CCL2 and receptor specific binding polypeptide thereof, and application of CCL2 and receptor specific binding polypeptide in prevention and treatment of cholestatic liver disease

Abstract

The invention discloses CCL2, a receptor specific binding polypeptide thereof and application of the receptor specific binding polypeptide in prevention and treatment of cholestatic liver diseases, and relates to the technical field of biological medicines. The CCL2 and the receptor specific binding polypeptide thereof, which are screened by a molecular simulation technology, can be specifically bound with a key molecule CCL2 for inducing diseases and a plurality of receptors thereof in a cholestatic liver disease occurrence process, so that liver function indexes are recovered; the traditional Chinese medicine composition can be used for relieving liver pathological injury, including inhibiting bile duct hyperplasia, relieving portal vein inflammatory response and relieving portal vein fibrosis, so that the pathological development process of fibrosis and inflammation caused by the liver injury is blocked.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to a CCL2 and its receptor-specific binding polypeptide and their application in the prevention and treatment of cholestatic liver diseases. Background Art

[0002] Cholestatic liver disease is a comprehensive disease caused by disorders in bile production, secretion, and excretion. Its inducing factors include various factors such as bile duct obstruction, infection, intestinal microbiota disorders, and abnormal gene expression. There are many clinical classifications of cholestatic liver diseases. Among them, the prevalence of primary biliary cholangitis is 14.6 per 100,000 people, and the prevalence of primary sclerosing cholangitis is 34.7 per 100,000 people. Among newborns, 1 in every 2,500 newborns has cholestasis. At the same time, 10.3% of inpatients with chronic liver diseases in China are accompanied by cholestasis, which is a common liver injury disease. Regardless of the inducing factor, cholestatic liver disease caused shows impaired liver function, bile duct hyperplasia, infiltration of inflammatory cells, and the occurrence of fibrosis, and ultimately develops into cirrhosis.

[0003] C-C motif chemokine ligand 2 (hereinafter referred to as CCL2 for short) is an important member of the CC chemokine family, which has obvious chemotactic effects on monocytes, T lymphocytes, natural killer cells, etc., and is also an activator of basophils and mast cells. In addition to its chemotactic effect on immune cells, CCL2 also mediates various biological functions such as cell activation, atherosclerosis, fibrosis, angiogenesis, and tumorigenesis. In the liver, CCL2 is involved in the occurrence and development of various liver diseases such as acute liver injury, chronic liver inflammation, fibrosis / cirrhosis, and liver cancer, and can be released by various cell types such as hepatocytes, macrophages, and cholangiocytes. CCR2 (C-C chemokine receptor 2) is a high-affinity receptor for CCL2.

[0004] Existing studies have shown that blocking the CCL2 / CCR2 signaling pathway can significantly reduce the inflammatory response and inhibit chronic liver diseases. CCL2 also binds to other non-classical receptors, such as CCR4 and CCR5, and also plays an important regulatory role in the occurrence and development of related diseases. Therefore, targeting the CCL2 / CCR2 signaling pathway and the signaling pathways of CCL2 and other receptors to study and obtain compounds or biological agents that can block these signaling pathways to treat related diseases is an effective approach. Summary of the Invention

[0005] The present invention provides a specific binding polypeptide of CCL2 and its receptors and its application in the prevention and treatment of cholestatic liver diseases. The specific binding polypeptide provided by the present invention can simultaneously target CCL2 and its multiple receptors CCR2, CCR4, and CCR5, thereby blocking multiple downstream signaling pathways of CCL2. Such a specific binding polypeptide is expected to target a variety of liver cell types and meet the intervention and treatment of different stages of liver lesions, mainly the inflammatory stage and the fibrotic stage. It is specifically achieved through the following technologies.

[0006] In the first aspect of the present invention, there is provided an application use of a specific binding polypeptide of CCL2 and its receptors, specifically for preparing a product for preventing, alleviating or treating cholestatic liver diseases, or for preparing a product for preventing, alleviating or treating bile duct hyperplasia, inflammatory response and fibrosis generated during the onset of the cholestatic liver diseases; the amino acid sequence of the specific binding polypeptide is as shown in SEQ ID NO.1.

[0007] Further, the product is a drug or a functional food.

[0008] In the second aspect of the present invention, there is provided an application use of a specific binding polypeptide of CCL2 and its receptors, specifically for preparing a detection product for detecting CCL2, CCR2, CCR4 or CCR5.

[0009] In the third aspect of the present invention, there is provided a specific binding polypeptide of CCL2 and its receptors, and the amino acid sequence of the specific binding polypeptide is as shown in SEQ ID NO.1.

