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, cFOS, 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 changes of osteoporosis by targeted inhibition of expression of cPLA2, and the discovery provides a new target for treatment of osteoporosis. According to the invention, cPLA2 is taken as a target spot, and a small molecule compound marein for treating osteoporosis is screened out.

Description

Technical Field

[0001] This invention relates to the application of cPLA2 inhibitors in the preparation of osteoporosis treatment drugs, and belongs to the field of biomedical research technology. Background Technology

[0002] Osteoporosis is a systemic chronic inflammatory disease caused by various factors that disrupt the balance between bone resorption and bone formation, resulting in decreased bone mass, reduced bone density, and increased susceptibility to fractures. With the further development of an aging society, the number of osteoporosis patients has surged, gradually eroding our health and placing a significant burden on families and society. Current clinical medications cannot provide comprehensive and effective intervention, and are insufficient to truly regulate bone metabolism balance. Therefore, the prevention and treatment of osteoporosis has become a pressing and challenging problem for the medical community.

[0003] Bone is a metabolically active tissue, constantly undergoing bone remodeling and renewal, primarily mediated by osteoblast-regulated bone formation and osteoclast-regulated bone resorption. RANK / RANKL / OPG is a crucial signaling pathway mediating the balance between bone resorption and bone formation, essential for maintaining bone metabolic homeostasis. RANKL, an essential factor for osteoclast differentiation, is secreted by osteocytes, pre-osteoblasts, or immune cells. It binds to RANK on the surface of osteoclast precursor cells, activating the NF-κB transcription factor and increasing the transcription and translation levels of osteoclast differentiation-related genes c-FOS and NFATc1. This promotes the fusion of osteoclasts into multinucleated cells and their activation into mature osteoclasts, which secrete hydrochloric acid and various hydrolytic enzymes to dissolve minerals and reduce bone matrix. Osteoclast inhibitory factor (OPG) competitively inhibits the RANKL-RANK system by binding to RANKL, thus suppressing osteoclast differentiation. Therefore, current research and drug development focus on inhibiting RANKL function.

[0004] Currently, the most commonly used drugs in clinical practice are zoledronic acid and denosumab, but both have significant limitations in clinical application and prevention. Zoledronic acid can inhibit osteoclast function and reduce bone loss by binding to osteoclasts, but it has significant side effects, including fever, hypocalcemia, gastrointestinal reactions, and kidney damage, making long-term effective intervention impossible. Denosumab is a monoclonal antibody against RANKL and has strong targeting properties. It can effectively bind to RANKL, preventing its activation of RANK on the surface of osteoclasts and their precursor cells, inhibiting osteoclast maturation and activation, and reducing bone loss. However, it has a strong withdrawal reaction; after discontinuation of denosumab, the number and activity of osteoclasts rebound, causing rapid bone loss and greatly increasing the risk of recurrent fractures. Domestic and international studies have revealed that the regulation of RANKL signaling in the development of osteoporosis is a complex process. Inflammatory responses, oxidative stress, lipid metabolism, and autophagy damage can all lead to increased RANKL-induced bone loss, while RANKL can also exacerbate these pathological processes, accelerating the rate of bone loss. Single-factor regulation cannot comprehensively inhibit the RANKL signaling network; therefore, identifying key targets that can achieve comprehensive regulation during the pathogenesis of osteoporosis is a crucial scientific question.

[0005] To achieve targeted and comprehensive regulation of bone metabolism, we discovered through combined transcriptomic and proteomic analysis that phospholipase A2 (cPLA2 / PLA2G4A) plays a crucial role in bone metabolism regulation. cPLA2, a widely distributed enzyme, participates in various cellular responses, such as cell proliferation, differentiation, and inflammation. Its 505th amino acid can be phosphorylated and activated by extracellular regulated kinase 1 / 2 (ERK1 / 2) and p38 mitogen-activated protein kinase (p38MAPK). Activated cPLA2 can perform the following functions: 1. Degrading phospholipids on the surface of lysosomes, leading to lysosomal damage and inhibiting their fusion with autophagosomes, thus blocking autophagic flux; 2. Catalyzing the hydrolysis of glycerol lipids to produce lysophospholipids and release arachidonic acid (AA), leading to lipid metabolism disorders; 3. Phosphorylation, transmitting inflammatory signals, further exacerbating inflammatory responses and oxidative stress. We believe that the properties of cPLA2 suggest it may serve as a target for regulating bone metabolism homeostasis. cPLA2 holds promise as a novel target for regulating autophagy and inflammatory responses, potentially enabling effective prevention and treatment of osteoporosis through multi-level and comprehensive regulation of RANKL signaling. Therefore, we hypothesize that cPLA2 can effectively regulate bone metabolism homeostasis and treat osteoporosis by effectively modulating autophagic flux, inflammation, and oxidative stress, while simultaneously intervening in osteoclast differentiation and function.

[0006] Currently, there are no studies on the regulatory role of cPLA2 in the pathogenesis of osteoporosis. Therefore, elucidating the role of cPLA2 in the pathogenesis of osteoporosis, providing new therapeutic targets and experimental evidence, designing simple and efficient drug formulations targeting cPLA2 activity, developing unknown functions of existing clinical drugs, expanding drug indications, and achieving rapid and efficient clinical translation of drugs are of great clinical significance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides the application of cPLA2 inhibitors in the preparation of osteoporosis treatment drugs, offering a new molecular target for the treatment of osteoporosis.

[0008] The technical solution of the present invention is as follows: Application of cPLA2 inhibitors in the preparation of drugs for the treatment of osteoporosis.

[0009] According to a preferred embodiment of the present invention, the cPLA2 inhibitor can increase bone density and stress resistance by reducing the expression level of cPLA2 or reducing the protein activity of cPLA2, thereby reducing the expression levels of osteoclast-related genes and inflammatory factors, and thus alleviating the pathogenesis of osteoporosis and achieving the effect of treating osteoporosis.

[0010] According to a preferred embodiment of the present invention, the cPLA2 inhibitor can increase bone density and reduce the expression level of inflammatory factors by reducing the expression level of cPLA2 or reducing the protein activity of cPLA2, thereby alleviating osteolysis and achieving the effect of treating osteoporosis.

[0011] According to a preferred embodiment of the present invention, the cPLA2 inhibitor can inhibit the effect of RANKL by reducing the expression level of cPLA2 or reducing the protein activity of cPLA2, thereby preventing osteoclast differentiation and achieving the effect of treating osteoporosis.

[0012] According to a preferred embodiment of the present invention, the cPLA2 inhibitor comprises marigin.

[0013] An osteoporosis treatment drug, including a cPLA2 inhibitor.

