Use of cpges as a target in developing or screening or preparing a drug for preventing and / or treating osteoarthritis

Abstract

This invention discloses the application of cPGES as a target in the development, screening, or preparation of drugs for the prevention and / or treatment of osteoarthritis. This invention is the first to propose cPGES as a drug target for osteoarthritis. Experiments show that intra-articular injection of cPGES knockdown virus significantly reduces joint pain in mice, protects against cartilage wear, improves bone metabolic imbalance, and inhibits the release of inflammatory factors. These results demonstrate that cPGES has a significant effect on improving osteoarthritis and can serve as a target for the treatment of osteoarthritis, which is of great significance for the future development and prevention of this type of disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of cytosolic prostaglandin E synthase (cPGES) as a target in the development, screening, or preparation of drugs for the prevention and / or treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis is a chronic degenerative joint disease characterized by progressive degeneration of articular cartilage, chronic inflammation of the synovium, abnormal subchondral bone remodeling, and osteophyte formation. Globally, approximately 500 million people are affected. With the accelerating aging of the population, osteoarthritis has become one of the leading causes of joint pain, functional impairment, and even disability in middle-aged and elderly individuals, severely impairing their quality of life and imposing a heavy personal, family, and socioeconomic burden.

[0003] Currently, clinical treatment for osteoarthritis primarily focuses on symptom relief. While commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) can provide short-term pain and inflammation reduction, long-term use is associated with adverse reactions such as gastrointestinal damage, cardiovascular risks, and liver and kidney toxicity. Chondroprotective agents, such as chondroitin sulfate, have a slow onset of action and limited efficacy in patients with advanced osteoarthritis whose structures have already undergone significant damage. The pathogenesis of osteoarthritis is complex, involving the interaction of multiple pathological processes, including the sustained release of pro-inflammatory cytokines, chondrocyte metabolic imbalance, excessive activation of extracellular matrix degrading enzymes (such as MMPs and ADAMTS), osteoclast-mediated enhanced bone resorption, and the involvement of multiple factors such as aging, mechanical stress, and metabolic abnormalities. However, the exact etiology and core molecular regulatory network of osteoarthritis are not yet fully elucidated, and there is currently a lack of radical treatments that can reverse or halt disease progression.

[0004] Therefore, in-depth analysis of the key signaling pathways and cellular metabolic interactions in the development of osteoarthritis, identification of novel molecular targets with intervention potential, and development of therapeutic strategies that combine safety, efficacy, and disease-modifying effects have become important directions that urgently need breakthroughs in the current field of osteoarthritis research. Summary of the Invention

[0005] The main objective of this invention is to provide an application of cPGES as a target in the development, screening, or preparation of drugs for the prevention and / or treatment of osteoarthritis, thereby overcoming the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: This invention provides the application of cPGES as a target in the development, screening, or preparation of drugs for the prevention and / or treatment of osteoarthritis.

[0007] This invention also provides the use of cPGES or downregulators of its encoding gene in the preparation of medicaments for the prevention and / or treatment of osteoarthritis.

[0008] This invention also provides a pharmaceutical composition for the prevention and / or treatment of osteoarthritis, comprising: cPGES or a downregulator of its encoding gene, and a pharmaceutically acceptable vector.

[0009] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention is the first to propose cPGES as a drug target for osteoarthritis. Experiments show that intra-articular injection of cPGES to knock down viruses can significantly reduce joint pain in mice, protect cartilage from wear, improve bone metabolic imbalance, and inhibit the release of inflammatory factors. These results indicate that cPGES has a significant effect on improving osteoarthritis and can serve as a target for the treatment of osteoarthritis, which is of great significance for the future drug development and prevention and treatment of this type of disease. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 This is a statistical graph showing the joint pain threshold of cPGES knockdown mice and control mice in an osteoarthritis model according to a typical embodiment of the present invention. Figure 2 This is a comparison of safranin-fast green staining in cPGES knockdown mice and control mice in an osteoarthritis model according to a typical embodiment of the present invention, and a statistical chart of OARSI scores based on safranin-fast green staining. Figure 3 This is a comparison of tartrate-resistant acid phosphatase-stained osteoclasts in a cPGES-knockdown mouse model and a control group mouse in a typical embodiment of the present invention. Figure 4 This is a comparison image and statistical analysis of Mirco-CT scans of cPGES knockdown mice and control mice in a typical embodiment of the present invention under an osteoarthritis model. Figure 5 This is a graph showing the expression levels of genes related to cartilage synthesis and metabolism after mRNA was extracted from joint tissues of cPGES knockdown mice and control mice in a typical embodiment of the present invention. Figure 6This is a graph showing the expression levels of inflammation-related genes after mRNA was extracted from joint tissues of cPGES knockdown mice and control mice in a typical embodiment of the present invention. Detailed Implementation

[0012] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention. The technical solution of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0013] Specifically, as one aspect of the technical solution of the present invention, it relates to the use of cPGES as a target in the development, screening or preparation of drugs for the prevention and / or treatment of osteoarthritis.

