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

Abstract

The present invention belongs to the field of biological preparations, and specifically relates to the use of the polypeptide Angio‑3 in the preparation of drugs for treating pulmonary fibrosis. The present invention provides a polypeptide Angio‑3 that has the effect of improving pulmonary fibrosis diseases. It can effectively inhibit the activation of lung fibroblasts and the deposition of extracellular matrix in an in vitro cell model induced by TGF‑β1, 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. The present invention provides the use of Angio‑3 in the preparation of drugs for pulmonary fibrosis diseases, providing effective drugs and new ideas for the clinical treatment of pulmonary fibrosis.

Description

Technical Field

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

[0002] Pulmonary fibrosis (PF) causes severe and irreversible changes in lung tissue, leading to loss of physiological function and is the primary clinical outcome of most chronic respiratory diseases. Chronic inflammation and repeated damage to alveolar epithelial cells lead to tissue scarring, which destroys lung structure and function. Clinical manifestations include progressive dyspnea, lung infection, worsening lung function, and impaired gas exchange, ultimately leading to respiratory failure and death. Due to its rapid progression and high mortality rate, PF is considered an intractable 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 PF, and are associated with significant adverse reactions. Given this situation, there is an urgent need to develop new anti-PF drugs with stable therapeutic effects, minimal side effects, and high safety.

[0003] The pathogenesis of pulmonary fibrosis is still unclear. It is currently believed that pulmonary fibrosis is mainly 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 ECM degradation, ultimately leading to pathological remodeling of lung tissue structure and impaired function.

[0004] Compared to small-molecule drugs and large-molecule protein drugs, peptide drugs offer high activity, low dosage, and low toxicity, making them a "unique drug" among large and small molecules. The harmless nature of peptide hydrolysis products and their high target affinity give them significant clinical application value and competitive advantages, particularly in the treatment of chronic diseases, where side effects and long-term therapeutic effects are crucial. The peptide Angio-3 (TPHTHNRTPE-NH2) is derived from the Kringle 3 domain of human plasminogen. Different combinations of plasminogen Kringle domains have distinct functions. For example, Kringle 1-5 domains reduced vascular length and density, inhibited vascular endothelial growth factor expression, and inhibited perivascular tumor invasion in a rat glioma model. Kringle 3-derived Angio-3 has been shown to inhibit the growth of breast cancer and melanoma by suppressing tumor angiogenesis and intratumoral vascular permeability. To date, there are no reports of Angio-3's use in the improvement and treatment of pulmonary fibrosis. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of Angio-3 in the preparation of drugs for pulmonary fibrosis, 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, which 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 using the Fmoc solid phase synthesis method and is obtained by cleaving the polypeptide resin for deprotection and purifying the crude polypeptide 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 and pathogenic microorganism infection and other factors.

[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 comprising the aforementioned polypeptide Angio-3 and a pharmaceutically acceptable carrier or excipient. Carriers can be those capable of reducing drug degradation and loss, and reducing side effects, such as micelles, microemulsions, and gels. Excipients can be materials added to prepare the drug into a suitable dosage form, such as buffers and lyophilization excipients. Liquid formulations typically include buffers, isotonic solutions, and aqueous solutions. Specifically, pharmaceutical compositions containing Angio-3 as the active ingredient are supplemented with pharmaceutically acceptable carriers and / or excipients.

[0015] The pharmaceutical combination of the present invention is suitable for various administration routes, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc. Depending on the administration route adopted, the polypeptide analog pharmaceutical combination of the present invention can be prepared into various suitable dosage forms, which contain at least an effective amount of the 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, pharmaceutical 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 silicon oxide; 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 carboxymethyl cellulose sodium; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0021] The antioxidants include sodium bisulfite, sodium metabisulfite, 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 include tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin application, aerosols, nasal sprays, and sterile solutions for injection. The drug of the present invention can be prepared as a solution or lyophilized powder for parenteral administration. The powder can be reconstituted by adding an appropriate solvent or other carrier before use. Liquid formulations are typically PBS buffer, isotonic saline solution, and aqueous solution.

[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, etc.

