Application of polypeptide Angio-3 in preparation of medicine for treating pulmonary fibrosis

Abstract

The invention belongs to the field of biological agents, and particularly relates to application of polypeptide Angio-3 in preparation of medicine for treating pulmonary fibrosis. The invention provides a polypeptide Angio-3 with an effect of improving pulmonary fibrosis diseases, the polypeptide Angio-3 can effectively inhibit activation of lung fibroblasts and deposition of extracellular matrixes in an in-vitro cell model induced by TGF-beta1, and the polypeptide Angio-3 has no obvious toxic effect on alveolar epithelial cells and fibroblasts; in a SiO2 induced silicosis model, Angio-3 can be used for inhibiting alveolar epithelial cell fibrosis and epithelial-mesenchymal transition. The invention provides application of Angio-3 in preparation of drugs for treating pulmonary fibrosis diseases, and provides effective drugs and new ideas for clinical anti-pulmonary fibrosis treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biological preparations, and specifically relates to the application of polypeptide Angio-3 in the preparation of medicines for treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis causes severe and irreversible changes in lung tissue and loss of its physiological function, and is the main clinical outcome of most chronic respiratory diseases. Chronic inflammation and repeated damage to alveolar epithelial cells cause tissue scarring, destroying lung structure and function. Clinical manifestations include progressive dyspnea, lung infection, deterioration of lung function, and obstruction of gas exchange, which eventually lead to respiratory failure and death. Due to its rapid progression and high mortality rate, pulmonary fibrosis is called a difficult lung disease by the World Health Organization and is listed as one of the preferred diseases for lung transplantation. Current therapeutic drugs are still limited in their ability to reverse and completely cure pulmonary fibrosis, and there are obvious adverse reactions after use. Based on this situation, it is urgent to develop new anti-pulmonary fibrosis drugs with stable therapeutic effects, few side effects and high safety.

[0003] The pathogenesis of pulmonary fibrosis is still unclear. It is currently believed that pulmonary fibrosis is mainly a lesion driven by repeated micro-injury of alveolar epithelial cells, which leads to abnormal communication between epithelial cells and fibroblasts, prompting abnormal deposition of extracellular matrix (ECM) in the basement membrane and interstitial tissue. This is the result of overexpression of ECM components (such as collagen, fibronectin, laminin and proteoglycans) or insufficient degradation of ECM, ultimately leading to pathological remodeling of lung tissue structure and impaired function.

[0004] Compared with small molecule drugs and large molecule protein drugs, peptide drugs have the characteristics of high activity, low dose, and low toxicity, becoming a "unique drug" between large and small molecules. For the side effects and long-term treatment effects that are very important in the treatment of chronic diseases, the harmless hydrolyzate of peptides and the high target affinity make them have great application value and competitive advantages in clinical practice. The peptide Angio-3 (TPHTHNRTPE-NH2) is a peptide derived from the Kringle 3 domain of human plasminogen. Different combinations of plasminogen Kringles domains have independent functions, such as Kringle 1-5 domains reduce blood vessel length and density in rat glioma models, inhibit the expression of vascular endothelial growth factor and the invasion of perivascular tumors. Kringle 3-derived Angio-3 can inhibit the growth of breast cancer and melanoma by inhibiting tumor angiogenesis and intratumoral vascular permeability. So far, there are no reports on the improvement and treatment of pulmonary fibrosis by Angio-3. Summary of the invention

[0005] The purpose of the present invention is to provide the application of Angio-3 in the preparation of drugs for pulmonary fibrosis diseases, and to provide effective drugs and new ideas for the clinical treatment of pulmonary fibrosis.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first object of the present invention is to provide a polypeptide, wherein the polypeptide is Angio-3;

[0008] The amino acid sequence of Angio-3 is Thr-Pro-His-Thr-His-Asn-Arg-Thr-Pro-Glu-CONH2.

