Letter to the Editor: Thymoquinone targets endoplasmic reticulum stress to rescue tendon stem cell identity - a new era for tendinopathy therapy

Abstract

Tendinopathy is a chronic and degenerative tendon disorder with limited therapeutic options. Recent insights into the cellular pathogenesis of tendinopathy have revealed that tendon-derived stem cells may aberrantly differentiate into chondrocytes under micro-injury-induced endoplasmic reticulum stress. In a recent study published by Tu et al in the World Journal of Stem Cells, thymoquinone - a bioactive compound from Nigella sativa - was demonstrated to inhibit the chondrogenic differentiation of tendon-derived stem cells both in vitro and in vivo, primarily through attenuation of the protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2/activating transcription factor 4/CCAAT/enhancer-binding protein homologous protein pathway. This article evaluates the significance of these findings within the broader field of tendon biology, highlights methodological strengths, and discusses future research directions for translating thymoquinone into a potential therapy for tendinopathy.

Key Words: Thymoquinone; Tendon-derived stem cells; Endoplasmic reticulum stress; Signaling pathway; Tendinopathy; Chondrogenic differentiation

Core Tip: This article critically analyzes the study by Tu et al, which demonstrated that tendon micro-injury activates endoplasmic reticulum (ER) stress signaling through the protein kinase RNA-like ER kinase/eukaryotic initiation factor 2/activating transcription factor 4/enhancer-binding protein homologous protein pathway, thereby promoting the chondrogenic differentiation of tendon-derived stem cells. Treatment with thymoquinone markedly alleviated ER stress and suppressed aberrant differentiation both in cultured cells and an in vivo rat model of treadmill-induced tendon injury. Collectively, these findings highlight thymoquinone as a promising therapeutic candidate for the prevention and treatment of tendinopathy by targeting ER stress-mediated cellular misdifferentiation.

TO THE EDITOR

Tendinopathy is a prevalent musculoskeletal disorder arising from the interplay of mechanical overload, microenvironmental disruption, and aberrant differentiation of tendon-derived stem cells (TDSCs)[1,2]. Tendon degeneration involves more than structural wear; it reflects dysregulated cellular stress responses and lineage decisions that drive fibrocartilaginous metaplasia[3]. Understanding the mechanisms that redirect TDSC fate is therefore central to advancing therapeutic strategies.

Recent work has highlighted the importance of paracrine communication and extracellular cues in shaping tendon cell identity. Mesenchymal stem cell-derived exosomes, for example, can modulate inflammation, matrix remodeling, and progenitor behavior[4]. Similarly, exosomes released from TDSCs promote repair through defined microRNA pathways and immune-matrix interactions[5], underscoring the sensitivity of tendon progenitors to both local biochemical signals and microenvironmental changes[6,7]. At the intracellular level, endoplasmic reticulum (ER) stress has emerged as a key regulator of stem cell fate[8]. Chen et al[9] in 2023 provide a comprehensive overview of the protein kinase RNA-like ER kinase (PERK)/eukaryotic initiation factor 2 (eIF2α)/activating transcription factor 4 (ATF4)/enhancer-binding protein homologous protein (CHOP) axis and its influence on differentiation programs, offering mechanistic context for Tu et al’s findings[10] published in the World Journal of Stem Cells that micro-injury-induced ER stress shifts TDSCs toward a chondrogenic phenotype. Together, these studies support the concept that ER stress represents a druggable node capable of redirecting pathological lineage decisions.

In clinical practice, rehabilitation and load management remain essential pillars of tendinopathy treatment. Evidence from Pavlova et al[11] in 2023 and foundational principles outlined by Rio et al[12] in 2016 highlight the necessity of optimizing mechanical stimuli to promote tendon adaptation. Integrating such approaches with molecular interventions - such as agents that mitigate ER stress - may provide synergistic benefits and more durable restoration of tendon homeostasis.

The aim of this article is to contextualize the findings of Tu et al[10] within the broader fields of tendon biology, ER stress, and regenerative therapeutics. Each subsequent section builds toward this objective: The methods and key findings validate the experimental framework; the critical appraisal synthesizes mechanistic insights and identifies the study’s contributions; and the future perspectives highlight the translational directions arising from this research.

Methods

Tu et al[10] combined in vitro and in vivo models to examine the effects of thymoquinone (TQ) on ER stress-mediated chondrogenesis. TDSCs were cultured on decellularized bovine tendon slices subjected to 6.4% strain to simulate tendon micro-injury[8,13]. In parallel, a rat treadmill-induced Achilles tendon injury model was established, followed by peri-tendinous injections of TQ. The expression of ER stress markers [glucose regulating protein 78 (GRP78), PERK, EIF2α, ATF4, CHOP] and chondrogenic markers (collagen II, aggrecan, SRY-box transcription factor 9) was assessed using reverse transcription-quantitative polymerase chain reaction, western blotting, and immunofluorescence. Comparisons with 4-phenylbutyric acid, a known ER stress inhibitor[14], provided mechanistic validation.

Key findings

The authors demonstrated several critical observations. First, tendon micro-injury (6.4% strain) induced ER stress and promoted chondrogenic differentiation of TDSCs, with marked upregulation of collagen II, aggrecan, and SRY-box transcription factor 9. TQ treatment (10 μM) significantly reduced ER stress marker expression - GRP78, ATF4, and CHOP - restoring tenogenic markers such as collagen I and tenomodulin. In vivo, TQ administration in treadmill-injured rats decreased ER stress components and chondrogenic markers while increasing collagen I and scleraxis expression. Notably, TQ showed similar efficacy to 4-phenylbutyric acid in attenuating the PERK/EIF2α/ATF4/CHOP pathway.

