Application of polypeptide HRH and analogue thereof 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 HRH and analogues thereof in preparation of drugs for treating pulmonary fibrosis. The amino acid sequence of the polypeptide HRH disclosed by the invention is NH2-His-Arg-His-Thr-Lys-Gln-Arg-His-Thr-Ala-Leu-His-CONH2. An in-vitro experiment proves that the polypeptide HRH can be used for effectively inhibiting pulmonary epithelial cell and fibroblast fibrosis induced by a transforming growth factor and silicosis fibrosis induced by silicon dioxide; in-vivo experiments further prove that the HRH can improve bleomycin-induced pulmonary fibrosis lesion of mice and improve weight loss and death rate of the mice, and meanwhile, the HRH has relatively high safety and no adverse toxic or side effect. The invention also provides an HRH polypeptide analogue which can more effectively inhibit the activation of fibroblasts. The polypeptide HRH prepared by the invention is low in dosage, safe and effective, and provides a potential new medicine for treating pulmonary fibrosis diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biological preparations, and specifically relates to the application of polypeptide HRH and analogs thereof in the preparation of drugs for treating pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis is a tissue scarring caused by chronic inflammation and repeated damage to alveolar epithelial cells, which leads to irreversible destruction of lung structure and loss of function, and ultimately causes respiratory failure and death. Due to unknown etiology, difficult diagnosis and poor prognosis, patients with idiopathic pulmonary fibrosis are already in the middle and late stages of the disease when diagnosed, with a median survival of only 3-5 years. The mortality rate exceeds that of many types of cancer, making it comparable to that of most tumors. Due to its rapid progression and high mortality rate, it is known as a difficult lung disease and is listed as one of the preferred diseases for lung transplantation. Epidemiological surveys show that the global incidence of IPF has increased year by year, which may be related to a variety of factors such as environmental exposure, viral infection, genetic susceptibility and drug intervention.

[0003] Clinical and experimental evidence suggests that pulmonary fibrosis is an epithelial-driven pathology that is a dysregulated repair response caused by repeated injury to alveolar epithelial cells and excessive extracellular matrix (ECM) accumulation. Chronic injury to alveolar epithelial cells and disruption of the alveolar basement membrane lead to the production of various pro-inflammatory and pro-fibrotic mediators that induce fibroblast differentiation into myofibroblasts, resulting in uncontrolled ECM synthesis and the development of pulmonary fibrosis. Although the exact mechanisms driving the development of fibrosis in most patients remain incompletely understood, multiple factors have been shown to contribute to changes in lung fibroblasts, such as inflammatory and immune mechanisms in the lung, endoplasmic reticulum stress, oxidative stress, and loss of balance between matrix metalloproteinases and their inhibitors.

[0004] At present, the treatment strategies for pulmonary fibrosis mainly focus on anti-fibrosis, inhibition of inflammation and immune response, antioxidant therapy and acid suppression. Although these treatments can delay the progression of the disease, they cannot reverse the fibrosis that has already formed and are often accompanied by serious toxic side effects, causing secondary damage to patients. Therefore, the development of new anti-pulmonary fibrosis drugs with stable therapeutic effects, few side effects and high safety is of great significance to improving the survival and quality of life of patients with pulmonary fibrosis.

[0005] Peptides have broad prospects in the development of new drugs due to their effectiveness, safety, and tolerance, as well as their low immunogenicity and production costs, which are not easy to accumulate in the body. Peptides are signaling molecules with many physiological functions and can act as agonists or antagonists by binding to receptors on the cell surface. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors. By binding to VEGF receptors (VEGFRs), it plays a vital role in physiological and pathological angiogenesis. Peptide HRH (HRHTKQRHTALH-NH 2 ) is a vascular endothelial growth factor receptor (VEGFR) binding peptide isolated through a phage display library. The peptide HRH can inhibit VEGF-stimulated endothelial cell proliferation and inhibit the formation of new blood vessels in vivo and in vitro. However, to date, there have been no reports on the research of HRH in improving and treating fibrosis-related diseases. Summary of the invention

