CN118903119A - Application of transcription factor ZBTB20 inhibitor in preparation of medicines for preventing and/or treating osteoarthritis - Google Patents

Abstract

The application relates to the field of osteoarthritis treatment, and particularly discloses application of a transcription factor ZBTB20 inhibitor in preparation of a medicament for preventing and/or treating osteoarthritis. Treatment of chondrocytes with the transcription factor ZBTB20 inhibitor TRULI inhibits nuclear translocation of ZBTB20, which is located in the cytosol and inhibits its activity. The compound TRULI can inhibit activation of inflammation signal in chondrocyte and restore cartilage matrix steady state. The co-culture of the compound TRULI can reduce the matrix loss phenomenon of the in vitro cultured cartilage tissue. The injection TRULI through the joint cavity can obviously relieve pain and dyskinesia caused by osteoarthritis, delay the damage of articular cartilage and have a therapeutic effect on the osteoarthritis.

Description

Use of transcription factor ZBTB20 inhibitors in the preparation of drugs for preventing and/or treating osteoarthritis

Technical Field

The present specification relates to the field of osteoarthritis treatment, and in particular to the use of transcription factor ZBTB20 inhibitors in the preparation of drugs for preventing and/or treating osteoarthritis.

Background Art

Osteoarthritis (OA) is a chronic disease that is prevalent in the elderly, seriously affecting the daily life and work of patients. It is currently one of the main causes of disability in the elderly. Osteoarthritis is a pan-joint disease that involves structural changes in articular cartilage, ligaments, joint capsules, synovium, and periarticular muscles, among which articular cartilage degeneration is one of the main pathological features. Currently, the clinical treatment of OA mainly relies on symptomatic treatments such as oral non-steroidal anti-inflammatory drugs (NSAIDs) and glucosamine to relieve joint swelling and pain, but most patients in the advanced stage still need surgical treatments such as joint replacement to restore joint function.

At present, the drug treatment of OA includes topical application, oral medication, and intra-articular injection. Topical NASIDs have a certain analgesic effect, but the effect is not as good as oral NASIDs. Oral medications include NSAIDs analgesics, opioid analgesics, slow-acting drugs that improve the condition, etc., but oral NSAIDs have potential adverse reactions in the gastrointestinal, cardiovascular and renal systems. Oral opioids have headaches, dizziness, drowsiness and possible addiction. Slow-acting drugs that improve the condition include glucosamine, chondroitin sulfate, diacerein, etc., and their effects are still controversial. Intra-articular injection drugs include sodium hyaluronate and glucocorticoid injections. Intra-articular sodium hyaluronate injection plays a physical role of lubrication by supplementing the content of hyaluronic acid in the joint, and has a certain effect of relieving pain, but its role in cartilage protection and delaying disease progression is still controversial; intra-articular injection of glucocorticoids relieves pain and swelling to a certain extent, but has no effect on delaying disease progression, and long-term use has a cartilage-destroying effect. Currently, drug treatment has a certain effect in relieving pain and restoring function, but it is still difficult to delay, prevent or even reverse the progression of the disease. Paying attention to the damage mechanism of articular cartilage and subchondral bone tissue, delaying cartilage damage and repairing cartilage have become one of the focuses of OA drug treatment research.

ZBTB20 (Zinc finger and BTB domain containing 20), a member of the BTB/POZ transcription factor family, participates in the regulation of multiple physiological processes such as metabolism, immune response and tumorigenesis by directly regulating the transcription level of target genes, and plays an important role in the development of the body and the maintenance of metabolic homeostasis. In cartilage development, ZBTB20 regulates the terminal differentiation process of hypertrophic chondrocytes by inhibiting the expression of Sox9 in them, but its function in articular chondrocytes is still unclear.

Current drugs for the treatment of osteoarthritis can relieve pain and swelling to a certain extent, but they cannot delay, prevent or even reverse cartilage damage, causing most patients in the advanced stage to still need surgical treatments such as joint replacements to restore joint function. In addition, there are many surgical complications and uncertain efficacy.

