Use of a drug, miltefosine, in promoting bone formation and preventing and treating osteoporosis

Abstract

The application discloses application of a medicine, miltefosine, in promoting bone formation and preventing and treating osteoporosis. In the bone formation process, the expression of protein phosphatase PPM1A in osteoblasts is significantly increased, the dephosphorylation of a core protein Smad2 of a TGF-beta signal path is regulated, the TGF-beta signal path is inhibited, the expression of an osteogenic marker is promoted, and the osteogenic ability of the organism is enhanced. The application discloses that the medicine, miltefosine, is used as a PPM1A enzyme activity catalyst, the in-vivo intervention of the medicine can significantly accelerate bone repair and delay bone loss of postmenopausal osteoporosis, a mechanism is that the enzyme activity of PPM1A of osteoblasts is catalyzed, the dephosphorylation of Smad2 protein is promoted, the TGF-beta / Smad2 signal path activity is inhibited, bone formation is promoted, bone repair is accelerated, and bone loss is prevented. Therefore, the miltefosine has a good application prospect in clinical treatment of promoting osteogenesis and preventing and treating osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of small molecule compound technology, and particularly relates to the application of the drug mitefocin in promoting bone formation and preventing osteoporosis. Background Technology

[0002] Postmenopausal osteoporosis (PMOP) is a common skeletal disease caused by estrogen deficiency after menopause, characterized primarily by bone loss and microstructural degradation. It is estimated that approximately 50% of postmenopausal women worldwide are affected by osteoporosis to varying degrees, with a fragility fracture rate as high as 40%. Due to its high incidence and serious harm, PMOP has become a global public health problem. PMOP is increasingly becoming a major threat to the health of postmenopausal women, making it urgent to actively explore its pathogenesis. Despite this, effective interventions to halt the progression of OP remain lacking, mainly because the cellular and molecular mechanisms of bone formation are not yet fully understood.

[0003] Existing research indicates that the transforming growth factor-β (TGF-β) signaling pathway plays a crucial role in maintaining bone homeostasis and osteoarthritis development. Increasing research shows that the TGF-β signaling pathway is involved in maintaining bone homeostasis, and Smad2 / 3 is considered a key component of the classic TGF-β signaling pathway. The core step of the TGF-β signaling pathway is the phosphorylation of Smad2 / 3 induced by TGF-β receptor I kinase (TGFβRI). Then, phosphorylated Smad2 / 3 (p-Smad2 / 3) binds to Smad4 and, as a complex, shuttles into the cell nucleus to participate in transcription.

[0004] Protein phosphatase magnesium-dependent 1A (PPM1A) is a member of the metal-dependent protein phosphatases (PPMs) family, capable of dephosphorylating phosphoserine and phosphothreonine residues. Notably, PPM1A has been identified as an effective protein phosphatase for a variety of substrates, including Smad2, a key regulator in the classical TGF-β signaling pathway. Considering that TGF-β signaling is a crucial pathway involved in regulating bone homeostasis and osteoporosis (OP), and PPM1A, as a p-Smad2 phosphatase, can participate in regulating TGF-β signaling activity, clarifying the regulatory role of PPM1A in osteoblasts is of great significance for discovering new targets and related drugs for the prevention and treatment of OP.

[0005] Miltefosine (MF) was approved by the U.S. Food and Drug Administration (FDA) on March 19, 2014, primarily for the treatment of three types of Rashmann disease (visceral Rashmann's disease, cutaneous Rashmann's disease, and mucosal Rashmann's disease), and is also used clinically for palliative care of several cancers that are difficult to treat with conventional therapies. Recently, miltefosine has been reported to act as a catalyst for the enzymatic activity of protein phosphatase PPM1A, improving the progression of Alzheimer's disease. However, the therapeutic effects of miltefosine on orthopedic diseases have not yet been reported. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a novel function of the drug mitefoxin, namely, the function of promoting bone formation and preventing osteoporosis. This invention aims to promote bone formation by catalyzing the PPM1A activity of osteoblasts, thereby inhibiting the excessive activation of the TGF-β signaling pathway in osteoblasts, and simultaneously providing the application of mitefoxin in the preparation of drugs for the prevention and treatment of osteoporosis.