[0014] According to a preferred embodiment of the present invention, the cPLA2 inhibitor can reduce the expression level of cPLA2 or reduce the protein activity of cPLA2.

[0015] According to a preferred embodiment of the present invention, the cPLA2 inhibitor comprises marigin.

[0016] According to a preferred embodiment of the present invention, the osteoporosis treatment drug further includes a pharmaceutically acceptable carrier or excipient.

[0017] According to a preferred embodiment of the present invention, the dosage form of the osteoporosis treatment drug includes, but is not limited to, tablets, powders, injections, capsules, or aerosols.

[0018] Application of cPLA2 as a target in screening drugs for the treatment of osteoporosis.

[0019] According to a preferred embodiment of the present invention, the screened osteoporosis treatment drug targets cPLA2 and can reduce the expression level of cPLA2 or reduce the protein activity of cPLA2.

[0020] According to a preferred embodiment of the present invention, the screened osteoporosis treatment drug targets cPLA2, and the drug binds to cPLA2 to increase the stability of the cPLA2 protein.

[0021] According to a preferred embodiment of the present invention, the osteoporosis treatment drug includes mariglycoside.

[0022] Beneficial effects: 1. This invention, through in vitro and in vivo experiments, confirms 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, cFOS, MMP9, DC-stamp) and inflammatory factors (IL-6, IL-1β, TNF-α) during the occurrence of osteoporosis. No researchers in this field have studied the relationship between cPLA2 and osteoclast-related genes and inflammatory factors and osteoporosis. The inventors' research on cPLA2 and osteoporosis models is groundbreaking.

[0023] 2. This invention discovers that cPLA2 plays a crucial role in the inflammation of osteoporosis and proposes reducing secondary pathological changes in osteoporosis by targeting and inhibiting cPLA2 expression. This discovery provides a new target for the treatment of osteoporosis, possessing significant clinical application potential and broad market prospects. This invention targets cPLA2 and screens for the small molecule compound marigin, which can be used to treat osteoporosis. Through in vitro and in vivo experiments, it was confirmed that marigin can exert its therapeutic effect on osteoporosis by targeting cPLA2 and reducing its protein activity. This discovery expands the pharmacological application scope of marigin, provides a new direction for its clinical development, and also provides a theoretical basis and experimental evidence for new clinical treatment strategies for osteoporosis. Attached Figure Description

[0024] Figure 1 These are representative 2D tomographic images of the femurs of four groups of mice in Example 1, obtained from miniature CT scans.

[0025] Figure 2These are representative miniature CT scan 3D reconstructed images of the femurs of four groups of mice in Example 1.

[0026] Figure 3 The bar chart shows the bone-related indices BMD, BV / TV, TB.TH, and TB.SP of the femur in four groups of mice in Example 1.

[0027] Figure 4 This is a bar chart showing the compressive strength of the three-point bending test of the femur in four groups of mice in Example 1.

[0028] Figure 5 HE staining images of femoral tissue from four groups of mice in Example 1.

[0029] Figure 6 The bar chart shows the expression levels of osteoclast-related genes ACP5, CTSK, and DC-stamp in the femoral tissues of four groups of mice in Example 1.

[0030] Figure 7 The bar chart shows the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of four groups of mice in Example 1.

[0031] Figure 8 These are representative miniature CT scan 3D reconstructed images of the skulls of four groups of mice in Example 2.

[0032] Figure 9 This is a bar chart showing the bone-related indicators TB.TH, TB.SP, Tb.N, and bone resorption area ratio of the skulls of four groups of mice in Example 2.

[0033] Figure 10 The bar chart shows the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in the serum of four groups of mice in Example 2.

[0034] Figure 11 The bar chart shows the expression levels of osteoclast-related genes ACP5, CTSK, cFOS, MMP9, and DC-stamp in the four groups of cells in Example 3.

[0035] Figure 12 This is a Western blotting image of osteoclast-related transcription factors in the two groups of cells in Experiment Example 3.

[0036] Figure 13 The image shows TRAP staining of four groups of cells in Experiment Example 3.

[0037] Figure 14 This is a schematic diagram of a virtual screening technology route for small molecule compounds targeting cPLA2 based on molecular docking.

[0038] Figure 15 This is a schematic diagram of the molecular docking between marisin and cPLA2 protein.

[0039] Figure 16 The diagram shows the molecular dynamics simulation of cPLA2 protein and malicin, with RMSD curve (a), Rg curve (b), SASA curve (c), HBONDS curve (d), and RMSF curve (e) in sequence.

[0040] Figure 17 The results of the DARTS experiment on marigin and cPLA2 protein are shown in the following figures: SDS-AGE electrophoresis (a) and Western blotting verification (b).

[0041] Figure 18 The results of the CETSA experiment on the binding of marigin and cPLA2 protein are shown in the following figures: Western blotting verification diagram and binding curve of marigin and cPLA2 protein at different temperatures with fixed doses (a), and Western blotting verification diagram and binding curve of marigin and cPLA2 protein at different doses with fixed temperatures (b).

[0042] Figure 19 These are representative miniature CT scan images of the femurs of six groups of mice in Example 6.

[0043] Figure 20 The bar chart shows the bone-related indices BV / TV, TB.TH, TB.SP, and TB.N of the femur in six groups of mice in Example 6.

[0044] Figure 21 This is a bar chart showing the compressive strength of the femur in six groups of mice in the three-point bending test, as described in Example 6.

[0045] Figure 22 This is a TRAP staining image of the femoral tissue of six groups of mice in Example 6.

[0046] Figure 23 The bar chart shows the expression levels of osteoclast-related genes ACP5, CTSK, and DC-stamp in the femoral tissue of six groups of mice in Example 6.

[0047] Figure 24 The survival curves of WT mouse BMMs under different concentrations of marigin treatment are shown.

[0048] Figure 25 The images show Western blotting results of WT mouse BMMs after treatment with malicin, in the order of (a) and (b) at different doses at a fixed time.

[0049] Figure 26This is a TRAP staining image of WT mouse BMMs after treatment with marinoside.

[0050] Figure 27 Image showing rhodamine-labeled phalloidin staining in WT mouse BMMs after treatment with marinoside.

[0051] Figure 28 Western blotting of osteoclast-related transcription factors in WT and KO mouse BMMs after treatment with marinoside.

[0052] In the significance analysis of 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 Implementation

[0053] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and consumables involved in the embodiments are all commercially available products; unless otherwise specified, the steps and experimental operations involved in the embodiments are all conventional technical operations in the art.

[0054] The parent mice used in these examples were sourced from: C57BL / 6J mice were purchased from Cyagen. 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 and ordinary C57BL / 6 mice.