[0009] Furthermore, the Angio-3 is derived from the plasminogen Kringle 3 domain;

[0010] Furthermore, the Angio-3 is synthesized from the carboxyl end to the amino end by Fmoc solid phase synthesis method, and is obtained by cutting the polypeptide resin for deprotection and purifying the crude polypeptide peptide chain.

[0011] The second object of the present invention is to provide the use of the aforementioned polypeptide Angio-3 in the preparation of a drug for treating pulmonary fibrosis.

[0012] Preferably, the polypeptide Angio-3 has a therapeutic and improving effect on bleomycin-induced pulmonary fibrosis, and can directly or indirectly treat diseases characterized by pulmonary fibrosis; wherein, pulmonary fibrosis includes idiopathic pulmonary fibrosis, occupational exposure, drugs, radioactive element damage, pathogenic microorganism infection and other factors causing pulmonary fibrosis.

[0013] Preferably, the drug inhibits the activation of lung fibroblasts, and / or the drug inhibits the deposition of extracellular matrix.

[0014] Another object of the present invention is to provide a composition, which contains the aforementioned polypeptide Angio-3, and also includes a pharmaceutically acceptable carrier or excipient. The carrier can be a carrier that can reduce drug degradation and loss and reduce side effects, such as micelles, microemulsions, gels, etc.; the excipient can be a material added to make the drug into a suitable dosage form, such as a buffer, an excipient for lyophilization, etc., and the liquid formula is generally a buffer, an isotonic solution, and an aqueous solution. Specifically, Angio-3 is used as an active ingredient to add a pharmaceutically acceptable carrier and / or excipient to support the pharmaceutical composition.

[0015] The drug combination of the present invention is suitable for various administration methods, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc. According to the administration method adopted, the polypeptide analog drug combination of the present invention can be prepared into various suitable dosage forms, which at least contain an effective amount of polypeptide Angio-3 and at least one pharmaceutically acceptable pharmaceutical excipient.

[0016] The auxiliary material is selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0017] The fillers or diluents include sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0018] The lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silica; micro powder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0019] The binder includes distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol and compounds similar to the above excipients.

[0020] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or cross-linked sodium carboxymethyl cellulose; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0021] The antioxidants include sodium bisulfite, sodium pyrosulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid and the like.

[0022] The preservatives or antibacterial agents include benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (parabens), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil and peppermint oil.

[0023] Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions for injection. The drug of the present invention can be made into solutions or lyophilized powders for parenteral administration. Before use, appropriate solvents or other carriers can be added to reconstitute the powders. Liquid formulations are generally PBS buffer, physiological saline isotonic solutions, and aqueous solutions.

[0024] The dosage of the polypeptide of the present invention in the pharmaceutical composition can vary within a wide range, and those skilled in the art can easily determine it based on objective factors such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, and the like.

[0025] The specific meanings of the abbreviations used in the present invention are as follows:

[0026] Thr (abbreviated as T) is serine, Pro (abbreviated as P) is proline, His (abbreviated as H) is histidine, Asn (abbreviated as N) is asparagine, Arg (abbreviated as R) is arginine, and Glu (abbreviated as E) is glutamic acid.

[0027] Beneficial effects of the present invention:

[0028] The present invention provides a polypeptide Angio-3 with the effect of improving pulmonary fibrosis. In an in vitro cell model induced by TGF-β1, it can effectively inhibit the activation of lung fibroblasts and the deposition of extracellular matrix, and has no obvious toxic effect on alveolar epithelial cells and fibroblasts. In a SiO2-induced silicosis model, Angio-3 can inhibit alveolar epithelial cell fibrosis and epithelial-mesenchymal transition (EMT).

[0029] In the animal model induced by bleomycin, Angio-3 can significantly increase the weight of mice and reduce the mortality rate, and significantly improve the fibrotic lesions in the lungs of mice; acute toxicity and subacute toxicity experiments prove that Angio-3 has high safety. Therefore, the polypeptide Angio-3 prepared by the present invention can effectively improve pulmonary fibrosis without significant drug toxicity, providing a good application prospect for the treatment of pulmonary fibrosis diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1The effect of Angio-3 on the survival rate of epithelial cells and fibroblasts. A is the survival rate of human lung epithelial cells (A549); B is the survival rate of embryonic fibroblasts (NIH3T3).

