Application of chrysin in the preparation of a drug for treating osteoarthritis

Abstract

The present invention belongs to the field of medicine, and discloses the application of chrysin in the preparation of a drug for treating osteoarthritis. The key target for chrysin to treat osteoarthritis is PPIA. Chrysin upregulates the expression of the key gene PPIA, improves the activity of chondrocytes, promotes the proliferation of chondrocytes, and affects the degeneration of chondrocytes to delay the progression of osteoarthritis, providing a new direction for the research and development of new drugs for osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of chrysin in the preparation of a drug for treating osteoarthritis. Background Art

[0002] Osteoarthritis is an orthopedic chronic disease that affects multiple joints, such as the knee joint, hip joint, wrist joint, etc. The main pathological changes include the degeneration of articular cartilage, osteophyte formation, changes in synovial fluid and joint capsule, manifested as symptoms such as joint pain, joint stiffness, joint swelling, joint deformation, etc. The exact cause of osteoarthritis is still unclear, but it is related to age, genetics, joint injury, obesity, lifestyle factors, and inflammatory joint diseases. Treatment methods include drug management, physical therapy, exercise and weight management, and surgical correction or joint replacement surgery when necessary. Advanced osteoarthritis seriously affects people's quality of life. Early intervention and management can help relieve pain, improve joint function, and enhance the quality of life. There are some limitations in the treatment methods of osteoarthritis because there is currently no method to reverse the degeneration of articular cartilage, and the treatment mainly focuses on symptom management, such as methods to relieve pain, stabilize the joint, and joint replacement. Commonly used drugs include non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, analgesics such as paracetamol, etc. And some drugs are considered to be able to improve the health of articular cartilage, but their effects are still under research, such as glucosamine and chondroitin sulfate. However, the treatment effects of these drugs are poor and they have side effects. Therefore, there is an urgent need to develop new drugs to relieve the degeneration of articular cartilage.

[0003] Periplaneta americana is considered a global health pest to humans and has attracted much attention because it is commonly found in polluted environments and may carry various bacteria, viruses, parasites, etc. that spread diseases. However, in recent years, some studies have found that Periplaneta americana also has potential effects in medicine and plays an important role in the treatment of diseases. The extract components of Periplaneta americana are used to treat a variety of diseases, including digestive system diseases, respiratory system diseases, cardiovascular diseases, etc. This may be because Periplaneta americana contains a variety of bioactive substances, such as polypeptides, proteins, enzymes, etc. These substances are considered to have various pharmacological activities such as antibacterial, anti-inflammatory, antioxidant, and antitumor activities, which contribute to the treatment and recovery of diseases.

[0004] Kangfuxin Liquid is extracted from Periplaneta americana and has been used in clinical treatment of various diseases, including but not limited to digestive system, respiratory system, and cardiovascular system diseases, such as the treatment of gastric bleeding, duodenal ulcer, trauma and other diseases. This may be because Kangfuxin Liquid contains a variety of bioactive components, such as polypeptides, proteins, and enzymes, etc. These components are considered to have various pharmacological activities, such as antibacterial, anti-inflammatory, antioxidant, and antitumor effects, which contribute to promoting the treatment and recovery of diseases.

[0005] Chrysin was first extracted from Kangfuxin Liquid prepared from Periplaneta americana. It is a plant flavonoid with a molecular structure consisting of two benzene rings (A and B) and an oxygen-containing heterocyclic ring, having hydroxyl groups on carbon atoms 5 and 7. Different from other flavones, it has no oxygen atom on ring A. Different substitutions of oxygen atoms on ring A result in various derivatives of chrysin, such as baicalein, baicalein and oroxylin A. Chrysin is widely present in nature, such as propolis, honey, passion fruit, etc., and is also widely used in pharmacological research. Chrysin has functions such as antioxidant, anti-apoptotic, anti-inflammatory, anti-tumor, etc. A variety of flavonoid compounds such as chrysin have significant anti-anxiety and anti-depressant activities in mammals and non-mammals, similar to clinically effective anti-anxiety drugs. Chrysin exerts an antioxidant effect through its protective effect on the neurodegeneration of a mouse model of Parkinson's disease induced by 6-hydroxydopamine, by scavenging free radicals and regulating the production of BDNF. Chrysin affects the tumor process by inhibiting the recruitment of the key NHEJ factor 53BP1 by inhibiting the expression of programmed death ligand 1. Therefore, we speculate that chrysin has a potential therapeutic effect on osteoarthritis. The present invention is based on this speculation for research. Summary of the Invention