[0055] Mariglycoside, purchased from MedChemExpress.

[0056] Example 1: In vivo experiments to investigate the key role of cPLA2 as a therapeutic target in osteoporosis In vivo experiments were conducted to verify the key role of cPLA2 as a novel target in osteoporosis, and cPLA2 was constructed. + / - C57BL / 6 mice, cPLA2 - / - C57BL / 6 mice were used as parent mice. After breeding, female mice were selected and ovariectomy (OVX) was performed to establish an osteoporosis model. The mice were observed for 3 months after the operation.

[0057] Experimental methods: An ovarian-neutered osteoporosis mouse model (OVX) was established. cPLA2 was constructed. + / -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 rearing, their tails were clipped to identify their genotype. After 8 weeks of rearing, female mice with suitable genotypes underwent bilateral ovariectomy. The specific steps were as follows: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (3 mg / mL, 0.1 mL / 10 g). After the back of the mice was shaved and prepared, they were fixed face down on the operating table. A long incision was made along the posterior midline of the back, centered at the intersection of a line 0.5 cm from the upper edge of the thigh root on both sides of the hind limbs and the posterior midline. A skin incision of approximately 0.5-1.0 cm was made. The incision was extended 0.5 cm to the left, revealing a white fat pad beneath the basal layer. The muscle layer above the fat pad was lifted with forceps, and the muscle layer was longitudinally incised along the lateral edge of the psoas major muscle using ophthalmic scissors, approximately 0.5 cm long. The fat pad was then lifted to reveal a red, mulberry-like ovary on the left side of the mouse. After removing the ovary with ophthalmic scissors, the incised muscle layer was sutured shut with No. 5 suture. The right ovary was located and removed using the same method. The muscle layer was sutured shut with No. 5 suture, and the skin incision was closed. Sodium penicillin was injected into both the muscle and skin incisions to prevent bacterial infection. The mice were placed in a warm environment and awaited recovery. In the sham surgery group, the back skin of the mice was incised, the fat pad next to the ovary was removed, and the skin incision was sutured. Sodium penicillin was injected into both the muscle and skin incisions to prevent bacterial infection. The mice were placed in a warm environment and awaited recovery. Postoperative observation lasted for 3 months, followed by testing.

[0058] Detection method: 1. Miniature CT Scan: Mice were anesthetized with sodium pentobarbital solution, then euthanized by cervical dislocation. Bilateral femurs were harvested, fat and muscle were removed, and the femurs were fixed in 4% paraformaldehyde at 4°C for 24 hours. The paraformaldehyde was then replaced with 70% ethanol and stored at 4°C. A 360-degree scan of the treated unilateral femur was performed using a miniature CT scanner with the following parameters: 90kV, 88mA. Finally, the bone volume fraction (BV / TV), trabecular bone number (TB.N), trabecular bone spacing (TB.SP), and trabecular bone thickness (TB.TH) were measured.

[0059] Mouse miniature CT scan results as follows Figure 1 , Figure 2 As shown, the OVX group mice had significantly reduced trabecular bone density and number compared to the Sham group mice, proving the successful establishment of the osteoporosis model mouse system. Sham group cPLA2 - / - mice compared to the Sham group cPLA2 + / - The density and number of trabecular bone in mice were significantly increased, and cPLA2 in the OVX group was also significantly increased. - / - Mice also showed cPLA2 levels compared to the OVX group. + / - The significant increase in mice indicates that cPLA2 knockout can alleviate the pathogenesis of osteoporosis.

[0060] Results of bone-related marker tests in mice are as follows: Figure 3 As shown, Sham group cPLA2 - / - Mice compared to the Sham group cPLA2 + / - Mice showed significantly increased bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone spacing (TB.SP), and trabecular bone thickness (TB.TH); OVX group cPLA2 - / - mice compared to the OVX group cPLA2 + / - The same trend is observed in mice.

[0061] 2. Three-point bending test: Each femur is placed on the bending support of the testing machine, and the machine is driven to apply a bending moment at a test speed of 5 mm / min until the femur is damaged. After the test, the printer automatically prints out the test data for each femur.

[0062] Experimental results are as follows Figure 4 As shown, the results demonstrate that the Sham group cPLA2 - / - The stress resistance of mice compared with that of the Sham group cPLA2 + / - No significant difference was observed in mice; cPLA2 in the OVX group - / - The mice's stress resistance was higher than that of the OVX group cPLA2 + / - The effect was significantly enhanced in mice.

[0063] 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 decalcification, the femurs were embedded in paraffin. The embedded specimens were cut into 5 μm sections and then stained with hematoxylin and eosin (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.

[0064] The results of HE staining of the tissue are as follows Figure 5 As shown, the results indicate that the Sham group cPLA2 - / - Mouse bone tissue compared to Sham group cPLA2 + / - Mice showed significant enhancement, with cPLA2 in the OVX group. - / - Mice also showed cPLA2 levels compared to the OVX group. + / - The number of mice has increased.

[0065] 4. Tissue RT-qPCR: Mouse femoral tissue was ground in liquid nitrogen, with 1 ml of Trizol reagent added per 50-100 mg of tissue, and homogenized. RNA was extracted from the tissue and reverse transcribed into cDNA using a reverse transcription kit. Osteoclast-related mRNAs (ACP5, CTSK, DC-stamp) were quantified using an RT-qPCR kit to observe whether osteoclast genes were downregulated after cPLA2 gene knockout.

[0066] RT-qPCR results are as follows Figure 6 As shown, the results indicate that the Sham group cPLA2 - / - mice compared to the Sham group cPLA2 + / - The levels of osteoclast-related factors CTSK and DC-stamp in mouse tissues were significantly decreased, while ACP5 showed no significant difference; cPLA2 in the OVX group - / - mice compared to the OVX group cPLA2 + / - The levels of osteoclast-related factors ACP5, CTSK, and DC-stamp in mouse tissues were significantly decreased.

[0067] 5. ELISA test: Collect serum from each group of mice and use the kit to measure IL-6, IL-1β and TNF-α levels.

[0068] The results are as follows Figure 7 As shown, the results indicate that the Sham group cPLA2 - / - mice compared to the Sham group cPLA2 + / - Serum levels of inflammatory markers IL-6 and TNF-α in mice were significantly decreased, while IL-1β showed no significant difference; cPLA2 in the OVX group - / - mice compared to the OVX group cPLA2 + / - The levels of inflammatory markers IL-1β, IL-6 and TNF-α in mouse serum were significantly decreased.

[0069] The above results indicate that cPLA2 plays a key role in the pathogenesis of osteoporosis and can serve as a therapeutic target to alleviate the progression of osteoporosis.