[0007] The technical solution adopted in this invention is:

[0008] The application of mitefoxin in promoting bone formation and preventing osteoporosis, wherein mitefoxin is hexadecyl 2-(trimethylamino)ethyl phosphate; its chemical formula is C 21 H 46 NO4P has the following structural formula:

[0009]

[0010] The mitefoxin is used to enhance osteogenic differentiation of osteoblasts;

[0011] The mitefoxin is used to increase the bone density of newly formed bone in the repair area;

[0012] The mitefoxin is used to increase the bone volume fraction of newly formed bone in the repair area;

[0013] The mitefoxin is used to enhance the trabeculae of newly formed bone in the repair area;

[0014] The mitefoxin is used to increase bone mineral density in the distal femur after menopause.

[0015] The mitefoxin is used to reduce trabecular separation in the distal femur after menopause.

[0016] The mitefoxin is used to reduce bone loss in the distal femur after menopause and to increase the number of osteoblasts on the surface of cancellous bone.

[0017] Furthermore, the drug is a bone-forming drug, and the drug is in the form of a solid powder.

[0018] Furthermore, the drug is a drug that catalyzes the activity of protein phosphatase PPM1A in osteoblasts.

[0019] Furthermore, the osteoporosis includes postmenopausal osteoporosis.

[0020] Furthermore, the effective dose of mitefoxin for promoting osteoproliferative effects is 5-10 mg / kg.

[0021] PPM1A is highly expressed in normal human osteoblasts, but its expression is downregulated in osteoporosis. Mitefocin, a catalyst for PPM1A enzyme activity, promotes bone formation by regulating the TGF-β / Smad2 signaling pathway in osteoblasts.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] Mitefocin can promote osteoblast differentiation and accelerate bone defect repair, while also improving bone loss in postmenopausal osteoporosis. Therefore, mitefoxin has the potential for clinical intervention in bone defects and the progression of osteoporosis. Attached Figure Description

[0024] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0025] Figure 1This is a diagram illustrating the mechanism of action of mitefotaxime in promoting osteoblast differentiation and accelerating bone repair in this invention; wherein, A is the chemical structural formula of mitefotaxime; B is the cell activity of primary osteoblasts under different concentrations of mitefotaxime intervention; C is the ALP staining of osteoblasts under different concentrations of mitefotaxime intervention; D is the mRNA expression level of osteoblast-related genes such as Sp7, Bglap, and Alpl in primary osteoblasts under different concentrations of mitefotaxime intervention; E is a representative Micro-CT scan image of mitefotaxime's effect on bone defect repair in a mouse model, scale bar. =1mm; F ​​is a quantitative analysis of bone microstructure in newly formed bone tissue 1 week after mitefoxanthioprine-treated bone defect surgery in mice; G is an SO / FG staining image of the repair site 1 week after mitefoxanthioprine-treated bone defect surgery in mice, scale bar = 1mm; H is a quantitative analysis of the area of ​​newly formed bone at the repair site 1 week after bone defect surgery in mice; I is an OCN immunohistochemical staining image of the repair site of bone defect in mice, scale bar = 1mm; J is a quantitative analysis of OCN immunohistochemical staining at the repair site of bone defect in mice; K is the effect of mitefoxanthioprine on wild-type (Ppm1a) + / + ) mice and Ppm1a knockout (Ppm1a - / - Micro-CT scans representing bone defect repair in a mouse model, scale bar = 50 μm. Quantitative analysis of bone microstructure in newly formed bone tissue of mice treated with different concentrations of mitefoxin one week after bone defect repair. Data are expressed as mean ± standard deviation, * = P < 0.05, ** = P < 0.01;

