Application of mPGES-2 as target spot in development, screening or preparation of medicine for preventing and / or treating osteoarthritis

Abstract

The invention discloses an application of mPGES-2 as a target spot in developing, screening or preparing a medicine for preventing and / or treating osteoarthritis. The invention proposes that mPGES-2 is a drug target of osteoarthritis for the first time. Experiments show that mPGES-2 knockout can significantly relieve joint swelling, protect cartilage wear, improve bone metabolism abnormality and inhibit release of inflammatory factors; meanwhile, the mPGES-2 has a remarkable effect on improving osteoarthritis, can be used as a target spot for treating osteoarthritis, and is of great significance to development, prevention and treatment of drugs for the diseases in the future.

Description

Technical Field

[0001] The present invention relates to the application of mPGES-2 (microsomal prostaglandin Esynthase-2) as a target in the development or screening or preparation of drugs for preventing and / or treating osteoarthritis, belonging to the technical field of biomedicine. Background Art

[0002] Osteoarthritis (OA) is a chronic disease characterized by articular cartilage degeneration, synovial inflammation, and abnormal subchondral bone remodeling. About 500 million patients worldwide are troubled by it, and with the aggravation of aging, more and more middle-aged and elderly people are seriously affected by joint pain, limb disability, functional impairment, and the quality of life in their later years, which brings a huge disease burden to individuals, families, and society. Although existing treatments (such as nonsteroidal anti-inflammatory drugs) can relieve symptoms in the short term, long-term use can easily lead to gastrointestinal damage and liver and kidney toxicity; cartilage protectors (such as chondroitin sulfate) have a slow onset and limited effect on patients in the middle and late stages. In addition, the pathogenesis of OA is complex, involving abnormal release of inflammatory factors (such as IL-6, TNF-α), excessive activation of osteoclasts, and degradation of cartilage matrix. At present, the exact cause and pathogenesis of OA are still unclear, and it is still impossible to fundamentally prevent and treat it. Therefore, it is urgent to develop new targets and intervention strategies for the prevention and treatment of OA. Summary of the invention

[0003] The main purpose of the present invention is to provide an application of mPGES-2 as a target in the development or screening or preparation of drugs for preventing and / or treating osteoarthritis, so as to overcome the deficiencies in the prior art.

[0004] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0005] The embodiments of the present invention provide the use of mPGES-2 as a target in the development, screening or preparation of drugs for preventing and / or treating osteoarthritis.

[0006] The embodiments of the present invention also provide the use of a down-regulator of mPGES-2 or its encoding gene in the preparation of a drug for preventing and / or treating osteoarthritis.

[0007] The embodiment of the present invention further provides a pharmaceutical composition for preventing and / or treating osteoarthritis, which comprises: a down-regulator of mPGES-2 or its encoding gene, and a pharmaceutically acceptable carrier.

[0008] Compared with the prior art, the beneficial effect of the present invention is that the present invention proposes for the first time that mPGES-2 is a drug target for osteoarthritis. Experiments have shown that knocking out mPGES-2 significantly reduces joint swelling, protects cartilage wear, improves bone metabolism imbalance, and inhibits the release of inflammatory factors. These results show that mPGES-2 has a significant effect on improving osteoarthritis and can be used as a target for the treatment of osteoarthritis, which is of great significance for the future drug development and preventive treatment of such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0010] Figure 1 A comparison of bone anatomy between mPGES-2 knockout (KO) mice and wild-type (WT) mice in a control group under an osteoarthritis model in a typical embodiment of the present invention;

[0011] Figure 2 This is a HE staining comparison diagram of mPGES-2 knockout (KO) mice and wild-type (WT) mice in a control group under an osteoarthritis model in a typical embodiment of the present invention;

[0012] Figure 3 This is a comparison of tartrate-resistant acid phosphatase (TRAP) stained osteoclasts in mPGES-2 knockout (K0) mice and wild-type (WT) mice in a control group under an osteoarthritis model in a typical embodiment of the present invention;

[0013] Figure 4-Figure 9 This is a graph showing the PCR results of osteoblastic and osteoclast markers in mPGES-2 knockout (KO) mice and wild-type (WT) mice in a control group under an osteoarthritis model in a typical embodiment of the present invention;

