Myristic acid beneficially modulates intervertebral disc degeneration by preventing endplate osteochondral remodeling and vertebral osteoporosis in naturally aged mice
Data files
Mar 21, 2025 version files 1.12 GB
-
RAW_data.7z
1.12 GB
-
README.md
12.21 KB
Abstract
Background: The origin of intervertebral disc degeneration (IDD) is highly complex, where both cartilage endplate remodeling and vertebral osteoporosis are of utmost importance. Myristic acid (MA), a saturated fatty acid derived from nutmeg, a traditional Chinese herb, has been shown to boost memory. Additionally, its isomers have been verified to have antiosteoporotic characteristics. However, the precise mechanism by which MA functions in relation to IDD remains unclear.
Methods: In vivo, a natural aging animal model was used. The drug—administration method of MA was intraperitoneal injection to mice aged 22 months at a dose of 2 mg/kg·d for 2 months. Micro-CT observed vertebral bone mass and endplate changes, followed by Hematoxylin‒eosin (H&E), Masson, and Safranin-O staining of tissues. TRAP staining counted osteoclasts; immunohistochemistry detected the expressions of Aggrecan and Collagen II. Bioinformatics explored MA’s anti-IDD mechanism. In vitro, MA-treated senescent endplate chondrocytes (induced by TBHP) were analyzed by Real-Time PCR (qPCR) and immunofluorescence (IF) for senescence and matrix synthesis markers. TRAP and F-actin detected MA’s effect on RAW264.7 osteoclast differentiation (induced by RANKL); qPCR examined the expressions of osteoclast genes.
Results: Using the natural aging model, we found that MA tended to improve vertebral osteoporosis and endplate osteochondral remodeling, decreased the TRAP activity of the endplate, and alleviated IDD in naturally aging mice. Bioinformatics analysis suggested that the relationships among IDD, osteoporosis, and endplate degeneration were mainly linked to cellular senescence. In vitro, MA postponed the senescence of TBHP-induced endplate chondrocytes by increasing the expression of aggrecan and decreasing the expressions of MMP-3, MMP-9, and the senescence markers p16 and p21. Additionally, MA notably inhibited osteoclast activity, as evidenced by a decrease in the number of osteoclasts and a significant suppression of F-actin formation. At the molecular level, MA efficiently reduced the expressions of osteoclast marker genes like ACP-5, CTSK, and DC-STAMP.
Conclusion: The findings of this research suggest that MA is capable of inhibiting endplate osteochondral remodeling and vertebral osteoporosis, diminishing osteoclastogenesis to preserve bone mass, and consequently delaying IDD in naturally aging mice. Hence, MA holds the potential to serve as an alternative therapeutic approach for IDD.
https://doi.org/10.5061/dryad.vhhmgqp4x
Description of the data and file structure
Usage notes
The following is a list of different data types in this repository categorized by file suffixes, along with brief descriptions of how to handle these data types.
.pdf: Portable Document Format, which is a fixed-layout electronic document format. It can be viewed through dedicated readers such as Adobe Acrobat Reader and Foxit Reader. It can also be opened and printed by office software like Microsoft Word and LibreOffice Writer. Additionally, it can be edited, converted in format (such as to Word or Excel), and annotated with the help of online tools.
.png: Portable Network Graphics format, which is a lossless compressed bitmap image format and is often used to store images with a transparent background. It can be viewed with common image viewers like Windows Photo Viewer and Mac Preview. It can also be edited, such as resizing, cropping, and adding special effects, using image editing software like Adobe Photoshop and GIMP.
.jpg (.jpeg): Joint Photographic Experts Group format, which is a lossy compressed bitmap image format and is commonly used to store photos and complex images. It can be viewed through the system’s built-in image viewing tools (such as Windows Photo Viewer and Mac Preview) or third-party software (such as IrfanView and FastStone Image Viewer). It can also have its color, contrast, brightness, etc. adjusted in software like Adobe Photoshop and PaintShop Pro. Although lossy compression will result in data loss, it can balance file size and quality, which is beneficial for network transmission and storage.
.tif (.tiff): Tagged Image File Format, which is a flexible bitmap format. It supports lossless compression and various color modes and is often used to store high-quality images such as scanned documents, photos, and scientific images. It can be viewed and edited with professional software like Adobe Photoshop and GIMP, and can also be simply viewed with Windows Photo Viewer and Mac Preview.
.FCS (Flow Cytometry Standard): The flow cytometry standard format mainly stores the data obtained from flow cytometry experiments, including parameter information such as cell size, internal structure, and fluorescence labeling intensity. It needs to be viewed and analyzed with specialized software such as FlowJo and FCS Express. These software can visualize the data, display the distribution of cell subsets, and perform cell counting and statistical analysis, helping researchers understand the characteristics and functions of cells.