[0006] The purpose of the present invention is to provide an application of HRH in the preparation of drugs for pulmonary fibrosis diseases, and to provide effective drugs and new ideas for clinical anti-pulmonary fibrosis treatment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The first object of the present invention is to provide a polypeptide, wherein the polypeptide is HRH; The amino acid sequence of the HRH is as follows, SEQ ID NO.12: NH 2 -His-Arg-His-Thr-Lys-Gln-Arg-His-Thr-Ala-Leu-His-CONH 2 .

[0008] Another object of the present invention is to provide analogs of the above polypeptide HRH; According to a specific embodiment of the present invention, the amino acid sequence of the HRH polypeptide analog is as follows: SEQ ID NO.1: NH 2 -ARHTKQRHTALH-CONH 2 ; SEQ ID NO.2: NH 2 -HAHTKQRHTALH-CONH 2 ; SEQ ID NO.5: NH 2 -HRHTAQRHTALH-CONH 2 ; SEQ ID NO.6: NH 2-HRHTKARHTALH-CONH 2 ; SEQ ID NO.9: NH 2 -HRHTKQRHAALH-CONH 2 .

[0009] Furthermore, the present invention also provides a general formula of a polypeptide HRH analog, the structure of which is as follows: NH 2 -His-Arg-His-Thr-Lys-Xaa6-Arg-His-Xaa9-Ala-Leu-His-CONH 2 , wherein Xaa6 is Ala or Gln; Xaa9 is Ala or Leu.

[0010] Preferably, Xaa6 is Ala.

[0011] Preferably, Xaa9 is Ala.

[0012] Preferably, the polypeptide HRH has a therapeutic and improving effect on bleomycin-induced pulmonary fibrosis, and can directly or indirectly treat diseases characterized by pulmonary fibrosis.

[0013] Among them, pulmonary fibrosis diseases include idiopathic pulmonary fibrosis, occupational exposure, drugs, radioactive element damage, pathogenic microorganism infection and other factors caused by pulmonary fibrosis diseases.

[0014] Another object of the present invention is to provide a composition, which contains the aforementioned polypeptide HRH and its analogs, 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.

[0015] Excipients can be materials added to make the drug into a suitable dosage form, such as buffers, excipients for lyophilization, etc. Liquid formulations are generally buffer solutions, isotonic solutions and aqueous solutions.

[0016] Specifically, HRH and its analogs are used as active ingredients and pharmaceutically acceptable carriers and / or excipients are added to support the pharmaceutical composition.

[0017] The auxiliary material is selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The specific meanings of the abbreviations used in the present invention are as follows: His (abbreviated H) is histidine, Arg (abbreviated R) is arginine, Thr (abbreviated T) is serine, Lys (abbreviated K) is lysine, Gln (abbreviated Q) is glutamine, Ala (abbreviated A) is alanine, and Leu (abbreviated L) is leucine.

[0027] Beneficial effects of the present invention: The present invention provides an application of polypeptide HRH in improving pulmonary fibrosis. In an in vitro cell model induced by TGF-β1, polypeptide HRH can effectively inhibit the activation of lung fibroblasts and epithelial-mesenchymal transition (EMT) of epithelial cells, and has no obvious toxic effect on alveolar epithelial cells and fibroblasts. 2 In the induced silicosis model, the polypeptide HRH can inhibit alveolar epithelial cell fibrosis and EMT; in the bleomycin-induced animal model, the polypeptide HRH can significantly increase the weight of mice and reduce the mortality rate, and significantly improve the fibrotic lesions of the mouse lungs; acute toxicity and subacute toxicity experiments prove that HRH has high safety. Therefore, the polypeptide HRH provided 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.