[0010] In the fourth aspect of the present invention, there is provided a biological material, and the biological material is any one of the following:

[0011] (1) A nucleic acid molecule encoding the specific binding polypeptide of CCL2 and its receptors;

[0012] (2) An expression vector or a vector group containing the nucleic acid molecule;

[0013] (3) A host cell containing the expression vector or the vector group.

[0014] In the fifth aspect of the present invention, there is provided a product for preventing, alleviating or treating a disease condition, and the product includes the specific binding polypeptide of CCL2 and its receptors; the disease condition is cholestatic liver diseases, or a product for bile duct hyperplasia, inflammatory response and fibrosis generated during the onset of the cholestatic liver diseases.

[0015] Further, the product contains pharmaceutically acceptable excipients.

[0016] The products provided by the present invention can be drugs or functional foods.

[0017] Further, the drug or functional food can be in the form of an oral preparation, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; it can also be in a dosage form for injection administration, such as injection solutions, powder for injection, etc., through intravenous, intraperitoneal, subcutaneous or intramuscular routes. All dosage form forms used are well-known to those of ordinary skill in the pharmaceutical art.

[0018] The products provided by the present invention can be administered to a subject by known routes in the art, including but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal, buccal, sublingual, intranasal, transdermal methods, etc.

[0019] Further, the dosage administered will depend on the age, health and weight of the recipient, the type of co-administered drugs, the treatment frequency, the administration route, etc. The drug can be administered in a single daily dose, or the total daily dose can be administered in divided doses two, three or four times a day. The dose can be administered once or multiple times, and the dosing time can be from a single day to several months or longer.

[0020] The products provided by the present invention can be administered to any human or animal (such as pets or livestock, etc.) that has or has had cholestatic liver disease and related disease conditions (such as bile duct hyperplasia, inflammatory response and fibrosis, etc.).

[0021] In a sixth aspect of the present invention, there is provided a detection preparation, which is characterized by comprising a specific binding polypeptide of the above-mentioned CCL2 and its receptor; the detection product is a detection reagent or a detection kit for detecting CCL2, CCR2, CCR4 or CCR5.

[0022] Further, it further comprises a chemical modifier conjugated to the specific binding polypeptide of CCL2 and its receptor.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] 1. Based on molecular simulation technology, the present invention discovers the amino acid residues where CCL2 interacts with its multiple receptors from the proteins that interact with CCL2, and replaces the cysteine in the polypeptide sequence with alanine or serine. By simulating the binding pocket of CCL2 and its receptor, a specific targeting polypeptide is designed. According to the Gibbs free energy strength of the specific targeting polypeptide with human and murine CCL2, screening is carried out, and finally a specific binding polypeptide of CCL2 and its receptor is obtained, and its amino acid sequence can bind to human and murine CCL2 and its related receptors with a relatively low binding energy.

[0025] 2. By evaluating the levels of ALT and AST in the serum, which are liver injury indicators, and the level of total bile acid (TBA) in the serum, it was found that in the mouse model of cholestatic liver disease, prophylactic treatment (administering the drug before injury) and therapeutic experiments (administering the drug after injury) with the CCL2 and its receptor-specific binding polypeptide of the present invention can significantly alleviate the liver injury in the mouse model.

[0026] 3. The present invention's research found that the CCL2 and its receptor-specific binding polypeptide can significantly alleviate bile duct hyperplasia, inflammatory response, and fibrosis during the process of cholestatic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is: a schematic diagram of the predicted binding of the CCL2 and its receptor-specific binding polypeptide of the present invention to human CCL2.

[0028] Figure 2 It is: a schematic diagram of the predicted binding of the CCL2 and its receptor-specific binding polypeptide of the present invention to human CCR2.

[0029] Figure 3 It is: a schematic diagram of the predicted binding of the CCL2 and its receptor-specific binding polypeptide of the present invention to human CCR4.

[0030] Figure 4 It is: a schematic diagram of the predicted binding of the CCL2 and its receptor-specific binding polypeptide of the present invention to human CCR5.

[0031] Figure 5 It is: a schematic diagram of the predicted binding of the CCL2 and its receptor-specific binding polypeptide of the present invention to murine CCR2.

[0032] Figure 6 It is: a schematic diagram of the predicted binding of the CCL2 and its receptor-specific binding polypeptide of the present invention to both CCR2 and CCL2.