Summary of the invention

In order to solve the above problems, the present application provides the use of a transcription factor ZBTB20 inhibitor in the preparation of a drug for preventing and/or treating osteoarthritis.

The present application also provides a pharmaceutical composition for treating osteoarthritis, comprising the transcription factor ZBTB20 inhibitor for the above purpose and a pharmaceutically acceptable carrier.

The beneficial effects brought about by this application include but are not limited to: (1) Compound TRULI treatment of chondrocytes can inhibit the nuclear translocation of ZBTB20, causing it to be located in the cytoplasm and inhibiting its activity. (2) Compound TRULI treatment of chondrocytes can inhibit the activation of inflammatory signals in chondrocytes and restore the homeostasis of cartilage matrix. (3) Adding compound TRULI for co-culture can reduce the loss of matrix in cultured cartilage tissue in vitro. (4) Intra-articular injection of TRULI can significantly reduce the pain and movement disorders caused by osteoarthritis, delay articular cartilage damage, and has a therapeutic effect on osteoarthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

The present application will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not restrictive, and include:

Figure 1. Transcription factor ZBTB20 plays an important role in the early development of osteoarthritis in joints. (A) Immunofluorescence staining of ZBTB20 in the Sham/DMM group at 2/4/8 weeks; (B) Statistical results of fluorescence signals and intracellular localization in (A); (C) Safranin O-fast green staining of articular cartilage sections in the Sham and DMM groups of the control group and Zbtb20-cKO mice; (D) OARSI scores, HC thickness, CC thickness, and HC/CC ratio of articular cartilage in each group in (C). The degree of articular cartilage damage was quantified by the OARSI score, and the higher the score, the greater the degree of damage. As OA progresses, the thickness of hyaline cartilage (HC) in the articular cartilage decreases, the thickness of calcified cartilage (CC) increases, and the HC/CC ratio decreases. (E) The manifestations of OA also include the formation of osteophytes and the increase in the thickness of the subchondral bone plate, which can be obtained by microCT analysis. MicroCT reconstruction images of the joints of the control group mice and the sham group and DMM group of Zbtb20-cKO mice. (F) (E) Statistical results of osteophyte volume and subchondral bone plate thickness in each group.

Figure 2 ZBTB20 regulates NF-κB signaling and cartilage matrix homeostasis in chondrocytes. The ability of OA joint chondrocytes to synthesize extracellular matrix mainly composed of type II collagen and proteoglycans (COL II, ACAN) is reduced, while the secretion of proteolytic enzymes including MMP13 and ADAMTS5 increases, resulting in an imbalance between extracellular matrix synthesis and catabolism. Therefore, the expression levels of COLII, ACAN, MMP13, and ADAMTS5 can be detected to evaluate the homeostasis of cartilage matrix decomposition and synthesis. (A) Immunofluorescence staining images of COL II, ACAN, MMP13, and ADAMTS5 in articular cartilage sections of the sham group and DMM group of the reference group mice and Zbtb20-cKO mice. (B) Statistical results of fluorescence signals in (A). The intracellular localization of transcription factor p65 can reflect the activation of the NF-κB signaling pathway: p65 nuclear localization indicates the activation of the NF-κB signaling pathway. (C) Immunofluorescence staining images of p65 in articular cartilage sections of the control group mice and the sham and DMM groups of Zbtb20-cKO mice. (D) Immunofluorescence staining images of p65 in primary chondrocytes transfected with Ad-mZbtb20-GFP and Ad-GFP. (E) Statistical results of fluorescence signals in (D).

Figure 3 Compound TRULI has the effect of inhibiting the nuclear translocation of ZBTB20. (A) Immunofluorescence staining images of ZBTB20 in primary chondrocytes treated with the reference group and IL-1β for 24 hours; (B) Statistical results of fluorescence signals in (A) Figure. (C) WB results of nuclear and cytoplasmic separation samples in each group; (D) Statistical results of ZBTB20 abundance in each cell component in (C) Figure; (E) Immunofluorescence staining images of ZBTB20 in primary chondrocytes treated with the reference group, IL-1β, and IL-1β+TRULI; (F) Statistical results of fluorescence signals in (E) Figure.