[0028] The present invention replaces the amino acid in the polypeptide HRH with alanine having a smaller methyl side chain to change its steric hindrance, and studies the anti-fibrosis activity of the synthesized analogs. It is found that after the 3rd, 4th, 7th, 8th, 11th, and 12th amino acids of HRH are replaced with alanine, the anti-fibrosis activity of HRH disappears, proving that the above sites are the key sites for HRH to exert its anti-fibrosis effect. After the amino acids at positions 1, 2, 5, 6, and 9 of HRH are replaced with alanine, the relative expression of α-SMA protein is significantly lower than that of the induced group at a concentration of 10 μM, indicating that the above HRH polypeptide analogs show a significant effect of inhibiting TGF-β1-induced α-SMA protein expression. In addition, the inhibitory effects of HRH analogs HRH-6 and HRH-9 on α-SMA protein expression are significantly higher than those of HRH, indicating that the anti-fibrosis effects of HRH-6 and HRH-9 are better than those of the parent peptide HRH. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1The figure shows the effect of HRH 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).

[0031] Figure 2 HRH inhibits the activation of fibroblasts induced by transforming growth factor TGF-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 HRH 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 Collagen I protein obtained by analysis; D is a bar graph of the relative expression of Fibronectin protein obtained by analysis.

[0032] Figure 3 HRH inhibits the expression of fibrosis-related proteins in A549 cells induced by TGF-β1; A is a Western blot analysis of the protein expression of α-SMA, Collagen I and Fibronectin in A549 cells after HRH action; 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; D is a bar graph of the relative expression of Fibronectin protein obtained by analysis.

[0033] Figure 4 HRH inhibits TGF-β1-induced EMT of epithelial cells; A is the protein expression of E-cadherin and Vimentin detected by Western blot after HRH acts on A549 cells; B is the bar graph of the relative expression of E-cadherin protein obtained by analysis; C is the bar graph of the relative expression of Vimentin protein obtained by analysis.

[0034] Figure 5 SiO for HRH inhibition 2Induced fibrosis and EMT of A549 cells; A is the protein expression diagram of α-SMA and Fibronectin detected by Western blot after HRH acted on A549 cells; B is the bar graph of the relative expression of α-SMA protein obtained by analysis; C is the bar graph of the relative expression of Fibronectin protein obtained by analysis; D is the protein expression diagram of E-cadherin and Vimentin detected by Western blot after HRH acted on A549 cells; E is the bar graph of the relative expression of E-cadherin protein obtained by analysis; F is the bar graph of the relative expression of Vimentin protein obtained by analysis.

[0035] Figure 6 HRH 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 status of mice in each group after 21 days; E is the HE, Masson, Collagen I, α-SMA and Collagen I immunohistochemical staining of lung tissues of mice in each group.

[0036] Figure 7 The figure shows the effect of HRH on the body weight and lung coefficient of mice with 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; Figure 8 Figure 3 shows the effect of HRH 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] Fig. 9 The figure shows the effect of HRH on the acute toxicity weight and lung coefficient of mice; 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; Fig.10 The effect of HRH 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.

[0038] Fig.11 HRH polypeptide analogs inhibit the expression of fibrosis marker proteins in NIH3T3 cells induced by TGF-β1; A is a Western blot analysis of α-SMA protein expression in NIH3T3 cells after HRH analogs acted on them; B and C are bar graphs of the relative expression levels of α-SMA proteins obtained by analysis. 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] The experimental conditions and methods described in the following examples are conventional conditions and methods unless otherwise specified, and the reagents and instruments described are all commercially available.

[0041] Among them, 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. 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.

[0042] Example 1 Preparation of Compounds 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: (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 four times. (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; (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; (4) Amino acid condensation reaction: Weigh an appropriate amount of the first amino acid at the C-terminus and 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 head with acetic anhydride (acetic anhydride: DIEA: DCM = 1: 1:2) for 30 min, wash with DMF four times and drain for use.