[0032] Figure 2 Angio-3 inhibits the activation of fibroblasts induced by transforming growth factor-β1. A is a Western blot analysis of the protein expression of α-smooth muscle actin (α-SMA), type I collagen (Collagen I), and fibronectin in NIH3T3 cells after Angio-3 acts on them; B is a bar graph of the relative expression of α-SMA protein obtained by analysis; C is a bar graph of the relative expression of Collagen I protein obtained by analysis; and D is a bar graph of the relative expression of Fibronectin protein obtained by analysis.

[0033] Figure 3 Angio-3 inhibits SiO2-induced epithelial cell EMT. A is a Western blot analysis of the protein expression of α-SMA and Fibronectin in A549 cells after Angio-3 treatment; B is a bar graph of the relative expression of α-SMA protein obtained by analysis; C is a bar graph of the relative expression of Fibronectin protein obtained by analysis; D is a Western blot analysis of the protein expression of E-cadherin and Vimentin in A549 cells after Angio-3 treatment; E is a bar graph of the relative expression of E-cadherin protein obtained by analysis; F is a bar graph of the relative expression of Vimentin protein obtained by analysis.

[0034] Figure 4 Angio-3 improves bleomycin-induced pulmonary fibrosis in mice. A is the general anatomy of the lungs of mice in each group; B is the lung coefficient of mice in each group; C is the weight change of mice in each group after 21 days; D is the survival of mice in each group after 21 days; E is the immunohistochemical staining of lung tissues of mice in each group, including HE, Masson, Collagen I, α-SMA and Collagen I.

[0035] Figure 5 The effect of Angio-3 on the body weight and lung coefficient of mice in subacute toxicity. A is the weight change of male mice; B is the weight change of female mice; C is the heart coefficient of each group of mice; D is the liver coefficient of each group of mice; E is the spleen coefficient of each group of mice; F is the lung coefficient of each group of mice; G is the left kidney coefficient of each group of mice; H is the right kidney coefficient of each group of mice;

[0036] Figure 6The effects of Angio-3 on subacute toxic pathology in mice. A is the general anatomical diagram of the organs of mice in each group; B is the HE staining diagram of the organs of mice in each group; C is the blood urea nitrogen (BUN) level of mice in each group; D is the blood creatinine (Cr) level of mice in each group; E is the aspartate aminotransferase (AST) level of mice in each group; F is the alanine aminotransferase (ALT) level of mice in each group.

[0037] Figure 7 The effects of Angio-3 on the acute toxicity of mice body weight and lung coefficient. A is the weight change of male mice; B is the weight change of female mice; C is the heart coefficient of each group of mice; D is the liver coefficient of each group of mice; E is the spleen coefficient of each group of mice; F is the lung coefficient of each group of mice; G is the left kidney coefficient of each group of mice; H is the right kidney coefficient of each group of mice;

[0038] Figure 8 The effects of Angio-3 on acute toxic pathology in mice. A is the general anatomical diagram of the organs of mice in each group; B is the HE staining diagram of the organs of mice in each group; C is the BUN level of mice in each group; D is the Cr level of mice in each group; E is the AST level of mice in each group; F is the ALT level of mice in each group. DETAILED DESCRIPTION

[0039] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0040] In this embodiment, the Western blot results were analyzed by grayscale analysis of protein expression using Evolution Capt software, and statistics and analysis were performed using GraphPad Prism 10.0 software. The data were expressed as mean ± standard deviation (Mean ± SD), and significant differences were analyzed using one-way analysis of variance and Tukey test. **** P < 0.0001, *** P < 0.001, ** P<0.01, *P<0.05.

