tests in mice with pulmonary fibrosis. Where A is the deep inspiratory volume, B is the dynamic resistance, C is the dynamic elasticity, and D is the dynamic compliance.

Figure 13 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin.

Figure 14 is a graph showing the results of TAT-A2 and Antp-A2 in reducing mortality in mice with pulmonary fibrosis induced by bleomycin. Among them: A is the sham operation group, B is the model group, C is the TAT-A2 group, and D is the Antp-A2 group.

Figure 15 is a graph showing the results of TAT-A2 and Antp-A2 in reducing lung weight index in mice with pulmonary fibrosis induced by bleomycin.

Figure 16 is a graph showing the results of TAT-A2 and Antp-A2 in reducing the number of inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin. Among them: A is the total number of white blood cells, B is the number of monocytes, C is the number of neutrophils, D is the number of lymphocytes, E is the number of eosinophils, and F is the number of basophils.

Figure 17 shows that TAT-A2 and Antp-A2 reduce the hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin. Fruit map.

detailed description

Example 1

Preparation of animal models of pulmonary fibrosis

1. Main reagents and experimental animals

The bleomycin used in the experiment was purchased from Nippon Kayaku, batch number X81040.

Pirfenidone, purchased from Dalian Meilun Biotechnology Co., Ltd., is a raw material drug with a pirfenidone content of >98.5%.

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

The peptides of the amino acid sequences shown in SEQ ID NO: 1 (A1), SEQ ID NO: 2 (A2) and SEQ ID NO: 3 (A3) of the Sequence Listing are ligated to the cell penetrating peptide pep2 (penetrating peptide pep2, respectively). The amino acid sequence is HLYVSPW, as shown in SEQ ID NO: 4 in the Sequence Listing, and constitutes the new derivatives pep2-A1, pep2-A2, pep2-A3, and the above polypeptides or chimeric peptides are all limited by Beijing Cypress Biotechnology. The company is synthesizing. A1, A2 and A3 are linked to the C-terminus of pep2 by two glutamic acid chains. The sequence structure of the above chimeric peptides is as follows:

The pep2-A1 sequence is:

"N"-His-Leu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Ser-Leu-Ser-Gln-Met-Leu-Ser-Met-"C" (the sequence is as SEQ ID in the sequence listing) NO: 10).

The sequence of pep2-A2 is:

"N"-His-Leu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Gly-Gly-Trp-Leu-Thr-Arg-Leu-Leu-Gln-Thr-Lys-"C" (its sequence As shown in SEQ ID NO: 11 in the Sequence Listing).

The sequence of pep2-A3 is:

"N"-His-Leu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Ile-Gly-Ala-Ala-Leu-Asp-Thr-Ile-"C" (the sequence is as SEQ ID in the Sequence Listing) NO: 12)).

SPF grade C57BL/6 mice (male, 6-8 weeks old, 16-18 g) used in the experiment were purchased from the Institute of Zoology of the Chinese Academy of Medical Sciences.

2. Preparation method

Male C57BL/6 (6-8 weeks old) mice were fasted overnight, anesthetized with sodium pentobarbital (45 mg/kg, i.p.), and intratracheally injected with bleomycin (5 U/kg). The specific scheme is as follows: the neck skin is cut with as little trauma as possible, the trachea is exposed with the assistance of the elbow ophthalmology, the trachea is pierced using a micro-injector, about 50 μl of bleomycin is injected into the trachea, and the tube is rapidly rotated. Stand upright for 5 minutes to allow bleomycin to enter the left and right lobe evenly. The entire operation is performed at a surgical table of about 60 °C. The sham operation group was intratracheally injected with an equal amount of physiological saline for injection.

