ODP599 Modulation of Wnt/b-catenin and Autophagy in an in vitro Model of Glucocorticoid-induced Osteoporosis
Authors: Mannino Federica , Doc Post 
Published: November 2022

Abstract

Background
During bone aging and osteoporosis, formation and resorption are not tightly coupled; the main cells involved in bone remodeling are osteoblasts and osteoclast but also osteocytes play a pivotal role in this process. Osteocytes are the most abundant cell type in bone and are mainly responsible for sensing mechanical signals on the bones, controlling osteoblast and osteoclast activities through cell-to-cell communication and via secreted factors. In particular, osteocytes regulate bone resorption, thanks to the production of RANKL, reducing osteoclast activity. Ellagic acid, a natural polyphenolic compound derived from pomegranate, could modulate cell function via specific estrogen receptor b; activation of ERb plays a critical role in bone remodeling suppressing osteoclast differentiation and function, promoting osteoblast proliferation through the Wnt/b-catenin pathway, increasing osteoprotegerin levels. In addition, stimulation of ERb plays an anti-inflammatory effect, increasing the expression of IL-10 and reducing the expression of proinflammatory cytokines, such as IL-1β and TNF-α, and can regulate the expression of autophagy inhibiting the PI3K/Akt/mTOR pathway. The hypothesis here tested is that ellagic acid, through ERb modulation, could induce Wnt/b-catenin and autophagy pathways in osteocytes challenged with dexamethasone to mimic glucocorticoid-induced osteoporosis.

Materials and methods
The osteocyte cells MLO-A5 were differentiated into osteocytes under appropriate culturing conditions. Cells were treated with Ellagic acid (1µM) following dexamethasone (1µM) challenge for 24h to induce an in vitro model of osteoporosis. At the end of the treatment period, cell viability was evaluated by MTT assay; qPCR was performed to evaluate the expression of the genes involved in osteocyte function (SOST, RANKL, Destrin and Dmp1) and Wnt/B-catenin signaling pathway (Wnt5a, Wnt10b, B-Catenin and DKK1); Western Blot was performed to evaluate the expression of proteins involved in apoptosis (cleaved-Caspase3) and autophagy (Beclin-1, LC3 and p62). In addition, the expression of Sclerostin and nuclear translocation of B-Catenin was evaluated by immunofluorescence.

Results
Ellagic acid reduced the gene expression of SOST, RANKL, Dmp1 and increased the expression of Destrin compared to untreated cells stimulated with dexamethasone; caused a significant increase of Wnt5a, Wnt10b and B-Catenin expression and reduced significantly the expression of DKK1. CGS21680 inhibited dexamethasone-induced apoptosis, reducing the expression of Caspase3, and increased the expression of Beclin-1 and LC3 compared to cell treated with dexamethasone. Finally, treatment with Ellagic acid stimulated the nuclear translocation of B-Catenin, promoting the transcription of genes involved in osteogenesis.

Conclusion
These preliminary data suggest that stimulation of ERb through Ellagic acid could modulate bone remodelling through activation of Wnt/b-catenin pathway and autophagy, providing evidence on the possible use of this natural compound as a new therapeutic approach for osteoporosis.
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