Application of mPGES-2 as target spot in screening or preparing medicine for preventing and / or treating primary osteoporosis

Abstract

The invention discloses an application of mPGES-2 as a target spot in screening or preparing a medicine for preventing and / or treating primary osteoporosis. The invention proposes that the mPGES-2 is a drug target of the osteoporosis disease for the first time. Experiments show that mPGES-2 knockout obviously improves bone trabecula structure disorder of old mice and ovariectomy mice, reduces bone loss, increases the number of osteoblasts and reduces osteoclast generation; meanwhile, the mPGES-2 has a remarkable effect on improving the osteoporosis, can be used as a target spot for treating the osteoporosis, and has important significance on drug development and prevention and treatment of the diseases in the future.

Description

Technical Field

[0001] The present invention relates to the use of mPGES-2 (microsomal prostaglandin E synthase-2) as a target in screening or preparing drugs for preventing and / or treating primary osteoporosis, and belongs to the field of biomedical technology. Background Art

[0002] Osteoporosis is a common and frequently-occurring disease among postmenopausal women, middle-aged and elderly people. The harm of osteoporosis is extremely great and extensive, having many serious impacts on the health and life of patients. Osteoporotic fractures are the most common and extremely harmful manifestation of osteoporosis. It is not only one of the main causes leading to disability in elderly patients, but also greatly increases the risk of death of patients. After a hip fracture occurs, about 20% of patients die due to various complications within one year, and about 50% of patients become disabled, with a significant decline in quality of life. At the same time, the treatment and care of osteoporosis and its fractures bring a heavy burden to families and society.

[0003] The prevention and treatment of osteoporosis face the severe challenges of a high prevalence rate but low awareness rate, diagnosis rate, and treatment rate, namely "one high and three lows". Exploring effective drug targets and developing targeted drugs for preventing or treating osteoporosis are of great significance for improving the quality of life of the elderly, reducing the occurrence probability of geriatric diseases, and prolonging lifespan. Summary of the Invention

[0004] The main object of the present invention is to provide the use of mPGES-2 as a target in screening or preparing drugs for preventing and / or treating primary osteoporosis, so as to overcome the deficiencies in the prior art.

[0005] To achieve the foregoing invention object, the technical solutions adopted by the present invention include:

[0006] The embodiments of the present invention provide the use of mPGES-2 as a target in screening or preparing drugs for preventing and / or treating primary osteoporosis.

[0007] The embodiments of the present invention also provide the use of an inhibitor of mPGES-2 or its encoding gene in preparing drugs for preventing and / or treating primary osteoporosis.

[0008] The embodiments of the present invention also provide a pharmaceutical composition for preventing and / or treating primary osteoporosis, which comprises: an inhibitor of mPGES-2 or its encoding gene, and a pharmaceutically acceptable carrier.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention firstly proposes that mPGES-2 is a drug target for osteoporosis diseases. Experiments have shown that mPGES-2 knockout significantly improves the disorder of trabecular bone structure in aged mice and ovariectomized mice, reduces bone loss, increases the number of osteoblasts, and decreases osteoclastogenesis. These results indicate that mPGES-2 has a significant effect on improving osteoporosis and can be used as a target for treating osteoporosis, which is of great significance for the future drug development and prevention and treatment of such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0011] Figures 1a - 1c It is a comparison diagram of bone HE of young and aged mPGES-2 knockout (KO) mice and wild-type (WT) mice, as well as a comparison diagram of bone HE of mPGES-2 knockout (KO) mice and control wild-type (WT) mice after bilateral ovariectomy in a typical embodiment of the present invention;

[0012] Figures 2a - 2d It is a comparison result diagram of bone CT of young and aged mPGES-2 knockout (KO) and wild-type (WT) mice, as well as a comparison result diagram of bone CT of mPGES-2 knockout (KO) and wild-type (WT) mice after bilateral ovariectomy in a typical embodiment of the present invention;

[0013] Figures 3a - 3c It is a comparison diagram of toluidine blue-stained chondrocytes of young and aged mPGES-2 knockout (KO) and wild-type (WT) mice, as well as a comparison diagram of toluidine blue-stained chondrocytes of mPGES-2 knockout (KO) and control wild-type (WT) mice after bilateral ovariectomy in a typical embodiment of the present invention;

[0014] Figures 4a - 4b It is a bone PCR result diagram of young and aged mPGES-2 knockout (KO) and wild-type (WT) mice in a typical embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have been able to propose the technical solution of the present invention. For the convenience of understanding this application, the following will describe this application in more detail. 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.

