Application of cPLA2 inhibitor in preparation of medicine for treating osteoporosis

Abstract

The invention relates to application of a cPLA2 inhibitor in preparation of a medicine for treating osteoporosis, and belongs to the technical field of biomedical research. In-vitro and in-vivo experiments confirm that the cPLA2 plays a key role in the pathological process of the osteoporosis, specifically, the cPLA2 is positively correlated with up-regulation of osteoclast related genes (ACP5, CTSK, cF0S, MMP9 and DC-stamp) and inflammatory factors (IL-6, IL-1beta and TNF-a) in the occurrence process of the osteoporosis, the cPLA2 plays a key role in inflammation of the osteoporosis, and the cPLA2 plays a role in treating osteoporosis. The invention provides reduction of secondary pathological change of osteoporosis by targeted inhibition of expression of cPLA2, which provides a new target for treatment of osteoporosis, and has important clinical application potential and wide market prospect.

Description

Technical Field

[0001] The invention relates to application of a cPLA2 inhibitor in preparing a drug for treating osteoporosis, and belongs to the technical field of biomedical research. Background Art

[0002] Osteoporosis is a systemic chronic inflammatory disease that is caused by a variety of reasons that lead to a disorder in the balance between bone resorption and bone formation, which in turn manifests as a decrease in bone mass, decreased bone density, and susceptibility to fractures. With the further development of the aging society, the number of osteoporosis patients has increased sharply, and osteoporosis has gradually "gnawed" at our health, while also placing a great burden on families and society. At this stage, clinical medication cannot achieve comprehensive and effective intervention, and it is difficult to truly regulate the balance of bone metabolism. Therefore, the prevention and treatment of osteoporosis has become a difficult problem that the medical community needs to solve urgently.

[0003] Bone is a highly metabolically active tissue that is constantly undergoing bone reconstruction and bone renewal, which is mainly achieved through bone formation regulated by osteoblasts and bone resorption regulated by osteoclasts. RANK / RANKL / OPG is an important signal that mediates the balance between bone resorption and bone formation, and is essential for maintaining bone metabolism balance. RANKL is an essential factor for osteoclast differentiation. It is secreted by osteocytes, preosteoblasts or immune cells. It binds to RANK on the surface of osteoclast precursor cells, activates NF-κB transcription factors, increases the transcription and translation levels of osteoclast differentiation-related genes c-FOS and NFATc1, promotes their fusion into multinuclear cells and activation into mature osteoclasts, secretes hydrochloric acid and various hydrolases to dissolve minerals and reduce bone matrix. Osteoclast inhibitory factor (OPG) inhibits osteoclast differentiation by binding to RANKL and competitively inhibiting the action of the RANKL-RANK system. Therefore, current research and drug development focus on how to inhibit the function of RANKL.

[0004] At present, the commonly used drugs in clinical practice are mainly zoledronic acid and denosumab, but they have great limitations in clinical application and prevention and treatment effects. Zoledronic acid can inhibit osteoclast function and reduce bone loss by binding to osteoclasts, but its side effects are large, which can cause fever, hypocalcemia, gastrointestinal reactions, renal damage, etc., and it is impossible to achieve long-term effective drug intervention. Desuozumab is a monoclonal antibody of RANKL, which has strong targeting and can effectively bind to RANKL to prevent it from activating RANK on the surface of osteoclasts and their precursor cells, inhibiting the maturation and activation of osteoclasts and reducing bone loss, but it has a strong withdrawal reaction. After discontinuation of denosumab, the number and activity of osteoclasts rebound, resulting in rapid bone loss, which is very likely to cause recurrent fractures. Domestic and foreign studies have found that in the development of osteoporosis, the regulation of RANKL signaling is a complex process, in which inflammatory response, oxidative stress, lipid metabolism, autophagy damage, etc. can all lead to increased RANKL-induced bone loss. RANKL can also aggravate the above pathological processes and increase the rate of bone loss. Single factor regulation cannot fully inhibit the RANKL signaling network. Therefore, finding important targets that can achieve comprehensive regulation in the development of osteoporosis is our key scientific issue.