Figure 4 Compound TRULI has the effect of inhibiting the activation of inflammatory signals and restoring the homeostasis of cartilage matrix. The ability of OA joint chondrocytes to synthesize extracellular matrix mainly composed of type II collagen and proteoglycans (COL II, ACAN) is reduced, while the level of secretion of proteolytic enzymes including MMP3, MMP13, ADAMTS5 increases, leading to an imbalance in the synthesis and catabolism of extracellular matrix. Therefore, the detection of COL II, ACAN, MMP13, and ADAMTS5 expression levels can evaluate the changes in cartilage matrix homeostasis. (A) WB results of IL-1β and TRULI-treated cell samples; (B) mRNA levels of Mmp3, Mmp13, Col2a1, and Acan in IL-1β and TRULI-treated cells; (C) Immunofluorescence staining images of COL II in primary chondrocytes treated with IL-1β and TRULI; (D) Statistical results of fluorescence signals in (C) Figure. The intracellular localization of transcription factor p65 can reflect the activation of the NF-κB signaling pathway: p65 nuclear localization indicates the activation of the NF-κB signaling pathway; (E) Immunofluorescence staining of p65 in primary chondrocytes treated with IL-1β and TRULI; (F) Statistical results of the fluorescence signal in (E) Figure.

FIG5 Compound TRULI has the effect of treating osteoarthritis. (A) Schematic diagram of the grouping and dosing schedule of TRULI in vivo administration for the treatment of OA, as shown in FIGA, 10-week-old C57BL/6 male mice were randomly divided into 4 groups, namely: 1_Sham group: a group in which the right knee received sham surgery; 2_Vehicle group: a group in which the right knee of mice received DMM surgery to create an OA model and received solvent injections at the same frequency and dose as the treatment group; 3_TRULI_2W: a group in which the right knee of mice received DMM surgery to create an OA model and then began to receive knee joint cavity TRULI injections 2 weeks later, with an injection frequency of twice a week; 4_TRULI_0W: a group in which the right knee of mice received DMM surgery to create an OA model and then began to receive knee joint cavity TRULI injections immediately, with an injection frequency of twice a week. Gait analysis was performed at 4, 6, and 8 weeks after surgery, and the knee joints were sampled and analyzed 8 weeks after surgery; (B) Statistical results of gait analysis of mice in each group. Figure B shows the statistical results of the ratio of various indicators (footprint angle, ground contact area, swing time, ground contact time) of the right leg (modeling side)/left leg (contralateral side) of mice in each group at each time point. The size of the dot represents -Log10(padj), and the larger the dot, the more significant the difference; the color of the dot indicates the size of the ratio, red means right side/left side>1, and purple means right side/left side<1; (C) Histological staining images of articular cartilage of mice in each group; (D) Statistical results of articular cartilage degeneration in (C); (E) Histological staining images of cartilage tissue cultured in vitro; (F) Statistical results of cartilage matrix loss in cartilage tissue cultured in vitro.

DETAILED DESCRIPTION

In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and/or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

The present application provides the use of a transcription factor ZBTB20 inhibitor in the preparation of a drug for preventing and/or treating osteoarthritis.

Transcription factors are a class of proteins that can bind to DNA molecules and regulate the transcription process of genes. Transcription factors can enhance or inhibit gene expression by recognizing specific DNA sequences. They play a vital role in cell differentiation, development, metabolism, and response to environmental changes.

Inhibitors are compounds or biomolecules that can reduce or block the activity of a biomolecule. They can be small molecule drugs, proteins, RNA molecules, or other types of molecules.

Transcription factor inhibitors are drugs that target the activity of transcription factors, which can regulate gene expression and have an important impact on many biological processes and diseases. Transcription factor inhibitors can interfere with the binding of transcription factors to DNA or their function, thereby inhibiting the expression of specific genes.

The term "prevention and/or treatment" (and grammatical variations thereof) refers to an attempt to alter the natural course of a disease in a treated individual, and may be a clinical intervention performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis.

In some embodiments, the transcription factor ZBTB20 inhibitor may be TRULI.