Example 2 Western-Blot detection of TRB3 protein expression in lung tissue of mice with pulmonary fibrosis

Prepare an appropriate amount of lung tissue from the obtained animal model in Example 1. Add lysis buffer [containing 0.1 mM EDTA (ethylenediaminetetraacetic acid), 0.1 mM EGTA (ethylene glycol diethyl ether diamine tetraacetic acid), 10 mM KCl (chlorine) Potassium) and 10 mM HEPEs (4-hydroxyethylpiperazineethanesulfonic acid) and 50 mM NaF (sodium fluoride), 0.1 M Na ₃ VO ₄ (sodium vanadate), 0.1 M Na ₄ P ₂ O ₇ (sodium pyrophosphate) , protease inhibitor 1μmol/L of Aprotinin (inhibitory enzyme), Trypsin inhibitor (trypsin inhibitor), PMSF (phenylmethanesulfonyl fluoride), Leupeptins (leucine) and DTT (dithiothreitol) After homogenization, place on ice for 15 min, or shake. Add 10% NP-40 quickly and mix at 4 ° C, 12000 rpm, and centrifuge for 5 min. The supernatant was taken, the protein concentration was determined by Coomassie Brilliant Blue method, and the protein concentration was adjusted to the same for Western Blot analysis to detect α-SMA and type I collagen. The color was measured by Amersham color developing solution (NBT/BCIP staining kit IK5030), and the optical density value of each band was measured by Western-blot analysis software (gelPro32). The results are shown in Fig. 1, and Fig. 1 is pulmonary fibrosis. High expression profile of TRB3 protein in mouse lung tissue. The results in Figure 1 indicate that the TRB3 protein in the lung tissue of mice with pulmonary fibrosis is highly expressed.

Example 3 Verification of the binding of p62 to protein TRB3 protein in pulmonary fibrosis tissue by immunoprecipitation

The immunoprecipitation reagents are as follows:

Lysate A solution: 0.6057 g Tris base, 1.7532 g NaCl, 0.1017 g MgCl ₂ ·6H ₂ O, 0.0742 g EDTA, 10 mL glycerol, 10 mL 10% NP40, deionized water to 150 mL, pH adjusted to 7.6 with HCl, The volume was adjusted to 191 mL, thoroughly mixed, filtered through a 0.45 μm filter, and stored at 4 °C.

Lysate B solution: 200 μL of 2 Mβ-glycerophosphate, 4 mL of 2.5 M NaF, 2 mL of 100 mM NaVO ₃ , 2 mL of 100 mM PMSF, 200 μL of 1 M DTT, 200 μL of 1 mg/mL of Leu, Pep, and Apr, total volume of 9 mL. The mother liquor was stored at -20 °C. Before use, the mother liquor of each component in the B solution was thawed, and added to the solution A in the above composition ratio and mixed.

Protein A/G Plus-Agarose was purchased from Santa Cruz, USA.

The specific steps are as follows:

(1) Remove the large leaves of the lung tissue and weigh them.

(2) The lung tissue of the lysate was co-immunoprecipitated, and the total protein of the cells was harvested by about 4-10 mg, and each group of proteins was adjusted to the same concentration. 200 μg of each group of proteins was taken, 200 μg of each group of proteins was taken as an input group of cell lysate, and the cell lysate of the obtained input group was used as a control.

(3) Residual protein 2 μg of P62 antibody (purchased from Sigma, number p0067) or the same common IgG antibody as P62 antibody (purchased from cell signaling, product number 2729), plus 10 μL of Protein A/G Plus-Agarose Resuspend thoroughly and shake slowly at 4 °C overnight. Centrifuge at 3000 rpm for 5 min at 4 ° C, carefully aspirate the supernatant, preferring to leave a small amount of supernatant and not to absorb Agarose. Add 0.5 mL of immunoprecipitation lysate, Mix well, let stand for 1 min in ice bath, centrifuge at 3000 °C for 30 sec at 4 °C, carefully aspirate the supernatant. The washing was repeated 5 times and allowed to stand for 5 min before the last centrifugation. The supernatant was carefully aspirated, 20-30 μL of 2×SDS gel loading buffer was added, mixed, denatured at 95 ° C for 3 min, and rapidly transferred to an ice bath for cooling. After centrifugation at 12,000 rpm for 2 min at room temperature, the supernatant was a precipitated protein sample, and some or all of it was subjected to SDS-polyacrylamide gel electrophoresis.

The results obtained are shown in Figure 2 (A) and Figure 2 (B), wherein Figure 2 (A) shows the protein content of TRB3 protein and P62 protein in the initial lung tissue lysate; Figure 2 (B) shows the lung tissue lysate through P62 The amount of TRB3 protein and P62 protein contained in the antibody or control antibody IgG after precipitation, as shown in Fig. 2 (A) and Fig. 2 (B), showed that the TRB3 protein and the P62 protein were combined with each other in the pulmonary fibrosis tissue. The input cell lysate preparation method is as shown above, and the input represents the protein content of TRB3 protein and P62 in the initial lung tissue lysate, that is, the protein stock solution passes through the P62 antibody or the control antibody IgG (since the P62 antibody is an IgG type antibody, The content of TRB3 protein and P62 protein before precipitation was selected by IgG antibody as control. The results showed that the contents of TRB3 protein and P62 protein in the input cell lysate were consistent.

