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TNF‑α induces premature senescence in tendon stem cells via the NF‑κB and p53/p21/cyclin E/CDK2 signaling pathways

  • Authors:
    • Hua Guo
    • Haixia Cao
    • Qian Lu
    • Zhifeng Gu
    • Guijuan Feng
  • View Affiliations / Copyright

    Affiliations: Department of Rheumatology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China, Department of Stomatology, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong, Jiangsu 226001, P.R. China
    Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 140
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    Published online on: July 10, 2025
       https://doi.org/10.3892/ijmm.2025.5581
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Abstract

Achilles tendinitis (AT) is a complex disorder that affects tendon tissue and often responds poorly to non‑steroidal anti‑inflammatory drugs. Tumor necrosis factor‑α (TNF‑α), a proinflammatory cytokine involved in cell death and immune regulation, serves a central role in AT progression. The present study investigated the effects of TNF‑α on tendon stem cells (TSCs) and evaluated potential therapeutic strategies for AT. TNF‑α‑induced changes in TSCs were determined by investigating markers of cellular senescence, reactive oxygen species (ROS) activity, DNA damage and the expression of key transcription factors, including NF‑κB (phosphorylated‑p65, p65), p53, p21, cyclin E and CDK2. To determine whether TNF‑α‑induced senescence could be reversed, TSCs were treated with etanercept, a TNF‑α‑specific inhibitor. TNF‑α stimulation induced significant senescence in TSCs, as evidenced by increased ROS production, DNA damage and altered expression of senescence‑associated transcription factors. TNF‑α activated the NF‑κB and p53/p21/cyclin E/CDK2 signaling pathways, promoting TSC senescence. Etanercept treatment effectively reversed these effects, decreasing TSC senescence, suppressing inflammatory cell infiltration, decreasing TNF‑α protein expression and mitigating collagen fiber degradation. TNF‑α promotes TSCs senescence through specific signaling pathways and etanercept can counteract these deleterious effects. These results provide insights into the pathogenesis of AT and highlight TNF‑α inhibition as a promising therapeutic approach. Targeting TNF‑α may offer a novel treatment strategy for individuals with AT.
View Figures

Figure 1

Identification of TSCs. (A) Alizarin
red S, (B) ALP and (C) Oil red O staining of TSCs. (D) Chondrogenic
differentiation of TSCs as detected by toluidine blue staining. (E)
Immunofluorescence staining of Oct4 and Nanog in TSCs. Scale
bar=100 µm. TSCs, tendon stem cells; ALP, alkaline
phosphatase.

Figure 2

Effects of TNF-α on senescence in
TSCs. (A) SA-β-gal staining following stimulation with TNF-α in
TSCs. Scale bar=200 µm. (B) Quantitative analysis of
SA-β-gal-positive cells in TSCs treated with TNF-α. (C)
Immunofluorescence revealed aberrant distribution of F-actin in
TSCs subjected to TNF-α treatment (20 ng/ml). Scale bar=100
µm. (D) Proliferation of TSCs decreased following
stimulation with TNF-α, as assessed using the EdU staining assay.
Scale bar=200 µm. (E) Quantitative analysis of EdU-positive
cells indicating decreased proliferation in TNF-α-treated TSCs. (F)
Cell cycle analysis with flow cytometry. Proportion of cells in the
G0/G1 phase increased in TSCs exposed to TNF-α. (G) Quantitative
summary showing an increased proportion of cells in the G0/G1 phase
in TNF-α-treated TSCs. **P<0.01,
***P<0.001,****P<0.0001. TSCs, tendon
stem cells; SA-β-gal, senescence-associated β-galactosidase;
F-actin, filamentous-actin.

