Structure and application of cubilose single peptide for enhancing and improving lung function and preventing pulmonary fibrosis

Abstract

The invention provides application of a cubilose-derived single peptide in preparation of a medicine. The single peptide comprises a peptide with an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% of homology thereof and / or a peptide with an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% of homology thereof; the medicine has at least one of the following purposes: enhancing and / or improving lung function; the pulmonary fibrosis is prevented and / or treated. The single peptide is derived from edible and medicinal bird's nests, is small in molecular weight and easy to absorb, and can be used for preparing medicines for enhancing or improving lung functions; the medicine and the health care product for preventing, treating or delaying pulmonary fibrosis have wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to the structure and application of a bird's nest single peptide for enhancing and improving lung function and preventing pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a chronic, progressive lung disease characterized by excessive accumulation of extracellular matrix in the damaged or inflamed tissues of the lungs, which increases fibrous connective tissue and causes permanent scarring that destroys normal structures, leading to severe lung dysfunction, decline, failure, and even death. Among them, idiopathic pulmonary fibrosis (IPF) is the most common form.

[0003] Pulmonary fibrosis cannot be cured or reversed, but drug treatment can help alleviate symptoms. At present, anti-inflammatory drugs are often used to slow the progression of pulmonary fibrosis, thereby improving the quality of life of patients, but the effect is usually limited, and there is a lack of fundamental treatment for the cause. Nintedanib and pirfenidone are two drugs used to treat idiopathic pulmonary fibrosis. They are multi-target tyrosine kinase inhibitors and anti-fibrotic drugs, respectively. They can inhibit or regulate the activity of growth factor receptors such as TGF-β1, PDGF and FGF, inhibit the proliferation of fibroblasts and the synthesis of extracellular matrix, thereby achieving anti-inflammatory and anti-fibrotic effects. However, nintedanib and pirfenidone cannot cure idiopathic pulmonary fibrosis, and the use of the above drugs is accompanied by common side effects, including gastrointestinal symptoms, rash and photosensitivity.

[0004] Bird's nest peptide is a hydrolysis product generated by protease hydrolysis of bird's nest. Bird's nest, the source of which, is a natural food. It is recorded in the Qing Dynasty's "Supplement to Compendium of Materia Medica" that bird's nest nourishes lung yin, resolves phlegm and stops coughing, and is a holy medicine for regulating deficiency and fatigue. Related literature also shows that bird's nest can relieve the symptoms of a variety of lung diseases, including: tuberculosis, asthma, dry cough, hemoptysis, dyspnea, general weakness caused by bronchial diseases, etc.

[0005] Therefore, screening out single bird's nest peptides that have the ability to resist pulmonary fibrosis and enhance lung function is of great significance for the application of bird's nest products in the development of medicines, health products or functional foods. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides the structure and application of bird's nest single peptides for enhancing and improving lung function and preventing pulmonary fibrosis. The single peptide of the present invention is derived from bird's nest, which is both a medicine and food, has a small molecular weight and is easily absorbed. It can be used to prepare medicines and health products that enhance or improve lung function; prevent, treat or delay pulmonary fibrosis, and has broad application prospects.

[0007] In the first aspect of the present invention, the present invention proposes a use of a single peptide in the preparation of a drug. According to an embodiment of the present invention, the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof; the drug has at least one of the following uses: enhancing and / or improving lung function; preventing and / or treating pulmonary fibrosis. The single peptide of the present invention is derived from the bird's nest, which is both a medicine and a food, has a small molecular weight and is easily absorbed. It can be used to prepare medicines and health products that enhance or improve lung function; prevent, treat or delay pulmonary fibrosis, and has broad application prospects.

[0008] According to an embodiment of the present invention, the use of the above-mentioned single peptide in the preparation of a drug may also have the following additional technical features:

[0009] According to an embodiment of the present invention, the single peptide is derived from bird's nest.