[0006] The object of the present invention is to provide the use of chrysin in the preparation of a drug for treating osteoarthritis, which improves the activity of chondrocytes, promotes the proliferation of chondrocytes, and affects the degeneration of chondrocytes to delay the progression of osteoarthritis by upregulating the expression of the key gene PPIA.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides the use of chrysin in the preparation of a drug for treating osteoarthritis.

[0009] In the above technical solution, the key target for chrysin to treat osteoarthritis is PPIA.

[0010] In the above technical solution, chrysin upregulates the expression of the key gene PPIA, improves the activity of chondrocytes, and promotes the proliferation of chondrocytes.

[0011] The present invention also provides a drug for treating osteoarthritis containing chrysin or a derivative that is converted into chrysin in vivo.

[0012] The beneficial effects of the present invention are as follows: Through network pharmacology, the present invention screened out the key target PPIA of osteoarthritis and chrysin, and found that the expression of PPIA was down-regulated in chondrocytes induced by IL-1β, and the expression of PPIA was up-regulated after treatment with chrysin, indicating that chrysin improved the activity of chondrocytes by up-regulating the expression of the key gene PPIA. Chrysin promoted the proliferation of chondrocytes and affected the degradation of chondrocytes by acting on the key target PPIA, thereby delaying the progression of osteoarthritis, providing a new direction for the research and development of new drugs for osteoarthritis. Brief Description of the Drawings

[0013] Figure 1 is the prediction experiment of the action targets of Chrysin,

[0014] Among them, (A) The action targets of Chrysin were predicted by 4 compound target prediction databases, (B-C) GO enrichment analysis of 332 action targets of Chrysin, (D) KEGG enrichment analysis of 332 action targets of Chrysin;

[0015] Figure 2 is the screening and functional analysis of OA-related differential genes,

[0016] Among them, (A-B) The 10 most significant up-regulated and down-regulated differential genes among the differentially expressed genes in OA, (C-D) The results of GSE enrichment analysis of the differential genes, (E) KEGG enrichment analysis of the differential genes, (F) GO enrichment analysis of the differential genes;

[0017] Figure 3 is the relationship between OA-related differential genes and immune infiltration,

[0018] Among them, (A) The correlation between OA-related differential genes and immune infiltration, (B) OA-related differential genes have significant correlations with T cellsCD4 memory resting, T cells regulatory (Tregs), Monocytes, and Dendritic cellsactivated;

[0019] Figure 4 is the experiment on the potential action targets of Chrysin in regulating the progression of OA,

[0020] Among them, (A) the intersection of the action targets of Chrysin and OA-related genes, (B) using the STRING database combined with the Cytoscape 3.8.2 software to draw the PPI network of the action targets of Chrysin and OA-related genes, (C) using the CytoNCA and MCODE functions in the Cytoscape 3.8.2 software to find the key cluster genes in the PPI network, (D) ELISA results show that the expression of PPIA decreases in the supernatant of chondrocytes induced by IL-1β, (E) KEGG enrichment analysis of Key clustergenes, (F) GO enrichment analysis of Key cluster genes, ****P<0.0001;

[0021] Figure 5 It is the docking study and related verification of Chrysin and its target molecule PPIA.