[0014] Fig.10 This is a comparison of TNF-α histochemical staining of mPGES-2 knockout (KO) mice and wild-type (WT) mice in a control group under an osteoarthritis model in a typical embodiment of the present invention;

[0015] Figure 11-Figure 12 This is a graph showing the PCR results of joint inflammation markers in mPGES-2 knockout (KO) mice and control wild-type (WT) mice in an osteoarthritis model in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0016] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. In order to facilitate the understanding of the present application, the present application will be described in more detail as follows. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0017] Specifically, as one aspect of the technical solution of the present invention, it involves the use of mPGES-2 as a target in the development, screening or preparation of drugs for preventing and / or treating osteoarthritis.

[0018] The medicine of the present invention can reduce arthritis swelling, protect cartilage, improve bone metabolism imbalance, and inhibit the release of inflammatory factors.

[0019] In some embodiments, the arthritis is mechanical anisotropy-induced bone and joint damage.

[0020] Furthermore, the drug can alleviate the degree of joint swelling caused by bone and joint injuries induced by uneven mechanical forces.

[0021] Furthermore, the drug can significantly inhibit abnormal bone cell metabolism in bone and joint injuries induced by uneven mechanical forces and maintain bone balance.

[0022] Furthermore, the drug can reduce the release of inflammatory factors in bone and joint injuries induced by uneven mechanical forces and the expression of inflammatory molecules in the joints.

[0023] In some embodiments, when the drug acts on a mouse model, it can at least inhibit the activity of mPGES-2 in the mouse model, and the mouse model is a mouse anterior cruciate ligament transection (ACLT) model.

[0024] Furthermore, the mouse ACLT model is a model in which the anterior cruciate ligament of the animal's knee joint is surgically cut, sometimes accompanied by damage to the medial collateral ligament or meniscus resection, to induce joint instability, thereby causing cartilage degeneration and osteoarthritis.

[0025] Furthermore, when the drug acts on a mouse model, it can at least reduce the degree of arthritis swelling and wear of the mice in the mouse model.

[0026] Furthermore, when the drug acts on a mouse model, it can at least promote the stabilization of the cartilage structure of the mice in the mouse model.

[0027] Furthermore, when the drug acts on a mouse model, it can at least improve the bone metabolism imbalance of the mice in the mouse model.

[0028] Furthermore, when the drug acts on a mouse model, it can at least inhibit the release of mouse inflammatory factors in the mouse model.

[0029] Furthermore, the inflammatory factors include TNF-α and / or IL-1β, but are not limited thereto.

[0030] As another aspect of the technical solution of the present invention, it relates to the use of a down-regulator of mPGES-2 or its encoding gene in the preparation of a drug for preventing and / or treating osteoarthritis.

[0031] In some embodiments, the down-regulator is selected from an interfering molecule that specifically interferes with the expression of a gene encoding mPGES-2 and / or a small molecule compound that specifically inhibits mPGES-2 or a gene encoding mPGES-2.

[0032] As another aspect of the technical solution of the present invention, it also relates to a pharmaceutical composition for preventing and / or treating osteoarthritis, which comprises: a down-regulator of mPGES-2 or its encoding gene, and a pharmaceutically acceptable carrier.

[0033] In some embodiments, the down-regulator is selected from an interfering molecule that specifically interferes with the expression of a gene encoding mPGES-2 and / or a small molecule compound that specifically inhibits mPGES-2 or a gene encoding mPGES-2.

[0034] The present invention is further described by the following examples: The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0035] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following examples are all well known in the art.

[0036] 1. Experimental steps

[0037] Experimental Animals:

[0038] The mPGES-2 heterozygous mice used in this example were obtained by Jiangsu Jicui Yaokang Biotechnology Co., Ltd. using CRISPRCas9 technology and were obtained by Ptges2 from Xuzhou Medical University. + / - The mPGES-2 wild-type (WT) mice and mPGES-2 knockout (KO) mice were obtained by hybridization.

[0039] Male C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., license number SCXK (Beijing) 2007-0001. The mice were placed under standard conditions of 50 ± 10% humidity and 23 ± 2 °C for 12 h per day and night to adapt to survival. The mice had free access to water and food. All animal management and handling 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.

