Publication: Heraclenin promotes the osteogenic differentiation of bone marrow stromal cells by activating the RhoA/ROCK pathway
Authors
Yu, Zuguang ; Yuan, Jun ; Yu, Yuanyuan
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Publisher
Universidad de Murcia. Departamento de Biología Celular e Histología
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DOI
https://doi.org/10.14670/HH-18-702
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info:eu-repo/semantics/article
Description
Abstract
Background. Osteoporosis is a devastating
skeletal disease, the pathogenesis of which is related to
abnormal bone metabolism, featured by the imbalance
between osteoblastic bone formation and osteoclastic
bone resorption. Stem cell-based therapies have been
demonstrated to improve osteoporosis treatment.
Previously, the linear furanocoumarin heraclenin was
reported to enhance osteoblast differentiation and
mineralization in mouse mesenchymal stem cells
(MSCs), suggesting its potential for osteogenic
differentiation and bone regeneration. Our study was
designed to confirm the promotive role of heraclenin on
osteogenic differentiation of human bone MSCs
(BMSCs) and explore the underlying mechanisms.
Methods. Human BMSCs were treated for 24, 48,
and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and
cell viability was determined by Cell Counting Kit-8
(CCK-8) assay. To further evaluate the cytotoxicity of
heraclenin, cell suspension obtained from BMSCs
treated with heraclenin (5, 10, and 20 μM) for 72h was
subjected to a MUSE™ cell analyzer for cell viability
and count assay. BMSCs were incubated in osteogenic
induction medium for 7 days. Then, osteogenic
differentiation and mineralization of BMSCs were
assessed through alkaline phosphatase (ALP) and
Alizarin Red S staining. The expression of osteogenesis
markers including ALP, osteocalcin (OCN), osterix
(OSX), and runt-related transcription factor 2 (RUNX2)
was detected via reverse transcription quantitative
polymerase chain reaction (RT-qPCR) and western
blotting. The effects of heraclenin on the RhoA/ROCK
pathway were estimated through western blotting. Y27632, the ROCK inhibitor, was used to confirm the role
of the RhoA/ROCK pathway in heraclenin-mediated
osteogenic differentiation of BMSCs.
Results. Heraclenin (5-80 μM) was non-toxic on
human BMSCs. Heraclenin treatment (5-20 μM) dosedependently enhanced ALP activity and calcium
deposition. Furthermore, heraclenin promoted ALP,
OCN, OSX, and RUNX2 mRNA and protein expression.
Mechanically, heraclenin treatment increased RhoA and
ROCK1 mRNA expression, stimulated the translocation
of ROCK from the cytosolic to the membrane fraction,
and elevated the protein levels of phosphorylated cofilin
(p-cofilin) and active RhoA. Additionally, treatment with
Y-27632 overturned the promotion of heraclenin on ALP
activity, calcium deposition, the expression of
osteogenesis markers, and the RhoA/ROCK signaling
pathway.
Conclusion. Heraclenin facilitates the osteogenic
differentiation of human BMSCs through the activation
of the RhoA/ROCK pathway.
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Citation
Histology and Histopathology, Vol.39, nº8, (2024)
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