[0041] Example 1 Preparation of polypeptide Angio-3 compound

[0042] Step 1: The compound was synthesized by Fmoc solid phase synthesis from the carboxyl end to the amino end. The specific steps are as follows:

[0043] (1) Activation of RINK resin: Weigh RINK resin (substitution degree 0.3 mmol / g) into a reactor and soak it with 50 mL of dichloromethane (DCM). After 2 h, wash the resin with nitrogen-dimethylformamide (DMF) and drain it. Repeat this process four times.

[0044] (2) Resin deprotection: Add DMF solution containing 20% ​​piperidine. Shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group, add DMF to wash 4 times, and remove the residual reagent;

[0045] (3) Indene test: Add ninhydrin: pyridine: phenol = 1:2:1 indene test reagent into a test tube, take a small amount of resin into the test tube, react at 100℃ for 1 min. If the indene test result is blue-purple, it proves that the protecting group has been removed;

[0046] (4) Amino acid condensation reaction: Weigh the first amino acid at the C-terminus and 1-hydroxy-benzotriazole (HOBT) in appropriate amounts, add 20 mL of DMF to dissolve, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor and then place it on a shaker at 30°C for 2 h. Seal the reaction mixture with acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for 30 min, wash with DMF four times, and drain for later use.

[0047] (5) Indole test: If the indole test result is yellow, it indicates that the condensation is successful;

[0048] (6) Repeat steps (2)(3)(4)(5) in the order of the amino acid sequence in the compound until all the amino acids in the compound to be synthesized are condensed to obtain a polypeptide chain: NH2-Thr-Pro-His-Thr-His-Asn-Arg-Thr-Pro-Glu-CONH2.

[0049] Step 2: Cutting the polypeptide chain: After the peptide chain is synthesized, it is deprotected, the resin is washed with DCM 4 times, and then the resin is drained with methanol. The polypeptide is cut from the resin with a cutting solution of TFA: Tis: H2O = 95: 2.5: 2.5. The cutting solution is evaporated with a rotary evaporator, precipitated with pre-cooled ice ether, extracted with deionized water, and the aqueous phase is collected and divided into 50 mL beakers. After freezing in a -80 ° C refrigerator overnight, it is freeze-dried to obtain a crude peptide.

[0050] Step 3: Purification of polypeptide chains:

[0051] (1) Weigh about 40 mg of crude peptide and dissolve it in deionized water to prepare a peptide solution. After it is completely dissolved, remove the insoluble matter with a 0.45 μm filter, and add 0.1% TFA to the elution solvent (acetonitrile and deionized water);

[0052] (2) High performance liquid chromatography using a C18 reverse preparative column with 100% acetonitrile flushed until the spectrum is stable, 5% acetonitrile initial concentration for equilibrium, and injection after setting the flow gradient;

[0053] (3) After injection, the absorption peak at 220 nm was detected, the main peak was collected, the collected components were marked, and the components were frozen at -80°C overnight and then freeze-dried to obtain the compound.

[0054] (4) After freeze-drying, a small amount of the compound was dissolved and eluted on a C18 reverse analytical column with 5%-95% acetonitrile / deionized water for 30 min. The purity was calculated by integrating the peak area of ​​the chromatogram at 220 nm. The purity of the compound was >95%. The separated product was characterized and identified by mass spectrometry to confirm the m / z value of the protonated molecular ion peak.

[0055]

[0056] Example 2 Cytotoxicity experiment of polypeptide Angio-3

[0057] Human lung epithelial cell A549 and mouse embryonic fibroblast NIH3T3 cell lines were selected to study and observe the effects of the test substances on the survival rates of the two cell lines.

[0058] A549 cells and NIH3T3 cells were cultured with RPMI 1640 + 10% FBS + 1% double antibody medium and DMEM + 10% FBS + 1% double antibody medium at 37°C, 5% CO2 for 24 hours, Angio-3 was added and incubated for 24 hours, then 10 μL of CCK-8 solution was added and incubated for 1 hour. After oscillation mixing, the absorbance at a wavelength of 450 nm was detected by a microplate reader.

