Abstract

Objective: To investigate the effects of verbascoside (VB; also known as acteoside) on tendon–bone healing in rats following anterior cruciate ligament (ACL) reconstruction, and to explore whether the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved.

Methods: Rats were randomly assigned to six groups: Sham, Model, low-dose VB (VB‑L), middle-dose VB (VB‑M), high-dose VB (VB‑H), and VB‑H + AG490 (a JAK2/STAT3 pathway inhibitor). Except for the Sham group, all groups underwent ACL reconstruction to establish the postoperative model.

Micro‑CT was used to assess bone microstructure parameters of the bone tunnel and to measure the cross‑sectional area of the femoral and tibial tunnel.

Biomechanical testing evaluated reconstructed knee stiffness and maximum load.

ELISA measured inflammatory cytokines and oxidative stress markers in knee lavage fluid.

H&E staining and Safranin O–Fast Green staining evaluated cartilage pathology.

Immunohistochemistry assessed expression of type I collagen (COL‑I) and matrix metalloproteinase‑13 (MMP13).

Western blot examined proteins related to the JAK2/STAT3 pathway.

Results: Compared with the Model group, VB‑L/VB‑M/VB‑H increased bone surface area to volume ratio, trabecular thickness, and BV/TV, and decreased the femoral and tibial bone tunnel cross‑sectional area, with the most significant changes in the VB‑H group (P<0.05P < 0.05P<0.05). VB‑L/VB‑M/VB‑H also increased stiffness and maximum load, again most prominently in VB‑H (P<0.05P < 0.05P<0.05). ELISA showed that VB reduced TNF‑α, IL‑6, IL‑1β, and MDA, while increasing SOD and GSH‑Px, with VB‑H showing the strongest effect (P<0.05P < 0.05P<0.05). Histology indicated improved cartilage damage with thicker cartilage layers, more orderly chondrocyte arrangement, reduced inflammatory infiltration, increased proteoglycan content, and clearer tidemark-most evident in VB‑H. Immunohistochemistry showed increased COL‑I and decreased MMP13 in VB groups (VB‑H strongest, P<0.05P < 0.05P<0.05). Western blot indicated increased p‑JAK2/JAK2 and p‑STAT3/STAT3 in VB groups (VB‑H strongest, P<0.05P < 0.05P<0.05). Compared with VB‑H, AG490 partially reversed these improvements.

Conclusion: Verbascoside may promote tendon–bone healing in rats after ACL reconstruction, potentially through activation of the JAK2/STAT3 signaling pathway.

Keywords: JAK2/STAT3 signaling pathway; verbascoside (acteoside); ACL reconstruction; tendon–bone healing

Introduction

ACL rupture is commonly caused by high‑intensity sport and mechanical trauma, typically presenting with pain, rapid joint swelling, instability, and reduced exercise tolerance-often leading to major functional limitation. ACL reconstruction is widely used clinically, but successful postoperative recovery depends heavily on tendon–bone integration at the graft–bone interface.

In recent years, researchers have explored multiple strategies to accelerate tendon–bone healing, such as physical stimulation (stretching, pressure, vibration), implantation of biomaterials, and use of cytokines or tissue‑engineering approaches. Even so, healing can be slow and outcomes remain variable, motivating interest in new adjunctive approaches, including bioactive compounds with anti‑inflammatory, antioxidant, and bone‑protective properties.

Verbascoside (acteoside) is a water‑soluble phenylethanoid glycoside found in various medicinal plants. It has been reported to exhibit anti‑inflammatory, antioxidant, and bone‑protective activities, including suppression of osteoclastogenesis and mitigation of bone erosion in inflammatory arthritis models.

A key mechanistic candidate is the JAK2/STAT3 pathway, a major intracellular signaling cascade involved in diverse physiological and pathological processes. Prior work suggests that activating JAK2/STAT3 can increase trabecular thickness, stiffness, and maximal load in bone injury contexts-supporting fracture healing.

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Where Cistanche fits

For readers exploring natural, herb-derived supportive strategies, it's useful to know that Cistanche (often discussed in traditional herbal contexts) is one of the plants frequently mentioned as containing phenylethanoid glycosides, including acteoside (verbascoside) in some preparations. This does not mean it is a proven or approved postoperative therapy for ACL reconstruction-rather, it provides a botanical context for why acteoside/verbascoside has attracted research attention. In controlled animal models like the one in this study, using a defined compound (VB) allows mechanistic testing-something whole‑herb supplements cannot guarantee due to variability in composition and dose.

