Application of atrial natriuretic peptide (ANP) in preparation of medicine for improving bone metabolism related diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of atrial natriuretic peptide (ANP) in preparation of a medicine for improving bone metabolism related diseases. Research finds that ANP promotes osteogenic differentiation related genes to be up-regulated, the ALP activity is remarkably improved, and mineralized nodule formation is enhanced; the result shows that ANP can promote osteogenic precursor cells to be differentiated into osteoblasts and increase mineralization ability. As a novel candidate factor for treating osteoporosis, the atrial natriuretic peptide ANP can improve osteoporosis by promoting osteogenic differentiation of osteogenic precursor cells.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of atrial natriuretic peptide (ANP) in the preparation of drugs for improving diseases related to bone metabolism. Background Art

[0002] Osteoporosis (OP) is a systemic disease characterized by low bone mass and degeneration of bone tissue microstructure, accompanied by bone fragility and increased risk of fractures. It is a common and frequently occurring disease that seriously affects the health of middle-aged and elderly people.

[0003] Bone tissue is a dynamic equilibrium system, and bone resorption and bone formation play an important role in the normal maintenance of bones. At present, a variety of drugs that act on osteoclasts have been developed to target the numerous risk factors for osteoporosis, such as RANKL (receptor activator of nuclear factorκB ligand) inhibitors and bisphosphonates, but only parathyroid hormone analogs, such as teriparatide, act on osteogenesis, and these drugs have many side effects. Therefore, it is still necessary to explore the pathogenesis of osteoporosis and discover new effective therapeutic targets.

[0004] Insufficient bone formation is one of the key factors leading to osteoporosis. The ability of osteoblast precursor cells to differentiate into osteocytes is regulated by multiple factors, among which the physiological hormone regulation mechanism has not yet been fully clarified. Atrial natriuretic peptide (ANP) is a polypeptide hormone mainly secreted by the atrium. It is known to play an important role in cardiovascular regulation, but it has not been used in bone metabolism. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention proposes the use of atrial natriuretic peptide (ANP) in the preparation of drugs for improving diseases related to bone metabolism.

[0006] Atrial natriuretic peptides (ANP), also known as atrial natriuretic peptides, is a peptide hormone synthesized and released by atrial myocytes. ANP activates the cGMP / PKG signaling pathway by binding to its receptors NPR-A and NPR-B, promoting the differentiation of osteoblast precursor cells. Specifically, after ANP binds to the receptor, it activates guanylate cyclase, increases the level of cyclic guanosine monophosphate (cGMP) in the cell, and then activates protein kinase G (PKG). PKG can phosphorylate a variety of proteins, thereby regulating the physiological functions of cells, including promoting the differentiation of osteoblast precursor cells into osteoblasts.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] Application of atrial natriuretic peptide (ANP) in the preparation of drugs for promoting differentiation of osteoblast precursor cells into osteocytes.

[0009] Furthermore, the atrial natriuretic peptide ANP is a peptide hormone with a ring structure consisting of 28 amino acids, and its amino acid sequence is SEQ ID NO: 1. The cysteine ​​residues at positions 7 and 23 form a disulfide bond, and the molecular weight is 3080.46.

[0010] Furthermore, the osteoblast precursor cells are mesenchymal stem cells or osteoblast precursors.

[0011] Furthermore, the drug is an injection, a sustained-release capsule or an implanted sustained-release material.

[0012] A method for constructing a pre-osteoblast cell line MC3T3-E1 cell differentiation model, the construction method comprising: inoculating MC3T3-E1 cells into a 6-well plate, and replacing the α-MEM complete culture medium containing an osteogenic inducing agent when the cell growth confluence reaches 50-60%; then adding ANP at a concentration of 100 nM for culturing, changing the medium every other day, and then performing ALP staining and ALP activity detection on day 7 of osteogenic induction, and performing Alizarin red staining on day 14 of induction to detect whether the pre-osteoblast cell line MC3T3-E1 cell differentiation model has been completed.