The compound TRULI, with a molecular formula of C ₁₈ H ₁₄ N ₄ OS and a molecular weight of 334.39, is an ATP-competitive kinase inhibitor that can promote the proliferation of mouse inner ear supporting cells, mouse cardiomyocytes, and Muller glial cells in human retinal organoids.

The structural formula of compound TRULI is:

In some embodiments, in the drug, the transcription factor ZBTB20 inhibitor can inhibit the nuclear translocation of the transcription factor ZBTB20 in chondrocytes, so that the transcription factor ZBTB20 is located in the cytoplasm, thereby inhibiting its transcription function.

In some embodiments, the drug can be used to inhibit the activation of inflammatory signals in chondrocytes and restore cartilage matrix homeostasis.

In some embodiments, the drug can be used to restore the homeostasis of cartilage matrix synthesis and catabolism. In some embodiments, preferably, the drug can inhibit the activation of inflammatory signals in chondrocytes by regulating the intracellular localization of ZBTB20, thereby restoring the homeostasis of cartilage matrix synthesis and catabolism.

In some embodiments, the medicament can be used to reduce cartilage tissue matrix loss.

In some embodiments, the drug can be used to inhibit the expression of cartilage matrix decomposing enzymes and promote the synthesis of cartilage matrix components.

In some embodiments, the drug can be used to alleviate osteoarthritis articular cartilage degeneration, injury, pain or impaired mobility. In some embodiments, preferably, the impaired mobility can include an increased angle of the affected side footprint, a decreased ground contact time and area.

In some embodiments, the drug can be used to restore bone and joint function, prevent aggravation of articular cartilage damage, and delay the progression of osteoarthritis. In some embodiments, preferably, the drug can restore bone and joint function, prevent aggravation of articular cartilage damage, and delay the progression of osteoarthritis by acting on early chondrocytes of osteoarthritis.

In some embodiments, the drug can be administered into the joint cavity, and preferably, the drug can be administered into the joint cavity by injection.

The present application also provides a pharmaceutical composition for treating osteoarthritis, comprising the transcription factor ZBTB20 inhibitor for the above purpose and a pharmaceutically acceptable carrier.

The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredients contained therein to be effective, and which contains no components which are unacceptably toxic to a subject to which the composition would be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is non-toxic to an individual. A pharmaceutically acceptable carrier may be a commonly used drug carrier, such as poly(ethylene glycol), including poly(ethylene glycol) having a molecular weight ranging from about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400 or PEG600), ethylene glycol, propylene glycol, glycerin, nonionic surfactant, tyloxapol, polysorbate 80, polyethylene glycol-15-hydroxy, stearate, phospholipids, lecithin, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, cyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxyethyl-γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin, dihydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, sulfobutyl ether-γ -cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, diglucosyl-β-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, maltotriosyl-β-cyclodextrin, maltotriosyl-γ-cyclodextrin, dimaltosyl-β-cyclodextrin, methyl-β-cyclodextrin, carboxyalkyl sulfide, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, vinyl acetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate or any combination thereof. Those skilled in the art can generally determine the formulation of the drug based on the dosage form requirements.

In some embodiments, the pharmaceutical composition of the present application can be administered in a monotherapy (e.g., without concomitant administration of any additional therapeutic agent, or without concomitant administration of any additional therapeutic agent against the same disease to be treated or prevented with the pharmaceutical composition of the present application). In some embodiments, the pharmaceutical composition of the present application can also be administered in combination with one or more other therapeutic agents or administered simultaneously.

The present application also provides a method for treating osteoarthritis, which comprises administering an effective amount of the pharmaceutical composition of the present application to a subject in need thereof.

The term "effective amount" refers to an amount or dosage of the pharmaceutical composition of the present invention that produces the desired effect in the treated patient after being administered to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of the mammal; its size, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific drug administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

In the present application, the pharmaceutical composition of the present application can be administered to a patient. A person skilled in the art can determine the appropriate mode of administration and dosage. In some embodiments, preferably, the pharmaceutical composition can be administered by intra-articular injection.