[0005] To achieve targeted and comprehensive regulation of bone metabolism, we found that phospholipase A2 (cPLA2 / PLA2G4A) plays a key role in the regulation of bone metabolism through combined transcriptomic and proteomic analysis. As a widely distributed enzyme, cPLA2 can participate in a variety of cellular reactions, such as cell proliferation, differentiation, and inflammation. Its amino acid 505 can be phosphorylated and activated by extracellular regulated kinase 1 / 2 (ERK1 / 2) and p38 mitogen-activated protein kinase (p38MAPK). Activated cPLA2 can produce the following effects: 1. Degrade phospholipids on the surface of lysosomes, causing lysosomal damage, thereby inhibiting their fusion with autophagosomes and blocking autophagic flow; 2. Catalyze the hydrolysis of glycerolipids to produce lysophospholipids and release arachidonic acid (AA), leading to lipid metabolism disorders; 3. Become phosphorylated, transmit inflammatory signals, and further aggravate inflammatory responses and oxidative stress. We believe that the action characteristics of cPLA2 determine that it may be used as a target for regulating bone metabolism balance. cPLA2 is expected to become a new target for regulating autophagy and inflammatory response, and achieve effective prevention and treatment of osteoporosis through multi-level and comprehensive regulation of RANKL signaling. Therefore, we speculate that cPLA2 can effectively regulate autophagy flow, inflammation and oxidative stress, while intervening in the differentiation and function of osteoclasts, thereby more comprehensively regulating bone metabolism balance and effectively treating osteoporosis.

[0006] At present, there is no relevant research on the regulatory role of cPLA2 in the pathogenesis of osteoporosis. Therefore, it is of great clinical significance to clarify the role of cPLA2 in the pathogenesis of osteoporosis, provide new targets and experimental basis for the treatment of osteoporosis, design simple and efficient drug preparations targeting cPLA2 activity, develop unknown functions of existing clinical drugs, broaden drug indications, and quickly and efficiently realize drug clinical transformation. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides the use of a cPLA2 inhibitor in the preparation of a drug for treating osteoporosis, thereby providing a new molecular target for the treatment of osteoporosis.

[0008] The technical solution of the present invention is as follows:

[0009] Application of cPLA2 inhibitors in the preparation of drugs for the treatment of osteoporosis.

[0010] Preferably according to the present invention, the cPLA2 inhibitor can reduce the expression level of cPLA2 or reduce the protein activity of cPLA2, thereby increasing bone density and compressive resistance, reducing the expression levels of osteoclast-related genes and inflammatory factors, and thus alleviating the pathogenesis of osteoporosis, thereby achieving the effect of treating osteoporosis.

[0011] Preferably, according to the present invention, the cPLA2 inhibitor can reduce the expression level of cPLA2 or the protein activity of cPLA2, thereby increasing bone density and reducing the expression level of inflammatory factors, thereby alleviating bone dissolution and achieving the effect of treating osteoporosis.

[0012] Preferably, according to the present invention, the cPLA2 inhibitor can inhibit the effect of RANKL and prevent osteoclast differentiation by reducing the expression level of cPLA2 or reducing the protein activity of cPLA2, thereby achieving the effect of treating osteoporosis.

[0013] A therapeutic drug for osteoporosis, comprising a cPLA2 inhibitor.

[0014] Preferably according to the present invention, the cPLA2 inhibitor can reduce the expression level of cPLA2 or reduce the protein activity of cPLA2.

[0015] Preferably according to the present invention, the osteoporosis therapeutic drug further comprises a pharmaceutically acceptable carrier or excipient.

[0016] Application of cPLA2 as a target in screening drugs for the treatment of osteoporosis.

[0017] Preferably, according to the present invention, the screened osteoporosis therapeutic drug takes cPLA2 as a target, and can reduce the expression level of cPLA2 or reduce the protein activity of cPLA2.

