Nutr Res Pract.  2016 Apr;10(2):148-153. 10.4162/nrp.2016.10.2.148.

Effects of Scytosiphon lomentaria on osteoblastic proliferation and differentiation of MC3T3-E1 cells

• Park MH
• Kim S
• Cheon J
• Lee J
• Kim BK
• Lee SH
• Kong C
• Kim YY
• Kim M

• KMID: 2313911
• DOI: http://doi.org/10.4162/nrp.2016.10.2.148

Abstract

BACKGROUND/OBJECTIVES
Bone formation and bone resorption continuously occur in bone tissue to prevent the accumulation of old bone, this being called bone remodeling. Osteoblasts especially play a crucial role in bone formation through the differentiation and proliferation. Therefore, in this study, we investigated the effects of Scytosiphon lomentaria extract (SLE) on osteoblastic proliferation and differentiation in MC3T3-E1 cells.
MATERIALS/METHODS
A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and protein expression analysis of osteoblastic genes were carried out to assess the osteoblastic proliferation and differentiation.
RESULTS
The results indicated that treatment of SLE promoted the proliferation of MC3T3-E1 cells and improved ALP activity. And, SLE treatment significantly promoted mineralized nodule formation compared with control. In addition, cells treated with SLE significantly upregulated protein expression of ALP, type 1 collagen, bone morphogenetic protein 2, runt-related transcription factor 2, osterix, and osteoprotegerin.
CONCLUSIONS
The results demonstrate that SLE promote differentiation inducement and proliferation of osteoblasts and, therefore may help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.

Keyword

Scytosiphon lomentaria; MC3T3-E1 cell; differentiation; alkaline phosphatase; mineralization

MeSH Terms

Alkaline Phosphatase
Bone and Bones
Bone Morphogenetic Protein 2
Bone Remodeling
Bone Resorption
Cell Proliferation
Collagen Type I
Osteoblasts*
Osteogenesis
Osteoprotegerin
Transcription Factors
Alkaline Phosphatase
Bone Morphogenetic Protein 2
Collagen Type I
Osteoprotegerin
Transcription Factors

Figure

• 

  Fig. 1 Effect of SLE on the proliferation in MC3T3-E1 cells. Cells were treated with SLE at 10, 50 and 100 µg/ml for 2 days. Cell proliferation was measured using the MTT assay. Data are expressed as percentage of control. Each value is expressed as mean ± SD (n = 3). a-dValues with different letters were significantly different at P < 0.05, as analyzed by Duncan's multiple range test. SLE: Scytosiphon lomentaria extract.

• 

  Fig. 2 Effect of SLE on alkaline phosphatase (ALP) activity in MC3T3-E1 cells. Cells were treated with SLE at 10, 50 and 100 µg/ml for 2 days. Data are expressed as percentage of control. Each value is expressed as mean ± SD (n = 3). a-cValues with different letters were significantly different at P < 0.05, as analyzed by Duncan's multiple range test. SLE: Scytosiphon lomentaria extract.

• 

  Fig. 3 Effect of SLE on mineralization in MC3T3-E1 cells. (A) The mineralized matrix was stained with Alizarin red S. (B) Quantitation of mineralization using Alizarin red staining, as described in the materials and methods section. Each value is expressed as mean ± SD (n = 3). a-cValues with different letters were significantly different at P < 0.05, as analyzed by Duncan's multiple range test. SLE: Scytosiphon lomentaria extract.

• 

  Fig. 4 Effects of SLE on osteoblast differentiation in MC3T3-E1 cells. Cells were treated with SLE at 10, 50 and 100 µg/ml for 2 days. (A) The protein expression of osteoblast differentiation factors, such as ALP, and Col 1 was detected by western blot. (B) Relative expression was quantified by densitometry using the Multi Gauge V3.1 and calculated according to the reference bands of β-actin. Each value is expressed as mean ± SD (n = 3). a-dValues with different letters were significantly different at P < 0.05, as analyzed by Duncan's multiple range test. SLE: Scytosiphon lomentaria extract.

• 

  Fig. 5 Effects of SLE on the OPG/RANKL ratio in MC3T3-E1 cells. Cells were treated with SLE at 10, 50 and 100 µg/ml for 2 days. (A) The protein expression of OPG/RANKL ratio, such as OPG and RANKL was detected by western blot. (B) Relative expression was quantified by densitometry using the Multi Gauge V3.1 and calculated according to the reference bands of β-actin. Each value is expressed as mean ± SD (n = 3). a-cValues with different letters were significantly different at P < 0.05, as analyzed by Duncan's multiple range test. SLE: Scytosiphon lomentaria extract.

• 

  Fig. 6 Effects of SLE on the BMP signaling pathway in MC3T3-E1 cells. Cells were treated with SLE at 10, 50 and 100 µg/ml for 2 days. (A): The protein expression of the BMP signaling pathway related markers, such as BMP-2, Runx2, and Osx was detected by western blot. (B): Relative expression was quantified by densitometry using the Multi Gauge V3.1 and calculated according to the reference bands of β-actin. Each value is expressed as mean ± SD (n = 3). a-dValues with different letters were significantly different at P < 0.05, as analyzed by Duncan's multiple range test. SLE: Scytosiphon lomentaria extract.

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