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

The relevant test methods in the examples of this application are as follows:

Reagents and Materials

1. Experimental Animals

P0 mice (used to extract primary chondrocytes) and wild-type C57BL/6 male mice were obtained from the Experimental Animal Center of the Air Force Medical University. All mice in this project were raised in the Experimental Animal Center of the Air Force Medical University, and their experimental environment met the requirements of the SPF sterile culture environment. All animal experimental operations in this project complied with the "Experimental Animal Welfare and Ethics".

2. Main reagents and consumables

TRULI:MCE, China

DNA extraction kit: Bio-Tech, China

2×Taq Master Mix: Vazyme, China

4% formaldehyde fixative: Zhonghui Hecai Biopharmaceutical Company, China

10% EDTA decalcification solution: Boster Biological Company, China

Sucrose: Tianjin Tianli Chemical Reagent Company, China

PBS powder: Wuhan Boster Biotechnology Co., Ltd., China

Cryoembedding medium: Leica, Germany

Improved Safranin O-Fast Green Cartilage Staining Solution: Beijing Solebow Technology Co., Ltd., China

Tamoxifen: Sigma

Immunofluorescence staining strong permeabilization solution: Bio-Tech Biotechnology, China

Immunofluorescence permeabilization solution: Biotime Biotechnology, China

QuickBlockTM Immunofluorescence Blocking Buffer/Primary Antibody/Secondary Antibody Diluent: Bio-Tech Biotechnology, China

DAPI staining solution: Thermo Fisher Scientific, US

Mounting fluid: Polysciences, USA

Fetal bovine serum: Hyclone

DMEM low glucose medium: Gibco, USA

Collagenase D: Roche, Switzerland

IL-1β: PeproTech, USA

RNA extraction kit: Takara, Japan

ReverTra qPCR RT Master Mix: TOYOBO, Japan

Green RT PCR Master Mix: TOYOBO, Japan

BeyoGelTM Plus PAGE precast gel: Beyotime Biopharmaceuticals, China

Electrophoresis buffer: Bio-Tech Biotechnology, China

Genshare CFAS Fast Transfer Buffer 10x: Zhonghui Hecai Biopharmaceutical Co., Ltd., China

20TBST buffer: Solebao Technology Co., Ltd., China

ZBTB20/MMP13 Antibody:Proteintech

ACAN antibody:Millipore

ADAMTS5 antibody:Abcam

COLII/p65 Antibody:Thermo

Experimental methods:

1. Genetic mice

In order to obtain Zbtb20 chondrocyte-inducible knockout mice, this study used chondrocyte-inducible expression Col2a1-CreER ^T2 mice to breed with Zbtb20 ^f/f mice to obtain Col2a1-CreER ^T2 ; Zbtb20 ^f/f mice.

Tamoxifen was injected intraperitoneally to knock out Zbtb20. Each mouse needs to be injected with 30 μL of tamoxifen each time, and the injection needs to be continued for 5 days. 1 mL of tamoxifen injection solution uses 50 mg of tamoxifen powder, 950 μL of corn oil and 50 μL of anhydrous ethanol, and vortexed and ultrasonicated to mix thoroughly. Store at -20℃ away from light.

2. Construction of mouse OA model by DMM

Before the operation, all instruments used were simply disinfected, and mice were anesthetized with 1% sodium pentobarbital by intraperitoneal injection. The surgical site of the mouse was shaved with a razor, and the surgical site was wiped with alcohol cotton pads. The mouse joint was placed under a stereoscope, and the field of view and focus were adjusted. The skin was cut from top to bottom on the inner side of the patellar ligament with a scalpel (about 5mm incision), and the incision was carefully deepened until the joint capsule was exposed. The joint capsule was cut open, and the position was adjusted to expose the joint cavity. After finding the meniscus and determining the position of the meniscus, the ligament connected to the meniscus was gently severed with a scalpel. Finally, the joint was sutured.

3. Knee joint harvesting and related treatment

After the mice were killed by injecting an overdose of anesthesia, the hind limbs were removed and fixed with 4% paraformaldehyde, washed with PBS after 3 days and replaced with decalcification solution. The decalcification was continued for two weeks, and the decalcification solution was replaced once a week. Finally, the samples were dehydrated with 30% sucrose for frozen sections.