[0010] According to an embodiment of the present invention, the pulmonary fibrosis includes but is not limited to: interstitial lung disease.

[0011] According to a specific embodiment of the present invention, the interstitial lung disease includes: chronic interstitial lung disease.

[0012] According to a more specific embodiment of the present invention, the chronic interstitial lung disease includes but is not limited to: at least one of idiopathic pulmonary fibrosis, systemic sclerosis-related interstitial lung disease, pulmonary lymphangioleiomyomatosis, sarcoidosis, progressive fibrosing interstitial lung disease, and α1-antitrypsin deficiency.

[0013] In the second aspect of the present invention, the present invention proposes a method for inhibiting the expression of transforming growth factor β1. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, and the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, the expression of the key pro-fibrotic cytokine TGF-β1 is inhibited by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, which helps to prevent or treat pulmonary fibrosis in the body; it can also be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc.

[0014] In the third aspect of the present invention, the present invention proposes a method for promoting the expression of E-cadherin. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, wherein the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, by co-culturing a cell sample or a tissue sample with a bird's nest single peptide of the present invention, the expression of E-cadherin is promoted, the characteristics of epithelial cells are maintained, and the epithelial-mesenchymal transition process is inhibited. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, helping to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for the treatment of pulmonary fibrosis, etc.

[0015] In the fourth aspect of the present invention, the present invention proposes a method for inhibiting the expression of neural cadherin. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, wherein the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention, the expression of neural cadherin is inhibited, thereby inhibiting epithelial-mesenchymal transition. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, helping to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc.

[0016] In the fifth aspect of the present invention, the present invention proposes a method for inhibiting the expression of type I collagen α1 chain. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, and the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, the expression of type I collagen α1 chain is inhibited by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, helping to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc.

[0017] In the sixth aspect of the present invention, the present invention proposes a method for inhibiting epithelial-mesenchymal transition. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, wherein the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the single peptide of the embodiment of the present invention, by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention, the expression level of E-cadherin is promoted, and the expression levels of neural cadherin, type I collagen α1 chain and transforming growth factor TGF-β1 are inhibited, thereby inhibiting epithelial-mesenchymal transition. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, which helps to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0019] Figure 1 This is a molecular weight distribution diagram of the bird's nest digest in Example 1 of the present invention;

[0020] Figure 2 Schematic diagrams of computer simulation molecular docking results of bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF with TGF-β1 and TβR2, respectively, in Example 2 of the present invention, wherein (A) is a schematic diagram of computer simulation molecular docking results of TGF-β1 and TβR2, (B) is a schematic diagram of computer simulation molecular docking results of bird's nest peptide 1-LAGAAHF with TGF-β1, (C) is a schematic diagram of computer simulation molecular docking results of bird's nest peptide 1-LAGAAHF with TβR2, (D) is a schematic diagram of computer simulation molecular docking results of bird's nest peptide 2-PTYGRTF with TGF-β1, and (E) is a schematic diagram of computer simulation molecular docking results of bird's nest peptide 2-PTYGRTF with TβR2;

[0021] Figure 3 The test results of the effects of different concentrations of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF on the cell viability of A549 cells in Example 3 of the present invention, wherein the concentrations of bird's nest extract and bird's nest digest are 1, 10, and 100 μg / mL, and the concentrations of bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF are 0.1, 1, and 10 μg / mL;

[0022] Figure 4This is a graph showing the transcription results of the TGF-β1 gene by the same concentrations of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF in Example 4 of the present invention, wherein the concentration is 10 μg / mL;

[0023] Figure 5 This is a graph showing the transcription results of the E-cadherin gene by the same concentrations of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF in Example 4 of the present invention, wherein the concentration is 10 μg / mL;

[0024] Figure 6 This is a graph showing the transcription results of the N-cadherin gene by the same concentrations of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF in Example 4 of the present invention, wherein the concentration is 10 μg / mL;

[0025] Figure 7 This is a graph showing the transcription results of the COL1A1 gene by the same concentrations of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF in Example 4 of the present invention, wherein the concentration is 10 μg / mL. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0027] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0028] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0029] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0030] Terms and Definitions

[0031] In this article, the term "EMT" refers to epithelial-mesenchymal transition, which is a process in which epithelial cells are transformed into mesenchymal cells through a specific program and is related to tissue fibrosis.