[0016] Specifically, as an aspect of the technical solution of the present invention, it relates to the application of mPGES-2 as a target in screening or preparing a drug for preventing and / or treating primary osteoporosis.

[0017] The drug in the present invention can improve the trabecular bone morphological structure, maintain bone mass, protect osteoblasts, reduce the number of osteoclasts, and alleviate cartilage damage.

[0018] In some embodiments, the primary osteoporosis includes senile osteoporosis and / or ovariectomy-simulated postmenopausal osteoporosis.

[0019] The drug in the present invention can improve the disorder of trabecular bone morphological structure caused by primary osteoporosis, inhibit the decrease of bone density and bone mineral content; can significantly inhibit the increase of osteoclasts caused by primary osteoporosis, and promote the number of osteoblasts to maintain bone balance; can protect chondrocytes and thus improve osteoporosis.

[0020] In some embodiments, when the drug acts on a mouse model, it can at least inhibit the activity of mPGES-2.

[0021] In some embodiments, when the drug acts on a mouse model, it can at least improve the trabecular bone morphological structure of the mice in the mouse model.

[0022] In some embodiments, when the drug acts on a mouse model, it can at least increase the bone density of the mice in the mouse model.

[0023] In some embodiments, when the drug acts on a mouse model, it can at least increase the bone mineral content of the mice in the mouse model.

[0024] In some embodiments, when the drug acts on a mouse model, it can at least alleviate the cartilage damage of the mice in the mouse model.

[0025] In some embodiments, when the drug acts on a mouse model, it can at least reduce the number of osteoclasts of the mice in the mouse model.

[0026] In some embodiments, when the drug acts on a mouse model, it can at least increase the number of osteoblasts of the mice in the mouse model.

[0027] The drug in the present invention can at least inhibit the activity of mPGES-2 to improve the osteoporosis condition; can at least improve the trabecular bone morphological structure, increase bone density and bone mineral content, and further improve osteoporosis; the drug can at least improve bone metabolism, inhibit the increase in the number of osteoclasts and the decrease in the number of osteoblasts caused by osteoporosis, and further improve osteoporosis; can at least protect chondrocytes, thereby helping to improve osteoporosis.

[0028] As another aspect of the technical solution of the present invention, it relates to the use of an inhibitor of mPGES-2 or its coding gene in the preparation of a drug for preventing and / or treating primary osteoporosis.

[0029] In some embodiments, the inhibitor is selected from interfering molecules that specifically interfere with the expression of the coding gene of mPGES-2 and / or small molecule compounds that specifically inhibit mPGES-2 or its coding gene.

[0030] As another aspect of the technical solution of the present invention, it also relates to a pharmaceutical composition for preventing and / or treating primary osteoporosis, which comprises: an inhibitor of mPGES-2 or its coding gene, and a pharmaceutically acceptable carrier.

[0031] In some embodiments, the inhibitor is selected from interfering molecules that specifically interfere with the expression of the coding gene of mPGES-2 and / or small molecule compounds that specifically inhibit mPGES-2 or its coding gene.

[0032] The present invention is further illustrated by the following examples: According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their 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.

[0033] Unless otherwise specified, all raw materials, reaction equipment, testing equipment and testing methods used in the following examples are well-known in the art.

[0034] The relevant experimental results and analysis of this example are as follows:

[0035] I. Experimental steps

[0036] Experimental animals:

[0037] The mPGES-2 heterozygous mice used in this example were obtained by Jiangsu Jicui Yakang Biotechnology Co., Ltd. using the CRISPR / Cas9 technology, and mPGES-2 wild-type (WT) mice and mPGES-2 knockout (KO) mice were obtained by hybridization through Ptges2 + / - to produce mPGES-2 wild-type (WT) mice and mPGES-2 knockout (KO) mice.

