Effects and Mechanism of Particulate Matter on Tendon Healing Based on Integrated Analysis of DNA Methylation and RNA Sequencing Data in a Rat Model

Abstract

Exposure to particulate matter (PM) has been linked with the severity of various diseases. To date, there is no study on the relationship between PM exposure and tendon healing. Open Achilles tenotomy of 20 rats was performed. The animals were divided into two groups according to exposure to PM: a PM group and a non-PM group. After 6 weeks of PM exposure, the harvest and investigations of lungs, blood samples, and Achilles tendons were performed. Compared to the non-PM group, the white blood cell count and tumor necrosis factor-alpha expression in the PM group were significantly higher. The Achilles tendons in PM group showed significantly increased inflammatory outcomes. A TEM analysis showed reduced collagen fibrils in the PM group. A biomechanical analysis demonstrated that the load to failure value was lower in the PM group. An upregulation of the gene encoding cyclic AMP response element-binding protein (CREB) was detected in the PM group by an integrated analysis of DNA methylation and RNA sequencing data, as confirmed via a Western blot analysis showing significantly elevated levels of phosphorylated CREB. In summary, PM exposure caused a deleterious effect on tendon healing. The molecular data indicate that the action mechanism of PM may be associated with upregulated CREB signaling.

Keywords: Achilles tendon; CREB signaling; DNA methylation; RNA sequencing; particulate matter; rat; tendon healing.

Figure 1

Evaluation of lung and systemic inflammation in particulate matter (PM)-exposed and control (non-PM) rats 6 weeks post-operation. (A) Gross and microscopic images of the lungs. Sections were stained with hematoxylin and eosin and observed under 10× and 400× magnification. The microscopic images revealed thickened interalveolar septa with increased numbers of lymphocytes and histiocytes. White arrow heads indicate the darkly spotted lesions in lungs of the PM group. Black arrow heads indicate the histiocytes in alveolar walls of the PM group. (B) White blood cell (WBC) and neutrophil densities in whole blood. (C) Serum levels of tumor necrosis factor-alpha (TNF-α). Data are presented as means ± SD. * p < 0.05, ** p < 0.005.

Figure 2

(A) Microscopic images of the Achilles tendons from native, control (non-PM), and PM-exposed rats 6 weeks post-operation. Additional images showing ossification (chondroid metaplasia in the marginal area and bone formation in the center) in the PM group are in the last row. Histological specimens were evaluated based on the modified Bonar score. The region with the greatest alteration in cell morphology was identified and evaluated. Images at 200× and 400× magnification of hematoxylin and eosin stain and 200× magnification of Masson’s trichrome stain and Alcian blue stain. Histological results showing the (B) total score, (C) cell morphology, (D) collagen fiber arrangement, (E) ground substance, (F) cellularity, and (G) vascularity of non-PM and PM groups. Data are presented as means ± SD. * p < 0.05, ** p < 0.005.

Figure 3

(A) Transmission electron microscopy (TEM) images of the collagen fibrils, (B) frequency of collagen fibril diameters, and (C) mean diameter of collagen fibrils in the Achilles tendons of control (non-PM) and PM-exposed rats 6 weeks post-operation. * p < 0.05.

Figure 4

(A–C) Gross images of the tendons in native, control (non-PM), and PM-exposed rats 6 weeks post-operation. (D) Biomechanical testing machine. (E) Cross-sectional area, (F) load to failure, (G) stiffness, and (H) stress outcomes. * p < 0.05.

Figure 5

(A) Kyoto Encyclopedia of Genes and Genome (KEGG) pathways associated with the significantly upregulated and downregulated differentially expressed genes (DEGs) between particulate matter (PM)-exposed and control (non-PM) rats (p < 0.05). (B) Canonical pathways with significant Z-scores (>2) identified using ingenuity pathway analysis. Heatmaps showing the DEGs in the (C) cAMP response element-binding protein (CREB) and (D) cAMP signaling pathways. (E) Schematic diagram showing the methods and criteria for gene analysis. (F) Venn diagram showing the combined DNA methylation and transcriptome data. The red circle indicates 67 genes exhibiting hypomethylated/upregulated expression. (G) The top five KEGG pathways associated with the hypomethylated/upregulated genes.

Figure 6

(A) Representative blots and (B) relative band intensities of cyclic adenosine monophosphate (cAMP), phosphorylated protein kinase B (p-Akt), phosphorylated cAMP response element-binding protein (p-CREB), and tumor necrosis factor-alpha (TNF-α). (C) Representative blots and (D) relative band intensities of collagen type I (COL1) and COL3 in the control (non-PM) and PM-exposed rats. The relative band intensity was normalized to β-actin. * p < 0.05, ** p < 0.01. (E) Proposed schematic diagram showing the action mechanism of PM exposure on tendon healing.

Figure 7

Overview of the experimental protocol. ELISA, enzyme-linked immunosorbent assay.

Figure 8

Methods followed for the PM exposure experiments. (A) Gross images of PM exposure using closed chambers and nebulizer systems. (B) Air inflow rate of the PM sample. (C) Dimensions of the closed chambers. (D) Simulations of the PM flow in the closed chamber 0, 20, 40, and 60 s after PM injection. d, diameter; w, width; l, length; h, height.