Files in the.pzfx format can be opened using GraphPad Prism 9 software. This software is a widely used tool for processing and analyzing scientific research data, drawing graphs, etc. With the functions of GraphPad Prism 9, in-depth analysis of the data inside, chart drawing, and various visualization operations can be carried out.
Bioinformatics analysis data
The “Bioinformatics analysis data” folder contains molecular docking diagrams of myristic acid with multiple proteins, results of GO and KEGG enrichment analyses, volcano plots, and classification clustering analysis diagrams related to intervertebral disc degeneration, endplate degeneration, and osteoporosis. The information of the files contained in it is as follows:
go_bubble: We conducted differential expression gene analysis on the datasets of the three diseases, namely “intervertebral disc degeneration”, “osteoporosis”, and “endplate degeneration”, and then carried out GO enrichment analysis of the differentially expressed genes.
heatmap1: A classification clustering analysis diagram of differentially expressed genes between the chondral endplate degeneration group and the normal group.
heatmap2: A classification clustering analysis diagram of differentially expressed genes between the intervertebral disc degeneration group and the normal group.
heatmap3: A classification clustering analysis diagram of differentially expressed genes between the SOP group and the normal group.
KEGG_bubble: We conducted differential expression gene analysis on the datasets of the three diseases, namely “intervertebral disc degeneration”, “osteoporosis”, and “endplate degeneration”, and then carried out KEGG pathway enrichment analysis of the differentially expressed genes.
volcano1: A volcano plot of differentially expressed genes between the chondral endplate degeneration group and the normal group.
Volcano2: A volcano plot of differentially expressed genes between the intervertebral disc degeneration group and the normal group.
Volcano3: A volcano plot of differentially expressed genes between the SOP group and the normal group.
ACAN-1: Obtain the protein structure in PDB format from the RCSB Protein Data Bank (http://www.rcsb.org/). After optimizing it with AutoDock software, perform molecular docking of MA and ACAN (PDB ID: 3B8Z), and finally visualize the docking results through PyMOL software (version 2.4.1).
ACAN-2: A partial enlarged view of ACAN-1.
MMP3-1: Obtain the protein structure in PDB format from the RCSB Protein Data Bank (http://www.rcsb.org/). After optimizing it with AutoDock software, perform molecular docking of MA and MMP3 (PDB ID: 4DPE), and finally visualize the docking results through PyMOL software (version 2.4.1).
MMP3-2: A partial enlarged view of MMP3-1.
MMP9-1: Obtain the protein structure in PDB format from the RCSB Protein Data Bank (http://www.rcsb.org/). After optimizing it with AutoDock software, perform molecular docking of MA and MMP9 (PDB ID: 1L6J), and finally visualize the docking results through PyMOL software (version 2.4.1).
MMP9-2: A partial enlarged view of MMP9-1.
P21-1: Obtain the protein structure in PDB format from the RCSB Protein Data Bank (http://www.rcsb.org/). After optimizing it with AutoDock software, perform molecular docking of MA and MMP9 (PDB ID: 5P21), and finally visualize the docking results through PyMOL software (version 2.4.1).
P21-2: A partial enlarged view of P21-1.
Data of the action mechanism diagram of MA
Mechanism diagram: A diagram illustrating the mechanism by which myristic acid (MA) retards intervertebral disc degeneration (IVDD) through inhibiting the remodeling of the chondral endplate and vertebral osteoporosis.
Data on the improvement of senescence of chondral endplate cells by MA
ACAN-IF: Immunofluorescence images of ACAN after myristic acid (MA) intervention in senescent chondral endplate cells. There are fluorescence images of three groups, namely the TBHP group, the 10 μmol MA group, and the 50 μmol MA group.
CCK8
MA-CCK8: Evaluate the cytotoxicity of MA to chondral endplate cells and screen out the appropriate drug concentration.
tbhp -cck8: Evaluate the optimal intervention concentration and intervention time of TBHP for creating a senescence model.
COL2A1-IF: Immunofluorescence images of COL2A1 after myristic acid (MA) intervention in senescent chondral endplate cells. There are fluorescence images of three groups, namely the TBHP group, the 10 μmol group, and the 50 μmol group.
galactose staining: Images of galactose staining of three groups, namely the TBHP group, the 10 μmol group, and the 50 μmol group, to verify that MA can inhibit the generation of senescent chondral endplate cells.
galactose straining-results：The statistical analysis results of the number of senescent positive cells among the TBHP group, the 10 μmol MA group, and the 50 μmol MA group.
IF and open field: It contains immunofluorescence images of ACAN and COL2A1 of chondral endplate cells, as well as images of chondral endplate cells under bright field background, aiming to verify that the cells we extracted are chondral endplate cells.
p16-IF: Immunofluorescence images of p16 after myristic acid (MA) intervention in senescent chondral endplate cells. There are fluorescence images of three groups, namely the TBHP group, the 10 μmol group, and the 50 μmol group.