[0018] Beneficial effects:

[0019] 1. The present invention confirms through in vitro and in vivo experiments that cPLA2 plays a key role in the pathological process of osteoporosis. Specifically, cPLA2 is positively correlated with the upregulation of osteoclast-related genes (ACP5, CTSK, cF0S, MMP9, DC-stamp) and inflammatory factors (IL-6, IL-1β, TNF-a) in the occurrence of osteoporosis. In this field, no researchers have studied the relationship between cPLA2 and osteoclast-related genes and inflammatory factors and osteoporosis. The inventor's research on cPLA2 and osteoporosis model is original.

[0020] 2. The present invention found that cPLA2 plays a key role in the inflammation of osteoporosis, and proposed to reduce the secondary pathological changes of osteoporosis by targeted inhibition of cPLA2 expression. This discovery provides a new target for the treatment of osteoporosis and has important clinical application potential and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Representative micro CT scan 2D tomographic display images of the femurs of the four groups of mice in Example 1.

[0022] Figure 2 Representative micro-CT scan 3D reconstructed images of the femurs of the four groups of mice in Example 1.

[0023] Figure 3 This is a bar graph of the bone-related indicators BMD, BV / TV, TB.TH, and TB.SP of the femurs of the four groups of mice in Example 1.

[0024] Figure 4 This is a bar graph of the compressive capacity of the femurs of the four groups of mice in the three-point bending test in Example 1.

[0025] Figure 5 This is the HE staining image of the femoral tissues of the four groups of mice in Example 1.

[0026] Figure 6 This is a bar graph of the expression levels of osteoclast-related genes ACP5, CTSK, and DC-stamp in the femoral tissues of the four groups of mice in Example 1.

[0027] Figure 7 This is a bar graph showing the expression levels of inflammatory factors IL-1β, IL-6 and TNF-a in the serum of the four groups of mice in Example 1.

[0028] Figure 8 Representative micro-CT scan 3D reconstructed images of the skulls of four groups of mice in Example 2.

[0029] Fig. 9 This is a bar graph of the bone-related indicators TB.TH, TB.SP, Tb.N, and bone dissolution area ratio of the skulls of the four groups of mice in Example 2.

[0030] Fig.10 This is a bar graph of the expression levels of inflammatory factors IL-1β, IL-6 and TNF-a in the serum of the four groups of mice in Example 2.

[0031] Fig.11 This is a bar graph of the expression levels of osteoclast-related genes ACP5, CTSK, cF0S, MMP9, and DC-stamp in the four groups of cells in Example 3.

[0032] Fig.12 This is a Western Blotting detection image of osteoclast-related transcription factors in two groups of cells in Experimental Example 3.

[0033] Fig.13 These are TRAP staining images of four groups of cells in Experimental Example 3.

[0034] In the significance analysis in the above figures, P>0.05 is ns, P<0.05 is *, P<0.01 is **, P<0.001 is ***, and P<0.0001 is ****. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and consumables involved in the embodiments, unless otherwise specified, are all common commercially available products; the steps and experimental operations involved in the embodiments, unless otherwise specified, are all conventional technical operations in the art.

[0036] The parental mice involved in the examples are from:

[0037] cPLA2 - / - C57BL / 6 mice were purchased from Cyagen, strain name: C57BL / 6J-Pla2g4a em1cyagen , Product Number: S-KO-03690. cPLA2 + / - C57BL / 6 mice are cPLA2 - / - A mouse strain resulting from the breeding of C57BL / 6 mice with normal C57BL / 6 mice.

[0038] Example 1: In vivo experiments to explore the key role of cPLA2 as a therapeutic target in osteoporosis

[0039] In vivo experiments verified the key role of cPLA2 as a new target in osteoporosis and constructed cPLA2 + / - C57BL / 6 mice, cPLA2 - / - C57BL / 6 mice were used as parental mice. Female mice were selected after breeding and underwent bilateral ovariectomy (OVX) to construct an osteoporosis model. The mice were observed for 3 months after surgery.