The output represents the amount of protein contained in the protein stock solution after precipitation by P62 antibody or control antibody IgG. Since the IgG antibody is used as a control antibody for the P62 antibody, the P62 protein cannot be precipitated, so the P62 protein blotting lane in the IgG antibody-treated cell lysate shows As a blank, the P62 antibody was used as an antibody of the experimental group to bind to the P62 protein and precipitated. Therefore, the cell lysate after the P62 antibody treatment showed that the P62 protein blotting lane was black. Because of the interaction between the P62 protein and the TRB3 protein, the TRB3 protein was also precipitated when the P62 protein was precipitated by the P62 antibody, so the TRB3 western blot of the cell lysate after the P62 antibody treatment showed black. Since the IgG antibody could not precipitate the P62 protein, the P62 interacting protein TRB3 protein could not be precipitated, so the TRB3 Western blotting lane of the IgG antibody-treated cell lysate was blank. The above experimental results fully demonstrate that TRB3 protein and P62 protein can directly interact

Example 4 Flow cytometry method to verify the phagocytosis of collagen by lung epithelial cells, and to verify the clearance of intracellular collagen by pep2-A1, pep2-A2, pep2-A3, and autophagy agonists and autophagy inhibitors.

(1) MLE-12 cells (MLE-12 cells were purchased from ATCC), and cells were added at a density of 1.2 × 10 ⁶ (wells/well), and cultured in a 6-well plate.

(2) FITC-labeled collagen (FITC-labeled collagen was purchased from Invitrogen) was added except for the control well, and the concentration was 1 μg/m for 1 hour.

(3) After 1 hour, the cells were washed 3 times with medium, and pep2-A1, pep2-A2, pep2-A3 (5 μM), and rapamycin (rapamycin purchased from Merck) (10 μM) and 3-MA (3-MA purchased from sigma) (2 mM).

(4) After 2 hours of stimulation, the cells were digested with trypsin and collected, and subjected to flow cytometry, excitation with a 488 nm laser, and detection by the FL1 channel.

The results are shown in Fig. 3(A) and Fig. 3(B), 3(A) is a flow result chart, and Fig. 3(B) is a flow result chart. As can be seen from Fig. 3(A) and Fig. 3(B), after collagen-FITC was added, the collagen content in MLE-12 cells was significantly increased, indicating the uptake of collagen. The intracellular collagen content was significantly reduced after using pep2-A1, pep2-A2, and pep2-A3. The use of the autophagy pathway agonist rapamycin also significantly reduced intracellular collagen content, while the use of the autophagy signal inhibitor 3-MA further aggravated intracellular collagen accumulation, with statistical mean ± SE. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

Example 5 Using pulmonary fibrosis animal model to verify p162-A1, pep2-A2, and pep2-A3 for pulmonary fibrosis

The animal models prepared in Example 1 were administered in groups after 10 days of modeling, and the grouping and administration were as shown in Table 1 (i.p. for intraperitoneal injection, i.g. for intragastric administration).

Table 1 Grouping of pulmonary fibrosis animal models after modeling

1. Determination of mouse mortality

From the 10th day after modeling, the deaths of each group of experimental animals were counted and calculated daily. The survival rate of death in a group of animals was calculated as 100%, and the survival rate of all animals in a group was 0%. 4 is shown. As can be seen from Figure 4, the survival rate of the model group was significantly reduced compared with the sham operation group. After drug treatment, the pep2-A1, pep2-A2, and pep2-A3 administration groups significantly improved the survival rate of fibrotic mice. The positive control drug pirfenidone administration group can reduce the death of experimental animals, but no statistics. difference. ##为p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group. The above experimental results show that the polypeptide and the derivative thereof of the present invention can effectively reduce the mortality of the mouse model of pulmonary fibrosis, and have the advantages of less toxic side effects and safer use.

