Thymoquinone inhibited the chondrogenic differentiation of tendon-derived stem cells caused by tendon injury.

BACKGROUND

The micro-injury of collagen fibers occurs as the tendon is stretched repeatedly between the strains of 4% and 8%, which results in the cumulative micro-damage in tendon. In prior studies, we have shown that micro-injured tendon slices with 6.4% strain promoted the chondrogenic differentiation of tendon-derived stem cells (TDSCs) through the activation of endoplasmic reticulum (ER) stress.

AIM: To investigate the potential of thymoquinone (TQ) to alleviate ER stress, and, consequently, to suppress the chondrogenic differentiation of TDSCs.

METHODS

Decellularized tendon slices, subjected to micro-injury with 6.4% strain, were prepared for the culture of TDSCs. Additionally, a rat model of Achilles tendon injury via treadmill running was established. The expression levels of tenocyte and chondrocyte markers, along with ER stress-related factors, were examined in TDSCs cultured on micro-injured tendon slices, and in injured rat tendons, using reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining, and western blot analysis. Furthermore, the inhibitory effects of TQ on ER stress, and the chondrogenic differentiation of TDSCs, were evaluated.

RESULTS

In both TDSCs on micro-injured tendon slices, and injured rat tendons, tenocyte-related markers were downregulated, whereas chondrocyte-related markers were upregulated. Treatment with TQ significantly reduced the expression of ER stress markers, including glucose-regulated protein 78 (3.59 ± 0.41 vs 1.18 ± 0.23, P < 0.001), activating transcription factor 4 (2.67 ± 0.26 vs 1.16 ± 0.13, P < 0.001), CCAAT/enhancer-binding protein homologous protein (2.90 ± 0.37 vs 1.24 ± 0.35, P < 0.001), as well as phosphorylated protein kinase RNA-like ER kinase, and phosphorylated eukaryotic initiation factor 2, thereby attenuating ER stress. Furthermore, TQ diminished the chondrogenic differentiation of TDSCs, as evidenced by decreased expression of collagen II (4.80 ± 0.47 vs 1.38 ± 0.28, P < 0.001), aggrecan (2.83 ± 0.26 vs 1.44 ± 0.19, P < 0.001), and SOX9 (4.13 ± 0.46 vs 1.26 ± 0.25, P < 0.001), effects comparable to those observed with 4-phenylbutyric acid.

CONCLUSION

These findings suggested that TQ inhibited the protein kinase RNA-like ER kinase/eukaryotic initiation factor 2/activating transcription factor 4/CCAAT/enhancer-binding protein homologous protein signaling pathway to alleviate ER stress, thereby reducing the chondrogenic differentiation of TDSCs, both in vitro and in vivo.