PCR
MA-treat: The expression results of mRNA of MMP3, MMP9, ACAN, and P21 after myristic acid (MA) intervention in senescent chondral endplate cells.
model of aging: The expression results of mRNA of senescence-related factors and matrix of chondral endplate cells after intervention with 100 μmol of TBHP. The mRNA expression levels of ACAN, col2a1, MMP3, MMP9, MMP13, p16, p21, p53, TS5, and SOX-9 in the normal group and the senescent group.
MA-IF: The quantitative analysis data of immunofluorescence of ACAN, COL2A1, and P16 after myristic acid (MA) intervention in senescent chondral endplate cells.
Data of bone microparameters and three-dimensional reconstruction images from animal experiments
Coronal CT FIG: Micro-CT vertebral body reconstruction diagram of L4-5.
CT-1: Comparative analysis of the microscopic parameters of vertebral bones and analysis of the intervertebral disc height among the young group, the senescent group, and the MA group.
CT-2: Microstructural analysis of the chondral endplates among the young group, the senescent group, and the MA group.
MA-1: Three-dimensional reconstruction diagram of the L4 vertebral body in the young group.
MA-2: Three-dimensional reconstruction diagram of the sagittal L4/5 vertebral body and the upper endplate of the L4/5 intervertebral disc in the young group.
OLD-1: Three-dimensional reconstruction diagram of the L4 vertebral body in the senescent group.
OLD-2: Three-dimensional reconstruction diagram of the sagittal L4/5 vertebral body and the upper endplate of the L4/5 intervertebral disc in the senescent group.
Young-1: Three-dimensional reconstruction diagram of the L4 vertebral body in the MA group.
Young-2: Three-dimensional reconstruction diagram of the sagittal L4/5 vertebral body and the upper endplate of the L4/5 intervertebral disc in the MA group.
Data on the inhibition of osteoclast differentiation by MA
F-ACTIN: The staining result images and analysis results of F-actin for the MA group, the 10 μmol MA group, and the 50 μmol MA group.
trap-cell: The staining result images and analysis results of trap for the MA group, the 10 μmol MA group, and the 50 μmol MA group.
PCR-cell: Myristic acid (MA) inhibits the expression of osteoclast-related indicators.
Data of animal slice staining on MA delaying IVDD
HE: The HE staining results of the intervertebral discs of the young group, the senescent group, and the MA group, as well as the tissue scoring results of the related annulus fibrosus, nucleus pulposus, endplate boundary, and the overall intervertebral disc.
Immunohistochemistry: The immunohistochemical staining images and quantitative analysis results of ACAN and COL2A1 in the intervertebral discs of the young group, the senescent group, and the MA group.
MASSON: The MASSON staining results of the intervertebral discs of the young group, the senescent group, and the MA group, as well as the quantitative analysis results of the thickness of the upper and lower chondral endplates.
SO: The safranin-O and fast green staining images of the intervertebral discs of the young group, the senescent group, and the MA group, as well as the quantitative analysis results of the number of NP cells.
Staining results of the five organs: The HE staining results of the liver, heart, spleen, lung, and kidney of the young group, the senescent group, and the MA group.
Trap: The trap staining results of the intervertebral discs of the young group, the senescent group, and the MA group, as well as the statistical analysis of the number of trap-positive cells at the position of the chondral endplate.
Data for the identification of chondral endplate cells by flow cytometry
Flow cytometry: The quantitative analysis results of flow cytometry for chondral endplate cells, including the expression analysis results of CD45, CD90, and CD29.
This research dataset focuses on the physiological mechanism of naturally aging mice, and the content is as follows: 1. Micro-CT data: It includes all the relevant data of naturally aging mice in the article, such as the microscopic structure of the cartilaginous endplate bone, the fine structural parameters of the vertebral bone, as well as the staining results of multiple methods like immunohistochemistry (IHC), hematoxylin-eosin (HE) staining, safranin O-fast green staining, and tartrate-resistant acid phosphatase (TRAP) staining. These data can reflect the morphological characteristics of the skeletal tissues and the cellular features of the mice. 2. Cell experiment data: Firstly, it includes the data of PCR, immunofluorescence (IF), Cell Counting Kit-8 (CCK8) assay, and flow cytometry identification of cartilaginous endplate cells, which conduct multi-dimensional analysis from aspects such as gene expression, protein expression, cell viability, and cell population characteristics. Secondly, it involves the data of F-actin staining, PCR, and TRAP staining related to the osteoclast differentiation of RAW264.7 cells, which helps to understand the mechanism of osteoclast differentiation. 3. Bioinformatics data: It covers the data related to molecular docking, Gene Ontology (GO) functional enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway. It explores the biological significance behind the data from the molecular level and biological pathways, providing support for revealing the molecular mechanism of aging.