[0043] (5) Indole test: If the indole test result is yellow, it indicates that the condensation is successful; (6) Repeat steps (2), (3), (4), and (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: NH 2 -Thr-Pro-His-Thr-His-Asn-Arg-Thr-Pro-Glu-CONH 2 ; Step 2: Cleavage of polypeptide chain: After peptide chain synthesis, deprotection was performed, and the resin was washed with DCM 4 times, and then the resin was drained with methanol. 2 The peptide was cut off from the resin with a cutting solution of 2:1:2:2.5. The cutting solution was evaporated with a rotary evaporator, precipitated with pre-cooled ice ether, extracted with deionized water, and the aqueous phase was collected and divided into 50 mL beakers, frozen in a -80°C refrigerator overnight, and then freeze-dried to obtain the crude peptide.

[0044] Step 3: Purification of polypeptide chains: (1) Weigh about 40 mg of crude peptide and dissolve it in deionized water to make 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); (2) HPLC used a C18 reverse preparative column with 100% acetonitrile to flush until the spectrum was stable, equilibrated with an initial concentration of 5% acetonitrile, and injected after setting the flow gradient; (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.

[0045] (4) After freeze-drying, 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 was calculated by integrating the peak area 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.

[0046] According to the method of Example 1, polypeptide HRH and its analogs were prepared. Table 1 shows the structures and measured m / z of the compounds synthesized in Example 1.

[0047] Table 1 Structure and mass spectrometry results of the compounds synthesized in Example 1

[0048] Example 2 Cytotoxicity test of the polypeptide HRH obtained in Example 1 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.

[0049] A549 cells and NIH3T3 cells were cultured with RPMI 1640 + 10% FBS + 1% double antibody culture medium and DMEM + 10% FBS + 1% double antibody culture medium at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 h, and different concentrations of HRH (10 μM, 20 μM, 40 μM, 80 μM, and 160 μM) were added. After incubation for 24 h, 10 μL of CCK-8 solution was added and incubated for 1 h. After oscillation mixing, the absorbance at a wavelength of 450 nm was detected by an enzyme-labeled instrument.

[0050] Figure 1 The results show that in the concentration range of 0-160 μM, after the peptide HRH acted on the cells, the cell survival rate remained basically the same as that of the control group, with no significant difference, indicating that the peptide HRH had no significant toxicity to the cells.

[0051] Example 3: HRH polypeptide inhibits TGF-β1-induced fibroblast activation NIH3T3 cells were plated in 6-well plates and cultured in DMEM + 10% FBS + 1% double-antibody medium at 37°C and 5% CO 2The cells were cultured under the same conditions for 24 h and then changed to serum-free medium for 12 h. TGF-β1 (5 ng / mL) and HRH (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 levels of α-SMA, Fibronectin and Collagen I were detected by Western blot.

[0052] Figure 2 The results of Western blot detection of α-SMA, Fibronectin and Collagen I protein expression after different concentrations of HRH were applied to NIH3T3 cells, as well as the relative expression levels of the proteins obtained by analysis.

[0053] Figure 2 A is the expression results of α-SMA, Fibronectin and Collagen I proteins in the blank control group, TGF-β1 induced group (5 ng / mL), and HRH added group (10 μM, 20 μM, 40 μM and 80 μM); Figure 2 BD is the relative expression levels of α-SMA, Collagen I and Fibronectin proteins in each group.

[0054] Western blot results showed that HRH concentrations of 10-160 μM could significantly reduce the expression of α-SMA, Collagen I and Fibronectin proteins induced by TGF-β1, indicating that polypeptide HRH exhibited an inhibitory effect on fibrosis markers in TGF-β1-induced NIH3T3 cells, suggesting that polypeptide HRH has good anti-fibrosis activity in fibroblasts.

