Application of azadirachtin in preparation of medicine for preventing and treating osteoporosis

Abstract

The invention discloses an application of azadirachtin in preparation of a medicine for preventing and treating osteoporosis. In-vitro and in-vivo experiments of the system prove that Nim remarkably inhibits differentiation of mouse bone marrow-derived mononuclear / macrophages induced by M-CSF and RANKL to osteoclasts. The Nim can also specifically induce the formed mature osteoclast to generate apoptosis. In the in-vivo osteoporosis model, Micro-CT three-dimensional reconstruction, bone parameter analysis and Hamp are carried out; the consistency of E dyeing results shows that Nim intervention effectively reverses bone trabecular structure damage and bone mass loss caused by ovariectomy, and partially recovers a bone microstructure. The invention proves that Nim effectively controls pathological bone resorption through double mechanisms of inhibiting osteoclast differentiation and promoting mature osteoclast apoptosis, and obviously improves bone loss caused by osteoporosis. The compound is clear in action effect and concentration-dependent, shows specific regulation and control capability on osteoclasts under an effective dose, and provides an important experimental basis for developing efficient anti-osteoporosis medicines.

Description

Technical Field

[0001] This invention belongs to the field of new uses of chemical drugs, specifically relating to the application of neem lactone in the preparation of drugs for the prevention and treatment of osteoporosis. Background Technology

[0002] Osteoporosis is a common chronic metabolic orthopedic disease. Studies have shown that bone health is directly affected by bone homeostasis. Bone homeostasis refers to the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption in bone tissue metabolism. When the balance between bone formation and bone resorption is disrupted, resulting in bone resorption exceeding bone formation, bone homeostasis is disrupted, leading to osteoporosis.

[0003] With the accelerating aging of the world's population, osteoporosis is becoming increasingly prevalent, affecting more than 200 million people worldwide. Osteoporosis is caused by a variety of factors leading to decreased bone mineral density and damage to bone microstructure, resulting in increased bone fragility and fracture susceptibility. Current treatments for osteoporosis include physical therapy and drug therapy. Drug therapy includes the use of bisphosphonates, calcitonin, and selective estrogen receptor modulators. While these drugs can restore bone strength, they can also lead to decreased bone strain, and some clinical agents are expensive, have serious side effects, and require long-term use. Therefore, there is an urgent need for researchers to gain a deeper understanding of osteoporosis and explore its pathogenesis in order to develop more effective drugs.

[0004] Osteoclasts are key cells in the bone marrow mononuclear / macrophage lineage that mediate bone resorption. When the bone marrow mononuclear / macrophage lineage is co-stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB (RANKL), it differentiates into osteoclasts. Subsequently, differentiated osteoclasts induce bone matrix degradation by secreting hydrogen ions, matrix metalloproteinases, and cathepsin K. Over-differentiated osteoclasts lead to excessive bone resorption, causing osteoporosis. Therefore, regulating osteoclast differentiation and apoptosis is considered an important approach to treating osteoporosis.

[0005] Nimbolilide is a triterpenoid active compound extracted from the flowers and leaves of the neem tree (Melaleuca indica), with the molecular formula C1. 27 H 30 O7 has a relative molecular mass of 466.52 and a CAS No. of 25990-37-8.

[0006] This compound has been shown to possess various biological activities, including anti-inflammatory, antioxidant, antitumor, and antibacterial activities. Recent studies have shown that in orthopedic diseases, neem lactone (Nim) can alleviate adjuvant-induced arthritis in rats by inhibiting the inflammatory response, reduce inflammatory cell infiltration through the Toll-like receptor pathway, and improve erythrocyte sedimentation rate by inhibiting rheumatoid factor. Currently, there are no reports on the prevention and treatment of osteoporosis using Nim. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of neem lactone in the preparation of drugs for the prevention and treatment of osteoporosis, thus providing a new approach for the prevention and treatment of osteoporosis.

[0008] To address the problems in the prior art, the technical solution adopted by this invention is as follows:

[0009] Application of neem lactone in the preparation of drugs for the prevention and treatment of osteoporosis.

[0010] Preferably, the osteoporosis prevention and treatment drug also includes pharmaceutically acceptable excipients such as neem lactone.