[0026] Figure 2This diagram illustrates the effect of mitefoxin on postmenopausal osteoporotic mice. A shows a representative Micro-CT scan of the distal femur in mice treated with mitefoxin for OVX at 8 weeks post-surgery (scale bar = 1 mm); B shows the quantitative analysis of bone mineral density (BMD) in the distal femur of mice treated with mitefoxin for OVX at 8 weeks post-surgery; C shows the quantitative analysis of the structural model index (SMI) of the distal femur in mice treated with mitefoxin for OVX at 8 weeks post-surgery; D... Image A shows the quantitative analysis of distal femoral cancellous bone connectivity density (Conn.Dn) 8 weeks after mitefocin intervention in OVX model mice; Image B shows the quantitative analysis of distal femoral cancellous bone volume fraction (BV / TV) 8 weeks after mitefocin intervention in OVX model mice; Image C shows the quantitative analysis of distal femoral trabecular bone number (Tb.N) 8 weeks after mitefocin intervention in OVX model mice; Image D shows the quantitative analysis of distal femoral trabecular bone thickness 8 weeks after mitefocin intervention in OVX model mice. Tb.Th) quantitative analysis map; H is the quantitative analysis map of trabecular separation (Tb.Sp) in the distal femur of mice 8 weeks after mitefossa intervention in OVX model mice; I is the representative map of ABH / OG staining in the distal femur of mice 8 weeks after mitefossa intervention in OVX model mice, scale bar = 0.5 mm; J is the quantitative analysis map of the proportion of trabecular area in the distal femur of mice 8 weeks after mitefossa intervention in OVX model mice; K is the quantitative analysis map of the proportion of trabecular area in the distal femur of mice 8 weeks after mitefossa intervention in OVX model mice. Quantitative analysis of distal osteoblast number; L is a representative immunofluorescence image of distal femoral OCN in mice 8 weeks after mitefocin intervention (OVX model), scale bar = 50 μm; M is a quantitative analysis of distal femoral OCN expression in mice 8 weeks after mitefocin intervention (OVX model); N is a representative immunohistochemical image of distal femoral ALP in mice 8 weeks after mitefocin intervention (OVX model); O is a quantitative analysis of distal femoral ALP expression in mice 8 weeks after mitefocin intervention (OVX model).

[0027] Figure 3 The diagrams show the effects and biosafety evaluation of mitefoxin on osteoclasts in postmenopausal mice. A represents TRAP staining of the distal femur in OVX model mice 8 weeks after mitefoxin intervention (scale bar = 0.5 mm); B represents quantitative analysis of osteoclast numbers in the distal femur of OVX model mice 8 weeks after surgery; C represents immunofluorescence of CTSK in the distal femur of OVX model mice 8 weeks after mitefoxin intervention (scale bar = 50 μm); D represents quantitative analysis of CTSK expression in the distal femur of OVX model mice 8 weeks after mitefoxin intervention; E represents immunohistochemical staining of Fabp4 in the distal femur of OVX model mice 8 weeks after mitefoxin intervention (scale bar = 50 μm); F represents quantitative analysis of Fabp4 expression in the distal femur of OVX model mice 8 weeks after mitefoxin intervention; G represents HE staining of the liver, heart, kidney, and spleen in OVX model mice 8 weeks after mitefoxin intervention. Detailed Implementation

[0028] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application.

[0029] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0030] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0031] Example 1

[0032] Research on the mechanism of action of mitifone in promoting bone formation:

[0033] 1.1 Methods;

[0034] 1.1.1 Establishment of a mouse bone defect model;

[0035] Ten-week-old mice were anesthetized with 2% isoflurane. After thorough anesthesia, the skin of the right knee joint was prepared by making an incision along the midline of the right knee joint and cutting along the medial border of the patellar ligament with ophthalmic scissors to expose the right knee joint cavity. The patella was placed in an externally dislocated position. A 26-gauge needle was then used to drill a hole in the intercondylar region of the femur, and a 0.6 mm diameter Kirschner wire was inserted from the femoral condyle to the proximal femur. After removing the Kirschner wire, the dislocated patella was reduced, and the skin was sutured closed. The mice were sacrificed 7 days postoperatively.

