[논문]보골지 추출물이 파골세포 분화 및 골흡수 관련 유전자 발현에 미치는 영향

류광현; 김엄지; 김민선; 김재현; 이유진; 진대환; 손영주; 정혁상

doi:10.14406/acu.2021.016

보골지 추출물이 파골세포 분화 및 골흡수 관련 유전자 발현에 미치는 영향
Psoraleae Semen Ethanol Extract Inhibits RANKL-Induced Osteoclast Differentiation and Osteoclast Specific Genes Expression 원문보기

Korean journal of acupuncture, v.38 no.3, 2021년, pp.140 - 150

류광현 (경희대학교 한의과대학 해부학교실) , 김엄지 (경희대학교 한의과대학 해부학교실) , 김민선 (경희대학교 한의과대학 해부학교실) , 김재현 (경희대학교 한의과대학 해부학교실) , 이유진 (경희대학교 한의과대학 해부학교실) , 진대환 (경희대학교 한의과대학 해부학교실) , 손영주 (경희대학교 한의과대학 해부학교실) , 정혁상 (경희대학교 한의과대학 해부학교실)

Abstract ▼ AI-Helper

Objectives : The increase of osteoclasts could cause osteoporosis and bone-related diseases. Also, the inhibition of osteoclast differentiation is important in treating bone-related diseases. Traditionally, Psoraleae Semen has been used for geriatric diseases, aging and musculoskeletal diseases. The purpose of this study is to investigate the effect of Psoraleae Semen ethanol extract (PS) on osteoclast differentiation and its function. Methods : To confirm the effect of PS on osteoclastogenesis and bone resorption activity, various levels of concentrations of PS (5, 10, 20 and 40 ㎍/ml) were tested on RAW 264.7 cells cultured with RANKL. We measured tartarate-resistant acid phosphatase (TRAP)-positive cells, TRAP activity, pit formation and F-actin ring formation. The expressions of nuclear factor of activated T-cells (NFATc1) and c-Fos were confirmed through western blot and reverse transcription- polymerase chain reaction (RT-PCR). Also, the expression of bone resorption and fusion-related genes in osteoclast was confirmed by RT-PCR. Results : PS decreased the number of TRAP-positive cells and the TRAP activity. In addition, PS significantly inhibited the formation of pit and F-actin ring. Furthermore, PS decreased the expression of osteoclast related genes. Conclusions : PS inhibits osteoclast differentiation and bone resorption ability through inhibition of the expression of osteoclast-related genes. This indicates that PS may be a potential therapeutic agent to osteoporosis by suppressing osteoclastogenesis.

주제어

표/그림 (7)

그림 Fig. 1. Effect of PS on the cell viability in RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations of PS (5, 10, 20 and 40 μg/ml) for 24 h. Cell viability was determined using the CCK-8 assay. Data are expressed as the mean±SEM of three independent experiments.
표 Table 1. Primer sequence for RT-PCR analysis
그림 Fig. 2. Effect of PS on RANKL-induced osteoclast differentiation and bone resorption. RAW 264.7 cells were treated with RANKL (100 ng/ml) and PS (5, 10, 20 and 40 μg/ml) for 5 days. (A) RANKL-induced osteoclasts were stained with a TRAP staining kit. Images were captured using by microscope (magnification, ×100; scale bar 200 μm). (B) The number of TRAP-positive cells were counted. (C) TRAP activity was measured by ELISA reader. (D) Images were captured using by microscope (magnification, ×200; scale bar, 100 μm). (E) The pit area was measured by Image J software. ##p<0.01 vs. the normal cells (RANKL-untreated cells) and *p<0.05 and **p<0.01 vs. the control cells (RANKL-treated cells). Data are expressed as the mean±SEM of three independent experiments.
그림 Fig. 3. Effect of the PS on RANKLinduced F-actin ring formation. RAW 264.7 cells were treated with RANKL (100 ng/ml) and PS (5, 10, 20 and 40 μg/ml) for 5 days. Factin ring was observed with a fluorescence microscope (magnification, ×200; scale bar, 100 μm).
그림 Fig. 4. Effect of PS on the protein expression levels of NFATc1 and c-Fos. RAW 264.7 cells were treated with RANKL (100 ng/ml) and PS (5, 10, 20 and 40 μg/ml) for 24 h. (A) The protein expression of the NFATc1 was analyzed by western blot and (B) normalized by actin. (C) The protein expression of the c-Fos was analyzed by western blot and (D) normalized by actin. #p<0.05 and ##p<0.01 vs. the normal cells (RANKL-untreated cells) and *p< 0.05 vs. the control cells (RANKLtreated cells). Data are expressed as the mean±SEM of three independent experiments.
그림 Fig. 5. Effect of PS on the expression of osteoclast differentiation-related genes. RAW 264.7 cells were treated with RANKL (100 ng/ml) and PS (5, 10, 20 and 40 μg/ml) for 4 days. (A) Expression of mRNA levels the NFATc1, c-Fos, TRAP and RANK were analyzed by RT-PCR. (B) The mRNA expression was normalized by GAPDH. #p<0.05 and ##p<0.01 vs. the normal cells (RANKL-untreated cells) and *p<0.05 and **p<0.01 vs. the control cells (RANKL-treated cells). Data are expressed as the mean±SEM of three independent experiments.
그림 Fig. 6. Effect of PS on the mRNA expression of bone resorption (CTK, MMP-9 and CA2) osteoclast fusion genes (OSCAR, ATP6v0d2 and DC-STAMP). RAW 264.7 cells were treated with RANKL (100 ng/ml) and PS (5, 10, 20 and 40 μg/ml) for 4 days. (A) The expression of bone resorption and osteoclast fusion genes were analyzed by RT-PCR. (B) The mRNA expression was normalized by GAPDH. #p<0.05 and ##p<0.01 vs. the normal cells (RANKL-untreated cells) and *p<0.05 and **p<0.01 vs. the control cells (RANKL-treated cells). Data are expressed as the mean±SEM of three independent experiments.

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