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.

Up-regulation of CREB-1 regulates tendon adhesion in the injury tendon healing through the CREB-1/TGF-β3 signaling pathway

“…TGF-β3 upregulates mRNA and protein expression of Smad7 and downregulates Smad3 expression during the tendon healing process [ 30 ], thereby reducing scar formation and adhesion around the tendon. CREB-1, as a key downstream factor of TGF-β, may play a role in fibrosis processes in various tissues [ 13 , 34 , 35 ]. Studies have shown that CREB-1 overexpression can antagonize the fibrotic effects of TGF-β1 [ 36 ].…”

“…CREB, as a transcription factor, can mediate a variety of physiological signals in cells [ 11 ], allowing it to participate in a variety of cell reaction processes. Of course, it can also regulate tissue fibrosis [ 12 ], and research on tendon tissue has revealed that the inflammation response of the tendon and CREB is closely linked [ 13 ]. TGF-β and CREB both play roles in the regulation of the inflammation during the tendon healing [ 14 ].…”

An Atelocollagen Injection Enhances the Healing of Nonoperatively Treated Achilles Tendon Tears: An Experimental Study in Rats

“…The biomechanical properties were determined using published methodology for the biomechanical assessment of ATRs in rats. 7 , 17 , 33 , 47 , 53 , 65 Each tendon was harvested with the attached calcaneal bone and musculotendinous portion. 1 Assuming an elliptical cross-section of the tendon, the tendon cross-sectional area (CSA; mm 2 ) at the midportion of the tendon callus was calculated based on 2 perpendicular axes of thickness (major axis = a and minor axis = b ) as follows: CSA = (π× a × b )/4.…”

“…Load to failure (N), stiffness (N/mm), and stress (N/mm 2 ) were recorded. 33 , 42 , 50 , 53 Stiffness was plotted by measuring the linear slope of the load-displacement curve before achieving the maximum load to failure. 21 , 53 Stress was calculated as load to failure divided by the CSA.…”

“…For the histological examination, specimens were collected from sacrificed rats as follows. 33 , 53 A 5 × 15–mm tissue block consisting of the regenerated tendon and peritendinous tissue at 5 mm proximal to the calcaneal insertion was obtained and fixed for 24 hours in 4% paraformaldehyde diluted in phosphate-buffered saline (pH 7.4). Then, fixed tissue was dehydrated with increasing concentrations of alcohol, cleared in xylene, and embedded in paraffin wax.…”