[0033] Figure 7 and Figure 8 They are respectively: in the mouse model of cholestatic liver disease, after the prophylactic treatment and intervention treatment experiments with the CCL2 and its receptor-specific binding polypeptide of the present invention, the levels of serum ALT, AST, and serum TBA, which are liver function indicators.

[0034] Figure 9 It is: in the mouse model of cholestatic liver disease, after the prophylactic treatment experiment with the CCL2 and its receptor-specific binding polypeptide of the present invention, the staining results of bile duct hyperplasia and fibrosis injury obtained.

[0035] Figure 10It is: the pathological damage assessment result diagram obtained after the preventive treatment experiment is carried out by using the CCL2 and its receptor specific binding polypeptide of the present invention in a mouse model of cholestatic liver disease.

[0036] Figure 11 It is: the staining result diagram of bile duct hyperplasia and fibrosis damage obtained after the intervention treatment experiment is carried out by using the CCL2 and its receptor specific binding polypeptide of the present invention in a mouse model of cholestatic liver disease.

[0037] Figure 12 It is: the pathological damage assessment result diagram obtained after the intervention treatment experiment is carried out by using the CCL2 and its receptor specific binding polypeptide of the present invention in a mouse model of cholestatic liver disease. Detailed implementation mode

[0038] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present invention.

[0039] The CCL2 and its receptor specific binding polypeptide provided by the present invention contains the amino acid sequence shown in SEQ ID NO.1 from the N-terminus to the C-terminus, specifically PDAINAPVTCCYNFTNRKIS.

[0040] The CCL2 and its receptor specific binding polypeptide can bind to CCL2 and prevent CCL2 from interacting with its receptors CCR2, CCR4, and CCR5. The CCL2 and its receptor specific binding polypeptide are predicted to bind to human CCL2, CCR2, CCR4, and CCR5 respectively, predicted to bind to murine CCR2, and the predicted binding structures with CCR2 and CCL2 are as Figures 1-6 shown.

[0041] The amino acids in the present invention refer to natural amino acids. Unless otherwise specified, any amino acid generally or specifically named by name includes D and L stereoisomers if their structures allow such stereoisomer forms. Natural amino acids include alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), cysteine (Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), leucine (Leu), isoleucine (Ile), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).

[0042] The CCL2 and its receptor-specific binding polypeptide provided by the present invention can specifically bind to CCL2, block the interaction between CCL2 and its receptors (CCR2, CCR4, CCR5), and effectively block the downstream signal transduction during the stage of massive expression of CCL2 and its receptors in the process of cholestatic liver disease injury.

[0043] The C57BL / 6 mice used in the following specific embodiments of the present invention were purchased from the Experimental Animal Research Center of Hubei Provincial Center for Disease Control and Prevention.

[0044] In the following specific embodiments of the present invention, the method for establishing a cholestatic liver disease mouse model is to ligate the bile duct of C57BL / 6 mice to induce cholestatic injury. The specific surgical protocol for model establishment is as follows: First, the abdominal skin of the mouse is prepared and the abdominal skin is wiped with a sterile gauze containing 75% ethanol. Then, the abdominal cavity of the mouse is opened along the midline of the abdomen with a sterilized scissors to expose the internal organs; the internal organs of the mouse abdominal cavity are gently pushed to one side with a cotton swab dipped in physiological saline to expose the common bile duct outside the liver; then the common bile duct outside the liver is carefully separated and a ligation thread is pre-placed; finally, the bile duct is ligated twice, the internal organs are repositioned, and the abdominal cavity of the mouse is sutured.

[0045] It is easy to think that the CCL2 and its receptor-specific binding polypeptide provided by the present invention can be artificially synthesized by using the commonly used polypeptide synthesis techniques in the art. Including but not limited to: solid-phase synthesis technology, liquid-phase synthesis technology, biosynthesis (such as enzymatic or engineered cell) technology, etc.

[0046] In the following specific embodiments, the CCL2 and its receptor-specific binding polypeptide are artificially synthesized according to the standard steps of solid-phase polypeptide synthesis technology. The synthesized polypeptide is purified by RP-HPLC (reverse-phase high performance liquid chromatography) using a C18 chromatographic column.

[0047] Specifically, the synthesis direction of the CCL2 and its receptor-specific binding polypeptide is from the C-terminus to the N-terminus. First, the resin (chlororesin) is swollen; then the first amino acid is linked to the resin; the first amino acid needs to be double-coupled to the resin, that is, the amino acid reacts with the resin twice; then the Fmoc group at the amino terminus of the amino acid is removed (deprotected); subsequently, the process of peptide chain extension is carried out. Through repeated condensation, washing, deprotection, washing, and condensation steps, amino acids are sequentially linked from the C-terminus to the N-terminus according to the polypeptide sequence until the required peptide fragment is synthesized; with the resin deswelling, drying, peptide segment excision, and purification of the crude peptide, the CCL2 and its receptor-specific binding polypeptide as shown in SEQ ID NO.1 can be synthesized.

