Application of erythrocyte differentiation regulation factor 1 in preparation of medicine for treating osteoporosis

Abstract

The invention discloses application of an erythrocyte differentiation regulation factor 1 in preparation of a medicine for treating osteoporosis, and belongs to the technical field of biology. According to the application of the erythrocyte differentiation regulatory factor 1 in preparation of the medicine for treating osteoporosis, an ERDR1 gene knockout mouse is constructed by utilizing a Cre-loxp system, and the skeletal development of the mouse is obviously inhibited after the ERDR1 gene is knocked out; in an ERDR1 gene knockout mouse, biomechanical parameters such as the maximum load, the elastic load, the rigidity, the elastic modulus, the maximum bending stress, the fracture work and the like are reduced to a certain degree, and it is prompted that the risk of fracture can be remarkably increased due to deletion of ERDR1; the ERDR1 gene knockout has an inhibiting effect on the osteogenic differentiation potential of the BMSCs, and the osteogenic differentiation capacity of the BMSCs is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to the application of erythrocyte differentiation regulatory factor 1 in the preparation of a drug for treating osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease that impairs bone strength, leading to bone brittleness and an increased risk of fractures. Current treatments for osteoporosis include bisphosphonates, calcitonin, parathyroid hormone, and vitamin D, but these all suffer from uncertain efficacy and significant adverse reactions. Therefore, the development of new drugs is urgent.

[0003] The erythroid differentiation regulator 1 (ERDR1) gene is a protein-coding gene highly conserved between humans and mice. Located in the pseudoautosomal regions of chromosomes X and Y, ERDR1 is involved in biological processes such as cell proliferation and apoptosis regulation, cell differentiation, cell migration and invasion, and signal transduction. However, the role of ERDR1 in osteoporosis has not been reported.

[0004] Therefore, providing an application of erythrocyte differentiation regulatory factor 1 in the preparation of drugs for treating osteoporosis is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the use of erythrocyte differentiation regulatory factor 1 in the preparation of a drug for treating osteoporosis.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention relates to the use of erythrocyte differentiation regulatory factor 1 in the preparation of a drug for treating osteoporosis, wherein the sequence of the erythrocyte differentiation regulatory factor 1 is shown in SEQ ID NO.1.

[0008] Furthermore, the erythroid differentiation regulatory factor 1 is used in preparing an osteoporosis model. The sequence of the erythroid differentiation regulatory factor 1 is shown in SEQ ID NO.1.

[0009] Erythroid differentiation regulator 1 (ERDR1) gene sequence:

[0010] ; SEQ ID NO.1.

[0011] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses the use of erythrocyte differentiation regulatory factor 1 in the preparation of drugs for the treatment of osteoporosis. ERDR1 gene knockout mice were constructed using the Cre-loxp system, and it was found that after ERDR1 gene knockout, the bone development of mice was significantly inhibited; in ERDR1 gene knockout mice, biomechanical parameters such as maximum load, elastic load, stiffness, elastic modulus, maximum bending stress, and fracture work all decreased to a certain extent, suggesting that the absence of ERDR1 may significantly increase the risk of fractures; ERDR1 gene knockout inhibited the osteogenic differentiation potential of BMSCs and reduced the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0013] Figure 1 Construction of ERDR1 knockout mouse model and verification of ERDR1 gene expression; A: PCR genotyping of ERDR1 CKO mice: flox allele, Prx1-Cre, and knockout verification; B: RT-qPCR detection of ERDR1 expression; ****P < 0.0001;

[0014] Figure 2 Results of Micro CT analysis of femoral cancellous bone in 4-week-old and 8-week-old mice; A: 3D reconstruction of femoral cancellous bone in 4-week-old and 8-week-old mice; A: 3D reconstruction of femoral trabeculae (left column) and 2D reconstruction of distal femur (right column) in 4-week-old and 8-week-old mice (left and right columns are different cross-sectional images); B: Statistical results of bone mineral density (BMD), trabecular volume fraction (BV / TV), trabecular connection density (Conn.D), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) measured by Micro CT in 4-week-old and 8-week-old mice; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0015] Figure 3 Results of the three-point bending test on the femur of 8-week-old mice; A: Load-displacement curve obtained from the three-point bending test; B: Biomechanical parameters of the femur of 8-week-old mice measured by the three-point bending test: elastic load, maximum load, stiffness, elastic modulus, maximum bending stress, and work of fracture; *P < 0.05, **P < 0.01, ***P < 0.001;

[0016] Figure 4 ALP (alkaline phosphatase) expression and mineralization of BMSCs cells in WT (wild type) and CKO (conditional knockout) mice; A: ALP staining and Alizarin red staining results of BMSCs cells after osteogenic induction; B: ALP activity and calcium nodule quantitative statistical results of BMSCs cells after osteogenic induction; ****P < 0.0001. DETAILED DESCRIPTION

[0017] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] Example 1 Construction of ERDR1 gene knockout mice

[0019] The ERDR1 knockout mice used in this study were constructed based on CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9) technology. CRISPR / Cas9-mediated loxP insertion and flox mice were generated. GRNA for the mouse ERDR1 gene (Table 1), ssDNA containing loxP sites, and Cas9 mRNA were co-injected into fertilized eggs to produce targeted conditional knockout offspring. F0 generation mice were identified by PCR and sequenced, and bred with WT (Wild Type) mice for germline transmission and reproduction to obtain F1 generation mice.

[0020] By crossing ERDR1 flox mice with Prx1-Cre transgenic mice, the offspring mice were screened by genotyping and other methods, and finally mesenchymal-specific ERDR1 gene knockout mice were obtained. flox / flox Mouse has a bright band at 402 bp, and both alleles carry loxP; ERDR1 + / + Mouse has a bright band at 346 bp, and the allele does not carry loxP; ERDR1 flox / + Mouse has two bright bands at 346 bp and 402 bp.