[0059] Figure 1 The results show that within the concentration range of 0-160 μM, after Angio-3 acted on cells, the cell survival rate remained at the same level as the control group, with no significant difference, indicating that Angio-3 had no significant toxicity to cells.

[0060] Example 3 Investigation of Angio-3 inhibiting TGF-β1-induced fibroblast activation

[0061] NIH3T3 cells were plated in 6-well plates and cultured in DMEM+10% FBS+1% double-antibody medium at 37°C, 5% CO2 for 24 h. After changing to serum-free medium and culturing for 12 h, TGF-β1 (5 ng / mL) and Angio-3 (10 μM, 20 μM, 40 μM and 80 μM) were added to the cells for 48 h, and then the total cell protein was extracted. The protein expression level of α-SMA was detected by Western blot.

[0062] Figure 2 The results of Western blot detection of α-SMA, Fibronectin and Collagen I protein expression after different concentrations of Angio-3 were applied to NIH3T3 cells, as well as the relative expression levels of the proteins obtained by analysis.

[0063] Figure 2 A is the expression results of α-SMA, Fibronectin and Collagen I proteins in the blank control group, TGF-β1 induced group, and Angio-3 added groups (10μM, 20μM, 40μM and 80μM); Figure 2 BD is the relative expression levels of α-SMA, Collagen I and Fibronectin proteins in each group obtained by analysis.

[0064] Western blot test results showed that Angio-3 at a concentration of 10-160 μM could significantly reduce the expression of α-SMA and Fibronectin proteins induced by TGF-β1, and Angio-3 at a concentration of 20-160 μM could significantly reduce the expression of Collagen I protein induced by TGF-β1, indicating that Angio-3 exhibited an inhibitory effect on fibrosis markers in TGF-β1-induced NIH3T3 cells, suggesting that Angio-3 has good anti-fibrosis activity in vitro.

[0065] Example 4 Investigating the effect of Angio-3 on inhibiting SiO2-induced epithelial cell EMT

[0066] A549 cells were plated in 6-well plates and cultured in RPMI 1640 + 10% FBS + 1% double antibody medium at 37°C, 5% CO2 for 24 hours, then changed to serum-free medium and cultured for 12 hours. 200 μg / mL SiO2 and different concentrations of Angio-3 (20 μM and 40 μM) were added to A549 cells for 24 hours, and then the total cell protein was extracted. Western blot was used to detect the protein expression levels of α-SMA, Fibronectin, E-cadherin and Vimentin.

[0067] Figure 3After A549 cells were treated with different concentrations of Angio-3, the protein expression results of α-SMA, Fibronectin, E-cadherin and Vimentin were detected by Western blot, and the relative expression levels of the proteins were analyzed.

[0068] Figure 3 A is the expression results of α-SMA and Fibronectin proteins in the blank control group, SiO2-induced group, and Angio-3-treated group (20 μM and 40 μM); Figure 3 B and Figure 3 C is the relative expression levels of α-SMA and Fibronectin proteins in each group obtained by analysis; Figure 3 D is the expression results of E-cadherin and Vimentin proteins in the blank control group, SiO2-induced group, and Angio-3-added group (20 μM and 40 μM); Figure 3 E and Figure 3 F is the relative expression levels of E-cadherin and Vimentin proteins in each group obtained by analysis.

[0069] Western blot results showed that after Angio-3 was treated with 20μM and 40μM concentrations in A549 cells, the relative expression levels of α-SMA and Fibronectin proteins were significantly lower than those in the SiO2-induced group ( Figure 3 AC), after Angio-3 acted on A549 cells at concentrations of 20μM and 40μM, compared with the SiO2-induced group, the expression of epithelial marker E-cadherin protein was significantly upregulated, and the expression of mesenchymal marker Vimentin protein was significantly downregulated, indicating that Angio-3 exhibited an inhibitory effect on fibrosis markers and EMT in SiO2-induced A549 cells, suggesting that Angio-3 has good anti-silicosis fibrosis activity in vitro.