[0032] In this article, the term "TGF-β1" refers to transforming growth factor β1, which plays a key role in tissue repair, immune regulation and fibrosis, and can induce epithelial-mesenchymal transition (EMT) and promote collagen synthesis.

[0033] In this article, the term "E-cadherin" refers to E-cadherin, which is mainly expressed in epithelial cells. It maintains the polarity and tissue structure of epithelial cells by forming intercellular adhesion connections. The downregulation of its expression is usually associated with the EMT process, resulting in reduced intercellular adhesion, thereby enhancing the migration and invasion ability of cells.

[0034] In this article, the term "N-cadherin" refers to neural cadherin, which is mainly expressed in mesenchymal cells and plays an important role in cell-to-cell adhesion and signal transduction. During the EMT process, the expression of N-cadherin is upregulated, replacing E-cadherin, thereby enhancing the migration and invasion ability of cells.

[0035] As used herein, the term "COL1A1" refers to the gene encoding the α1 chain of type I collagen in humans. Type I collagen is the most abundant type of collagen in the body, and increased expression of COL1A1 is often associated with tissue fibrosis and repair processes.

[0036] In this article, the term "IM412 drug" refers to a small molecule compound drug that has anti-fibrosis and inhibitory effects on epithelial-mesenchymal transition (EMT). It works by inhibiting the TGF-β1 signaling pathway, reducing the expression of TGF-β1-induced fibrosis markers to inhibit cell migration and invasion, and is often used to treat pathological fibrotic conditions.

[0037] In this article, the term "bird's nest single peptide" refers to the bird's nest small molecule protein peptide with a smaller molecular weight obtained by treating the protein macromolecules in the bird's nest with enzymatic technology, breaking them into short peptide chains, and finally extracting and separating them.

[0038] In this article, combined with the above context, "bird's nest single peptide" is equivalent to "bird's nest peptide".

[0039] The present invention proposes the use of a single peptide in the preparation of a drug, a method for promoting the expression of E-cadherin, a method for inhibiting the expression of transforming growth factor β1, neural cadherin, and type I collagen α1 chain, and a method for inhibiting epithelial-mesenchymal transition, which will be described in detail below.

[0040] use

[0041] The present invention proposes a use of a single peptide in the preparation of a drug. According to an embodiment of the present invention, the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereto; the drug has at least one of the following uses: enhancing and / or improving lung function; preventing and / or treating pulmonary fibrosis. The single peptide according to an embodiment of the present invention is derived from bird's nest, which is both a medicine and a food, has a small molecular weight and is easily absorbed, and can be used to prepare medicines and health products that enhance and / or improve lung function; prevent and / or treat and / or delay pulmonary fibrosis, and has broad application prospects.

[0042] According to an embodiment of the present invention, the single peptide is derived from bird's nest. According to the single peptide of the embodiment of the present invention, a new development path for bird's nest products is opened up, the economic value of bird's nest is increased, and the application of bird's nest products in multiple fields is promoted.

[0043] According to an embodiment of the present invention, the pulmonary fibrosis includes but is not limited to: interstitial lung disease.

[0044] According to a specific embodiment of the present invention, the interstitial lung disease includes: chronic interstitial lung disease.

[0045] According to a more specific embodiment of the present invention, the chronic interstitial lung disease includes but is not limited to: idiopathic pulmonary fibrosis, systemic sclerosis-related interstitial lung disease, pulmonary lymphangioleiomyomatosis, sarcoidosis, progressive fibrosing interstitial lung disease, at least one of α1-antitrypsin deficiency. The single peptide according to the embodiment of the present invention is derived from the bird's nest, which is both a medicine and a food. It is green and safe and can be widely used in the development of drugs and health products to improve lung function and prevent or treat various pulmonary fibrosis.

