Building on the well-documented osteogenic effects reported for extracts of Eucommiae Cortex [6,7] and the shared biological characteristics between bone and tendon tissues, we hypothesized that this medicinal material might also harbor therapeutic potential for tendinopathy. Here, we provide the first comprehensive evidence supporting this hypothesis. We identify aucubin (AU), a predominant iridoid glycoside from Eucommiae Cortex, as an active constituent that confers multi-faceted efficacy against tendinopathy: it protects tenocytes from oxidative stress-induced apoptosis, enhances their migration, promotes structured collagen remodeling in vivo, and orchestrates a restorative tissue microenvironment. Most notably, integrated pharmacological evidence establishes that these therapeutic effects are mediated, at least in part, through an estrogen receptor β (ERβ) mechanism. This finding not only elucidates a key mechanistic pathway but also positions AU as a promising phytoestrogenic candidate for tendon repair.

Our bioactivity-guided fractionation strategy successfully traced the cytoprotective and promigratory activities of the aqueous fraction derived from the ethanolic extract of Eucommiae Cortex to AU, an iridoid constituent whose selection for validation was guided by its chemical predominance and a literature-based prediction of mechanistic relevance to tendinopathy. The protective effects of AU were unequivocal: it significantly restored the viability of tenocytes under oxidative assault (Figure 2C and Figure 3A) and markedly reduced H₂O₂-induced apoptosis and necrosis (Figure 3B,C). Mechanistically, this cytoprotection was underpinned by AU’s potent capacity to scavenge ROS, reduce lipid peroxidation (MDA), enhance endogenous antioxidant activity (SOD), and suppress pro-inflammatory cytokines (IL-1β, TNF-α) (Figure 3D–I). These multi-target actions directly counteract the intertwined pathological drivers of tendinopathy—including oxidative stress, apoptosis, and chronic inflammation [1,2,3]. While confirming AU’s broad cytoprotective potential, our work specifically delineates its efficacy and mechanisms within tenocytes, the primary effector cells in tendinopathy. This aligns with and contextually extends AU’s reported protective effects in other cell types [11,12,13].

A pivotal mechanistic insight from this work is the implication of the estrogen receptor β (ERβ) pathway in AU’s action. This conclusion is supported by a consistent pharmacological profile in which AU’s core cytoprotective effects—restoring cell viability (Figure 3A), suppressing apoptosis (Figure 3B,C), mitigating oxidative stress (Figure 3D–G), and reducing IL-1β (Figure 3H)—were mimicked by 17β-estradiol (E2) and, importantly, reversed by the selective ERβ antagonist (R, R)-THC. Although the modulation of TNF-α presented a more complex pattern, the collective data strongly suggest that ERβ signaling plays a major role in the primary mechanism of AU within tenocytes, aligning with its reported phytoestrogenic activity [14].

Importantly, this well-defined cellular mechanism provides a plausible molecular foundation for the multi-faceted therapeutic efficacy of AU, which we subsequently evaluated in vivo using a well-controlled experimental design. First, we exclusively employed male Sprague-Dawley rats to eliminate the confounding effects of the fluctuating endogenous estrogen cycle in females, thereby establishing a stable baseline to clearly attribute any therapeutic effects to AU’s pharmacological action via ERβ. Second, we utilized the rat collagenase-induced Achilles tendinopathy model. While this model simulates an acute injury rather than chronic overuse, it was selected for its high reproducibility and faithful recapitulation of core pathological hallmarks of human tendinopathy, including collagen degradation, MMP overexpression, and inflammatory infiltration [21,24]. Furthermore, our protocol incorporated a 7-day recovery period after the final collagenase injection to allow the resolution of the initial acute inflammatory phase, thereby shifting the therapeutic intervention window to a more persistent, pathology-driven stage. Finally, we adopted a local subcutaneous injection regimen to directly target the injured Achilles tendon, a strategy justified by the limited vascular supply of tendon tissue and employed in other studies of natural product-derived therapeutics [25,26].

Within this optimized model framework, AU treatment effectively translated the cellular benefits into multi-level tissue repair. At the gross and imaging level, AU-treated tendons exhibited improved structural integrity, as evidenced by more uniform echogenicity on ultrasound compared to the heterogeneous, hypoechoic areas in the Model group (Figure 4C). Histologically, AU treatment led to a significant reduction in total histopathological scores (Figure 4D,E) and promoted better-organized collagen fiber architecture in both H&E and Masson’s Trichrome stains (Figure 4B) [27]. Quantification of Masson’s staining confirmed a significant decrease in the area of immature collagen (blue staining) following AU treatment (Figure 4F). This local AU administration effectively promoted substantial morphological and organizational recovery, offering a potential therapeutic strategy distinct from current palliative options like NSAIDs or corticosteroid injections, which fail to promote regeneration and may carry risks [5].

