CN115006382A - Application of myristic acid in preparation of medicine for resisting senile osteoporosis - Google Patents

Abstract

The invention relates to the field of pharmaceutical compounds, in particular to application of myristic acid in preparation of a medicine for resisting senile osteoporosis. The invention utilizes a mouse model of Senile Osteoporosis (SOP) constructed by a natural aging mouse to evaluate the influence and action mechanism of Myristic Acid (MA) on the Senile Osteoporosis (SOP). Experiments show that myristic acid can improve the bone mass of an SOP model mouse, promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) of the mouse, inhibit osteoclastic differentiation of bone marrow hematopoietic stem cells (BMMs) of the mouse, provide a new medicine selection for treating senile osteoporosis, and provide a new direction for research on treatment of the senile osteoporosis. The myristic acid has wide source, simple preparation process, safety as a medicament and no obvious toxic or side effect.

Description

Application of myristic acid in preparation of medicine for resisting senile osteoporosis

Technical Field

The invention relates to the field of pharmaceutical compounds, in particular to application of myristic acid in preparation of a medicine for resisting senile osteoporosis.

Background

With the accelerated aging of the world population, aging-related diseases such as Senile Osteoporosis (SOP) are increasingly developed year by year in various countries throughout the world. Senile osteoporosis, one of the major degenerative diseases of the elderly, has become an "invisible killer" around the elderly, and is easily overlooked at ordinary times due to the hidden symptoms of osteoporosis. Osteoporosis fracture is one of the most common and serious complications of Senile Osteoporosis (SOP), 1000 cases of osteoporosis fracture occur every hour all over the world, the disability rate is as high as 50%, the fatality rate is as high as 42%, and huge burden is brought to patients, families and socioeconomic performance. Therefore, senile osteoporosis has become an important health problem affecting the life of old people, and how to prevent and treat senile osteoporosis has become an important subject requiring long-term research of human beings. Although the existing anti-senile osteoporosis medicines can reduce the fracture risk to a certain extent, the medicines have limited effects and mostly have certain side effects, and no medicines for specifically improving senile osteoporosis exist. Therefore, the research and development of novel therapeutic drugs for resisting senile osteoporosis are urgent.

Myristic acid (MA, IUPAC, also known as myristic acid, molecular formula C) ₁₄ H ₂₈ O ₂ CAS #: 544-63-8) is a saturated fatty acid, mainly found in cardamom oil, coconut oil and palm kernel oil. Previous researches show that myristic acid can promote proliferation and differentiation of neural stem cells in vitro and improve cognitive function decline caused by aging. And the aging process is accompanied by a decrease in MA, which suggests MA is under treatmentCan play an important role in aging-related diseases. Myristic Acid (MA) can also inhibit the expression of toll-like receptor-2 (TLR2) on the cell membrane surface of mouse bone marrow-derived macrophages (BMDMs), and has an anti-inflammatory effect. In addition, myristic acid, isomer myristoleic acid, inhibits osteoclastogenesis and bone resorption by inhibiting RANKL activation. However, the effect of myristic acid on osteoclastogenic differentiation has not been reported in the prior art at present.

Disclosure of Invention

In order to solve the problems, on the basis of the existing research on treating neurodegenerative diseases by using myristic acid, the invention aims to provide the application of myristic acid in preparing the anti-senile osteoporosis medicine and provide a new direction for the research on treating senile osteoporosis.

In order to achieve the purpose, the invention adopts the following technical scheme.

In particular to application of myristic acid in preparing a medicine for resisting senile osteoporosis.

In some embodiments, the medicament may promote osteoblast differentiation, inhibiting osteoclast differentiation.

In some embodiments, the osteoblasts are bone marrow mesenchymal stem cells (BMSCs).

In some embodiments, the osteoclasts are bone marrow hematopoietic stem cells (BMMs).

In some embodiments, the drug may increase bone mass.

The invention also provides a medicine for treating senile osteoporosis, which comprises myristic acid and pharmaceutically acceptable auxiliary materials.