[0046] Method for inhibiting the expression of transforming growth factor β1

[0047] The present invention proposes a method for inhibiting the expression of transforming growth factor β1. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, wherein the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, the expression of the key pro-fibrotic cytokine TGF-β1 is inhibited by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, helping to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc.

[0048] Methods for promoting expression of E-cadherin

[0049] The present invention proposes a method for promoting the expression of E-cadherin. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, wherein the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention, the expression of E-cadherin is promoted, the characteristics of epithelial cells are maintained, and the epithelial-mesenchymal transition process is inhibited. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, helping to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for the treatment of pulmonary fibrosis, etc.

[0050] Methods for inhibiting the expression of neural cadherin

[0051] The present invention proposes a method for inhibiting the expression of neural cadherin. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, wherein the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention, the expression of neural cadherin is inhibited, thereby inhibiting epithelial-mesenchymal transition. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, helping to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc.

[0052] Method for inhibiting the expression of type I collagen alpha 1 chain

[0053] The present invention proposes a method for inhibiting the expression of type I collagen α1 chain. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, and the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, the expression of type I collagen α1 chain is inhibited by co-culturing a cell sample or a tissue sample with the bird's nest single peptide of the present invention. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, helping to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc.

[0054] Methods to inhibit epithelial-mesenchymal transition

[0055] The present invention proposes a method for inhibiting epithelial-mesenchymal transition. According to an embodiment of the present invention, it includes: co-culturing a cell sample or a tissue sample with a single peptide, wherein the single peptide includes: a peptide having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereof and / or a peptide having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereof. According to the method of an embodiment of the present invention, by co-culturing a cell sample or a tissue sample with a bird's nest single peptide of the present invention, the expression level of E-cadherin is promoted, and the expression levels of neural cadherin, type I collagen α1 chain and transforming growth factor TGF-β1 are inhibited, thereby inhibiting epithelial-mesenchymal transition. Exemplarily, the method can be performed in vitro, and can be for therapeutic purposes, which helps to prevent or treat pulmonary fibrosis in the body, or can be for non-therapeutic purposes, such as studying the mechanism of pulmonary fibrosis, screening drugs for treating pulmonary fibrosis, etc.

[0056] The amino acid sequences involved in the present invention are shown in Table 1.

[0057] Table 1

[0058] SEQ ID NO: Amino acid sequence Remark 1 LAGAHF <![CDATA[Edible bird's nest peptide EBN P3 The amino acid sequence of which is denoted as edible bird's nest peptide 1 - LAGAAHF]]> 2 PTYGRTF <![CDATA[Edible bird's nest peptide EBN P4 The amino acid sequence of which is denoted as edible bird's nest peptide 2 - PTYGRTF]]>

[0059] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0060] Example 1: Extraction and purification of bird's nest peptide

[0061] Pretreatment: Weigh a certain amount of dry bird's nest sample and weigh it, add water according to the weight ratio of dry bird's nest sample: water = 1:100, soak it at room temperature overnight, filter it, and obtain the bird's nest soaked material; cut it into pieces, mix it evenly, and dry the bird's nest soaked material to obtain dry bird's nest.

[0062] Preparation of bird's nest extract: weigh the dry weight of the above-mentioned dry bird's nest, add water according to the weight ratio of dry bird's nest: water = 1:30, stew for 8 hours, vacuum filter to obtain bird's nest extract; freeze-dry to obtain a white bird's nest extract.

[0063] Preparation of bird's nest digest: weigh the above-mentioned bird's nest extract, add pepsin at a weight ratio of bird's nest extract: pepsin = 1:1, and adjust its pH value to 3.0 with 6M hydrochloric acid to simulate the gastric digestion environment, hydrolyze for 8 hours, freeze-dry, and obtain bird's nest digest.