[0013] Furthermore, the components of the α-MEM complete culture medium containing osteogenic inducing agents include: 10 mmol / L β-glycerophosphate, 50 μg / mL L-ascorbic acid, and 10 nM dexamethasone.

[0014] Furthermore, the steps of ALP staining are as follows: the MC3T3-E1 cells induced to differentiate for 7 days were washed with 1×PBS buffer, fixed with 4% paraformaldehyde for 10 minutes, added with BCIP / NBT staining working solution, and incubated at room temperature in the dark for 20-30 minutes; the cells were washed three times with deionized water; air-dried, stored at room temperature in the dark; and photographed and recorded.

[0015] Furthermore, the steps of ALP activity detection are as follows: after 7 days of induction and differentiation culture of MC3T3-E1 cells, the culture medium is removed, a lysis buffer without protease inhibitors is added, the cells are centrifuged at 12000g for 10 min at 4°C, the supernatant is taken, and the ALP activity is detected using an alkaline phosphatase activity detection kit.

[0016] Furthermore, the steps of Alizarin Red staining are as follows: after 14 days of induced differentiation culture of MC3T3-E1 cells, the cells were washed with 1×PBS buffer, fixed with 4% paraformaldehyde for 10 minutes, added with 2% Alizarin Red dye solution, incubated at room temperature in the dark for 20 minutes, washed with PBS, air-dried, stored at room temperature in the dark, and photographed to record and observe the formation of calcium nodules.

[0017] A reagent comprising atrial natriuretic peptide (ANP).

[0018] The beneficial effects of the present invention are as follows: ANP promotes the upregulation of osteogenic differentiation-related genes, significantly increases ALP activity, and enhances the formation of mineralized nodules; this indicates that ANP can promote the differentiation of osteogenic precursor cells into osteoblasts and increase mineralization capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 is the effect of ANP deficiency on mouse bone mass in Example 1, wherein Figure 1 A represents micro-CT tomography scans of femurs of 8-week-old WT and Nppa (ANP encoding gene) knockout (KO) mice, and parallel three-dimensional reconstruction to observe the changes in bone microstructure and count the trabecular area; Figure 1 B represents paraffin sections of mouse femurs, H&E staining, and calculation of the percentage of trabecular bone; Figure 1 C represents the osteoclast-specific marker tartrate-resistant acid phosphatase (TRAP) staining, and the TRAP-positive area was counted.

[0021] Figure 2 The expression of osteoblast differentiation-related marker genes was reduced in Nppa KO mice; mRNA was extracted from the femoral diaphysis of Nppa KO and WT mice, and qPCR was performed to detect osteoblast marker genes (Bmp2, Runx2, Alp, Bglap and col1a1) (A) and osteoclast marker genes (Opg, Rankl, Rank, Ctsk and Nfatc1) (B), respectively.

[0022] Figure 3 The effect of ANP on the pre-osteoblast cell line MC3T3-E1 cells was verified: ANP can promote the differentiation of osteoblast precursor cells into osteoblasts and increase mineralization capacity; ANP deficiency leads to reduced differentiation capacity of mouse osteoblast precursor cells. Osteoblast precursor cells were extracted from Nppa KO and WT mice, and osteogenic differentiation was induced in vitro. Alkaline phosphatase (ALP) staining (A) and Alizarin red staining (B) were performed before induction (0 day), 7 days and 14 days of induction to identify the differentiation and mineralization capacity of osteoblast precursor cells. (C) The expression of genes related to osteogenic differentiation was detected by qPCR.