[0022] Among them, (A) Binding model of Chrysin and PPIA molecular active pocket, (B) Amino acid residues of the covalent bond between Chrysin and PPIA protein, the red dotted line indicates the covalent bond, and the figure is drawn by the Pymol software, (C-D) CCK8 experiment shows that Chrysin inhibits the proliferation of chondrocytes. When the concentration of Chrysin is 15uM and the action time is 72h, the action effect is the best, (E) Elisa experiment shows that Chrysin promotes the expression of PPIA in damaged chondrocytes, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0023] Figure 6 It is the calculation result of the binding free energy of MM / PBSA. Specific implementation mode

[0024] In order to better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will convey the concept of the present invention fully to those skilled in the art. The present invention will be defined only by the claims.

[0025] 1. Experimental process

[0026] 1. Screening of Chrysin targets

[0027] Search for the most matching drug information of chrysin in PubChem, and then download the chrysin-related targets in 4 drug databases (http: / / ctdbase.org / , http: / / www.swisstargetprediction.ch / , http: / / bindingdb.org / bin d / index.jsp, http: / / targetnet.scbdd.com / home / index / ). Use VENN software to perform Venn analysis on the drug targets of the four databases, and perform GO and KEGG analyses on all drug target genes through the microsignal website (http: / / www.bioinformatics.com.cn / ).

[0028] 2. Screen differential genes of osteoarthritis

[0029] Download the osteoarthritis dataset GSE48556 from the Gene Expression Omnibus (GEO) database (http: / / www.ncbi.nlm.nih.gov / geo / ). To find the differential genes of osteoarthritis, we used 100 linear models of the limma package for microarray data in R, and selected genes that met the criteria of |log2 fold change (FC)| > 0 and P value < 0.05 as DEGs. Use "xiantao" (www.xiantao.love) to visualize the differential genes.

[0030] 3. Functional enrichment

[0031] Use the GO (Gene Ontology) database: The functions of genes are divided into three categories: biological process (BP), cellular component (CC), and molecular function (MF). The KEGG (Kyoto Encyclopedia of Genes and Genomes) database is used to annotate the differential genes. These analyses are performed using the clusterProfiler package in R 4.2.1 for enrichment analysis, and the org.Hs.eg.db package is used for ID conversion. Use "xiantao" (www.xianta o.love) to visualize the results of GO and KEGG enrichment analyses.

[0032] 4. GSEA analysis

[0033] Perform Gene Set Enrichment Analysis (GSEA) on the differential genes of osteoarthritis. After performing ID conversion on the molecules in the input data using the gene set database: MSigDB Collections (https: / / www.gsea-msigdb.org / gsea / msigdb / collections.jsp), perform enrichment analysis using the clusterProfiler package. Use the bioinformatics analysis tool of "xiantao" ( www.xiantao.love ) for visualization.

[0034] 5. Immune infiltration

[0035] Download the osteoarthritis dataset GSE48556 from the GEO database (http: / / www.ncbi.nlm.nih.gov / geo / ), which includes 33 normal cases and 106 osteoarthritis cases. Use the "reshape2" package in R language to process and analyze the data, and calculate the infiltration abundance of the normal group and the osteoarthritis group. Use the R package "ggplot2" to draw the infiltration map of the immune cell differences between the above groups. The Spearman algorithm is used for the correlation between immune cells with significantly different infiltration abundances between the two groups.

[0036] 6. PPI network of osteoarthritis and chrysin targets

[0037] Construct a PPI network for 1725 differential genes related to OA and 201 chrysin action targets on the string software, import it into the CytoNCA mode in the Cytoscape software for degree analysis, then screen out the Keycluster in the MCODE mode, and perform GO and KEGG analysis on the Keycluster using the bioinformatics analysis tool "xiantao" (www.xiantao.love).