[0064] Chromatographic system: using Superdex TM The bird's nest digest was separated into bird's nest proteins according to their molecular weight using a 75Increase column. A liquid chromatograph equipped with a diode array detector was used with detection wavelengths of 214nm and 280nm; a dextran gel column of 10×300mm; and a flow rate of about 0.4mL / min to obtain bird's nest peptides. The elution process is shown in Table 2.

[0065] Table 2: Elution process

[0066] Time (minutes) Normal saline (%, v / v) Elution method 0-90 100 Isocratic

[0067] Determination of bird's nest peptide sequence: The bird's nest peptide obtained in the above steps was labeled with TMT10plex and analyzed by Orbitrap mass spectrometry. The bird's nest peptide sequence was determined using the Chaetura pelagica, Aerodramus and Apodidae databases in NCBI protein.

[0068] The molecular weight distribution of the bird's nest digest in Example 1 is shown in Figure 1 .

[0069] The results showed that two bird's nest peptide sequences were obtained, both consisting of 7 amino acids, namely bird's nest peptide 1 (EBN p3 ): LAGAAHF; bird's nest peptide 2 (EBN p4 ):PTYGRTF.

[0070] Example 2: Analysis of the affinity of bird's nest peptides for TGF-β1 and TβR2 using computer simulation molecular docking technology

[0071] The inventors used computer simulation molecular docking technology to perform affinity analysis on the docking of the two bird's nest peptides 1-LAGAAHF and bird's nest peptide 2-PTYGRTF obtained in Example 1 with transforming growth factor-β1 (Transforming Growth Factor Beta 1, TGF-β1) and transforming growth factor-β type II receptor (Transforming Growth Factor Beta Type 2 Receptor, TβR2), respectively.

[0072] The computer simulation molecular docking results of bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF with TGF-β1 and TβR2 in Example 2 are shown in FIG. Figure 2 The computer simulation predicted molecular docking analysis results of bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF with TGF-β1 and TβR2 are shown in Table 3.

[0073] Table 3 Computer simulation prediction molecular docking analysis results of bird's nest peptide 1-LAGAAHF, bird's nest peptide 2-PTYGRTF with TGF-β1 and TβR2 respectively

[0074]

[0075]

[0076] The results showed that the docking of TGF-β1 and TβR2 was a positive control, and bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF had strong affinity with TGF-β1 and TβR2.

[0077] The above results show that: from the protein structure level, it is proved that bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF can interfere with TGF-β1-induced epithelial-mesenchymal transition by regulating the TGF-β1 signaling pathway. It is speculated that bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF have the effect of enhancing lung function and preventing or treating pulmonary fibrosis.

[0078] Example 3: Cytotoxicity test

[0079] In order to evaluate the cytotoxicity of the bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF samples obtained in Example 1, as well as the cytotoxicity of bird's nest extracts and bird's nest digests, the inventors conducted cytotoxicity tests on human non-small cell lung cancer A549 cells to screen and confirm the safe concentration without cytotoxicity and ensure the smooth progress of subsequent experiments.

[0080] The MTT method was used to detect the effects of bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF on the survival rate of A549 cells. The investigation method is as follows:

[0081] A549 cells were cultured in DMEM medium containing 10% (v / v) FBS (fetal bovine serum) and 1% (w / v) P / S (penicillin / streptomycin). The adherent cells at the bottom of the culture dish were digested with 0.25% (w / v) trypsin solution to prepare a cell suspension, which was counted and diluted to a cell concentration of 1×10 5 The suspension was inoculated into a 96-well plate and placed in a CO2 incubator (5% CO2, 37°C) for 24 hours.