4. Safranin staining of mouse joint sections

The mouse joints were frozen sections with a thickness of 8 μm, and stained with the Bio-Tech modified safranin staining kit, AB solution, acid differentiation solution, fast green staining solution, weak acid solution and safranin-O staining solution, respectively, and finally xylene was used for transparency and resin sealing.

5. Immunofluorescence staining of mouse joint sections

Mouse joints were frozen and sectioned at 8 μm thick. The sections were permeabilized with Beyotime immunofluorescence staining strong permeation solution, and then immunostained with Beyotime immunofluorescence staining blocking solution for 1 hour at room temperature. After blocking, the sections were incubated with primary antibody at 4°C overnight. After the primary antibody incubation, the sections were washed with PBS and fluorescently labeled secondary antibodies were used to visualize specific proteins. Finally, the cell nuclei were stained with DAPI. The sections were sealed with anti-quenching sealing agent. The staining results were observed using a laser confocal microscope.

6. microCT Analysis

After the tissue was fixed with 4% paraformaldehyde, it was placed in the SKYSCAN 1276 instrument and scanned according to the parameter settings. Subsequently, the scan files were reconstructed and analyzed using three-dimensional reconstruction processing software and dedicated bone analysis software.

7. Extraction and in vitro culture of primary chondrocytes

The rib cartilage cells of P0 mice were obtained and digested with collagenase D for 40 minutes to treat the soft tissue. The calcified cartilage was then removed and the rib cartilage was digested into chondrocytes by treating with collagenase D for 4 hours. The cells were cultured in DMEM low-glucose medium containing 10% fetal bovine serum.

8. Construction of OA model by treating cells with inflammatory factors

IL-1β is an important inflammatory factor in osteoarthritis articular cartilage degeneration. This study used IL-1β to induce an OA inflammation model in vitro. Preliminary experimental results showed that 1ng/mL IL-1β can induce an inflammatory response in primary chondrocytes.

9. Adenovirus Transfection of Primary Chondrocytes

The viruses used in this study were adenovirus vectors (Ad-mZbtb20-GFP) and control virus vectors (Ad-GFP) designed and prepared by Hanheng Biotechnology (Shanghai) Co., Ltd. The cells were infected when the confluency was 60%. Half of the serum-free medium was used for treatment for 6 hours, and the solution was supplemented. After 2 hours, the virus suspension was replaced with normal serum-containing culture medium. Culture for 48-72 hours.

10. Immunofluorescence staining of primary chondrocytes

Collect cells and fix them in 4% PFA for 15 minutes, permeabilize them with Beyotime immunofluorescence staining strong permeation solution, and then use Beyotime immunofluorescence staining blocking solution for immunostaining blocking at room temperature for 1 hour. After blocking, incubate with primary antibody at 4°C overnight. After the primary antibody incubation, wash with PBS and use fluorescently labeled secondary antibodies to visualize specific proteins. At the same time, the cell nucleus is stained with DAPI. Finally, the anti-quenching sealing agent is used to seal the slice. The staining results are observed using a laser confocal microscope.

11. Western Blotting

Wash the culture dish with PBS, lyse the cells on ice for 30 minutes with Denature Lysis Buffer containing protease inhibitors and phosphatase inhibitors, scrape the protein solution with a cell scraper and place it in an EP tube, boil it at 95°C for 8 minutes, add 5× loading buffer, boil it at 95°C for 10 minutes, and store it directly at -20°C.

Select SDS-PAGE gel of appropriate concentration according to the size of the target protein, load 3-6μl for electrophoresis, then transfer the protein to PVDF membrane at 400mA, block with skim milk for 1 hour, add primary antibody and incubate overnight at 4℃, wash with TBST, add HRP-labeled secondary antibody for 1 hour, and develop after washing with TBST.

12. RNA Extraction

Wash the culture dish with PBS, add 350μl Buffer RL (add 50% TDT solution in advance) to the well plate, and then purify RNA according to the TAKARA kit experimental steps. Wash with 75% ethanol, RWA, and RWB in sequence, and finally elute RNA with 50μl RNase Free dH2O.

