Polypeptide for treating osteoarthritis aiming at AHR target spot and application thereof

Abstract

The invention relates to the technical field of biological medicines, and discloses a polypeptide for treating osteoarthritis by aiming at AHR (Aryl Hydrocarbon Receptor), the amino acid sequence of the polypeptide is GFQQSDVIHQSVYELISINHTEDRA, and the amino acid sequence of the polypeptide is GFQQSDVIHQSVYELISINHTEDRA, and the amino acid sequence of the polypeptide is GFQQSDVIHQSVYELISINHTEDRA. The polypeptide is prepared by a solid-phase polypeptide synthesis method; the solid-phase polypeptide synthesis method specifically comprises the following steps: synthesizing from a C end to an N end, connecting a carboxyl group of a first amino acid to insoluble resin, then adding other amino acids one by one to form a peptide chain, and carrying out protection and deprotection reactions of a side chain in each step. The polypeptide is composed of natural amino acids and has good biocompatibility and degradability, and experimental results show that no obvious toxic reaction occurs in the cell level and the animal level, so that the polypeptide has conditions for further development of pharmacokinetic research and preclinical tests, and a feasible basis is provided for conversion to clinical application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a polypeptide for treating osteoarthritis by targeting aryl hydrocarbon receptor and application thereof. BACKGROUND

[0002] Osteoarthritis (OA) is the most common joint disease in the elderly, and the incidence of OA is increasing year by year. It is reported that about 40% of people over 70 years old suffer from OA. There is still a lack of effective drugs for OA treatment, and end-stage patients have to undergo joint replacement surgery, which brings heavy economic and psychological burden to families and society. Therefore, finding effective targets for treating OA is of great significance for treating OA. In recent years, scientists have found that more and more metabolites are involved in the pathogenesis of OA, but the research on metabolites in the joint cavity microenvironment is not complete. These metabolites are directly produced by joint cartilage tissue or metabolized by joint surrounding tissues such as synovial membrane, fat pad, etc., and generally have their fixed physiological characteristics and are involved in some important physiological processes. In the process of inflammation or aging, the production or degradation of some metabolites changes, and they accumulate in the joint cavity microenvironment, promote the expression of matrix metalloproteinase-3 (MMP3), matrix metalloproteinase-13 (MMP13), etc. in cartilage tissue, and decrease the expression of Aggrecan (Agg) and Collagen Type II Alpha 1 (Col2a1). Cartilage aging and damage further affect bone remodeling under the cartilage, which is the bone structure under the cartilage layer and provides mechanical support and nutrient supply for the cartilage. Under the condition of OA, bone remodeling under the cartilage is intensified, accompanied by the invasion of H-type blood vessels and nerves, which aggravates cartilage destruction and pain.

[0003] The present application prevents the combination of Kyn, an intermediate product in the metabolism of tryptophan (Trp), and its receptor-AHR at the cellular level and in the joint cavity, prevents Kyn from playing a downstream effect of promoting joint damage, and protects the joint from damage. SUMMARY

[0004] To achieve the above object, the present application is implemented by the following technical scheme: a polypeptide for treating osteoarthritis by targeting aryl hydrocarbon receptor AHR, the amino acid sequence of the polypeptide is GFQQSDVIHQSVYELIHTEDRA.

[0005] Preferably, the polypeptide is prepared by solid-phase polypeptide synthesis.

[0006] Preferably, the specific steps of the solid-phase polypeptide synthesis method are as follows: Synthesis from C-terminal to N-terminal, the carboxyl group of the first amino acid is connected to an insoluble resin, and then other amino acids are added one by one to form a peptide chain, and each step is accompanied by side chain protection and deprotection reactions; After adding an amino acid each time, the resin is thoroughly washed to remove unreacted reagents and byproducts, and after the entire peptide chain is assembled, a deprotection reaction is performed to release the synthesized polypeptide; The polypeptide is cleaved from the resin and purified using high-performance liquid chromatography technology; Mass spectrometry is used to analyze and characterize the structure and composition of the polypeptide, and the purified polypeptide is stored by freeze-drying; Wherein the C-terminal is the carboxyl terminal, and the N-terminal is the amino terminal.

[0007] Preferably, the polypeptide blocks the binding of Kyn to AHR and inhibits AHR-mediated inflammatory response and cartilage matrix degradation, thereby protecting the joint cartilage from damage.