[0082] Different concentrations of bird's nest extract (1, 10, 100 μg / mL), bird's nest digest (1, 10, 100 μg / mL), bird's nest peptide 1-LAGAAHF (0.1, 1, 10 μg / mL) and bird's nest peptide 2-PTYGRTF (0.1, 1, 10 μg / mL) were added in a gradient, and after 48 hours of continuous culture, the medium in the 96-well plate was removed, and 0.5 mg / mL of MTT solution was added, and the plate was placed in an incubator for 2 hours. DMSO solution was added and shaken on a shaker for 15 minutes, and then the absorbance value was measured at 570 nm using an enzyme marker.

[0083] The test results of the effects of different concentrations of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF on the cell viability of A549 cells in Example 3 are shown in Figure 3 .

[0084] The results showed that A549 cells did not show a decrease in the number of A549 cells when the concentrations of bird's nest extract and bird's nest digest were 1, 10, and 100 μg / mL, and the concentrations of bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF were 0.1, 1, and 10 μg / mL.

[0085] The above results indicate that the bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF in the present invention will not induce A549 cell death within a concentration range of ≤10 μg / mL, and their safety is reliable.

[0086] Example 4: Effect of bird's nest peptide on epithelial-mesenchymal transition induced by TGF-β1

[0087] Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial rare lung disease involving the failure of alveolar epithelial repair, resulting in excessive secretion of a variety of cytokines and growth factors that promote the differentiation of myofibroblasts. At the same time, these myofibroblasts secrete a large amount of extracellular matrix (ECM), such as collagen, which acts on fibrotic tissue remodeling in pulmonary fibrosis. Among them, transforming growth factor β1 (TGF-β1), epithelial cadherin (E-cadherin), neural cadherin (N-cadherin) and type I collagen α1 chain (COL1A1) are all involved in the process of epithelial-mesenchymal transition (EMT). By measuring the changes in their transcriptional activity levels, the effect of bird's nest peptides on TGF-β1-induced epithelial-mesenchymal transition can be evaluated. The specific investigation methods are as follows:

[0088] 1. Cell culture

[0089] (1) Bird's nest extract group, bird's nest digest group, bird's nest peptide 1-LAGAAHF group, and bird's nest peptide 2-PTYGRTF group: A549 cells were prepared at a cell concentration of 1×10 5 The cells were suspended in 12-well plates and placed in CO 2 The cells were cultured in an incubator (5% CO2, 37°C) for 24 hours, and then 10 μg / mL of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF were added. After 2 hours of culture, 5 ng / mL of exogenous TGF-β1 was added and cultured for another 24 hours.

[0090] (2) Blank control group: A549 cells were prepared at a cell concentration of 1×10 5 The cells were suspended in 12-well plates and placed in CO 2 The cells were cultured in an incubator (5% CO2, 37°C) for 24 hours, and then the culture medium was replaced with new medium and cultured for another 24 hours.

[0091] (3) IM412 drug control group: A549 cells were made into a cell concentration of 1×10 5 The cells were suspended in a 12-well plate at a concentration of 100 μg / mL and inoculated into a CO2 incubator (5% CO2, 37°C) for 24 hours. IM412 was then added and cultured for 2 hours. 5 ng / mL of exogenous TGF-β1 was then added and cultured for another 24 hours.

[0092] (4) Model group: A549 cells were cultured at a cell concentration of 1×10 5 The cells were suspended in a 12-well plate at a concentration of 100 μg / mL and inoculated into the plate. After culturing in a CO2 incubator (5% CO2, 37°C) for 26 hours, 5 ng / mL of exogenous TGF-β1 was added and cultured for another 24 hours.

[0093] 2. Total RNA extraction

[0094] After 24 hours of incubation, use RT RNA Total RNA was extracted from A549 cells cultured under the above conditions, and DNase I was used to remove the contaminated DNA. The quality and quantity of total RNA were determined by A260 / A280 and A260 / A230 measurements (the A260 / A280 ratio needs to be greater than 1.8, indicating that the extracted RNA sample is relatively pure). According to the experimental manual, Prime-Script TM 500 ng of RNA sample was reverse transcribed into cDNA using RT kit (Takara).

