CN107629114B

CN107629114B - Polypeptide, derivative thereof and application thereof in preparation of anti-pulmonary fibrosis drugs

Info

Publication number: CN107629114B
Application number: CN201710713498.XA
Authority: CN
Inventors: 胡卓伟; 吕晓希; 刘姗姗
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-08-18
Filing date: 2017-08-18
Publication date: 2021-04-09
Anticipated expiration: 2037-08-18
Also published as: CN107629114A

Abstract

The invention discloses a polypeptide specifically binding to TRB3 or a derivative thereof and application thereof in preparing a medicament for treating and/or preventing pulmonary fibrosis. The amino acid sequence of the polypeptide is shown as a sequence table SEQ ID No.12, or is shown as a non-natural amino acid with a side chain capable of being connected by replacing two or more than two amino acids in the amino acid sequence shown as the sequence table SEQ ID No. 12; the derivative comprises a chimeric peptide formed by connecting the polypeptide and a cell-penetrating peptide and a fusion peptide formed by the polypeptide and a virus. The polypeptide and the peptide derivatives thereof can be specifically combined with TRB3, so that the interaction between TRB3 and MDM2 proteins is blocked, and the polypeptide and the peptide derivatives thereof are applied to the preparation of medicaments for treating and preventing pulmonary fibrosis. The prepared medicine has the advantages of obvious curative effect, less toxic and side effects and safe use in treating pulmonary fibrosis diseases.

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 a medicament for treating and/or preventing pulmonary fibrosis.

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. Pulmonary fibrosis is one of four major diseases of respiratory diseases, has complex etiology and unknown pathogenesis, and the existing medicaments and methods for treating pulmonary fibrosis are very limited, have poor and humanized curative effects and extremely poor prognosis, and the 5-year survival rate is only 50 percent.

TRB3 (Tribbeles Homologue 3) is one of the homologous protein family members of the human pseudokinase Tribbeles, has the function of adaptor protein and is involved in the assembly of various protein complexes. Multiple studies suggest that TRB3 can interact with various transcription factors, ubiquitin ligase, cell membrane receptors, and MAPK, PI3K signal pathway member proteins, and participate in cell differentiation, apoptosis, autophagy, and metabolic regulation. Recently, various evidences suggest that TRB3 exhibits high expression in pulmonary fibrosis tissues and plays an important promoting role in the development of pulmonary fibrosis. MDM2 is an E3 ubiquitin ligase in the ubiquitin proteasome system.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a polypeptide, a derivative thereof and application thereof in preparing a medicament for treating and/or preventing pulmonary fibrosis, aiming at the current situation that a safe and effective anti-pulmonary fibrosis medicament is lacked at present. The polypeptide targets the interaction between TRB3 and MDM2, and can be used for preparing a medicament for preventing and/or treating pulmonary fibrosis with high activity and high selectivity.

Through intensive research and repeated experiments, the inventors of the present invention found that TRB3 inhibits the degradation of the transformation protein SLUG of the epithelial mesenchymal stem cell by interacting with MDM2, thereby promoting pulmonary fibrosis, and therefore, the interaction between TRB3 and MDM2 is a potential site for preventing and/or treating pulmonary fibrosis. It follows that blocking TRB3 interaction with MDM2 is a potential approach for preventing and/or treating pulmonary fibrosis, and there is a need to develop drugs that can block TRB3 interaction with MDM 2. The inventor obtains a polypeptide MR2 (the amino acid sequence of which is shown in a sequence table SEQ ID No. 12) which targets the interaction of TRB3 and MDM2 protein; meanwhile, the inventor finds that if the amino acid residue at a specific position in the polypeptide MR2 is replaced by an unnatural amino acid with a side chain capable of being connected, such as S-pentenoic alanine (S5), R-pentenoic alanine (R5) or R-octenylalanine (R8), the modified polypeptide has a stable secondary structure of alpha helix, thereby having extremely high affinity, stability against enzymolysis and cell penetrating property. Therefore, the modified polypeptide has high alpha helix stability, TRB3 binding capacity and metabolic stability, and can be applied to preparation of drugs for treating and/or preventing pulmonary fibrosis. Based on the research work of the inventor, the invention provides the following technical scheme.

One of the technical schemes provided by the invention is as follows: a polypeptide which specifically binds to TRB3, or a derivative of the polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID No.12 of the sequence table, or the derivative comprises a chimeric peptide formed by connecting the polypeptide and a cell-penetrating peptide, a fusion peptide formed by the polypeptide and a virus, a methylated polypeptide, a glycosylated polypeptide and a pegylated polypeptide, wherein two or more amino acids in the amino acid sequence shown as SEQ ID No.12 of the sequence table are replaced by unnatural amino acids with connectable side chains.

The polypeptide with the amino acid sequence shown in the sequence table SEQ ID No.12 is called MR2 polypeptide.

The unnatural amino acid to which the other side chain is attached is an unnatural amino acid that is conventional in the art, preferably S-pentenylalanine (S5), R-pentenylalanine (R5), or R-octenylalanine (R8). In the polypeptide, the number of the substituted amino acids is two, and the positions of the substituted amino acids are the ith position and the (i + 3) th position, or the ith position and the (i + 4) th position, or the ith position and the (i + 7) th position respectively, wherein i is more than or equal to 1 and less than or equal to 11, and i is a positive integer. Wherein, i +3, i +4 and i +7 all increase the helix rate of the polypeptide, thereby improving the stability of the polypeptide. Preferably, the unnatural amino acid substituted at position i is R-pentenylalanine, S-pentenylalanine or R-octenylalanine, and the unnatural amino acid substituted at position i +3, i +4 or i +7 is S-pentenylalanine.

More preferably, the amino acid sequence of the polypeptide is as shown in any one of sequence table SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25, SEQ ID No.26, SEQ ID No.27 or SEQ ID No. 28.

Wherein, the amino acid sequences shown in SEQ ID No. 1-SEQ ID No.11 and SEQ ID No. 23-SEQ ID No.28 can be appropriately substituted, deleted or added, as long as the modified amino acid sequence can still be specifically combined with TRB3 and the activity before modification is kept.

Among them, the cell-penetrating peptide of the present invention is a cell-penetrating peptide that is conventional in the art as long as it can assist in delivering the polypeptide into a cell to function. Generally, the cell-penetrating peptide is a short peptide molecule consisting of 10-30 amino acids. Preferably, the cell-penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide, more preferably to the N-terminus of the polypeptide. The cell-penetrating peptide and the polypeptide or polypeptide derivative that specifically binds TRB3 are preferably linked by two glycines (Gly-Gly).

Preferably, the cell-penetrating peptide is any one or more of a TAT peptide (amino acid sequence of which is shown in SEQ ID NO: 13) of an HIV-1 virus reverse transcription activator (Tat) protein, a transcription factor Antp peptide (amino acid sequence of which is shown in SEQ ID NO: 14) of drosophila antennapedia homeoprotein, a Pep-1 peptide (amino acid sequence of which is shown in SEQ ID NO: 15), an MPG peptide (amino acid sequence of which is shown in SEQ ID NO: 16) and an RGD peptide (amino acid sequence of which is shown in SEQ ID NO: 17). More preferably, the cell-penetrating peptide is Pep2 peptide (the amino acid sequence of which is shown in the sequence table SEQ No: 18).