[0025] The abbreviations used in the present invention have the following specific meanings:

[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 that has the effect of improving pulmonary fibrosis. It can effectively inhibit the activation of lung fibroblasts and the deposition of extracellular matrix in an in vitro cell model induced by TGF-β1, 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 a bleomycin-induced animal model, Angio-3 significantly increased mouse body weight and reduced mortality, and significantly improved lung fibrosis. Acute and subacute toxicity experiments confirmed the high safety of Angio-3. Therefore, the polypeptide Angio-3 prepared by this invention can effectively improve pulmonary fibrosis without significant drug toxicity, offering promising application prospects for the treatment of pulmonary fibrosis. 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any 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 TGF-β1-induced fibroblast activation. A is a Western blot analysis of α-smooth muscle actin (α-SMA), type I collagen (Collagen I), and fibronectin protein expression in NIH3T3 cells after Angio-3 treatment; B is a bar graph showing the relative expression of α-SMA protein; C is a bar graph showing the relative expression of Collagen I protein; and D is a bar graph showing the relative expression of Fibronectin protein.

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

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

[0035] Figure 5 The effects of Angio-3 on the body weight and lung coefficients 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 mice in each group; D is the liver coefficient of mice in each group; E is the spleen coefficient of mice in each group; F is the lung coefficient of mice in each group; G is the left kidney coefficient of mice in each group; H is the right kidney coefficient of mice in each group;

[0036] Figure 6The effects of Angio-3 on subacute toxic pathology in mice. A is a gross anatomical diagram of the organs of mice in each group; B is a HE-stained image 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 body weight and lung coefficients of mice after acute toxicity. A is the weight change of male mice; B is the weight change of female mice; C is the heart coefficient of mice in each group; D is the liver coefficient of mice in each group; E is the spleen coefficient of mice in each group; F is the lung coefficient of mice in each group; G is the left kidney coefficient of mice in each group; H is the right kidney coefficient of mice in each group;

[0038] Figure 8 The effects of Angio-3 on acute toxic pathology in mice. A is the gross anatomical diagram of the organs of mice in each group; B is the HE staining 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 objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0040] In this example, Western blot results were analyzed using Evolution Capt software for grayscale analysis of protein expression, and GraphPad Prism 10.0 software was used for statistical analysis and analysis. Data were expressed as mean ± standard deviation (mean ± SD), and significant differences were analyzed by one-way analysis of variance and Tukey's 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 using the Fmoc solid-phase synthesis method from the carboxyl end to the amino end. The specific steps are as follows:

[0043] (1) Activation of RINK resin: Weigh RINK resin (degree of substitution 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 decolorization shaker for 20 minutes to remove the Fmoc protecting group. Wash with DMF four times and remove any residual reagents.

[0045] (3) Indene test: Add ninhydrin: pyridine: phenol = 1:2:1 indene test reagent to a test tube, take a small amount of resin into the test tube, and react at 100℃ for 1 minute. 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 an appropriate amount of 1-hydroxybenzotriazole (HOBT), 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 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 to dryness for later use.

[0047] (5) Indene test: If the indene 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: Cleavage of the Peptide Chain: After peptide synthesis, deprotect the peptide chain and rinse the resin four times with DCM. The resin was then drained with methanol. The peptide was cleaved from the resin using a cleavage solution of TFA:TiS:H₂O = 95:2.5:2.5. The cleavage solution was evaporated using a rotary evaporator, and the product was precipitated with pre-chilled ether. Extraction was performed with deionized water. The aqueous phase was collected and aliquoted into 50 mL beakers. The product was frozen at -80°C overnight and freeze-dried to yield the crude peptide.

[0050] Step 3: Purification of polypeptide chains:

[0051] (1) Weigh approximately 40 mg of crude peptide and dissolve it in deionized water to prepare a peptide solution. After complete dissolution, remove insoluble matter using a 0.45 μm filter. Add 0.1% TFA to the elution solvent (acetonitrile and deionized water);

[0052] (2) HPLC was performed using a C18 reverse preparative column flushed with 100% acetonitrile until the spectrum was stable, equilibrated with an initial concentration of 5% acetonitrile, and the flow gradient was set before injection;

[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 lyophilization, a small amount of compound was dissolved and eluted on a C18 reverse analytical column with 5%-95% acetonitrile / deionized water for 30 min. The purity of the chromatogram was statistically analyzed by integrating the peak area at 220 nm. The purity of the compound was >95%. The isolated 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 Peptide 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 after incubation for 24 hours, 10 μL of CCK-8 solution was added and incubated for 1 hour. After shaking and mixing, the absorbance at a wavelength of 450 nm was detected by microplate reader.

