Peptides having anti-inflammatory and anti-fibrotic activities and uses thereof

Abstract

The peptide of the present invention has an activity of suppressing the expression of inflammatory cytokine genes, inflammatory cytokine proteins, or proteins involved in inflammatory cytokine signaling in lung cells where an inflammatory response is induced. The peptide can also reduce the number of neutrophils in lung tissue where an inflammatory response is induced. Therefore, the peptide of the present invention may be used as an active substance useful for treating, preventing, or improving pulmonary inflammatory diseases.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS]

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0040544, filed on March 31, 2022, and all contents disclosed in the documents of the Korean patent application are incorporated herein by reference.

[0003] [Technical field]

[0004] The present invention relates to a peptide having anti-inflammatory and anti-fibrotic activity and its use. More specifically, the present invention relates to a peptide having activity for treating, preventing, or ameliorating pulmonary inflammatory disease or pulmonary fibrosis and its use. [Background technology]

[0005] The human lungs, which account for 6% of the body volume, are composed of numerous small gas sacs called alveoli. The primary purpose of the lungs is to facilitate the exchange of gases with the systemic circulatory system. When the lungs are stimulated by any cause and an inflammatory response is induced, cytokines and growth factors are secreted from immune cells in the lungs, such as neutrophils, macrophages, neutrophilic leukocytes, mast cells, and various lung cells. Inflammatory cytokines are known to be important mediators of inflammatory lung diseases, and pulmonary epithelial cells play an important role in the cytokine network at the local inflammatory site by producing and secreting interleukins, chemokines, colony stimulating factors, and growth factors in response to cytokines secreted by inflammatory cells.

[0006] Asthma and chronic obstructive pulmonary disease (COPD), which are representative lung inflammatory diseases, are both induced by chronic airway inflammation and are very common worldwide, with their prevalence increasing rapidly. In asthma patients, eosinophils, mast cells and CD4+T cells invade the mucosa and submucosa layers of the airways, and these cells produce and secrete IL-4, IL-5 and IL-13. It is also known that pulmonary fibrosis is caused by an inflammatory response in the lungs. Most organs undergo inflammation and healing processes after tissue damage, so if the damage is minor, normal structure and function are maintained, but if there is continuous damage, the tissue undergoes a process of fibrosis during the healing process.

[0007] Currently, corticosteroids are the most commonly used anti-inflammatory drugs for reducing inflammation, and other drugs targeting immune responses and cytokines are being developed to activate Th1 responses or suppress Th2 responses, as well as cytokine inhibitors such as anti-IL-2, anti-IL-4, anti-IL-5, anti-IL-9, anti-IL-13, anti-PGD2, anti-TNF-α, mast cell inhibitors, PPAR-γ promoters, anti-IgE, and anti-CD23. However, these drugs have side effects in actual clinical practice or are only weakly effective, so there is a pressing need to develop new drugs that are safer, more effective, and more cost-effective. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2011 / 029931 Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have conducted research and made efforts to develop a peptide having anti-inflammatory activity, particularly activity capable of suppressing inflammation in the lungs and suppressing the progression to pulmonary fibrosis. As a result, they have demonstrated through lung cell and animal experiments that the peptide of the present invention significantly alleviates inflammatory reactions in the lungs and effectively suppresses the progression to pulmonary fibrosis, thereby completing the present invention.

[0010] It is therefore an object of the present invention to provide a pharmaceutical composition for the treatment or prevention of pulmonary inflammatory diseases.

[0011] Another object of the present invention is to provide a functional food composition for improving or preventing pulmonary inflammation.

[0012] It is yet another object of the present invention to provide a functional food composition for improving or preventing pulmonary fibrosis. [Means for solving the problem]

[0013] To achieve the above object, one aspect of the present invention provides a pharmaceutical composition for treating or preventing a pulmonary inflammatory disease, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0014] Another aspect of the present invention provides a functional food composition for improving or preventing pulmonary inflammation, comprising a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0015] Another aspect of the present invention provides a functional food composition for improving or preventing pulmonary fibrosis, comprising a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0016] The present invention will be described in detail below.

[0017] 1. Peptides and their activities

[0018] According to one aspect of the present invention there is provided a peptide comprising the amino acid sequence disclosed in SEQ ID NO:1.

[0019] As used herein, the term "peptide" refers to a linear molecule formed by the joining of amino acid residues to one another by peptide bonds.

[0020] The peptide of the present invention comprising the amino acid sequence of SEQ ID NO:1 may be used without modification, but amino acid mutants or fragments having a different sequence due to deletion, insertion, substitution, or a combination of amino acid residues may be used, as long as the original activity of the peptide, such as antidiabetic and antiobesity activity, is not affected.

[0021] The peptide of the present invention may be modified by phosphorylation, sulfuration, acylation, glycosylation, methylation, farnesylation, etc., within the range that does not change its activity.

[0022] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to the peptide comprising the amino acid sequence of SEQ ID NO: 1, and a variant or an active fragment thereof. The substantially identical amino acid sequence means an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more, respectively, with the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may additionally include a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically manufactured to increase the half-life or peptide stability.

[0023] The peptide of the present invention may be modified at the N-terminus and / or C-terminus in order to increase its activity by selecting a portion of the amino acid sequence. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" includes not only in vivo stability that protects the peptide of the present invention from attack by in vivo protease, but also storage stability (e.g., room temperature storage stability).

[0024] The N-terminal modification may be a protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and a polyethylene glycol (PEG) attached to the N-terminus of the peptide. The C-terminal modification may be a protecting group selected from the group consisting of a hydroxyl group (-OH), an amino group (-NH 2 ), azide (-NHNH 2 ) and the like may be combined, but are not limited thereto.

[0025] The peptides of the present invention can be prepared by various methods well known in the art, for example, the peptides of the present invention can be prepared by chemical synthesis methods well known in the art, in particular solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid synthesis techniques (U.S. Pat. No. 5,516,891).

[0026] The peptide of the present invention has activity of suppressing the expression of inflammatory cytokine genes, inflammatory cytokine proteins, or proteins involved in inflammatory cytokine signaling in lung cells in which an inflammatory response is induced.