[0011] Preferably, the mass percentage of the neem lactone in the osteoporosis prevention and treatment drug is 99.79%.

[0012] Preferably, the osteoporosis prevention and treatment drug is a drug that can inhibit osteoclast differentiation.

[0013] Preferably, the osteoporosis prevention and treatment drug is a drug that can increase bone density.

[0014] Preferably, the osteoporosis prevention and treatment drug is a drug that can increase the ratio of bone volume to total volume.

[0015] Preferably, the osteoporosis prevention and treatment drug is a drug that can increase the number of trabeculae.

[0016] Preferably, the osteoporosis prevention and treatment drug is a drug that can reduce trabecular separation.

[0017] Preferably, the osteoporosis is caused by oophorectomy.

[0018] Beneficial effects:

[0019] This invention demonstrates through systematic in vitro and in vivo experiments that neem lactone (Nim) significantly inhibits osteoclast differentiation and promotes mature osteoclast apoptosis, thereby effectively improving osteoporosis symptoms and exhibiting good targeting properties.

[0020] In in vitro experiments, Nim (100 nM and 200 nM) significantly inhibited the differentiation of mouse bone marrow-derived monocytes / macrophages (BMMs) into osteoclasts induced by M-CSF and RANKL. This was manifested by a significant downregulation of the mRNA expression levels of key osteoclast differentiation markers (MMP9, CTSK, ACP5, Trap, NFATc1, c-Fos, Oscar) (P<0.01). TRAP staining further confirmed that the number of multinucleated TRAP-positive osteoclasts formed after Nim treatment was significantly reduced, and the effect was concentration-dependent (200 nM was superior to 100 nM). More importantly, Nim could also specifically induce apoptosis in already formed mature osteoclasts. Annexin V-FITC assay showed a significantly increased apoptosis rate (P<0.01), and molecular mechanism studies confirmed that Nim drives the apoptosis program by upregulating the expression of pro-apoptotic genes (Bax, Bak, cytochrome c, caspase 3) and downregulating the expression of anti-apoptotic gene (Bcl2).

[0021] In an in vivo osteoporosis model (OVX mice), Micro-CT three-dimensional reconstruction and bone parameter analysis (significantly increased bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular bone number (Tb.N), and significantly decreased trabecular bone separation (Tb.Sp, P<0.01)) and H&E staining results consistently showed that Nim intervention effectively reversed the destruction of trabecular bone structure and bone loss caused by ovariectomy, and partially restored bone microstructure.

[0022] In summary, this invention demonstrates that Nim effectively controls pathological bone resorption and significantly improves bone loss in osteoporosis through a dual mechanism—inhibiting osteoclast differentiation and promoting mature osteoclast apoptosis. Its effects are well-defined and concentration-dependent, exhibiting specific regulatory capacity on osteoclasts at effective doses, providing important experimental evidence for the development of highly effective anti-osteoporosis drugs. Attached Figure Description

[0023] Figure 1 Images of TRAP staining in each group, scale bar: 100 μm.

[0024] Figure 2 The number of TRAP-positive cells in each group was quantitatively counted. The significance of the unstretched portion compared with the osteoclast-induced group was statistically significant. a p<0.05, b p<0.01, n=4.

[0025] Figure 3 The relative expression levels of osteoclast differentiation-related genes in each group are shown. The un-underlined portions are significant markers compared to the osteoclast-induced group. a p<0.05, bp<0.01, n=4.

[0026] Figure 4 The graphs show the flow cytometry results and quantitative statistics for each group. The unlined portions are significant markers compared to the osteoclast-induced group. a p<0.05, b p<0.01, n=4.

[0027] Figure 5 The relative expression levels of osteoclast apoptosis-related genes in each group are shown. The un-underlined portions are significant markers compared to the osteoclast-induced group. a p<0.05, b p<0.01, n=4.

[0028] Figure 6 The relative expression levels of Fas and FasL genes in each group are shown. The unstamped portion is a significance marker compared to the osteoclast-induced group. a p<0.05, b p<0.01, n=4.

