Asiatic acid methyl ester, a new asiaticoside derivative, induces osteogenic differentiation of hPDLCs

Abstract

Objective: Asiaticoside has the capacity to induce osteogenic differentiation of human periodontal ligament cells (hPDLCs) through Wnt (Wingless-related integration site) signaling. A modified chemical structure (by removing glycoside side chain), referred to as asiatic acid methyl ester (AA1), has been constructed and evaluated for its capacity to induce osteogenic differentiation.

Design: hPDLCs viability was determined by MTT assay. The mRNA expression were analyzed by using quantitative real time PCR. The subcellular localization of β-catenin was demonstrated by immunofluorescence staining. hPDLCs were cultured in osteogenic medium and the deposited calcium was detected by alizarin red staining. The protein expressions were examined by immunofluorescence and western blot.

Results: AA1 at a concentration 50 µM or below was not toxic to hPDLCs whereas asiaticoside had revealed no impact on cell viability at any concentration tested. The results showed that 2.5 µM of AA1 and 100 µM of asiaticoside significantly induced gene expression of osterix and dentin matrix protein1 (DMP1), concomitant with a formation of mineralized nodules. AA1 activated Wnt signaling as shown by an increase of WNT3A expression and nuclear translocation of β-catenin. This activation was effectively inhibited when cells were pretreated with recombinant human Dickkopf1 (rhDKK1). rhDKK1 significantly attenuated the AA1-induced expression of DMP1 at both mRNA and protein level.

Conclusions: These results confirm a common mechanism between asiaticoside and AA1 in fostering osteogenic differentiation of hPDLCs with a higher potency of AA1. In summary, this study has introduced a novel derivative of asiaticoside, which potentially enhance therapeutic effectiveness for periodontal regeneration.

Keywords: Asiatic acid methyl ester; Asiaticoside; Osteogenic differentiation; Periodontal ligament cells; Wnt.