In some embodiments, the pharmaceutical dosage form is granules, capsules, tablets, solutions, pills, powders.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention innovatively discovers that Myristic Acid (MA) can improve the bone mass of a mouse model with Senile Osteoporosis (SOP), specifically improves the senile osteoporosis, can be used for preparing the anti-senile osteoporosis medicine, and provides a new effective medicine selection for treating the Senile Osteoporosis (SOP).

2. The invention innovatively discovers that Myristic Acid (MA) can promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro and inhibit osteoclastic differentiation of bone marrow hematopoietic stem cells (BMMs), provides a new research direction for treating senile osteoporosis, and develops a new treatment idea.

3. The compound myristic acid disclosed by the invention is wide in source, simple in preparation process, low in cost and convenient for large-scale production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of the results of bone mass of SOP model mice provided by intraperitoneal injection of myristic acid.

FIGS. 2 to 5 are graphs showing the results of the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) promoted by myristic acid.

FIGS. 6 to 10 are graphs showing the results of the inhibition of osteoclastic differentiation by myristic acid on bone marrow hematopoietic stem cells (BMMs).

Detailed Description

Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Myristic acid (Myristic acid), molecular formula: c ₁₄ H ₂₈ O ₂ CAS #: 544-63-8, has a molecular weight of 228.37, and is white to yellowish white hard solid, occasionally glossy crystalline solid, or white to yellowish white powder, and odorless. Myristic acid exists in vegetable oil such as cardamom oil (content is 70-80%), palm oil (content is 1-3%), coconut oil (content is 17-20%) in the form of glyceride in nature. Can be used for preparing various edible flavors, and also can be used as additive, metal processing and flavor solvent. The structural formula is as follows:

the beneficial therapeutic effect of myristic acid in the invention for specifically improving senile osteoporosis will be demonstrated by actual research data as follows:

data and method

Mice:

22-month-old C57BL/6 mice were used and purchased from the university of Chinese medicine, Guangzhou laboratory animal center (GZUCM) (animal Productivity certificate # (Yue) 20180034). All animal experiments were approved by the ethical committee of the first subsidiary hospital of the university of traditional Chinese medicine in Guangzhou (approval document TCMF 1-2021026). Commercially available 22-month-old mice were randomly divided into two groups: 22-month-old mice (22M, i.p. solvent equivalent) and MA (MA +22M, 2 mg/kg. d MA i.p.). The drug was administered by intraperitoneal injection for 2 months.

micro-CT detection

micro-CT is adopted to detect the density, the biomechanics, the bone microstructure and the morphological change of the lumbar vertebrae of the mouse: SOP mice were taken, and the tibia was dissociated and subjected to micro-CT scanning. The scanning range is 1mm below the tibial growth plate and 1.5mm near the tibia, 180 layers are scanned, the thickness of each layer is set to be 14 micrometers, and the voltage and the current are respectively set to be 55kV and 145mA during scanning. After scanning, micro-CT self-contained analysis software μ CT80 Evaluation Program (v6.5-1, Scanco Medical, Switzerland) is used for analysis, the region of interest is set to be a spongy bone region (with the center of the centrum in the vertebral body as the center) in a circular range with the diameter of 2mm in the tibia, and bone microstructure parameters of the region of interest are obtained.

Tartrate-resistant acid phosphatase (TRAP) staining

Reference to TRAP staining kit instructions, paraffin sections of tissue were routinely dewaxed, stained with TRAP solution containing sodium acetate, sodium tartrate, napthol-AS-TR-phosphate, N, N-dimethyl formamide, and fast red (Sigma Aldrich, Germany), incubated at 37 ℃ for 50min, counterstained with hematoxylin (Fa Shandon, Thermoscientific) for 30s, mounted with glycerogelatin (Merck) and observed under the microscope.