[0070] Example 2: In vivo experiments to investigate the key role of cPLA2 as a therapeutic target in osteolysis. Experimental methods: A lipopolysaccharide (LPS)-induced inflammatory mouse model of craniolysis was constructed, and cPLA2 was developed. + / - 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 rearing, their tails were clipped to identify their genotype. After 5-6 weeks of rearing, male mice with suitable genotypes were selected for LPS surgery. The specific steps are as follows: 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 of the mouse skull was removed and the skin was prepared, the mouse was fixed face down on the operating table. A skin incision of about 0.5-1.0 cm was made along the midline of the skull, with the midline of the line connecting the two ears of the mouse as the center. The skin incision faced forward. The muscle layer of the epidermis was dissected with forceps, and the meningeal layer on the surface of the skull was cut open. LPS solution, which had been dissolved in PBS buffer beforehand, was dripped in at 12.5 mg / g. After the liquid had seeped in, the skin incision was sutured with No. 5 suture. The mouse was placed in a warm environment to wait for awakening. In the sham surgery group (Sham group), the skin on the upper side of the skull of mice was incised, and the same volume of PBS buffer was dripped in. The skin incision was then sutured, and the mice were placed in a warm environment to await recovery. They were observed for one week post-surgery, and tests were performed one week later.

[0071] Detection method: 1. Miniature CT Scan: Mice were anesthetized with sodium pentobarbital solution, then euthanized by cervical dislocation. Skull bones were collected, fat and muscle were removed, and the skull was fixed in 4% paraformaldehyde at 4°C for 24 hours. The paraformaldehyde was then replaced with 70% ethanol and stored at 4°C. A 360-degree scan was performed on the treated skull using a miniature CT scanner with the following parameters: 90kV, 88mA. Finally, trabecular thickness (TB.TH), trabecular spacing (TB.SP), trabecular number (TB.N), and osteolysis area were measured.

[0072] Mouse miniature CT scan results as follows Figure 8 As shown, the LPS group mice had significantly lower bone trabecular density and number compared to the Sham group mice, proving the successful establishment of the osteolysis model in mice. Sham group cPLA2 - / - mice compared to the Sham group cPLA2 + / - The density and number of trabecular bone in mice were significantly increased, and cPLA2 in the LPS group was also significantly increased. - / - Mice also showed cPLA2 levels compared to the LPS group. + / - The number of mice increased significantly. This indicates that cPLA2 knockout can alleviate the pathogenesis of LPS-induced inflammatory osteolysis.

[0073] Results of bone-related marker tests in mice are as follows: Figure 9 As shown, Sham group cPLA2 - / - mice compared to the Sham group cPLA2 + / - In mice, the intertrabecular space (TB.SP) was significantly reduced, while there were no significant differences in trabecular thickness (TB.TH), trabecular number (Tb.N), and osteolysis area; cPLA2 in the LPS group - / - mice compared to the LPS group cPLA2+ / - The trabecular bone thickness (TB.TH) of mice was significantly increased, the intertrabecular bone spacing (TB.SP) was significantly decreased, the number of trabecular bone (Tb.N) was significantly increased, and the area of ​​bone resorption was significantly reduced.

[0074] 2. ELISA test: Serum from each group of mice was collected, and IL-6, IL-1β and TNF-α levels were measured using a kit.

[0075] The results are as follows Figure 10 As shown, the results indicate that the Sham group cPLA2 - / - mice compared to the Sham group cPLA2 + / - Serum levels of the inflammatory marker IL-1β in mice were significantly decreased, while levels of IL-6 and TNF-α showed no significant difference; LPS group cPLA2 - / - Compared with mice, the LPS group cPLA2 + / - The levels of inflammatory markers IL-6, IL-1β and TNF-α in mouse serum were significantly decreased.

[0076] The above results indicate that cPLA2 plays a key role in the pathogenesis of osteolysis and can serve as a therapeutic target to alleviate osteolysis.

[0077] Example 3: In vitro functional experiments to investigate the function of cPLA2 Experimental methods: (1) In vitro culture of mouse bone marrow-derived macrophages The genotype of 8-week-old infants was selected as cPLA2. - / - and cPLA2 + / - C57BL / 6 mice were used. After removing the femur and tibia, excess tissue was cleaned off, the ends were cut off, and the cells were placed in perforated EP tubes. 50 μL of osteoclast induction medium was added, and the cells were centrifuged at 12,000 rpm for 1 min. The cells were then resuspended and centrifuged again at 1,000 rpm for 5 min. The supernatant was discarded, the cells were resuspended, and the cells were seeded into a plate. 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 added again. After 6 days, the cells were successfully cultured into macrophages (BMMs).

[0078] (2) Intervention in the differentiation of macrophages into osteoclasts After most monocytes fused into macrophages, RANKL (100 ng / mL) was used to stimulate the inflammatory phenotype of BMMs, measure inflammation-related markers, and induce osteoclast differentiation to measure osteoclast-related markers.

[0079] Detection method: 1. One day after inducing osteoclast differentiation in bone marrow-derived macrophages, cells were collected and detected using real-time quantitative PCR (RT-qPCR). Total RNA was extracted from cultured cells using TRIZOL reagent, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. The mRNA (ACP5, CTSK, cFOS, MMP9, DC-stamp) was quantified using an RT-qPCR kit to observe whether there was downregulation of osteoclast genes after cPLA2 gene knockout.

[0080] RT-qPCR results are as follows Figure 11 As shown, one day after induction of osteoclast differentiation of bone marrow-derived macrophages, cPLA2... + / - Compared to the group of cells, cPLA2 - / - The expression levels of osteoclast-related mRNAs (ACP5, CTSK, cFOS, MMP9, DC-stamp) in the group cells were significantly decreased, confirming that cPLA2 knockout can inhibit the effect of RANKL and prevent normal osteoclast differentiation.

[0081] 2. Macrophages derived from bone marrow were induced to undergo osteoclastogenesis for 0, 1, 3, and 5 days. Cell proteins were extracted, and Western blotting was used to detect protein bands. A gel imaging system was used to visualize the protein bands.

[0082] Test results as follows Figure 12 As shown, with cPLA2 + / - Compared to the group of cells, cPLA2 - / - The expression levels of osteoclast-related transcription factors such as NFATc1, cFOS, 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.

[0083] 3. After inducing osteoclast differentiation in bone marrow-derived macrophages for 7-9 days, collect the cells, perform TRAP staining, and examine the size of the stained (purple-red) area with the naked eye and under a microscope to detect the osteoclast differentiation level of the cells.