2. Detection of the interaction between TRB3 protein and p62 protein in lung of mice with pulmonary fibrosis induced by bleomycin by immunoprecipitation

The specific operation method is the same as that in the third embodiment. The results are shown in Fig. 5(A) and Fig. 5(B), wherein Fig. 5(A) shows the protein content of TRB3 protein and P62 protein in the initial lung tissue lysate; Fig. 5(B) shows that the lung tissue lysate passed pep2 respectively. After treatment with -A1, pep2-A2, and pep2-A3, the protein amount of the obtained TRB3 protein and P62 protein was precipitated by P62 antibody or control antibody IgG, and TRB3 in pulmonary fibrosis tissue was observed from Fig. 5 (A) and Fig. 5 (B). The protein and p62 protein are present in combination with each other. After drug treatment, pep2-A1, pep2-A2, and pep2-A3 all reduced the binding of TRB3 protein to p62 protein. The input represents the protein content of the TRB3 protein and the P62 protein in the initial lung tissue lysate, that is, the protein stock solution is precipitated before being precipitated by the P62 antibody or the control antibody IgG (the IgG antibody is selected as the control because the P62 antibody is an IgG type antibody). The protein levels of TRB3 protein and P62 protein are identical. The output represents the amount of protein contained in the protein stock after precipitation by the P62 antibody or the control antibody IgG. After adding pep2-A1, pep2-A2, and pep2-A3, since the above three polypeptides can affect the interaction between P62 and TRB3 proteins, the color of the TRB3 protein imprinting lane becomes lighter. The IgG antibody could not precipitate the P62 protein as a control antibody, and the P62 interacting protein TRB3 protein could not be precipitated accordingly, and the TRB3 protein imprinting lane was blank. The above experimental results fully demonstrate that the polypeptides and derivatives thereof of the present invention are capable of blocking the mutual binding between the TRB3 protein and the P62 protein.

3. Detection of lung weight index in mice

The lungs of the mice were finely stripped and the wet weight was weighed. The lung weight (mg) was divided by the body weight (g) of the mice to obtain the lung weight index. The results are shown in Fig. 6. Fig. 6 shows the decrease of pep2-A1, pep2-A2, and pep2-A3. The lung weight index results of mice with pulmonary fibrosis induced by bleomycin showed that the lung weight index of mice was significantly increased after administration of bleomycin compared with the sham operation group. After administration of pep2-A1, pep2-A2 and pep2-A3, the lung weight index of fibrotic mice was significantly decreased. The positive control drug pirfenidone slightly decreased the lung weight index of the experimental animals, but there was no statistical difference. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

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

The mice were dissected in the neck and the organs were exposed for intubation. The PBS lavage volume was 0.8 ml, and the lavage frequency was 3-5 times. The recovered lavage fluid was centrifuged at 1500 rpm for 10 minutes at 4 ° C, and the supernatant was collected at -20 ° C for cytokine detection; the cell pellet was resuspended in 1 ml of PBS containing 1% BSA, and 10 μl of the suspension was taken for cell counting. Analysis was performed using a blood cell analyzer. The results are shown in Figure 7. Figure 7 shows the results of pep2-A1, pep2-A2, and pep2-A3 in reducing the number of inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin. Among them: A is the total number of white blood cells, B is the number of monocytes, C is the number of neutrophils, D is the number of lymphocytes, E is the number of basophils, and F is the number of eosinophils. As can be seen from the results of the figure, total leukocytes in the alveolar lavage fluid of mice after administration of bleomycin were compared with the sham operation group. The number, number of monocytes, number of basophils, number of eosinophils, and number of neutrophils were extremely significantly increased. After administration of pep2-A1, pep2-A2, pep2-A3 and the positive control drug pirfenidone, the number of inflammatory cells in the alveolar lavage fluid of mice with pulmonary fibrosis was reduced to some extent. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

Example 6 Pathological evaluation of pulmonary fibrosis

1. Pathomorphological analysis of pulmonary fibrosis induced by bleomycin

HE staining, hematoxylin-eosin staining. Hematoxylin dyeing solution is an alkaline dyeing solution, which makes the chromatin in the nucleus and the ribosome in the cytoplasm purple-blue; eosin is an acid dye, which mainly makes the components in the cytoplasm and extracellular matrix red. General tissue changes and tissue products can be visualized by this staining method, which is the most commonly used staining method for morphology.