[0040] Experimental methods: An ovarian ovariectomized osteoporosis mouse (OVX) model was established. + / - C57BL / 6 mice, cPLA2 - / - C57BL / 6 mice were used as parent mice. Female mice were selected after breeding, and their birth time was recorded. After 4 weeks of feeding, their tails were cut to identify their genotypes. After 8 weeks of feeding, female mice with suitable genotypes were selected for bilateral ovariectomy. The specific steps were as follows: the mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution. The concentration of sodium pentobarbital was 3 mg / mL and the injection volume was 0.1 mL / 10 g. After the hair on the back of the mice was removed and the skin was prepared, they were fixed face down on the operating table. A long cut was made along the posterior midline of the back with the intersection of 0.5 cm on the line connecting the upper edges of the thigh roots on both sides of the mouse's hind limbs and the posterior midline of the back as the center. The skin incision was about 0.5-1.0cm. The skin incision was freed to the left by 0.5cm. White fat mass was visible through the base layer. The muscle layer on the surface of the fat mass was lifted with forceps. The muscle layer was cut longitudinally with ophthalmic scissors along the outer edge of the psoas major muscle. The fat mass was about 0.5cm long. The red mulberry-like ovary on the left side of the mouse was seen when the fat mass was turned over. After the ophthalmic scissors were used to remove the ovary, the cut muscle layer was sutured with No. 5 thread. The right ovary of the mouse was found and removed in the same way. After the muscle layer was sutured with No. 5 thread, the skin incision was sutured, and sodium penicillin was injected into the muscle layer incision and skin incision to prevent bacterial infection. The mice were placed in a warm environment and waited for awakening. In the sham group, after the back skin of the mice was cut, the fat mass next to the ovary was cut, the skin incision was sutured, sodium penicillin was injected into the muscle layer incision and skin incision to prevent bacterial infection, and the mice were placed in a warm environment and waited for awakening. The mice were observed for 3 months after surgery and tested after 3 months.

[0041] Detection method:

[0042] 1. Micro CT scanning: Mice were anesthetized with sodium pentobarbital solution and then killed by cervical dislocation. The bilateral femurs were collected, fat and muscle were removed, and fixed with 4% paraformaldehyde in a 4°C refrigerator for 24 hours. The paraformaldehyde was then replaced with 70% ethanol and stored at 4°C. The unilateral femur treated above was scanned 360 degrees using micro CT. The scanning parameters were as follows: 90 kV, 88 mA. Finally, the bone volume fraction BV / TV, the number of trabeculae TB.N, the trabecular gap TB.SP, and the trabecular thickness TB.TH were detected.

[0043] Mouse micro-CT scan results Figure 1 , Figure 2 As shown in the figure, the density and number of trabecular bone in OVX group mice were significantly reduced compared with those in Sham group mice, which proved that osteoporosis model mice were successfully established. - / - Compared with the sham group, the cPLA2 + / - The density and number of trabecular bone in mice increased significantly. - / - The cPLA2 + / - The expression of cPLA2 in the mice was significantly increased, indicating that cPLA2 knockout can alleviate the pathogenesis of osteoporosis.

[0044] The results of bone-related index detection in mice are as follows Figure 3 As shown, cPLA2 in the Sham group - / - Compared with the sham group, cPLA2 + / - The bone density (BMD) and bone volume fraction (BV / TV) of mice increased significantly, the trabecular space (TB.SP) decreased significantly, and the trabecular thickness (TB.TH) increased significantly. - / - Compared with OVX group, cPLA2 + / - The same trend was observed in mice.

[0045] 2. Three-point bending test: Place each femur on the bending support of the testing machine and drive the machine to apply bending moment at a test speed of 5mm / min until the femur is broken. After the test, the printer automatically prints out the test data of each femur.