[0027] In one embodiment, the inflammatory cytokine gene whose expression is inhibited may be one or more selected from the group consisting of TNFα, IL-1β, IL-6, IL-8 and COX-2 genes.

[0028] In one embodiment, the inflammatory cytokine protein or protein involved in inflammatory cytokine signaling whose expression is inhibited may be one or more selected from the group consisting of IL-1β, IL-6, COX2, IL-8, p-JNK, p-ERK, p-PI3K, p-AKT, p-ERK, and myeloperoxidase.

[0029] The peptide of the present invention suppresses the expression of fibrotic genes in lung cells in which an inflammatory response is induced.

[0030] In one embodiment, the fibrosis gene whose expression is inhibited may be one or more selected from the group consisting of α-SMA, Collagen 1A1, Fibronectin 1, and Tenascin C genes.

[0031] The peptides of the present invention are capable of reducing the number of neutrophils in lung tissue where an inflammatory response is induced.

[0032] Since the above-mentioned peptide of the present invention has the above-mentioned activity, it can exert a very excellent efficacy in treating, preventing or ameliorating pulmonary inflammatory diseases.

[0033] 2. Composition for treating or preventing pulmonary inflammatory diseases

[0034] Pharmaceutical Compositions

[0035] According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing a pulmonary inflammatory disease, comprising a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0036] As described above, the peptide of the present invention has excellent therapeutic or preventive activity against pulmonary inflammatory diseases because it inhibits the expression of inflammatory cytokine genes, proteins, or proteins involved in signal transduction in lung cells in which an inflammatory response is induced.

[0037] In one embodiment of the present invention, the pulmonary inflammatory disease may be a disease selected from the group consisting of asthma, allergic asthma, bronchitis, chronic bronchitis, acute bronchitis, bronchiolitis, diffuse panbronchiolitis, bronchiectasis, pleurisy, alveolitis, vasculitis, pneumonia, hypersensitivity pneumonitis, chronic obstructive pulmonary disease, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, acute respiratory distress syndrome, acute lung injury, smoke inhalation lung injury, and thermal lung injury, but is not limited thereto.

[0038] In the pharmaceutical composition for treating or preventing a pulmonary inflammatory disease of the present invention, the peptide can suppress the expression of inflammatory cytokine genes, inflammatory cytokine proteins, or proteins involved in inflammatory cytokine signaling in lung cells in which an inflammatory response is induced.

[0039] The inflammatory cytokine gene may be one or more selected from the group consisting of TNFα, IL-1β, IL-6, IL-8 and COX-2 genes.

[0040] The inflammatory cytokine protein or protein involved in inflammatory cytokine signaling may be one or more selected from the group consisting of IL-1β, IL-6, COX2, IL-8, p-JNK, p-ERK, p-PI3K, p-AKT, p-ERK, and myeloperoxidase.

[0041] In the pharmaceutical composition for treating or preventing a pulmonary inflammatory disease of the present invention, the peptide can suppress the expression of fibrotic genes in lung cells in which an inflammatory response is induced.

[0042] The fibrosis gene may be one or more selected from the group consisting of α-SMA, Collagen 1A1, Fibronectin 1, and Tenascin C genes.

[0043] In the pharmaceutical composition for treating or preventing a pulmonary inflammatory disease of the present invention, the peptide can reduce the number of neutrophils in lung tissue where an inflammatory response is induced.

[0044] The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of the above-mentioned peptide and a pharma- ceutically acceptable carrier.

[0045] The term "therapeutically effective amount" means an amount sufficient for the peptide, which is the active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient for achieving efficacy in treating or preventing pulmonary inflammatory diseases.

[0046] The pharma- ceutically acceptable carriers are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0047] The pharmaceutical composition of the present invention may further contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited thereto.

[0048] Suitable pharma- ceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (19th ed., 1995, Williams & Wilkins).

[0049] The pharmaceutical composition of the present invention may be administered by any suitable route for treating pulmonary inflammatory diseases, for example, orally or parenterally. In the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, etc.

[0050] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, and may be variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity.

[0051] The pharmaceutical composition of the present invention may be prepared in the form of a unit dose by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner easily practiced by a person skilled in the art to which the present invention pertains, or may be prepared in a multi-dose container. The dosage form may be a solution, suspension or emulsion in an oil or aqueous medium, or an extract, powder, granules, tablet or capsule, and may further include a dispersant or stabilizer.

[0052] Food Composition

[0053] According to another aspect of the present invention, there is provided a functional food composition for improving or preventing pulmonary inflammation, comprising a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0054] The pulmonary inflammation may be pulmonary inflammation caused by a disease selected from the group consisting of asthma, allergic asthma, bronchitis, chronic bronchitis, acute bronchitis, bronchiolitis, diffuse panbronchiolitis, bronchiectasis, pleurisy, alveolitis, vasculitis, pneumonia, hypersensitivity pneumonitis, chronic obstructive pulmonary disease, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, acute respiratory distress syndrome, acute lung injury, smoke inhalation lung injury, and heat-induced lung injury, but is not limited thereto.

[0055] According to yet another aspect of the present invention, there is provided a functional food composition for improving or preventing pulmonary fibrosis, which comprises a peptide comprising the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0056] In the functional food composition for improving or preventing pulmonary inflammation or pulmonary fibrosis of the present invention, the peptide can suppress the expression of inflammatory cytokine genes, inflammatory cytokine proteins, or proteins involved in inflammatory cytokine signaling in lung cells in which an inflammatory response is induced.

[0057] The inflammatory cytokine gene may be one or more selected from the group consisting of TNFα, IL-1β, IL-6, IL-8 and COX-2 genes.

[0058] The inflammatory cytokine protein or protein involved in inflammatory cytokine signaling may be one or more selected from the group consisting of IL-1β, IL-6, COX2, IL-8, p-JNK, p-ERK, p-PI3K, p-AKT, p-ERK, and myeloperoxidase.

[0059] In the functional food composition for improving or preventing pulmonary inflammation or pulmonary fibrosis of the present invention, the peptide can suppress the expression of fibrotic genes in lung cells in which an inflammatory response is induced.

[0060] The fibrosis gene may be one or more selected from the group consisting of α-SMA, Collagen 1A1, Fibronectin 1, and Tenascin C genes.

