Plumbagin modulates RANKL-induced osteoclast and osteoblast differentiation

Abstract

Background: Bone remodeling is a balanced, dynamic function of two counteractive processes: osteoblasts-mediated bone formation and osteoclasts-dependent bone resorption. An imbalance between these two processes causes bone diseases. Herein, the effects and molecular modus operandi of plumbagin- a bioactive phytometabolite from Plumbago sp. have been investigated on osteoblast and osteoclast differentiation. Material and Methods: Cell viability assay and Alizarin red-S staining assessed the plumbagin effects on pre-osteoblastic MC3T3-E1 cell viability and differentiation. Alkaline phosphatase activity, the osteoblast-specific genes, and protein expression were investigated by colorimetry, quantitative-PCR and immunoblotting, respectively. TRAP assay and osteoclast-specific proteins' expression assessed plumbagin's effect on osteoclastogenesis. During plumbagin-driven osteoclastogenesis, we used paired-end RNA-sequencing data to identify the key up-/down-regulated DEGs, validation by qPCR, and bioinformatics-based pathway analysis. Results: Plumbagin at a very low dose (0.25 µM) significantly promoted osteoblast differentiation, as evident from alkaline phosphatase, matrix mineralization, and enhanced expression of osteoblast-specific markers, including Runx2 and its target genes. The reduced TRAP activity, osteoclast-specific protein expression, and RNA sequencing suggested RANKL-induced osteoclastogenesis inhibition by plumbagin at 2 µM.. Conclusion: Plumbagin significantly enhances osteoblast differentiation by enhanced matrix mineralization and osteoblast-specific gene expression but suppresses the RANKL-induced osteoclastogenesis by regulating key osteoclast-specific DEGs and protein expression. Plumbagin can thus significantly influence bone biology and affect bone diseases.

Keywords

• Bone cell differentiation
• Plumbagin
• MC3T3-E1 cell line
• NFATc1
• RNA sequencing