Preferably, the chimeric peptide is a chimeric polypeptide Pep2-MR2 (the amino acid sequence of which is shown in SEQ ID No.19 of the sequence table) formed by connecting the Pep2 peptide to the MR2 polypeptide, a chimeric polypeptide TAT-MR2 (the amino acid sequence of which is shown in SEQ ID No.20 of the sequence table) formed by connecting the TAT polypeptide to the MR2 polypeptide, or a chimeric polypeptide Antp-MR2 (the amino acid sequence of which is shown in SEQ ID No.21 of the sequence table) formed by connecting the Antp peptide to the MR2 polypeptide.

The second technical scheme provided by the invention is as follows: use of a polypeptide that specifically binds to TRB3 or a derivative of said polypeptide in the manufacture of a medicament for the treatment and/or prevention of pulmonary fibrosis.

The "pulmonary fibrosis" described in the present invention is pulmonary fibrosis which is conventional in the art. Preferably, the pulmonary fibrosis is characterized by a pathological change in idiopathic pulmonary fibrosis, resulting from a variety of factors. Wherein, the pulmonary fibrosis preferably refers to pulmonary fibrosis of human or animals. More preferably, the symptoms of pulmonary fibrosis include: pulmonary inflammation and deterioration of lung function caused by pulmonary fibrosis. Preferably, the pulmonary fibrosis is caused by lung injury, dust or drugs. The medicine is conventional in the field, and can induce pulmonary fibrosis. Preferably bleomycin.

Wherein the pulmonary fibrosis is preferably primary (specific) pulmonary fibrosis, i.e. pulmonary fibrosis of unknown cause; or preferably secondary pulmonary fibrosis, i.e. pulmonary fibrosis secondary to the original disease. More preferably, pulmonary function deterioration, pulmonary inflammation and pulmonary injury in pulmonary fibrosis. The disease of pulmonary fibrosis preferably comprises Chronic Obstructive Pulmonary Disease (COPD), idiopathic pulmonary fibrosis or interstitial pneumonia.

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.

The third technical scheme provided by the invention is as follows: a pharmaceutical composition for anti-pulmonary fibrosis, which contains the polypeptide specifically binding to TRB3 or a derivative of the polypeptide as an active ingredient.

The active component refers to a compound with the function of preventing or treating pulmonary fibrosis. In the pharmaceutical composition, the polypeptide specifically binding to TRB3 or the derivative of the polypeptide may be used as an active ingredient alone or together with other compounds having anti-pulmonary fibrosis activity.

The route of administration of the pharmaceutical composition of the present invention is preferably injection administration or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.

Preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers. The medicinal carrier is a conventional medicinal carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable medicinal auxiliary material. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the protein and 0.01-99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

Preferably, the pharmaceutical composition is administered in an effective amount, which is an amount that alleviates or delays the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the result sought.

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 positive progress effects of the invention are as follows: the polypeptide or the polypeptide derivative can be specifically combined with TRB3, and the interaction between TRB3 and MDM2 proteins is blocked, so that the polypeptide or the polypeptide derivative can be applied to the preparation of medicaments for treating and preventing pulmonary fibrosis. The prepared medicine has the advantages of obvious curative effect, less toxic and side effects and safe use in treating pulmonary fibrosis diseases.

Drawings

FIG. 1 shows the TRB3, MDM2 and SLUG expression increasing map in pulmonary tissues of pulmonary fibrosis mice.

FIG. 2 is a graph of TRB3 interaction with MDM2 in lung tissue of lung fibroid mice. Wherein the output shows the protein amounts of TRB3 protein and MDM2 protein contained in the lung tissue lysate after the lung tissue lysate is subjected to MDM2 antibody or control antibody IgG precipitation; wherein the input shows the protein content of TRB3 protein and MDM2 protein in the original lung tissue lysate.

FIG. 3 is a graph showing that human lung epithelial cell over-expression TRB3 induces SLUG protein expression increase.

FIGS. 4-1 and 4-2 demonstrate the binding ability of the polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 to the TRB3 protein by the surface plasmon resonance method. A is the kinetic curve of polypeptide MR2 and TRB3 protein; b is the binding kinetic curve of the polypeptide S1 and the TRB3 protein; c is the binding kinetic curve of the polypeptide S2 and TRB3 protein; d is a binding kinetic curve of the polypeptide S3 and the TRB3 protein; e is a binding kinetic curve of the polypeptide S4 and the TRB3 protein; f is a binding kinetic curve of the polypeptide S5 and the TRB3 protein; g is the binding kinetic curve of the polypeptide S6 and the TRB3 protein; h is the binding kinetic curve of the polypeptide S7 and the TRB3 protein; i is a binding kinetic curve of the polypeptide S8 and TRB3 protein; j is the binding kinetic curve of the polypeptide S9 and TRB3 protein; k is a binding kinetic curve of the polypeptide S10 and TRB3 protein; l is the binding kinetic curve of the polypeptide S11 and TRB3 protein. Wherein the abscissa is the reaction time in seconds. The ordinate represents the reaction intensity of the reaction chip surface with the polypeptide, and the unit is RU.

FIG. 5 shows that the polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 interfere with the interaction pattern of TRB3 and MDM2 protein. Wherein, the output shows the protein amount of TRB3 protein and MDM2 protein contained in the cell lysate after the cell lysate is subjected to MDM2 antibody or control antibody IgG precipitation; the input showed the protein content of TRB3 protein and MDM2 protein in the initial cell lysate.

FIGS. 6-1 and 6-2 are graphs showing the results of the survival rate test of mice with pulmonary fibrosis caused by bleomycin reduction by the polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11.

FIG. 7 is a graph showing the results of polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 for reducing pulmonary fibrosis index of mice with pulmonary fibrosis caused by bleomycin.

FIG. 8 is a pathological examination (HE) pattern of polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 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 MR2 group, D is a bleomycin and S1 group, E is a bleomycin and S2 group, F is a bleomycin and S3 group, G is a bleomycin and S4 group, H is a bleomycin and S5 group, I is a bleomycin and S6 group, J is a bleomycin and S7 group, K is a bleomycin and S8 group, L is a bleomycin and S9 group, M is a bleomycin and S10 group, and N is a bleomycin and S11 group.

FIG. 9 is a graph of the results of the pathological examination scores of polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 for reducing pulmonary fibrosis caused by bleomycin.

FIG. 10 is a graph showing the results of polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 reducing the content of hydroxyproline in lung tissues of mice with pulmonary fibrosis caused by bleomycin.

FIG. 11 shows the results of lung function tests of mice with pulmonary fibrosis improved by the polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11. 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. 12 is a graph showing the survival assay results of mice with pulmonary fibrosis caused by bleomycin reduction by the polypeptides S19, S20 and S21.

FIG. 13 is a graph showing the results of polypeptides S19, S20, and S21 for decreasing pulmonary fibrosis mouse lung weight index caused by bleomycin.

FIG. 14 is a pathological examination (HE) pattern of polypeptides S19, S20, S21 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 S19 group, D is a bleomycin and S20 group, and E is a bleomycin and S21 group.

FIG. 15 is a graph of the results of pathological examination scores of polypeptides S19, S20, S21 for reducing pulmonary fibrosis caused by bleomycin.

FIG. 16 is a graph showing the results of polypeptides S19, S20 and S21 reducing the content of hydroxyproline in lung tissues of mice with pulmonary fibrosis caused by bleomycin.