[0061] In the functional food composition for improving or preventing pulmonary inflammation or pulmonary fibrosis of the present invention, the peptide can reduce the number of neutrophils in lung tissue where an inflammatory response is induced.

[0062] The peptide in the functional food composition of the present invention may be included in an appropriate amount selected within the range of 0.0001% by weight to 10% by weight based on the weight of the total composition, but is not limited thereto.

[0063] In one embodiment, the functional food composition of the present invention may comprise a nutrient-effective amount of the peptide and a nutrient-acceptable carrier.

[0064] The food composition of the present invention may contain, as the active ingredient, not only peptides but also ingredients that are usually added during the production of food, such as proteins, carbohydrates, fats, nutrients, seasonings and flavorings. Examples of the carbohydrates mentioned above are monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as dextrin, cyclodextrin, and other common sugars, and sugar alcohols, such as xylitol, sorbitol, erythritol, etc. As flavorings, natural flavorings, thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (saccharin, aspartame, etc.) may be used. The ratio of the carbohydrates may be generally about 1 to 20 g, preferably about 5 to 12 g, per 100 g of the food composition of the present invention, but is not limited thereto.

[0065] In addition to the above-mentioned ingredients, the functional food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, colorants and thickeners (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. In addition, it may contain natural fruit juice and fruit pulp for producing fruit juice drinks and vegetable drinks. For example, when the functional food composition of the present invention is manufactured as a drink, it may additionally contain citric acid, isomerized sugar, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, licorice extract, etc. in addition to the peptide, which is the active ingredient of the present invention.

[0066] In another aspect of the present invention, there is provided a method for treating, preventing or ameliorating a pulmonary inflammatory disease, comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:1 or a pharmaceutical composition comprising said peptide to a patient suffering from the pulmonary inflammatory disease.

[0067] In yet another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the treatment, prevention, or amelioration of pulmonary inflammatory diseases.

[0068] In yet another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 as described above for producing a medicament or functional food composition for the treatment, prevention, or amelioration of pulmonary inflammatory diseases.

[0069] The above-described peptide and pharmaceutical composition of the present invention may be similarly applied to the method for treating, preventing, or improving pulmonary inflammatory diseases, and will not be described in detail again to avoid overly complicating the specification. Effect of the Invention

[0070] The peptide of the present invention has an activity of suppressing the expression of inflammatory cytokine genes, inflammatory cytokine proteins, or proteins involved in inflammatory cytokine signaling in lung cells where an inflammatory response is induced. The peptide can also reduce the number of neutrophils in lung tissue where an inflammatory response is induced. Therefore, the peptide of the present invention can be used as an active substance useful for treating, preventing, or improving pulmonary inflammatory diseases.

[0071] However, the effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief description of the drawings]

[0072] [Figure 1]FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on gene expression of inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 in MRC-5 cells in which an inflammatory response was induced by LPS treatment. [Diagram 2] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on gene expression of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and COX-2 in A549 cells in which an inflammatory response was induced by LPS treatment. [Diagram 3] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression of IL-1β, IL-6, pJNK, and pERK in MRC-5 cells in which an inflammatory response was induced by LPS treatment. [Figure 4a] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Production Example 1 on the expression of Phospho-PI3K, Phospho-AKT, and Phospho-ERK in A549 cells in which an inflammatory response was induced by LPS treatment. [Figure 4b] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression of IL-1β, IL-6, and COX2 in A549 cells in which an inflammatory response was induced by LPS treatment. [Figure 5a] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on IL-6 expression in MRC-5 cells in which an inflammatory response was induced by LPS treatment. [Figure 5b] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on IL-8 expression in MRC-5 cells in which an inflammatory response was induced by LPS treatment. [Figure 6] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression levels of the fibrotic genes α-SMA and Collagen 1A1 gene in MRC-5 cells in which an inflammatory response was induced by LPS treatment. [Figure 7]FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression levels of the fibrotic genes α-SMA, Fibronectin 1, Collagen 1A1, and Tenascin C in TGF-β1-treated MRC-5 cells. [Figure 8] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression levels of genes for the inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and COX-2 in IL-1β-treated A549 cells. [Figure 9a] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression level of the inflammatory cytokine IL-1β in IL-1β-treated A549 cells. [Figure 9b] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression level of the inflammatory cytokine IL-6 in IL-1β-treated A549 cells. [Figure 9c] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression level of the inflammatory cytokine IL-8 in IL-1β-treated A549 cells. [Figure 10a] FIG. 1 shows the results of measuring the effects of treatment with the peptide of Preparation Example 1 on the total cell count, neutrophil count, and macrophage count in bronchial lavage fluid from mice in which pulmonary inflammation was induced by LPS. [Figure 10b] FIG. 1 shows the results of measuring the effects of treatment with the peptide of Preparation Example 1 on the total cell count, neutrophil count, and macrophage count in bronchial lavage fluid from mice in which pulmonary inflammation was induced by LPS. [Figure 11a] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression level of TNF-α in bronchial lavage fluid of mice with LPS-induced pulmonary inflammation. [Figure 11b]FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression level of IL-1β in bronchial lavage fluid of mice with pulmonary inflammation induced by LPS. [Figure 11c] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression level of IL-6 in bronchial lavage fluid from mice with pulmonary inflammation induced by LPS. [Figure 11d] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the expression level of myeloperoxidase (MPO) in bronchial lavage fluid from mice with pulmonary inflammation induced by LPS. [Figure 12a] FIG. 1 shows the results of observing lung tissues from mice with pulmonary inflammation induced by LPS after H&E staining. [Figure 12b] This figure shows the results of measuring the thickness of the alveolar wall, the total cell count, and the number of alveoli after H&E staining of lung tissue from mice in which pulmonary inflammation was induced by LPS. [Figure 13a] FIG. 1 shows the results of measuring the body weight of mice in which pulmonary fibrosis was induced by BLM (bleomycin). [Figure 13b] FIG. 1 shows the results of measuring the effect of treatment with the peptide of Preparation Example 1 on the numbers of macrophages and lymphocytes in bronchial lavage fluid from mice in which pulmonary fibrosis was induced by BLM (bleomycin). [Figure 13c] FIG. 1 shows the results of microscopic observation of cells in bronchial lavage fluid from mice in which pulmonary fibrosis was induced by BLM (bleomycin). [Figure 13d] FIG. 1 shows the results of measuring the total cell count in bronchial lavage fluid from mice in which pulmonary fibrosis was induced by BLM (bleomycin). [Figure 14a] FIG. 1 shows the results of observing H&E staining of lung tissue from mice in which pulmonary fibrosis was induced by BLM (bleomycin). [Figure 14b]FIG. 1 shows photographs of H&E stained lung tissue from mice with pulmonary fibrosis induced by BLM (bleomycin), showing the results of measuring the number of alveoli, the total cell count, and the thickness of the alveolar wall in each treatment group. [Figure 15a] FIG. 1 shows the results of Trichrome staining of lung tissue from mice in which pulmonary fibrosis was induced by BLM (bleomycin). [Figure 15b] The amount of collagen in the normal group is taken as 100%, and the change in the amount of collagen in the remaining treatment groups is shown in percentage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0074] Production Example 1: Production of peptides