[0008] Preferably, the polypeptide forms hydrogen bonds with the amino acid residues GLU158, ASP150, SER149, SER67, GLN154 of AHR, with distances of 2.9 Å, 3.2 Å, 3.9 Å, 4.1 Å, and 3.0 Å, respectively, and forms a salt bridge with the ARG70 residue of AHR with a distance of 3.7 Å, and is hydrophobic with the LEU159, ALA10, PHE146, and VAL151 residues.

[0009] Preferably, the polypeptide is used to act with Kyn at the cellular level, inhibiting the expression of matrix metalloproteinase 3 and matrix metalloproteinase 13 induced by Kyn, while promoting the expression of cartilage matrix proteins collagen type 2 and chondroitin sulfate.

[0010] Preferably, the polypeptide is used for cell experiments with wild-type mice.

[0011] Preferably, the wild-type mice are 3-5 day old newborn wild-type mice, and the joint cartilage tissue is isolated, digested with collagenase and trypsin, and then a single cartilage cell suspension is prepared, the cells are plated, and after the cells are fully grown, the polypeptide pre-incubated cartilage cells are formed and passaged for use.

[0012] Preferably, the polypeptide stored by the freeze-drying method is ground into a powder and dissolved in dimethyl sulfoxide, and the polypeptide pre-incubated cartilage cells are given at 10 nM and 20 nM, respectively, and then stimulated with 100 mg / ml Kyn for 24 hours, and the changes in the expression of matrix metalloproteinase 3 and matrix metalloproteinase 13 induced by Kyn are detected by Western Blots.

[0013] Preferably, after the polypeptide is administered to pre-incubate the chondrocytes, the joint tissue is taken after the operation, and then the joint tissue is subjected to decalcification, paraffin embedding and sectioning treatment, and then the degree of joint cartilage damage is evaluated by using the safranin-fast green staining method; And the joint damage level of different experimental groups is scored using the OARSI scoring system.

[0014] The application provides a polypeptide for treating osteoarthritis by targeting AHR and an application thereof. 1、The polypeptide in the application is composed of natural amino acids, has good biocompatibility and degradability, and experimental results show that no obvious toxic reaction is found in cell and animal levels, which indicates that the polypeptide has high safety, and the polypeptide has conditions for further developing pharmacokinetic research and preclinical tests, and provides a feasibility basis for clinical application.

[0015] 2、The polypeptide provided in the application can block the combination of Kyn and AHR, and experimental results show that the polypeptide has an effect better than that of a known AHR inhibitor SR1, and has less systemic side effects, and there is no drug for treating osteoarthritis by targeting AHR in the current clinic, so the polypeptide is effective and reduces systemic toxic side effects, and provides a new technical approach for treating osteoarthritis.

[0016] 3、The polypeptide used in the application has the characteristics of small molecular weight, clear structure and batch preparation by a solid-phase synthesis method, so that the polypeptide is convenient for structural modification for different target sequences, and the polypeptide with cartilage targeting ability can be designed according to needs, so that the systemic exposure and non-targeting effect are reduced under the premise of ensuring the drug efficacy. DETAILED DESCRIPTION

[0017] Figure 1 It is a schematic diagram of the effect of Kyn on chondrocytes, macrophages and hemangioblasts in the application; Figure 2 It is a schematic diagram of the effect of kyn on arthritis in mice in the application; Figure 3 It is a schematic diagram of the AHR table of arthritis patients and models in the application; Figure 4 It is a schematic diagram of the three-dimensional interaction between the target protein AHR and the ligand Kyn in the application; Figure 5 It is a schematic diagram of the polypeptide relieving arthritis in mice in the application. DETAILED DESCRIPTION

[0018] The technical solutions of the present application will be described clearly and completely below in combination with the drawings of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0019] Please refer to the drawings of the present application Figure 1 - the drawings of the present application Figure 5 The embodiments of the present application provide a polypeptide for treating osteoarthritis by targeting AHR, and the amino acid sequence of the polypeptide is GFQQSDVIHQSVYELIHTEDRA.