[0036] 1.1.2 Animal sample processing and slide preparation;

[0037] Mice were sacrificed by CO2 one week post-surgery, and tissue samples were harvested from the knee joints. After tissue separation, the animal samples were fixed in 4% paraformaldehyde (PFA) at room temperature for 3 days, followed by decalcification in 14% ethylenediaminetetraacetic acid (EDTA) solution for 2 weeks, with the decalcification solution changed every other day. After decalcification, the samples were dehydrated using an automated dehydrator. All samples were embedded in paraffin to prepare paraffin blocks, which were then sectioned to a thickness of 4 μm.

[0038] 1.1.3 Imaging observation;

[0039] All samples were scanned using a high-resolution micro-CT (μ-CT) scanner with scanning parameters set to 45 kV, 500 μA, and an exposure time of 780 ms. Regions of interest (ROIs) were selected from the distal femur to represent areas of bone repair. Quantitative analysis of bone microstructure parameters, including bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular bone number (Tb.N), was performed on the ROIs.

[0040] 1.1.4 Histological observation;

[0041] Before staining, the sample sections were baked in a 60℃ oven for 4 hours to increase the adhesion between the sample tissue and the glass slide. After removing the sections, they were allowed to stand for 15 minutes to return to room temperature. Then, the sections were dewaxed and rehydrated with xylene and graded alcohols. After that, Safranin-O / Fast Green (SO / FG) staining was performed. The specific staining steps were as follows: first, the sections were placed in 0.05% Fast Green staining solution for 3 minutes, then in 3% glacial acetic acid solution for 10 seconds, and then in 2.5% Safranin staining solution for 10 minutes. After that, the sections were rinsed with pure water 3 times, 3 minutes each time, and dehydrated and cleared step by step. The sections were then mounted with a mounting medium.

[0042] 1.1.5 Immunohistochemical analysis;

[0043] Tissue slides were placed in a 60°C oven overnight. The next day, the slides were removed and allowed to stand for 15 minutes to return to room temperature. Then, the slides were dewaxed and rehydrated using xylene and graded alcohol solutions. A heat retrieval method was employed, in which tissue slides were immersed in sodium citrate buffer and placed in a 60°C oven for 4 hours to complete antigen retrieval and fully expose the antigenic epitopes. The tissue samples were permeabilized with 0.3% Triton X-100 solution. Then, 100 μL of endogenous peroxidase inhibitor was added to each tissue sample, and the sample was incubated at room temperature for 10 minutes. Next, 100 μL of blocking normal goat serum working solution was added, and the sample was incubated at room temperature for 15 minutes. After discarding the serum, 100 μL of primary antibody was added, and the sample was incubated overnight at 4°C. The following day, the sample slides were removed and brought to room temperature. The primary antibody reagent was washed away with PBS buffer, and 100 μL of biotin-labeled goat anti-rabbit IgG polymer and 100 μL of horseradish enzyme-labeled streptavidin working solution were added sequentially. Then, an appropriate amount of freshly prepared DAB solution was added for staining. After staining, the slides were placed in hematoxylin staining solution for 20 seconds, differentiated with 1% hydrochloric acid alcohol, rinsed with tap water to return to blue, and finally dehydrated and cleared by sequentially passing through a gradient of alcohols and xylene. The sample slides were mounted with neutral resin.

[0044] 1.1.6 Isolation and culture of primary osteoblasts;

[0045] Mice aged 3-5 days were euthanized with CO2 and then disinfected by immersion in 70% alcohol for 15 minutes. Afterward, the mice were removed, and the skin on the top of the skull was carefully peeled off. Skull tissue blocks were then placed in α-Modified Eagle Medium (α-MEM) containing 1 mg / mL collagenase-A and 1% penicillin / streptomycin, and digested at 37°C for 6 hours, with the tissue blocks agitated every hour. After digestion, the tissue blocks were filtered through a 70 μm cell sieve, centrifuged at 1000 rpm for 5 minutes, resuspended in α-MEM, and seeded into wells. Osteoblasts were harvested and cultured in complete α-MEM medium containing 10% fetal bovine serum. Once osteoblasts achieved 60-70% complete adherence and confluence, osteogenic differentiation was induced using osteogenic medium containing ascorbic acid (50 μg / ml) and sodium β-glycerophosphate (10 mM). Cells were collected after 7 days of induction for further experiments.