[0055] Example 4 HRH polypeptide inhibits TGF-β1-induced epithelial cell fibrosis and EMT A549 cells were plated in 6-well plates and cultured in RPMI 1640 + 10% FBS + 1% double-antibody medium at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 h, then changed to serum-free medium for 12 h, TGF-β1 (5 ng / mL) and HRH (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 levels of α-SMA, Fibronectin, Collagen I, E-cadherin and Vimentin were detected by Western blot.

[0056] Figure 3The results of Western blot detection of α-SMA, Collagen I and Fibronectin protein expression after A549 cells were treated with different concentrations of HRH, as well as the relative expression levels of the proteins obtained by analysis.

[0057] Figure 3 A is the expression results of α-SMA, Fibronectin and Collagen I proteins in the blank control group, TGF-β1 induced group (5 ng / mL), and HRH added group (10 μM, 20 μM, 40 μM and 80 μM); Figure 3 BD is the relative expression levels of α-SMA, Collagen I and Fibronectin proteins in each group.

[0058] Western blot results showed that HRH concentrations of 10-160 μM could significantly reduce the expression of α-SMA, Collagen I and Fibronectin proteins induced by TGF-β1, indicating that polypeptide HRH exhibited an inhibitory effect on fibrosis markers in TGF-β1-induced A549 cells, suggesting that polypeptide HRH has good anti-fibrosis activity in lung epithelial cells.

[0059] Figure 4 The results of Western blot detection of E-cadherin and Vimentin protein expression after A549 cells were treated with different concentrations of HRH, as well as the relative expression levels of the proteins obtained by analysis.

[0060] Figure 4 A is the expression results of E-cadherin and Vimentin proteins in the blank control group, TGF-β1 induced group (5 ng / mL), and HRH added group (10 μM, 20 μM, 40 μM, and 80 μM); Figure 4 BC is the relative expression levels of E-cadherin and Vimentin proteins in each group.

[0061] Western blot results showed that after the peptide HRH acted on A549 cells at concentrations of 20 μM and 40 μM, the expression of epithelial marker E-cadherin protein was significantly increased, and the expression of mesenchymal marker Vimentin protein was significantly decreased, indicating that HRH has the effect of inhibiting TGF-β1-induced EMT of A549 cells. Example 5 Peptide HRH inhibits SiO2-induced epithelial cell fibrosis and EMT A549 cells were plated in 6-well plates and incubated with RPMI 1640 + 10% FBS + 1% double-antibody medium at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 h, then the medium was changed to serum-free medium for 12 h, and 200 μg / mL SiO 2 After A549 cells were treated with different concentrations of HRH (20 μM and 40 μM) for 24 h, total cell protein was extracted, and the protein expression levels of α-SMA, Fibronectin, E-cadherin and Vimentin were detected by Western blot.

[0062] Figure 5 The results of Western blot detection of α-SMA, Fibronectin, E-cadherin and Vimentin protein expression after A549 cells were treated with different concentrations of HRH, as well as the relative expression levels of the proteins obtained by analysis.

[0063] Figure 5 A is the blank control group, SiO 2 The results of α-SMA and Fibronectin protein expression in the induction group (200 μg / mL) and HRH-addition group (20 μM and 40 μM); Figure 5 BC is the relative expression levels of α-SMA and Fibronectin proteins in each group obtained by analysis; Figure 5 D is the blank control group, SiO 2 The results of E-cadherin and Vimentin protein expression in the induction group and HRH addition group (20 μM and 40 μM); Figure 5 EF is the relative expression levels of E-cadherin and Vimentin proteins in each group obtained by analysis.