Critical appraisal

Tu et al[10] have provided compelling mechanistic evidence linking TQ-mediated ER stress suppression to inhibition of pathological TDSC differentiation. Their dual-model approach strengthens the translational relevance, bridging molecular findings with in vivo outcomes. The observed downregulation of GRP78 and CHOP parallels previous findings where TQ alleviated ER stress in hepatic[15,16] and neuronal tissues[17,18], supporting its role as a cross-tissue ER modulator. Moreover, this study complements Soltanfar et al[19], who demonstrated that TQ improves tendon repair by enhancing biomechanical integrity and collagen alignment.

This study distinguishes itself from prior investigations by linking intracellular ER stress directly with pathologic lineage deviation of TDSCs[9]. Whereas previous studies have examined tendon degeneration through inflammatory or matrix-remodeling pathways, the integration of ER stress biology with stem cell fate regulation represents a unique mechanistic framework[4,9]. By demonstrating that TQ can recalibrate the PERK/eIF2α/ATF4/CHOP axis and prevent chondrogenic misdifferentiation, the work provides an innovative conceptual bridge between cell-intrinsic stress responses and tissue-level degeneration.

However, this study is limited by several factors. The ex vivo tendon slice model cannot fully reproduce the complex neurovascular and immunological dynamics present in vivo. The treadmill-running model introduces variability in injury severity that is difficult to standardize. Additionally, the study focuses on molecular markers without evaluating long-term functional outcomes such as tensile strength, fascicle alignment, and collagen fiber organization. Mechanistically, although TQ attenuates ER stress, the interactions among oxidative stress, cytokine signaling, and unfolded protein response activation remain incompletely defined[20]. Finally, translational applicability requires future exploration of TQ’s pharmacokinetics, tendon residency time, dose-response profiles, and potential off-target effects.

Future perspectives

Future investigations should aim to integrate multi-omics analyses to map the global transcriptomic and proteomic shifts induced by TQ during tendon healing. Evaluating TQ’s pharmacokinetics and delivery formulations, such as liposomal encapsulation[21], may enhance local bioavailability and therapeutic efficiency. Combining TQ with mechanical loading rehabilitation or scaffold-based tendon engineering could further enhance repair outcomes. Moreover, identifying TQ analogs with improved selectivity for ER stress signaling may yield novel, tendon-specific small molecules.

Conclusions

Tu et al[10] have convincingly established TQ as a modulator of ER stress signaling in TDSCs, thereby mitigating aberrant chondrogenic differentiation following tendon injury. By targeting the PERK/EIF2α/ATF4/CHOP axis, TQ restores tendon cell homeostasis and presents a promising candidate for the prevention and treatment of tendinopathy. This study not only deepens our mechanistic understanding of tendon pathobiology but also opens a new translational avenue for small-molecule therapies in degenerative tendon disease (Figure 1).

Figure 1 Graphic: Thymoquinone attenuates endoplasmic reticulum stress-induced chondrogenic drift of tendon-derived stem cells. This conceptual illustration depicts how mechanical micro-injury initiates endoplasmic reticulum (ER) stress in tendon-derived stem cells (TDSCs) and how thymoquinone counteracts this process to restore tenogenic fate. Left panel: Repetitive treadmill loading and mechanical overload create micro-tears within tendon collagen fibers, leading to extracellular matrix disruption, cytoskeletal disorganization, and increased reactive oxygen species. These biomechanical and oxidative insults damage resident TDSCs and initiate the unfolded protein response. Middle panel: ER stress is characterized by misfolded protein accumulation, ER lumen overload, and loss of protein homeostasis, activating the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response. The PERK → phosphorylated eukaryotic initiation factor 2 → activating transcription factor 4 → enhancer-binding protein homologous protein (CHOP) signaling cascade is shown vertically, with CHOP driving transcriptional programs that promote SRY-box transcription factor 9 (SOX9) upregulation and pathological differentiation. Right-upper panel: Sustained ER stress redirects TDSCs toward a chondrocyte-like phenotype, characterized by rounded cell morphology, cartilaginous matrix deposition, and increased expression of SOX9, collagen type II alpha-1, and ACAN. These changes contribute to fibrocartilaginous metaplasia and loss of tenogenic identity. Right-lower panel: Thymoquinone exerts protective effects by attenuating PERK activation, reducing eukaryotic initiation factor 2 phosphorylation, suppressing activating transcription factor 4 activity, and downregulating CHOP. Inhibitory interactions are indicated by blockade symbols (⊣). By dampening ER stress, thymoquinone prevents SOX9 induction, restores spindle-shaped TDSC morphology, and promotes tenogenic marker expression, including TNMD, MKX, and SCX. Color coding distinguishes pathological signaling (red-orange-yellow) from thymoquinone-mediated rescue (blue-green). ECM: Extracellular matrix; TDSC: Tendon-derived stem cell; ROS: Reactive oxygen species; ER: Endoplasmic reticulum; PERK: Protein kinase RNA-like endoplasmic reticulum kinase; P-eIF2α: Phosphorylated eukaryotic initiation factor 2; ATF4: Activating transcription factor 4; UPR: Unfolded protein response; CHOP: Enhancer-binding protein homologous protein; SOX9: SRY-box transcription factor 9; COL2A1: Collagen type II alpha-1; TQ: Thymoquinone.