[0029] Figure 7 3D modeling images of the distal femur after Micro-CT scans for each group. The significance of the unmarked areas compared to the OVX model group (ns: no statistically significant difference). a p<0.05, b p<0.01, n=6.

[0030] Figure 8 Images of the distal femur stained with H&E in each group.

[0031] Figure 9 The graphs show the statistical significance of bone tissue parameters for each group. The unlined portions are not statistically significant compared to the OVX model group. (ns: no statistical significance) a p<0.05, b p<0.01, n=6. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] I. Materials and Methods

[0034] 1. Materials

[0035] 1.1 Reagents and Experimental Equipment

[0036] 1.1.1 Main Medicines and Reagents

[0037] Nim, with the molecular formula C 27 H 30 O7, relative molecular mass is 466.52, CAS No. is 25990-37-8, purchased from MedChemExpress, China; paraformaldehyde, sterile PBS, hematoxylin, eosin, anhydrous ethanol, distilled water, 3% sodium pentobarbital, fetal bovine serum (FBS), α-MEM medium, RANKL, M-CSF, total RNA extraction reagent, SYBR Green dye, cDNA reverse transcription kit, Annexin V-FITC apoptosis detection kit, tartrate-resistant acid phosphatase kit.

[0038] 1.1.2 Main Instruments

[0039] Cell culture incubator (Thermo Scientific, USA), flow cytometer (Thermo Scientific, USA), Micro-CT (SkyScan 1176, Belgium), paraffin microtome (Leica 2135, Germany), slide burner (Leica 1120, Germany), paraffin embedding machine (Leica 1150, Germany), Axiovert 40C optical microscope (Zeiss, Germany), a set of surgical instruments, etc.

[0040] 1.2 Laboratory Animals

[0041] Twenty-four healthy female C57BL / 6J mice, weighing 20–25g, 6–8 weeks old, SPF grade, were provided by the Animal Experiment Center of Soochow University. The rearing conditions were as follows: 6 mice per cage, room temperature 18–20℃, humidity 50–60%, good ventilation, and free access to food and water.

[0042] 2. Experimental Methods

[0043] 2.1 Cell Experiments

[0044] 2.1.1 Cell extraction and culture

[0045] Six- to eight-week-old C57BL / 6J mice were rapidly euthanized by cervical dislocation. The bilateral femurs and tibias were then separated using sterile surgical instruments and washed in sterile PBS. Under sterile conditions, the separated bones were sequentially immersed, washed, and disinfected for 3 minutes each in 75% ethanol, sterile PBS, 75% ethanol, and sterile PBS. The ends of the tibia and femur were then cut off with sterile surgical scissors to expose the medullary cavity. The medullary cavity was flushed with α-MEM medium containing 10% FBS using a 1ml syringe until it turned white. The flushing solution was then transferred to a 15ml centrifuge tube and centrifuged at 1200 rpm for 3 minutes, discarding the supernatant. Red blood cell lysis buffer was added to lyse the bone marrow red blood cells, which were resuspended and lysed for 3 minutes. The cells were then centrifuged at 1200 rpm for 3 minutes, discarding the supernatant. Finally, the bone marrow cell pellet was resuspended in complete culture medium and seeded into cell culture dishes. After culturing in a 5% CO2, 37°C cell culture incubator for 16-24 hours, the medium was completely replaced, and the cells were rinsed with preheated medium. The medium was then collected and centrifuged at 1200 rpm for 3 minutes to obtain bone marrow-derived macrophages (BMMs). The cells were then thoroughly mixed with α-MEM medium containing 30 ng / ml macrophage colony-stimulating factor M-CSF, 10% FBS, and 1% penicillin-dextrin antibiotics, and seeded into culture dishes for subsequent experiments.

[0046] 2.1.2 Osteoclast Differentiation Induction

[0047] BMM cells were seeded evenly in cell culture plates, and then cultured in complete medium containing 30 ng / ml M-CSF for 8 hours. After cell attachment, the old medium was removed, and the cells were divided into four intervention groups: the control group was cultured in complete medium containing M-CSF, while the osteoclast induction group, the low-dose neem lactone group, and the high-dose neem lactone group were cultured in complete medium containing M-CSF and 50 ng / mL RANKL. Based on previous literature, 100 nM and 200 nM were selected as intervention concentrations for the low-dose and high-dose neem lactone groups, respectively. The cells were treated accordingly 3-5 days after intervention.

