Use of nfia enhancer in the preparation of anti-osteoporosis drugs for inhibiting bone resorption

Abstract

The present application discloses a method for preparing anti-osteoporosis drugs for inhibiting bone resorption by regulating NFIA. The present application finds that NFIA inhibits osteoclast differentiation and its bone resorption activity by inhibiting the transcription of RANKL. The cancellous bone mass of mice with NFIA gene specifically knocked out in osteogenic precursor cells and mesenchymal cells is reduced; the adeno-associated virus overexpressing NFIA injected through the tail vein can relieve the bone loss induced by ovariectomy in mice. The drugs prepared according to the present application can inhibit osteoclast differentiation, reduce bone resorption and increase the bone mass of the body by enhancing the expression of NFIA or enhancing the protein function of NFIA, and have the effect of preventing and treating osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of NFIA enhancers in the preparation of anti-osteoporosis drugs that inhibit bone resorption. Background Technology

[0002] Osteoporosis is a systemic bone disease characterized by low bone mass and destruction of bone tissue ultrastructure. As a result, bone fragility increases significantly, and even minor external forces can induce fractures, posing a serious threat to the health of patients.

[0003] With the accelerating aging of the world's population, the prevalence of osteoporosis and the incidence of osteoporotic fractures are rising rapidly. Related data shows that fractures of the hip, vertebrae, and other parts of the body caused by osteoporosis in middle-aged and elderly people not only lead to pain and limited mobility for patients, but also significantly increase the burden of family care and social medical costs, becoming a significant public health issue that urgently needs attention and response.

[0004] Currently, drugs used clinically to treat osteoporosis primarily fall into two categories based on their mechanisms of action: promoting bone formation and inhibiting bone resorption. Among these, drugs that effectively promote bone formation are relatively few. Teriparatide, a commonly used osteoproliferative drug, has a lifelong use limit of no more than two years. Meanwhile, more widely used drugs that inhibit bone resorption may cause various adverse reactions with long-term use, including osteonecrosis of the mandible and atypical fractures, thus limiting their clinical application. Against this backdrop, the limitations of existing treatments are becoming increasingly apparent. Developing new osteoporosis treatments with higher safety and more precise mechanisms has become a crucial direction urgently needing breakthroughs in both clinical and research fields.

[0005] Transcription factor NFIA is a member of the nuclear factor I (NFI) family, which includes NFIB, NFIC, and NFIX. These are important transcription factors containing highly conserved N-terminal DNA-binding regions. They bind to the same palindromic DNA-binding sequence (TTGGC(N5)GCCAA) in heterodimer and homodimer forms, activating or inhibiting the transcription of target genes and playing a crucial role in the development of the central nervous system, bones, lungs, and other tissues and organs. However, research on NFIA regulating osteoclast differentiation and thus bone homeostasis has not been reported in the literature. Currently, there are no reports of studies using targeted NFIA to prevent or treat osteoporosis. Summary of the Invention

[0006] In view of the shortcomings of existing technologies and practical needs, this invention proposes the application of NFIA enhancers in the preparation of anti-osteoporosis drugs that inhibit bone resorption.

[0007] This invention is the first to discover that NFIA inhibits osteoclast differentiation and bone resorption activity by suppressing RANKL transcription. Mice with specific knockout of the NFIA gene in osteoblast progenitor cells and mesenchymal cells showed reduced cancellous bone mass; tail vein injection of adeno-associated virus overexpressing NFIA alleviated ovariectomy-induced bone loss in mice. These findings suggest that NFIA helps increase bone mass and is a potential drug for the prevention and treatment of osteoporosis.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] This invention discloses for the first time a method for preparing an anti-osteoporosis drug that inhibits bone resorption by regulating NFIA. The prepared drug inhibits osteoclast differentiation of bone marrow osteoclast precursor cells by enhancing NFIA expression or its protein function, thereby reducing bone resorption and increasing bone mass, and has the effect of preventing and treating osteoporosis. Attached Figure Description

[0010] Figure 1 The results of μCT scans of the tibia in mice with NFIA gene knockout specifically targeting osteogenic progenitor cells are shown. A is a 3D reconstruction of the cancellous bone in the metaphysis of the tibia in female mice; B is the trabecular bone volume fraction in female mice; C is the trabecular bone mineral density in female mice; D is the number of trabeculae in female mice; E is the trabecular bone separation in female mice; F is a 3D reconstruction of the cancellous bone in the metaphysis of the tibia in male mice; G is the trabecular bone volume fraction in male mice; H is the trabecular bone mineral density in male mice; I is the number of trabeculae in male mice; J is the trabecular bone separation in male mice.