[0075] A peptide having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was isolated using C18 reverse phase high performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was an ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm, Waters Co, USA).

[0076] [Table 1]

[0077] Subsequently, the efficacy of the peptide of SEQ ID NO:1 prepared above was evaluated.

[0078] Experimental Example 1: Suppression of LPS-induced inflammatory cytokine gene expression

[0079] An experiment was carried out to determine whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 suppresses the expression of inflammatory cytokine genes in lung cells.

[0080] 1.MRC-5 cells

[0081] MRC-5 cells (lung fibroblast cells) were placed in a 12-well plate at 3 × 10 5 Cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells were treated with active substances and drugs for the following groups: a normal group (no treatment), a group treated with 5 μg / ml LPS (Sigma Aldrich) (negative control group), a group treated with 5 μg / ml LPS and the peptide of Preparation 1 (10, 50, 100, 500 μM), and a group treated with 5 μg / ml LPS and 10 μM dexamethasone (DEX) (positive control group), and RNA was extracted after 24 hours and RT-PCR was performed. RNA was extracted from MRC-5 cells using easy-BLUETM Total RNA extraction kit (Qiagen, Germany). The extracted RNA was converted to cDNA using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). A reaction mixture was prepared using PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for each gene, and PCR was performed using a PCR machine (Eppendorf, Germany). Then, the mRNA expression pattern was determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are shown in Table 2 below. As a result of the experiment, as shown in FIG. 1, the expression of the genes of inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 was increased by treatment with LPS, an inflammation-inducing factor, but it was confirmed that the increased expression of the genes was reduced in a concentration-dependent manner when treated with the peptide of Preparation Example 1.

[0082] 2.A549 cells

[0083] A549 cells (human alveolar basal epithelial cells) were cultured in a 6-well plate at 3 × 10 5 The cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells were treated with active substances and drugs for the following groups: a normal group (no treatment), a group treated with 5 μg / ml LPS (Sigma Aldrich) (negative control group), a group treated with 5 μg / ml LPS and the peptide of Preparation 1 (10, 50, 100, 500 μM), and a group treated with 5 μg / ml LPS and 10 μM dexamethasone (DEX) (positive control group), and RNA was extracted after 3 hours and RT-PCR was performed. RNA was extracted from the A549 cells of the treatment groups using easy-BLUETM Total RNA extraction kit (Qiagen, Germany). The extracted RNA was converted to cDNA using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). A reaction mixture was prepared using PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for each gene, and PCR was performed using a PCR machine (Eppendorf, Germany). Then, the mRNA expression pattern was determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are shown in Table 2 below. As a result of the experiment, as shown in FIG. 2, the expression of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and COX-2 genes was increased by treatment with LPS, an inflammation inducer, but it was confirmed that the increased expression of the above genes was reduced in a concentration-dependent manner when treated with the peptide of Preparation Example 1.

[0084] [Table 2]

[0085] Experimental Example 2: Inhibition of LPS-induced inflammatory cytokine protein expression

[0086] An experiment was carried out to determine whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 suppresses the expression of inflammatory cytokine proteins in lung cells.

[0087] 1. Western Blotting Analysis 1: MRC-5 Cells

[0088] MRC-5 cells (lung fibroblast cells) were seeded at 2 x 105 cells / well in a 6-well plate for use in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with a medium without FBS to induce starvation for 4 hours. The cells were treated with the active substances and drugs and cultured for 24 hours on the following groups: a normal group (no treatment of cells), a group treated with 5 μg / ml LPS (Sigma Aldrich) (negative control group), a group treated with 5 μg / ml LPS and the peptide of Preparation 1 (10, 50, 100, 500 μM), and a group treated with 5 μg / ml LPS and 10 μM dexamethasone (DEX) (positive control group). The cells were cultured for each treatment group and the control group, dissolved in a lysis buffer, and centrifuged at 4°C at 12,000 rpm for 30 minutes to obtain proteins, which were then quantified using a BCA Protein Assay kit (Sigma). Proteins were electrotransferred onto a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membrane with the attached proteins was blocked by treating it with 5% skim milk, and then reacted with the primary antibody at 4℃ overnight. After washing with PBS-T, the membrane was reacted with the secondary antibody at room temperature for 1 hour, washed again with PBS-T, and visualized using Gel Doc (Bio-Rad, Hercules, CA, USA) and Western detection reagent (Elpis Biotech, Daejeon, Korea). The antibodies used in the experiments were as follows: anti-IL-1β antibody (Cell signaling technology (CST), USA), anti-IL-6 antibody (Santa Cruz Biotechnology, USA), anti-pJNK antibody (Santa Cruz Biotechnology, USA), anti-pERK antibody (Cell signaling technology (CST), USA), and anti-β-Actin antibody (Santa Cruz Biotechnology, USA).As a result of the experiment, as shown in Figure 3, the expression of IL-1β, IL-6, pJNK, and pERK increased when treated with LPS, but the expression of IL-1β, IL-6, pJNK, and pERK again decreased in a concentration-dependent manner when treated with the peptide of Example 1.