Fig. 17 shows the results of lung function tests on mice with pulmonary fibrosis improved by the polypeptides S19, S20, and S21. 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. 18 is a graph showing the results of polypeptides S5 and S22 reducing pulmonary fibrosis mouse lung weight index caused by bleomycin.

Fig. 19 is a graph of the results of pathological examination scoring of polypeptides S5 and S22 to reduce pulmonary fibrosis caused by bleomycin.

FIG. 20 is a graph showing the results of polypeptides S5 and S23-S28 for reducing pulmonary fibrosis mouse lung weight index caused by bleomycin.

FIG. 21 is a graph of the results of pathological examination scoring of polypeptides S5 and S23-S28 for reducing 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.

The PBS solution described in the examples refers to a phosphate buffer solution at a concentration of 0.1M and a pH of 7.2.

The room temperature described in the examples is a room temperature which is conventional in the art, and is preferably 15 to 30 ℃.

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.

EXAMPLE 1 Synthesis of the polypeptide

The amino acid sequence of the polypeptide MR2 is shown in a sequence table SEQ ID No. 12. The polypeptide MR2 was synthesized and purified by Beijing Saibaosheng Gene technology, Inc.

Two unnatural amino acids S-pentenylalanine (S5) were introduced for solid phase polypeptide chain synthesis. And after the synthesis of the solid-phase polypeptide chain is finished, performing olefin metathesis (RCM) cyclization by using ruthenium as a catalyst to obtain the target polypeptide. Finally, the target polypeptide is cleaved from the resin and purified. The above-mentioned steps for solid phase peptide chain synthesis and purification are carried out by Zhongji peptide Biochemical Co., Ltd. Wherein, two S-pentenyl alanines are inserted into the i th and i +4 th positions in the polypeptide MR2 amino acid sequence, thereby obtaining the modified polypeptide with different sequences (the amino acid sequence is shown in the sequence table SEQ ID No. 1-SEQ ID No.11), and the specific insertion sites are as follows:

S1：S5-Leu-Met-Ala-S5-Phe-Thr-Cys-Ala-Lys-Lys-Leu-Lys-Lys-Arg

S2：His-S5-Met-Ala-Cys-S5-Thr-Cys-Ala-Lys-Lys-Leu-Lys-Lys-Arg

S3：His-Leu-S5-Ala-Cys-Phe-S5-Cys-Ala-Lys-Lys-Leu-Lys-Lys-Arg

S4：His-Leu-Met-S5-Cys-Phe-Thr-S5-Ala-Lys-Lys-Leu-Lys-Lys-Arg

S5：His-Leu-Met-Ala-S5-Phe-Thr-Cys-S5-Lys-Lys-Leu-Lys-Lys-Arg

S6：His-Leu-Met-Ala-Cys-S5-Thr-Cys-Ala-S5-Lys-Leu-Lys-Lys-Arg

S7：His-Leu-Met-Ala-Cys-Phe-S5-Cys-Ala-Lys-S5-Leu-Lys-Lys-Arg

S8：His-Leu-Met-Ala-Cys-Phe-Thr-S5-Ala-Lys-Lys-S5-Lys-Lys-Arg

S9：His-Leu-Met-Ala-Cys-Phe-Thr-Cys-S5-Lys-Lys-Leu-S5-Lys-Arg

S10：His-Leu-Met-Ala-Cys-Phe-Thr-Cys-Ala-S5-Lys-Leu-Lys-S5-Arg

S11：His-Leu-Met-Ala-Cys-Phe-Thr-Cys-Ala-Lys-S5-Leu-Lys-Lys-S5

example 2Western-Blot detection of expression of TRB3, MDM2 and SLUG proteins in pulmonary tissues of pulmonary fibrosis mice

Preparation of animal model

Bleomycin was purchased from Japan Chemicals, lot No. 640412.

The compound used in example 2 was purchased from Sigma, unless otherwise specified.

SPF grade C57BL/6 mice (male, 6-8 weeks old, 16-18 g) were purchased from Experimental animals technology, Inc. of Wei Tony Hua, Beijing.

Male C57BL/6 mice were fasted overnight, anesthetized with 45mg/kg sodium pentobarbital by intraperitoneal injection (i.p.), and injected intratracheally with bleomycin at a dose of 3U/kg. The specific operation steps are as follows: the skin of the neck of a mouse is cut with a wound as small as possible, the trachea is exposed with the help of elbow ophthalmic forceps, a micro-injector is used for puncturing the trachea, about 50 mu L of bleomycin is injected into the trachea, and the bleomycin is rapidly rotated and erected for 5 minutes so as to uniformly enter the left and right lung lobes, thereby constructing the animal model. The whole operation is carried out at a surgical operating table at about 60 ℃. Mice in the sham group were injected intratracheally with equal amounts of normal saline for injection.

The mice in the constructed animal model are raised in an SPF animal room, the mice are sacrificed after the lung of the mice is changed by fibrosis pathology by the 10 th day, and the lung tissues of the mice are taken for subsequent experiments. Mice in the sham group were also housed in SPF-grade animals and sacrificed by day 10 and lung tissue was taken for follow-up experiments.

Second, Western-Blot detection of pulmonary fibrosis mouse lung tissue TRB3, MDM2 and SLUG protein expression

Taking a proper amount of lung tissue of the animal model obtained in the first step, adding lysis buffer [ containing 0.1mM Ethylene Diamine Tetraacetic Acid (EDTA), 0.1mM ethylene glycol diethyl diamine tetraacetic acid (EGTA), 10mM KCl, 10mM 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 50mM NaF, 0.1M Na3VO4, 0.1M Na3PO4, 1mM/L Aprotinin (Aprotinin), 1mM/L Trypsin inhibitor (Trypsin inhibitor), 1mM/L phenylmethylsulfonyl fluoride (PMSF), 1mM/L leupeptin (Leupeptins) and 1mM/L Dithiothreitol (DTT) ] for homogenizing, and then placing on ice for 15min or shaking. Quickly adding 10% (v/v) ethylphenylpolyethylene glycol (NP-40), mixing, and centrifuging at 12000rpm for 5min at 4 deg.C. The supernatant was taken and the protein concentration was determined by Coomassie Brilliant blue and adjusted to the same for Western Blot analysis.

Western Blot analysis detected TRB3, MDM2 and SLUG. The bands were developed using Amersham developing solution (NBT/BCIP staining kit IK5030, available from Huamei Co.), and analyzed by Western-blot analysis software (gelPro32) for densitometric value, and the results are shown in FIG. 1. FIG. 1 shows that TRB3, MDM2 and SLUG proteins are in a high expression state in lung tissues of the pulmonary fibrosis mouse.

Example 3 demonstration of binding of MDM2 to TRB3 protein in pulmonary fibrotic tissues by Co-immunoprecipitation

The reagents used for co-immunoprecipitation were as follows:

lysate a: 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl 2.6H2O, 0.0742g EDTA, 10mL glycerol and 10mL 10% (v/v) NP40 were weighed, deionized water was added to 150mL, the pH was adjusted to 7.6 with HCl, the volume was adjusted to 191mL, mixed well, filtered through a 0.45 μm filter membrane, and stored at 4 ℃.