This study therefore investigates whether verbascoside can improve tendon–bone healing in a rat ACL reconstruction model and whether this effect relates to JAK2/STAT3 activation.

Materials and Methods

1. Animals

Male SPF SD rats (7 weeks old, 200–220 g) were obtained from the Experimental Animal Department of Hebei Medical University (license: SCXK (Hebei) 2025‑005). Housing conditions: humidity 55%–65%, temperature 22–24 °C, 12‑h light/dark cycle, free access to food and water. The study was approved by the Animal Ethics Committee of Cangzhou Medical College (approval no. 2202130618).

2. Drugs and Reagents

Verbascoside (purity 99.8%, HY‑N0021‑107208), AG490 (purity 99.0%, HY‑12000‑09922): Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.

ELISA kits for TNF‑α (ml002859), IL‑6 (ml102828), IL‑1β (ml003057), SOD (EK‑M26101), MDA (EK‑M29370), GSH‑Px (EK‑M27911): Shanghai Enzyme‑linked Biotechnology Co., Ltd.

Antibodies: JAK2 (FNab04432), STAT3 (FNab08298) from Wuhan Fine Biotech; COL‑I (GTX82720), MMP13 (GTX100665), p‑JAK2 (GTX32203) from GeneTex; p‑STAT3 (bsm‑52210R) from Bioss; β‑actin (48139) from Nanjing Saigowe; HRP‑goat anti‑rabbit IgG (P0948) from Beyotime.

3. Instruments

Microplate reader (EnVision), microscope (Olympus BX51), gel imaging system (Bio‑Rad GelDoc Go).

4. Model Establishment, Grouping, and Administration

Modeling (ACL reconstruction): Rats were anesthetized with intraperitoneal 10% chloral hydrate (4 mL/kg). A medial parapatellar incision was made to expose the ACL, which was transected (positive drawer and Lachman tests). Tibial and femoral tunnels were drilled at the native ACL footprint. A fixed tendon graft was passed through the tibial tunnel into the joint cavity and into the femoral tunnel, then sutured and fixed on the lateral femoral soft tissue near the tunnel outlet. With knee flexed at 30°, the tibial end was sutured and fixed to the tibial tunnel bone bridge and surrounding soft tissue. Negative drawer and Lachman tests and good graft tension indicated successful reconstruction. The incision was closed, disinfected, and penicillin was injected for 3 days to prevent infection. Success criteria: negative drawer/Lachman and good graft tension.

Grouping: 120 rats were used. Eighteen were randomly selected as Sham (incision and closure only). Of the remaining 102 modeled rats, 90 were successfully established (success rate 88.24%) and randomly assigned (18 per group) to: Model, VB‑L, VB‑M, VB‑H, and VB‑H+AG490.

Dosing regimen (once daily for 8 weeks):

VB‑L/VB‑M/VB‑H: 50/100/200 mg/kg VB by gavage + equal‑volume saline intraperitoneally.

VB‑H+AG490: 200 mg/kg VB by gavage + AG490 5.0 mg/kg intraperitoneally.

Sham/Model: equal‑dose saline by gavage + saline intraperitoneally.

5. Micro‑CT

After euthanasia, intact knee joints with adjacent femur and tibia were harvested and fixed in 4% paraformaldehyde at 4 °C for 24 h. Samples were wrapped in moist lint‑free paper and scanned with 360° Micro‑CT (10 μm resolution; 70 kV; 114 μA; 400 ms exposure; 0.5 mm Al filter). Parameters analyzed: bone surface area/volume ratio, BV/TV, trabecular thickness; and cross‑sectional area of femoral and tibial tunnels.

6. Biomechanical Testing

Reconstructed knees were mounted in a material testing machine. A 0.8 N preload was applied to remove slack, followed by tensile testing at 5 mm/min until the load dropped ~25% from the peak. Load–displacement was recorded and stiffness calculated.

7. ELISA for Inflammatory and Oxidative Stress Factors in Knee Lavage

Knees were opened at the suprapatellar pouch and washed with saline three times to collect lavage fluid. After centrifugation (2,000 r/min, 15 min), supernatants were used to quantify TNF‑α, IL‑6, IL‑1β, and oxidative stress markers SOD, GSH‑Px, MDA by ELISA.