[0048] 2.1.3 Tartrate-resistant phosphatase (TRAP) staining

[0049] BMMs cells were divided into 2×10 4Cells were seeded at a density of 10 cells / well in 24-well plates and then treated as described in section 2.1.2. After 5 days of treatment, the culture medium was removed, and the cells were rinsed three times with pre-chilled PBS buffer, followed by fixation with 4% paraformaldehyde for 15 minutes. After removing the paraformaldehyde, the cells were rinsed three times with pre-chilled PBS buffer. The cells were stained using the TRAP kit according to the reagent instructions. After staining, osteoclast images were observed and recorded under a microscope. Positive staining results indicated multinucleated cells (≥3 nuclei), i.e., osteoclasts. Finally, the number of osteoclasts in each group was quantitatively analyzed using ImageJ.

[0050] 2.1.4 Real-time quantitative polymerase chain reaction (RT-qPCR)

[0051] Cells were seeded at a density of 4 × 10⁵ cells / well in 6-well plates and subjected to appropriate intervention. After 3 days of intervention, the culture medium was removed, and the cells were washed three times with PBS buffer. Then, 1 mL of Trizol reagent was added to each well to lyse the cells, followed by total RNA extraction using chloroform. The RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer. Then, 1 μg of total RNA was used to construct a 20 μL reverse transcription system according to the cDNA reverse transcription kit manual, and cDNA was synthesized by reverse transcription. Mix SYBR Green dye, upstream and downstream primers, and DEPC water according to the amplification kit instructions. Add the mixture to a 96-well PCR plate for quantitative PCR. Then, add the cDNA obtained from reverse transcription, mix thoroughly, and run the qPCR. A three-step qPCR amplification process is used: the initial denaturation stage is maintained at 95°C for 10 min to achieve double-strand dissociation, followed by a cyclic amplification stage. Each cycle consists of a 15-second denaturation at 95°C followed by a 60-second annealing / extension step at 60°C, for a total of 40 cycles. Standardization calibration is performed using β-actin as an endogenous reference. -ΔΔCT Calculate the relative expression levels of genes.

[0052] 2.1.5 Annexin V-FITC / PI cell apoptosis detection

[0053] BMMs were seeded in 6-well plates at a density of 4 × 10⁵ cells / well and cultured in complete medium containing M-CSF and RANKL. After 3 days, the medium was replaced with fresh medium and neem lactone was added for a second 3-day intervention. After the intervention, the cell pellet was collected after trypsin digestion, washed three times with pre-chilled PBS buffer, and centrifuged again to obtain the cells. The cells were then stained according to the Annexin V-FITC / PI apoptosis detection kit instructions, and the staining was performed using flow cytometry.

[0054] 2.2 Animal Experiments

[0055] 2.2.1 Grouping of experimental animals

[0056] Twenty-four C57BL / 6J mice were randomly divided into the following four groups:

[0057] (1) Sham surgery group: 6 animals; fat around the ovaries was scraped off. 200 µl of sterile PBS solution was injected intraperitoneally every two days for 8 consecutive weeks, after which the animals were sacrificed.

[0058] (2) OVX model group: 6 animals were included in the OVX model group; bilateral oophorectomy was performed after ligation of the fallopian tubes. 200 µl of sterile PBS solution was injected intraperitoneally every two days for 8 consecutive weeks, after which the animals were sacrificed.

[0059] (3) OVX model + 5 mg / kg Nim group: 6 animals, which were treated with low dose of Nim. The modeling method was the same as that of the model group. Every two days, 200 µl of sterile PBS solution containing 5 mg / kg Nim was injected into the peritoneum. After 8 weeks of continuous injection, the animals were sacrificed.

[0060] (4) OVX model + 10 mg / kg Nim group: 6 animals, which were treated with high doses of Nim. The modeling method was the same as that of the model group. Every two days, 200 µl of sterile PBS solution containing 10 mg / kg Nim was injected into the peritoneum. After 8 weeks of continuous injection, the animals were sacrificed.