[0011] Figure 2 The results of μCT scans of the tibia in mice with NFIA gene knockout specifically in mesenchymal cells are shown. A is a 3D reconstruction of the cancellous bone in the metaphysis of the tibia of female mice; B is the trabecular bone volume fraction in female mice; C is the trabecular bone mineral density in female mice; D is the number of trabeculae in female mice; E is the trabecular bone separation in female mice; F is a 3D reconstruction of the cancellous bone in the metaphysis of the tibia of male mice; G is the trabecular bone volume fraction in male mice; H is the trabecular bone mineral density in male mice; I is the number of trabeculae in male mice; J is the trabecular bone separation in male mice.

[0012] Figure 3 The results show the staining of tartrate-resistant acid phosphatase (TRAP) in the tibial metaphysis of mice with NFIA gene knockout specifically for osteoblast progenitor cells, and the measurement of type I collagen C-terminal peptide (CTX-1) in serum. A: TRAP staining results in the tibial metaphysis of female mice; B: TRAP-positive cell count in female mice; C: TRAP staining results in the tibial metaphysis of male mice; D: TRAP-positive cell count in male mice; E: CTX-1 measurement results in the serum of female mice; F: CTX-1 measurement results in the serum of male mice.

[0013] Figure 4This study investigated the effects of NFIA gene knockout in osteoblast precursor cells on promoting osteoclast differentiation in mouse bone marrow and simultaneously increasing RANKL expression in bone marrow stromal cells. A shows TRAP staining results after osteoclast differentiation was induced in bone marrow cells from gene knockout mice; B shows the TRAP-positive cell count after osteoclast differentiation was induced in bone marrow cells from gene knockout mice; C shows the mRNA expression of osteoclast differentiation markers NFATC1, CTSK, and TRAP after osteoclast differentiation was induced in bone marrow cells from gene knockout mice; D shows the protein expression of osteoclast differentiation markers NFATC1, CTSK, and MMP9 after osteoclast differentiation was induced in bone marrow cells from gene knockout mice; E shows the RANKL mRNA expression in bone marrow stromal cells; and F shows the RANKL protein expression in bone marrow stromal cells.

[0014] Figure 5 The results show the effects of AAV-NFIA virus on alleviating bone loss in ovariectomized (OVX) mice. A is a 3D reconstruction of the cancellous bone in the tibial metaphysis of female mice; B is the volume fraction of trabecular bone in female mice; C is the mineral density of trabecular bone in female mice; D is the number of trabecular bones in female mice; E is a 3D reconstruction of the cortical bone in the tibial bone of female mice; F is the thickness of the cortical bone in female mice. Detailed Implementation

[0015] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides further illustrative information. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit its scope.

[0016] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0017] The materials and methods involved in the embodiments of the present invention are described below.

[0018] (1) Laboratory animals

[0019] Nfia fx / wt Mice were purchased from GemPharmatech (Nanjing, China), Osx-Cre mice from Biocytogen (storage number: #110131), and Prx1-Cre mice from Jackson Lab (storage number: #005584). Osx-Cre mice and Nfia... fx / wt Mice mating produces Osx-Cre; Nfia fx / wt Further with Nfia fx / fx Mice were bred to obtain osteoblast progenitor cell-specific knockout NFIA gene mice (KO-Nfia).Osx ); by Prx1-Cre mice and Nfia fx / wt Prx1-Cre; Nfia was obtained by breeding mice. fx / wt Further with Nfia fx / fx Mice were bred to obtain mesenchymal cell-specific knockout NFIA gene mice (KO-Nfia). Prx1 ).

[0020] (2) uCT analysis

[0021] Mouse tibial specimens were scanned using a Scanco viva CT80 (Scanco Medical AG, Switzerland) with scanning parameters of 55 kVp and 145 μA. Three-dimensional reconstruction was performed using Scanco software. The region of interest in cancellous bone was selected as a 1 mm region starting 0.1 mm below the growth plate. Bone morphostatic parameters included: trabecular bone volume fraction (BV / TV, %) and trabecular bone mineral density (Tb.BMD, mg / cm³). 3 The parameters included the number of trabeculae (Tb.N, / mm) and trabecular separation (Tb.Sp, mm). The region of interest for cortical bone was selected as a 0.5 mm region starting 2 mm below the growth plate. Cortical bone parameters included cortical bone thickness (Cort.Th).

[0022] (3) TRAP staining of mouse bone tissue sections

[0023] Mouse tibias and femurs were isolated and fixed in 10% formaldehyde for 3 days. Samples were decalcified in 14% EDTA (pH 7.4) for 21 days, dehydrated by gradients of ethanol (70%-100%) and xylene, embedded in paraffin, and cut into 4 μm thick sagittal serial sections. To assess osteoclast formation, TRAP staining was performed, and the number of TRAP-positive cells was counted in a region extending 100 μm below the growth plate of the tibial metaphysis to 1 mm distal to the growth plate.