[0023] Figure 4:ANP promotes osteogenic differentiation of MC-3T3 cells; MC-3T3 cells were induced to differentiate in vitro, and human ANP was added. The expression of osteoblast differentiation-related genes (such as Runx2, Bmp2, Col1a1 and Alp) was detected 7 days after induction (A); (B) ALP staining was performed, the ALP staining area was counted, and the ALP activity was detected. (C) Alizarin red staining was performed on day 14, and the mineralized nodule area was counted. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0025] Example 1: ANP deficiency leads to bone loss in mice

[0026] Nppa (ANP encoding gene) knockout (KO) mice were constructed (from Nanjing Jicui Pharmaceutical Co., Ltd., China, using CRISPR / Cas9 technology to knock out exons 1-3 of the mouse Nppa gene), and the femurs of 8-week-old wild-type (WT) mice were collected for micro-CT tomography and three-dimensional reconstruction to observe the changes in bone density and bone microstructure. Paraffin sections were made at a thickness of 5 μm, followed by H&E staining and tartrate-resistant acid phosphatase (TRAP) staining for osteoclast-specific marker enzymes; femoral diaphysis mRNA was extracted and then reverse transcribed for qPCR to detect osteoblast marker genes (Bmp2, Runx2, Alp, Bglap, and col1a1) and osteoclast marker genes (Opg, Rankl, Rank, Ctsk, and Nfatc1).

[0027] The specific steps are as follows:

[0028] Preparation of Nppa (ANP encoding gene) KO mice: The method used was CRISPR / Cas9 technology to knock out exons 1-3 of the mouse Nppa gene, which was provided by Nanjing Jicui Pharmaceutical Biotechnology Co., Ltd.

[0029] Micro CT detection: The femur of 8-week-old mice was fixed with 4% paraformaldehyde, and the samples were scanned under the same conditions using the instrument Skyscan1176μCTscanner (Bruker, Aartselaar, Belgium), and relevant processing and three-dimensional reconstruction were performed. Scanning conditions: tube voltage 50kV, tube current 500μA, scanning thickness 9μm, 3D reconstruction and data analysis using NRecon v1.6 and CTAnv1.13.11.0 software; after scanning, the analysis software was used to select the region of interest (ROI), and the ROI was selected from 540μm from the distal end of the femur to 1.35mm from the proximal end, and then the trabecular area was counted using Image J software.

[0030] Tissue section preparation:

[0031] The femoral tissue of 8W mice was removed and fixed with 4% paraformaldehyde overnight, then decalcified with 0.5M EDTA for 7 consecutive days, gradient dehydrated and paraffin embedded, and then serial 5 μm sections were prepared with a microtome (RM2245, Leica) and placed on adhesive slides (188105, Jiangsu Shitai Experimental Equipment Co., Ltd.), and H&E staining and Trap staining were performed respectively.

[0032] H&E staining:

[0033] (1) Dewaxing: Place the sections in xylene I, II, and III, respectively, for 5 min each at room temperature;

[0034] (2) Hydration: Place the sections in anhydrous ethanol, 95%, 80%, and 70% ethanol at room temperature for 3 min each time;

[0035] (3) Washing: Rinse the sections in running water for 5 min and then soak in distilled water for 3 min.

[0036] (4) Hematoxylin staining: add hematoxylin solution for 5 min, and rinse with running water for 15 min;

[0037] (5) Eosin staining: add eosin solution and stain for 5 seconds;

[0038] (6) Dehydration: gradient dehydration in 70% ethanol, 80% ethanol, and anhydrous ethanol, 5 s each time;

[0039] (7) Sealing: After the slices are dried, they are sealed with neutral gum and observed and photographed under an upright microscope.

[0040] TRAP staining:

[0041] Paraffin sections of mouse femur were taken and stained with a tartrate-resistant acid phosphatase staining kit (G1492, Beijing Solebow Technology Co., Ltd.). The sections were first dewaxed and rehydrated, fixed with 2-8°C TRAP fixative for 1 min, washed with water, and then dripped with TRAP incubation solution, incubated at 37°C for 1 h, washed with distilled water, counterstained with methyl green for 2-3 min, sealed with neutral resin, photographed under a microscope, and the TRAP-positive area was counted.