[0084] TRAP staining results are as follows Figure 13 As shown, with cPLA2 + / - Compared to the group of cells, cPLA2 - / - The cells in this group showed a lighter purplish-red staining and fewer osteoclasts, confirming that cPLA2 knockout can inhibit osteoclast differentiation.

[0085] The above results indicate that cPLA2 plays a key role in osteoclast differentiation, and cPLA2 knockout can inhibit osteoclast differentiation, thereby treating osteoporosis.

[0086] Example 4: Computer-based virtual screening of cPLA2-targeting inhibitors A schematic diagram of the virtual screening technology route for small molecule compounds targeting cPLA2 based on molecular docking is shown below. Figure 14 As shown, specifically: 1) Protein Preparation: Download the 3D structure of Human cPLA2 (PDB ID: 1CJY) from the RCSB PDB website. Use the Protein Preparation Wizard module to remove water molecules, add hydrogen, remove the B chain, remove other small molecules and ligands, and add missing atoms. Then perform energy optimization (OPLS2005 force field, RMSD 0.30 Å). Create a grid file of the processed protein using the Receptor Grid Generation module, with a box size of 20 Å × 20 Å × 20 Å.

[0087] 2) Compound preparation: The 2D formats of HY-L001P MCE Bioactive Compound Library Plus (containing 21.1K compounds), HY-L111 Novel Bioactive Compound Library (containing 1201 compounds), and HY-L901MCE 50K Diversity Library (containing 50,000 compounds) were processed by hydrogenation, energy optimization, etc. in the LigPrepModule of Schrödinger software to output 3D structures for virtual screening.

[0088] 3) Molecular docking: The Virtual Screening Workflow module is used for virtual screening. The prepared compounds are imported, and the Glide module is used for molecular docking, where the acceptor and ligand molecules dock with each other through geometric and energy matching. HY-L001P MCE Bioactive Compound Library Plus: First, the high-throughput screening (HTVS) mode in the Glide module was used to screen the small molecule compounds prepared in the database. The top 15% of the small molecule compounds with the highest scores were selected for a second round of screening using the standard (SP) mode. Then, the top 15% of the small molecule compounds with the highest scores were selected for a third round of screening using the high-precision (XP) mode to obtain the ranking of the small molecule compounds by score. The screening results of the top 20 small molecule compounds are shown in Table 1.

[0089] Table 1. Compound screening results of HY-L001P MCE Bioactive Compound Library Plus

[0090] HY-L111 Novel Bioactive Compound Library: First, the small molecule compounds prepared in the database are screened using the standard (SP) mode in the Glide module. The top 50% of the small molecule compounds with the highest scores are selected for a second round of screening using the high precision (XP) mode to obtain the ranking of the small molecule compounds by score. The screening results of the top 20 small molecule compounds are shown in Table 2.

[0091] Table 2. Compound screening results of HY-L111 Novel Bioactive Compound Library

[0092] HY-L901 MCE 50K Diversity Library: First, the high-throughput screening (HTVS) mode in the Glide module was used to screen the small molecule compounds prepared in the database. The top 10% of the small molecule compounds with the highest scores were selected for a second round of screening using the standard (SP) mode. Then, the top 10% of the small molecule compounds with the highest scores were selected for a third round of screening using the high-precision (XP) mode to obtain the ranking of the small molecule compounds by score. The screening results of the top 20 small molecule compounds are shown in Table 3.

[0093] Table 3. Compound screening results of HY-L901 MCE 50K Diversity Library

[0094] Based on the review of target-compound binding affinity and compound structure, and considering the ranking results and prior research on small molecule compounds, marein was selected as a candidate molecule that may bind to the target protein cPLA2. The chemical structure of marein is as follows:

[0095] Example 5: Affinity test of marigin-cPLA2 (1) Computer simulation of molecular docking experiment (MOE-Dock experiment): The two-dimensional structure of marigin was downloaded from PubChem and converted into a three-dimensional structure in MOE through energy minimization to serve as a ligand. The cPLA2 protein sequence was found on Uniprotkb and imported into the SWISS-MODLE website to find the optimal structure. Energy minimization was performed on the selected ligand to reduce the energy in the system after docking and achieve a stable state. The ligand was optimized using DS software. The protein file was imported into PYMOL software, redundant ligands and water molecules were removed, and the protein was hydrogenated and charged, then set as a receptor. Small molecules were hydrogenated and charged, then set as ligands. Twist angles and torsion bonds were checked, and the grid was set to the entire protein region for ligand binding. After grid setting, 50 Autodock dockings were performed, and the conformation with the highest reproducibility and best binding energy was selected.

[0096] The conformation of marigin binding to cPLA2 protein is as follows: Figure 15 As shown, marigin exhibits a binding interaction deep within the cPLA2 binding pocket. Within the binding pocket, the 3D model reveals that marigin forms four hydrogen bonds with the GLU710, ASP608, and GLU615 amino acid residues on the cPLA2 protein. The 3D model also shows that the ligand exists within the cavity on the protein surface, consistent with the ligand structure, which is beneficial for binding. In the 2D model, the ligand interacts with the amino acids on the protein via van der Waals forces, essential for the stability of the binding site. MOE-Dock simulations determined the binding affinity of marigin to cPLA2, with a docking fraction of -7.5 kcal / mol, indicating a strong interaction between marigin and cPLA2.

[0097] (2) cPLA2 molecular dynamics simulation experiment of malicin Molecular dynamics simulations were performed using GROMACS 2022, with force field parameters generated using the pdb2gmx tool in GROMACS. The topology file for the GAFF2 force field was generated based on the ligand structure using sobtop_1.0 (dev3.1) software, and charge distribution of the ligands was performed using the RESP method to ensure the charge distribution conformed to physicochemical properties. For the receptor protein, AMBER14SB force field parameters were used. During the simulation, solvation of the system was performed using the TIP3P water model, employing a 1 nm cubic water box to ensure adequate solvation and electroneutrality. To ensure electroneutrality, Na+ was added to the system using the gmx genion tool in GROMACS. + and Cl Ions were used, with an ion concentration of 0.15 M NaCl. Charge neutralization and ion strength were set to normal physiological conditions during the simulation. Long-range electrostatic interactions were handled using the Particle Mesh Ewald (PME) method, with a cutoff distance of 1 nm. Force field parameters and PME method settings were optimized according to GROMACS specifications. Bond constraints were performed using the LINCS algorithm. Prior to molecular dynamics simulations, the system underwent energy optimization. Energy minimization included 3000 steps of the steepest descent method followed by 2000 steps of the conjugate gradient method. The optimization process consisted of three stages: first, constraining the solute and minimizing the energy of water molecules; then, constraining the counter ions and minimizing the energy; and finally, unconstrained energy minimization of the entire system. During simulation, the temperature was maintained at 310 K using a Nosé–Hoover isothermal coupler; the pressure was maintained at 1 bar using a Parrinello–Rahman isobaric coupler. The simulation time was set to 100 ns, using an NPT (isobaric isothermal) set with an integration step size of 2 fs. During the simulation, we used the GROMACS tools gmx_rmsd, gmx_rmsf, ​​gmx_hbond, gmx_Rg, and gmx_sasa to calculate the root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonds (HBonds), radius of gyration (Rg), and solvent accessible surface area (SASA) to analyze the system's stability, structural changes, and solvent effects.