8. H&E and Safranin O–Fast Green Staining

Knees were fixed, decalcified, paraffin‑embedded, sectioned at 4 μm, deparaffinized, rehydrated, and stained with H&E and Safranin O–Fast Green for cartilage pathology evaluation.

9. Immunohistochemistry for COL‑I and MMP13

After routine processing, antigen retrieval and blocking were performed. Sections were incubated with primary antibodies against COL‑I and MMP13 overnight at 4 °C, then with HRP‑conjugated secondary antibody for 1 h at room temperature. DAB was used for color development, hematoxylin counterstaining performed, and expression observed microscopically.

10. Western Blot for JAK2/STAT3 Pathway Proteins

Total protein was extracted from knee tissues, quantified, denatured, separated by electrophoresis, transferred to PVDF, and blocked. Membranes were incubated with primary antibodies against p‑JAK2, JAK2, p‑STAT3, STAT3, and β‑actin overnight at 4 °C, followed by HRP‑secondary antibody for 1 h. Bands were visualized and analyzed by densitometry.

 

Table 6. Comparison of expression levels of JAK2/STAT3 signaling pathway-related proteins in the knee joint tissues of each group of rats (n=6n=6n=6, mean ± SD)

Group	p‑JAK2/JAK2	p‑STAT3/STAT3
Sham	0.95 ± 0.10	0.88 ± 0.10
Model	0.34 ± 0.04a^{a}a	0.25 ± 0.04a^{a}a
VB‑L	0.52 ± 0.06b^{b}b	0.43 ± 0.05b^{b}b
VB‑M	0.71 ± 0.08bc^{bc}bc	0.61 ± 0.07bc^{bc}bc
VB‑H	0.92 ± 0.10bcd^{bcd}bcd	0.83 ± 0.09bcd^{bcd}bcd
VB‑H + AG490	0.55 ± 0.06e^{e}e	0.46 ± 0.05e^{e}e

11. Statistical Analysis

Data were normally distributed and expressed as mean ± SD. SPSS 29.0 was used. One‑way ANOVA was applied for multi‑group comparisons, and SNK‑q test for pairwise comparisons. P<0.05P < 0.05P<0.05 indicated statistical significance.

Results

1. Bone Tunnel Microstructure and Tunnel Cross‑Sectional Area

Compared with Sham, the Model group showed decreased bone surface area/volume ratio, trabecular thickness, and BV/TV, and increased femoral and tibial tunnel cross‑sectional area (P<0.05P < 0.05P<0.05). Compared with Model, VB‑L/VB‑M/VB‑H increased bone microstructure parameters and decreased tunnel cross‑sectional areas (P<0.05P < 0.05P<0.05), with VB‑H being most significant. Compared with VB‑H, VB‑H+AG490 reduced these improvements and increased tunnel areas (P<0.05P < 0.05P<0.05).

2. Knee Stiffness and Maximum Load

Compared with Sham, the Model group exhibited reduced stiffness and maximum load (P<0.05P < 0.05P<0.05). VB‑L/VB‑M/VB‑H significantly increased both outcomes vs Model (P<0.05P < 0.05P<0.05), strongest in VB‑H. AG490 partially reversed the VB‑H benefits (P<0.05P < 0.05P<0.05).

3. Inflammatory Cytokines in Knee Lavage

TNF‑α, IL‑6, and IL‑1β were elevated in the Model group vs Sham (P<0.05P < 0.05P<0.05). VB treatment decreased these cytokines vs Model (P<0.05P < 0.05P<0.05). AG490 increased cytokine levels vs VB‑H (P<0.05P < 0.05P<0.05).

4. Oxidative Stress Markers in Knee Lavage

Model rats had increased MDA and decreased SOD and GSH‑Px vs Sham (P<0.05P < 0.05P<0.05). VB lowered MDA and raised SOD and GSH‑Px vs Model (P<0.05P < 0.05P<0.05). AG490 partially reversed these effects vs VB‑H (P<0.05P < 0.05P<0.05).