[0020] Specifically, osteoarthritis (OA) is the most common joint disease in the elderly. Scientists have found that more and more metabolites are involved in the pathogenesis of OA, but the research on metabolites in the joint cavity microenvironment is not complete. Under the OA state, the bone remodeling of subchondral bone is intensified, accompanied by the invasion of H-type blood vessels and nerves, which aggravates the destruction of cartilage and pain. Tryptophan metabolism plays a complex regulatory role in the body. Studies have found that kynurenine (Kyn), an intermediate product of tryptophan (Trp) metabolism, is prone to accumulate in the joint cavity, acts on the articular cartilage, accelerates the production of matrix metalloproteinases MMP3 and MMP13, speeds up the loss of collagen, and promotes the occurrence and development of arthritis. Its target aryl hydrocarbon receptor (AHR) is expressed at a higher level in the cartilage of OA patients and in the articular cartilage of Destabilization of the Medial Meniscus (DMM) operation mice. It is proved that the accumulation of Kyn in the joint cavity mediates the progression of osteoarthritis.

[0021] Tryptophan is the substrate of protein synthesis and many biological metabolites in vivo, which has important physiological functions and is involved in the development of many diseases. There are three main pathways of tryptophan metabolism: 1) Kyn metabolism pathway; 2) serotonin (5-hydroxytryptamine, 5-HT) metabolism pathway; 3) indole metabolism pathway. In previous experiments, we found that the concentration of Kyn in serum increased significantly at 2 and 4 weeks after DMM surgery due to meniscus instability causing articular cartilage damage, while the concentrations of other tryptophan metabolites did not change significantly. We also found that the concentration of Kyn increased in the synovial fluid of OA patients. By treating primary chondrocytes with different concentrations of Kyn, we found that Kyn activated the Kyn target aryl hydrocarbon receptor (AHR) in a concentration-dependent manner, significantly reduced the levels of collagen Col2a1 and Aggrecan, and increased the expression levels of matrix-degrading enzymes MMP3 and MMP13, thereby accelerating cartilage destruction. In the joint cavity microenvironment, there is a large number of macrophage infiltration in synovial tissue, and the number of macrophages increases significantly in the inflammatory environment of OA. In cultured macrophages, we found that Kyn increased macrophage migration. In the pathological condition of OA, the infiltration of H-type blood vessels in subchondral bone is also one of the pathological phenotypes of OA. We cultured HUVEC cells, and found that Kyn increased HUVEC tube formation and accelerated HUVEC migration in a concentration-dependent manner. Therefore, we speculate that Kyn exacerbates cartilage and surrounding cell damage at the cellular level; To further verify the role of Kyn in OA, we injected Kyn into the joint cavity to observe its effect on OA in vivo. The results showed that 20 mg / kg Kyn significantly exacerbated articular cartilage destruction in DMM mice after 4 weeks of intra-articular injection, and the ratio of hyaline cartilage (HC) to calcified cartilage (CC) decreased. Micro-CT showed that the bone volume / total volume (BV / TV) and trabecular number (Tb.N) of subchondral bone in the Sham group were significantly reduced. After 8 weeks of Kyn administration, cartilage destruction was further exacerbated, and Kyn more obviously accelerated the progression of cartilage destruction. These results indicate that Kyn significantly accelerates articular cartilage destruction in vivo; Previous studies have shown that Kyn acts on its receptor AHR. To further verify the role of AHR in OA, we detected the expression level of AHR in the articular cartilage of OA patients and DMM mice. The results showed that the expression level of AHR increased significantly in both OA patients and DMM mice under the inflammatory state of OA, suggesting that AHR plays a key role in Kyn-induced cartilage destruction. We have identified that Kyn significantly exacerbates cartilage destruction through AHR, but it is not feasible to knock out AHR in humans, and the existing inhibitors are unknown for safety, forcing us to find effective drugs to inhibit AHR, especially biological agents that can prevent its ligands from entering the articular cartilage. Small molecule polypeptides are short-chain compounds formed by connecting 2-50 amino acids through peptide bonds, with the characteristics of small molecular weight, easy absorption, high biological activity, and widely involved in physiological regulation. Small molecule polypeptides are ubiquitous in nature and living organisms, with structures between amino acids and proteins. According to the number of amino acids, they can be divided into oligopeptides and polypeptides, of which 2-10 amino acids are called oligopeptides, and 10-50 amino acids are called polypeptides. Compared with proteins, small molecule polypeptides have smaller molecular weight and simpler spatial structure, and their biological activity is closely related to the amino acid sequence. Polypeptides with specific sequences can exert effects similar to hormones, neurotransmitters, or enzyme inhibitors. For example, glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine, with antioxidant function; enkephalin is an endogenous analgesic pentapeptide. In the field of medicine, small molecule polypeptide drugs have been used for the treatment of diabetes, tumors, immune regulation, etc., such as insulin analogues, thymopentin, and the global hit semaglutide. Some small molecule polypeptides can also be used as nutritional supplements, such as collagen peptides, which are helpful for skin health. The popular polypeptide preparations on the market are mostly agonists of receptor classes, and so far polypeptide products as therapeutic drugs are still blank in the field of OA, while polypeptide drugs will be the mainstream direction of future treatment. Our previous research has developed a polypeptide that blocks the binding of Kyn to AHR, and its effectiveness has been verified in mouse experiments; The polypeptide can block the interaction between Kyn and AHR at the molecular level by binding to the AHR target point, thereby avoiding the activation of the downstream signaling pathway caused by the combination of Kyn and AHR. The polypeptide is designed based on the molecular docking results of Kyn-AHR, can competitively bind to AHR, and block the interaction between Kyn and AHR, thereby avoiding the downstream signaling pathway reaction triggered by the activation of AHR by Kyn. Kyn is an intermediate product of tryptophan metabolism, which significantly accumulates in the articular cartilage tissue in the pathological state of osteoarthritis, can induce the activation of AHR, promote the expression of matrix metalloproteinase 3 and matrix metalloproteinase 13, and inhibit the generation of type II collagen and cartilage aggregation protein, leading to the degradation of cartilage matrix and the destruction of joint structure. The polypeptide can reduce the binding capacity of Kyn by binding to the key amino acid residue site of AHR, and weaken the AHR signal transduction. The cell experiment verifies that the polypeptide can significantly inhibit the activation of AHR and the expression change of downstream related proteins induced by Kyn; in the animal experiment, the articular cavity injection can improve the subchondral bone structure and reduce the cartilage destruction. The polypeptide realizes the inhibition of the Kyn-AHR pathway in a clear molecular binding blocking manner, has the characteristics of clear structure, strong targeting and being obtainable by chemical synthesis, and provides an implementable technical means for the molecular intervention of osteoarthritis.

