WO2022041311A1 - Application of 2-bromopalmitic acid in preparation of drug for prevention and treatment of bone loss-related disease - Google Patents

Abstract

The present invention relates to the technical field of medicine, and discloses an application of 2-bromopalmitic acid (2-BP) in preparation of a drug for prevention and treatment of a bone loss-related disease. In the present invention, by means of multiple experimental methods such as tartrate-resistant acid phosphatase staining, phalloidin fluorescent staining, bone plate bone resorption evaluation, reverse transcription real-time fluorescence-based quantitative PCR, and western blotting, it is made clear for the first time that 2-BP can inhibit an osteoclast differentiation process when BMMs are stimulated by RANKL. Moreover, by constructing an ovariectomized mouse osteoporosis model, it is made clear that 2-BP also has the function of inhibiting osteoclast differentiation and activation in vivo, and can significantly alleviate the pathological process of a large bone loss caused by estrogen deficiency; a in vivo experimental data basis for 2-BP in prevention and treatment of bone loss-related diseases is provided.

Description

Application of 2-bromopalmitic acid in the preparation of medicines for preventing and treating bone loss related diseases technical field

The invention relates to the application of 2-bromopalmitic acid in the preparation of a medicine for preventing and treating bone loss-related diseases, and belongs to the technical field of medicine.

Background technique

As an important part of the motor system, bone provides structural support and protection for the body, and participates in physiological processes such as hematopoiesis, calcium metabolism, and endocrine regulation in the body. Bone remodeling occurs throughout human life, and most diseases related to bone metabolism affect the process of bone remodeling. Bone remodeling in normal body depends on the direct dynamic balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The imbalance between the two will lead to excessive bone formation and sclerosis-related diseases. Or bone insufficiency, excessive resorption related diseases. Osteoporosis (Osteoporosis) is a systemic bone disease that leads to the decrease of bone density and bone quality, the destruction of bone microstructure, and the increase of bone fragility, which is prone to fracture. It is closely related to the aging of the body. In 2006, there were nearly 70 million osteoporosis patients in my country, and more than 200 million people with osteopenia. Although the latest epidemiological data is lacking, it is estimated that the number of osteoporosis and osteopenia in my country has far exceeded the above figures.

The serious consequence of osteoporosis is the occurrence of osteoporotic fractures, which is one of the main causes of disability and death in elderly patients. Epidemiological surveys based on imaging show that the prevalence of vertebral fractures in women over the age of 50 in my country is about 15%, and the prevalence of vertebral fractures increases with age after the age of 50. The incidence of osteoporotic vertebral fractures can be as high as 36.6%; while hip fractures are the most serious osteoporotic fractures, studies have shown that within 1 year after hip fractures, 20% of patients will die of various complications, About 50% of the patients are disabled, the quality of life is significantly reduced, and a heavy burden is placed on the family and society. In addition, osteoclasts and their mediated bone resorption not only play an important role in the occurrence and development of osteoporosis, but also play an important role in bone loss diseases such as periprosthetic osteolysis, rheumatoid osteoarthritis, and tumor bone metastasis. It is also crucial in the prevention and treatment of such diseases.

At present, the prevention of osteoporosis mainly includes adjustment of lifestyle, calcium and vitamin D, and its treatment mainly focuses on inhibiting osteoclast-mediated bone resorption or/and promoting osteoblast-mediated bone formation. Bisphosphonates are the most widely used anti-osteoporosis drugs in clinical practice. They have high affinity with skeletal hydroxyapatite and can specifically bind to the bone surface with active bone remodeling, inhibiting the function of osteoclasts and thus inhibiting Bone resorption. However, the use of bisphosphonates is associated with gastrointestinal adverse effects, transient "flu-like" symptoms, renal toxicity, and the risk of osteonecrosis of the jaw and atypical femoral fractures. Anti-osteoporosis drugs such as calcitonin, estrogen receptor modulators (raloxifene), menopausal hormone therapy, parathyroid hormone analogs (teriparatide), and RANKL (denosumab) are all available. There are different indications, usage, dosage, side effects, and some drugs are expensive and difficult to be widely used. Therefore, it is particularly important to develop new drugs aimed at inhibiting the differentiation and maturation of osteoclasts.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides an application of 2-bromopalmitic acid in the preparation of a medicine for preventing and treating bone loss-related diseases.