[0064] Western blot results showed that after HRH 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 SiO 2 Induction group ( Figure 3 AC), HRH at concentrations of 20 μM and 40 μM in A549 cells compared with SiO 2 In the 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 the polypeptide HRH was activated in SiO 2 The induced A549 cells showed an inhibitory effect on fibrosis markers and EMT, suggesting that HRH has good anti-silicotic fibrosis activity in vitro. Example 6 Peptide HRH improves bleomycin-induced pulmonary fibrosis in mice 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), HRH administration group (0.1 mg / kg); Establishment of pulmonary fibrosis model and administration scheme: C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (50 mg / kg), and the neck skin of mice was disinfected with 75% alcohol. The skin was incised about 1 cm with sterile surgical instruments, 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, and pulmonary fibrosis mice were obtained. The normal group, saline group, and bleomycin group were subcutaneously injected with an equal volume of sterile PBS, and the mice in the administration group were intraperitoneally injected with 100 μL of HRH solution. After 21 days of continuous administration, blood samples and lung tissue samples were collected for pathological observation and extraction of lung tissue proteins to detect the expression of pulmonary fibrosis-related proteins.

[0065] Figure 6 This is the effect of HRH on pulmonary fibrosis in mice after 21 days of administration. Figure 6 A is a general picture of the lung dissection of mice in each group. The results show that the lung tissue of mice in the normal group and the normal saline control group was pink, without edema and pathological phenomena. The lung tissue of mice induced by bleomycin showed edema, tissue consolidation and hemorrhagic transformation. When the HRH dosage was 0.1 mg / kg, the degree of lung damage caused by bleomycin was alleviated. Figure 6 B is the lung coefficient of mice in each group. The results showed that the lung coefficient of mice in the HRH administration group was significantly lower than that in the bleomycin model group, indicating that HRH can effectively alleviate pathological changes such as edema caused by bleomycin; Figure 6 C is the weight change of mice in each group, and the results show that HRH can effectively improve the weight loss of mice caused by bleomycin; Figure 6 D shows the survival rate of mice in each group. The results show that HRH can effectively improve the survival rate of mice with pulmonary fibrosis; Figure 6 E is the HE, Masson and immunohistochemical staining of the pathological sections of mice in each group. The results showed that after HRH was administered at a dose of 0.1 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 polypeptide HRH can effectively improve bleomycin-induced pulmonary fibrosis.

[0066] Example 7 Subacute toxicity evaluation of polypeptide HRH on C57BL / 6 mice 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 h before administration and had free access to water.

[0067] Grouping and dosing: PBS control group (100 μL PBS intraperitoneally injected) and HRH administration group (0.1 mg / kg, 100 μL intraperitoneally injected), administered once a day, observed and recorded the mice's eating and drinking, body weight, behavior, mortality, mouse status, and the onset, duration, and recovery time of abnormal symptoms. After 21 days, samples were collected for gross anatomical examination, serological index detection, and pathological observation.

[0068] Figure 7 Figure 2 shows the body weight changes and organ coefficients of mice in each group. Figure 7 A and Figure 7 B shows the weight changes of male and female mice. The results show that HRH has no effect on the weight of mice; Figure 7 C-7H was the coefficient of heart, liver, spleen, lung and kidney of each group of mice. The results showed that HRH did not cause enlargement, congestion and atrophy of the above organs after administration at a dose of 0.1 mg / kg.

[0069] 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 HRH, administered at a dose of 0.1 mg / kg for 21 days, caused slight vacuolar degeneration in the liver tissue of female mice, but did not cause obvious lesions in the liver of male mice and the heart, spleen, lung and kidney of mice in each group; Figure 8 C-8F 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 HRH administration compared with the control group, indicating that HRH had no significant effect on the liver and kidney function of mice.

[0070] Example 8 Evaluation of acute toxicity of polypeptide HRH to C57BL / 6 mice 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 h before administration and had free access to water.

[0071] Grouping and dosing: PBS control group (100 μL PBS intraperitoneally injected) and HRH administration group (3000 mg / kg, 100 μL intraperitoneally injected), single administration, observed and recorded the mice's eating and drinking, body weight, behavior, mortality, mouse status, and the onset, duration, and recovery time of abnormal symptoms. After 14 days, samples were collected for gross anatomical examination, serological index detection, and pathological observation.