[0046] 1.1.7 Cell mRNA extraction and RT-qPCR detection;

[0047] Total RNA was harvested and extracted from osteoblast cultures using TRIzol reagent. The TaqMan reverse transcription kit (Biomake) was used to reverse transcribe the mRNA into complementary DNA. Subsequently, SYBR premix Ex Taq was used. TM II. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed. Primer sequences are shown in Table 1, providing a basis for quantitative analysis using β-actin as an internal reference gene. The RT-qPCR gene primer sequences are shown in Table 1.

[0048] Table 1

[0049]

[0050] 1.2 Results;

[0051] 1.2.1 Effects of mitefoxin on osteoblast proliferation and differentiation in vitro;

[0052] The chemical formula of mitefocin is C21H46NO4P ( Figure 1 A) According to the CCK-8 assay, mitefoxin at concentrations from 0.5 μM to 20 μM had no significant effect on the activity of primary osteoblasts in vitro, while a concentration of 50 μM caused the death of approximately 50% of primary osteoblasts. Figure 1 B), subsequent in vitro studies used drug concentrations of 2.5 μM, 5.0 μM, and 10 μM. Alkaline phosphatase staining results indicated that mitefoxin intervention significantly enhanced the osteogenic differentiation capacity of osteoblasts in vitro. Figure 1 C), and RT-PCR results showed that mitefoxin upregulated the expression of osteogenic genes Sp7, Bglap, and Alpl, enhancing bone formation (C). Figure 1 D). In summary, in vitro intervention with mitefoxin can effectively promote osteogenic differentiation of osteoblasts.

[0053] 1.2.2 Effects of mitefoxin on bone defect repair in mice;

[0054] A mouse model of distal femoral bone defect was established, and mitefocrine was administered to investigate its in vivo effect on bone repair. μ-CT scans of femoral samples one week after modeling showed that mitefocrine intervention effectively accelerated bone repair. Figure 1 E) Further CT analysis results suggested that mitefoxin can increase bone mineral density (BMD), bone volume fraction (BV / TV), and trabecular bone number (Tb.N) in the repaired area. Figure 1 F). Simultaneously, SO / FG staining confirmed that the area of ​​new bone formation at the bone defect site in mice treated with mitefoxin was significantly larger than that in control mice. Figure 1 G and H). Meanwhile, compared to the control group, the OCN expression level in the bone defect repair area of ​​the intervention group was significantly increased (G and H). Figure 1 I and J). Importantly, mitefoxin failed to exert the aforementioned osteogenic effect in Ppm1a knockout (Ppm1a- / -) mice. Figure 1 This phenomenon is also consistent with the analysis results of μ-CT (K). Figure 1 Therefore, these data indicate that in vivo intervention with mitefoxin can effectively accelerate bone defect repair in mice, and this effect depends on the protein phosphatase PPM1A.

[0055] 1.3 Conclusion;

[0056] 1.3.1 Mitefocin can promote osteogenic differentiation of osteoblasts in vitro;

[0057] 1.3.2 Mitefocin can accelerate the repair of bone defects in mice.

[0058] Example 2

[0059] A study on the effects of mitifone intervention on postmenopausal osteoporosis:

[0060] 2.1 Methods;

[0061] 2.1.1 Construction of the mouse OVX model;

[0062] All mice underwent bilateral ovariectomy (OVX) to establish an estrogen-deficient osteoporosis model. Twelve-week-old mice were anesthetized via intraperitoneal injection of 2% isoflurane. After thorough anesthesia, the skin and peritoneum were incised along the lumbar region. The fallopian tubes were then ligated, and both ovaries were removed. In the sham-operated group, the ovaries and fallopian tubes were preserved intact; only the skin and peritoneum were incised, followed by disinfection and suturing.