[0089] 2. Western Blotting Analysis 2: A549 Cells

[0090] A549 cells (human alveolar basal epithelial cells) were cultured in a 6-well plate at 3 × 10 5The cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells were treated with the active substances and drugs and cultured for 24 hours for the following groups: a normal group (no treatment of the cells), a group treated with LPS (Sigma Aldrich) (5 μg / ml) (negative control group), a group treated with LPS (5 μg / ml) and the peptide of Preparation Example 1 (10, 50, 100, 500 μM), and a group treated with LPS (5 μg / ml) and dexamethasone (DEX) (10 μM) (positive control group). The cells were dissolved in a lysis buffer and centrifuged at 4°C at 12,000 rpm for 30 minutes, and the protein obtained was quantified using a BCA Protein Assay kit (Sigma). Proteins were electrotransferred onto a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membrane with the attached proteins was blocked by treating it with 5% skim milk, and then reacted with the primary antibody at 4℃ overnight. After washing with PBS-T, the membrane was reacted with the secondary antibody at room temperature for 1 hour, washed again with PBS-T, and visualized using Gel Doc (Bio-Rad, Hercules, CA, USA) and Western detection reagent (Elpis Biotech, Daejeon, Korea).The antibodies used in the experiments are as follows: anti-IL-1β antibody: (Cell signaling technology (CST), USA), anti-IL-6 antibody: (Santa Cruz Biotechnology, USA), anti-COX2 antibody: (Santa Cruz Biotechnology, USA), anti-Phospho-PI3K antibody: (Cell signaling technology (CST), USA), anti-Phospho-AKT antibody: (Cell signaling technology (CST), USA), anti-Phospho-ERK antibody: (Cell signaling technology (CST), USA), anti-β-Actin antibody: (Santa Cruz Biotechnology, USA). As a result of the experiment, as shown in Figures 4a and 4b, the expression of Phospho-PI3K, Phospho-AKT, Phospho-ERK, IL-1β, IL-6, and COX2 increased when treated with LPS, but the protein expression levels decreased in a concentration-dependent manner when treated with the peptide of Preparation Example 1.

[0091] 3.ELISA analysis

[0092] MRC-5 cells (lung fibroblast cells) were placed in a 12-well plate at 2 × 10 5The cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells were treated with the active substances and drugs and cultured for 72 hours for the following groups: a normal group (no cell treatment), a group treated with 5 μg / ml LPS (Sigma Aldrich) (negative control group), a group treated with 5 μg / ml LPS and the peptide of Preparation Example 1 (10, 50, 100, 500 μM), and a group treated with 5 μg / ml LPS and 10 μM dexamethasone (DEX) (positive control group). After cell culture, the culture medium was centrifuged at 3,000 rpm at 4℃ for 10 minutes to obtain a supernatant, which was then subjected to ELISA analysis using an IL-6 Quantikine ELISA Kit (R&D systems) and an IL-8 Quantikine ELISA kit (R&D systems) according to the instructions in the kit, and the absorbance was measured at 450 nm using a microplate reader. As a result, as shown in Figures 5a and 5b, the expression levels of IL-6 and IL-8 increased when treated with LPS, but the expression levels of IL-6 and IL-8 decreased in a concentration-dependent manner when treated with the peptide of Preparation Example 1.

[0093] Experimental Example 3: Inhibition of LPS-induced fibrosis gene expression

[0094] An experiment was carried out to see whether the peptide of SEQ ID NO: 1 produced in Production Example 1 inhibits the expression of fibrotic genes induced by LPS in lung cells. MRC-5 cells (lung fibroblast cells) were placed in a 12-well plate at 2 × 10 5The cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells were treated with the above active substances and drugs on the following groups: a normal group (no cells were treated), a group treated with 5 μg / ml LPS (Sigma Aldrich) (negative control group), a group treated with 5 μg / ml LPS and the peptide of Preparation Example 1 (10, 50, 100, 500 μM), and a group treated with 5 μg / ml LPS and 10 μM dexamethasone (DEX) (positive control group), and RNA was extracted after 3 hours and RT-PCR was performed. RNA was extracted from MRC-5 cells using easy-BLUETM Total RNA extraction kit (Qiagen, Germany). The extracted RNA was converted to cDNA using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). After preparing a reaction mixture with PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for each gene, PCR was performed using a PCR machine (Eppendorf, Germany). Then, the mRNA expression pattern was determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are listed in Table 3 below.

[0095] [Table 3]

[0096] As a result of the experiment, as shown in Figure 6, it was confirmed that the expression levels of α-SMA and Collagen 1A1 genes increased when treated with LPS, but the expression levels of α-SMA and Collagen 1A1 genes decreased again in a concentration-dependent manner when treated with the peptide of Preparation Example 1.

[0097] Experimental Example 4: Suppression of fibrotic gene expression induced by TGF-β1

[0098] An experiment was conducted to determine whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 inhibits the expression of fibrosis genes induced by TGF-β1 in lung cells. MRC-5 cells (lung fibroblast cells) were placed in a 12-well plate at 2 × 10 5 The cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells were treated with the active substances and drugs for the following groups: a normal group (no treatment), a group treated with 5 ng / ml TGF-β1 (negative control group), a group treated with 5 ng / ml TGF-β1 and the peptide of Preparation Example 1 (10, 50, 100, 500 μM), and a group treated with 5 ng / ml TGF-β1 and 10 μM dexamethasone (DEX) (positive control group), and RNA was extracted after 3 hours and RT-PCR was performed. RNA was extracted from MRC-5 cells using easy-BLUETM Total RNA extraction kit (Qiagen, Germany). The extracted RNA was converted to cDNA using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). After preparing a reaction mixture with PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for each gene, PCR was performed using a PCR machine (Eppendorf, Germany). The mRNA expression pattern was then determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are listed in Table 4 below.

[0099] [Table 4]

[0100] As a result of the experiment, as shown in Figure 7, when cells were treated with TGF-β1, the expression of the genes α-SMA, Fibronectin1, Collagen1A1, and Tenascin C increased, but when cells were treated with the peptide of Preparation Example 1, the gene expression decreased in a concentration-dependent manner.