[0040] Example 1 Construction of osteoarthritis model

[0041] Six young male mPGES-2 wild-type (WT) and mPGES-2 knockout (KO) mice were selected.

[0042] The anterior cruciate ligament transection model (ACLT) was selected to simulate human osteoarthritis. The mouse was fixed in a supine position, the left hind limb was flexed 90°, and the surgical area was thoroughly disinfected. A smooth incision was made at the knee joint, and the skin and muscle were bluntly separated to expose the trochlear groove of the knee joint. The joint capsule was dissected along the inner edge of the patellar ligament, and the intra-articular adipose tissue was bluntly cleaned. The mouse knee joint was bent to 90°, the anterior cruciate ligament was exposed, the ligament was cut with a microscissor, and a drawer test was performed to verify that the ligament had been successfully cut.

[0043] Example 2 Observation of bone tissue by H&E staining

[0044] Specific experimental methods include:

[0045] 1. Preparation of paraffin sections

[0046] (1) Fixation of tissue specimens: bone tissues of mice in each group in Example 1 were fixed in 4% paraformaldehyde at room temperature for 24 hours, wrapped with gauze, marked, and rinsed with running water overnight;

[0047] (2) Dehydration and transparency: Place the dehydration box in a dehydrator and dehydrate with graded alcohols 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, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes

[0048] (3) Wax dipping and embedding: melt paraffin I at 65° for 1 hour, melt paraffin II at 65° for 1 hour, and melt paraffin III at 65° for 1 hour. Embed the wax-dipping tissue in an embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20° freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block;

[0049] (4) Sectioning and spreading: Cut slices with a microtome to a thickness of 5 μm, spread the slices in a 50°C water bath, pick up the slices and mount them on a clean glass slide, and bake them in a 60°C oven overnight. After sectioning, mark them and store them for later use.

[0050] 2. H&E staining

[0051] (1) Dewaxing and rehydration: The sections were dewaxed twice in xylene (15 min / time), dehydrated in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0052] (2) Hematoxylin staining: The sections were placed in hematoxylin staining solution for 15 minutes, rinsed with tap water for 3 minutes, and separated with hydrochloric acid alcohol (70% alcohol 99mk + concentrated hydrochloric acid 1ml) for 10 seconds;

[0053] (3) Blueing and dehydration: Rinse with tap water for 10 minutes to turn it blue. Dehydrate the slices in 50%, 70%, 80%, and 90% alcohol for 5 minutes each;

[0054] (4) Eosin counterstaining: stain with 1% eosin solution for 2 minutes, dehydrate in 95% alcohol and 100% alcohol for 3 minutes respectively until the boundary is clear;

[0055] (5) Transparency and sealing: After xylene is transparent for 3 minutes, the slides are sealed with neutral gum;

[0056] (6) After sealing, the slides were placed in a 50°C oven for drying and the changes in the tissue structures were observed under a light microscope.

[0057] Example 3 TRAP staining for bone tissue observation

[0058] 1. Preparation of paraffin sections

[0059] Same as the method in 2.1 above.

[0060] 2. TRAP staining

[0061] (1) Dewaxing and rehydration: sections were dewaxed twice in xylene (15 min / time), dehydrated in 100%, 95%, 90%, 80%, 70%, and 50% alcohol for 5 min each, and finally rehydrated in distilled water for 3 min.

[0062] (2) TRAP staining: Add TRAP staining solution to a 50 ml slide staining jar, place the slices in it, 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 appropriately extended to 60 minutes or until the color develops to the expected depth under a microscope);

[0063] (3) Counterstaining: staining with hematoxylin for 5-8 minutes or methyl green for 2-3 minutes as a contrast stain for the cell nucleus;

[0064] (4) Dehydration, transparency, and sealing: Dehydrate the sections in 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 5 minutes each. Transparentize with xylene for 3 minutes, and then seal the sections with neutral gum.

[0065] (5) After sealing, the slides were placed in a 50°C oven for drying and the changes in tissue structures were observed under a light microscope. TRAP-positive osteoclasts appeared red, and the nuclei appeared blue-purple or green.