13. Fluorescence quantitative qPCR

Reverse transcription of RNA: reaction system: 4DN Master 2μl + RNA 6μl, incubate at 37℃ for 5min. Add 2μl 5DNMaster, incubate at 37℃ for 15min, 50℃ for 5min, 98℃ for 5min, store at 4℃.

Reaction system (20 μl):

Add the real-time PCR system in each tube into a specific 96-well plate and place it in a real-time quantitative PCR instrument for amplification.

14. Statistical Analysis

The experimental results in this paper were repeated 3 times or more. GraphPad Prism 9.5.0 software was used to perform statistical analysis and plot all experimental data, and all data were expressed as mean ± standard error. P < 0.05 was considered statistically significant.

Example 1 - Transcription factor ZBTB20 plays an important role in articular chondrocytes in the progression of osteoarthritis

As shown in Figure 1A-B, a mouse osteoarthritis model was established by destabilization of the medial meniscus (DMM). Samples were collected and analyzed at 2/4/8 weeks after surgery. ZBTB20 was significantly nuclearized in the articular chondrocytes of mice 2 weeks after DMM surgery, and the expression of ZBTB20 in the articular cartilage of mice 4-8 weeks after DMM surgery was significantly reduced, indicating that ZBTB20 was first transported into the nucleus in the early stage of OA, and then the expression level gradually decreased with the progression of OA, suggesting that it may play an important role in the early stage of OA. As shown in Figure 1C-F, compared with the control group mice, the articular cartilage damage of Zbtb20 chondrocyte-specific knockout mice after DMM surgery was milder, and the growth of osteophytes and the remodeling of subchondral bone plates were all reduced. In summary, ZBTB20 plays an important role in articular chondrocytes in the early stage of osteoarthritis.

In Figure 1D, the OARSI score is a scoring system used to assess the severity of cartilage damage in osteoarthritis (OA) and is scored based on the degree of damage to the articular cartilage, including the degree of cartilage loss, surface irregularities, cracks, and sclerosis. Grade 0: The surface is intact, the cartilage surface is smooth, the cells are arranged normally, and there is no obvious proliferative change. Grade 1: Fibrosis appears on the surface of the articular cartilage without other damage. Grade 2: Vertical cracks extend down to the layers below the surface and some loss of the surface layer. Grade 3: Vertical cracks/erosions in the calcified cartilage extend to <25% of the articular surface. Grade 4: Calcified cartilage has vertical cracks/erosions that extend to 25-50% of the articular surface. Grade 5: Calcified cartilage has vertical cracks/erosions that extend to 50-75% of the articular surface. Grade 6: Calcified cartilage has vertical cracks/erosions that extend to >75% of the articular surface.

Articular cartilage damage is closely related to the decomposition and anabolism homeostasis of cartilage matrix. As shown in Figure 2A-B, Zbtb20 knockout enhanced the synthesis of OA cartilage matrix and inhibited the expression of OA cartilage matrix degrading enzymes, indicating that it restored the homeostasis of cartilage cell extracellular matrix synthesis and catabolism in OA articular cartilage. NF-κB signaling in chondrocytes regulates cartilage matrix homeostasis. As shown in Figure 2C-E, Zbtb20 knockout inhibited the activation of NF-κB signaling in OA articular cartilage, while overexpression caused NF-κB signaling activation, indicating that ZBTB20 caused cartilage matrix disorder by activating the NF-κB signaling pathway and aggravated cartilage degeneration. In summary, these results indicate that the transcription factor ZBTB20 plays an important role in the early stage of OA, activating the NF-κB signaling pathway in chondrocytes, resulting in aggravated articular cartilage damage.

Example 2—Compound TRULI has the effect of inhibiting ZBTB20 nuclear translocation

In vitro OA models were constructed by stimulating primary chondrocytes cultured in vitro with IL-1β. As shown in Figure 3A-D, ZBTB20 was diffusely distributed in the control group cells, and ZBTB20 was translocated into the nucleus in the articular chondrocytes treated with IL-1β, indicating that ZBTB20 was significantly translocated into the nucleus in the early stage of OA stimulated by inflammatory factors. As shown in Figure 3E-F, the cells were further treated with the compound TRULI, which significantly inhibited the nuclear translocation of ZBTB20 in OA chondrocytes, indicating that the compound TRULI has the effect of inhibiting the nuclear translocation of ZBTB20.