[0038] 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.

[0039] Example 1 Construction of osteoporosis in the elderly

[0040] Aged mice spontaneously develop osteoporosis. mPGES-2 knockout (KO) and wild-type (WT) mice on a C57 background were raised naturally from birth to 2 years old and used in the experiment.

[0041] Example 2 Construction of osteoporosis model after ovariectomy

[0042] Eight-week-old female mPGES-2 wild-type (WT) and mPGES-2 knockout (KO) mice were selected.

[0043] 1. Animal anesthesia and fixation: Anesthetize the mouse and fix it in the lateral position in preparation for surgery.

[0044] 2. Abdominal surgery: After abdominal skin preparation and disinfection, the skin is incised along the midline of the abdomen, the subcutaneous tissue is bluntly separated, the muscle layer and peritoneum are incised, and the abdominal cavity is opened.

[0045] 3. Ovariectomy: Push aside the fat layer to find the uterus, gently pull out the ovary along the fallopian tube, clamp the fallopian tube under the ovary with tissue forceps, ligate it and cut off the ovary, and remove the other ovary in the same way.

[0046] 4. Postoperative treatment: Suture the muscles and skin layer by layer, and disinfect the sutures with iodine.

[0047] Example 3 Observation of bone tissue by H&E staining

[0048] Specific experimental methods include:

[0049] 1. Preparation of paraffin sections

[0050] (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;

[0051] (2) Dehydration and clearing: Place the dehydration box into the dehydrator and dehydrate it successively with gradient ethanol, 75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, absolute ethanol I for 30 min, absolute ethanol II for 30 min, ethanol-benzene for 5 - 10 min, xylene I for 5 - 10 min, xylene II for 5 - 10 min;

[0052] (3) Wax infiltration and embedding: Melt paraffin I at 65°C for 1 h, melt paraffin II at 65°C for 1 h, melt paraffin III at 65°C for 1 h. Embed the wax-impregnated tissue in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take out the tissue from the dehydration box, place it into the embedding frame according to the requirements of the embedding surface, and attach the corresponding label. Cool it on a -20°C freezing table. After the wax solidifies, take out the wax block from the embedding frame and trim the wax block;

[0053] (4) Sectioning and spreading: Section the tissue at a thickness of 5 μm with a microtome, spread the sections in a 50°C water bath, pick up and paste the sections onto clean glass slides, and bake the slides in an oven at 60°C overnight. After sectioning, make marks and save for later use.

[0054] 2. H&E staining

[0055] (1) Dewaxing and rehydration: Dewax the sections twice with xylene (15 min / time), dehydrate them in 100%, 95%, 90%, 80%, 70%, 50% ethanol for 5 min each, and finally rehydrate them in distilled water for 3 min;

[0056] (2) Hematoxylin staining: Place the sections in hematoxylin staining solution for 15 min, rinse them with tap water for 3 min, and differentiate them with hydrochloric acid alcohol (99 ml of 70% alcohol + 1 ml of concentrated hydrochloric acid) for 10 s;

[0057] (3) Blueing and dehydration: Rinse with tap water for 10 min to make them turn blue. Place the sections successively in 50%, 70%, 80%, 90% ethanol for 5 min each;

[0058] (4) Eosin counterstaining: Stain with 1% eosin staining solution for 2 min, and dehydrate and differentiate them in 95% and 100% ethanol for 3 min each until the boundaries are clear;

[0059] (5) Clearing and mounting: After clearing with xylene for 3 min, mount the sections with neutral balsam;

[0060] (6) After mounting, put them into an oven at 50°C to dry, and observe the changes in the tissue structures under a light microscope.

[0061] Example 4 Bone CT Observation

[0062] (1) Prepare mouse bone samples. Take the bones of the required parts of the mouse, remove the surrounding substances irrelevant to the research, place them in 4% paraformaldehyde for fixation for 24 h, and rinse them with running water overnight.