[0022] The polypeptide is prepared by solid-phase polypeptide synthesis.

[0023] Specifically, the solid-phase polypeptide synthesis method uses insoluble resin as a carrier for step-by-step assembly, which can remove unreacted components and by-products after each reaction step through washing, thereby effectively controlling the specificity of the reaction and the consistency of the reaction site. In the synthesis process, side chain protection and deprotection reactions can be used to prevent non-specific condensation and improve the accuracy of peptide chain assembly. The solid-phase polypeptide synthesis method has clear operating conditions and controllable reaction process, is suitable for the preparation of polypeptides with long amino acid sequences and complex structures, and can ensure the repeatability and high purity of the product.

[0024] The specific steps of the solid-phase polypeptide synthesis method are as follows: Synthesis from C-terminal to N-terminal, the carboxyl group of the first amino acid is connected to the insoluble resin, and then other amino acids are added one by one to form a peptide chain, and each step is accompanied by side chain protection and deprotection reactions; After adding one amino acid each time, the resin is thoroughly washed to remove unreacted reagents and by-products, and after the entire peptide chain is assembled, a deprotection reaction is performed to release the synthesized polypeptide; The polypeptide is cleaved from the resin and purified using high-performance liquid chromatography technology; Mass spectrometry is used to analyze and characterize the structure and composition of the polypeptide, and the purified polypeptide is stored by freeze-drying; The C-terminal is the carboxyl terminal, and the N-terminal is the amino terminal.

[0025] Specifically, by connecting amino acid monomers in turn from the carboxyl end to the amino end, and coordinating with side chain protection and deprotection reactions during synthesis, the reaction site can be effectively limited to prevent non-specific condensation, ensuring the correct assembly of the peptide chain. After each reaction, thorough washing can remove unreacted reagents and byproducts, thereby reducing impurity formation and improving product purity. After the peptide chain assembly is complete, the target polypeptide is released by deprotection reaction, and then purified by high performance liquid chromatography and verified by mass spectrometry analysis, to obtain a target polypeptide with a clear structure and accurate molecular weight. The purified polypeptide is stored by freeze-drying, which can maintain its chemical stability and biological activity. The polypeptide sample with accurate structure, high purity and good reproducibility can be obtained in a short synthesis period, which is suitable for the preparation of sequence-specific polypeptides designed in the present application.