The first object of the present invention is to provide an application of 2-bromopalmitic acid in the preparation of a medicine for preventing and treating bone loss-related diseases.

Further, the bone loss-related disease is osteoporosis, wear particle-mediated osteolysis, rheumatoid arthritis or tumor bone destruction.

Further, the medicine for preventing and treating bone loss-related diseases is a medicine for inhibiting the differentiation and maturation of osteoclasts.

Further, the medicine for preventing and treating bone loss-related diseases is a medicine for inhibiting the expression level of characteristic genes in the process of osteoclast differentiation.

Further, the characteristic genes include one or more of Nfatc1, C-fos, Ctsk, Acp5, Oscar, Dc-stamp, Atp6v0a3, and Atp6v0d2.

Further, the drug for preventing and treating bone loss-related diseases is a drug for inhibiting the activation of the c-Fos/NFATc1 signaling pathway in the process of osteoclast differentiation.

Further, the dosage forms of the medicine for preventing and treating bone loss-related diseases are injections, capsules, oral preparations, and microcapsule preparations.

Further, the dosage of the medicine for preventing and treating bone loss-related diseases is 1-10 mg/kg.

Beneficial effects of the present invention:

The present invention utilizes various experimental methods such as tartrate-resistant acid phosphatase staining, phalloidin fluorescent staining, evaluation of bone resorption function of bone plate, reverse transcription real-time fluorescent quantitative PCR, western blotting and other experimental methods, and for the first time it is clear that 2-BP can inhibit BMMs in RANKL. Osteoclast differentiation process under stimulation. And by constructing a mouse model of ovariectomized osteoporosis, it is clear that 2-BP also has the function of inhibiting the differentiation and activation of osteoclasts in vivo, which can significantly alleviate the pathological process of a large number of bone loss caused by estrogen deficiency, and provide a mechanism for preventing and treating bone loss. Loss of the in vivo experimental data base for related diseases.

Description of drawings

Figure 1 shows the cytotoxicity of 2-BP on monocytes and macrophages detected by CCK-8 method;

Figure 2 shows the effect of 2-BP on the osteoclast differentiation of monocyte-macrophages detected by tartrate-resistant acid phosphatase staining;

Figure 3 shows the effect of 2-BP on the production of the characteristic F-actin ring during the osteoclast differentiation of monocyte-macrophages detected by fluorescent staining of phalloidin;

Figure 4 is a reverse transcription real-time fluorescence quantitative PCR detection of the expression of related characteristic genes in the process of osteoclast differentiation;

Figure 5 shows the effect of 2-BP on the expression of transcription factors related to the osteoclast differentiation process detected by western blot;

Figure 6 is a three-dimensional image of tibial cancellous bone and analysis of related bone mass parameters.

detailed description

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

Example 1: 2--BP inhibits osteoclast differentiation without obvious cytotoxicity

(1) Isolation and culture of primary bone marrow-derived mononuclear macrophages BMMs

Take one C57BL/6 mouse aged 5 to 8 weeks, immerse it in 75% medical alcohol for 3 minutes after pulling the neck, dissect the lower limbs of the mouse in an ultra-clean workbench, and separate the skin, fascia and joint capsule of the knee and hip joints. Mouse femurs and tibias were obtained and placed in bacterial culture dishes to which sterile phosphate buffered saline was previously added. Use sterile scissors to cut the upper end of the femur and the lower end of the tibia, separate the bone marrow by transient centrifugation at 10,000 rpm, add 2 ml of red blood cell lysis buffer to resuspend the bone marrow, and let stand for 5 minutes to lyse the red blood cells. Then centrifuge horizontally at room temperature (1000rpm, 5 minutes), discard the liquid, add 10ml of complete Alpha MEM medium containing 30ng/ml M-CSF, mix well by gentle pipetting, transfer to a 10cm sterile cell culture dish, and store at 37°C. , Incubate in a 5% CO ₂ incubator. On the 2nd day, the medium was changed to remove the non-adherent foreign cells, and the culture was continued until the bone marrow-derived mononuclear macrophages grew to a density of more than 80%.