[0024] (4) Mouse serum ELISA assay

[0025] The level of CTX-1 in mouse serum was determined using a CTX-1 ELISA kit (BBI, Shanghai, China).

[0026] (5) Cell culture and induced differentiation

[0027] Primary bone marrow stromal cell (BMSCs) isolation: Primary bone marrow stromal cells were isolated using the whole bone marrow adherent culture method. After euthanizing 4-6 week old mice, the bilateral femurs and tibias were aseptically removed, the epiphyses were removed, and the medullary cavity was exposed. The medullary cavity was flushed multiple times with 5 mL of DMEM culture medium using a syringe, and cells were collected and then seeded into α-MEM containing 10% fetal bovine serum. The medium was changed for the first time after 3 days of culture to remove non-adherent cells. After washing with PBS, the medium was replaced with fresh medium. The culture medium was changed every 3 days. Cells were passaged when they reached 80% confluence. Third-generation cells were used for subsequent experiments.

[0028] Primary bone marrow cell (BM) isolation and osteoclast induction: Bone marrow cells were collected from 10-14 day old mice and cultured at 8 × 10⁻⁶ cells / year. 6 The cells were seeded at a density of 1 / mL in 48-well plates in α-MEM containing 10% fetal bovine serum (FBS). The cells were cultured for 3 days at 20 ng / mL M-CSF, and then... -7 Osteoclast formation was induced in the presence of mmol / L 1,25(OH)2VitD3 and 20 ng / ml M-CSF. The culture medium was changed every 3 days. After 8-12 days of cell culture, cells were fixed with 4% paraformaldehyde and then stained with TRAP. TRAP-positive osteoclasts containing ≥3 nuclei were counted.

[0029] (6) Quantitative real-time polymerase chain reaction (qRT-PCR)

[0030] Total RNA was extracted from cells using a total RNA extraction kit (Omega, America). Reverse transcription was performed using a first-strand cDNA synthesis kit (PerfectStart Uni RT&qPCR Kit, TransGen, Beijing, China). Real-time quantitative PCR was performed using the SYBR Green fluorescent PCR method, with β-actin as an internal control. -ΔΔCt The calculation method yields the relative expression level of each target gene: ΔCt = Ct value of target gene - Ct value of internal reference gene; ΔΔCt = ΔCt of experimental group - ΔCt of control group.

[0031] (7) Western blotting

[0032] Total protein was extracted from cells using RIPA lysis buffer and separated by SDS-PAGE gel electrophoresis. The protein was then transferred to a nitrocellulose membrane, blocked with 5% skim milk powder at room temperature for 1 h, and incubated overnight at 4°C with primary antibody. After incubation with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature, the expression of the target protein was detected using an ECL chemiluminescence kit (ABclonal, Wuhan, China).

[0033] (8) Construction of ovariectomized (OVX) osteoporosis mouse model and AAV delivery

[0034] Three-month-old female C57BL / 6 mice underwent ovariectomy (OVX) or sham surgery under anesthesia. To construct the AAV-NFIA expression vector, an NFIA fusion cDNA with a C-terminus linked to the coding sequences of 10XHis and P2A was cloned into the pcAAV-CMV-EGFP-tWPA vector. The Nfia-10xHis-P2A fusion cDNA sequence is shown in SEQ ID NO.1.

[0035]

[0036] The virus was packaged using an AAV9 capsid. Three days post-surgery, 150 μL (8 × 10^11 vg) of AAV-NFIA or AAV-EGFP virus was delivered systemically via tail vein injection. Tibial bone was harvested eight weeks post-injection for μCT analysis.

[0037] (9) Statistical analysis

[0038] All experiments were repeated at least three times, and data were analyzed using GraphPad Prism 8. Data are expressed as mean ± standard deviation (mean ± SD). The relative levels of mRNA, protein, and luciferase activity in the control group were set to 1. Student's t-test was used for comparisons between two groups, with Welch correction applied when variances were unequal. One-way or two-way ANOVA was used for comparisons among multiple groups, followed by Dunnett's test or Tukey's test for post-hoc comparisons. p A value <0.05 is considered statistically significant.

[0039] Example 1

[0040] This embodiment analyzes mice with osteoblast progenitor-specific NFIA gene knockout (KO-Nfia). Osx Bone mass changes. μCT was used to analyze KO-Nfia. Osx Three-dimensional reconstruction of the tibial cancellous bone microstructure of control mice was performed. Figure 1 A: Female; Figure 1 F: Male). Based on measured bone morphometric static parameters, compared to the control group, 5-month-old KO-Nfia... Osx Female mice and 8-month-old KO-Nfia Osx In male mice, trabecular bone volume fraction (Tb. BV / TV), trabecular bone mineral density (Tb. BMD), and trabecular bone number (Tb. N) were all significantly decreased, while trabecular bone separation (Tb. Sp) was increased. Figure 1 BE: Female; Figure 1 GJ: Male). These results indicate that knocking out the NFIA gene specifically in osteoblast precursor cells leads to a reduction in cancellous bone mass in adult mice.