[0014] The drug in this invention can relieve pain associated with arthritis, protect cartilage, improve bone metabolism imbalance, and inhibit the release of inflammatory factors.

[0015] In some implementations, the arthritis is mechanically uneven induced bone and joint damage.

[0016] Furthermore, the drug can relieve pain associated with bone and joint injuries induced by uneven mechanical forces.

[0017] Furthermore, the drug can significantly inhibit abnormal osteocyte metabolism in bone and joint injuries induced by uneven mechanical force, and maintain bone balance.

[0018] Furthermore, the drug can reduce the release of inflammatory factors and the expression of inflammatory molecules in joints during bone and joint injuries induced by uneven mechanical forces.

[0019] In some preferred embodiments, when the drug is applied to a mouse model, it can inhibit the activity of cPGES in the mouse model, wherein the mouse model is a transverse anterior cruciate ligament (mouse ACLT model).

[0020] Furthermore, the mouse ACLT model involves surgically severing the anterior cruciate ligament of the knee joint in animals, sometimes accompanied by damage to the medial collateral ligament or meniscus removal, inducing joint instability, which in turn leads to cartilage degeneration and osteoarthritis.

[0021] Furthermore, when the drug is applied to a mouse model, it can alleviate joint pain in the mice.

[0022] Furthermore, when the drug is applied to a mouse model, it can promote the stabilization of cartilage structure in the mouse model.

[0023] Furthermore, when the drug is applied to a mouse model, it can improve the bone metabolism imbalance in the mouse model.

[0024] Furthermore, when the drug is applied to a mouse model, it can slow down the fibrosis process of cartilage in the joint area.

[0025] Furthermore, when the drug is applied to a mouse model, it can inhibit the release of inflammatory factors in the joint area of ​​the mouse model.

[0026] Another aspect of the present invention provides the use of cPGES or a downregulator of its encoding gene in the preparation of medicaments for the prevention and / or treatment of osteoarthritis.

[0027] In some preferred embodiments, the downregulator is selected from interfering molecules that specifically interfere with the expression of the gene encoding cPGES and / or small molecule compounds that specifically inhibit cPGES or its encoding gene.

[0028] Another aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of osteoarthritis, comprising: cPGES or a downregulator of its encoding gene, and a pharmaceutically acceptable vector.

[0029] In some preferred embodiments, the downregulator is selected from interfering molecules that specifically interfere with the expression of the gene encoding cPGES and / or small molecule compounds that specifically inhibit cPGES or its encoding gene.

[0030] Further, the downregulator comprises a cPGES knockdown virus (pAAV-U6-shRNA(Ptges3)-CMV-EGFP-WPRE(Obio)), which has the sequence shown in SEQ ID NO.1, specifically as follows: (5'to3'): GGAGAATCCGGCCAGTCATTT.

[0031] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0032] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0033] I. Experimental Procedure Laboratory animals: The male C57BL / 6 mice used in this example were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd., with the license number SCXK(Beijing)2007-0001. The mice were placed under standard conditions with a humidity of 50±10% and a temperature of 23±2°C, and were allowed to adapt to survival for 12 hours each during the day and night. The mice had free access to drinking water and food. All animal management and treatment protocols were approved by the Animal Ethics Committee of Xuzhou Medical University. All experiments were conducted in accordance with the recommendations of the Ethical Guidelines for the Care and Use of Animals.

[0034] Example 1 Construction of an osteoarthritis model C57BL / 6 mice (male, 12 weeks old) were used and randomly divided into a control group (n = 5) and a knockdown group. In the knockdown group, cPGES knockdown virus (pAAV-U6-shRNA(Ptges3)-CMV-EGFP-WPRE(Obio)) was locally injected into the joint cavity. The cPGES knockdown virus was prepared by Heyuan Biotech Co., Ltd., and its sequence was as follows: (5' to 3'): GGAGAATCCGGCCAGTCATTT. The anterior cruciate ligament transection model was used to simulate human osteoarthritis. The mice were fixed in the supine position, with the left hind limb flexed at 90°. The surgical area was thoroughly disinfected. A smooth incision was made at the knee joint, and the skin and muscles were bluntly dissected to expose the trochlear groove of the knee joint. The joint capsule was opened along the inner edge of the patellar ligament, and the intra-articular adipose tissue was bluntly dissected. The knee joint of the mouse was bent to 90° to expose the anterior cruciate ligament, which was cut with micro scissors, and a drawer test was performed to verify that the ligament had been successfully cut.