(2) Digestion and enumeration of primary bone marrow-derived mononuclear macrophages

Discard the medium, wash twice with sterile phosphate-buffered saline, add 3 ml of trypsin for 10 minutes, and add 1 ml of serum-containing medium when the cells are slightly shrunken under the microscope. Horizontal centrifugation (1000 rpm, 5 minutes). Discard the supernatant, add an appropriate amount of serum-containing medium to resuspend the cells, and mix by gently pipetting; at the same time, take 100 μl of the cell suspension to an EP tube, add 1900 μl of 0.4% trypan blue staining solution, observe under a microscope within 3 minutes, and count out Count the number of cells in the 4 large compartments of the plate that are not stained with the dye solution. The number of cells in the cell suspension/ml=(the total number of cells in 4 large cells/4)×dilution factor (20 times)×10 ⁴ . (Note: Dead cells will be stained blue, live cells will not be stained)

(3) The cytotoxicity of 2-BP on monocytes and macrophages was detected by CCK-8 method (Fig. 1)

Mononuclear macrophages were seeded into 96-well plates with a cell density of 5×10 ³ cells/well, and each plate had a total of 24 wells (1 control group, 7 drug-added groups with different concentrations, 3 duplicate wells in each group), The highest concentration of 2-BP was 200 μM; the medium was complete alpha MEM containing 30 ng/ml M-CSF, cultured for 48, 72, and 96 hours, respectively, and the medium was changed every other day. After the corresponding time point, add 10 μl of CCK-8 reagent to each well, incubate it in a cell incubator for 1 hour in the dark, take it out, shake it on the microplate reader and measure the absorbance at 450 nm (the more active cells, the higher the absorbance). The experimental results showed that the concentration of 2-BP below 200 μM had no obvious inhibitory effect on the proliferation of monocyte-macrophages.

(4) The effect of 2-BP on the osteoclast differentiation of monocyte-macrophages was detected by tartrate-resistant acid phosphatase staining (Fig. 2)

The control group and 3 drug-treated groups were set up, each group had 3 replicate wells, the mononuclear macrophages were seeded in 24-well plates (10×10 ⁴ cells/well), and the medium was changed to containing 50ng/ml every other day. Complete Alpha MEM medium of RANKL and 30ng/ml M-CSF, 6.25, 12.5 and 25 μM 2-BP were added to the drug treatment group respectively, and the control group was added with the same amount of dimethyl sulfoxide as the 25 μM 2-BP group. , the medium was changed every other day and cultured for 6 days. When the control group had obvious mature and hypertrophic osteoclasts, it was terminated. After the medium was aspirated, the phosphate buffered saline solution was washed once, and 500ul/well of 4% paraformaldehyde was added to fix it. Cells were stained with tartrate-resistant acid phosphatase for 20 minutes using the kit, photographed under an inverted microscope, and the number of osteoclasts with more than 3 nuclei, positive tartrate-resistant acid phosphatase staining and the spreading area of each osteoclast were calculated later. It is clear that 2-BP has an inhibitory effect on osteoclast differentiation. The experimental results showed that the number of osteoclasts and the area of each osteoclast were significantly reduced under the treatment of 6.25μM 2-BP, the degree of inhibition was more obvious under the treatment of 12.5μM, and the differentiation of osteoclasts was basically not formed under the treatment of 25μM.

(5) The effect of 2-BP on the production of the characteristic F-actin ring during the osteoclast differentiation of monocyte-macrophages was detected by fluorescent staining of phalloidin (Fig. 3)