[0059] Figure 1 The results show the effect of Angio-3 at different concentrations on the survival rate of A549 and NIH3T3 cells. The results show that within the concentration range of 0-160μM, the cell survival rate after Angio-3 treatment remained at the same level as the control group, with no significant difference, indicating that Angio-3 has no significant toxicity to cells.

[0060] Example 3: Investigating the effect of Angio-3 on the inhibition of 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 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 and the relative expression levels of the proteins obtained after treatment of NIH3T3 cells with different concentrations of Angio-3.

[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-fibrotic 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. The culture medium was then changed to serum-free medium 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 3The results of Western blot detection of α-SMA, Fibronectin, E-cadherin, and Vimentin protein expression after A549 cells were treated with different concentrations of Angio-3, as well as the relative expression levels of the proteins obtained by analysis.

[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-treated 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: Angio-3 improves bleomycin-induced pulmonary fibrosis in mice

[0071] Male C57BL / 6J mice (8 weeks old, weighing approximately 18 g) were randomly divided into 4 groups, with 8 mice in each group, including: normal group (Normal), saline group (Saline), bleomycin group (BLM, 5 mg / kg), and Angio-3 treatment 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). The neck skin of the mice was disinfected with 75% alcohol. The skin was incised approximately 1 cm using sterile surgical instruments. The muscles were bluntly separated to expose the trachea. Pulmonary fibrosis was induced by intratracheal injection of 50 μL of bleomycin solution (5 mg / kg). Mice with pulmonary fibrosis were obtained.

[0074] Mice with pulmonary fibrosis were subcutaneously injected with an equal volume of sterile PBS in the normal, saline, and bleomycin groups. Mice in the treatment group received an intraperitoneal injection of 100 μL of Angio-3 solution. After 21 days of continuous administration, blood and lung tissue samples were collected for pathological observation and lung tissue protein extraction 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 gross anatomy of the lungs of mice in each group. The results showed that the lung tissues of mice in the normal group and the normal saline control group were pink, without edema or pathological phenomena. The lung tissues of mice induced by bleomycin showed edema, tissue consolidation and hemorrhagic transformation. When the Angio-3 dose 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 increased compared with that of mice 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 decreased, 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 Angio-3 administration was significantly improved (80%), indicating that Angio-3 can effectively improve the survival rate of mice with pulmonary fibrosis.

[0079] Figure 4E is the pathological sections of mice in each group, HE, Masson and immunohistochemical staining. The results showed that after 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 pulmonary fibrosis.

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

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

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

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

[0084] Figure 6 The diagrams show the gross anatomy, HE staining and liver and kidney function levels of the organs of each group of mice. Figure 6 A-6B are the gross anatomy 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 renal and liver function test results of each group of mice, 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 approximately 18 g) were randomly divided into two groups, with 10 mice in each group, half male and half female. The mice were fasted for 12 hours before administration and had free access to water.

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

[0088] Figure 7 Figure 3 shows the body weight changes and organ coefficients of mice in each group. Figure 7 A and Figure 7 B shows the changes in body weight 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 represents the coefficients of the 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 the heart, spleen, kidney and lung.

[0089] Figure 8 The diagrams show the gross anatomy, HE staining and liver and kidney function levels of the organs of each group of mice. Figure 8 A-8B are 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, mild vacuolar degeneration occurred in the liver of female mice, but no obvious lesions were caused in the heart, liver, spleen, and lungs of male mice. Figure 8 C-8F shows the renal and liver function test results for each group of mice. These results show that after administration of 5000 mg / kg of Angio-3, there were no significant differences in BUN, Cr, AST, and ALT levels compared to the control group, indicating that Angio-3 had no significant effect on the liver and kidney function of mice. Furthermore, no mice died at the 5000 mg / kg dose of Angio-3, and according to the Global Harmonized Toxicity Scale (GHS) standard, Angio-3 toxicity was not classified. These results indicate that Angio-3 is a less toxic drug with fewer subacute and acute side effects and a larger safety window.

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

Images