Example 3 - Compound TRULI can delay the degeneration of articular cartilage and has the effect of treating osteoarthritis

The effect of TRULI was detected in an in vitro OA model. As shown in Figure 4A-D, TRULI can restore the level of cartilage matrix synthesis and inhibit the level of cartilage matrix degradation. As shown in Figure 4E-F, the detection of p65 subcellular localization in chondrocytes showed that TRULI can inhibit the activation of NF-κB signaling. This part of the effect is consistent with the effect of ZBTB20 knockout on chondrocytes, indicating that the mechanism of action of TRULI inhibits the nuclear entry of ZBTB20.

The therapeutic effect of TRULI was analyzed in the mouse OA model, and the administration method was intra-articular injection. The group setting is shown in Figure 5A. The pain caused by OA can lead to impaired motor ability of mice, which is manifested in the increase of the angle of the footprint on the affected side, the decrease of the ground contact time and area, etc. As shown in Figure 5B, the gait analysis results of each group of mice showed that intra-articular injection of TURLI effectively alleviated the behavioral abnormalities and impaired motor ability of OA mice. As shown in Figure 5C-D, the histological staining results showed that TURLI alleviated the damage to OA joint cartilage. In vitro culture of cartilage tissue can be used to observe and analyze the effects of drugs on the loss of cartilage matrix. As shown in Figure 5E-F, in cartilage tissue cultured in vitro, co-culture with TURLI can effectively inhibit the loss of cartilage matrix caused by in vitro culture. In summary, this part of the data shows that TURLI inhibits the loss of cartilage matrix, reduces cartilage degeneration, relieves joint pain, and has the effect of treating osteoarthritis.

The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and/or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values is as accurate as possible within the feasible range.

Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims (10)

1. Use of a transcription factor ZBTB20 inhibitor for the preparation of a medicament for the prevention and/or treatment of osteoarthritis.

2. The use of claim 1, wherein the inhibitor of transcription factor ZBTB20 is TRULI.

3. The use according to claim 1, wherein the inhibitor of the transcription factor ZBTB20 is capable of inhibiting nuclear translocation of the transcription factor ZBTB20 within chondrocytes, locating the transcription factor ZBTB20 in the cytosol, inhibiting its transcriptional function in the medicament.

4. The use according to claim 1, wherein the medicament is for inhibiting activation of inflammatory signals in chondrocytes, restoring cartilage matrix homeostasis.

5. Use according to claim 1, wherein the medicament is for restoring cartilage matrix anabolic homeostasis, preferably wherein the medicament inhibits activation of inflammatory signals in chondrocytes by modulating ZBTB20 intracellular localization, thereby restoring cartilage matrix anabolic homeostasis.

6. The use according to claim 1, wherein the medicament is for reducing cartilage tissue matrix loss.

7. The use according to claim 1, wherein the medicament is for inhibiting the expression of cartilage matrix degrading enzymes and promoting the synthesis of cartilage matrix components.

8. Use according to claim 1, wherein the medicament is for alleviating osteoarthritis articular cartilage degeneration, injury, pain or impaired locomotor ability, preferably comprising an increased angle of the affected side footprint, a decreased touchdown time and area.

9. Use according to claim 1, wherein the medicament is for restoring osteoarticular function, preventing the exacerbation of articular cartilage damage, delaying the progression of an osteoarthritic disease, preferably wherein the medicament is for restoring osteoarticular function, preventing the exacerbation of articular cartilage damage, delaying the progression of an osteoarthritic disease by acting on chondrocytes in an early stage of osteoarthritic disease.

And/or the administration mode of the medicine is joint cavity administration, preferably, the administration mode of the medicine is joint cavity injection.

10. A pharmaceutical composition for the treatment of osteoarthritis comprising a transcription factor ZBTB20 inhibitor for use according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.