[0035] Example 2 Pain measurement experiment in mice The Von Frey filament method was classically used to measure pain in mice: A set of nylon monofilaments with calibrated intensities was used, and they were manually applied vertically to the animal's skin. The "up-and-down method" was used to determine the 50% mechanical withdrawal threshold (50% MWT).

[0036] (1) Animal adaptation: Three days before the experiment, the mice were placed in the experimental room to adapt to the environment. On the test day, the mice were individually placed in the test device (such as a transparent plexiglass box with a metal mesh bottom) and adapted for 15 - 60 minutes in a quiet and dark environment until they stopped exploring and remained relatively stationary.

[0037] (2) Equipment and environment: Check whether the Von Frey filaments are intact. Strictly control the experimental environment: The temperature is maintained at 22 - 25°C, the humidity is 40 - 60%, and the environment is quiet (noise less than 40 decibels) to avoid interference of these factors on the pain threshold.

[0038] (3) The stimulation site is the central area of the plantar surface of the hind paw, and the thick part of the footpad should be avoided.

[0039] (4) Stimulation application: Hold the monofilament of the selected strength with tweezers, press it gently perpendicular to the skin surface to bend the monofilament and hold it for 1-2 seconds before removing it.

[0040] (5) Observe the reaction: immediately observe whether the animal exhibits a clear pain response such as retracting its claws, shaking its claws, or licking its claws.

[0041] (6) “Up-down” sequence: Start with a monofilament of medium intensity. If the animal does not respond, use a monofilament of higher intensity for the next stimulation; if it responds, use a monofilament of lower intensity for the next stimulation. The stimulation interval should be at least 30 seconds.

[0042] (7) Termination and Recording: Repeat the above steps until there are two consecutive "responses" or "no responses", or the preset number of measurements is completed (e.g., 6 effective stimuli). Record the intensity of each stimulus and the animal's response (positive √ or negative ×) in detail.

[0043] (8) Threshold calculation: Using the classic Dixon formula or dedicated software, the 50% mechanical withdrawal response threshold (50% MWT) is calculated based on the last few stimulus sequences recorded by the "up and down method".

[0044] Example 3: Observation of bone tissue by safranin-fast green staining Specific experimental methods include: 1. Preparation of paraffin sections (1) Fixation of tissue specimens: Bone tissue from each group of mice in Example 1 was fixed in 4% paraformaldehyde at room temperature for 24 hours, wrapped in gauze, marked, and rinsed with running water overnight; (2) Dehydration and transparency: Place the dehydration box into the dehydrator and dehydrate it with alcohol in sequence: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, and xylene II for 5-10 minutes. (3) Paraffin infiltration and embedding: 65°C melted paraffin I for 1 hour, 65°C melted paraffin II for 1 hour, 65°C melted paraffin III for 1 hour. The paraffin-infiltrated tissues were embedded in the embedding machine. First, the molten paraffin was placed into the embedding frame. Before the paraffin solidified, the tissues were removed from the dehydration box and placed into the embedding frame according to the embedding surface requirements, and the corresponding labels were affixed. The tissues were cooled on a -20°C freezing stage. After the paraffin solidified, the paraffin blocks were removed from the embedding frame and the paraffin blocks were trimmed. (4) Sectioning and spreading: Section the slides to a thickness of 5μm using a microtome, spread them in a 50℃ water bath, pick up the slides and mount them on clean glass slides, and bake them in a 60℃ oven overnight. After sectioning, mark the slides and store them for later use.

[0045] 2. Safranin-Fixed Green Staining (1) Dewaxing and rehydration: The sections were dewaxed twice with xylene (15 minutes each time), and then dehydrated for 5 minutes each in 100%, 95%, 90%, 80%, 70%, and 50% alcohol, respectively. Finally, they were rehydrated in distilled water for 3 minutes. (2) Safranin staining: Add safranin staining solution, incubate at room temperature for 2 hours, and rinse briefly with running water; (3) Fast Green staining: Fast Green staining for about 1 minute; (4) Dehydration, clearing, and mounting: Place the sections in 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 5 minutes each, clear with xylene for 3 minutes, and then mount with neutral resin. (5) After sealing, place the slide in a 50°C oven to dry and observe the changes in each tissue structure under a light microscope.