[0046] The experimental results are as follows Figure 4 As shown, the results showed that cPLA2 - / - The stress resistance of mice and the expression of cPLA2 in sham group + / - There was no significant difference in cPLA2 in OVX group - / - The stress resistance of mice was higher than that of OVX group cPLA2 + / - Mice were significantly enhanced.

[0047] 3. Histological examination: The femurs of each group of experimental mice were first fixed for 24 hours and then decalcified in a 10% EDTA solution prepared with PBS buffer. After the decalcification process, the femurs were embedded in paraffin. The embedded specimens were cut into 5 μm sections and then stained with HE for general histological examination. High-resolution images of the stained sections were captured using an Olympus optical microscope (Japan) and then processed to obtain the corresponding morphological data.

[0048] The results of tissue HE staining are as follows Figure 5 As shown, the results suggest that cPLA2 - / - Compared with the sham group, the cPLA2 + / - The cPLA2 expression in OVX group was significantly enhanced.- / - The cPLA2 + / - Mice have increased.

[0049] 4. Tissue RT-qPCR: Grind the mouse femur tissue in liquid nitrogen, add 1 ml of Trizol reagent to every 50-100 mg of tissue, and break it with a homogenizer. Extract RNA from the tissue, reverse transcribe RNA into cDNA using a reverse transcription kit, and quantify osteoclast-related mRNA (ACP5, CTSK, DC-stamp) using an RT-qPCR kit to observe whether osteoclast genes are downregulated after cPLA2 gene knockout.

[0050] RT-qPCR results Figure 6 As shown, the results suggest that cPLA2 - / - Compared with the sham group, the cPLA2 + / - The levels of osteoclast-related factors CTSK and DC-stamp in mouse tissues decreased significantly, while ACP5 showed no significant difference. - / - Compared with OVX group, cPLA2 + / - The levels of osteoclast-related factors ACP5, CTSK, and DC-stamp in mouse tissues were significantly decreased.

[0051] 5. ELISA test: Collect the serum of each group of mice and use the kit to measure the IL-6, IL-1β and TNF-a indicators.

[0052] The results are as follows Figure 7 As shown, the results showed that cPLA2 - / - Compared with the sham group, the cPLA2 + / - The levels of inflammatory markers IL-6 and TNF-a in the serum of mice decreased significantly, while there was no significant difference in IL-1β. - / - Compared with OVX group, cPLA2 + / - The levels of inflammatory markers IL-1β, IL-6 and TNF-a in the mouse serum were significantly decreased.

[0053] The above results indicate that cPLA2 plays a key role in the pathogenesis of osteoporosis and can be used as a therapeutic target to alleviate the pathogenesis of osteoporosis.

[0054] Example 2: In vivo experiments to explore the key role of cPLA2 as a therapeutic target in osteolysis

[0055] Experimental methods: A lipopolysaccharide (LPS)-induced inflammatory craniolysis model was established in mice, and cPLA2 + / - C57BL / 6 mice, cPLA2 - / -C57BL / 6 mice were used as parent mice. Male mice were selected after breeding, and their birth time was recorded. After 4 weeks of feeding, their tails were cut to identify their genotypes. After 5-6 weeks of feeding, male mice with suitable genotypes were selected for LPS surgery. The specific steps were as follows: the mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution. The concentration of sodium pentobarbital was 3 mg / mL and the injection volume was 0.1 mL / 10 g. After the mouse's head was depilated and skinned, it was fixed face down on the operating table. A skin incision about 0.5-1.0 cm long was made along the midline of the skull with the midpoint of the line connecting the bilateral ears of the mouse as the center. The skin incision was forward, and the muscle layer of the epidermis was peeled off with tweezers. The meningeal layer on the surface of the skull was cut open, and a 12.5 mg / g LPS solution dissolved in PBS buffer in advance was dripped. After the liquid infiltrated, the skin incision was sutured with No. 5 thread, and the mouse was placed in a warm environment and waited for awakening. In the sham operation group, the mice were incised on the upper side of the skull, and then the same volume of PBS buffer was dripped. The skin incision was sutured, and the mice were placed in a warm environment and waited to wake up. The mice were observed for 1 week after surgery, and then tested 1 week later.