[0070] Example 5 Investigation of Angio-3 to Improve Bleomycin-Induced Pulmonary Fibrosis in Mice

[0071] Male C57BL / 6J mice (8 weeks old, weighing about 18 g) were randomly divided into 4 groups, 8 mice in each group, including: normal group (Normal), saline group (Saline), bleomycin group (BLM, 5 mg / kg), Angio-3 administration group (0.5 mg / kg);

[0072] Pulmonary fibrosis model establishment and drug administration regimen:

[0073] C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg), and the neck skin of the mice was disinfected with 75% alcohol. The skin was incised about 1 cm with a sterile surgical instrument, and the muscles were bluntly separated to expose the trachea. 50 μL of bleomycin solution (5 mg / kg) was injected into the trachea to induce pulmonary fibrosis, thereby obtaining mice with pulmonary fibrosis.

[0074] The normal group, saline group, and bleomycin group were subcutaneously injected with an equal volume of sterile PBS into the mice with pulmonary fibrosis, and the mice in the drug group were intraperitoneally injected with 100 μL of Angio-3 solution. After 21 days of continuous drug administration, blood samples and lung tissue samples were collected for pathological observation and lung tissue protein was extracted to detect the expression of pulmonary fibrosis-related proteins.

[0075] Figure 4 This is the effect of Angio-3 on pulmonary fibrosis in mice after 21 days of administration. Figure 4 A is a general dissection of the lungs of mice in each group. The results showed that the lung tissues of mice in the normal group and the saline control group were pink, without edema and pathological phenomena. The lung tissues of mice induced by bleomycin showed edema, tissue consolidation and hemorrhagic transformation. When the dose of Angio-3 was 0.5 mg / kg, the degree of lung damage caused by bleomycin was alleviated.

[0076] Figure 4 B is the lung coefficient of each group of mice. The results showed that the lung coefficient of mice in the bleomycin model group was significantly higher than that in the control group. After intraperitoneal administration of Angio-3 at a dose of 0.5 mg / kg, the lung coefficient of mice was significantly reduced, indicating that Angio-3 can effectively reduce pathological changes such as edema caused by bleomycin.

[0077] Figure 4 C is the weight change of mice in each group. The results show that the weight of mice in the Angio-3 administration group was significantly increased compared with the bleomycin model group, indicating that Angio-3 can effectively improve the changes such as weight loss of mice caused by bleomycin.

[0078] Figure 4 D is the survival rate of mice in each group. Compared with the survival rate of mice in the bleomycin model group (69.5%), the survival rate of mice after administration of Angio-3 was significantly improved (80%), indicating that Angio-3 can effectively improve the survival rate of mice with pulmonary fibrosis.

[0079] Figure 4E is the HE, Masson and immunohistochemical staining of the pathological sections of mice in each group. The results showed that after the administration of Angio-3 at a dose of 0.5 mg / kg, the lung tissue structure was more complete than that in the bleomycin model group, collagen deposition was reduced, α-SMA and Fibronectin levels were reduced, and the degree of fibrosis was significantly alleviated, indicating that Angio-3 can effectively improve bleomycin-induced lung fibrosis.

[0080] Example 6 Investigation of the subacute toxicity of Angio-3 to C57BL / 6 mice

[0081] C57BL / 6 mice (8 weeks old, weighing about 18 g) were randomly divided into 2 groups, 10 mice in each group, half of which were male and half were female. They were fasted for 12 hours before administration and had free access to water.

[0082] Grouping and dosing: PBS control group (100 μL PBS, intraperitoneal injection) and Angio-3 treatment group (0.5 mg / kg, 100 μL, intraperitoneal injection), drug administration once a day, observe and record the mice's eating and drinking, body weight, behavior, mortality, mouse status, and the appearance, duration, and recovery time of abnormal symptoms. After 21 days, samples were collected for gross anatomical examination, serological index detection, and pathological observation.