[0095] 3. Design primer sequences

[0096] The primer nucleotide sequences involved are designed as follows:

[0097] E-cadherin F-Forward Primer: 5'-GCC TCC TGAAAA GAG AGT GGAAG-3'

[0098] E-cadherin R-Reverse Primer: 5'-TGG CAG TGT CTC TCC AAA TCC G-3'

[0099] N-cadherin F-Forward Primer: 5'-CCT CCA GAG TTT ACT GCC ATG AC-3'

[0100] N-cadherin R - reverse primer: 5'-GTA GGA TCT CCG CCA CTG ATT C-3'

[0101] COL1A1 F-Forward Primer: 5'-TCT GCG ACA ACG GCA AGG TG-3'

[0102] COL1A1 R - Reverse Primer: 5'-GAC GCC GGT GGT TTC TTG GT-3'

[0103] TGF-β1F-Forward Primer: 5'-TAC CTG AAC CCG TGT TGC TCT C-3'

[0104] TGF-β1R - reverse primer: 5'-GTT GCT GAG GTA TCG CCA GGA A-3'

[0105] GAPDH F-Forward Primer: 5'-ACA ACT TTG GTA TCG TGG AAG G-3'

[0106] GAPDH R - Reverse Primer: 5'-GCC ATC ACG CCA CAG TTTC-3'

[0107] 4. Real time PCR

[0108] pass 480 was used for relative gene quantification to detect the expression of TGF-β1, E-cadherin, N-cadherin and COL1A1 genes. Amplification was performed for 45 cycles: each cycle consisted of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 20 seconds. The 3-phosphoglyceraldehyde dehydrogenase (GAPDH) gene was used as an internal reference. -△△CT Methods Results were calculated to determine mRNA levels.

[0109] (1) TGF-β1 gene transcription results

[0110] The results of the same concentrations of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF on TGF-β1 gene transcription in Example 4 are shown in Figure 4 .

[0111] The results showed that compared with the A549 cells in the blank control group and the IM412 drug control group, the mRNA expression level of TGF-β1 in the A549 cells in the TGF-β1 model group was significantly increased; compared with the A549 cells in the model group, the mRNA expression level of TGF-β1 in the A549 cells in the bird's nest extract group, bird's nest digest group, bird's nest peptide 1-LAGAAHF group and bird's nest peptide 2-PTYGRTF group was decreased.

[0112] The above results show that bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF can all inhibit the expression of TGF-β1 in A549 cells, among which the inhibitory effects of bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF are stronger than those of bird's nest extract and bird's nest digest.

[0113] (2) E-cadherin gene transcription results

[0114] The results of the same concentration of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF on E-cadherin gene transcription are shown in Figure 5 .

[0115] The results showed that compared with the A549 cells in the blank control group and the IM412 drug control group, the mRNA expression level of E-cadherin induced by TGF-β1 in the A549 cells of the E-cadherin model group was significantly decreased; compared with the A549 cells in the model group, the mRNA expression levels of E-cadherin induced by TGF-β1 in the A549 cells of the bird's nest extract group, bird's nest digest group, bird's nest peptide 1-LAGAAHF group and bird's nest peptide 2-PTYGRTF group were all increased.

[0116] The above results indicate that bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF can all promote the expression of E-cadherin induced by TGF-β1 in A549 cells, among which bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF are more effective in promoting the expression of E-cadherin induced by TGF-β1 than bird's nest extract and bird's nest digest.

[0117] (3) N-cadherin gene transcription results

[0118] The results of the same concentration of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF on N-cadherin gene transcription are shown in Figure 6 .

[0119] The results showed that compared with the A549 cells in the blank control group and the IM412 drug control group, the expression level of TGF-β1-induced N-cadherin in the A549 cells in the model group was significantly increased; compared with the A549 cells in the model group, the mRNA expression levels of TGF-β1-induced N-cadherin in the A549 cells in the bird's nest extract group, bird's nest digest group, bird's nest peptide 1-LAGAAHF group and bird's nest peptide 2-PTYGRTF group were all decreased.