[0026] The application of the polypeptide in treating osteoarthritis, the polypeptide blocks the binding of Kyn to AHR and inhibits the inflammatory response and cartilage matrix degradation mediated by AHR, so as to protect the articular cartilage from damage.

[0027] Specifically, the polypeptide can competitively block the interaction of Kyn with AHR by binding to AHR, thereby reducing the activation of downstream signaling pathways by Kyn. Kyn is an intermediate product in the tryptophan metabolic pathway and can induce inflammatory response and cartilage matrix degradation under the pathological conditions of osteoarthritis. The polypeptide can inhibit AHR-mediated inflammatory signaling by blocking the binding of Kyn to AHR, reduce the expression of matrix metalloproteinase 3 and matrix metalloproteinase 13, maintain the structural stability of the extracellular matrix of chondrocytes, prevent the decrease of type II collagen and cartilage aggregate protein content, thereby reducing cartilage degeneration and abnormal remodeling of subchondral bone, and playing a role in protecting the structural integrity of articular cartilage.

[0028] The polypeptide forms hydrogen bonds with the amino acid residues GLU158, ASP150, SER149, SER67, GLN154 of AHR, with a distance of 2.9 Å, 3.2 Å, 3.9 Å, 4.1 Å, and 3.0 Å, respectively, and forms a salt bridge with the ARG70 residue of AHR with a distance of 3.7 Å, and hydrophobic with LEU159, ALA10, PHE146, and VAL151 residues.

[0029] Specifically, the three-dimensional structure file of the target protein AHR is found and downloaded using the Uniptot database. The molecular structure file of the ligand Kyn is downloaded using the Pubchem database. The optimal conformation of the small molecule is optimized by molecular mechanics using Chem3D software, and the optimal conformation with minimum energy is finally obtained.

[0030] Docking process: The target protein and the target protein were pretreated using AutoDockTools 1.5.6 to obtain a pdbqt file, and the target protein and the ligand molecule were simulated and docked using AutoDockVinav.1.2.0 software, the docking algorithm was Lamarck genetic algorithm, the docking mode was semi-flexible docking, the exhaustiveness was set to 8, the maximum number of output conformations was set to 9, and the docking results are shown in Table 1 below; The binding energy is less than -5 kcal / mol, the binding is excellent, and the binding is strong less than -7 kcal / mol. Therefore, the small molecule and AHR can be stably combined, and the docking results are processed by 3D visualization using PyMOL2.3.0 software; Visual analysis shows that kynurenine forms hydrogen bond interactions with GLU158, ASP150, SER149, SER67, GLN154 of AHR, with hydrogen bond distances of 2.9 Å, 3.2 Å, 3.9 Å, 4.1 Å and 3.0 Å, respectively; at the same time, it forms a salt bridge with arginine residue 70, with a distance of 3.7 Å; and forms a hydrophobic interaction with leucine residue 159, alanine residue 10, phenylalanine residue 146 and valine residue 151. The existence of multiple point interactions improves the stability and affinity of the polypeptide and AHR binding, so that the polypeptide can occupy the Kyn binding site in a higher binding energy state, thereby achieving competitive blocking of Kyn and AHR binding at the molecular level, reducing AHR-mediated inflammatory signal activation and cartilage matrix degradation response, providing a molecular binding basis for subsequent joint cartilage protection in cell and animal experiments. After the sequence is determined, in cooperation with Wuhan Tiande Biotechnology Co., Ltd., we refer to the software optimization and determine the sequence of the polypeptide GFQQSDVIHQSVYELIHTEDRA. This polypeptide is named AHR binding Peptide (ABP) and can inhibit the binding of kyn and AHR.

[0031] Inhibiting the expression of matrix metalloproteinase 3 and matrix metalloproteinase 13 induced by Kyn, while promoting the expression of cartilage matrix proteins collagen type 2 and chondroitin sulfate.