[0083] Figure 5 Figure 2 shows the body weight changes and organ coefficients of mice in each group. Figure 5 A and Figure 5 B shows the weight changes of male and female mice. The results show that Angio-3 has no effect on the weight of mice. Figure 5 C-5H are the coefficients of the heart, liver, spleen, lung and kidney of each group of mice. The results showed that Angio-3 did not cause enlargement, congestion and atrophy of the above organs after administration at a dose of 0.5 mg / kg.

[0084] Figure 6 Shown are the gross anatomical diagram of the organs, HE staining, and liver and kidney function levels of each group of mice. Figure 6 A-6B are the gross anatomical images and HE staining images of the organs of mice in each group. The results show that Angio-3 administered at a dose of 0.5 mg / kg for 21 days did not cause obvious lesions in the heart, liver, spleen, lung and kidney of mice in each group; Figure 6 C-6F is the test results of renal and liver function of mice in each group, showing that there was no significant difference in BUN, Cr, AST and ALT levels after Angio-3 administration compared with the control group, indicating that Angio-3 had no significant effect on the liver and kidney function of mice.

[0085] Example 7 Investigating the acute toxicity of Angio-3 to C57BL / 6 mice

[0086] C57BL / 6 mice (8 weeks old, weighing about 18 g) were randomly divided into 2 groups, 10 mice in each group, half of which were male and half were female. They were fasted for 12 hours before administration and had free access to water.

[0087] Grouping and dosing: PBS control group (100 μL PBS intraperitoneal injection) and Angio-3 treatment group (5000 mg / kg, 100 μL intraperitoneal injection), single administration, observe and record the mice's eating and drinking, body weight, behavior, death, mouse status, and the appearance, duration, and recovery time of abnormal symptoms. After 14 days, samples were collected for gross anatomical examination, serological index detection, and pathological observation.

[0088] Figure 7 Figure 2 shows the body weight changes and organ coefficients of mice in each group. Figure 7 A and Figure 7 B is the weight change of male and female mice. The results show that Angio-3 can cause weight loss in mice when administered at a single dose of 5000 mg / kg. Figure 7 C-7H are the coefficients of heart, liver, spleen, lung and kidney of each group of mice. The results showed that Angio-3 at a dose of 5000 mg / kg caused liver enlargement in female mice, but had no effect on the coefficients of heart, spleen, kidney and lung.

[0089] Figure 8 Shown are the gross anatomical diagram of the organs, HE staining, and liver and kidney function levels of each group of mice. Figure 8 A-8B are the gross anatomical images and HE staining images of the organs of mice in each group. The results show that after administration of Angio-3 at a dose of 5000 mg / kg, slight vacuolar degeneration occurred in the liver of female mice, but no obvious lesions were caused in the heart, liver, spleen and lung of male mice; Figure 8 C-8F is the test results of renal and liver function of each group of mice, showing that there was no significant difference in BUN, Cr, AST and ALT levels after administration of Angio-3 at a dose of 5000 mg / kg compared with the control group of mice, indicating that Angio-3 had no significant effect on the liver and kidney function of mice. In addition, no mice died when the dose of Angio-3 was 5000 mg / kg, and according to the Global Harmonized Toxicity Grading System (GHS) standard, Angio-3 toxicity was not classified. The above results show that Angio-3 is a less toxic drug, with fewer subacute and acute toxic side effects and a larger safety window.

[0090] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Application of polypeptide Angio-3 in the preparation of drugs for the treatment of pulmonary fibrosis.

2. The use according to claim 1, characterized in that: The drug inhibits the activation of lung fibroblasts, and / or, the drug inhibits the deposition of extracellular matrix.

3. A drug for treating pulmonary fibrosis, characterized in that: The main component of the drug is polypeptide Angio-3.

4. The drug according to claim 3, characterized in that The drug also includes pharmaceutically acceptable excipients.

5. The drug according to claim 4, characterized in that The pharmaceutically acceptable excipients include any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, and stabilizers.

6. The drug according to claim 3, characterized in that The drug inhibits the activation of lung fibroblasts, and / or, the drug inhibits the deposition of extracellular matrix.

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