[0101] Experimental Example 5: Inhibition of inflammatory cytokine gene expression induced by IL-1β

[0102] An experiment was conducted to determine whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 suppresses the expression of inflammatory cytokine genes in lung cells. A549 cells (human alveolar basal epithelial cells) were cultured in a 6-well plate at 3 × 10 5 The cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells were treated with active substances and drugs for the following groups: a normal group (no treatment), an IL-1β (2ng / ml) treatment group (negative control group), an IL-1β (2ng / ml) and Preparation Example 1 peptide (10, 50, 100, 500μM) treatment group, and an IL-1β (2ng / ml) and dexamethasone (DEX) 10μM treatment group (positive control group), and RNA was extracted after 3 hours and RT-PCR was performed. RNA was extracted from the A549 cells of each group using easy-BLUETM Total RNA extraction kit (Qiagen, Germany). The extracted RNA was converted to cDNA using RT-PCR premix (iNtRON Biotechnology, Seongnam, Korea). A reaction mixture was prepared using PCR premix (iNtRON Biotechnology, Seongnam, Korea) and primers for each gene, and PCR was performed using a PCR machine (Eppendorf, Germany). Then, the mRNA expression pattern was determined by agarose gel electrophoresis. The base sequences of the primers used in the experiment are shown in Table 2. As a result of the experiment, as shown in FIG. 8, it was confirmed that the expression levels of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8 and COX-2 genes were increased by IL-1β treatment, but the expression levels of the genes were decreased in a concentration-dependent manner when treated with the peptide of Preparation Example 1.

[0103] Experimental Example 6: Inhibition of IL-1β-induced inflammatory cytokine protein expression

[0104] An experiment was conducted to determine whether the peptide of SEQ ID NO: 1 prepared in Preparation Example 1 inhibits the expression of inflammatory cytokine proteins induced by IL-1β in lung cells. A549 cells (human alveolar basal epithelial cells) were cultured in a 12-well plate at 2 × 10 5 The cells were seeded at 1 cell / well and used in the experiment. After culturing in a medium containing 10% FBS for 1 day, the medium was replaced with one not containing FBS and starvation was induced for 4 hours. The cells in the following groups were treated with the active substances and drugs and cultured for 72 hours: a normal group (NON) in which the cells were not treated with anything, an IL-1β (2 ng / ml) treatment group (negative control group, NC), an IL-1β (2 ng / ml) and peptide (10, 50, 100, 500 μM) treatment group of Preparation Example 1, and an IL-1β (2 ng / ml) and dexamethasone (DEX) (10 μM) treatment group (positive control group). After cell culture, the culture medium was centrifuged at 4℃ at 3,000 rpm for 10 minutes to obtain a supernatant, which was then subjected to ELISA analysis using an IL-6 Quantikine ELISA Kit (R&D systems), an IL-8 Quantikine ELISA Kit (R&D systems), and an IL-1β Quantikine ELISA Kit (R&D systems) according to the instructions in the kit, and the absorbance was measured at 450 nm using a microplate reader. As a result of the experiment, as shown in Figures 9a, 9b, and 9c, it was confirmed that the expression levels of IL-1β, IL-6, and IL-8 increased when IL-1β was treated, but the expression levels of IL-1β, IL-6, and IL-8 decreased in a concentration-dependent manner when the peptide of Preparation Example 1 was treated.

[0105] Experimental Example 7: Suppression of acute pulmonary inflammation induced by LPS

[0106] 1. Animal Experiments

[0107] For the animal experiment, BALB / c mice (female / 7W) were used and an acute lung inflammation model was established by injecting LPS. Seven-week-old BALB / c female mice were divided into 5 groups of 5 mice each as shown in Table 5 below, and the experiment was carried out. On the first day, LPS (10μg / 50μl / mouse) was administered through the nasal cavity to groups G2 to G5, and 1 hour after LPS administration, the peptide of Preparation 1 (0.5mg, 1mg) was administered through the nasal cavity to groups G3 and G4, and dexamethasone (100μg) was administered intraperitoneally to the G5 positive control group. 24 hours after administration of the active substance and drug, the mice were sacrificed to obtain bronchial lavage fluid and lung tissue, which were used for the following analysis.

[0108] [Table 5]

[0109] In [Table 5], peptide: peptide of Production Example 1, DEX: dexamethasone, IN: intranasal administration, IP: intraperitoneal administration.

[0110] 2. Collection of bronchial lavage fluid (BALF) and cell counting

[0111] After anesthetizing the mouse, the treatment site was disinfected with 70% ethanol. Then, the skin of the mouse was incised from the lower lip to the chest, and the airway was opened. 1 ml of PBS was injected and withdrawn twice through an IV catheter (BD, USA) to collect bronchoalveolar lavage fluid (BALF). The collected BALF was centrifuged at 3000 rpm for 10 minutes to separate cells, and the separated cells were resuspended in 0.5 ml PBS and diluted with trypan blue solution at a 1:1 ratio, and the total cell count was measured using a hematocytometer. The remaining 10 μl of the cell suspension was smeared on a slide glass, and the cells were stained with Diff-Quik staining solution (Sysmex, Japan) for differential cell count. After staining, the cells were observed and counted under a microscope, and the immune cell count was converted to the total cell count and shown in a graph. As can be seen from the experimental results shown in Figures 10a and 10b, the number of neutrophils was increased by LPS treatment, and the increased number of neutrophils was significantly decreased in a concentration-dependent manner by treatment with the peptide of Preparation Example 1. There was no significant increase in macrophages by LPS treatment, and there was no increase or decrease by treatment with the peptide of Preparation Example 1.

[0112] 3. Analysis of the expression of pro-inflammatory proteins in bronchial lavage fluid (BALF)

[0113] The collected BALF was subjected to ELISA analysis for pro-inflammatory proteins. The collected BALF was centrifuged at 3000 rpm for 10 minutes to obtain a supernatant, which was analyzed using a TNF-α Quantikine ELISA kit (R&D systems), an IL-1β Quantikine ELISA kit (R&D systems), an IL-6 Quantikine ELISA kit (R&D systems), and a myeloperoxidase (MPO) Quantikine ELISA kit (R&D systems) according to the usage guidelines, and the absorbance was measured at 450 nm using a microplate reader. As shown in Figures 11a to 11d, the expression levels of TNF-α, IL-1β, IL-6, and myeloperoxidase (MPO) were increased by administration of LPS, and the increased expression levels of the cytokines were reduced in a concentration-dependent manner by treatment with the peptide of Preparation Example 1.