[0038] 7. Molecular docking of chrysin and key target genes and calculation of MM / PBSA binding free energy

[0039] Obtain the three-dimensional structure of the main active ingredient of chrysin from the PubChem data (https: / / pubchem.ncbi.nlm.nih.gov / ). The three-dimensional structure of the corresponding target protein comes from the RCSB PDB database (www.rcsb.org / ). Use the PyMOL 2.4.0 software to remove the solvent and separate the original ligand in the target protein. Use the AutoDock tool to determine the active pocket of these target proteins. The PyMOL 2.4.0 software is used for the visualization of the docking results.

[0040] Docking programs provide a simplified scoring function for predicting the binding affinity of ligands to receptors. However, these programs have some limitations in accurately predicting binding energy. To validate the binding affinity of the compounds under study, we employed the Molecular Mechanics Poisson-Boltzmann Surface Area (MM / PBSA) and Molecular Mechanics Generalized Born Surface Area (MM / GBSA) methods. These methods have been implemented in AMBER MM / PBSA.

[0041] 8, CCK-8

[0042] Chondrocytes were seeded in 96-well plates, with 5×10 ^3 cells per well, and cultured at 37 °C for 12 hours. The next day, 10 μL of CCK-8 solution (Solarbio, CA1210) was added to each independent experimental well and incubated at 37 °C for 1 hour. Then, the absorbance value was read using a Bio-RAD microplate at a 450 nm absorbance filter.

[0043] 9, ELISA

[0044] Samples were seeded in 96-well plates, the anti-PPIA antibody was added to the wells, and then incubated overnight at 4 °C to allow the antigen or antibody to adsorb onto the plate. The plate was washed 3 times with buffer, blocked with blocking solution bovine serum albumin, the treated samples were added, incubated, the plate was washed again with buffer, and the fluorescently labeled antibody was added. Incubated, the plate was washed with buffer, the substrate was added, and the signal intensity was measured using a microplate reader, and the absorbance value was read at a wavelength of 450 nm.

[0045] II Experimental Results

[0046] 1. Prediction of the action targets of Chrysin

[0047] To screen for the action targets of Chrysin, we predicted a total of 332 action targets of Chrysin through 4 compound target prediction databases (http: / / ctdbase.org / , http: / / www.swisstargetprediction.ch / , http: / / bindingdb.org / bind / index.jsp, http: / / targetnet.scbdd.com / home / index / ). After removing redundant targets and creating a VENN diagram, we obtained 7 key targets ( Figure 1 A). Next, we performed functional enrichment on these 332 genes.

[0048] GO enrichment analysis based on BP, CC, and NFs showed that these 332 Chrysin action targets were mainly related to positive regulation of MAPK cascade, positive regulation of protein kinase activity, positive regulation of MAPK cascade, G protein-coupled serotonin receptor signaling pathway, phospholipase C-activating G protein-coupled receptor signaling pathway, and adenylate cyclase-activating G protein-coupled receptor signaling pathway, etc.( Figure 1 B-C). In addition, according to the KEGG pathway enrichment analysis, the 332 genes were mainly enriched in Nitrogen metabolismTh17, cell differentiation, MAPK signaling pathway, Th1and Th2 cell differentiation, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, Drug metabolism-cytochrome P450, Osteoclast differentiationIL-17 signaling pathway and other pathways( Figure 1 D).

