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

doi:10.4252/wjsc.v17.i11.112393

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Nov 26, 2025 (publication date) through Nov 26, 2025

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World Journal of Stem Cells

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1948-0210

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World J Stem Cells. Nov 26, 2025; 17(11): 112393
Published online Nov 26, 2025. doi: 10.4252/wjsc.v17.i11.112393

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

Yi-Jun Tu, Yun-Qi Liu, Yan-Yan Pan, Hong-Yang Cai, Chang Liu

Yi-Jun Tu, Yun-Qi Liu, Yan-Yan Pan, Hong-Yang Cai, Chang Liu, Central Hospital of Dalian University of Technology, Dalian 116033, Liaoning Province, China

Author contributions: Tu YJ performed the experiments and acquired the data; Tu YJ and Liu C interpreted the data; Liu YQ, Pan YY, and Cai HY helped perform the experiments; Liu C provided the conception, designed the research, drafted, and revised the manuscript. All authors have read and approved the final submitted manuscript.

Supported by the Department of Science & Technology of Liaoning Province, No. 2024-MS-285; and National Natural Science Foundation of China, No. 82303572.

Institutional animal care and use committee statement: The animal protocol was reviewed and agreed by the Ethic Committee of Central Hospital of Dalian University of Technology, in accordance with the general rules for the welfare of laboratory animals (No. GB_T42011-2022). The animal protocol was designed to minimize pain or discomfort to the animals. The animals were acclimatized to laboratory conditions (23 °C, 12 hours/12 hours light/dark, 50% humidity, ad libitum access to food and water), for 2 weeks prior to experimentation. All animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for tissue collection.

Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.

ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.

Data sharing statement: The datasets generated and/or analyzed during the current study are available in the ﬁgshare repository (https://10.6084/m9.figshare.29486207).

Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/

Corresponding author: Chang Liu, PhD, Affiliate Associate Professor, Central Hospital of Dalian University of Technology, No. 826 Southwest Road, Dalian 116033, Liaoning Province, China. liuch2024@dlut.edu.cn

Received: July 31, 2025
Revised: August 18, 2025
Accepted: November 3, 2025
Published online: November 26, 2025
Processing time: 119 Days and 3.7 Hours

Abstract

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.

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

Core Tip: In our previous investigation, we demonstrated that micro-injured tendon with 6.4% strain promoted the chondrogenic differentiation of tendon-derived stem cells, by activating endoplasmic reticulum (ER) stress. To further validate this phenomenon in the animal model, we established a rat Achilles tendon injury model via treadmill running. In the present study, tendon injury was found to activate ER stress by upregulating the protein kinase RNA-like ER kinase/eukaryotic initiation factor 2/activating transcription factor 4/CCAAT/enhancer-binding protein homologous protein pathway. And thymoquinone was shown to alleviate ER stress, while inhibiting the chondrogenic differentiation of tendon-derived stem cells in both in vitro and in vivo models. These findings suggest that thymoquinone may hold potential as a preventive and therapeutic agent for tendinopathy.