[0061] 2.2.2 Preparation of the Ovarian-Free Mouse (OVX) Model

[0062] First, experimental mice were intraperitoneally injected with 3% sodium pentobarbital (60 mg / kg). After anesthesia took effect, the hair in a 5*5cm area along the midline of the back was shaved. After disinfection with povidone-iodine, the skin was incised along the midline of the back, and the skin and muscle layers were separated layer by layer. The ovaries were located in the fat pad below the kidneys, and the fallopian tubes were ligated before bilateral ovarian removal. Mice with the fat around the ovaries removed were designated as the sham-operated group. All groups were randomized, with a sample size of 6. After surgery, the surgical incisions of the mice were sutured layer by layer, and the mice continued to be housed in their original environment. Postoperatively, mice in the low-dose azirnonidene group (5 mg / kg) and the high-dose azirnonidene group (10 mg / kg) were intraperitoneally injected with Nim solution, while the model group and the sham-operated group were injected with an equal volume of sterile PBS, once every 2 days.

[0063] 2.2.3 Specimen Collection

[0064] Eight weeks after administration, all mice were euthanized by rapid cervical dislocation and femoral tissue was collected: the bilateral femurs of the mice were separated, the surface soft tissue was removed, and the femurs were fixed in 10% paraformaldehyde solution for 48 h. After Micro-CT scanning, the tissue sections were decalcified with 10% EDTA and stained.

[0065] 2.2.4 Micro-CT Detection

[0066] Bone tissue samples were scanned and quantitative analysis of bone microstructure was performed using a high-precision microtomography system (SkyScan 1076, Bruker MicroCT, Belgium). Samples were neatly arranged within the equipment to avoid contact with the test tube walls. Scanning parameters were set as follows: current 800 μA, voltage 50 kV, resolution 18 μm. After scanning, 3D reconstruction of the 2D images was performed using SkyScan RECON software. Femoral data were collected from the same area in all samples, and 2D and 3D image reconstructions were performed using 3D image processing software. Skeletal condition was observed, and morphological parameters including bone density, bone volume ratio, number of trabeculae, and trabecular separation were analyzed.

[0067] 2.2.5 Histological staining

[0068] After decalcification with EDTA decalcification solution, the femoral specimen was trimmed. Finally, the paraffin-embedded specimen was sectioned using a histome to produce 5μm thick paraffin sections. HE staining steps:

[0069] (1) After dewaxing paraffin sections with xylene (10 min × 3 times), they were successively passed through 100%, 100%, 95%, 90%, 85% ethanol to water, each pass lasting 5 min.

[0070] (2) Rinse with distilled water for 3 min, stain with hematoxylin solution for 5 min, and rinse with tap water for 5 min;

[0071] (3) Differentiate with 1% hydrochloric acid alcohol solution for 60s, then rinse with tap water for 1min;

[0072] (4) Blue in 10% ammonia solution for 60 seconds, then rinse with tap water for 1 minute;

[0073] (5) Counterstain with 1% eosin solution for 3 min, then rinse with tap water for 1 min;

[0074] (6) Routine dehydration, clearing, and sealing.

[0075] (7) Observe and photograph the morphological changes of femoral bone tissue under a light microscope.

[0076] 2.3 Statistical Analysis

[0077] All statistical analyses were performed using GraphPad Prism version 10.0. Continuous variables were described using mean ± standard deviation (SD). Normality was first tested. For normally distributed data, one-way ANOVA was used to determine statistical significance between groups. For non-normally distributed data, a nonparametric Kruskal-Wallis H test was used. Significance markers were uniformly placed at the top of the control group data series (ns < 0.05). a p<0.05, b p<0.01).

[0078] II. Results

[0079] 1. Results of Nim in vitro cell intervention experiments

[0080] 1.1 Nim inhibits RANKL-induced osteoclast differentiation of BMMs

[0081] Five days after inducing osteoclast differentiation in mouse BMMs cells, Trap staining results showed that RANKL intervention successfully promoted the fusion of BMMs cells into a large number of TRAP-positive multinucleated osteoclasts in the osteoclast-induced group. Figure 1 Compared with the osteoclast-induced group, Nim (100 nM and 200 nM) intervention significantly reduced osteoclast formation, with statistical significance (p < 0.01), and the inhibitory effect of 200 nM neem lactone was significantly better than that of 100 nM. Figure 2 ).