[0098] RMSD is a good indicator of conformational stability of proteins and ligands, and also an indicator of the degree of deviation of atomic positions from their initial positions. The smaller the deviation, the better the conformational stability. Therefore, RMSD was used to evaluate the balance of the simulation system. Figure 16 As shown in Figure a, the marisin-cPLA2 protein complex system reached equilibrium after 20 ns, eventually fluctuating around 1.5 Å. This indicates that marisin exhibits high stability when binding to the cPLA2 protein. Rg can be used to describe changes in the overall structure and can be used to characterize the tightness of protein structure, such as... Figure 16 As shown in Figure b, the complex system exhibits slight fluctuations during motion, indicating a conformational change occurred. SASA is an indicator for assessing protein surface area; this simulation calculated the solvent-accessible surface area between the target protein and the small molecule, such as... Figure 16 As shown in Figure c, the results indicate that the complex system exhibits slight fluctuations. This demonstrates that the binding of small molecules affects the binding microenvironment and leads to some degree of change in SASA. Hydrogen bonds play a crucial role in the binding of ligands to proteins. The number of hydrogen bonds between small molecules and target proteins during the kinetic process is shown in Figure c. Figure 3As shown in Figure d, the number of hydrogen bonds between marisin and the cPLA2 protein ranges from 0 to 4, with approximately 2 hydrogen bonds in most cases. This indicates that the marisin-cPLA2 protein complex exhibits good hydrogen bond interactions. RMSF can represent the flexible size of amino acid residues in a protein, such as... Figure 16 As shown in Figure e, the RMSF values ​​of the marisin-cPLA2 protein complex are relatively low (mostly below 4 Å), thus exhibiting lower flexibility and higher stability.

[0099] In summary, the marisin-cPLA2 protein complex system is stable and exhibits good hydrogen bonding, thus demonstrating good binding between marisin and cPLA2 protein.

[0100] (3) Stability test of marigin binding to cPLA2 protein (DARTS test) 1) Prepare DARTS cell lysis buffer (1 mL): 730 μL M-PER mammalian protein extraction reagent, 10 μL protease inhibitor, 10 μL 200 mM phosphatase inhibitor sodium orthovanadate, 50 μL 1 M sodium fluoride solution, 100 μL 100 mM β-glycerophosphate sodium solution, and 100 μL 50 mM sodium pyrophosphate solution; Prepare 10×TNC (1 mL): pH 8.0, 500 μL 1 M Tris-HCl buffer, 100 μL 5 M sodium chloride solution, 100 μL 1 M calcium chloride solution, and 300 μL sterile deionized water. Before use, dilute 10×TNC 10 times with sterile deionized water; the protease concentration should be 10 mg / mL.

[0101] 2) Wash 293T cells twice with pre-cooled phosphate-buffered saline (PBS). After removing the PBS, add 500 μL of DARTS cell lysis buffer. Scrape cells and transfer them to 1.5 mL EP tubes. Incubate at 4°C on a shaker for 1.5 hours. Centrifuge at 13000 rpm for 15 minutes at 4°C. Collect the supernatant protein and add 225 μL of supernatant protein to two separate new EP tubes. Add 5 μL of DMSO (drug solvent, control group) to one tube and 5 μL of marigin (final drug concentration 200 μM, drug group) to the other. Incubate at room temperature in the dark for 1.5 hours. Divide each protein tube into four portions (8 tubes total), and fill each tube with 50 μL of DMSO solution. μL; Prepare protease solutions of different concentrations using 1×TNC, according to protease / protein ratios of 0, 1:1600, 1:800, and 1:400 (mass ratios). Add the prepared protease solutions to the corresponding protein tubes (groups are -0, +0, -1:1600, +1:1600, -1:800, +1:800, -1:400, +1:400; - is the control group, + is the drug group; the order of sample loading for gel electrophoresis is also the same). Group 0 is added with 1×TNC. Incubate at room temperature for 10 min, add 1 μL of cocktail to each tube, and incubate on ice for 5 min to terminate digestion. Add loading buffer at a protein:loading buffer ratio of 4:1, boil in a water bath for 10 min, and then store at -80℃ or directly perform SDS-PAGE electrophoresis. The loading order was consistent with the grouping order. After SDS-PAGE electrophoresis, Coomassie blue staining was performed directly without membrane transfer for 1 h. The destaining solution was changed and eluted until clear bands appeared. The results were verified by Western blotting.

[0102] SDS-PAGE electrophoresis image as follows Figure 17 As shown in Figure a, a difference band appears around 100 kDa. Western blotting validation results are as follows... Figure 17 Figure b shows that the differential band corresponds to the cPLA2 protein. Marigold may improve protein stability and resist protease digestion by binding to the cPLA2 protein.

[0103] (4) Cell heat transfer assay (CETSA assay) 1) Binding of drug to target protein at fixed doses and different temperatures: 293T cells in logarithmic growth phase were cultured in medium (a-MEM, 10% FBS, 1% penicillin-drug antibody) until 70%-80% confluence. The control group received DMSO, while the drug-treated group received mariglobulin (DMSO solvent, final concentration 10 μM). Cells were incubated for 1 hour. Cells were resuspended and aliquoted into nine 200 μL PCR tubes (100 μL / tube, approximately 3 million cells per tube). Each tube was labeled with specified temperatures: 40, 43, 46, 49, 52, 55, 58, and 61 °C. The tubes were heated for 3 minutes at each of the nine temperatures using a 96-well plate thermal cycler, immediately removed, and incubated at room temperature for 3 minutes; then rapidly frozen in liquid nitrogen. The cells were frozen and thawed twice using liquid nitrogen and a thermal cycler set to 25 °C to ensure uniform temperature between tubes. Briefly vortex the tube and centrifuge at 2000 g for 20 min at 4°C to precipitate cell debris and aggregated proteins. Add 40 μL of cell lysis supernatant and 20 μL of educating loading buffer to a new 200 μL PCR tube, vortex, briefly centrifuge, and heat at 100°C for 10 min; vortex the tube again, briefly centrifuge, load the sample, and perform Western blotting to detect the target protein.