Lysate B: 200. mu.L of 2 M.beta. -phosphoglycerol, 4mL of 2.5M NaF, 2mL of 100mM NaVO3, 2mL of 100mM PMSF, 200. mu.L of 1M DTT, 200. mu.L of 1mg/mL Leu, 200. mu.L of 1mg/mL Pep, and 200. mu.L of 1mg/mL Apr were weighed out in a total volume of 9 mL. Storing at-20 deg.C. When the cell lysate is used, the lysate B is unfrozen, and the lysate B is added into the lysate A and uniformly mixed according to the volume ratio of the lysate B to the lysate A of 1:100, so that the cell lysate is obtained.

Co-immunoprecipitation lotion: comprising 1% (v/v) NP40, 150mM NaCl, 20mM HEPES, 10% (v/v) glycerol pH7.5 and 1mM EDTA.

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

The specific operation steps are as follows:

(1) and (3) taking down the lung tissue big leaves with proper amount of the animal model obtained by the step one of the step 2, and weighing the lung tissue.

(2) Lung tissue weighed in the step (1) was lysed by a coprecipitation lysate, and about 10mg of total cellular protein was harvested, and the lung tissue protein in both the animal model and the sham operation group was adjusted to a concentration of 10. mu.g/mL. The lung tissue protein 200. mu.g of the animal model and the sham-operated group were used as input groups, and the input groups were used as controls.

(3) An equal amount of the remaining 9800. mu.g of Protein from each group was added separately to 2. mu.g of MDM2 antibody (purchased from Abcam, accession number ab16895) and to a common IgG antibody of the same species as MDM2 antibody (purchased from Cell Signaling, commercial number 2729), while adding 10. mu.L of Protein A/G Plus-Agarose (purchased from Santa Cruz, accession number SC-2003) and resuspended thoroughly and shaken slowly at 4 ℃ overnight with gentle rotation. Centrifuge at 3000rpm for 5min at 4 ℃ and carefully aspirate the supernatant. Adding 0.5mL of co-immunoprecipitation lysate, mixing, standing in ice bath for 1min, centrifuging at 4 ℃ and 3000rpm for 30 s, and carefully removing the supernatant by suction. Washing was repeated 5 times, and left for 5min before the final centrifugation. Carefully remove the supernatant, add 30. mu.L of 2 XSDS gel loading buffer, mix well, denature for 3min at 95 ℃ and quickly transfer to an ice bath to cool. Centrifuging at 12000rpm for 2min at room temperature to obtain supernatant as precipitated protein sample, and performing SDS-polyacrylamide gel electrophoresis on part of the protein sample.

The results are shown in FIG. 2, and it can be seen from FIG. 2 that the TRB3 protein and MDM2 protein are combined with each other in the pulmonary fibrosis tissue. Wherein the "input" is the cell lysate described above; "input" represents the initial TRB3 and MDM2 levels in the protein sample, i.e., TRB3 and MDM2 levels in the protein sample prior to precipitation with MDM2 antibody or control antibody IgG (since MDM2 antibody is an IgG type antibody, IgG antibody was chosen as the control). The results showed that the TRB3 protein and MDM2 protein were present in the same amount in the lysates of the cells of the input groups.

"output" represents the TRB3 and MDM2 content of the protein sample after precipitation of the protein sample with MDM2 antibody or IgG control antibody. Since the MDM2 protein could not be precipitated by IgG antibody as a control antibody to MDM2 antibody, the MDM 2western blot lane in IgG antibody treated protein samples was shown as blank; whereas the MDM2 antibody, as a panel antibody, binds to and precipitates MDM2 protein, the result of the protein sample treated with MDM2 antibody is that the MDM 2Western blot lane appears black. Because of the interaction between the MDM2 protein and the TRB3 protein, the MDM2 antibody can precipitate TRB3 protein when precipitating MDM2 protein, so the TRB3 Western blot lane of the cell lysate treated with MDM2 antibody is shown in black. The TRB3 Western blot lane of IgG antibody-treated cell lysates was blank, since the MDM2 protein could not be precipitated by IgG antibody, and therefore the MDM2 interacting protein TRB3 could not be precipitated by the IgG antibody. The above results are sufficient to demonstrate that TRB3 protein and MDM2 protein are capable of direct interaction.

Example 4TRB3 elicits an increase in lung epithelial cells SLUG.

(1) mu.L of enucleated enzyme water was added to the tube, shaken for 10 seconds, and the transfection reagent (purchased from Invitrogen, model lipofectamine 2000) was diluted.

(2) Transfection reagents were mixed with plasmid DNA [ control plasmid (purchased from aoruidongyuan, model PS 100001); the TRB3 overexpression plasmid (purchased from AoRunto, model RC206687) was expressed as 1: 2, the ratio of the amount (mL) of the transfection reagent to the mass (mg) of the DNA, and standing for 15 minutes at room temperature to obtain a mixed solution.

(3) M199 medium was aspirated from the plates of BEAS-2B cells (purchased from the institute of basic medicine, national academy of medical sciences) and washed once with PBS.

(4) 400. mu.L of the mixture was added, and the cells were returned to the incubator and cultured at 37 ℃ for 24 hours.

(5) Transfected BEAS-2B cells were lysed using RIPA lysate (purchased from Biyunyan, model P0013B) and intracellular SLUG protein expression was detected using the Western-blot method of example 3.

The results are shown in FIG. 3. FIG. 3 shows that the expression of Slug in BEAS-2B cells overexpressing TRB3 is significantly increased.

Example 5 detection of the binding Capacity of the polypeptide S1-S11 to the TRB3 protein by surface plasmon resonance

The surface plasmon resonance experiment was performed in a surface plasmon resonance instrument Biacore T200, and the procedure was performed according to the specification of the surface plasmon resonance instrument Biacore T200. The method comprises the following specific steps:

1. the purified TRB3 protein (from RD) was coupled to a CM5 chip (from GE) via an amino group, unbound protein was removed by elution at a flow rate of 10. mu.L/min, and the chip surface was equilibrated for 2 hours. The specific steps of amino coupling, elution and equilibration are described in the relevant specification of the chip CM5, GE.

2. 250 μ L of the S1-S11 and MR2 polypeptide fragments prepared in example 1 at different concentrations (800, 400, 200, 50, 12.5, 6.25 and 3.125nM) were injected automatically and the whole surface plasmon resonance experiment was performed at 25 ℃. The buffer used was HBS-EP buffer [0.01M HEPES, 0.15M NaCl, 3mM EDTA and 0.005% (w/w) surfactant TWEEN20 ]. Binding curves of different concentrations of the polypeptide to TRB3 were simulated using Biacore T200 self-contained analysis software and the results are shown in FIGS. 4-1, 4-2 and Table 1. FIGS. 4-1, 4-2 and Table 1 demonstrate that peptide fragments MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 can interact with TRB3 protein.

TABLE 1 affinity test of the polypeptides S1-S11 and MR2 with TRB3 protein

Polypeptide name	Affinity constant (KD) to TRB3 protein
		MR2	4.33×10^-8M
S1	2.02×10^-8M
		S2	1.91×10^-8M
S3	4.27×10^-8M
		S4	9.63×10^-9M
S5	1.15×10^-8M
		S6	4.96×10^-8M
S7	8.95×10^-9M
		S8	3.59×10^-8M
S9	3.71×10^-8M
		S10	2.93×10^-8M
S11	3.77×10^-8M

Example 6 circular dichroism method for detecting alpha helix rate of polypeptide S1-S11

The alpha helix rate of the polypeptide was measured by circular dichroism spectroscopy (purchased from Jasco, Japan). The polypeptides MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, and S11 prepared in example 1 were dissolved in PBS solution, and the on-machine concentration of the circular dichroism spectrometer was adjusted to 1mg/mL, and the results are shown in table 2. Table 2 shows that the alpha-helix ratios of the polypeptides S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11 are significantly higher than that of the polypeptide MR 2. Wherein, the alpha helix ratio refers to the percentage of the number of peptide fragments of the polypeptide which maintain the alpha helix of the secondary structure to the number of peptide fragments of the total polypeptide.