[0049] 2. Differentially expressed genes in osteoarthritis

[0050] We analyzed the expression profiles of each sample in the GSE48556 dataset. After gene annotation and removal of duplicate gene symbols, we obtained 1725 differentially expressed genes, including 747 up-regulated genes and 978 down-regulated genes( Figure 2 A-B). Among them, the 10 most significantly up-regulated differentially expressed genes were GZMH, HBA2, PRF1, EDG1, GPR18, ID3, FAM43A, FLJ14213, ADRB2, and HSPA1B( Figure 2A), the 10 most significantly down-regulated differential genes were H3F3B, SNORD13, IL8, LOC649841, C6orf111, EGR1, TRIB1, RPS4Y1, ATHL1, and ZNF148( Figure 2 A). Next, we performed functional enrichment analysis on these differential genes. The GSE enrichment results showed that these genes were related to Cell Cycle, DNA Replication, Activation of ATR In Response To Replication Stress, G2 M Checkpoints, and MitoticG1 Phase and G1 S Transition, etc.( Figure 2 C-D). KEGG analysis showed that these differential genes were enriched in signal pathways such as positive regulation of cell adhesion regulation of protein modification, Tcell receptor signaling pathway, and T cell differentiation( Figure 2 E). GO enrichment analysis showed that the 747 key genes related to OA were mainly related to positive regulation of cell adhesion, positive regulation of cytokine production, leukocyte cell-cell adhesion, and regulation of T cell activation, etc.( Figure 2 F).

[0051] Notably, the 747 key genes related to OA were related to B cells memory, B cells Dendritic cells resting, Eosinophils, Macrophages M0, Macrophages M1, MacrophagesM2, Mast cells activated, Mast cells resting, Monocytes, Neutrophils, NK cellsactivated, NK cells resting, Plasma cells, T cells CD4 memory activated, T cellsCD4 memory resting, T cells CD4 CD8 T cells, follicular helper T cells, gamma delta T cells, and regulatory T cells (Tregs) are correlated. Figure 3 A). Among them, these genes are significantly correlated with CD4 memory resting T cells (p < 0.001), regulatory T cells (Tregs) (p = 0.0462), monocytes (p = 0.04567), and activated dendritic cells (p < 0.001). Figure 3 B).

[0052] 3. PPI network of key cluster genes

[0053] We performed VENN mapping on 1725 OA-related differential genes and 201 Chrysin action targets, with a total of 1715 genes counted, including 11 overlapping genes. The intersection of drug targets and disease genes suggests that Chrysin may be a drug for treating OA. Figure 4 A). We used the STRING database to Figure 4 perform PPI network analysis on all genes in A to predict protein interactions. Figure 4 B), and found the core gene PPIA in the key-cluster. Figure 4 C). Moreover, we found that the expression of PPIA decreased in IL-1β-induced chondrocytes, indicating that PPIA is one of the markers of damaged chondrocytes. Figure 4 D). PPIA serves as a key target for Chrysin to treat OA.

[0054] We performed functional enrichment analysis on key-cluster genes. The KEGG results showed significant enrichment in pathways such as steroid hormone biosynthesis, linoleic acid metabolism, arachidonic acid metabolism, DNA adducts, chemical carcinogenesis, drug metabolism cytochrome P450, metabolism of xenobiotics, and serotonergic synapse. Figure 4E). GO enrichment analysis showed that the key-cluster genes were mainly related to olefinic compound metabolic process, long-chain fatty acid metabolic, arachidonic acid metabolic process, steroid metabolic process, unsaturated fatty acid metabolic process, etc. Figure 4 F).

[0055] 4. Molecular docking study of Chrysin with its target PPIA and related verification

[0056] Chrysin achieved deep binding with PPIA in the active pocket and produced abundant hydrophilic and hydrophobic contacts. Figure 5 A - B). Chrysin docked deeply in the pocket and formed hydrogen bond contacts with GLY-71, GLN110, and GLN-62. Figure 5 B). The binding free energy was calculated by MM / PBSA, and the detailed information of the binding free energy was as Figure 6 shown. After co-culturing Chrysin with chondrocytes through the CCK8 experiment, we found that Chrysin had a promoting effect on the proliferation of chondrocytes. Figure 5 C). When the concentration was 15 μM and co-cultured for 72 h, the proliferation of chondrocytes was obvious. Figure 5 D). The results of the Elisa experiment also demonstrated that Chrysin promoted the proliferation of chondrocytes. Figure 5 E).

[0057] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of this invention.

Claims

1. Use of chrysin in the preparation of a reagent for non-therapeutically increasing the expression of peptidylprolyl isomerase A in chondrocytes.

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