[0056] Detection method:

[0057] 1. Micro CT scanning: The mice were anesthetized with sodium pentobarbital solution and then killed by cervical dislocation. The skull was collected, fat and muscle were removed, and fixed with 4% paraformaldehyde in a 4°C refrigerator for 24 hours. The paraformaldehyde was then replaced with 70% ethanol and stored at 4°C. The skull treated above was scanned 360 degrees using micro CT. The scanning parameters were as follows: 90 kV, 88 mA. Finally, the trabecular thickness TB.TH, trabecular spacing TB.SP, trabecular number TB.N, and osteolysis area were detected.

[0058] Mouse micro-CT scan results Figure 8 As shown in the figure, the density and number of trabeculae in the LPS group were significantly reduced compared with those in the Sham group, which proved that the osteolysis model was successfully established. - / - Compared with the sham group, the cPLA2 + / - The density and number of trabecular bone in mice increased significantly. - / - Compared with the LPS group, cPLA2 + / - The expression of cPLA2 in the mitochondria was significantly increased in mice, indicating that cPLA2 knockout can alleviate the pathogenesis of LPS-induced inflammatory osteolysis.

[0059] The results of bone-related index detection in mice are as follows Fig. 9 As shown, cPLA2 in the Sham group - / - Compared with the sham group, the cPLA2 + / - The trabecular space TB.SP of mice was significantly reduced, and there was no significant difference in trabecular thickness TB.TH, trabecular number Tb.N, and osteolysis area; cPLA2 - / -Compared with the LPS group, cPLA2 + / - The thickness of mouse trabeculae TB.TH was significantly thickened, the trabecular space TB.SP was significantly reduced, the number of trabeculae Tb.N was significantly increased, and the area of ​​osteolysis was significantly reduced.

[0060] 2. ELISA test: Collect the serum of each group of mice and use the kit to measure the IL-6, IL-1β and TNF-a indicators.

[0061] The results are as follows Fig.10 As shown, the results showed that cPLA2 - / - Compared with the sham group, the cPLA2 + / - The level of inflammatory marker IL-1β in the serum of mice decreased significantly, while there was no significant difference in the levels of IL-6 and TNF-a. - / - Compared with mice, cPLA2 + / - The levels of inflammatory markers IL-6, IL-1β and TNF-a in the mouse serum were significantly decreased.

[0062] The above results indicate that cPLA2 plays a key role in the pathogenesis of osteolysis and can be used as a therapeutic target to alleviate osteolysis.

[0063] Example 3: In vitro functional experiments to explore the function of cPLA2

[0064] Experimental methods:

[0065] (1) In vitro culture of mouse bone marrow-derived macrophages

[0066] The genotype of the 8-week-old cPLA2 - / - and cPLA2 + / - C57BL / 6 mice were taken out, the femur and tibia were removed, the excess tissue was cleaned, the two ends were cut off and placed in a perforated EP tube, 50 μL of osteoclast induction medium was added, and the cells were centrifuged at 12000 rpm for 1 min, then the cells were resuspended, and centrifuged again at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended and plated, and M-CSF (macrophage colony stimulating factor, 10 ng / mL) was added for induction; the medium was changed after 3 days, and M-CSF (10 ng / mL) was continued to be added. After 6 days, macrophages (BMMs) were successfully cultured.

[0067] (2) Intervention of macrophage differentiation into osteoclasts

[0068] After most of the monocytes were fused into macrophages, RANKL (100 ng / mL) was used to stimulate the inflammatory phenotype of BMMs and measure inflammation-related indicators, and to induce osteoclast differentiation and measure osteoclast-related indicators.