The control group and 3 drug-treated groups were set up, each group had 3 replicate wells, the mononuclear macrophages were seeded in 24-well plates (10×10 ⁴ cells/well), and the medium was changed to containing 50ng/ml every other day. Complete Alpha MEM medium of RANKL and 30ng/ml M-CSF, 6.25, 12.5 and 25 μM 2-BP were added to the drug treatment group respectively, and the control group was added with the same amount of dimethyl sulfoxide as the 25 μM 2-BP group. , the medium was changed every other day and cultured for 6 days. When the control group had obvious mature and hypertrophic osteoclasts, it was terminated. After the medium was aspirated, the phosphate buffered saline solution was washed once, and 500ul/well of 4% paraformaldehyde was added to fix it. Cells were incubated for 20 minutes, washed twice with phosphate-buffered saline, and then drained. Add 300 μl/well of 0.1% Triton X-100 and incubate for 5 minutes, wash twice with phosphate buffered saline, and then drain, incubate with 300 μl/well of 1% bovine serum albumin for 5 minutes and then drain, add 1% phalloidin and incubate for 20 min, after exhaustion, DAPI counterstaining, finally removing the staining solution and adding phosphate buffered saline solution, and taking pictures under an inverted fluorescence microscope. The experimental results suggest that 2-BP can inhibit the formation of characteristic F-actin ring during the osteoclast differentiation of monocyte-macrophages in a concentration-dependent manner.

Example 2: 2-BP inhibits the expression of related characteristic genes during osteoclast differentiation

(1) Cell processing and RNA collection and isolation

The control group and 3 drug-treated groups were set up, each group had 3 replicate wells, and the monocyte-macrophages were seeded in 6-well plates (40×10 ⁴ cells/well), and the medium was changed to containing 50ng/ml every other day. Complete Alpha MEM medium of RANKL and 30ng/ml M-CSF, 6.25, 12.5 and 25 μM 2-BP were added to the drug treatment group respectively, and the control group was added with the same amount of dimethyl sulfoxide as the 25 μM 2-BP group. , the medium was changed every other day, cultured for 6 days, and terminated when the control group had obvious mature and hypertrophic osteoclasts. The total RNA was extracted with an RNA extraction kit, and after the concentration was determined, 500 ng of RNA was taken from the reaction system to synthesize cDNA. The reaction system includes reverse transcription buffer, primers, dNTPs, reverse transcriptase, DEPC water, and RNA template (total volume 10 μl). After the synthesis, the samples were diluted with ultrapure water to 100 μl and stored in a -80°C refrigerator for later use.

(2) Reverse transcription real-time fluorescence quantitative PCR to detect the expression of related characteristic genes in the process of osteoclast differentiation (Figure 4)

A 20 μl reaction system was used for reverse transcription real-time fluorescent quantitative PCR, and 2 μl of cDNA, 0.25 μl of upstream and downstream primers, 10 μl of SYBR Green qPCR Mix, and 7.5 μl of ddH2O were added to each system. Perform the following reaction conditions on the Bio-Rad CFX96 instrument: pre-denaturation at 95°C for 3 minutes, then cycle 40 times at 95°C for 10 seconds and 60°C for 30 seconds, and judge whether the reaction of each system is normal according to the melting curve, and finally use fluorescence quantitative PCR The software provided by the instrument analyzes the results to obtain the CT value, and uses the 2 ^-ΔΔCT method to analyze and compare the mRNA expression levels of the genes directly corresponding to each sample and perform statistical analysis. The experimental results showed that the expression of osteoclast-related characteristic genes Nfatc1, c-Fos, Ctsk, Acp5, Oscar, Dc-stamp, Atp6v0a3, Atp6v0d2 decreased in a concentration-dependent manner with the increase of 2-BP concentration.

Example 3: 2-BP inhibits the expression of important downstream transcription factors NFATc1 and c-Fos in the signaling pathway during osteoclast differentiation

(1) Protein sample processing and extraction

The mononuclear macrophages were seeded in 6-well plates (40×10 ⁴ cells/well, 30ng/ml M-CSF in complete Alpha MEM medium), and divided into control group and drug-treated group. After overnight, the medium was changed to Complete Alpha MEM medium containing 50ng/ml RANKL and 30ng/ml M-CSF, 25μM 2-BP was added to the drug treatment group, and the control group was added with the same amount of dimethyl sulfoxide solvent, and the medium was changed every other day, respectively. Protein samples were collected on days 0, 1, 3, and 5.

Extraction and concentration determination of protein samples: After culturing to the corresponding time point, the supernatant was quickly discarded, washed once with phosphate buffered saline at 4°C, and the 6-well plate was placed on ice. 100 μl of RIPA cell lysate containing enzyme inhibitor, fully pipetted and lysed on ice for 20 minutes, transfer the lysate to a 1.5 ml EP tube, pre-cool the centrifuge at 4°C in advance, centrifuge at 13,000 rpm for 15 minutes, and collect the supernatant into a new EP tube , use the BCA protein concentration kit to measure the protein concentration of each sample, and store it in a -80 ℃ refrigerator.