The lung tissue of the right lower lobe of the experimental animals was taken, and 4% paraformaldehyde was fixed and embedded in paraffin. The largest cross section of the wax block embedded in the lung tissue was observed by HE staining. The results are shown in Fig. 8. Among them, A is a sham operation group, B is a model group, C is a pep2-A1 group, D is a pep2-A2 group, E is a pep2-A3 group, and F is a positive control drug pirfenidone group. It can be seen from Fig. 8 that the HE stained tissue in the lungs of the sham-operated mice is clearly visible, the alveolar structure is intact, and no pathological changes of inflammation and fibrosis are observed. After administration of pep2-A1, pep2-A2 and pep2-A3, it can alleviate lung inflammation caused by bleomycin, and effectively improve lung injury and restore normal lung structure. Especially in the pep2-A2 group, the lung inflammation disappeared and the lung tissue structure was clear.

The inflammatory grade was observed according to the results of HE staining, and the criteria were as follows (grade 0-5): grade 0: normal tissue. Level 1: Very small inflammatory changes. Grade 2: mild to moderate inflammatory changes with no apparent destruction of lung tissue structure. Grade 3: Moderate inflammatory damage (alveolar decidua thickening). Grade 4: Moderately severe inflammatory damage, formation of tissue mass, or localized pneumonia area disrupts the normal structure of lung tissue. 5: severe inflammatory injury, severe destruction of the local lung tissue structure caused by lumen closure. Fig. 9 is a graph showing the results of case analysis of inflammatory grading. As can be seen from Fig. 9, significant inflammation occurred in the lungs of mice after administration of bleomycin as compared with the sham operation group. Administration of pep2-A1, pep2-A2, pep2-A3, and the positive control drug pirfenidone significantly reduced lung inflammation caused by bleomycin. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

2. Pathological imaging analysis of Sirius red special staining

Sirius red can specifically stain fibrotic collagen, which is a special staining method for common tissue sections.

The lung tissue of the right lower lobe of the animal was taken, and 4% paraformaldehyde was fixed and embedded in paraffin. The largest cross section of the wax block embedded in the lung tissue was observed by Sirius red staining. A high-resolution pathological picture of Sirius Red special staining (200 times) was obtained using the high-resolution color pathology analysis system Spot Advanced 3.0. The stained area of collagen after Sirius red staining and the area of lung tissue in the field of view were measured using Image-Pro Plus 5.1. With colored area, The ratio of the colored area to the area of the lung tissue of the field of view indicates the relative amount of the collagen. Ten specimens were analyzed in each group of bleomycin-induced pulmonary fibrosis experiments. Each specimen was randomly divided into six fields. The mean value represents the relative content and expression intensity of collagen tissue in an animal's lung tissue. The absolute area of collagen in the field of view was compared by parameter analysis of variance. The results are shown in Figure 10 and Figure 11. Figure 10 shows that pep2-A1, pep2-A2, and pep2-A3 reduce the pathological examination of bleomycin-induced pulmonary fibrosis (Sirius Red), where A is the sham operation group and B is In the model group, C is the pep2-A1 group, D is the pep2-A2 group, E is the pep2-A3 group, and F is the positive control drug pirfenidone group. Figure 11 is a graph showing the effect of pep2-A1, pep2-A2, and pep2-A3 on collagen content in lung tissue of mice with pulmonary fibrosis. As can be seen from Figures 10 and 11, there was no significant collagen staining in Sirius red staining of sham-operated mice. After administration of bleomycin, significant collagen accumulation occurred in the lungs of mice, and a large amount of fibrotic tissue was formed. Administration of pep2-A1, pep2-A2, and pep2-A3 can alleviate pulmonary collagen accumulation caused by bleomycin and improve pulmonary fibrosis. ##为p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

Example 7 Evaluation of pulmonary function in lung fibrosis mice

Pulmonary function is a gold indicator for clinical detection of pulmonary fibrosis in patients. The decline in lung function is often accompanied by an increase in fibrosis, and the improvement in lung function often also represents the recovery of lung tissue structure.

The lung fibrosis model mice obtained in Example 1 were anesthetized with sodium pentobarbital (45 mg/kg, ip), and the lung function test was performed by Flexivent small animal pulmonary function meter. The detection method was TLC, SnapShots. (For the detection method, please refer to: Lv X, Wang X, Li K, et al. Rupatadine Protects against Pulmonary Fibrosis by Attenuating PAF-Mediated Senescence in Rodents [J]. PloS one, 2013, 8(7): e68631.).

