[논문]MC3T3-E1 조골세포주와 RAW 264.7 파골세포주에서 길경을 함유한 한약재 추출물의 항골다공증 효과

정재인; 이현숙; 김형준; 김용민; 김수현; 유동진; 김은지

doi:10.15204/jkobgy.2018.31.4.001

MC3T3-E1 조골세포주와 RAW 264.7 파골세포주에서 길경을 함유한 한약재 추출물의 항골다공증 효과
Anti-osteoporotic Activity of Mixed Herbal Extract Involving Platycodon Grandiflorum Root in Osteoblastic MC3T3-E1 and Osteoclastic RAW 264.7 Cells 원문보기

大韓韓方婦人科學會誌 = The Journal of Korean obstetrics & gynecology, v.31 no.4, 2018년, pp.1 - 15

정재인 (한림대학교 식의약품 효능평가 및 기능성소재개발센터) , 이현숙 (동서대학교 식품영양학과) , 김형준 (세명대학교 한의과대학) , 김용민 (세명대학교 화장품과학과) , 김수현 (구운식품 기업부설연구소) , 유동진 (구운식품 기업부설연구소) , 김은지 (한림대학교 식의약품 효능평가 및 기능성소재개발센터)

초록
AI-Helper

목 적: 본 연구는 길경(Platycodon grandiflorum root)를 함유한 복합 한약재 추출물(ExMH-PGR)의 골다공증 예방 및 치료효과를 알아보기 위해 MC3T3-E1 조골세포주와 RAW 264.7 파골세포주를 이용하여 in vitro 수준에서 검증하였다. 방 법: 배양액에 다양한 농도의 ExMH-PGR를 첨가하여 MC3T3-E1 세포와 RAW 264.7 세포의 세포증식을 비교하였다. MC3T3-E1 세포에서 Alkaline phosphatase (ALP) 활성, 콜라겐 합성, 오스테올칼신 생성, 무기질 축적을 분석하였다. RAW 264.7 세포에서 Tartrate-resistant acid phosphatase (TRAP) 활성과 actin ring 형성 정도를 분석하였다. 결 과: ExMH-PGR는 $25{\mu}g/mL$ 농도까지 유의한 수준으로 ALP 활성, 콜라겐 합성, 그리고 오스테올칼신 형성을 증가시켰다. $50{\sim}200{\mu}g/mL$의 ExMH-PGR은 TRAP 활성과 actin ring 형성을 유의하게 억제했다. 결 론: ExMH-PGR은 조골세포의 활성을 촉진하고 파골세포의 활성을 억제하는 효과가 있어 골다공증 치료에 유용할 가능성이 있다.

Abstract ▼ AI-Helper

Objectives: Osteoporosis is considered a serious human disease. We developed an extract of mixed herbs containing root of Platycodon grandiflorum (ExMH-PGR), which is expected to be effective in preventing or treating osteoporosis. The aim of this study was to investigate the anti-osteoporotic effect of ExMH-PGR in osteoblastic MC3T3-E1 cells and osteoclastic RAW 264.7 cells. Methods: To examine the anti-osteoporotic effect of ExMH-PGR, osteoblast and osteoclast differentiation were induced and cultured with various concentrations of ExMH-PGR. Alkaline phosphatase (ALP) activity, collagen synthesis, osteocalcin production, and mineralization in MC3T3-E1 cells were analyzed. Tartrate-resistant acid phosphatase (TRAP) activity and the formation of actin ring in RAW 264.7 cells were analyzed. Results: ExMH-PGR at concentration up to $25{\mu}g/mL$ significantly increased ALP activity, collagen synthesis, osteocalcin production, and mineralization in MC3T3-E1 cells. ExMH-PGR at 50 to $200{\mu}g/mL$ significantly inhibited TRAP activity and the formation of actin ring in RAW 264.7 cells. Conclusions: These results demonstrate that ExMH-PGR stimulates osteoblastic activities and inhibits osteoclastic activities in in vitro systems, suggesting that ExMH-PGR might be considered as an anti-osteoporotic candidate for treatment of osteoporosis disease.

주제어

표/그림 (7)

