Macrophage Changes and High-Throughput Sequencing in Aging Mouse Intervertebral Disks

Abstract

Background: Intervertebral disk (IVD) degeneration is associated with lower back pain and aging; however, the mechanisms underlying age-related changes and the changes in macrophage polarization in aging intervertebral disks require further elucidation. The aim of this study was to evaluate changes in macrophages, the differential expression of senescence genes, and their relationship with hub genes in IVDs during aging in mice.

Methods: Twenty-eight male wild C57 mice aged 4 weeks were divided into two groups. Four mice per group were selected for high-throughput sequencing and 10 for tail IVD immunohistochemical analysis. Adult and aged mouse IVD specimens were stained with hematoxylin-eosin, Fast Green, and Alcian Blue to determine collagen (Col) 1, Col2, proteoglycan, P16, P21, P53, CD11b, CD86, CD206, IL-1, TGF-β, and IL-4 expression. High-throughput sequencing was performed on adult and aged mouse IVD tissues.

Results: Aged mouse IVDs showed reduced height and marked degeneration, with decreased Col2 and proteoglycan expression and increased Col1 expression. The expression of senescence markers, senescence-associated IL-1, TGF-β, and IL-4, and macrophage-related markers, CD11b, CD86, and CD206, increased markedly with age. High-throughput sequencing revealed 1975 differentially expressed genes in adult and aged mice, with 797 genes showing upregulated expression (top five: Kcna7, Mmp9, Panx3, Myl10, and Bglap) and 1178 showing downregulated expression (top five: Srd5a2, Slc38a5, Gm47283, Npy, and Pcdh8). Gene Ontology and pathway enrichment analyses highlighted aging-related cellular components, biological processes, and metabolic pathways. The identified hub genes included Cox5a, Ndufs6, and Ndufb9.

Conclusions: Disk senescence and reduced height in aged mice are linked to upregulated expression of senescence-associated phenotypes and macrophage polarization markers. These findings suggest that macrophages and differential gene expression play key roles in age-related IVD degeneration, indicating that they can be used as potential targets for therapeutic intervention.

Keywords: high‐throughput sequencing; intervertebral disk; macrophages; senescence.

FIGURE 1

| Mouse IVD images showing tissue staining and DR showing the disk height statistics. (A) Saffron O, Fast Green, and Alcian Blue staining (scale bar: 200 μm, magnification ×10); (B, C) DR and caudal vertebrae of adult and aged mice and height statistics; (D) IVD altered histological scoring statistic (*p < 0.05, ****p < 0.0001). Each point on the statistical chart represents a mouse.

FIGURE 2

| Expression of biomarkers related to senescence and the extracellular matrix in IVDs. Immunohistochemical staining for P16, P21, and P53, and proportion of positively stained cells per high‐power field to total cell count; immunohistochemical staining for Col1, Col2, and Agg, and average optical density; ****p < 0.0001, ***p < 0.001, **p < 0.01. Scale bar: 200 μm. IVD, intervertebral disk.

FIGURE 3

| Expression of macrophage, SASP, and inflammation‐related markers in IVDs. Immunohistochemical staining for CD206, CD11b, and CD86, and proportion of positively stained cells per high‐power field to total cell count; immunohistochemical staining for IL‐1, IL‐4, and TGF‐β, and average optical density; *p < 0.05, **p < 0.01, ****p < 0.0001. Scale bar: 200 μm. SASP, senescence‐associated secretory phenotype.

FIGURE 4

| Volcano plot and clustering chart of differentially expressed genes related to aging. (A) Volcano plot. Horizontal axis, log2 value of fold difference; vertical axis, negative logarithm p‐value. Red and blue points represent differentially expressed genes with upregulated and downregulated expression, respectively; gray points represent genes with no significant differences in expression during aging; (B) Clustering chart.

FIGURE 5

| GO enrichment statistical chart and pathway enrichment factor chart. (A) Statistical chart of the top 30 GO entries; (B) Pathway enrichment factor chart. GO, Gene Ontology.

FIGURE 6

| Protein–protein interaction network in aging mouse IVDs. Nodes represent hub genes and edges represent protein interactions. The size represents the rating, and the higher the score, the larger the node.