[0041] Example 2

[0042] This embodiment analyzes mesenchymal cell-specific knockout NFIA gene mice (KO-Nfia). Prx1 Bone mass changes were observed using μCT on KO-Nfia. Prx1 Three-dimensional reconstruction of the tibial cancellous bone microstructure of control mice was performed. Figure 2 A: Female; Figure 2 F: Male). Based on measured bone morphometric static parameters, compared to the control group, 5-month-old KO-Nfia... Prx1 Female mice and 8-month-old KO-Nfia Prx1 In male mice, the trabecular bone volume fraction (Tb.BV / TV), trabecular bone mineral density (Tb.BMD), and trabecular bone number (Tb.N) of the tibia were significantly decreased, while trabecular bone separation (Tb.Sp) was increased. Figure 2 BE: Female; Figure 2 (GJ: male), indicating that specific knockout of the NFIA gene in mesenchymal cells leads to a reduction in cancellous bone mass in adult mice.

[0043] Example 3

[0044] This embodiment analyzes osteoblast progenitor cell-specific knockout NFIA gene mice (KO-Nfia). Osx Results of osteoclast staining at the tibial metaphysis and measurement of serum bone resorption marker CTX-1. TRAP staining was performed on osteoclasts. Figure 3 A: Female; Figure 3 C: Male). Quantitative results show that female and male KO-Nfia Osx The number of osteoclasts in mice was significantly increased. Figure 3 B: Female; Figure 3 D: Male). ELISA testing showed that, compared with the control group, both female and male KO-Nfia Osx The level of CTX-1 in mouse serum was elevated ( Figure 3 E: Female; Figure 3 F: Male).

[0045] Example 4

[0046] This embodiment analyzed the results of NFIA gene knockout in osteoblast progenitor cells promoting osteoclast differentiation in mouse bone marrow and simultaneously promoting RANKL expression in bone marrow stromal cells. After osteoclast induction in isolated and cultured bone marrow cells, compared with the control group, KO-Nfia... Osx Osteoclast differentiation in mice was promoted, and the number of TRAP-positive cells increased. Figure 4 A, B), increased expression of osteoclast differentiation marker genes NFATC1, CTSK, and TRAP mRNA ( Figure 4 C), increased expression of NFATC1, CTSK, and MMP9 proteins ( Figure 4 (D) indicates that NFIA gene knockout in osteoblast precursor cells promotes osteoclast differentiation in mouse bone marrow. Bone marrow stromal cells were isolated, and compared to the control group, KO-Nfia... Osx Increased RANKL mRNA and protein expression in mouse bone marrow stromal cells ( Figure 4 (E, F) indicates that NFIA inhibits osteoclast differentiation by downregulating RANKL expression in bone marrow stromal cells.

[0047] Example 5

[0048] This study analyzed the ameliorative effect of AAV-NFIA virus on bone loss in OVX mice. Compared with the Sham / AAV-EGFP group, the OVX / AAV-EGFP group showed a significant decrease in bone mass, indicating successful OVX modeling. Specifically, this was manifested in reduced Tb.BV / TV, Tb.BMD, and Tb.N in trabecular bone, and reduced Ct.Th in cortical bone. Compared with OVX / AAV-EGFP mice, bone loss was alleviated in the OVX / AAV-NFIA group, as shown by increased Tb.BV / TV, Tb.BMD, and Tb.N in trabecular bone, and increased Ct.Th in cortical bone. Figure 5 These results indicate that NFIA can alleviate bone loss caused by OVX.

[0049] In summary, NFIA can inhibit osteoclast differentiation, reduce bone resorption, and increase bone mass.

[0050] Therefore, drugs prepared by enhancing NFIA gene expression or enhancing its protein function have the potential to prevent and treat osteoporosis.

[0051] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. The application of NFIA enhancers in the preparation of anti-osteoporosis drugs that inhibit bone resorption, characterized by: The NFIA enhancer is an overexpression vector targeting the NFIA-encoding gene. It inhibits osteoclast differentiation in bone marrow, reduces bone resorption, and increases bone mass by enhancing NFIA expression.

2. The application according to claim 1, characterized in that, The overexpression vector of the NFIA encoding gene includes a virus or cell that overexpresses NFIA.

3. The application according to any one of claims 1–2, characterized in that, The dosage form of the drug includes injections.

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