[0069] Detection method:

[0070] 1. After inducing osteoclast differentiation of bone marrow-derived macrophages for 1 day, the cells were collected and detected by real-time fluorescence quantitative PCR (RT-qPCR). The total RNA of the cultured cells was extracted using TRIZOL reagent, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. The mRNA (ACP5, CTSK, cF0S, MMP9, DC-stamp) was quantified using an RT-qPCR kit to observe whether there was downregulation of osteoclast genes after cPLA2 gene knockout.

[0071] RT-qPCR results Fig.11 As shown, bone marrow-derived macrophages were induced to differentiate into osteoclasts for 1 day and then expressed in vitro with cPLA2 + / - Compared with the cells in the cPLA2 - / - The expression levels of osteoclast-related mRNA (ACP5, CTSK, cF0S, MMP9, DC-stamp) in the group of cells were significantly decreased, confirming that cPLA2 knockout can inhibit the effect of RANKL and prevent normal osteoclast differentiation.

[0072] 2. After inducing osteoclast differentiation of bone marrow-derived macrophages for 0, 1, 3, and 5 days, the cells were collected, the cell proteins were extracted, and the protein was detected by Western blotting. The protein bands were visualized using a gel imaging system.

[0073] Test results such as Fig.12 As shown, with cPLA2 + / - Compared with the cells in the cPLA2 - / - The expression levels of osteoclast-related transcription factors such as NFATc1, c-FOS, and MMP9 in the group cells were significantly decreased, confirming that cPLA2 knockout can significantly reduce the expression levels of osteoclast differentiation-related transcription factors.

[0074] 3. After inducing osteoclast differentiation of bone marrow-derived macrophages for 7-9 days, the cells were collected and stained with TRAP. The size of the stained (purple-red) area was examined with the naked eye and under a microscope to detect the level of osteoclast differentiation of the cells.

[0075] TRAP staining results Fig.13 As shown, with cPLA2 + / - Compared with the cells in the cPLA2 - / - The purple-red color of the cells in the group was lighter, and the number of osteoclasts was smaller, confirming that cPLA2 knockout could inhibit osteoclast differentiation.

[0076] The above results indicate that cPLA2 plays a key role in osteoclast differentiation, and cPLA2 knockout can inhibit osteoclast differentiation and thus treat osteoporosis.

Claims

1. Application of cPLA2 inhibitors in the preparation of drugs for the treatment of osteoporosis.

2. The use according to claim 1, characterized in that The cPLA2 inhibitor can reduce the expression of cPLA2 or reduce the protein activity of cPLA2, thereby increasing bone density and compressive resistance, and reducing the expression levels of osteoclast-related genes and inflammatory factors, thereby alleviating the pathogenesis of osteoporosis and achieving the effect of treating osteoporosis.

3. The use according to claim 1, characterized in that The cPLA2 inhibitor can reduce the expression level of cPLA2 or the protein activity of cPLA2, thereby increasing bone density and reducing the expression level of inflammatory factors, thereby alleviating bone dissolution and achieving the effect of treating osteoporosis.

4. The use according to claim 1, characterized in that The cPLA2 inhibitor can inhibit the effect of RANKL and prevent osteoclast differentiation by reducing the expression of cPLA2 or reducing the protein activity of cPLA2, thereby achieving the effect of treating osteoporosis.

5. A drug for treating osteoporosis, characterized in that: Including cPLA2 inhibitors.

6. The osteoporosis therapeutic drug according to claim 5, characterized in that: The cPLA2 inhibitor can reduce the expression level of cPLA2 or reduce the protein activity of cPLA2.

7. The osteoporosis therapeutic drug according to claim 5, characterized in that: The osteoporosis therapeutic drug also includes a pharmaceutically acceptable carrier or excipient.

8. Application of cPLA2 as a target in screening drugs for the treatment of osteoporosis.

9. The use according to claim 8, characterized in that The screened osteoporosis therapeutic drugs target cPLA2 and can reduce the expression level of cPLA2 or reduce the protein activity of cPLA2.

Images