Beyond general tissue repair, AU promoted a qualitatively superior form of healing characterized by enhanced collagen maturity, which is central to functional recovery. The therapeutic correction of an aberrant collagen composition is a key goal in tendinopathy, given that human tendinopathic tissue is characterized by an excessive production of immature type III collagen over mature type I collagen [28]. Our data demonstrate that AU effectively addresses this pathological hallmark. Sirius Red staining under polarized light revealed that AU treatment significantly increased the proportion of mature, thick collagen type I (orange-red birefringence) relative to immature collagen type III (green birefringence) (Figure 5A,B). This shift towards a mature collagen profile was corroborated by immunohistochemistry, showing upregulated Col I and downregulated Col III expression (Figure 5C–F). As the primary structural component of tendon (60–85% dry weight), the restoration of a high Col I/Col III ratio is critical, as type III collagen deposited during early healing forms a disorganized, biomechanically inferior “patch” that persists in tendinopathy [27]. Most importantly, at the ultrastructural level, transmission electron microscopy demonstrated that AU treatment significantly increased the mean diameter of collagen fibrils and shifted the fibril diameter distribution towards larger sizes (Figure 5G–I), which is directly associated with superior biomechanical strength [29]. These findings collectively indicate that AU not only restores collagen content but critically reverses a key pathological feature. By promoting a mature collagen composition (elevated Col I/Col III ratio), AU facilitates the assembly of a higher-quality extracellular matrix, as evidenced by the significant increase in collagen fibril diameter—an ultrastructural hallmark of superior biomechanical strength. This coordinated improvement steers the healing process away from scar formation towards a more regenerative outcome.

To elucidate the molecular underpinnings of this improved repair, we analyzed key regulators of the tendon microenvironment. AU treatment created a more anabolic and less catabolic milieu. It significantly suppressed the expression of matrix-degrading enzymes (MMP-3 and MMP-13) and the pro-apoptotic marker Cleaved Caspase-3 (Figure 6A–F). Elevated caspase-3 is a feature of degenerative human and overload-induced rat tendinopathy [30], and its reduction by AU underscores an anti-apoptotic effect. Given that MMP-3 and MMP-13 can degrade multiple extracellular matrix components, including collagen, and mediate the development of painful tendinopathy [31], their downregulation likely contributes directly to the observed increase in collagen fibril diameter and the shift towards a mature Col I/Col III ratio. This effect may be both a direct consequence of AU’s action and indirectly mediated through its anti-inflammatory and antioxidant properties. Concurrently, the mitigation of oxidative stress (reduced MDA, enhanced SOD activity) and inflammation (lowered IL-1β and TNF-α) within the tendon tissue (Figure 6G–J) supports a pro-regenerative niche by removing key pathological drivers known to exacerbate tissue damage and impair healing. Therefore, AU orchestrates a multifaceted restorative environment by coordinately inhibiting matrix degradation, suppressing apoptosis, and mitigating oxidative stress and inflammation.

While this study provides a multi-faceted characterization of AU’s tendon-reparative effects, several limitations should be acknowledged to guide future research. First, regarding the animal model, the collagenase-induced tendinopathy model, despite our inclusion of a recovery period, simulates an acute injury rather than the chronic mechanical overload typical of human disease. Future studies in validated chronic overuse models are essential to confirm AU’s efficacy under more etiologically relevant conditions. Second, while we documented robust improvements at histological, biochemical, and ultrastructural levels, our study lacks direct biomechanical or functional assessments. Conclusive evidence of true functional recovery requires complementary evaluations, such as measuring the ultimate tensile strength of healed tendons or performing functional gait analysis. Third, concerning mechanistic specificity, our data robustly implicate the ERβ pathway as a central, though not necessarily exclusive, mediator of AU’s effects. The consistent reversal by the selective antagonist (R, R)-THC establishes a strong pharmacological dependency. However, tendon repair is a complex process likely involving crosstalk between multiple signaling pathways. Future studies employing genetic approaches (e.g., tenocyte-specific ERβ knockdown) are warranted not only to confirm its non-redundant role but also to map its interaction with other repair-relevant pathways (e.g., TGF-β, IGF-1), thereby delineating the broader mechanistic network through which AU operates. Looking toward therapeutic translation, future studies should define the local pharmacokinetics of AU within tendon tissue to optimize dosing regimens for topical or injectable delivery. Furthermore, the potential synergistic effects of AU with other constituents of Eucommiae Cortex, such as geniposidic acid [32,33,34,35], warrant exploration to fully appreciate the holistic efficacy of the crude extract.