[0072] Fig. 9 Figure 2 shows the body weight changes and organ coefficients of mice in each group. Fig. 9 A and Fig. 9 B is the weight change of male and female mice. The results show that HRH can cause weight loss in mice when administered at a single dose of 3000 mg / kg. Fig. 9 C-9H was the coefficient of heart, liver, spleen, lung and kidney of each group of mice. The results showed that HRH at a dose of 3000 mg / kg caused liver enlargement in male and female mice, but had no effect on the coefficients of heart, spleen, kidney and lung.

[0073] Fig.10 Shown are the gross anatomical diagram of the organs, HE staining, and liver and kidney function levels of each group of mice. Fig.10 A-10B is the gross anatomical diagram and HE staining diagram of the organs of mice in each group. The results show that after HRH was administered at a dose of 3000 mg / kg, slight vacuolar degeneration occurred in the liver of female and male mice, but no obvious lesions were caused in the heart, liver, spleen and lung of male mice; Fig.10 C-10F 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 HRH was administered at a dose of 3000 mg / kg compared with the control group, indicating that HRH had no significant effect on the liver and kidney function of mice. In addition, half of the mice died when the HRH dose was 3000 mg / kg. According to the Global Harmonized Toxicity Grading System (GHS) standard, the acute toxicity level of HRH is Class 5. The above results show that HRH is a less toxic drug, with fewer subacute and acute toxic side effects and a larger safety window.

[0074] Example 9 Activity screening of HRH polypeptide analogs NIH3T3 cells were plated in 6-well plates and cultured in DMEM + 10% FBS + 1% double-antibody medium at 37°C and 5% CO 2 The cells were cultured under the same conditions for 24 h, and then the medium was changed to serum-free medium for 12 h. Then, 5 ng / mL TGF-β1 and HRH peptide analogs (HRH, HRH-1, HRH-2, HRH-3, HRH-4, HRH-5, HRH-6, HRH-7, HRH-8, HRH-9, HRH-10 and HRH-11) were added to the cells for 48 h, and the total protein of the cells was extracted. The protein expression level of α-SMA was detected by Western blot.

[0075] Fig.11After NIH3T3 cells were treated with HRH polypeptide analogs (HRH, HRH-1, HRH-2, HRH-3, HRH-4, HRH-5, HRH-6, HRH-7, HRH-8, HRH-9, HRH-10 and HRH-11), the expression of α-SMA protein was detected by Western blot and the relative expression level of the protein was analyzed.

[0076] Fig.11 A is the expression results of α-SMA protein in the blank control group, TGF-β1 induced group (5 ng / mL), and HRH peptide analog added group (10 μM); Fig.11 BC is the relative expression level of α-SMA protein in each group obtained by analysis.

[0077] The results showed that the peptide HRH showed an inhibitory effect on fibrosis markers in TGF-β1-induced NIH3T3 cells, indicating that the relative expression of α-SMA protein in HRH-1, HRH-2, HRH-5, HRH-6, and HRH-9 at a concentration of 10 μM was significantly lower than that in the induced group, indicating that the above compounds showed a significant inhibitory effect on TGF-β1-induced α-SMA protein expression. In addition, the inhibitory effect of HRH analogs HRH-6 and HRH-9 on α-SMA protein expression was significantly higher than that of HRH, indicating that the anti-fibrosis effect of HRH-6 and HRH-9 is better than that of the parent peptide HRH.

[0078] 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 HRH 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 epithelial-mesenchymal transition of epithelial cells.

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

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.

7. A polypeptide HRH analogue, characterized in that: Its amino acid sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.

9.

8. Use of the polypeptide HRH analogue according to claim 7 in the preparation of a drug for treating pulmonary fibrosis.

9. A drug for treating pulmonary fibrosis, characterized in that: The main component of the drug is one or more of the polypeptide HRH analogs described in claim 7.

10. The drug for treating pulmonary fibrosis according to claim 9, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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