[0048] The CCL2 and its receptor-specific binding polypeptide provided by the present invention can effectively alleviate hepatocyte death, bile duct hyperplasia, portal vein inflammatory response, and portal vein fibrosis in diseases such as cholestatic liver disease.

[0049] Example 1: Screening of CCL2 and its receptor-specific binding polypeptide

[0050] The specific screening method is as follows:

[0051] (1) Perform molecular docking of human CCL2 and human CCR2 (https: / / alphafold3.org / ), and determine their interaction regions according to the docking results.

[0052] (2) Design a series of CCL2-specific binding polypeptides according to the amino acid residues with interactions.

[0053] (3) Dock the designed polypeptide group with human and murine CCL2 again, and screen for polypeptides with high affinity for both.

[0054] (4) Dock the screened polypeptides with human CCR2, CCR4, and CCR5, and murine CCR2 again, and screen for polypeptides that interact with all three receptors according to the Gibbs free energy from low to high. The amino acid sequence is shown as SEQ ID NO.1.

[0055] The structures of CCL2 and its receptor-specific binding polypeptide predicted to bind to human CCL2, CCR2, CCR4, and CCR5, murine CCR2, and CCR2 and CCL2 are as Figures 1-6 shown.

[0056] Use the screened CCL2 and its receptor-specific binding polypeptide for further experiments on cholestatic liver disease mice.

[0057] Example 2: Preventive treatment test of CCL2 and its receptor-specific binding polypeptide on cholestatic liver injury

[0058] Select the CCL2 and its receptor-specific binding polypeptide screened in Example 1 and evaluate its preventive treatment effect on cholestatic liver injury. The specific test method is as follows:

[0059] (1) Weigh 10 mg of the polypeptide accurately, add 200 μL of DMSO to dissolve it, transfer 10 μL each time, and dilute it with 1 mL of physiological saline to finally obtain a stock solution with a final concentration of 0.3 mg / ml (polypeptide stock solution), and store it in small tubes at -80°C in the refrigerator.

[0060] (2)Dosing at 3 mg / kg: Directly take the polypeptide stock solution with a final concentration of 0.3 mg / mL and perform tail vein injection according to the injection volume of mouse body weight (g) × 10 μL.

[0061] Dosing at 1 mg / kg: Take the 0.3 mg / mL polypeptide stock solution, dilute it with physiological saline at a ratio of 1:2, and perform tail vein injection according to the injection volume of mouse body weight (g) × 10 μL.

[0062] Dosing at 0.3 mg / kg: Take the 0.3 mg / mL polypeptide stock solution, dilute it with physiological saline at a ratio of 1:9, and perform tail vein injection according to the injection volume of mouse body weight (g) × 10 μL.

[0063] (3)Before inducing cholestatic injury in mice by bile duct ligation (i.e., before modeling), perform polypeptide tail vein injection on mice treated as control (3‰ DMSO) and different doses (0.3 mg / kg, 1 mg / kg, 3 mg / kg) once a day for three consecutive days.

[0064] After the mouse modeling is completed, perform polypeptide tail vein injection on mice with different doses once a day. Collect the serum and liver tissues of the mice on the seventh day of modeling.

[0065] Among them, the serum is used to detect the levels of liver injury indicators ALT and AST, as well as the total bile acid (TBA) level. The liver tissue is used to make paraffin sections for evaluating the pathological injury, bile duct hyperplasia, and the occurrence of liver fibrosis.

[0066] The results are as Figure 7 、 9 and shown in Figure 10. The abscissas P1-0.3, P1-1, and P1-3 are the CCL2 and its receptor-specific binding polypeptides at concentrations of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, respectively.

[0067] Figure 7 It is pointed out that the CCL2 and its receptor-specific binding polypeptide at 3 mg / kg can significantly relieve the liver function injury induced by cholestasis, that is, slow down the increase in serum ALT level; the CCL2 and its receptor-specific binding polypeptides at each concentration (including 0.3 mg / kg, 1 mg / kg, 3 mg / kg) can significantly relieve the increase in serum total bile acid level induced by cholestasis.