[0066] Example 4 Immunohistochemical staining for bone tissue observation

[0067] (1) Place the paraffin sections in an oven and bake at 60°C for 40 minutes to dry.

[0068] (2) Dewax the sections in sequence: first soak in xylene for 10 minutes, then soak in clean xylene for the same 10 minutes. Then soak in anhydrous ethanol, 95% ethanol, and 80% ethanol for 5 minutes each, and finally soak in distilled water for 5 minutes.

[0069] (3) Antigen repair: Soak the slices in 200 mL of sodium citrate buffer, place in a microwave oven, heat on high for 5 minutes, then on low for 5 minutes. Remove and let stand at room temperature for one hour. Gently shake dry the slices and draw circles around the tissue with a histochemical pen to prevent the loss of liquid in subsequent steps. Add an appropriate amount of 3% hydrogen peroxide solution to each tissue, incubate at room temperature for 20 minutes to block endogenous peroxidase activity, and then wash three times with PBS, 3 minutes each time.

[0070] (4) Blocking: Add an appropriate amount of 5% BSA solution to each tissue and incubate in a 37°C oven for 30 minutes.

[0071] (5) Primary antibody incubation: Gently shake dry the sections, add an appropriate amount of diluted primary antibody, and then place the sections in a humidified chamber and incubate at 4°C overnight.

[0072] (6) Take the slide out of 4°C, warm it up at room temperature for 30 minutes, and then wash it three times with PBS, 3 minutes each time. Then, add an appropriate amount of reaction enhancement solution, incubate it at room temperature for 20 minutes, and wash it again with PBS three times, 3 minutes each time.

[0073] (7) Add an appropriate amount of enhanced enzyme-labeled goat anti-mouse / rabbit immunoglobulin G (IgG) polymer to each tissue, incubate at room temperature for 25 minutes, and then wash three times with PBS, each time for 3 minutes.

[0074] (8) DAB staining: Add 50 μL of freshly prepared DAB staining solution to each tissue and incubate at room temperature for 1-3 minutes. Terminate the staining process as appropriate.

[0075] (9) Hematoxylin staining of cell nuclei: After rinsing with running water for 10 minutes, add hematoxylin and incubate at room temperature for 20 seconds, then rinse with running water for 10 minutes.

[0076] (10) Dehydrate the sections in sequence: soak in 80% ethanol, 95% ethanol, and anhydrous ethanol for 5 minutes each, then soak in xylene for 10 minutes, and then soak in clean xylene for another 10 minutes. Finally, seal the sections with neutral gum.

[0077] Example 5 qRT-PCR

[0078] (1) Place 0.02 g of tissue and 500 μL of Trizol lysis buffer into a grinding tube, add nuclease-free grinding beads, and use a homogenizer to homogenize until the tissue is completely broken. Let it stand at room temperature for 10 minutes to allow it to be fully lysed.

[0079] (2) After adding 100 μL of chloroform, the mixture was rapidly shaken up and down for 15 seconds. After standing at room temperature for 10 minutes, the sample was placed in a centrifuge and centrifuged at 12,000 rpm for 15 minutes.

[0080] (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. Place the sample in a centrifuge and centrifuge at 12,000 rpm for 10 minutes.

[0081] (4) After centrifugation, discard the supernatant, add 500 μL of pre-cooled 75% ethanol to the precipitate, wash it with a pipette, place the sample in a centrifuge, and centrifuge it at 12,000 rpm for 10 minutes. Repeat this process.

[0082] (5) After centrifugation, discard the supernatant, leave the EP tube open at room temperature for 10 minutes, and add 80 μL of nuclease-free water to the tube to measure the RNA concentration.

[0083] (6) Prepare the reverse transcription working solution according to Table 1.

[0084] Table 1: RNA reverse transcription reaction system

[0085]

[0086] Reverse transcription conditions: 37°C, 15 min → 85°C, 5 s → 4°C hold;

[0087] (7) Prepare the amplification system according to Table 2, with a total reaction volume of 10.00 μL.