TABLE 2 circular dichroism method for determining alpha helix rate of polypeptide

Example 7 Co-immunoprecipitation method to verify that S1-S11 and MR2 polypeptides inhibit binding of protein MDM2 and TRB3 at the cellular level

1. Human lung epithelial BEAS-2B cells (purchased from the institute of basic medicine of Chinese academy of medical sciences) were plated on a 90mm2 culture dish, TRB3 expression plasmid was transferred to the cells after the cells were attached, 1mg/mL of the polypeptide MR2, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 or S11 prepared in example 1 was added after 24 hours, and the cells were collected after 12 hours of incubation in an incubator at 37 ℃.

2. Co-immunoprecipitation was performed with MDM2 antibody as described in example 3, followed by detection using TRB3 antibody.

The results are shown in FIG. 5. The results in FIG. 5 show that the interference of the polypeptides S1-S11 on the TRB3/MDM2 protein interaction is significantly stronger than that of MR 2.

Example 8 determination of the Effect of the Polypeptides S1-S11 on mortality in mice with pulmonary fibrosis

Animal models of pulmonary fibrosis were made according to the method described in example 2, with polypeptide therapy starting on day 10 after molding (treatment groups are shown in table 3), and time was calculated from day 10 after molding to count mortality. The death condition of each group of experimental animals is counted and calculated every day, if a certain group of animals do not die, the survival rate is 100%, and if all the animals die, the survival rate is 0%. The survival rate is the percentage of the number of surviving mice in the group of mice. The results are shown in table 4 of fig. 6-1 and 6-2, and fig. 6-1 and 6-2 illustrate that the survival rate of the model group is significantly reduced compared with that of the sham operation group. After drug treatment, the S1-S11 administration groups can obviously improve the survival rate of the fibrosis mice. In FIGS. 6-1 and 6-2, # # is p <0.01 compared to the sham group; p is <0.05 compared to the bleomycin group; p <0.01 compared to the bleomycin group; i.p. refers to intra-abdominal injection. The results show that the polypeptide can effectively reduce the death rate of the pulmonary fibrosis model mouse, and has the advantages of less toxic and side effects and safer use.

TABLE 3 grouping administration after model building of pulmonary fibrosis animal model

TABLE 4 Effect of the Polypeptides S1-S11 on mortality in pulmonary fibrosis mice

Example 9 detection of the Effect of the Polypeptides S1-S11 on the Lung weight index of mice with pulmonary fibrosis

Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 3. After 28 days, the lung of the mice was finely stripped and the wet weight was taken, and the lung weight (mg) was divided by the body weight (g) of the mice to obtain a lung weight index, and the results are shown in FIG. 7 and Table 5. As can be seen from fig. 7, the lung weight index of mice was significantly increased after administration of bleomycin compared to the sham group. Therefore, the lung weight index of the fibrotic mice can be obviously reduced after the administration of the polypeptides S1-S11. In fig. 7, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 5 Effect of the Polypeptides S1-S11 on the Lung weight index of Lung-Retention mice

Group of	Index of lung weight
		Artificial operation group	6.7875
Bleomycin group	12.7375
		MR2 administration group	11.4625
S1 administration group	8.675
		S2 administration group	9.35
S3 administration group	8.8
		S4 administration group	8.925
S5 administration group	8.575
		S6 administration group	9.725
S7 administration group	7.675
		S8 administration group	8.775
S9 administration group	7.4125
		S10 administration group	8.8875
S11 administration group	9.2875

Example 10 determination of the Effect of the Polypeptides S1-S11 on the pathomorphology of bleomycin-induced pulmonary fibrosis

General tissue changes and tissue products can be shown by HE staining, the hematoxylin-eosin staining, which is the most commonly used staining method for morphology. Wherein the 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. Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 3. After 28 days, the lung tissue of the right lower lobe of the experimental animal was taken and fixed with 4% (v/v) paraformaldehyde and then embedded in paraffin. In the largest cross-sectional section of the wax block embedding lung tissue HE staining was observed for basic pathological changes. The results are shown in FIG. 8. FIG. 8 shows that HE stained tissues in the lung of the sham operated mice are clearly visible, alveolar structures are intact, and inflammatory and fibrotic pathological changes are not seen. The lungs of the bleomycin mice are obviously inflamed, inflammatory cells are infiltrated in a large quantity, and the lung tissue structure is seriously damaged. After the polypeptide S1-S11 is administrated, the lung inflammation caused by bleomycin can be relieved, lung injury is effectively improved, and the normal structure of the lung is recovered.

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.

The results are shown in FIG. 9 and Table 6. The results show that significant inflammation of the lungs of mice occurred after bleomycin administration compared to sham operated groups. The administration of the polypeptides S1-S11 can obviously reduce the lung inflammation caused by bleomycin. In fig. 9, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 6 inflammatory score of Polypeptides S1-S11 on bleomycin-induced pulmonary fibrosis

Group of	Inflammatory score
		Artificial operation group	0
Bleomycin group	4.333333
		MR2 administration group	4
S1 administration group	2.666667
		S2 administration group	2.666667
S3 administration group	2.5
		S4 administration group	2.833333
S5 administration group	2.333333
		S6 administration group	3
S7 administrationGroup of	2
		S8 administration group	2.833333
S9 administration group	2.166667
		S10 administration group	3
S11 administration group	3

Example 11 determination of the Effect of the Polypeptides S1-S11 on the hydroxyproline content in mice with pulmonary fibrosis

Hydroxyproline accounts for 13.4% (w/w) of collagen, very little of elastin and none of the other proteins, and therefore the content of collagen was determined by hydroxyproline. Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 3. And detecting the content of the hydroxyproline in the left lung of the animal after 28 days, and evaluating the condition of the pulmonary fibrosis.

The specific method comprises the following steps: feeding mice in the animal model prepared in the example 2 in an SPF animal room, performing polypeptide treatment according to the administration scheme shown in the table 3, taking all lung lobes on the left side of the animal to be tested on the 28 th day after the animal model is constructed, recording wet weight, ultrasonically homogenizing physiological saline to prepare 10% (w/w) tissue homogenate, taking about 150 mu L of homogenate supernatant, adding 500 mu L of alkali hydrolysate (provided by a hydroxyproline alkali hydrolysis kit built by bioengineering technology, Inc. of Nanjing), uniformly mixing, performing alkali hydrolysis treatment for 40min at 120 ℃ and 0.1Kpa, adjusting pH value, fixing volume, and taking supernatant after active carbon treatment. Hydroxyproline was measured by chloramine-T method (the procedure of this example is described in the kit of Nanjing Biotechnology engineering Co., Ltd.). The results are shown in FIG. 10 and Table 7. As can be seen from fig. 10, the bleomycin group had an increased hydroxyproline content and its significance compared to the sham group, indicating that the fibrotic pathology was severely altered. The content of hydroxyproline in the lung of the fibrotic mouse can be obviously reduced after the administration of S1-S11. In fig. 10, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 7 Effect of the Polypeptides S1-S11 on hydroxyproline content in mice with pulmonary fibrosis

Group of	Hydroxyproline content (μ g/mg protein)
		Artificial operation group	0.42125
Bleomycin group	1.0875
		MR2 administration group	0.90875
S1 administration group	0.70875
		S2 administration group	0.70625
S3 administration group	0.82875
		S4 administration group	0.65
S5 administration group	0.6575
		S6 administration group	0.83375
S7 administration group	0.655
		S8 administration group	0.825
S9 administration group	0.72125
		S10 administration group	0.81625
S11 administration group	0.7725

Example 12 determination of the Effect of the Polypeptides S1-S11 on pulmonary function in mice with pulmonary fibrosis

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. Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 3. On 28 days after the animal model was constructed, mice were anesthetized with pentobarbital sodium (45mg/kg, i.p.), and lung function was detected by a Flexitent small animal lung function apparatus (TLC, Snapslots (see: Lv X, Wang X, Li K, et al. Rupatadine Protections against PAF-media science by anchoring PAF-media sensing in Rodents [ J ]. ploS one,2013,8(7): e 68631.).