[0120] The above results show that bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF can all inhibit the expression of N-cadherin induced by TGF-β1, among which bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF have stronger effects on inhibiting the expression of N-cadherin induced by TGF-β1 than bird's nest extract and bird's nest digest.

[0121] (4) COL1A1 gene transcription results

[0122] The results of the same concentration of bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF on COL1A1 gene transcription are shown in Figure 7 .

[0123] The results showed that compared with the A549 cells in the blank control group and the IM412 drug control group, the TGF-β1-induced COL1A1 expression level in the A549 cells in the model group was significantly increased; compared with the A549 cells in the model group, the TGF-β1-induced COL1A1 mRNA expression level in the A549 cells in the bird's nest extract group, bird's nest digest group, bird's nest peptide 1-LAGAAHF group and bird's nest peptide 2-PTYGRTF group was decreased.

[0124] The above results show that bird's nest extract, bird's nest digest, bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF can all inhibit the expression of COL1A1 induced by TGF-β1, among which bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF have stronger ability to inhibit the expression of COL1A1 induced by TGF-β1 than bird's nest extract and bird's nest digest.

[0125] Both bird's nest peptide 1-LAGAAHF and bird's nest peptide 2-PTYGRTF showed the ability to counteract the gene expression of epithelial-mesenchymal transition strongly induced by TGF-β1. They can upregulate the E-cadherin gene expression of A549 lung cells, inhibit the mRNA transcription levels of N-cadherin, collagen COL1A1 and transforming growth factor TGF-β1, thereby inhibiting epithelial-mesenchymal transition. In theory, they can be used to prevent the treatment of idiopathic pulmonary fibrosis (IPF), and to develop drugs, health products or foods for improving and enhancing lung function.

[0126] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of a single peptide in preparing a drug, characterized in that: The single peptide includes: a peptide having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence at least 80% homologous thereto and / or a peptide having an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence at least 80% homologous thereto; The drug has at least one of the following uses: Enhance and / or improve lung function; Prevention and / or treatment of pulmonary fibrosis.

2. The use according to claim 1, characterized in that The single peptide is derived from bird's nest.

3. The use according to claim 1, characterized in that The pulmonary fibrosis includes interstitial lung disease; Optionally, the interstitial lung disease comprises: chronic interstitial lung disease; Optionally, the chronic interstitial lung disease comprises at least one of idiopathic pulmonary fibrosis, systemic sclerosis-associated interstitial lung disease, pulmonary lymphangioleiomyomatosis, sarcoidosis, progressive fibrosing interstitial lung disease, and α1-antitrypsin deficiency.

4. A method for inhibiting the expression of transforming growth factor β1, characterized in that: include: The cell sample or tissue sample is co-cultured with a single peptide, wherein the single peptide comprises: a peptide having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto and / or a peptide having an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereto.

5. A method for promoting the expression of E-cadherin, characterized in that: include: The cell sample or tissue sample is co-cultured with a single peptide, wherein the single peptide comprises: a peptide having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto and / or a peptide having an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereto.

6. A method for inhibiting the expression of neural cadherin, characterized in that: include: The cell sample or tissue sample is co-cultured with a single peptide, wherein the single peptide comprises: a peptide having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto and / or a peptide having an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereto.

7. A method for inhibiting the expression of type I collagen α1 chain, characterized in that: include: The cell sample or tissue sample is co-cultured with a single peptide, wherein the single peptide comprises: a peptide having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto and / or a peptide having an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereto.

8. A method for inhibiting epithelial-mesenchymal transition, characterized in that: include: The cell sample or tissue sample is co-cultured with a single peptide, wherein the single peptide comprises: a peptide having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto and / or a peptide having an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence with at least 80% homology thereto.

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