The results are shown in Fig. 12. Fig. 12 is a graph showing the results of lung function tests in mice with pulmonary fibrosis, where A is a deep inspiratory volume, B is a dynamic resistance, C is a dynamic elasticity, and D is a dynamic compliance. As can be seen from Fig. 12, compared with the sham operation group, the deep inspiratory volume of the bleomycin-induced pulmonary fibrosis mice was significantly reduced, the dynamic resistance and dynamic elasticity of the lungs were increased, and the compliance was significantly reduced. Pulmonary function was significantly restored after treatment with pep2-A1, pep2-A2, and pep2-A3. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

Example 8 Determination of hydroxyproline content in lung fibrosis mice

Hydroxyproline accounts for 13.4% of collagen, and it is a very small amount in elastin, and it is not present in other proteins. Therefore, the content of collagen is detected by hydroxyproline. The content of hydroxyproline in the left lobe of the animals was examined to evaluate the condition of pulmonary fibrosis. The specific method is as follows: the whole lung lobe of the left side of the model animal prepared in Example 1 was prepared, the wet weight was recorded, and 10% of the tissue homogenate was prepared by ultrasonic homogenization of physiological saline, and about 150 μl of the homogenate supernatant was added, and 500 μl of alkali was hydrolyzed. After mixing with liquid and vortex, the mixture was treated by alkaline hydrolysis at 120 ° C for 0.1 min, and the pH was adjusted to volume. After the activated carbon treatment, the supernatant was taken. The hydroxyproline assay was carried out according to the instructions (chloramine T method). The results are shown in Figure 13. Figure 13 is a graph showing the results of pep2-A1, pep2-A2, and pep2-A3 in reducing hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin. The results showed that compared with the sham operation group, the hydroxyproline content in the model group was significantly increased, indicating that the pathological changes of fibrosis were severe. The administration of pep2-A1, pep2-A2, and pep2-A3 significantly reduced the content of hydroxyproline in the lungs of fibrotic mice. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

In this experiment, pathological examination, pathological imaging and other means of analysis found that pep2-A1, pep2-A2, pep2-A3 can significantly inhibit bleomycin-induced pulmonary fibrosis; significantly reduce lung fibrosis in mice Mortality; significantly reduced lung weight index in mice with pulmonary fibrosis; significantly reduced the number of various inflammatory cells in alveolar lavage fluid of mice with pulmonary fibrosis; and effectively improved lung injury and restored normal lung structure; significantly reduced Hydroxyproline and collagen content in lung tissue of mice with pulmonary fibrosis; significantly improved lung function in mice with pulmonary fibrosis. The above experimental results prove that pep2-A1, pep2-A2, and pep2-A3 have excellent therapeutic prospects in pulmonary fibrosis.

Example 9 Using an animal model of pulmonary fibrosis to verify the effects of TAT-A2 and Antp-A2 on pulmonary fibrosis

1. Main reagents and experimental animals

The bleomycin used in the experiment was purchased from Nippon Kayaku Co., Ltd., batch number X90147.

Pirfenidone, purchased from Dalian Meilun Biotechnology Co., Ltd., is a raw material drug with a pirfenidone content of >98.5%.

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

The peptide of the amino acid sequence shown in SEQ ID NO: 2 (A2) of the Sequence Listing is ligated to the TAT peptide (YGRKKRRQRRR, respectively) of the cell transmembrane peptide HIV-1 viral trans-activator transcription (Tat) protein. Its amino acid sequence is shown in SEQ ID NO: 5), the transcription factor Antp peptide of Drosophila tentacles homologous protein (RQIKIWFQNRRMKWKK, whose amino acid sequence is shown as SEQ ID NO: 6), constitutes a new derivative TAT-A2. Antp-A2, the above polypeptide or chimeric peptide was synthesized by Beijing Saibaisheng Gene Technology Co., Ltd. A2 is linked to the C-terminus of the TAT peptide and the Antp peptide by two glutamic acid chains. The sequence structure of the above chimeric peptides is as follows:

The TAT-A2 sequence is:

"N"-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-Gly-Gly-

Trp-Leu-Thr-Arg-Leu-Leu-Gln-Thr-Lys-"C" (the sequence of which is shown in SEQ ID NO: 13 in the Sequence Listing)

The sequence of Antp-A2 is:

"N"-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Gly-Gly-Gly-Gly-Trp-Leu-Thr- Arg-Leu-Leu-Gln-Thr-Lys-"C" (the sequence of which is shown in SEQ ID NO: 14 in the Sequence Listing)

SPF grade C57BL/6 mice (male, 6-8 weeks old, 16-18 g) used in the experiment were purchased from the Institute of Zoology of the Chinese Academy of Medical Sciences.

