Modulation of EGR1 Expression by Hyperglycemia in Swine Rotator Cuff Tendons

Abstract

Diabetes mellitus is known to impair tendon structure and function, yet the molecular mechanisms linking hyperglycemia to tendon degeneration remain poorly understood. This study investigated the expression of early growth response-1 (EGR1) and its association with toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) signaling pathways in the rotator cuff tendons of hyperglycemic swine, a model chosen for its anatomical similarity to humans. Rotator cuff tendon tissues were collected from normal and hyperglycemic swine and analyzed using histology, qRT-PCR, Western blotting, and immunohistochemistry. Histological evaluation revealed altered tenocyte morphology and increased cellularity in hyperglycemic tendons. qRT-PCR results showed significant transcriptional upregulation of EGR1, TLR4, and NF-κB in hyperglycemic samples, suggesting activation of inflammatory and stress-response pathways. However, protein analysis revealed a non-significant decrease in EGR1 levels and modest increases in TLR4 and NF-κB, indicating possible post-transcriptional regulation. This discrepancy between mRNA and protein levels of EGR1 may be attributed to altered stress granule dynamics under hyperglycemic conditions. These findings elucidate a novel interplay among metabolic stress, innate immune signaling, and translational regulation in tendon tissue, proposing that targeting TLR4 signaling or stress granule formation may offer therapeutic potential for preserving tendon integrity in diabetic patients.

Keywords: Diabetes; Early growth response-1; Hyperglycemia; Inflammation; Rotator cuff tendon; Stress granules; Toll-like receptor.

Figure 1:

Representative hematoxylin and eosin (H&E) stained sections of rotator cuff tendons from normoglycemic (A) and hyperglycemic (B) swine. Black arrows indicate elongated tenocyte nuclei; arrowhead indicates rounded tenocyte nuclei; green arrows denote regions with absent or poorly visible nuclei; red arrows highlight areas of increased cellularity. Images are representative of all histology analyses for n = 6 (Normal swine) and n = 6 (Hyperglycemic swine).

Figure 2:

Quantitative real-time PCR (qRT-PCR) analysis of gene expression levels in rotator cuff tendons under hyperglycemic and normoglycemic conditions. (A) Expression of EGR1. (B) Expression of TLR-4. (C) Expression of NF-κB. Gene expression was normalized to 18S rRNA and calculated using the ^ΔΔCt method. Data are presented as mean ± SD. n = 6 for normal swine and n = 6 for hyperglycemic swine. *p < 0.05, **p < 0.01.

Figure 3:

Western blot analysis of protein expression in rotator cuff tendon lysates from normoglycemic and hyperglycemic swine. (A) Representative immunoblots for early growth response protein 1 (EGR1; 57 kDa), Toll-like receptor 4 (TLR4; 110 kDa), nuclear factor kappa B (NF-κB; 60 kDa), and 14–3-3-ζ (52 kDa) as a loading control. (B–D) Densitometric quantification of EGR1, TLR4, and NF-κB normalized to 14–3-3-ζ. Data are presented as mean ± SD. Sample sizes: n = 6 for normal swine and n = 6 for hyperglycemic swine.

Figure 4:

Immunohistochemical analysis of EGR1, TLR4, and NF-κB expression in RCT under normoglycemic and hyperglycemic conditions. (A, C, E) Representative IHC images of tendon tissue sections stained for EGR1 (A), TLR4 (C), and NF-κB (E) in normoglycemic (left) and hyperglycemic (right) groups. (B, D, F) Quantification of mean staining intensity for EGR1 (B), TLR4 (D), and NF-κB (F). Data are presented as mean ± SD. Images are representative of all IHC analyses for n = 6 for normal swine and n = 6 for hyperglycemic swine.