[0021] BMSCs were successfully extracted and purified from the femurs and tibias of WT and CKO (conditional knockout) mice. RNA was extracted and the expression of ERDR1 was detected by RT-qPCR. It was found that ERDR1 was expressed in BMSCs. After gene knockout, the mRNA level of ERDR1 was significantly decreased, indicating that a mesenchymal-specific ERDR1 gene knockout mouse model was basically successfully constructed ( Figure 1 ).

[0022] Table 1 gRNA target sequences

[0023]

[0024] Example 2 Effect of ERDR1 gene knockout on bone mass in mice

[0025] After constructing ERDR1 gene knockout mice using the Cre-loxp system, Micro CT scans were performed on the femurs of mice at 4 and 8 weeks of age. This study found that after ERDR1 gene knockout, the bone development of mice was significantly inhibited. Bone mineral density (BMD) reflects the bone mineral content per unit area and is a key indicator for measuring bone strength. Compared with the wild-type (WT) group, the femoral bone mineral density (Tb.BMD) of the gene knockout (CKO) group mice decreased significantly; the bone volume fraction that directly reflects the change in bone mass, that is, the ratio of bone volume to tissue volume (BV / TV), decreased significantly, and the trabecular connection density (Conn.D) that reflects the tightness of the connection of the trabecular network decreased significantly; the number of trabeculae (Tb.N) and thickness (Tb.Th) representing the spatial structure of trabeculae decreased, while the trabecular separation (Tb.Sp) increased significantly, indicating that after the gene knockout, the trabeculae of mice developed abnormally, the spatial distance between each other increased, the trabeculae decreased significantly, the structure was sparse, and the cancellous bone was obviously dysplastic ( Figure 2 ).

[0026] Example 3 Effects of ERDR1 gene knockout on mouse femoral biomechanics

[0027] The bone biomechanical properties of 8-week-old mice were assessed using a three-point femoral bending test. Using a universal testing machine, the femurs were continuously loaded until fracture, and the load and displacement were recorded. Subsequently, load-displacement curves were plotted and analyzed to determine a series of bone biomechanical parameters, including maximum load, elastic load, stiffness, elastic modulus, maximum bending stress, and fracture energy.

[0028] Maximum load refers to the maximum external force a femur can withstand in a three-point bending test. It directly reflects the overall strength of the bone, specifically its ability to resist fracture under extreme loading conditions. Elastic load refers to the maximum external force a bone can withstand while undergoing only elastic deformation, revealing its initial flexibility and elastic recovery. Stiffness is the ratio of load to displacement and characterizes the bone's ability to resist deformation. A bone with higher stiffness requires a greater external force to produce a given degree of deformation, but excessive stiffness can affect energy dissipation and lead to brittle fracture under sudden external forces. Elastic modulus is a fundamental material property that describes the stress-strain relationship of bone within its elastic range. It reflects the inherent stiffness of the bone material and is independent of its geometry. A higher elastic modulus indicates a greater resistance to elastic deformation at the material level. Maximum bending stress occurs at the site of highest stress concentration during femoral bending, quantifying the bone's internal resistance to bending forces. Work of fracture represents the total energy absorbed by the femur from stress application to fracture. A higher work of fracture indicates a greater ability of the bone to buffer and absorb energy under sudden, high-intensity external forces, thus delaying or preventing fracture. Compared with wild-type mice of the same age, the above biomechanical parameters in ERDR1 knockout mice decreased to a certain extent, suggesting that the loss of ERDR1 may significantly increase the risk of fractures ( Figure 3 ).

[0029] Example 4 Effect of ERDR1 gene knockout on alkaline phosphatase (ALP) expression and mineralization in bone marrow mesenchymal stem cells (BMSCs)

[0030] Well-grown BMSCs from wild-type (WT) and CKO mice were digested, counted, and seeded into corresponding culture plates. BMSCs were induced to differentiate into osteoblasts using an osteogenic induction solution (consisting of DMEM complete medium containing 1 μM dexamethasone, 50 μg / mL vitamin C, and 10 mM β-glycerophosphate). A series of assays were then performed to observe differences between wild-type (WT) and CKO mice. Alkaline phosphatase (ALP) staining was performed on the 7th day of induction. The results showed that compared with WT BMSCs, CKO BMSCs stained lighter, with significantly fewer areas of intense dark blue staining, suggesting significantly lower ALP enzyme activity and a relatively small number of cells involved in early osteogenic differentiation. ALP activity assay data further quantified this difference, indicating that ALP activity in CKO BMSCs may be much lower than that in WT BMSCs during the early stages of osteogenic differentiation. Alizarin red (ARS) staining was performed on the 21st day of osteogenic induction. As the osteogenic differentiation process progressed, comparative observations revealed that, compared with WT BMSCs, CKO BMSCs had fewer calcium nodules, a duller color, and a more scattered distribution after staining with alizarin red, and their ability to form mineralized nodules was significantly reduced. Further quantitative analysis of calcium nodules was performed, and the results were consistent with those of alizarin red staining. The above results indicate that ERDR1 gene knockout inhibited the osteogenic differentiation potential of BMSCs and reduced the osteogenic differentiation ability of BMSCs ( Figure 4 ).

[0031] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of erythrocyte differentiation regulatory factor 1 in the preparation of a drug for treating osteoporosis, characterized in that: The sequence of the erythrocyte differentiation regulatory factor 1 is shown in SEQ ID NO.

1.

2. Application of erythroid differentiation regulatory factor 1 in preparing an osteoporosis model, characterized in that: The sequence of the erythrocyte differentiation regulatory factor 1 is shown in SEQ ID NO.1.

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