[0088] Table 2: qRT-PCR reaction system

[0089]

[0090]

[0091] 2. Experimental Results

[0092] 1. Knockout of mPGES-2 significantly improves bone and joint swelling in arthritic mice

[0093] Osteoarthritis is the most common form of arthritis, also known as "wear and tear" of the joints, which is the wear and tear of the cartilage within the joints. This causes bones to rub against each other and the joints to become painful, stiff, or swollen. An osteoarthritis model was established by cutting the anterior cruciate ligament of the animal's knee joint, and samples were taken for analysis 8 weeks after surgery. Through bone and joint dissection, it was found that compared with wild-type (WT) mice, the degree of joint swelling in mPGES-2 knockout (KO) mice was significantly reduced, such as Figure 1 These results suggest that knockout of mPGES-2 may have a protective effect against osteoarthritis.

[0094] 2. Knockout of mPGES-2 significantly reduces cartilage structural damage

[0095] The development of osteoarthritis is often accompanied by significant changes in the morphology of bone tissues such as cartilage and subchondral bone. HE staining showed that the cartilage morphology of mice in the mPGES-2 knockout group was more regular, with clear boundaries, and the swelling and wear were significantly reduced compared with wild-type mice. Figure 2 As shown, this suggests that mPGES-2 knockout has a significant protective effect on cartilage damage in osteoarthritis.

[0096] 3. Knockout of mPGES-2 significantly inhibits abnormal bone metabolism in osteoarthritis mice

[0097] Osteoarthritis is essentially a type of arthritis that damages the articular cartilage or subarticular bone. The reduction of cartilage drives the subchondral bone to begin to change. Under the stimulation of growth factors and cytokines, osteoblasts and osteoclasts are activated, leading to new bone, thickening and stiffening of the subchondral plate, and endochondral ossification. Subsequently, the effect of mPGES-2 on bone metabolism in arthritis was detected. The study found that knocking out mPGES-2 significantly inhibited the activation of osteoblasts and osteoclasts, which was manifested by a significant reduction in the expression of their marker molecules, such as Figure 3-Figure 9 The above results suggest that targeting mPGES-2 can improve abnormal bone metabolism during arthritis.

[0098] 4. Knockout of mPGES-2 significantly inhibits inflammation in arthritis mice

[0099] Subsequently, the effects of mPGES-2 knockout on indicators related to osteoarthritis in mice were further analyzed. The test found that after knocking out mPGES-2, the infiltration of inflammatory cells in the joint cavity was significantly reduced, and the expression of inflammatory factors (such as TNF-α and IL-1β) in the serum was also significantly reduced. Figure 10-12The above results indicate that targeting mPGES-2 can significantly improve the symptoms of osteoarthritis and play a protective role against osteoarthritis.

[0100] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0101] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Use of mPGES-2 as a target in the development, screening or preparation of drugs for the prevention and / or treatment of osteoarthritis.

2. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least inhibit the activity of mPGES-2 in the mouse model, and the mouse model is a mouse anterior cruciate ligament transection model.

3. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least reduce the arthritis swelling and wear of the mice in the mouse model.

4. The use according to claim 2, characterized in that: When the drug acts on a mouse model, it can at least promote and stabilize the cartilage structure of the mice in the mouse model.

5. The use according to claim 2, characterized in that: When the drug acts on a mouse model, it can at least improve the bone metabolism imbalance of the mice in the mouse model.

6. The use according to claim 2, characterized in that: When the drug acts on a mouse model, it can at least inhibit the release of mouse inflammatory factors in the mouse model; preferably, the inflammatory factors include TNF-α and / or IL-1β.

7. Use of mPGES-2 or a down-regulator of its encoding gene in the preparation of a medicament for preventing and / or treating osteoarthritis.

8. The use according to claim 7, characterized in that: The down-regulator is selected from interfering molecules that specifically interfere with the expression of the gene encoding mPGES-2 and / or small molecule compounds that specifically inhibit mPGES-2 or its encoding gene.

9. A pharmaceutical composition for preventing and / or treating osteoarthritis, characterized in that: include: A down-regulator of mPGES-2 or its encoding gene, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that: The down-regulator is selected from interfering molecules that specifically interfere with the expression of the gene encoding mPGES-2 and / or small molecule compounds that specifically inhibit mPGES-2 or its encoding gene.

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