5. Cartilage Pathology (H&E and Safranin O–Fast Green)

H&E showed severe cartilage injury in the Model group with loss of layered structure, damaged and disorganized surface cells, and marked inflammatory infiltration. VB groups showed明显 improvement: thicker cartilage, more chondrocytes with orderly arrangement, and reduced inflammatory infiltration; VB‑H had the clearest improvement.
Safranin O–Fast Green indicated severe proteoglycan loss and irregular/absent tidemark in Model, while VB restored proteoglycan staining and tidemark integrity; AG490 worsened pathology relative to VB‑H.

6. COL‑I and MMP13 Expression

Compared with Sham, Model showed decreased COL‑I and increased MMP13 (P<0.05P < 0.05P<0.05). VB increased COL‑I and decreased MMP13 vs Model (P<0.05P < 0.05P<0.05), strongest in VB‑H. AG490 partially reversed these changes (P<0.05P < 0.05P<0.05).

7. JAK2/STAT3 Pathway Proteins

Model rats had reduced p‑JAK2/JAK2 and p‑STAT3/STAT3 vs Sham (P<0.05P < 0.05P<0.05). VB increased both ratios vs Model (P<0.05P < 0.05P<0.05), strongest in VB‑H. AG490 decreased both ratios vs VB‑H (P<0.05P < 0.05P<0.05).

 

Discussion 

Tendons connect muscle to bone and are essential for restoring joint function. In the ACL reconstruction setting, tendon–bone integration is a major determinant of mechanical stability and functional recovery. In this model, surgery impaired bone microarchitecture and biomechanics (lower BV/TV, trabecular thickness, stiffness, maximum load; larger tunnel areas), consistent with delayed interface maturation.

Inflammation plays a dual role in tendon–bone healing: a controlled inflammatory response can clear debris and support cellular proliferation and collagen formation, whereas excessive inflammation promotes tissue damage, adhesions, and scar formation. Here, TNF‑α, IL‑6, and IL‑1β increased markedly after reconstruction, indicating a pro‑inflammatory environment likely unfavorable for organized interface healing.

Oxidative stress is similarly important. Elevated MDA with reduced SOD and GSH‑Px suggests increased lipid peroxidation and reduced antioxidant defense. Oxidative stress can disrupt osteogenesis and impair matrix remodeling. In parallel, increased MMP13 and reduced COL‑I imply accelerated extracellular matrix degradation, which may delay stabilization of the tendon–bone interface.

Why verbascoside (acteoside) is interesting in "natural" strategies

Verbascoside is a plant-derived compound with anti‑inflammatory and antioxidant properties, which aligns with the idea that modulating inflammatory and oxidative microenvironments could help interface healing. In this study, VB improved bone microstructure, reduced tunnel enlargement, enhanced mechanical strength, reduced inflammatory cytokines, improved redox markers, increased COL‑I, and lowered MMP13-suggesting a coordinated shift toward constructive remodeling rather than destructive inflammation.

JAK2/STAT3 as a mechanistic hub

The JAK2/STAT3 pathway is activated when extracellular signals bind cytokine receptors, promoting JAK2 phosphorylation, recruitment/phosphorylation of STAT3, STAT3 dimerization, and nuclear translocation to regulate genes involved in inflammation, oxidative stress response, and bone formation. The present results showed decreased pathway activation post‑modeling, but VB increased p‑JAK2/JAK2 and p‑STAT3/STAT3. Importantly, blocking the pathway with AG490 partially reversed VB's benefits across micro‑CT, biomechanics, inflammation/oxidative markers, matrix remodeling proteins, and histology-supporting pathway involvement.

 

Practical "natural" context: Cistanche and acteoside-rich botanicals

For audiences interested in herb-derived strategies, Cistanche is often discussed as a botanical source of phenylethanoid glycosides, including acteoside/verbascoside in some preparations. From a translational perspective, the key scientific point is that a defined dose of purified VB can be mechanistically validated in animal models, whereas whole‑herb products can vary in composition, extraction method, and bioavailability. Therefore, while Cistanche is relevant to the conversation around acteoside-rich botanicals, the evidence in this manuscript specifically supports verbascoside as a compound in a controlled postoperative rat model, not a clinical recommendation.

Overall conclusion: Verbascoside may enhance tendon–bone healing after ACL reconstruction in rats, potentially by activating JAK2/STAT3 signaling and improving the inflammatory/oxidative microenvironment while favoring collagen matrix integrity.

 

References

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