Extraction, culture and osteogenic differentiation induction of mouse BMSC

After anesthesia, the cervical vertebrae were removed and the mice were sacrificed and soaked in 75% alcohol for 5 min. The feet of the mice are clamped by bending forceps, the skin of the lower limbs is peeled off, and the lower limbs are immersed in 75% alcohol after hip joints and ankle joints are disconnected. And (5) rapidly transferring the lower limbs of the mice to a cell room super clean bench, and continuously cleaning the residual attached muscles of the mice. The femur or tibia is clamped with curved forceps, the shaft is removed with scissors, the medullary cavity is exposed, and a 10ml syringe is used to flush the medullary cavity into α -MEM containing 20% FBS until the medullary cavity is whitish. Repeatedly sucking and blowing bone marrow suspension with syringe to obtain single cell suspension, transferring to 25cm ² In the petri dish, the petri dish was cultured in an incubator at 37 ℃ with 5% CO 2. After the BMSCs (bone marrow mesenchymal stem cells) of P4 or P5 generation were grown to 90%, they were seeded in a 12-well plate (1X 104/cm) ² ) Inducing osteogenesis 80% after cell fusionAnd (4) performing ALP or alizarin red staining.

Mouse BMM extraction, culture, induced osteoclast differentiation

The obtained mouse hematopoietic bone marrow cells were purified using a 40 μ M cell filter, differentiated for 3 days with 50ng/ml mouse macrophage stimulating colony factor (M-CSF, R & D) in alpha-MEM medium of 10% Fetal Bovine Serum (FBS), and then differentiated for 5-7 days with 25ng/ml M-CSF and 50ng/ml RANKL (R & D), to obtain mature osteoclasts, stained for TRAP, and counted.

qPCR detection

Mouse BMSCs and BMMs were inoculated into 6-well plates, and the number of cells was 2X 10 ⁵ . After inducing osteoclast differentiation, 1mL TRIzol reagent was added to each well to extract total cellular RNA. Total RNA is extracted by a TAKARA MiniBEST Universal extraction kit, the concentration and purity of the RNA are analyzed by a Thermo full-wavelength microplate reader, a PrimeScript RTMaster Mix reverse transcription box carries out cDNA synthesis reaction, according to the operation steps of SYBR PremixEx TaqTM II, a Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument is used for carrying out two-step fluorescence quantitative analysis, the reaction conditions are set to 95 ℃ for 30s, 95 ℃ for 5s, 60 ℃ for 1min for extension, 40 cycles, and each sample is provided with 3 technical repeat holes.

Werstern blob detection

Mouse BMSCs, BMMs were seeded on 100mm dishes containing 2X 106 cells. After inducing osteoclast differentiation, 200. mu.L of RIPA lysis buffer (Beyotime) prepared from phosphatase inhibitor and protease inhibitor was added to extract protein at a final concentration of 4.0. mu.g/. mu.l, and 5-fold diluted protein sample buffer was added thereto at 95 ℃ for 5 minutes. Preparing gel according to the molecular weight of the target protein, and conventionally loading, electrophoresing and transferring the membrane. After membrane transfer, the membranes were completely immersed in 5% BSA-TBST and incubated for 6 hours at room temperature in a horizontal shaker for blocking. Primary antibody was diluted with 5% BSA-TBST and incubated overnight at 4 ℃ in a horizontal shaker. The following day, 3 membrane washes with TBST, and then incubation with the corresponding secondary antibody at room temperature for 40 min (1: 10000). After washing the membrane 3 times with TBST, ECL development and exposure of the film are carried out. Imaging by a Tanon-2500R type full-automatic digital gel imaging system, analyzing the gray value of a protein electrophoresis band by Scion Image software, calibrating by taking GAPDF as internal reference protein, and expressing the relative protein expression level of a target gene by adopting the gray value of the target protein/the gray value of the GAPDF band.

Grouping intervention and detection index

BMSC grouping and intervention:

control group: extracting BMSC of a control group mouse, and inducing osteogenic differentiation;

SOP group: extracting BMSCs of mice in an SOP group, and inducing osteogenic differentiation;

③ SOP + myristic acid group: extracting BMSCs of mice in the SOP group, inducing osteogenic differentiation and intervening with myristic acid;

BMM grouping and intervention:

control group: extracting BMM of a control group mouse, and inducing osteoclast differentiation;

SOP group: extracting BMM of mice in an SOP group, and inducing osteoclast differentiation;

③ SOP + myristic acid group: the SOP group mouse BMM was extracted, osteoclast differentiation was induced, and intervention with myristic acid was performed.