The results are shown in fig. 11 and table 8, where a is the amount of deep breathing, B is the dynamic resistance, C is the dynamic elasticity, and D is the dynamic compliance. Fig. 11 shows that compared with the sham-operated group, bleomycin-induced pulmonary fibrosis mice have significantly reduced deep inspiratory capacity, increased dynamic resistance and dynamic elasticity of the lung, and significantly reduced compliance. The lung function is obviously recovered after the treatment of the polypeptide S1-S11. In fig. 11, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 8 Effect of the Polypeptides S1-S11 on pulmonary function in pulmonary fibrosis mice

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

Example 13 Synthesis of polypeptide derivatives S19-21 (chimeric polypeptide of the polypeptide MR2+ cell-penetrating peptide)

S19: the chimeric polypeptide Pep2-MR2 (the amino acid sequence of which is shown in a sequence table SEQ ID No. 19), S20: the amino acid sequence of the chimeric polypeptide TAT-MR2 (shown as a sequence table SEQ ID No. 20), S21: the chimeric polypeptide Antp-MR2 (the amino acid sequence of which is shown in the sequence table SEQ ID No. 21) is synthesized and purified by Beijing Saibaosheng Gene technology Co.

Example 14 determination of the Effect of S19-21 on mortality in mice with pulmonary fibrosis

Animal models of pulmonary fibrosis were made according to the method described in example 2, with polypeptide therapy administered starting on day 10 post-molding (treatment groups are shown in table 9), and time was calculated to count mortality starting on day 10 post-molding. The death condition of each group of experimental animals is counted and calculated every day, if a certain group of animals do not die, the survival rate is 100%, and if all the animals die, the survival rate is 0%. The results are shown in FIG. 12 and Table 10. Fig. 12 illustrates that the survival rate of the model group was significantly reduced compared to the sham-operated group. After drug treatment, the S19-S21 administration groups can obviously improve the survival rate of the fibrosis mice. In fig. 12, # # is p <0.01 compared to sham; p is <0.05 compared to the bleomycin group; p is <0.01 compared to the bleomycin group. The results show that the polypeptide derivative can effectively reduce the death rate of the pulmonary fibrosis model mouse, and has the advantages of less toxic and side effects and safer use.

TABLE 9 grouping administration after model building of pulmonary fibrosis animal model

TABLE 10 influence of S19-21 on mortality in mice with pulmonary fibrosis

Example 15 detection of the Effect of S19-21 on the pulmonary weight index of mice with pulmonary fibrosis

Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 9. After 28 days, the lung of the mice was finely stripped and the wet weight was taken, and the lung weight (mg) was divided by the body weight (g) of the mice to obtain a lung weight index, and the results are shown in FIG. 13 and Table 11. As can be seen from fig. 13, the lung weight index of mice was significantly increased after administration of bleomycin compared to the sham group. Therefore, the lung weight index of the fibrosis mouse can be obviously reduced after the administration of S19-S21. In fig. 13, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 11 influence of S19-21 on pulmonary weight index in pulmonary maintenance mice

Group of	Index of lung weight
		Artificial operation group	6.7875
Bleomycin group	12.7375
		S19 administration group	8.675
S20 administration group	10.0625
		S21 administration group	11.2125

Example 16 measurement of the Effect of S19-21 on the pathomorphology of pulmonary fibrosis caused by bleomycin

Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 9. After 28 days, the lung tissue of the right lower lobe of the experimental animal was taken and fixed with 4% (v/v) paraformaldehyde and then embedded in paraffin. In the largest cross-sectional section of the wax block embedding lung tissue HE staining was observed for basic pathological changes. The results are shown in FIG. 14. FIG. 14 shows that the administration of S19-S21 can reduce the inflammation of the lung caused by bleomycin, effectively improve the lung injury and restore the normal structure of the lung.

The results are shown in FIG. 15 and Table 12. The results show that significant inflammation of the lungs of mice occurred after bleomycin administration compared to sham operated groups. The administration of S19-S21 can significantly reduce the pulmonary inflammation caused by bleomycin. In fig. 15, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 12S 19-21 inflammatory scores for bleomycin-induced pulmonary fibrosis

Example 17 determination of the Effect of S19-21 on hydroxyproline content in mice with pulmonary fibrosis

Hydroxyproline accounts for 13.4% (w/w) of collagen, very little of elastin and none of the other proteins, and therefore the content of collagen was determined by hydroxyproline. Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 9. And detecting the content of the hydroxyproline in the left lung of the animal after 28 days, and evaluating the condition of the pulmonary fibrosis.

The specific method comprises the following steps: feeding mice in the animal model prepared in the example 2 in an SPF animal room, performing polypeptide treatment according to the administration scheme shown in the table 9, taking all lung lobes on the left side of the animal to be tested on the 28 th day after the animal model is constructed, recording wet weight, ultrasonically homogenizing physiological saline to prepare 10% (w/w) tissue homogenate, taking about 150 mu L of homogenate supernatant, adding 500 mu L of alkali hydrolysate (provided by a hydroxyproline alkali hydrolysis kit built by bioengineering technology, Inc. of Nanjing), uniformly mixing, performing alkali hydrolysis treatment for 40min at 120 ℃ and 0.1Kpa, adjusting pH value, fixing volume, and taking supernatant after active carbon treatment. Hydroxyproline was measured by chloramine-T method (the procedure of this example is described in the kit of Nanjing Biotechnology engineering Co., Ltd.). The results are shown in FIG. 16 and Table 13. As can be seen from fig. 16, the bleomycin group had an increased hydroxyproline content and its significance compared to the sham group, indicating that the fibrotic pathology was severely altered. And the content of hydroxyproline in the lung of the fibrotic mouse can be obviously reduced after the S19-S21 administration. In fig. 16, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

Table 13S 19-21 Effect on hydroxyproline content in mice with pulmonary fibrosis

Group of	Hydroxyproline content (μ g/mg protein)
		Artificial operation group	0.42125
Bleomycin group	0.915
		S19 administration group	0.56375
S20 administration group	0.744286
		S21 administration group	0.74875

Example 18 determination of the Effect of S19-21 on pulmonary function in mice with pulmonary fibrosis

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. Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 9. After 28 days, mice were anesthetized with pentobarbital sodium (45mg/kg, i.p.), and lung function was measured by a Flexitent small animal lung function apparatus (TLC, SnaPs, see Lv X, Wang X, Li K, et al, Rupatadine Protection against plasmid Pulmonary Fibrosis by assay PAF-Mediated sensing in Rodents [ J ]. ploS one,2013,8(7): e 68631.).

