The Fruit of Poncirius Trifoliata Promotes Mesenchymal Stem Cell Differentiation into Osteoblasts in Glucocorticoid-induced Osteoporosis Model

Abstract

The fruit of Poncirus trifoliata is known to have effects on digestive system, cardiovascular system, and kidney function. The authors studied the effects of ethyl acetate extract of poncirus trifoliata on the activities of osteoblast and animal model. Effects of osteoblastic differentiation were measured by alkaline phosphatase (ALP) activity, osteopontin (OPN) protein expression and mineral nodule formation in dexamethasone (DEX)-treated MC3T3-E1 cells and primary bone marrow mesenchymal stem cells (BMSCs). Five fractions of ethyl acetate extract of poncirus trifoliata through column chromatography over a silica gel eluting in gradient system and combined based on their thin layer chromatography (TLC) pattern. Fourth subfraction (E4) of ethyl acetate extract was further purified by column chromatography over MCI gel eluting, then it was purified by repeated silica gel column chromatography to give naringin and poncirin, respectively. Bone mineral density (BMD) was measured before and after treatment with ethyl acetate extract, E4 and poncirin in glucocorticoid-induced osteoporotic (GIO) mice. In evaluation of bone microarchitecture, it was tested whether E4 and poncirin affects trabecular bone structure in GIO mice using micro-CT. The E4 and poncirin significantly increased trabecular bone structure parameters in GIO mice. A biomarker of bone-formation, bone-alkaline phosphatase (BALP), osteocalcin (OC), and a biomarker of bone-resorption, C-terminal telopeptides of type I collagen (CTX), were evaluated in the serum of the GIO mice. We surmised that if poncirin induce mesenchymal stem cell differentiation into osteoblast, it might contribute to increase bone mass. To address this issue, poncirin treated in C3H10T1/2 mesenchymal stem cell lines and primary BMSCs. Treatment of C3H10T1/2 cells and BMSCs with poncirin induced osteoblast differentiation through runt-related transcription factor2 (Runx2) expression and transcriptional activity, transcriptional coactivator with PDZ-binding motif (TAZ), OC expression and mineral nodule formation. Poncirin prevented adipocyte differentiation via inhibition of lipid droplet accumulation, PPAR-γ, and C/EBP-β expression. Also poncirin prevented osteoclast differentiation via inhibition of TRAP-positive MNCs, and regulation of OPG/RANKL ratio. During mesenchymal stem cell differentiation, DEX inhibited osteoblast differentiation through inhibition of ALP activity, Runx2, OC, osteoprotegerin (OPG) expression and mineral nodule formation. However, poncirin induced mesenchymal stem cell differentiation into osteoblasts in DEX-treated C3H10T1/2 cells. It was tested that whether DEX inhibited Runx2 expression through smurf1 expression in C3H10T1/2 cells. DEX induced Runx2 degradation through enhancement of smurf1 expression in the levels of mRNA and protein. DEX-induced Runx2 degradation was recovered by transfection of Smad ubiquitination regulatory factor1 (Smurf1) siRNA.