[0068] Figure 9 and Figure 10It was pointed out that CCL2 and its receptor-specific binding polypeptides at each concentration (0.3 mg / kg, 1 mg / kg, 3 mg / kg) could significantly alleviate liver pathological damage (H&E), bile duct hyperplasia (CK19 + ), and fibrosis (Collagen IV + ).

[0069] Example 3: Interventional treatment trial of CCL2 and its receptor-specific binding polypeptides on cholestatic liver injury

[0070] Select the CCL2 and its receptor-specific binding polypeptides obtained by screening in Example 1, and evaluate their interventional treatment effects on cholestatic liver injury. The specific test methods are as follows:

[0071] (1) One day after inducing cholestatic injury by bile duct ligation in mice, different doses (0.3 mg / kg, 1 mg / kg, 3 mg / kg) of the treated polypeptides were administered to the cholestatic mice by tail vein injection. Inject once a day for six consecutive days. That is, on the seventh day after modeling, the mouse serum and liver tissues were collected.

[0072] Among them, the serum was used to detect the levels of liver injury indicators ALT and AST, as well as the total bile acid (TBA) level. The liver tissues were used to make paraffin sections for evaluating the occurrence of pathological damage, bile duct hyperplasia, and liver fibrosis.

[0073] The test results are shown in Figure 8 、 11 and 12.

[0074] Figure 8 It was pointed out that CCL2 and its receptor-specific binding polypeptides at 0.3 mg / kg could significantly alleviate cholestasis-induced liver function injury, that is, slow down the increase in serum ALT and AST levels; CCL2 and its receptor-specific binding polypeptides at each concentration (0.3 mg / kg, 1 mg / kg, 3 mg / kg) could significantly alleviate the increase in serum total bile acid (TBA) levels induced by cholestasis.

[0075] From Figure 11 and Figure 12 It was pointed out that CCL2 and its receptor-specific binding polypeptides at each concentration (0.3 mg / kg, 1 mg / kg, 3 mg / kg) could significantly alleviate liver pathological damage (H&E), bile duct hyperplasia (CK19 + ), and fibrosis (Collagen IV + ).

[0076] From the test results of Examples 2-3 above, it can be seen that the CCL2 and its receptor-specific binding polypeptide screened and artificially synthesized by the present invention can effectively alleviate the increase in serum ALT, AST, and total bile acid levels induced by cholestasis and liver pathological damage, including the functions of inhibiting bile duct hyperplasia, reducing portal vein inflammatory response, and alleviating portal vein fibrosis.

[0077] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. Use of a specific binding polypeptide of CCL2 and its receptor, characterized in that, For preparing a product for preventing, alleviating or treating cholestatic liver disease, or for preparing a product for preventing, alleviating or treating bile duct hyperplasia, inflammatory response and fibrosis generated during the pathogenesis of the cholestatic liver disease; the amino acid sequence of the specific binding polypeptide is as shown in SEQ ID NO.

1.

2. Use of the specific binding polypeptide of CCL2 and its receptor according to claim 1, characterized in that, The product is a drug or a functional food.

3. Use of a specific binding polypeptide of CCL2 and its receptor, characterized in that, For preparing a detection product for detecting CCL2, CCR2, CCR4 or CCR5.

4. A specific binding polypeptide of CCL2 and its receptor, characterized in that, The amino acid sequence of the specific binding polypeptide is as shown in SEQ ID NO.

1.

5. A biological material, characterized in that, The biological material is any one of the following: (1) A nucleic acid molecule encoding the specific binding polypeptide of CCL2 and its receptor as claimed in claim 4; (2) An expression vector or a vector group containing the nucleic acid molecule; (3) A host cell containing the expression vector or the vector group.

6. A product for preventing, alleviating or treating a disease condition, characterized in that, The product includes the specific binding polypeptide of CCL2 and its receptor as claimed in claim 5; the disease condition is cholestatic liver disease, or a product of bile duct hyperplasia, inflammatory response and fibrosis generated during the pathogenesis of the cholestatic liver disease.

7. The product for preventing, alleviating or treating cholestatic liver disease according to claim 6, wherein The product contains pharmaceutically acceptable excipients.

8. A detection product, characterized in that, Includes the specific binding polypeptide of CCL2 and its receptor as claimed in claim 5; the detection product is a detection reagent or a detection kit for detecting CCL2, CCR2, CCR4 or CCR5.

9. The specific binding polypeptide of CCL2 and its receptor according to claim 8, characterized in that, Also includes a chemical modifier conjugated with the specific binding polypeptide of CCL2 and its receptor.

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