Paeonolide as a Novel Regulator of Core-Binding Factor Subunit Alpha-1 in Bone-Forming Cells

Abstract

Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects; paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 μM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 μM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 μM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 μM) increased the bone morphogenetic protein (BMP)–Smad1/5/8 and Wnt–β-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 μM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.

Biological Mechanisms of Paeonoside in the Differentiation of Pre-Osteoblasts and the Formation of Mineralized Nodules

“…It was also reported as a protective compound against sepsis-induced lethality [16]. Our group recently reported that another bioactive compound, paeonolide, identified from P. suffruticosa regulates core-binding factor subunit alpha 1 in bone-forming cells [17]. However, the biological effects and mechanisms of PASI have not yet been demonstrated in osteoblasts.…”

“…Differentiation was induced as previously described [17]. Briefly, the cells were incubated in OS containing 50 µg/mL L-AA and 10 mM β-GP, and fresh medium was replaced every two days.…”

“…Cell viability was measured using an MTT assay to detect NADH-dependent dehydrogenase activity as previously described [17].…”

“…Osteoblast differentiation was induced using OS containing with PASI for seven days. ALP activity and staining assay were performed as previously described [17].…”

“…Paeonoside (PASI) Colorless amorphous powder; EI-MS m/z = 328.31 [M] + , molecular formula C 15 H 20 O 8 ; 1 H-NMR (500 MHz, CD 3 OD) δ: 7.71 (1H, dd, J = 1.4, 8.9 Hz, H-6), 6.83 (1H, s, H-3), 6.62 (1H, dd, J = 2.3, 8.9 Hz, H-5), 5.04 (1H, d, J = 7.8 Hz, H-1 ), 3.90 (1H, d, J = 12.1 Hz, H-6 b), 3.82 (3H, s, -OCH3), 3.68 (1H, m, H-6 a), 3.51 (3H, m, H-2 , 5 , 3 ), 3.37 (1H, m, H-4 ), 2.62 (3H, s, H-8); 13 C-NMR (125 MHz, CD 3 OD) δ: 200.7 (C-7), 166.4 (C-4), 160.7 (C-2), 133.1 (C-6), 122.4 (C-1), 109.6 (C-5), 102.7 (C-3), 102.6 (C-1 ), 78.6 (C-3 ), 78.4 (C-5 ), 74.9 (C-2 ), 71.4 (C-4 ), 62.7 (C-6 ), 56.3 (4-OCH3), 32.3 (C-8).4.4. Cell Culture of Pre-Osteoblast MC3T3-E1MC3T3-E1 cells were purchased from the American Type Culture Collection (ATCC, CRL-2593) (Manassas, VA, USA) and cultured as previously described[17].…”