(2) Western blotting to detect the effect of 2-BP on the expression of transcription factors related to osteoclast differentiation (Figure 5)

Western blot was used to detect the protein expression changes of NFATc1 and c-Fos transcription factors during the osteoclast differentiation of long-term protein samples to further verify the role of 2-BP in regulating osteoclast differentiation. The experimental results showed that 2-BP could inhibit osteoclast differentiation by affecting the expression levels of NFATc1 and c-Fos transcription factors under RANKL stimulation.

Example 4: 2-BP prevents and treats osteoporosis induced by ovariectomy in mice

(1) Drug dissolution, grouping of mice and post-treatment of OVX modeling: 20% DMSO+80% PBS solvent was prepared, and 2-BP was added to prepare a solution with a final concentration of 1 mg/ml. Twenty-eight C57BL/6 mice were randomly divided into 4 groups with 7 mice in each group. The blank control group was bred routinely. The positive OVX control group was given intraperitoneal injection of the above-mentioned solvent only after successful modeling. After successful modeling, intraperitoneal injection of 2-BP 2.5 mg/kg and 5 mg/kg was given once a day, respectively.

(2) Ovariectomized osteoporosis model in OVX mice: after anesthesia in the sham-operated group, a longitudinal incision was made on the back, and the subcutaneous fascia and muscle were bluntly separated, and the muscle was cut at the projection of the left and right dorsal kidneys. Muscle, fascia, and skin are sutured. The OVX control group, the low-concentration drug group and the high-concentration drug group underwent modeling surgery. After successful anesthesia, a longitudinal incision was made on the back under sterile conditions, and the subcutaneous fascia and muscles were bluntly separated, and cut at the projection of the left and right dorsal kidneys. Muscle, the fallopian tube and ovary were clipped, and the ovary was cut off after ligation below the ovary, and the muscle, fascia, and skin were sutured. After 4 weeks of spraying, they were sacrificed by dislocation of the neck. The tibia was dissected and fixed in 4% paraformaldehyde for 2 days, and then stored in 75% alcohol.

(3) Micro-CT scans the skulls of each group and obtains three-dimensional images of tibial cancellous bone through 2D construction, 3D reconstruction and other operations, and analyzes related bone mass parameters. The results showed that the OVX control group had obvious osteopenia phenotype compared with the sham-operated group, and the model was successfully constructed. At the same time, the osteopenia in the low-concentration drug group and the high-concentration drug group were significantly improved compared with the OVX control group. 2-BP The process of bone loss caused by ovariectomy can be significantly alleviated (Figure 6).

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims (8)

1. An application of 2-bromopalmitic acid in the preparation of a medicine for preventing and treating bone loss-related diseases.
2. The use according to claim 1, wherein the bone loss-related disease is osteoporosis, wear particle-mediated osteolysis, rheumatoid arthritis or tumor bone destruction.
3. The application according to claim 1, wherein the medicine for preventing and treating bone loss-related diseases is a medicine for inhibiting the differentiation and maturation of osteoclasts.
4. The application according to claim 1, wherein the medicine for preventing and treating bone loss-related diseases is a medicine for inhibiting the expression level of characteristic genes in the process of osteoclast differentiation.
5. The application according to claim 4, wherein the characteristic gene comprises one or more of Nfatc1, C-fos, Ctsk, Acp5, Oscar, Dc-stamp, Atp6v0a3, and Atp6v0d2.
6. The application according to claim 1, wherein the drug for preventing and treating bone loss-related diseases is a drug for inhibiting the activation of the c-Fos/NFATc1 signaling pathway during osteoclast differentiation.
7. The application according to claim 1, wherein the dosage form of the medicine for preventing and treating bone loss-related diseases is injection, capsule, oral preparation, and microcapsule preparation.
8. The application according to claim 1, wherein the dosage of the medicine for preventing and treating bone loss-related diseases is 1-10 mg/kg.

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