[0046] Example 4: Observation of bone tissue by TRAP staining 1. Preparation of paraffin sections Same as method 3.1 above.

[0047] 2. TRAP staining (1) Dewaxing and rehydration: The sections were dewaxed twice with xylene (15 minutes each time), and then dehydrated for 5 minutes each in 100%, 95%, 90%, 80%, 70%, and 50% alcohol, respectively. Finally, they were rehydrated in distilled water for 3 minutes. (2) TRAP staining: Add TRAP staining solution to a 50 ml glass slide staining jar, place the slide in the jar, and incubate at 37°C in the dark for 45 minutes (if the TRAP activity in the cells is low, the incubation time can be extended to 60 minutes or the color can be developed under a microscope to the expected depth). (3) Counterstaining: stain with hematoxylin for 5-8 minutes or methyl green for 2-3 minutes as a contrast stain for cell nuclei; (4) Dehydration, clearing, and mounting: Place the sections in 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 5 minutes each, clear with xylene for 3 minutes, and then mount with neutral resin. (5) After mounting, place the slide in a 50°C oven to dry. Observe the changes in the tissue structure under a light microscope. TRAP-positive osteoclasts are red and the nuclei are blue-purple or green.

[0048] Example 5: Micro-CT scan analysis of bone structure changes in mice (1) Scanning: The sample is taken out of the fixative, the excess liquid is wiped off with gauze, the sample is placed on the instrument scanning bed and scanning is started. The original image is obtained after scanning.

[0049] (2) Reconstruction: The original image was reconstructed using the 3D reconstruction software Recon.

[0050] (3) Analysis: The target region ROI was analyzed using the data analysis software Avatar. All samples were analyzed in the same region to obtain the required parameter values ​​and export the data.

[0051] Example 6 qRT-PCR (1) Place 0.02g of tissue and 500 μL of Trizol lysis buffer into a grinding tube, add nuclease-free grinding beads, homogenize with a homogenizer until the tissue is completely broken, and let stand at room temperature for 10 minutes to allow it to fully lyse.

[0052] (2) After adding 100 μL of chloroform, shake rapidly up and down for 15 seconds, let stand at room temperature for 10 minutes, and then put the sample into a centrifuge and centrifuge at 12000 rpm for 15 minutes.

[0053] (3) Carefully transfer the upper aqueous phase to a new EP tube, add 300 μL of isopropanol and let stand at room temperature for 10 minutes. Then put the sample into a centrifuge and centrifuge at 12000 rpm for 10 minutes.

[0054] (4) After centrifugation, discard the supernatant, add 500 μL of pre-cooled 75% ethanol to the precipitate, wash it with a pipette, put the sample into a centrifuge, centrifuge at 12000 rpm for 10 minutes, and repeat once.

[0055] (5) After centrifugation, discard the supernatant, open the EP tube and place it at room temperature for 10 minutes. Add 80 μL of nuclease-free water to the tube and measure the RNA concentration.

[0056] (6) Prepare reverse transcription working solution according to Table 1.

[0057] Table 1: RNA reverse transcription reaction system

[0058] Reverse transcription conditions: 37℃, 15 min → 85℃, 5 s → 4℃ (hold); (7) Prepare the amplification system according to Table 2. The total volume of the reaction system is 10.00 μL.

[0059] Table 2: qRT-PCR reaction system

[0060] II. Experimental Results (1) Targeted knockdown of cPGES significantly improved arthritis in mice, alleviating joint pain. Osteoarthritis is the most common type of arthritis, also known as "wear and tear" of the joints, where the cartilage within the joint wears down. This causes bones to rub against each other, leading to pain, stiffness, or swelling in the joint. An osteoarthritis model was constructed by severing the anterior cruciate ligament of the knee joint in animals, and tissue samples were collected for analysis 8 weeks post-surgery. Three days before tissue collection, a pain test was performed on the mice. The results showed that the control group mice had a decreased mechanical withdrawal threshold, exhibiting typical symptoms of exacerbated arthritis pain. Conversely, the knockdown group mice had an increased mechanical withdrawal threshold, such as... Figure 1 As shown, knocking down cPGES suggests that it is a target for relieving arthritis pain.