[0063] 2.1.2 Animal sample processing and slide preparation;

[0064] Eight weeks post-surgery, all mice were sacrificed by CO2, and bilateral femoral samples were collected. These samples were fixed in 4% PFA at room temperature for 3 days, followed by decalcification in 14% EDTA solution for 2 weeks, with the decalcification solution changed every other day. After decalcification, the samples were dehydrated using an automated dehydrator. All samples were embedded in paraffin to prepare paraffin blocks, which were then sectioned to a thickness of 4 μm.

[0065] 2.1.3 Imaging observation;

[0066] All samples were scanned using a high-resolution μ-CT scanner with scanning parameters set to 45 kV, 500 μA, and an exposure time of 780 ms. The distal femoral metaphysis was selected as the region of interest (ROI) for analysis. Quantitative analysis of bone microstructure parameters, including bone mineral density (BMD), bone structure model index (SMI), connectivity density (Conn.Dn), bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp), was performed on the ROI.

[0067] 2.1.4 Histological morphological observation;

[0068] Before staining, the sample sections were baked in a 60℃ oven for 4 hours to increase the adhesion between the sample tissue and the glass slide. After removing the sections, they were allowed to stand for 15 minutes to return to room temperature. Then, the sections were dewaxed and rehydrated using xylene and graded alcohols. After that, Alcian blue hematoxylin / Orange G (ABH / OG) staining was performed. The specific staining steps were as follows: First, the sections were placed in 1% hydrochloric acid alcohol for differentiation for 30 seconds. Then, they were placed in Alcian blue hematoxylin solution for staining for 1 hour. After that, they were rinsed with pure water 3 times, 3 minutes each time. Then, the sections were placed in 1% hydrochloric acid alcohol for differentiation for 3 seconds. After rinsing with pure water 3 times, 1 minute each time, they were placed in 0.5% ammonia solution for 15 seconds for inversion. Then, the sections were placed in 95% alcohol solution for 1 minute. After that, they were transferred to Eosin / Orange G working solution for staining for 1 minute and 30 seconds. The sections were then dehydrated and cleared step by step. Finally, the sections were mounted with a mounting medium.

[0069] 2.1.5 Immunohistochemical analysis;

[0070] Tissue slides were placed in a 60°C oven overnight. The next day, the slides were removed and allowed to stand for 15 minutes to return to room temperature. Then, the slides were dewaxed and rehydrated using xylene and graded alcohol solutions. A heat retrieval method was employed, in which tissue slides were immersed in sodium citrate buffer and placed in a 60°C oven for 4 hours to complete antigen retrieval and fully expose the antigenic epitopes. The tissue samples were permeabilized with 0.3% Triton X-100 solution. Then, 100 μL of endogenous peroxidase inhibitor was added to each tissue sample, and the sample was incubated at room temperature for 10 minutes. Next, 100 μL of blocking normal goat serum working solution was added, and the sample was incubated at room temperature for 15 minutes. After discarding the serum, 100 μL of primary antibody was added, and the sample was incubated overnight at 4°C. The following day, the sample slides were removed and brought to room temperature. The primary antibody reagent was washed away with PBS buffer, and 100 μL of biotin-labeled goat anti-rabbit IgG polymer and 100 μL of horseradish enzyme-labeled streptavidin working solution were added sequentially. Then, an appropriate amount of freshly prepared DAB solution was added for staining. After staining, the slides were placed in hematoxylin staining solution for 20 seconds, differentiated with 1% hydrochloric acid alcohol, rinsed with tap water to return to blue, and finally dehydrated and cleared by sequentially passing through a gradient of alcohols and xylene. The sample slides were mounted with neutral resin.