[0042] RNA extraction and quantitative PCR (qPCR) detection

[0043] The femur of mice was taken, RNA was extracted with Trizol reagent (15596018, Thermo Fisher Scientific), and cDNA was generated by reverse transcription using the reverse transcription kit of Nanjing Novozyme Biotechnology Co., Ltd. after quantification. Real-time fluorescence quantitative PCR (qPCR) was performed using the QuantStudio 6 real-time PCR system (A25742, Thermo Fisher Scientific) to detect the expression levels of osteoblast marker genes (Bmp2, Runx2, Alp, Bglap and col1a1) and osteoclast marker genes (Opg, Rankl, Rank, Ctsk and Nfatc1). The results were standardized with the expression level of Gapdh (3-phosphoglyceraldehyde dehydrogenase). The oligonucleotide primer sequences used in qPCR are SEQ ID NO: 2-SEQ ID NO: 23, as shown in Table 1.

[0044] Table 1: SEQ ID NO: 2-SEQ ID NO: 23 sequence list

[0045]

[0046]

[0047] result: Figure 1 A The results showed that the content of trabecular bone (white) in the femur of ANP KO mice was significantly reduced compared with that of WT mice. Figure 1 H&E staining and statistical results in B further showed that the proportion of trabecular area in ANP KO mice was reduced, showing osteoporotic phenotypes such as significantly reduced bone density and reduced number of trabeculae. However, TRAP staining results showed that there was no significant difference in osteoclast index between ANP KO mice and WT mice ( Figure 1 C). Figure 2 The expression levels of osteoblast differentiation and osteoclast-related genes in the femur of mice were detected. The results showed that the expression of osteoblast differentiation-related genes in ANP KO mice was reduced ( Figure 2 A), while the expression levels of osteoclast differentiation-related genes in ANP KO mice were not significantly different from those in WT mice ( Figure 2B) These results further suggest that ANP deficiency leads to decreased osteogenic differentiation in mice, thus causing bone loss.

[0048] Example 2: ANP deficiency leads to delayed differentiation of osteoblast precursor cells in mice

[0049] Osteoblast precursor cells were extracted from Nppa KO and WT mice and induced to differentiate into osteoblasts in vitro. Alkaline phosphatase (ALP) staining and Alizarin red staining were performed before induction (0 day), 7 days and 14 days after induction to identify the differentiation and mineralization abilities of osteoblast precursor cells.

[0050] The specific steps are as follows:

[0051] Isolation of primary osteoblast precursor cells:

[0052] After the newborn Nppa KO and WT mice were killed, the limbs were separated under sterile conditions, and the excess skin, muscle, fat and other tissues were removed, leaving only the long bones of the limbs; the epiphysis was removed, and the bone marrow was rinsed with PBS until the femur was completely rinsed and the color was white and slightly transparent; the rinsed bone shaft was transferred to a culture dish and cut into 1-2 mm 2 The bone pieces were divided into pieces of different sizes and rinsed with PBS for 3 times; all the washed bone pieces were transferred to a 50ml centrifuge tube containing 0.2% collagenase II solution, and digested for 2 hours in a 37℃ shaker (200rpm), and the pieces were taken out and manually shaken for observation every 1 hour. After 2 hours, an equal amount of MEM-α culture medium was added to terminate the digestion, and the pieces were centrifuged at 800rpm for 5 minutes, and the liquid was discarded; the digested bone pieces were transferred to a culture dish containing PBS and rinsed repeatedly to remove the excess soft tissue after digestion; then the broken bone pieces were inoculated in α-MEM culture medium containing 20% ​​serum and incubated in a 37℃ incubator. The bone pieces will stick tightly to the bottom of the bottle, and the culture medium will be changed every 2 days. After 3-5 days, the osteoblast precursor cells will crawl out of the bone pieces; after the cell fusion is 70-80%, the first subculture is carried out, and the culture medium is changed every 2 days. The experiment was carried out with the third generation of primary cells.