The results are shown in fig. 17 and table 14, where a is the amount of deep breathing, B is the dynamic resistance, C is the dynamic elasticity, and D is the dynamic compliance. Fig. 17 shows that bleomycin-induced pulmonary fibrosis mice have significantly reduced deep inspiratory capacity, increased pulmonary dynamic resistance and dynamic elasticity, and significantly reduced compliance, compared with the sham group. The lung function is obviously recovered after the treatment of the polypeptide S19-S21. In fig. 17, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 14 influence of S19-21 on pulmonary function of pulmonary fibrosis mice

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

Example 19 Synthesis of comparative example polypeptide S22 (amino acid sequence is shown in CN201410826820.6 SEQ ID NO:11 in the sequence Listing)

A chimeric polypeptide Pep-A2 (the amino acid sequence of which is shown in a sequence table SEQ ID No. 22) capable of inhibiting the combination of TRB3 and protein P62 is synthesized and purified by Beijing Sibuthih gene technology Co.

S22：His-Leu-Tyr-Val-Ser-Pro-Trp-Gly-Gly-Gly-Gly-Trp-Leu-Thr-Arg-Leu-Leu-Gln-Thr-Lys

(H-L-Y-V-S-P-W-G-G-G-G-W-L-T-R-L-L-Q-T-K)

Example 20 circular dichroism method for detecting alpha helix rate of polypeptide S22

The alpha helix rate of the polypeptide was measured by circular dichroism spectroscopy (purchased from Jasco, Japan). The polypeptides MR2, S5 and S22 prepared in example 1 were dissolved in PBS solution, and the on-board concentration of the circular dichroism spectrometer was adjusted to 1mg/mL, and the results are shown in Table 15. Table 15 shows that the alpha-helix rate of the polypeptide S5 is significantly higher than that of the polypeptides MR2 and S22. Wherein, the alpha helix ratio refers to the percentage of the number of peptide fragments of the polypeptide which maintain the alpha helix of the secondary structure to the number of peptide fragments of the total polypeptide.

TABLE 15 determination of alpha-helix rate of polypeptide by circular dichroism

Example 21 testing the effects of S5 and S22 on the lung weight index of pulmonary fibrosis mice

Pulmonary fibrosis models were made as described in example 2 and group dosing was performed as per table 16. After 28 days, the lung of the mice was finely stripped and the wet weight was taken, and the lung weight (mg) was divided by the body weight (g) of the mice to obtain a lung weight index, and the results are shown in FIG. 18 and Table 17. As can be seen from fig. 18, the lung weight index of mice was significantly increased after administration of bleomycin compared to the sham group. The lung weight index of the fibrosis mice can be obviously reduced after the administration of S5 and S22, wherein the preferred is S5. In fig. 18, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 16 grouping administration after model building of pulmonary fibrosis animal model

TABLE 17 Effect of S5 and S22 on pulmonary weight index in Lung-Retention mice

Group of	Index of lung weight
		Artificial operation group	5.8827
Bleomycin group	11.1804
		S5 administration group	7.9196
S22 administration group	9.2866

Example 22 determination of the Effect of S5 and S22 on the pathomorphology of bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis models were made as described in example 2 and group dosing was performed as per table 16. After 28 days, the right lower lobe lung tissue of the experimental animal was taken and fixed with 4% paraformaldehyde and embedded in paraffin. In the largest cross-sectional section of the wax block embedding lung tissue HE staining was observed for basic pathological changes. 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.

The results are shown in FIG. 19 and Table 18. The results show that significant inflammation of the lungs of mice occurred after bleomycin administration compared to sham operated groups. Both S5 and S22 administration significantly reduced pulmonary inflammation caused by bleomycin, with S5 being preferred. In fig. 19, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

Tables 18S 5 and S22 inflammatory scores for bleomycin-induced pulmonary fibrosis

Group of	Inflammatory score
		Artificial operation group	0
Bleomycin group	4.167
		S5 administration group	1.5
S22 toMedicine group	2.5

Example 23 Synthesis of Polypeptides S23 to S28

Two unnatural amino acids S-pentenylalanine (S5) or R-pentenylalanine (R5) or R-octenylalanine (R8) were introduced for solid phase polypeptide chain synthesis. And after the synthesis of the solid-phase polypeptide chain is finished, performing olefin metathesis (RCM) cyclization by using ruthenium as a catalyst to obtain the target polypeptide. Finally, the target polypeptide is cleaved from the resin and purified. The above-mentioned steps for solid phase peptide chain synthesis and purification are carried out by Zhongji peptide Biochemical Co., Ltd. Wherein, S-pentenyl alanine (S5) or R-pentenyl alanine (R5) or R-octenyl alanine (R8) is inserted into the i, i +3 position or the i, i +7 position in the polypeptide MR2 amino acid sequence, thereby obtaining the modified polypeptide with different sequences (the amino acid sequence is shown in the sequence table SEQ ID No. 23-SEQ ID No.28), and the specific insertion site is shown as follows:

S23：R5-Leu-Met-S5-Cys-Phe-Thr-Cys-Ala-Lys-Lys-Leu-Lys-Lys-Arg

(R5-L-M-S5-C-F-T-C-A-K-K-L-K-K-R)

S24：His-Leu-Met-Ala-R5-Phe-Thr-S5-Ala-Lys-Lys-Leu-Lys-Lys-Arg

(H-L-M-A-R5-F-T-S5-A-K-K-L-K-K-R)

S25：His-Leu-Met-Ala-Cys-Phe-Thr-Cys-Ala-R5-Lys-Leu-S5-Lys-Arg

(H-L-M-A-C-F-T-C-A-R5-K-L-S5-K-R)

S26：R8-Leu-Met-Ala-Cys-Phe-Thr-S5-Ala-Lys-Lys-Leu-Lys-Lys-Arg

(R8-L-M-A-C-F-T-S5-A-K-K-L-K-K-R)

S27：His-Leu-Met-R8-Cys-Phe-Thr-Cys-Ala-Lys-S5-Leu-Lys-Lys-Arg

(H-L-M-R8-C-F-T-C-A-K-S5-L-K-K-R)

S28：His-Leu-Met-Ala-Cys-Phe-R8-Cys-Ala-Lys-Lys-Leu-Lys-S5-Arg

(H-L-M-A-C-F-R8-C-A-K-K-L-K-S5-R)

example 24 detection of alpha helicity of Polypeptides S23-S28 by circular dichroism

The alpha helix rate of the polypeptide was measured by circular dichroism spectroscopy (purchased from Jasco, Japan). The polypeptides MR2, S5 and S23-S28 prepared in example 1 were dissolved in PBS solution, and the on-machine concentration of the circular dichroism spectrometer was adjusted to 1mg/mL, and the results are shown in Table 19. Table 18 shows that the alpha-helix ratios of the polypeptides S5 and S23-S28 are significantly higher than that of the polypeptide MR 2. Wherein, the alpha helix ratio refers to the percentage of the number of peptide fragments of the polypeptide which maintain the alpha helix of the secondary structure to the number of peptide fragments of the total polypeptide.