2. Preparation method is the same as in the first embodiment

The animal models prepared in Example 1 were administered in groups after 10 days of modeling, and the grouping and administration were as shown in Table 2 (i.p. For intraperitoneal injection, i.g. for intragastric administration):

Table 2 Grouping of pulmonary fibrosis animal models after modeling

1. Determination of mouse mortality

From the 10th day after modeling, the deaths of each group of experimental animals were counted and calculated daily. The survival rate of death in a group of animals was calculated as 100%, and the survival rate of all deaths in a group of animals was 0%. 14 can be seen, Figure 14 is a graph showing the results of TAT-A2 and Antp-A2 in reducing mortality of mice with pulmonary fibrosis induced by bleomycin. Among them: A is the sham operation group, B is the model group, C is the TAT-A2 group, and D is the Antp-A2 group. Compared with the sham operation group, the survival rate of the model group was significantly reduced. The results showed that after drug treatment, both TAT-A2 and Antp-A2 administration groups significantly improved the survival rate of fibrotic mice. ## is p<0.01 compared with the sham operation group, and * is p<0.05 compared with the model group. The above experimental results indicate that the polypeptide A2 of the present invention is linked to a known cell penetrating peptide to effectively reduce the mortality of a mouse model of pulmonary fibrosis.

2. Detection of lung weight index in mice

The lungs of the mice were finely stripped and weighed. The lung weight (mg) was divided by the body weight (g) of the mice to obtain the lung weight index. The results are shown in Figure 15. Figure 15 shows that TAT-A2 and Antp-A2 reduce bleomycin. Pulmonary weight index results of lung fibrosis in mice. As can be seen from the results shown in the figure, the lung weight index of the mice was significantly increased after administration of bleomycin as compared with the sham operation group. Both TAT-A2 and Antp-A2 significantly reduced the lung weight index of fibrotic mice after administration. Where ## is p<0.01 compared with the sham operation group, and ** is p<0.01 compared with the model group.

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

The mice were dissected in the neck and the organs were exposed for intubation. The PBS lavage volume was 0.8 ml, and the lavage frequency was 3-5 times. The recovered lavage fluid was centrifuged at 1500 rpm for 10 minutes at 4 ° C, and the supernatant was collected at -20 ° C for cytokine detection; the cell pellet was resuspended in 1 ml of PBS containing 1% BSA, and 10 μl of the suspension was taken for cell counting. Application of blood cell analyzer Line analysis. The results are shown in Fig. 16. Fig. 16 is a graph showing the results of TAT-A2 and Antp-A2 in reducing the number of various inflammatory cells in the alveolar lavage fluid of mice with pulmonary fibrosis induced by bleomycin. Among them: A is the total number of white blood cells, B is the number of monocytes, C is the number of neutrophils, D is the number of lymphocytes, E is the number of eosinophils, and F is the number of basophils. From the results shown in the figure, the total number of white blood cells, the number of monocytes, the number of basophils, the number of eosinophils, and the number of eosinophils in the alveolar lavage fluid of mice after administration of bleomycin were compared with those of the sham operation group. The number of neutrophils is extremely significant. TAT-A2 and Antp-A2 can reduce the number of inflammatory cells in the alveolar lavage fluid of mice with pulmonary fibrosis to varying degrees. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