[0053] Osteogenic differentiation:

[0054] Primary osteoblast precursor cells and MC3T3-E1 cells (CL-0378, Wuhan Punosai) were inoculated into 6-well plates. When the cell confluence reached 50-60%, α-MEM complete medium containing osteogenic induction agents (10mmol / L β-glycerophosphate, 50μg / mL L-ascorbic acid, 10nM dexamethasone) was replaced every other day. Alkaline phosphatase (ALP) staining and Alizarin red staining were performed at 7d and 14d of osteogenic induction to identify osteoblast differentiation and mineralization abilities.

[0055] Alkaline phosphatase (ALP) staining:

[0056] After 7-14 days of osteogenic differentiation, alkaline phosphatase staining was performed. The cells were washed with 1× PBS buffer, fixed with 4% paraformaldehyde for 10 min, and BCIP / NBT staining working solution (Biyuntian, Shanghai, China) was added. The cells were incubated at room temperature in the dark for 20-30 min. The cells were washed three times with deionized water, air-dried, and stored at room temperature in the dark. The cells were photographed and recorded.

[0057] Alizarin red staining:

[0058] Alizarin red staining was performed 7-14 days after osteogenic differentiation. The cells were washed with 1× PBS buffer, fixed with 4% paraformaldehyde for 10 min, and 2% alizarin red dye solution (pH 4.2, Biyuntian, Shanghai, China) was added. The cells were incubated at room temperature in the dark for 20 min, washed with PBS, air-dried, and stored at room temperature in the dark. The formation of calcium nodules was recorded and photographed.

[0059] RNA extraction and qPCR detection

[0060] Cells were collected before and after osteogenic differentiation induction, RNA was extracted, reverse transcribed into cDNA, and the expression of osteogenic differentiation-related genes was detected by qPCR. The specific method was the same as above.

[0061] result: Figure 3 The results showed that with the increase of induction time, the primary cells from WT and Nppa KO mice showed an increase in alkaline phosphatase and alizarin red staining, and the osteogenic differentiation marker genes Bmp2 and Runx2 were also significantly upregulated, but this upward trend in Nppa KO mice was not obvious compared with WT. The experimental results suggest that the osteogenic differentiation ability and mineralization ability of osteoblast precursor cells from Nppa KO mice are significantly reduced.

[0062] Example 3: ANP promotes differentiation of pre-osteoblast cell line MC3T3-E1 cells

[0063] MC3T3-E1 cells were induced to differentiate in vitro and treated with human ANP. The expression of osteoblast differentiation-related genes (such as Runx2 and Bmp2) was detected 7 days after induction, ALP staining was performed, and ALP activity was detected. Alizarin red staining was performed on the 14th day, and the area of ​​mineralized nodules was counted.

[0064] Effect of ANP on osteogenic differentiation of pre-osteoblast cell line MC-3T3E1 cells: MC3T3-E1 cells were seeded into 6-well plates. When the cell confluence reached 50-60%, the α-MEM complete medium containing osteogenic inducing agents was replaced. ANP (AS-20648, Anaspec) was added at a concentration of 100 nM to the ANP treatment group, and the medium was changed every other day. ALP staining and ALP activity detection were performed at 7 days of osteogenic induction. Alizarin red staining was performed at 14 days of induction. The ALP staining and Alizarin red staining methods were the same as before.

[0065] ALP activity detection:

[0066] After 7 days of cell differentiation induction, the culture medium was removed, lysis buffer (without protease inhibitors) was added, and the cells were centrifuged at 12000g for 10 min at 4°C. The supernatant was taken and the ALP activity was detected using the Bio-Tech Alkaline Phosphatase Activity Assay Kit (Cat. No.: P0321S).

