Curculigoside regulates proliferation, differentiation, and pro-inflammatory cytokines levels in dexamethasone-induced rat calvarial osteoblasts

Abstract

Background: Curculigoside (CCG), one of the main bioactive phenolic compounds isolated from the rhizome of Curculigo orchioides Gaertn., is reported to prevent bone loss in ovariectomized rats. However, the underlying molecular mechanisms are largely unknown. Therefore, we investigated the effects of CCG on proliferation and differentiation of calvarial osteoblasts and discussed the related mechanisms.

Materials and methods: Osteoblasts were incubated with dexamethasone (DEX) in the absence or presence of CCG concentrations for 24-72 h. Cell proliferation was evaluated by Cell Counting Kit-8 assay. Mitochondria membrane potential (MMP) and reactive oxygen species (ROS) were assessed by flow cytometry. We assessed the anti-inflammatory responses of CCG on DEX-induced osteoblasts by an enzyme-linked immunosorbent assay (ELISA). Relative protein expression of BMP-2, b-catenin, RANKL, OPG and RANK was measured using Western blotting.

Results: It was found that osteoblasts proliferation decreased significantly after treated with 1 μM of dexamethasone (DEX), compared with untreated osteoblasts and the cytotoxic effect of DEX was reversed remarkably when pretreatment with 25-100 μg/ml of CCG. Pretreatment with 25-100 μg/ml of CCG increased MMP level and decreased ROS production in osteoblasts induced by DEX. In addition, DEX-induced inhibition of differentiation markers such as alkaline phosphatase (ALP), OPG, BMP-2, β-catenin, IGF-1 and M-CSF level, and promotion of differentiation markers such as RANKL and RANK was significantly reversed in the presence of CCG. CCG also reversed DEX-induced production of pro-inflammatory cytokines.

Conclusions: These results provide new insights into the osteoblast-protective mechanisms of CCG through inducing proliferation and differentiation and reducing the inflammatory responses, indicating that CCG may be developed as an agent for the prevention and treatment of osteoporosis.

Keywords: Curculigoside; calvarial osteoblasts; osteoporosis; pro-inflammatory cytokines; proliferation.

Figure 1

The chemical structure of curculigoside (CCG, C₂₂H₂₆O₁₁).

Figure 2

Protective effects of CCG on DEX-induced cell injury in osteoblasts. A. Dose-dependent effect of DEX on cell proliferation. DEX at 1 μM significantly reduced cell proliferation after 72 h of incubation. B. Pretreatment with CCG (25, 50 and 100 μg/ml, 24 h) alleviated DEX-induced cell injury. The data were presented as the mean ± SD (n=3); #P<0.05 vs. control without any treatment group, *P<0.05 vs. the DEX only group.

Figure 3

The activity of ALP is altered by CCG under DEX condition. A. The cells were treated with medium containing 0, 0.01, 0.1, 1 or 10 μM of DEX and the activity of ALP was decreased. B. The cells were pretreated with 25, 50 and 100 μg/ml of CCG for 24 h, and then incubated with an additional 1 μM of DEX. The data were presented as the mean ± SD (n=3); #P<0.05 vs. control without any treatment group, *P<0.05 vs. the DEX only group.

Figure 4

The effects of CCG on the expression of osteoblastic differentiation associated markers. A, B. Western blotting (left) and quantification (right) of BMP-2, b-catenin, RANKL, OPG and RANK expression profiles in five treatment groups. C, D. ELISA analyzed IGF-1 and M-CSF protein expression in five treatment groups. The data were presented as the mean ± SD (n=3); #P<0.05 vs. control without any treatment group, *P<0.05 vs. the DEX only group.

Figure 5

The effects of CCG on MMP and ROS in DEX-induced osteoblasts. A. DEX-induced osteoblasts were pretreated with CCG for 24 h at 25, 50 and 100 μg/mL respectively, then incubated with TMRM and analyzed by flow cytometry. B. DEX-induced osteoblasts were pretreated with CCG for 24 h at 25, 50 and 100 μg/mL respectively, and fluorescence probe DCFH-DA was used to determine the levels of ROS production. The data were presented as the mean ± SD (n=3); #P<0.05 vs. control without any treatment group, *P<0.05 vs. the DEX only group.

Figure 6

The effects of CCG on the expression of pro-inflammatory cytokines. ELISA analysis of TNF-α (A), IL-1b (B), IL-6 (C) and COX-2 (D) protein expression in five treatment groups. The data were presented as the mean ± SD (n=3); #P<0.05 vs. control without any treatment group, *P<0.05 vs. the DEX only group.