TABLE 19 determination of alpha-helix rate of polypeptide by circular dichroism

Example 25 examination of the Effect of S5 and S23-S28 on the pulmonary weight index of mice with pulmonary fibrosis

Pulmonary fibrosis models were made as described in example 2 and administered in groups as per table 20. After 28 days, the lung of the mice was finely stripped and the wet weight was taken, and the lung weight (mg) was divided by the body weight (g) of the mice to obtain a lung weight index, and the results are shown in FIG. 20 and Table 21. As can be seen from fig. 20, the lung weight index of mice was significantly increased after administration of bleomycin compared to the sham group. After being administrated, the S5 and S23-S28 can obviously reduce the lung weight index of the fibrosis mouse. In fig. 20, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

TABLE 20 grouping administration after model building of pulmonary fibrosis animal model

TABLE 21 influence of S5 and S23-S28 on pulmonary weight index in pulmonary maintenance mice

Group of	Index of lung weight
		Artificial operation group	6.0783
Bleomycin group	12.2862
		S5 administration group	8.1153
S23 administration group	8.0072
		S24 administration group	8.6614
S25 administration group	9.1148
		S26 administration group	7.8693
S27 administration group	8.8105
		S28 administration group	8.3711

Example 26 the effect of S5 and S23-S28 on the pathomorphology of bleomycin-induced pulmonary fibrosis was determined.

The results are shown in FIG. 21 and Table 22. The results show that significant inflammation of the lungs of mice occurred after bleomycin administration compared to sham operated groups. The administration of both S5 and S23-S28 can significantly reduce pulmonary inflammation caused by bleomycin. In fig. 21, # # is p <0.01 compared to the sham group, p <0.05 compared to the bleomycin group, and p <0.01 compared to the bleomycin group.

Table 22S 5 and S23-S28 inflammatory scores for bleomycin-induced pulmonary fibrosis

Group of	Inflammatory score
		Artificial operation group	0
Bleomycin group	4.333
		S5 administration group	1.667
S23 administration group	1.833
		S24 administration group	1.500
S25 administration group	1.500
		S26 administration group	1.833
S27 administration group	1.667
		S28 administration group	2.000

The results of the above examples show that the polypeptide derivative of the present invention has significant effect on pulmonary fibrosis resistance, and is superior to TRB3 and p62 interaction inhibitory peptide. Meanwhile, the polypeptide of the invention can also be used as an active ingredient for preparing anti-pulmonary fibrosis drugs.

Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely examples and that many variations or modifications may be made to these embodiments without departing from the principles and spirit of the invention, and equivalents may fall within the scope of the invention as defined by the appended claims.

Sequence listing

<110> Huzhuowei

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

<130> P1711110C

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S1

<400> 1

S5 Leu Met Ala S5 Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S2

<400> 2

His S5 Met Ala Cys S5 Thr Cys Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S3

<400> 3

His Leu S5 Ala Cys Phe S5 Cys Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 4

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S4

<400> 4

His Leu Met S5 Cys Phe Thr S5 Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 5

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S5

<400> 5

His Leu Met Ala S5 Phe Thr Cys S5 Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 6

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S6

<400> 6

His Leu Met Ala Cys S5 Thr Cys Ala S5 Lys Leu Lys Lys Arg

1 5 10 15

<210> 7

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S7

<400> 7

His Leu Met Ala Cys Phe S5 Cys Ala Lys S5 Leu Lys Lys Arg

1 5 10 15

<210> 8

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S8

<400> 8

His Leu Met Ala Cys Phe Thr S5 Ala Lys Lys S5 Lys Lys Arg

1 5 10 15

<210> 9

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S9

<400> 9

His Leu Met Ala Cys Phe Thr Cys S5 Lys Lys Leu S5 Lys Arg

1 5 10 15

<210> 10

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S10

<400> 10

His Leu Met Ala Cys Phe Thr Cys Ala S5 Lys Leu Lys S5 Arg

1 5 10 15

<210> 11

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S11

<400> 11

His Leu Met Ala Cys Phe Thr Cys Ala Lys S5 Leu Lys Lys S5

1 5 10 15

<210> 12

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> MR2 polypeptide

<400> 12

His Leu Met Ala Cys Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 13

<211> 11

<212> PRT

<213> Human immunodeficiency virus type 1

<220>

<223> TAT peptide (S13)

<400> 13

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210> 14

<211> 16

<212> PRT

<213> Drosophila melanogaster

<220>

<223> Antp peptide (S14)

<400> 14

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

1 5 10 15

<210> 15

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Pep-1 peptide (S15)

<400> 15

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> 16

<211> 27

<212> PRT

<213> Human immunodeficiency virus

<220>

<223> MPG peptide (S16)

<400> 16

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> 17

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> RGD peptide (S17)

<400> 17

Arg Gly Asp

1

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Pep2 peptide (S18)

<400> 18

His Leu Tyr Val Ser Pro Trp

1 5

<210> 19

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> chimeric polypeptide Pep2-MR2 (S19)

<400> 19

His Leu Tyr Val Ser Pro Trp Gly Gly His Leu Met Ala Cys Phe Thr

1 5 10 15

Cys Ala Lys Lys Leu Lys Lys Arg

20

<210> 20

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> chimeric polypeptide TAT-MR2 (S20)

<400> 20

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly His Leu Met

1 5 10 15

Ala Cys Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg

20 25

<210> 21

<211> 33

<212> PRT

<213> Artificial sequence

<220>

<223> chimeric polypeptide Antp-MR2 (S21)

<400> 21

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

1 5 10 15

Gly Gly His Leu Met Ala Cys Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg

20 25 30

<210> 22

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Pep-A2(S22)

<400> 22

His Leu Tyr Val Ser Pro Trp Gly Gly Gly Gly Trp Leu Thr Arg Leu

1 5 10 15

Gln Thr Lys

<210> 23

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S23

<400> 23

R5 Leu Met S5 Cys Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 24

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S24

<400> 24

His Leu Met Ala R5 Phe Thr S5 Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 25

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S25

<400> 25

His Leu Met Ala Cys Phe Thr Cys Ala R5 Lys Leu S5 Lys Arg

1 5 10 15

<210> 26

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S26

<400> 26

R8 Leu Met Ala Cys Phe Thr S5 Ala Lys Lys Leu Lys Lys Arg

1 5 10 15

<210> 27

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S27

<400> 27

His Leu Met R8 Cys Phe Thr Cys Ala Lys S5 Leu Lys Lys Arg

1 5 10 15

<210> 28

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> S28

<400> 28

His Leu Met Ala Cys Phe R8 Cys Ala Lys Lys Leu Lys S5 Arg

1 5 10 15

Claims

1. A polypeptide specifically binding to TRB3 is characterized in that the amino acid sequence of the polypeptide is shown in any one of sequence tables SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10 or SEQ ID No. 11.

2. Use of a polypeptide that specifically binds TRB3 according to claim 1 in the manufacture of a medicament for the treatment and/or prevention of pulmonary fibrosis.

3. The use of claim 2, wherein the pulmonary fibrosis is primary pulmonary fibrosis or secondary pulmonary fibrosis.

4. The use of claim 2, wherein the pulmonary fibrosis is bleomycin-induced pulmonary fibrosis.

5. A pharmaceutical composition against pulmonary fibrosis comprising the polypeptide of claim 1 that specifically binds TRB 3.

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

7. The pharmaceutical composition according to claim 5 or 6, which comprises the polypeptide that specifically binds TRB3 according to claim 1 together with a compound having anti-pulmonary fibrosis activity as an active ingredient.