표 Table 1. Used to Prepare the Extract of Herbs Mixture [ExMH-PGR*]
그림 Fig. 1. Effect of ExMH-PGR on ALP activity in MC3T3-E1 cells. MC3T3-E1 cells were plated at 2×10⁴ cells/well and incubated for 24 h. After 24 h of incubation, cells were incubated for 5 days in OB-DIM containing various concentrations of ExMH-PGR. ALP activity was measured. Each bar represents the mean±SE. **P<0.01 significantly different from OB-DIM untreated group. Means without a common letter differ significantly at P<0.05.
그림 Fig. 2. Effect of ExMH-PGR on collagen synthesis in MC3T3-E1 cells. MC3T3-E1 cells were plated at 2×10⁴ cells/well and incubated for 24 h. After 24 h of incubation, cells were incubated for 8 days in OB-DIM containing various concentrations of ExMH-PGR. Collagen synthesis was measured. Each bar represents the mean±SE. ***P<0.001 significantly different from OB-DIM untreated group. Means without a common letter differ significantly at P<0.05.
그림 Fig. 3. Effect of ExMH-PGR on osteocalcin production in MC3T3-E1 cells. MC3T3-E1 cells were plated at 2×10⁴ cells/well and incubated for 24 h. After 24 h of incubation, cells were incubated for 14 days in OB-DIM containing various concentrations of ExMH-PGR. Media conditioned for 24 h were collected. Levels of osteoblast-secreted osteocalcin in 24 h-conditioned media were measured. Each bar represents the mean±SE. ***P<0.001 significantly different from OB-DIM untreated group. Means without a common letter differ significantly at P<0.05
그림 Fig. 4. Effect of ExMH-PGR on mineralization in MC3T3-E1 cells. MC3T3-E1 cells were plated at 2×10⁴ cells/well and incubated for 24 h. After 24 h of incubation, cells were incubated for 14 days in OB-DIM containing various concentrations of ExMH-PGR. The degree of mineralization was measured. Each bar represents the mean±SE. ***P<0.001 significantly different from OB-DIM untreated group. Means without a common letter differ significantly at P<0.05.
그림 Fig. 5. Effect of ExMH-PGR on TRAP activity in RAW 264.7 cells. RAW 264.7 cells were plated at 5×10⁴ cells/well and incubated for 24 h. After 24 h of incubation, cells were incubated for 5 days in OC-DIM containing various concentrations of ExMH-PGR. TRAP activity was measured. Each bar represents the mean±SE. **P<0.01 significantly different from OC-DIM untreated group. Means without a common letter differ significantly at P<0.05.
그림 Fig. 6. Effect of ExMH-PGR on actin ring formation in RAW 264.7 cells. RAW 264.7 cells were seeded into 24-well plates containing cover glass at 1×10⁴ cells/well and incubated for 24 h. After 24 h of incubation, cells were incubated for 5 days in OC-DIM containing various concentrations of ExMH-PGR. After 5 days, cells were fixed with 4% paraformaldehyde containing 0.1% Triton X-100 and stained with Alexa Fluor 594 Phalloidin and DAPI to stain actin and nuclei, respectively. Morphological changes of actin ring in cells were observed via microscopy (n=3; magnification, 200×).

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

제안 방법

Alizarin Red S, an anthraquinonoid derivative, was used to measure the amount of calcium deposited in osteoblasts to investigate mineralization of osteoblasts. MC3T3-E1 cells were plated and treated with ExMH-PGR as described above.
The amount of calcified is proportional to the degree of Alizarin Red staining²⁰⁾. Therefore, Alizarin red staining was performed in this study to determine the effect of ExMH-PGR on mineralization in MC3TC-E1 cells. As demonstrated in Fig.
We developed an aqueous extract of mixed herbs containing PGR (ExMH-PGR) during our search for useful ingredient to develop dietary supplement or beverage that could be effective in preventing or treating osteoporosis using natural foods and ingredients. This study was conducted to investigate the anti-osteoporotic effect of ExMH-PGR by measuring its effect on bone metabolism related biomarkers in in vitro systems using osteoblastic MC3T3-E1 cells and osteoclastic RAW 264.7 cells.
To investigate the effect of ExMH-PGR on osteoclast differentiation, we incubated RAW 264.7 cells in OC-DIM to induce osteoclast differentiation and treated cells with various concentrations of ExMH-PGR. We then measured TRAP activity as osteoclast differentiation marker.
. To investigate the effect of ExMH-PGR on the function of osteoclast, we analyzed TRAP activity and actin ring formation in RAW 264.7 cells. Osteoclasts are the cells involved in bone matrix resorption and bone and calcium homeostasis.
To investigate whether ExMH-PGR affects late process of osteoblast differentiation, we measured the production of osteocalcin and the degree of mineralization as late-stage markers of osteoblast differentiation in MC3TC-E1 cells. As shown in Fig.

대상 데이터

MC3T3-E1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in α-MEM supplemented with 10% FBS, 100,000 U/L penicillin, and 100 mg/L streptomycin at 37℃ in a humidified atmosphere of 5% CO2.
PGR and other herbs were purchased from a local herbal medicine drugstore (Wonju, Korea). Compositions and contents of herbs used in ExMH-PGR production are shown in Table 1.
Morphological changes of actin ring in cells were observed via microscopy. Photographs were taken with a Carl Zeiss AxioImager microscope (Carl Zeiss, Jena, Germany).
The reagents and materials in this study were purchased from the indicated suppliers:α-minimal essential medium (α-MEM), fetal bovine serum (FBS), penicilin/streptomycin, trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA), and Alexa Fluor 594 Phalloidin from Thermo Fisher Scientific Inc. (Waltham, MA, USA); Dulbecco’s Modified Eagle’s Medium (DMEM) from Welgene (Daegu, Korea); 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), ascorbic acid, β-glycerophosphate, and RANKL from Sigma-Aldrich Co. (St. Louis, MO, USA); TRACP & ALP assay kit and osteocalcin ELISA Kit from Takara Bio Inc. (Kusatsu, Japan); Sirius red collagen detection kit from Chondrex Inc. (Redmond, WA, USA); Osteogenesis assay kit from Millipore (Burlington, MA, USA); PD98059 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

데이터처리

Statistical significance was determined using ANOVA followed by Duncan’s multiple range test to express the difference between experimental groups.

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증