[0032] Specifically, by reducing the content of matrix metalloproteinase 3 and matrix metalloproteinase 13, the decomposition rate of proteoglycans and collagen in the extracellular matrix of cartilage cells can be reduced, thereby reducing the cartilage degradation process. At the same time, promoting the synthesis of collagen type II and cartilage aggregate protein helps to maintain the integrity and elastic characteristics of the extracellular matrix in the cartilage tissue, so that the cartilage structure maintains a normal functional state. Achieving inhibition of Kyn-induced degenerative signals at the cellular level, maintaining the dynamic balance of cartilage metabolism, and providing a clear basis for preventing cartilage degeneration of osteoarthritis.

[0033] The polypeptide is subjected to a cell experiment by using a wild type mouse.

[0034] Specifically, the polypeptide is subjected to a cell experiment by using a wild type mouse. The cartilage cells derived from a wild type mouse are used as the experimental object, and the cell reaction consistent with the physiological state can be obtained under the condition that the genetic background is stable and no exogenous gene intervention is performed, thereby ensuring the authenticity and repeatability of the experimental data. The polypeptide provides a reliable basis for verifying the regulation of the AHR related pathway under the natural physiological condition.

[0035] The wild type mouse is a 3-5 day old newborn wild type mouse, and the articular cartilage tissue is isolated. After being digested by collagenase and trypsin, a single cartilage cell suspension is prepared, and the cells are plated to form polypeptide pre-incubated cartilage cells and passaged for use.

[0036] Specifically, the experiment uses the articular cartilage tissue derived from a 3-5 day old newborn wild type mouse for cell separation and culture. The single cartilage cell suspension is obtained by combined digestion of collagenase and trypsin, which can improve the sufficiency of tissue digestion and the dispersion of cells under the premise of maintaining the biological characteristics of cells, and obtain a large number of primary cartilage cells with high activity. The obtained cells are plated and cultured to form a monolayer of cartilage cells after adherent growth, thereby providing a uniform cell model for subsequent polypeptide pre-incubation experiments. The cells are passaged after being fully grown to maintain the proliferation ability and phenotype stability of the cells, thereby ensuring the repeatability and comparability of the results of polypeptide action experiments. The polypeptide pre-incubated cartilage cells prepared have high consistency in structure and function with the in vivo cartilage cells, and can be used to evaluate the molecular effect of the polypeptide under Kyn stimulation, thereby providing an experimental basis for subsequent in vitro verification.

[0037] The polypeptide stored by freeze-drying is ground into powder and dissolved with dimethyl sulfoxide. The cartilage cells are pre-incubated with 10 nM and 20 nM polypeptide, respectively, and then stimulated with 100 mg / ml Kyn for 24 hours. The expression changes of matrix metalloproteinase 3 and matrix metalloproteinase 13 induced by Kyn are detected by Western Blots.

[0038] Specifically, freeze-drying and powdering can maintain the structural stability of the polypeptide, prevent degradation during storage and dissolution, and thus ensure the accuracy of the polypeptide concentration in the solution and the repeatability of the experiment. Using dimethyl sulfoxide as a solvent can improve the solubility of the polypeptide in the culture system, so that it can fully contact with the cells. Different concentrations of polypeptide pre-incubation settings can help to determine the dose-dependent relationship of the polypeptide to the Kyn-induced effect. The expression of matrix metalloproteinase 3 and matrix metalloproteinase 13 is detected by Western Blotting, which can directly reflect the degree of inhibition of the polypeptide on the degradation signal pathway mediated by Kyn, thereby providing verification basis for evaluating the molecular effect of the polypeptide in the in vitro cartilage protection.

[0039] After the polypeptide is given to pre-incubate the chondrocytes, the joint tissue is decalcified, paraffin-embedded and sectioned, and then the safranin-fast green staining method is used to evaluate the degree of joint cartilage damage; And the OARSI scoring system is used to score the joint damage level of different experimental groups.