Figure 3

Effects of the NF-κB and
p53/p21/cyclin E/CDK2 signaling pathways on senescence in
TNF-α-treated TSCs. (A) ROS staining of TSCs using DCF fluorescence
probe, showing intracellular ROS distribution. Scale bar=100
µm. (B) Quantitative analysis of DCF fluorescence intensity,
demonstrating TNF-α-induced elevation of ROS levels. (C)
Immunofluorescence staining of γ-H2A.X. Following stimulations with
TNF-α (20 ng/ml, six times), the proportion of γ-H2A.X-positive
TSCs exhibited a considerable increase. Scale bar=100 µm.
(D) Quantitative analysis of γ-H2A.X-positive TSCs following TNF-α
treatment. (E) Expression of γ-H2A.X, H2A.X, p-p65 and p65
following TNF-α stimulation as assessed by western blot. GAPDH was
used as a control. (F) Bar groups showed the relative density of
γ-H2A.X, H2A.X, p-p65 and p65. (G) Expression of p53, p21, cyclin E
and CDK2 following TNF-α stimulation as assessed by western blot.
GAPDH was used as a control. (H) Relative density of p53, p21,
cyclin E and CDK2. (I) Immunofluorescence examination of p65, p53
and p21 expression was consistent with western blotting. Scale
bar=100 µm. *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001. ROS, reactive
oxygen species; TSCs, tendon stem cells; p-, phosphorylation; ns,
not significant.

Figure 4

Effects of etanercept on the NF-κB
and p53/p21/cyclin E/CDK2 signaling pathways in tendon stem cell
senescence. (A) ROS staining of TSCs. ROS generation was markedly
elevated following repeated TNF-α stimulation and subsequently
reduced after repeated administration of etanercept. Scale bar=100
µm. (B) Quantitative analysis of DCF fluorescence intensity.
(C) Expression of γ-H2A.X, H2A.X, p53, p21, p-p65 and p65 after
stimulation with TNF-α + etanercept as assessed by western
blotting. GAPDH was used as a control. (D) Bar groups showed the
relative density of γ-H2A.X, H2A.X, p53, p21, p-p65 and p65. (E)
Immunofluorescence of γ-H2A.X and p65 yielded consistent results
with those from western blotting. Scale bar=100 µm. (F)
Immunofluorescence of p53 and p21 yielded consistent results with
those from western blotting. Scale bar=100 µm..
**P<0.01, ***P<0.001,
****P<0.0001. ROS, reactive oxygen species; p-,
phosphorylation; ns, not significant.

Figure 5

Effects of etanercept on senescence
in TSCs. (A) Senescence of TSCs was analyzed by SA-β-gal staining.
Scale bar=200 µm. (B) Quantitative analysis of
SA-β-gal-positive cells in TSCs. (C) Aberrant distribution of
F-actin in TSCs subjected to TNF-α treatment as revealed by
immunofluorescence. Effects were reversed by etanercept. Scale
bar=100 µm. (D) Substantial reduction in the proliferation
rate of TNF-α-treated TSCs, as indicated by a EdU staining
experiment. Proliferation recovered following treatment with
etanercept. Scale bar=200 µm. (E) Quantitative analysis of
EdU-positive TSCs. (F) Proportion of cells in the G0/G1 phase of
the cell cycle increased following treatment with TNF-α, as
detected using flow cytometry. This rise is subsequently reversed
after treatment with etanercept. (G) Quantitative summary showing
an decreased proportion of cells in the G0/G1 phase in
etanercept-treated TSCs. *P<0.05,
**P<0.01, ****P<0.0001. TSCs, tendon
stem cells; SA-β-gal, senescence-associated β-galactosidase;
F-actin, filamentous-actin.