[0114] 4. Measurement of histological changes in lung tissue

[0115] Lung tissues obtained from mice used in animal experiments were washed with PBS at room temperature and fixed with 4% paraformaldehyde (PBS dilution). The fixed tissues were washed three times with PBS and dehydrated using a 70% to 100% concentration gradient ethanol series. The lung tissue samples were then embedded and cut into 4 μm-thick slices. The 4 μm-sized slices placed on slides were dewaxed and hydrated using a concentration gradient ethanol series, and stained with hematoxylin solution for 1 minute and eosin solution for 10 seconds. After staining, the tissue slides were immersed in 90% and 100% ethanol, and finally immersed in xylene twice for 5 minutes each, and then the mounting of the tissue slides was performed.

[0116] The results of observing H&E staining of lung tissue are shown in FIG. 12a.

[0117] The graph in Figure 12b shows the results of measuring the thickness of the alveolar wall (the thickness of the alveolar wall in the normal group is set to 100% and the degree of increase in thickness in the remaining treatment groups is shown as a percentage), the results of measuring the number of cells in photographs of stained lungs (the number of cells in the normal group is set to 100% and the degree of increase in cell number in the remaining treatment groups is shown as a percentage), and the results of measuring the number of alveoli in lung photographs (the number of alveoli in the normal group is set to 100% and the increase or decrease in alveolar number in the remaining treatment groups is shown as a percentage).

[0118] Experimental Example 8: Inhibition of BLM-induced pulmonary fibrosis

[0119] 1. Animal model of BLM-induced pulmonary fibrosis

[0120] Eight-week-old BALB / c male mice were divided into four groups of five mice each, as shown in Table 6 below. On day 1, a bleomycin solution (10 ng / 50 μl / mouse) was administered through the oropharynx, and after one BLM induction, from day 7, the peptide of Preparation Example 1 (1 mg / mouse) was administered intranasally three times a week for 14 days, and pirfeninone (2 mg / mouse), the positive control group, was orally administered three times a week for 14 days. On day 21 after BLM induction, the mice were sacrificed to obtain bronchial lavage fluid and lung tissue, which were used for the following analysis.

[0121] [Table 6]

[0122] In the above Table 6, Peptide: the peptide of Production Example 1, IN: intranasal administration, and Oral: oral administration.

[0123] 2. Weight change

[0124] The results of measuring the body weight of the mice subjected to the experiment are shown in Figure 13a. The mice with BLM-induced pulmonary fibrosis lost body weight, and the administration of the peptide of Preparation 1 restored the lost body weight.

[0125] 3. Collection of bronchial lavage fluid (BALF) and cell counting

[0126] The mice used in the experiment were anesthetized, and the treatment site was disinfected with 70% ethanol. The skin of the mouse was then incised from the lower lip to the chest, and the airway was opened. 1 ml of PBS was injected and withdrawn twice through an IV catheter (BD, USA) to collect bronchoalveolar lavage fluid (BALF). The collected BALF was centrifuged at 3000 rpm for 10 minutes to separate cells, and the separated cells were resuspended in 0.5 ml PBS and diluted with trypan blue solution at a 1:1 ratio, and the total cell count was measured using a hemocytometer. The remaining 10 μl of the cell suspension was smeared on a slide glass, and the cells were stained with Diff-Quik staining solution (Sysmex, Japan) for differential cell count. After staining, the cells were observed and counted under a microscope, and the immune cell count was converted to the total cell count and shown in a graph. As can be seen from the experimental results shown in Figure 13b, the numbers of macrophages and lymphocytes were increased by BLM treatment, and the increased numbers of macrophages and lymphocytes were decreased by treatment with the peptide of Preparation Example 1. Figures 13c and 13d show the results of observing the cells under a microscope and the results of measuring the total cell count.

[0127] 4. Measurement of histological changes in lung tissue - H&E staining

[0128] Lung tissues obtained from mice used in animal experiments were washed with PBS at room temperature and then fixed with 4% paraformaldehyde (PBS dilution). The fixed tissues were washed three times with PBS and dehydrated using a series of ethanol gradients from 70% to 100%. The lung tissue samples were then embedded and cut into 4 μm-thick slices. The 4 μm-sized slices placed on slides were dewaxed and hydrated using a series of ethanol gradients, and stained in hematoxylin solution for 1 minute and eosin solution for 10 seconds. After staining, the tissue slides were immersed in 90% and 100% ethanol, and finally in xylene for 5 minutes twice, and then the mounting of the tissue slides was performed.

[0129] FIG. 14a shows the results of observing H&E staining of lung tissue.

[0130] Figure 14b shows the results of measuring the number of alveoli in each treatment group in photographs of stained lung tissue, the results of measuring the total cell number in photographs of stained lung tissue (the total cell number in the normal group is set to 100%, and the degree of increase in total cell number for the remaining treatment groups is shown as a percentage), and the results of measuring the thickness of the alveolar wall in each treatment group in photographs of stained lung tissue (the alveolar wall thickness in the normal group is set to 100%, and the degree of increase in thickness for the remaining treatment groups is shown as a percentage).

[0131] 5. Measurement of histological changes in lung tissue - Masson's Trichrome staining

[0132] Lung tissues obtained from mice used in animal experiments were washed with PBS at room temperature and then fixed with 4% paraformaldehyde (PBS dilution). Fixed tissues were washed three times with PBS and dehydrated using a series of ethanol gradients from 70% to 100%. Lung tissue samples were then embedded and cut into 4 μm-thick slices. The 4 μm-sized slices placed on slides were dewaxed and hydrated using a series of ethanol gradients. Staining was performed with Weigert's iron hematoxlin, Biebrich scarlet-acid fuchsin solution, phosphomolybdic-phosphotungstic acid solution, and aniline blue solution for 10 minutes each. After staining, the tissue slide was immersed in 1% acetic acid solution for 2 minutes and then washed with DW. The tissue slide was immersed in 95% and 100% ethanol, and finally in xylene for 5 minutes twice, and then mounted. Figure 15a shows the results of collagen staining using trichrome staining. Figure 15b shows the change in collagen amount in the remaining treatment groups, expressed as %, with the amount of collagen in the normal group taken as 100%. The degree of pulmonary fibrosis induced by BLM treatment was examined through changes in collagen content, and it was found that the degree of collagen deposition in lung tissue was significantly reduced by treatment with the peptide of Preparation Example 1.