[0061] (2) Targeted knockdown of cPGES significantly reduced cartilage structure damage. During the development and progression of osteoarthritis, significant changes in the morphology of bone tissues such as cartilage and subchondral bone are often observed. Safranin-Fix-Green staining results showed that, compared to the control group mice, the knockdown group mice exhibited more regular cartilage morphology, clearer boundaries, and significantly reduced swelling and wear (e.g., Figure 2 The OARSI scoring system can objectively and quantitatively assess the severity of cartilage damage. Statistical analysis results show that ( Figure 2 Compared to the control group, the knockdown group mice showed a significantly lower score for articular cartilage damage. In the OARSI scoring system, a lower score indicates more intact cartilage structure and milder pathological damage. These findings suggest that cPGES knockdown may have a protective effect against osteoarthritis-related cartilage damage.

[0062] (3) Targeted knockdown of cPGES inhibits abnormal bone metabolism in osteoarthritis mice The pathological essence of osteoarthritis lies in the damage to articular cartilage and the underlying bone tissue. In the early stages of the disease, cartilage degeneration and loss can lead to structural and metabolic changes in subchondral bone: studies have found that targeted knockdown of cPGES can significantly inhibit abnormal activation of osteoclasts (such as...). Figure 3 This was manifested by a significant reduction in osteoclasts in the knockdown group, as indicated by osteoclast-specific staining. Micro-CT scans showed that, compared to the control group, the knockdown group had relatively higher bone volume fraction (BV / TV) and bone mineral density (BMD). Under the stimulation of various growth factors and cytokines, osteoblasts and osteoclasts are abnormally activated, thereby promoting abnormal osteophyte formation, subchondral bone plate thickening and sclerosis, and intrachondral ossification, among other processes (e.g., Figure 4 ).

[0063] (4) Targeted knockdown of cPGES can slow down the fibrosis process of cartilage in the joint area. Analysis of mRNA extracted from joint tissue using qRT-PCR revealed that targeted knockdown of cPGES increased levels of type II collagen gene (Col2a1), SRY-associated HMG box gene 9 (Sox9), and agglutinin gene (Acan), which are markers promoting collagen formation, while decreasing levels of type I collagen α1 chain gene (Col1a1), matrix metalloproteinase 13 (Mmp13), and matrix metalloproteinase 3 (Mmp3), which are markers promoting collagen degradation. This resulted in the promotion of cartilage collagen synthesis while inhibiting collagen degradation, thus slowing down the fibrosis process of joint cartilage. Figure 5 .

[0064] (5) Targeted knockdown of cPGES significantly inhibited inflammation in arthritic mice. Subsequently, the effects of targeted knockdown of cPGES on inflammation-related indicators of osteoarthritis in mice were further analyzed. qRT-PCR analysis of mRNA extracted from joint tissues revealed that targeted knockdown of cPGES significantly reduced the expression of inflammatory factors such as interleukin-1β (Il-1β) and tumor necrosis factor-α (TnF-α). Figure 6 The above results indicate that targeted knockdown of cPGES can significantly improve osteoarthritis symptoms and play a protective role against osteoarthritis.

[0065] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0066] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. The use of cPGES as a target in the development, screening, or preparation of drugs for the prevention and / or treatment of osteoarthritis.

2. The application according to claim 1, characterized in that: When the drug is applied to a mouse model, it can inhibit the activity of cPGES in the mouse model, wherein the mouse model is a transverse anterior cruciate ligament rupture.

3. The application according to claim 2, characterized in that: When the drug is applied to a mouse model, it can relieve joint pain in the mice.

4. The application according to claim 2, characterized in that: When the drug is applied to a mouse model, it can promote the stabilization of cartilage structure in the mouse model.

5. The application according to claim 2, characterized in that: When the drug is applied to a mouse model, it can improve the bone metabolism imbalance in the mouse model.

6. The application according to claim 2, characterized in that: When the drug is applied to a mouse model, it can slow down the fibrosis process of cartilage in the joint area. And / or, when the drug is applied to a mouse model, it can inhibit the release of inflammatory factors in the joint area of ​​the mouse model.

7. The use of cPGES or downregulators of its encoding gene in the preparation of medicaments for the prevention and / or treatment of osteoarthritis.

8. The application according to claim 7, characterized in that: The downregulator is selected from interfering molecules that specifically interfere with the expression of the gene encoding cPGES and / or small molecule compounds that specifically inhibit cPGES or its encoding gene.

9. A pharmaceutical composition for the prevention and / or treatment of osteoarthritis, characterized in that, include: cPGES or downregulators of their encoding genes, and pharmaceutically acceptable vectors.

10. The pharmaceutical composition according to claim 9, characterized in that: The downregulator is selected from interfering molecules that specifically interfere with the expression of the gene encoding cPGES and / or small molecule compounds that specifically inhibit cPGES or its gene encoding; preferably, the downregulator comprises a cPGES knockdown virus having the sequence shown in SEQ ID NO. 1.

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