[0067] result: Figure 4 A results showed that after 7 days, ANP stimulation significantly upregulated the expression of osteogenic marker genes Bmp2, Runx2, Col1a1 and Alp in MC3T3-E1 cells; Figure 4 B and 4C show that ANP significantly increased the alkaline phosphatase staining, viability and alizarin red staining of MC3T3-E1 cells. These results suggest that ANP promotes the upregulation of osteogenic differentiation-related genes, significantly increases ALP activity, and enhances the formation of mineralized nodules. This indicates that ANP can promote the differentiation of osteogenic precursor cells into osteoblasts and increase mineralization capacity.

[0068] In combination with the above examples, it can be seen that atrial natriuretic peptide ANP, as a new candidate factor for treating osteoporosis, can improve osteoporosis by promoting osteogenic differentiation of osteoblast precursor cells.

[0069] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0070] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Application of atrial natriuretic peptide (ANP) in the preparation of drugs for promoting the differentiation of osteoblast precursor cells into osteocytes.

2. The use according to claim 1, characterized in that The atrial natriuretic peptide ANP is a peptide hormone with a ring structure consisting of 28 amino acids, and its amino acid sequence is SEQ ID NO:

1. The cysteine ​​residues at positions 7 and 23 form a disulfide bond, and the molecular weight is 3080.

46.

3. The use according to claim 1, characterized in that: The osteoblast precursor cells are mesenchymal stem cells or osteoblast precursors.

4. The use according to claim 1, characterized in that The medicine is an injection, a sustained-release capsule or an implanted sustained-release material.

5. A method for constructing a differentiation model of pre-osteoblast cell line MC3T3-E1 cells, characterized in that: The construction method comprises the following steps: MC3T3-E1 cells are inoculated into a 6-well plate, and when the cell growth confluence reaches 50-60%, an α-MEM complete culture medium containing an osteogenic inducing agent is replaced; then ANP with a concentration of 100 nM is added for culture, and the medium is changed every other day, and then ALP staining and ALP activity detection are performed on day 7 of osteogenic induction, and Alizarin red staining is performed on day 14 of induction to detect whether the differentiation model of the pre-osteoblastic cell line MC3T3-E1 cells is completed.

6. The method for constructing the pre-osteoblast cell line MC3T3-E1 cell differentiation model according to claim 5, characterized in that: The components of the α-MEM complete culture medium containing osteogenic inducing agent include: 10 mmol / L β-glycerophosphate, 50 μg / mL L-ascorbic acid, and 10 nM dexamethasone.

7. The method for constructing the pre-osteoblast cell line MC3T3-E1 cell differentiation model according to claim 5, characterized in that: The steps of ALP staining are as follows: wash the MC3T3-E1 cells induced to differentiate for 7 days with 1×PBS buffer, fix with 4% paraformaldehyde for 10 minutes, add BCIP / NBT staining working solution, and incubate at room temperature in the dark for 20-30 minutes; wash the cells three times with deionized water; air dry, store at room temperature in the dark; and take pictures and record.

8. The method for constructing the pre-osteoblast cell line MC3T3-E1 cell differentiation model according to claim 5, characterized in that: The steps of ALP activity detection are as follows: after 7 days of induction and differentiation culture of MC3T3-E1 cells, the culture medium is removed, lysis buffer without protease inhibitors is added, centrifuged at 12000g for 10 min at 4°C, the supernatant is taken, and ALP activity is detected using an alkaline phosphatase activity detection kit.

9. The method for constructing the pre-osteoblast cell line MC3T3-E1 cell differentiation model according to claim 5, characterized in that: The steps of Alizarin Red staining are as follows: after 14 days of induction and differentiation culture of MC3T3-E1 cells, the cells were washed with 1×PBS buffer, fixed with 4% paraformaldehyde for 10 minutes, added with 2% Alizarin Red dye solution, incubated at room temperature in the dark for 20 minutes, washed with PBS, air-dried, stored at room temperature in the dark, and photographed to record the formation of calcium nodules.

10. A drug for promoting differentiation of osteoblast precursor cells into osteocytes, characterized in that: The medicine contains atrial natriuretic peptide ANP.

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