[0133] Preparation Example 2: Preparation of pharmaceutical composition

[0134] 2-1. Manufacturing of powders

[0135] Peptide of the present invention 2g Lactose 1g The above ingredients were mixed and filled into an airtight cloth to prepare a powder.

[0136] 2-2. Tablet manufacturing

[0137] Peptide of the present invention 100mg Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0138] The above ingredients were mixed and then compressed into tablets according to a conventional tablet manufacturing method.

[0139] 2-3. Capsule manufacturing

[0140] Peptide of the present invention 100mg Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0141] The above ingredients were mixed and then filled into a gelatin capsule according to a conventional capsule manufacturing method to prepare a capsule.

[0142] 2-4. Manufacturing of pills

[0143] Peptide of the present invention 1g Lactose 1.5g 1g glycerin Xylitol 0.5g

[0144] The above ingredients were mixed and then formulated into pills of 4 g each according to a conventional method.

[0145] 2-5. Granule manufacturing

[0146] Peptide of the present invention 150mg Soybean extract 50mg Glucose 200mg Starch 600mg

[0147] After mixing the ingredients, 100mg of 30% ethanol was added and dried at 60 degrees Celsius to form granules, which were then filled into the fabric.

[0148] Production Example 3: Production of functional food composition

[0149] 3-1. Health food manufacturing

[0150] Peptide of the present invention 500 μg Vitamin mixture (as needed) Vitamin A Acetate 70mg Vitamin E 1.0mg Vitamin D 0.13 mg Vitamin B2 0.15mg Vitamin B6 0.5mg Vitamin B12 0.2 mg Vitamin C 10mg Biotin 10mg Nicotinamide 1.7mg Folic acid 50mg Calcium pantothenate 0.5mg Inorganic mixture appropriate amount Ferrous sulfate 1.75mg Zinc oxide 0.82mg Magnesium carbonate 25.3mg Dipotassium phosphate 15mg Dicalcium phosphate 55mg Potassium citrate 90mg Calcium carbonate 100mg Magnesium chloride 24.8mg

[0151] The composition ratio of the vitamin and mineral mixture is a preferred embodiment in which ingredients relatively suitable for health foods are mixed together, but the mixing ratio may be varied as desired. After mixing the ingredients according to a conventional health food manufacturing method, granules can be produced and used to manufacture a health food composition according to a conventional method.

[0152] 3-2. Manufacturing of health drinks

[0153] Peptide of the present invention 500 μg Citric acid 1000mg Oligosaccharides 100g Plum concentrate 2g Taurine 1g Add purified water to make a total of 900ml

[0154] The ingredients were mixed according to a typical method for producing health drinks, then heated and stirred at 85°C for about 1 hour, and the resulting solution was filtered and collected in a sterilized container, sealed and sterilized, then refrigerated and stored before being used to produce a health drink composition. The above composition ratio was created by mixing ingredients relatively suitable for health drinks as a preferred embodiment, but the composition ratio can be freely modified according to regional and ethnic preferences, such as demand class, demand country, and use purpose.

[0155] Although the above describes exemplary embodiments of the present application, the scope of the present application is not limited to the specific embodiments described above, and a person having ordinary knowledge in the field may make appropriate modifications within the scope of the claims of the present application.

Claims

1. A pharmaceutical composition for treating or preventing a pulmonary inflammatory disease, comprising as an active ingredient a peptide comprising the amino acid sequence of SEQ ID NO:

1.

2. The pharmaceutical composition for treating or preventing a pulmonary inflammatory disease according to claim 1, wherein the pulmonary inflammatory disease is a disease selected from the group consisting of asthma, allergic asthma, bronchitis, chronic bronchitis, acute bronchitis, bronchiolitis, diffuse panbronchiolitis, bronchiectasis, pleurisy, alveolitis, vasculitis, pneumonia, hypersensitivity pneumonitis, chronic obstructive pulmonary disease, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, acute respiratory distress syndrome, acute lung injury, smoke inhalation lung injury, and thermal lung injury.

3. The pharmaceutical composition for treating or preventing a pulmonary inflammatory disease according to claim 1, wherein the peptide suppresses the expression of an inflammatory cytokine gene, an inflammatory cytokine protein, or a protein involved in inflammatory cytokine signaling in lung cells in which an inflammatory response is induced.

4. The pharmaceutical composition for treating or preventing a pulmonary inflammatory disease according to claim 3, wherein the inflammatory cytokine gene is one or more selected from the group consisting of TNFα, IL-1β, IL-6, IL-8 and COX-2 genes.

5. The pharmaceutical composition for treating or preventing a pulmonary inflammatory disease according to claim 3, wherein the inflammatory cytokine protein or a protein involved in inflammatory cytokine signaling is one or more selected from the group consisting of IL-1β, IL-6, COX2, IL-8, p-JNK, p-ERK, p-PI3K, p-AKT, p-ERK, and myeloperoxidase.

6. The pharmaceutical composition for treating or preventing a pulmonary inflammatory disease according to claim 1, wherein the peptide suppresses the expression of fibrotic genes in lung cells in which an inflammatory response is induced.

7. The pharmaceutical composition for treating or preventing a pulmonary inflammatory disease according to claim 6, wherein the fibrosis gene is one or more selected from the group consisting of α-SMA, Collagen 1A1, Fibronectin 1, and Tenascin C genes.

8. The pharmaceutical composition for treating or preventing a pulmonary inflammatory disease according to claim 1, wherein the peptide reduces the number of neutrophils in lung tissue where an inflammatory response is induced.

9. A functional food composition for improving or preventing pulmonary inflammation, comprising as an active ingredient a peptide having the amino acid sequence of SEQ ID NO:

1.

10. A functional food composition for improving or preventing pulmonary fibrosis, comprising a peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

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