Figure 6

Histological changes in rats with
tendinitis. (A) Injection schedule of PBS, collagenase I and
etanercept in Sprague-Dawley rats. (B) H&E and Masson's
trichrome staining 2 weeks post-injection of PBS, collagenase I +
PBS and collagenase I + etanercept into tendon tissues. Red denotes
collagen fibers; blue denotes the collapse of the collagen matrix.
(C) Immunostaining for TNF-α on PBS-treated tendon tissue sections
revealed negligible staining. Collagenase I-treated tendon tissue
sections exhibited substantial positive staining (brown). (D) The
percentages of TNF-α positive cells in tendon tissue. (E) SA-β-gal
staining reveals little positive staining in PBS and collagenase I
+ etanercept groups, but substantial staining in collagenase I +
PBS tendon tissues. (F) SA-β-gal-positive area (%) of tendon
tissues. (G) Co-localization of CD44 (green) with p53 (red) in
tendon tissues. The nucleus inside the tendon tissues is labeled
with DAPI (blue). Scale bar=50, 100, 200 µm.
*P<0.05, ***P<0.001. H&E,
hematoxylin and eosin, SA-β-gal, senescence-associated
β-galactosidase.

Figure 7

Impact of TNF-α on TSCs in normal
tendon tissues. Under physiological conditions, TSCs exhibit
typical functionality, characterized by regular cell cycles, intact
F-actin structures, low levels of ROS and normal expressions of
transcription factors. Following stimulation by TNF-α, TSCs
experience increased ROS production and DNA damage, activation of
the NF-κB signaling pathway (resulting in elevated levels of p-p65
and p65, leading to p65 translocation to the nucleus) and
modulation of the p53/p21/cyclin E/CDK2 signaling pathways
(resulting in upregulation of p53 and p21 and downregulation of
cyclin E and CDK2). These changes induce senescence in TSCs,
characterized by alterations such as enlarged cell volume and
disrupted F-actin structures. Etanercept, a TNF-α inhibitor,
mitigates these effects by binding to TNF-α, thereby inhibiting the
activation of signaling pathways. This inhibition leads to reduced
ROS levels, mitigates DNA damage, decreases expression of p53, p21
and p-p65, normalizes cyclin E and CDK2 expression and ultimately
reverses senescence in TSCs, thereby restoring normal cellular
functions. Figure created using BioRender (app.biorender.com/illustrations). TSC, tendon stem
cells; ROS, reactive oxygen species; F-actin, filamentous-actin;
p-, phosphorylation; TNFR, tumor necrosis factor receptor;
SA-β-gal, senescence-associated β-galactosidase.
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Copy and paste a formatted citation
Spandidos Publications style
Guo H, Cao H, Lu Q, Gu Z and Feng G: TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways. Int J Mol Med 56: 140, 2025.
APA
Guo, H., Cao, H., Lu, Q., Gu, Z., & Feng, G. (2025). TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways. International Journal of Molecular Medicine, 56, 140. https://doi.org/10.3892/ijmm.2025.5581
MLA
Guo, H., Cao, H., Lu, Q., Gu, Z., Feng, G."TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways". International Journal of Molecular Medicine 56.3 (2025): 140.
Chicago
Guo, H., Cao, H., Lu, Q., Gu, Z., Feng, G."TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways". International Journal of Molecular Medicine 56, no. 3 (2025): 140. https://doi.org/10.3892/ijmm.2025.5581
Copy and paste a formatted citation
x
Spandidos Publications style
Guo H, Cao H, Lu Q, Gu Z and Feng G: TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways. Int J Mol Med 56: 140, 2025.
APA
Guo, H., Cao, H., Lu, Q., Gu, Z., & Feng, G. (2025). TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways. International Journal of Molecular Medicine, 56, 140. https://doi.org/10.3892/ijmm.2025.5581
MLA
Guo, H., Cao, H., Lu, Q., Gu, Z., Feng, G."TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways". International Journal of Molecular Medicine 56.3 (2025): 140.
Chicago
Guo, H., Cao, H., Lu, Q., Gu, Z., Feng, G."TNF‑&alpha; induces premature senescence in tendon stem cells via the NF‑&kappa;B and p53/p21/cyclin E/CDK2 signaling pathways". International Journal of Molecular Medicine 56, no. 3 (2025): 140. https://doi.org/10.3892/ijmm.2025.5581
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