[0063] (2) Decalcification treatment. Place the bone sample into nitric acid decalcifying solution, with the liquid level about 3 ml higher than the specimen. Judgment of the decalcification end point: It is considered qualified when a thick pin can be easily inserted.

[0064] (3) Micro-CT scanning. Place the decalcified bone sample on the sample stage of the Micro-CT scanner, fix the position, select appropriate resolution and scanning parameters, start scanning, observe the image quality during the scanning process, and save the original data after the scanning is completed.

[0065] (4) Image reconstruction and analysis. Use special software to reconstruct the original data to obtain three-dimensional reconstructed images, perform threshold segmentation on the region of interest, extract two-dimensional and three-dimensional images of cortical bone and cancellous bone, quantitatively analyze parameters such as bone morphology and density, and output the analysis results and reports.

[0066] Example 5 Observation of bone tissue by toluidine blue staining

[0067] 1. Preparation of paraffin sections

[0068] Same as Example 3.

[0069] 2. Toluidine blue staining

[0070] (1) Dewax the paraffin sections to distilled water.

[0071] (2) Immerse in cartilage staining solution (toluidine blue method) for 30 min.

[0072] (3) Wash with running water for 2 min and blot dry with filter paper.

[0073] (4) Differentiate with acetone until the chondrocytes are clearly visible in purple-blue.

[0074] (5) Dehydrate, clear, and mount the paraffin sections with resin routinely.

[0075] Example 6 qRT-PCR

[0076] (1) Put 0.02 g of tissue and 500 μL of Trizol lysate into a grinding tube, add nuclease-free grinding beads, and homogenize with a homogenizer until the tissue is completely broken, and let it stand at room temperature for 10 minutes to fully lyse.

[0077] (2) Add 100 μL of chloroform and quickly shake up and down for 15 seconds, let it stand at room temperature for 10 minutes, then put the sample into a centrifuge and centrifuge at a speed of 12000 rpm for 15 minutes.

[0078] (3) Carefully transfer the upper aqueous phase to a new EP tube. Add 300 μL of isopropanol and let it stand at room temperature for 10 minutes. Place the sample in a centrifuge and centrifuge at 12,000 rpm for 10 minutes.

[0079] (4) After centrifugation, discard the supernatant. Add 500 μL of pre-cooled 75% ethanol to the pellet, pipette to wash, place the sample in a centrifuge and centrifuge at 12,000 rpm for 10 minutes, and repeat once.

[0080] (5) After centrifugation, discard the supernatant. Open the lid of the EP tube and let it stand at room temperature for 10 minutes. Add 80 μL of nuclease-free water to the tube to measure the RNA concentration.

[0081] (6) Prepare the reverse transcription working solution according to Table 1.

[0082] Table 1 RNA reverse transcription reaction system

[0083]

[0084] Reverse transcription conditions: 37 °C, 15 min → 85 °C, 5 s → hold at 4 °C;

[0085] (7) Prepare the amplification system according to Table 2. The total volume of the reaction system is 10.00 μL.

[0086] Table 2 qRT-PCR reaction system

[0087]

[0088] II. Experimental results

[0089] 1. mPGES-2 knockout significantly improves primary osteoporosis

[0090] Osteoporosis is the most common bone disease in middle-aged and elderly people. Osteoporosis mainly occurs in the elderly or postmenopausal women, which is also known as osteoporosis in postmenopausal women and osteoporosis in the elderly. To investigate the effect of mPGES-2 on senile osteoporosis, naturally fed young (8-week-old) and old (2-year-old) mPGES-2 wild-type (WT) and knockout (KO) male mice were selected. Through HE staining, it was found that knocking out mPGES-2 had no obvious effect on the bone structure of young mice, but significantly improved the symptoms of osteoporosis such as trabecular bone structure disorder and cartilage degeneration in old mice, as Figures 1a - 1b shown. Further, to investigate whether mPGES-2 has an effect on osteoporosis in postmenopausal women, ovariectomy was used to simulate osteoporosis in postmenopausal women. The HE results showed that knocking out mPGES-2 could effectively reduce the bone damage caused by ovariectomy, as Figure 1cAs shown above. The above results suggest that targeting mPGES-2 is very likely to be an effective means for the treatment of osteoporosis.