[0040] Specifically, after the polypeptide is given to pre-incubate the chondrocytes, the joint tissue is decalcified, paraffin-embedded and sectioned, and then the safranin-fast green staining method is used to evaluate the tissue structure of the joint cartilage, which can effectively remove the inorganic salt components in the bone tissue, so that the staining agent fully penetrates into the cartilage matrix area, thereby clearly showing the cartilage cell hierarchy and matrix distribution in the section. The safranin-fast green staining can distinguish between hyaline cartilage and calcified cartilage tissue, so that the cartilage damage area and the normal area have clear boundaries in the microscopic structure, which is convenient for quantitative or semi-quantitative analysis of the damage degree. By using the OARSI scoring system to score the sections of different experimental groups, the changes in the cartilage structure of the polypeptide treatment group and the control group can be compared under standardized indicators, and the inhibitory effect of the polypeptide on joint cartilage damage under the stimulation of Kyn can be objectively reflected. Combined with histological observation and quantitative evaluation, it is used to judge the actual influence of the polypeptide on the cartilage protection effect in the osteoarthritis model.

[0041] Table 1 Top four docking results for affinity Protein Compound Binding Energy (kcal·mol -1 )]]> AHR kynurenine -6.1 ; Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A polypeptide for treating osteoarthritis against AHR, characterized in that, The amino acid sequence of the polypeptide is GFQQSDVIHQSVYELIHTEDRA.

2. The polypeptide for treating osteoarthritis by targeting AHR according to claim 1, wherein, The polypeptide is prepared by solid-phase polypeptide synthesis.

3. The polypeptide for treating osteoarthritis with AHR according to claim 1, characterized in that, The specific steps of the solid-phase polypeptide synthesis are as follows: From C-terminal to N-terminal, the carboxyl group of the first amino acid is connected to an insoluble resin, and then other amino acids are added one by one to form a peptide chain, and each step is accompanied by side chain protection and deprotection reactions; After adding one amino acid at a time, the resin is thoroughly washed to remove unreacted reagents and byproducts, and after the entire peptide chain is assembled, a deprotection reaction is performed to release the synthesized polypeptide; The polypeptide is cleaved from the resin and purified using high-performance liquid chromatography technology; Mass spectrometry is used to analyze and characterize the structure and composition of the polypeptide, and the purified polypeptide is stored by freeze-drying; The C-terminal is the carboxyl terminal, and the N-terminal is the amino terminal.

4. Use of a polypeptide according to any one of claims 1 to 3 for the treatment of osteoarthritis, characterized in that, The polypeptide blocks the binding of Kyn to AHR, inhibits AHR-mediated inflammatory response and cartilage matrix degradation, and protects the joint cartilage from damage.

5. Use according to claim 4, characterized in that, The polypeptide forms hydrogen bonds with the amino acid residues GLU158, ASP150, SER149, SER67, GLN154 of AHR, with distances of 2.9 Å, 3.2 Å, 3.9 Å, 4.1 Å, and 3.0 Å, respectively, and forms a salt bridge with the ARG70 residue of AHR with a distance of 3.7 Å, and also undergoes hydrophobicity with the LEU159, ALA10, PHE146, and VAL151 residues.

6. Use according to claim 4, characterized in that, The polypeptide is used to act with Kyn at the cellular level, inhibits the expression of matrix metalloproteinase 3 and matrix metalloproteinase 13 induced by Kyn, and promotes the expression of cartilage matrix proteins collagen type 2 and chondroitin sulfate.

7. The polypeptide for treating osteoarthritis with AHR according to claim 2, characterized in that, The polypeptide is subjected to a cell experiment by wild-type mice.

8. The polypeptide for treating osteoarthritis by targeting AHR according to claim 7, wherein, The wild-type mice use 3-5 day old wild-type mice, isolate joint cartilage tissue, digest with collagenase and trypsin, prepare a single chondrocyte suspension, plate the cells, and after they grow, form polypeptide pre-incubated chondrocytes and pass them on.

9. The polypeptide for treating osteoarthritis with AHR according to claim 7, characterized in that, After the polypeptide stored by freeze-drying is ground into a powder and dissolved in dimethyl sulfoxide, 10 nM and 20 nM polypeptide pre-incubated chondrocytes are administered, and then 100 mg / ml Kyn is used to stimulate the cells for 24 hours, and the changes in the expression of matrix metalloproteinase 3 and matrix metalloproteinase 13 induced by Kyn are detected by Western Blots.

10. The polypeptide for treating osteoarthritis with AHR according to claim 7, characterized in that, After the polypeptide pre-incubated chondrocytes are administered, the joint tissue is decalcified, paraffin-embedded, and sectioned, and then the Safranin O-Fast Green staining method is used to evaluate the degree of joint cartilage damage; And the OARSI scoring system is used to score the joint damage levels of different experimental groups.

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