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.

Chondrogenic differentiation; Endoplasmic reticulum stress; Tendinopathy; Tendon-derived stem cells; Thymoquinone.