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풀무치 에탄올 추출물이 MG-63 조골세포 분화에 미치는 영향

Osteoblastogenic Activity of Locusta migratoria Ethanol Extracts on Pre-Osteoblastic MG-63 Cells

  • 백민희 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 서민철 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 이준하 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 김인우 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 김미애 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 황재삼 (농촌진흥청 국립농업과학원 농업생물부)
  • Baek, Minhee (Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Seo, Minchul (Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Lee, Joon Ha (Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Kim, In-Woo (Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Kim, Mi-Ae (Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Hwang, Jae-Sam (Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration)
  • 투고 : 2018.09.10
  • 심사 : 2018.11.07
  • 발행 : 2018.12.30

초록

최근 들어 곤충을 식품 및 바이오 소재로 이용한 연구가 활발히 진행되고 있다. 그러나 곤충을 이용한 조골세포 활성 및 분화에 따른 골 형성 촉진 효과에 대한 연구는 아직 미흡한 실정이다. 뿐만 아니라 풀무치를 이용한 기능성 연구는 거의 이루어지지 않고 있다. 따라서 본 연구에서는 골 형성 촉진 효능을 가진 새로운 천연물 소재 개발을 위해 풀무치 추출물의 MG-63 조골세포의 분화 촉진 효과를 연구하였다. 조골세포에서 풀무치 추출물의 독성 및 증식 효과를 평가하기 위하여 MTS assay를 진행한 결과, $1,000{\mu}g/ml$ 농도까지 세포 독성이 나타나지 않았으며, 48시간 배양했을 때 $500-1,000{\mu}g/ml$ 농도에서 105%와 116%의 세포 증식 효능을 확인 하였다. 풀무치 추출물이 조골세포 분화에 미치는 영향을 확인하기 위하여 3일 및 5일간 풀무치 추출물을 MG-63 조골세포에 처리한 후 ALP 활성을 측정하였다. 그 결과 $100{\mu}g/ml$ 농도에서 positive control로 사용한 조골세포 분화배지(DM)군과 유사한 정도로 분화가 증가하였으며 500 및 $1,000{\mu}g/ml$ 농도에서는 2-3배까지 조골세포 분화가 촉진되었다. 이 결과는 ALP staining에서도 유사하게 나타났다. mRNA 발현량의 변화를 측정한 결과, Alpl과 Runx2 유전자 발현량이 증가하였고, 단백질 발현량을 측정했을 때에도 유사한 결과를 확인하였다. 이를 통해 ALP와 Runx2 유전자 및 단백질 발현에 의해서 ALP 활성이 증가하고 조골세포 분화가 촉진되었을 것으로 판단되며, 풀무치 추출물을 이용한 골 형성 촉진에 따른 골다공증 예방 및 치료 기능성 소재 개발에 대한 가능성을 확인하였다.

Insects have been investigated as a novel source of food and biomaterial in several recent studies. However, their osteoblastogenic cell activity has not been sufficiently researched and so, to investigate the potential of this natural material for promoting osteoblastogenesis, we studied the activity of Locusta migratoria ethanol extract (LME) on MG-63 pre-osteoblast cells. The cytotoxicity and proliferation effects of LME on MG-63 cells were measured by MTS assay, and there was no cytotoxicity up to $1,000{\mu}g/ml$. With LME treatment of 500 and $1,000{\mu}g/ml$ for 48 hr, cell proliferation increased to 105% and 116% versus control, respectively. The osteoblastogenic activity of the LME was measured through alkaline phosphatase (ALP) staining at three and five days. As a result, both 500 and $1,000{\mu}g/ml$ LME concentrations were seen to increase ALP activity by more than three times compared with control at three and five days. In addition, the expression level of the osteogenic markers ALP and RUNX2 was markedly increased after LME treatment. These results demonstrate that Locusta migratoria ethanol extract promotes osteoblastogenesis as evidenced by the increased osteogenic markers and suggest that LME may be a potential agent for bone formation and osteoporosis prevention.

키워드

SMGHBM_2018_v28n12_1448_f0001.png 이미지

Fig. 1. Effect of Locusta migratoria extract on the osteoblastic cell cytotoxicity.

SMGHBM_2018_v28n12_1448_f0002.png 이미지

Fig. 2. Effect of Locusta migratoria extract on the ALP activity and staining of osteoblastic cells.

SMGHBM_2018_v28n12_1448_f0003.png 이미지

Fig. 3. Effect of Locusta migratoria extract on osteoblatogenesis related gene expression.

SMGHBM_2018_v28n12_1448_f0004.png 이미지

Fig. 4. Effect of Locusta migratoria extract on osteoblatogenesis related protein expression.

Table 1. The sequences of primers for RT-PCR

SMGHBM_2018_v28n12_1448_t0001.png 이미지

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