Eucommia ulmoides Oliver polysaccharide alleviates glucocorticoid-induced osteoporosis by stimulating bone formation via ERK/BMP-2/SMAD signaling

Abstract

Osteoporosis (OP) is a metabolic disease characterized by low bone mineral mass owing to osteoblast dysfunction. Eucommia ulmoides Oliver (EuO) is a Chinese herbal medicine traditionally used to treat OP. Here, a polysaccharide purified from the EuO cortex (EuOCP3) was administered to OP mice constructed with dexamethasone (Dex) to investigate its anti-OP activity. EuOCP3 significantly improved Dex-induced bone microarchitecture destruction, increased osteoblast numbers and surface, and stimulated an increase in the expression of osteoblast differentiation markers in the femurs of OP mice. Furthermore, EuOCP3 was applied to MC3T3-E1 cells to further explore its effects on osteoblast differentiation. EuOCP3 significantly promoted osteoblast differentiation and increased the level of phosphorylated extracellular signal-regulated kinase1/2 (ERK1/2) and SMAD1/5/8. The EuOCP3-mediated enhancement of osteoblast differentiation-related proteins and phosphorylated SMAD1/5/8 expression levels was strongly suppressed by an ERK inhibitor (PD98059), which confirmed the critical role of ERK signaling in EuOCP3-induced osteoblast differentiation. In summary, EuOCP3 can stimulate bone formation by improving osteoblast differentiation via ERK/BMP-2/SMAD signaling, indicating the potential use of EuOCP3 as a functional ingredient in food products for anti-OP treatment.

Keywords: Eucommia ulmoides Oliver; ERK/BMP-2/SMAD signaling; Osteoblast differentiation; Osteoporosis; Polysaccharide.

Fig. 1

EuOCP3 improved Dex-induced microarchitecture destruction in femoral tissue. (a) Sagittal section images and (b) trabecular bone images in femoral tissue. (c) OCN staining (100 × ; scale bar: 100 μm and 400 × ; scale bar: 20 μm). (d) Tb.BV/TV, (e) Tb.BS/BV and (f) Tb.Th (n = 3). The osteoblasts in femurs were analyzed via histomorphometric analysis parameters including (g) N.Ob/B.Pm and (h) Ob.S/BS (n = 6). Data are analyzed using a one-way ANOVA and shown as the mean ± SEM. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. CTRL mice; * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. vehicle-treated OP mice.

Fig. 2

EuOCP3 promoted osteoblast differentiation in OP mice. EuOCP3 increased the levels of (a) BMP-2, (b) PICP, and (c) OCN in the serum of OP mice (n = 8). EuOCP3 increased the expression of (d) RUNX2, (e) Osterix, and (f) OPN in the femurs of OP mice (400 × ; scale bar: 20 μm). Quantification of IHC staining include (g) RUNX2, (h) Osterix, and (i) OPN (n = 6). Data are analyzed using a one-way ANOVA and shown as the mean ± SEM. ^# P < 0.05, ^### P < 0.001 vs. CTRL mice; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. vehicle-treated OP mice.

Fig. 3

EuOCP3 regulated the factors related to osteoblast differentiation in femurs of OP mice. EuOCP3 enhanced the expressions of RUNX2, Osterix, OPN and OCN in femur of OP mice analyzed via western blotting. Data are analyzed using a one-way ANOVA and shown as the mean ± SEM (n = 3). ^### P < 0.001 vs. CTRL mice; ** P < 0.01, and *** P < 0.001 vs. vehicle-treated OP mice.

Fig. 4

EuOCP3 promoted osteoblast differentiation in MC3T3-E1 cells. EuOCP3 increased the expression of ALP in MC3T3-E1 cells after (a) 7 day (n = 3) and (b) 14-day co-culture. Data are analyzed using a one-way ANOVA and shown as the mean ± SEM. *** P < 0.001 vs. CTRL cells.

Fig. 5

EuOCP3 promoted osteoblast differentiation in MC3T3-E1 cells. EuOCP3 promoted the expression levels of (a) osteoblast differentiation markers and increased the levels of (b) phosphorylated ERK1/2 and SMAD1/5/8 in MC3T3-E1 cells at doses of 10 and 20 μg/mL. Data are analyzed using a one-way ANOVA and shown as the mean ± SEM (n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. CTRL cells.

Fig. 6

ERK inhibitor prevented the osteoblast differentiation induced by EuOCP3 in MC3T3-E1 cells. ERK inhibitor (PD98059) prevented the expression of (a) ALP and (b) BMP-2, RUNX2, Collagen I, OPN, and OCN in EuOCP3-exposed MC3T3-E1 cells. (c) PD98059 prevented the phosphorylation of ERK1/2 and SMAD1/5/8 in EuOCP3-exposed MC3T3-E1 cells. Data are analyzed using a one-way ANOVA and shown as the mean ± SEM (n = 3). *** P < 0.001 vs. CTRL cells; ^### P < 0.001 vs. EuOCP3-treated cells.