4, mouse lung tissue hydroxyproline detection

Take all the lungs on the left side of the animal, record the wet weight, prepare a 10% tissue homogenate by ultrasonic homogenization, and take about 150 μl of the homogenate supernatant, add 500 μl of alkali hydrolyzate, vortex and mix, 120 degrees. Under the condition of 0.1Kpa, the alkali hydrolysis treatment was carried out for 40 minutes, the pH value was adjusted, and the volume was adjusted. After the activated carbon treatment, the supernatant was taken. The hydroxyproline assay was carried out according to the instructions (chloramine T method). The results are shown in Fig. 17. Fig. 17 is a graph showing the results of TAT-A2 and Antp-A2 in reducing the hydroxyproline content in lung tissue of mice with pulmonary fibrosis induced by bleomycin. From the results shown in the figure, it can be seen that compared with the sham operation group. The hydroxyproline content of the model group and its significant increase, indicating that the pathological changes of fibrosis are serious. TAT-A2 and Antp-A2 can significantly reduce the content of hydroxyproline in the lungs of fibrotic mice. Where ## was p<0.01 compared with the sham operation group, * was p<0.05 compared with the model group, and ** was p<0.01 compared with the model group.

In this experiment, it was found by experiments that TAT-A2 and Antp-A2 can significantly inhibit bleomycin-induced pulmonary fibrosis; significantly reduce the mortality of mice with pulmonary fibrosis; significantly reduce the lungs of mice with pulmonary fibrosis Heavy index; significantly reduced the number of various inflammatory cells in the alveolar lavage fluid of mice with pulmonary fibrosis; significantly reduced the hydroxyproline content in the lung tissue of mice with pulmonary fibrosis. The above experimental results prove that TAT-A2 and Antp-A2 also have excellent therapeutic prospects in pulmonary fibrosis.

The experimental results in Example 8 and Example 9 were expressed as mean ± standard error. After parameter or non-parametric variance test, a significant difference was considered by comparison p < 0.05, and p < 0.01 was considered to have an extremely significant difference. The statistics of pathological grading data were analyzed by chi-square test. After comparison, p<0.05 was considered to have significant difference, and p<0.01 was considered to have extremely significant difference.

The PBS used in the examples of the present invention, that is, a phosphate buffer solution, had a concentration of 0.1 M and a pH of 7.2.

The results of the above examples show that the polypeptide of the present invention or the derivative of the polypeptide has a remarkable effect of treating pulmonary fibrotic diseases, and can be used as an active ingredient for the preparation of a medicament for preventing or treating pulmonary fibrosis.

While the invention has been described with respect to the preferred embodiments of the embodiments of the embodiments of the invention modify. Accordingly, the scope of the invention is defined by the appended claims.

Claims (8)

1. Use of a polypeptide capable of specifically binding to a TRB3 protein or a derivative thereof for the preparation of a medicament for preventing or treating pulmonary fibrosis, characterized in that the polypeptide which specifically binds to the TRB3 protein is as SEQ ID NO in the sequence listing A polypeptide represented by any one of SEQ ID NO: 2 and SEQ ID NO: 3; the derivative of the polypeptide which specifically binds to the TRB3 protein is a polypeptide which specifically binds to the TRB3 protein and is contiguous with a cell penetrating peptide The chimeric peptide formed.
2. The use according to claim 1, wherein the cell penetrating peptide is as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID in the Sequence Listing. NO: 8 and the polypeptide of any one of SEQ ID NO: 9.
3. The use according to claim 1 or 2, wherein the chimeric peptide is a polypeptide as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing. The resulting peptide is ligated to the cell penetrating peptide at the N-terminus or C-terminus of the sequence.
4. The use according to at least one of claims 1 to 3, wherein the amino acid sequence of the chimeric peptide is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ in the Sequence Listing. ID NO: 13 and SEQ ID NO: 14 are shown.
5. The use according to at least one of claims 1 to 4, wherein the polypeptide which specifically binds to the TRB3 protein or a derivative thereof is used as a single active ingredient in the medicament for preventing or treating pulmonary fibrosis Alternatively, it may be combined with other compounds having activity for preventing or treating pulmonary fibrosis as an active ingredient.
6. The use according to at least one of claims 1 to 5, characterized in that the pulmonary fibrosis is primary pulmonary fibrosis or secondary pulmonary fibrosis.
7. The use according to claim 6 wherein said secondary pulmonary fibrosis is bleomycin-induced pulmonary fibrosis.
8. The use according to at least one of claims 1 to 7, wherein the drug for treating pulmonary fibrosis is one of symptoms of pulmonary inflammation, pulmonary function deterioration and lung injury caused by pulmonary fibrosis. Or a variety of drugs.

Images