[0091] 2. mPGES-2 knockout significantly alleviates the trabecular bone structure damage and bone loss caused by osteoporosis

[0092] Osteoporosis is a systemic bone metabolic disorder disease in which bone density and bone quality decrease due to various reasons, the microstructure of bone tissue is damaged, and bone fragility increases, resulting in an increased susceptibility to fractures. At the microscopic level, compared with normal and healthy bones, the bones of osteoporosis mice tend to be honeycombed, with large bone pores, and a decrease in bone density and bone mass. Subsequently, micro-CT scans were performed on old mice, ovariectomized mice, and their control mice, and relevant indicators were analyzed. The results showed that compared with young mice, the arrangement of trabecular bone structure in old mice was irregular and the number was significantly reduced, and the bone density was significantly decreased, as Figures 2a - 2b shown. On the contrary, after mPGES-2 knockout, the number of trabecular bones in old mice increased significantly, the trabecular bone structure improved, and the degree of bone mineralization increased, as Figures 2a - 2b shown. Similarly, consistent results were obtained in ovariectomized mice, as Figures 2c - 2d shown. These results indicate that mPGES-2 knockout can effectively counteract osteoporosis caused by aging and insufficient estrogen secretion.

[0093] 3. mPGES-2 knockout alleviates cartilage damage in osteoporosis mice

[0094] During the occurrence and development of osteoporosis, cartilage wear often occurs, which will further aggravate the progression of osteoporosis. Toluidine blue staining can clearly show the structure of the cartilage morphological tide line and observe whether there is damage to the cartilage tissue. Subsequently, through staining, it was found that mPGES-2 knockout had no obvious effect on the cartilage of young mice, but after mPGES-2 knockout in old mice, the boundary of cartilage in bone tissue was clearer and the positive color of cartilage was deeper, as Figures 3a - 3b shown, indicating that mPGES-2 knockout plays a protective role on cartilage. Similarly, a consistent phenomenon was also observed in ovariectomized mice, as Figure 3c shown. The above results show that targeting mPGES-2 can effectively counteract cartilage damage caused by osteoporosis.

[0095] 4. mPGES-2 knockout significantly improves abnormal bone metabolism

[0096] Osteoporosis is essentially an abnormality of bone metabolism, that is, the influence of osteoclasts in the body is greater than that of osteoblasts, and the rate of bone resorption exceeds the rate of bone formation. Through detection, it was found that compared with old wild-type mice, the expression level of osteoblast markers in old mPGES-2KO mice increased significantly, while the expression of osteoclast markers decreased, and the abnormal bone metabolism was significantly improved, asFigure 4a As shown. Consistent results were also observed in the ovariectomized mouse model, such as Figure 4b As shown. These results indicate that mPGES-2 knockout can significantly improve bone metabolic imbalance and play a good protective role against primary osteoporosis.

[0097] In addition, the inventors of this case also referred to the foregoing embodiments and conducted tests with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0098] 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 by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and all these changes and improvements fall within the scope of the present invention claimed.

Claims

1. Use of mPGES-2 as a target in screening or preparing drugs for preventing and / or treating primary osteoporosis.

2. The use according to claim 1, characterized in that: The primary osteoporosis includes senile osteoporosis and / or ovarian removal surgery simulating postmenopausal osteoporosis.

3. 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.

4. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least improve the morphological structure of the trabecular bones of the mice in the mouse model; And / or, when the drug acts on a mouse model, it can at least increase the bone density of mice in the mouse model; And / or, when the drug acts on the mouse model, it can at least increase the bone mineral content of the mice in the mouse model.

5. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least reduce cartilage damage in the mice in the mouse model.

6. The use according to claim 1, characterized in that: When the drug acts on a mouse model, it can at least reduce the number of mouse osteoclasts in the mouse model; And / or, when the drug acts on a mouse model, it can at least increase the number of mouse osteoblasts in the mouse model.

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

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 primary osteoporosis, 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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