[0071] 2.2 Results;

[0072] 2.2.1 Mitefocin intervention can improve bone loss in postmenopausal mice;

[0073] Micro-CT scans showed that 8 weeks after OVX modeling, the bone loss in the distal femoral metaphysis of mice in the model group was significantly reduced, while the bone loss in the distal femoral metaphysis of mice in the low-dose and high-dose mitefocin intervention groups was significantly less than that in the control group. Figure 2 A). Further bone microstructure analysis showed that mitefoxine intervention significantly increased distal femoral bone density (BMD). Figure 2 B), and improve bone tissue SMI ( Figure 2 C) and Conn.Dn( Figure 2 D). Simultaneously, analysis of cancellous bone revealed that mice treated with mitefoxin showed significantly increased bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th) compared to the control group, while trabecular bone separation (Tb.Sp) was significantly reduced. Figure 2 EH). Histomorphological analysis also showed that mitefoxine intervention reduced bone loss in the distal femur. Figure 2 IJ), and increases the number of osteoblasts on the surface of cancellous bone ( Figure 2 K). Furthermore, immunofluorescence staining of OCN and immunohistochemical staining of ALP both showed that mitefocin significantly promoted bone formation in OVX mice. Figure 2 LO). TRAP staining of distal femoral sections showed that low-dose mitefoxin intervention did not affect the number of TRAP-positive cells in OVX mice, while high-dose treatment slightly reduced the number of TRAP-positive cells. Figure 3 AB), the expression level of the mature osteoclast marker CTSK was not significantly different between the control group and the mitefoxin-treated group. Figure 3 CD). Meanwhile, Fap4 immunohistochemical staining results indicated that mitefoxin had no significant effect on adipocyte formation. Figure 3 The results indicate that mitefoxin intervention can effectively reduce bone loss and improve bone microstructure in postmenopausal mice, and this effect is mainly achieved by enhancing bone formation.

[0074] 2.2.2 Biosafety evaluation of mitefoxin;

[0075] Eight weeks after mitefocin intervention in the OVX model, the liver, heart, kidney, spleen, and other core organs of mice were collected. The histological morphology of these organs was observed by HE staining. The integrity, tissue structure, and morphology of all major organs in the low-dose and high-dose mitefocin intervention groups were similar to those in the control group, and no obvious lesions or organic changes were found. Figure 3 G). The results showed that mitefoxin intervention for 8 weeks had no significant toxic side effects on the liver, heart, kidneys, and spleen of OVX model mice, indicating that mitefoxin intervention for 8 weeks is biosafety in OVX model mice.

[0076] 2.3 Conclusion;

[0077] 2.3.1 Mitefocin intervention can promote osteogenic function in postmenopausal mice and effectively improve bone loss;

[0078] 2.3.2 Mitefocin intervention is biosafety in postmenopausal mice.

[0079] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein.

[0080] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. The application of mitefoxin in the preparation of drugs for the prevention and treatment of osteoporosis, characterized in that, The mitefoxin is hexadecyl 2-(trimethylamino)ethyl phosphate; its chemical formula is C. 21 H 46 NO4P has the following structural formula: ； The mitefoxin is used to enhance osteogenic differentiation of osteoblasts; The mitefoxin is used to increase the bone density of newly formed bone in the repair area; The mitefoxin is used to increase the bone volume fraction of newly formed bone in the repair area; The mitefoxin is used to enhance the trabeculae of newly formed bone in the repair area; The drug is a drug that catalyzes the activity of protein phosphatase PPM1A in osteoblasts; The effective dose of mitifone for promoting osteoproliferation is 5-10 mg / kg.

2. The application of mitefoxin according to claim 1 in the preparation of drugs for the prevention and treatment of osteoporosis, characterized in that, The drug is a bone formation promoting drug, and it is in the form of a solid powder.

3. The application of mitefoxin according to claim 1 in the preparation of drugs for the prevention and treatment of osteoporosis, characterized in that, The osteoporosis mentioned refers to postmenopausal osteoporosis.

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