[0104] Target protein detection results as follows Figure 18 As shown in Figure a, at different temperatures (49℃-61℃), maricin can reduce the denaturation of the target protein cPLA2 compared to dimethyl sulfoxide (DMSO), especially at 49℃, indicating that maricin can bind to cPLA2 and inhibit its denaturation.

[0105] 2) Drug binding to target protein at different doses at a fixed temperature: A suspension of 293T cells in logarithmic growth phase was taken, with a cell suspension concentration of approximately 4 × 10⁻⁶. 7Cells / mL were aliquoted into seven 200μL PCR tubes (100μL / tube). Different concentration gradients of maligosin solution (DMSO) were added to each tube to achieve final maligosin concentrations of 0, 0.001, 0.01, 0.1, 1, 10, and 100μM. The tubes were incubated at 37°C, 5% CO2, and saturated humidity for 30 min, agitated every 10 min. The PCR tubes were then placed in a Techne thermal cycler and heated to 75°C for 3 min, followed by incubation at room temperature for 3 min, and then rapidly frozen in liquid nitrogen. The cells were frozen and thawed twice using liquid nitrogen and a thermal cycler set to 25°C to ensure uniform temperature between tubes. The tubes were briefly shaken and centrifuged at 4°C, 2000g for 20 min to precipitate cell debris and aggregated proteins. Add 40 μL of cell lysis supernatant and 20 μL of reducing loading buffer to a new 200 μL PCR tube, vortex, briefly centrifuge, heat at 100℃ for 10 min; vortex the tube again, briefly centrifuge, load the sample, and detect the target protein using Western blotting.

[0106] Target protein detection results as follows Figure 18 As shown in Figure b, at 49℃, maricin inhibited cPLA2 denaturation in a dose-dependent manner, indicating that the inhibitory effect of maricin on cPLA2 denaturation is drug dose-dependent.

[0107] Example 6: In vivo experiments to investigate the therapeutic effect of marigin on osteoporosis by targeting cPLA2. In vivo experiments validated the key roles of cPLA2 as a novel target and maricin as its inhibitor in osteoporosis. The study selected ordinary C57BL / 6 and cPLA2... - / - C57BL / 6 mice were divided into WT and KO groups. Osteoporosis model was established by bilateral ovariectomy (OVX). The WT and KO groups were further divided into ovariectomy and intraperitoneal injection of PBS solution group (OVX group), ovariectomy and intraperitoneal injection of maligosin group (OVX-MAR group, drug solvent is PBS solution), and sham operation group and intraperitoneal injection of PBS solution group (Sham group), for a total of 6 groups, with 10 mice in each group.

[0108] Experimental Methods: An ovariectomized osteoporosis mouse model (OVX) was established. Female mice from the WT and KO groups were selected and fed for 8 weeks before undergoing bilateral ovariectomy, following the same procedures as in Example 1. Postoperatively, the OVX+MAR group received daily intraperitoneal injections of 50 μM / kg marigline (solvent: PBS solution), while the OVX and Sham groups received the same volume of PBS solution daily via intraperitoneal injection. After 60 days, the mice were sacrificed, and tissue samples were collected for micro-CT scanning, three-point bending test, and tissue RT-qPCR detection of osteoclast-related indicators, using the same methods as in Example 1. Histological examination (TRAP staining) was also performed.

[0109] 1) Miniature CT scan: Results of miniature CT scans of mice are as follows Figure 19 As shown, the OVX+MAR group in WT mice showed a significant increase in trabecular bone density and number compared to the OVX group, demonstrating that maricin has a significant therapeutic effect on OVX-induced osteoporosis. However, the difference in trabecular bone density and number between the OVX+MAR group and the OVX group in KO mice was not significant compared to that in WT mice, indicating that the therapeutic effect of maricin on osteoporosis in mice after cPLA2 knockout in vivo was not significant, proving that the pharmacological effect of maricin is achieved through targeted action on cPLA2.

[0110] Results of bone-related marker tests in mice are as follows: Figure 20 As shown, the WT mouse OVX+MAR group showed a significant increase in bone volume fraction (BV / TV), a significant decrease in trabecular bone spacing (TB.SP), a significant increase in trabecular bone thickness (TB.TH), and a significant increase in the number of trabecular bones (TB.N) compared to the OVX group; while there were no significant differences in BV / TV, TB.SP, TB.TH, and TB.N between the KO mouse OVX+MAR group and the OVX group.

[0111] 2) Three-point bending test: Experimental results are as follows Figure 21 As shown, the results demonstrate that the OVX+MAR group of WT mice had significantly enhanced stress resistance compared to the OVX group; while there was no significant difference in stress resistance between the OVX+MAR group and the OVX group in KO mice.

[0112] 3) Histological examination: The fixed femur was decalcified with 10% EDTA at 4℃ for 30 days. Afterwards, sagittal longitudinal sections were paraffin-embedded, and a 5mm thick section from the center was used for TRAP staining. According to the instructions, the mixed TRAP staining solution was preheated to 37℃; treated with xylene, graded alcohol, and ddH2O; incubated at 37℃ with TRAP staining solution for 30 min; rinsed three times with PBS solution; treated with graded alcohol and xylene; air-dried; and mounted with neutral resin. Osteoclasts were TRAP-positive multinucleated cells on the surface of the adjacent trabeculae. After fixation, TRAP staining was performed according to the instructions. Osteoclasts in bone tissue appeared wine-red.

[0113] Experimental results are as follows Figure 22 As shown, the number of osteoclasts in the WT mouse OVX group was significantly higher than that in the OVX+MAR group and the SHAM group, while there was no significant difference in the number of osteoclasts among the three KO mouse groups.

[0114] 4) Tissue RT-qPCR: RT-qPCR results are as follows Figure 23 As shown, the results indicate that the levels of osteoclast-related factors ACP5, CTSK, and DC-stamp in the OVX+MAR group of WT mice were significantly lower than those in the OVX group, while there was no significant difference in the levels of osteoclast-related factors ACP5, CTSK, and DC-stamp between the OVX+MAR group and the OVX group in KO mice.

[0115] The above results indicate that cPLA2 plays a key role in the pathogenesis of osteoporosis, and that marigin can alleviate the progression of osteoporosis by targeting cPLA2.

[0116] Example 7: In vitro experimental verification of the function of maricin in inhibiting osteoclast differentiation (1) In vitro culture of mouse bone marrow-derived macrophages Select 8-week-old regular C57BL / 6 (WT) or cPLA2. - / - C57BL / 6 (KO) mice were used to culture macrophages (BMMs) according to the experimental method in Example 3.