[0082] Gene expression was detected using RT-qPCR. Results showed that 3 days of combined intervention with M-CSF and RANKL successfully induced osteoclast differentiation in mouse BMMs. Osteoclast differentiation markers such as MMP9, CTSK, ACP5, Trap, NFATc1, c-Fos, and Oscar were significantly increased compared to the control group (P < 0.01). However, after Nim (100 nM and 200 nM) intervention, osteoclast differentiation markers significantly decreased compared to the osteoclast induction group (p < 0.01), and the 200 nM concentration of azadirachtin showed significantly better inhibitory effects than the 100 nM concentration. Figure 3 )

[0083] 1.2 Nim promotes apoptosis of mature osteoclasts

[0084] Further investigation was conducted to examine the effect of Nim on mature osteoclast apoptosis. Annexin V-FITC apoptosis assay results showed that, compared with the osteoclast induction group, Nim intervention significantly promoted mature osteoclast apoptosis, and the apoptosis-inducing effect of 200 nM azadirachtin was significantly better than that of 100 nM intervention (p < 0.01). Annexin V-FITC apoptosis assay results showed that, compared with the osteoclast induction group, Nim intervention significantly promoted mature osteoclast apoptosis (P < 0.01). Figure 4 ).

[0085] Gene expression analysis showed that, compared with the osteoclast-induced group, Nim intervention significantly promoted the expression of apoptosis-related genes such as Bax, Bak, cytochrome c, and caspase 3 (p < 0.01), while significantly inhibiting the expression of the apoptosis-inhibiting gene Bcl2 (p < 0.01). Figure 5 Further analysis of the mechanism by which Nim promotes osteoclast apoptosis revealed that, compared with the osteoclast-induced group, Nim intervention significantly promoted the expression of Fas and FasL, and this effect also showed a concentration-dependent effect. Figure 6 ).

[0086] 2. Nim inhibits excessive bone loss caused by estrogen deficiency.

[0087] Micro-CT scans were performed on the femur, and two-dimensional and three-dimensional reconstructions were conducted. The extent of trabecular bone structure damage and bone parameters were analyzed. Results showed that compared to the sham-operated group, the OVX group mice exhibited significantly increased trabecular bone sparsity, indicating that bilateral ovariectomy successfully induced osteoporosis symptoms in the mice. After Nim intervention, partial recovery of the trabecular bone structure was observed, and the degree of osteoporosis was significantly improved. Figure 7 The same phenomenon was also observed in H&E staining of bone tissue sections. Figure 8 Further analysis of bone parameters showed that the bone mineral density, bone volume to total volume ratio, and number of trabeculae were significantly decreased in the OVX model group mice, while the trabeculae separation was significantly increased (p < 0.01). Figure 9 ).

Claims

1. Application of neem lactone in the preparation of drugs for the prevention and treatment of osteoporosis.

2. The application according to claim 1, characterized in that, The osteoporosis prevention and treatment drugs also include pharmaceutically acceptable excipients such as neem lactone.

3. The application according to claim 1, characterized in that, The mass percentage of the neem lactone in the osteoporosis prevention and treatment drug is 99.79%.

4. The application according to claim 1, characterized in that, The aforementioned osteoporosis prevention and treatment drug is one that can inhibit osteoclast differentiation.

5. The application according to claim 1, characterized in that, The aforementioned osteoporosis prevention and treatment drugs are those that can increase bone density.

6. The application according to claim 1, characterized in that, The osteoporosis prevention and treatment drugs are those that can increase the proportion of bone volume to total volume.

7. The application according to claim 1, characterized in that, The osteoporosis prevention and treatment drugs mentioned are those that can increase the number of trabeculae.

8. The application according to claim 1, characterized in that, The osteoporosis prevention and treatment drugs mentioned are those that can reduce trabecular separation.

9. The application according to claim 1, characterized in that, The osteoporosis was caused by the removal of the oophores.

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