Detecting the growth and proliferation capacity of BMSC and BMM, the differentiation capacity of osteoblast and osteoclast, and detecting the marker gene and protein related to osteoclast and osteoblast.

Second, experimental results

All data were checked for normality and homogeneity of variance. All data are expressed as mean ± standard deviation. All data fit a normal cycle and are equally diverse. The independent sample t-test is used to compare two different samples. The comparison of univariate samples among groups was performed using one-way analysis of variance. Bivariate samples between groups were compared using two-way analysis of variance (ANOVA) (p <0.05 is statistically significant for differences).

The bone mass of SOP model mice treated with intraperitoneal myristic acid is shown in fig. 1: A. intraperitoneal injection of myristic acid into female and male mice, each group of micro-CT representative images; B. bone tissue of both female and male groups of mice was the BV/TV value. As is evident from the figure, the MA-treated mice in the experimental group have a significant improvement in bone mass as compared with the SOP model group.

The effect of myristic acid in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is shown in FIGS. 2-5, FIG. 2 is that CCK8 detects the effect of myristic acid on the proliferation of BMSCs, and the concentration of myristic acid in the case of not more than 100 [ mu ] mol/L neither promotes nor inhibits the proliferation of BMSCs; FIG. 3 shows the effect of myristic acid on promoting osteogenic differentiation of BMSCs, as measured by alkaline phosphatase (ALP) and Alizarin Red (ARS) staining; FIGS. 4 and 5 show the expression of osteogenic differentiation specific proteins RUNX2 and OPG detected by qPCR and Western-blot.

The effect of myristic acid on osteoclast differentiation inhibition of bone marrow hematopoietic stem cells (BMMs) is shown in fig. 6-10: FIG. 6 is a diagram of CCK8 testing the effect of myristic acid on the proliferation of BMMs, at concentrations not exceeding 100. mu. mol/L, with no promotion or inhibition of the proliferation of BMMs; fig. 7 and 8 show that the TRAP staining and bone pit formation experiments identify the influence of myristic acid on the osteoclastic differentiation and osteoclastic bone resorption capacity of BMMs, and the results indicate that the number of mature osteoclasts is obviously reduced and the bone resorption capacity is inhibited with the increase of the myristic acid concentration, thereby proving that myristic acid can inhibit the osteoclastic differentiation of BMMs. FIGS. 9 and 10 show the expression of the osteoclast differentiation specific proteins CTSK and TNF alpha detected by qPCR and Western-blot.

The results show that 2 mg/kg.d intraperitoneal injection of Myristic Acid (MA) can improve the bone mass of an SOP mouse after 2 months of administration, can promote osteogenic differentiation in vitro, inhibit osteoclastic differentiation, can obviously improve senile osteoporosis symptoms of an SOP mouse model, and can be used for preparing an anti-senile osteoporosis medicine.

In the description herein, references to the description of the term "one embodiment," "another embodiment," or "first through xth embodiments," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, method steps, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The above-mentioned serial numbers of the embodiments of the present invention are merely for description and do not represent the merits of the embodiments.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims

The present invention has been further described with reference to specific embodiments, but it should be understood that the detailed description should not be construed as limiting the spirit and scope of the present invention, and various modifications made to the above-described embodiments by those of ordinary skill in the art after reading this specification are within the scope of the present invention.

Claims (7)

1. Application of myristic acid in preparing medicine for treating senile osteoporosis is provided.

2. The use of claim 1, wherein the medicament promotes osteoblast differentiation and inhibits osteoclast differentiation.

3. The use of claim 2, wherein the osteoblast cells are mesenchymal stem cells.

4. The use of claim 2, wherein the osteoclast is a bone marrow hematopoietic stem cell.

5. The use of claim 1, wherein the medicament increases bone mass.

6. The medicine for treating senile osteoporosis is characterized by comprising myristic acid and pharmaceutically acceptable auxiliary materials.

7. The medicament of claim 6, wherein the medicament is in the form of granules, capsules, tablets, solutions, pills, powders.

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