[0117] (2) The cytotoxicity of malicin to mouse BMMs was detected by CCK-8 assay. The CCK-8 assay kit can be used to detect cell proliferation and cytotoxicity. Its principle is based on the reduction of WST-8 in the CCK-8 reagent to an orange-yellow formazan product by dehydrogenases in the mitochondria of cells. The color intensity of the formazan product is directly proportional to the number of viable cells. The OD value can be measured at 450 nm using a microplate reader, indirectly reflecting cell viability and metabolic activity. After WT mouse BMMs reached the logarithmic growth phase, a cell suspension was prepared and seeded at a density of 5000 cells / well in 96-well plates, with 6 replicates per group. After cell adhesion at 37℃ and 5% CO2, the original culture medium was discarded, and cells were treated with different final concentrations (0-200 μM) of malicin. The control group was treated with complete culture medium without malicin. After incubation for 24 h, 48 h, and 72 h respectively, the original culture medium was discarded, and 90 μL of fresh culture medium and 10 μL of CCK-8 solution were added to each well. The cells were cultured in the dark in a cell culture incubator for 2 h, and the absorbance at 450 nm was measured using a microplate reader to calculate the cell viability.

[0118] Experimental results are as follows Figure 24As shown, the results indicate that maricin concentrations below 100 μM are not toxic to BMM cells. Furthermore, under the conditions of this study, maricin treatment did not exhibit significant cytotoxicity.

[0119] (3) Detection of osteoclast-related factors in BMMs of WT mice after treatment with marigin 1) Osteoclastogenesis was induced in WT mouse BMMs (100 ng / mL RANKL stimulation). Simultaneously, different final concentrations (0, 1, 10, 50 μM) of malicin were administered for 1 day. Cells were then collected, cell proteins were extracted, and Western blotting was performed to detect protein bands. A gel imaging system was used to develop and image the protein bands.

[0120] Test results as follows Figure 25 As shown in Figure a, the expression levels of osteoclast-related transcription factors such as NFATc1, cFos, and CTSK in BMMs decreased in a concentration-dependent manner after treatment with marijuana, confirming that marijuana can significantly reduce the expression levels of osteoclast differentiation-related transcription factors with increasing concentration. Furthermore, compared with the RANKL stimulation group alone, RANKL stimulation followed by marijuana treatment (50 μM) reduced the phosphorylation level of protein cPLA2 and decreased cPLA2 protein activity.

[0121] 2) WT mouse BMMs were induced to undergo osteoclastogenesis for 1, 3, and 5 days with or without malicin (50 μM). After induction, cells were collected, cell proteins were extracted, and Western blotting was used to detect protein bands. A gel imaging system was used to visualize the protein bands.

[0122] Test results as follows Figure 25 As shown in Figure b, Western blot analysis revealed that the expression levels of osteoclast-related factors such as NFATc1, cFos, and CTSK significantly increased with increasing induction time. Simultaneously, the phosphorylation level of protein cPLA2 increased with increasing induction time; while mariganin reduced both. This further demonstrates that mariganin can reduce the expression levels of osteoclast differentiation-related transcription factors, as well as decrease the phosphorylation level and activity of protein cPLA2.

[0123] 3) Macrophages derived from bone marrow of WT mice were induced to undergo osteoclast differentiation for 7 days with or without malicin (1, 10, 50 μM). Cells were then collected and subjected to TRAP staining. The size of the stained (purple-red) area was examined by the naked eye and under a microscope to detect the osteoclast differentiation level of the cells.

[0124] Test results as follows Figure 26As shown, compared with the control group, the cells in the maricin-treated group had a lighter purplish-red staining, fewer osteoclasts, and showed a dose-dependent effect, confirming that maricin can inhibit osteoclast differentiation.

[0125] 4) Macrophages derived from bone marrow of WT mice were induced to undergo osteoclast differentiation for 7 days under conditions with or without marigin (10 μM). The effects of marigin on the actin cytoskeleton and osteoclast morphology were evaluated using the rhodamine-labeled phalloidin staining method.

[0126] Test results as follows Figure 27 As shown, RANKL induced the formation of adhesion bands in mature osteoclasts, and malicin significantly disrupted the formation of F-actin rings and reduced the number of multinucleated osteoclasts.

[0127] 5) Target protein knockout validation: Eight-week-old WT mice and KO mice were selected, and bone marrow markers (BMMs) were extracted and cultured as described above. Osteoclastogenesis was induced (100 ng / mL RANKL stimulation). Based on whether or not malocclusion was added, they were divided into four groups: WT+RANKL group, WT+RANKL+MAR group, KO+RANKL group, and KO+RANKL+MAR group. The WT+RANKL+MAR group and the KO+RANKL+MAR group were treated with malocclusion (drug solvent: DMSO, final concentration: 50 μM), while the WT+RANKL group and the KO+RANKL group were treated with the same volume of DMSO. The mice were incubated for 24 h. Western blotting was used to verify whether the drug could still inhibit osteoclastogenesis after knocking out the target protein cPLA2 encoding gene.

[0128] Western blotting results are as follows: Figure 28 As shown, the results indicate that knocking out the cPLA2 encoding gene weakens the inhibitory effect of maricin on osteoclast-related factors NFATc1, cFos, ACP5, and CTSK in BMMs cells, suggesting that the inhibitory effect of maricin on osteoclast differentiation is weakened after the target protein cPLA2 is knocked out.

Claims

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

2. The application as described in claim 1, characterized in that, The cPLA2 inhibitor can increase bone density and stress resistance by reducing the expression level or protein activity of cPLA2, and reduce 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 application as described in claim 1, characterized in that, The cPLA2 inhibitor can increase bone density and reduce the expression level of inflammatory factors by reducing the expression level or protein activity of cPLA2, thereby alleviating osteolysis and achieving the effect of treating osteoporosis.

4. The application as described in claim 1, characterized in that, The cPLA2 inhibitor can inhibit the effect of RANKL by reducing the expression level or protein activity of cPLA2, thereby preventing osteoclast differentiation and achieving the effect of treating osteoporosis.

5. The application as described in claim 1, characterized in that, The cPLA2 inhibitor includes marigin.

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

7. The application as described in claim 6, characterized in that, The selected osteoporosis treatment drugs target cPLA2 and can reduce the expression level or protein activity of cPLA2.

8. The application as described in claim 6, characterized in that, The selected osteoporosis treatment drugs target cPLA2, and the drugs bind to cPLA2 to increase the stability of the cPLA2 protein.

9. The application as described in claim 6, characterized in that, The osteoporosis treatment drugs include mariglycosides.

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