Objectives : Osteoporosis is the most common bone disease and osteoporosis fracture is the leading cause of decreased life. Bisphosphonate and selective estrogen receptor modulators are the best choice of treatment for osteoporosis. However, when used for a long time, they increase the probability o...
Objectives : Osteoporosis is the most common bone disease and osteoporosis fracture is the leading cause of decreased life. Bisphosphonate and selective estrogen receptor modulators are the best choice of treatment for osteoporosis. However, when used for a long time, they increase the probability of side effect such as osteonecrosis of the jaw. Thus, it is crucial to develop alternative medicine to treat osteoporosis. Gentianae Macrophyllae Radix, a herbal medicine, is mainly to treat rheumatoid arthritis. However, the effect of the water extract of Gentianae Macrophyllae Radix (w-GM) on osteoporosis has not been investigated. Thus, we examine whether w-GM can inhibit osteoclast differentiation and bone resorption on receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-treated RAW 264.7 cells. In this study, RAW 264.7 cells were used as an osteoclast differentiation model by treating them with RANKL. Methods : RAW 264.7 cells were used to determine the effect of w-GM on osteoclast differentiation and bone resorption. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells, TRAP activity and pit formation assay were examined. In addition, protein expressions were measured by western blot and mRNA expressions were analyzed by reverse transcription polymerase chain reaction. Results : Treatment with w-GM inhibited the number of TRAP-positive cells, TRAP activity and pit area. In addition, w-GM decreased protein expression such as mitogen-activated protein kinase, NF-κB, c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). It also inhibited the mRNA levels such as c-Fos, NFATc1, TRAP, NF-κB, calcitonin receptor and cathepsin K in RANKL-treated RAW 264.7 cells. Conclusions : These results suggest that w-GM has inhibitory effects via osteoclast differentiation, thus it could be a new medication for osteoporosis.
Objectives : Osteoporosis is the most common bone disease and osteoporosis fracture is the leading cause of decreased life. Bisphosphonate and selective estrogen receptor modulators are the best choice of treatment for osteoporosis. However, when used for a long time, they increase the probability of side effect such as osteonecrosis of the jaw. Thus, it is crucial to develop alternative medicine to treat osteoporosis. Gentianae Macrophyllae Radix, a herbal medicine, is mainly to treat rheumatoid arthritis. However, the effect of the water extract of Gentianae Macrophyllae Radix (w-GM) on osteoporosis has not been investigated. Thus, we examine whether w-GM can inhibit osteoclast differentiation and bone resorption on receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-treated RAW 264.7 cells. In this study, RAW 264.7 cells were used as an osteoclast differentiation model by treating them with RANKL. Methods : RAW 264.7 cells were used to determine the effect of w-GM on osteoclast differentiation and bone resorption. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells, TRAP activity and pit formation assay were examined. In addition, protein expressions were measured by western blot and mRNA expressions were analyzed by reverse transcription polymerase chain reaction. Results : Treatment with w-GM inhibited the number of TRAP-positive cells, TRAP activity and pit area. In addition, w-GM decreased protein expression such as mitogen-activated protein kinase, NF-κB, c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). It also inhibited the mRNA levels such as c-Fos, NFATc1, TRAP, NF-κB, calcitonin receptor and cathepsin K in RANKL-treated RAW 264.7 cells. Conclusions : These results suggest that w-GM has inhibitory effects via osteoclast differentiation, thus it could be a new medication for osteoporosis.
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문제 정의
본 연구는 淸虛熱, 淸濕熱 등의 효능을 지닌 진교가 골다공증에 있어 주요 발병 원인인 과도한 파골세포의 분화 억제에 효과가 있을 것이라 생각되어 진행하였고, 유의미한 결과를 얻었기에 보고하는 바이다.
가설 설정
(A) TRAP-positive osteoclasts and pit area were identified with microscope (40×). (B) TRAP-positive cells were counted as osteolcast. (C) Supernatants were collected, and the TRAP activity in the supernatants was determined in 405 nm by ELISA reader.
제안 방법
5×103 cells/well의 밀도로 96-well plate에 분주하고, 24시간 배양한 후, RANKL(100 ng/ml)과 w-GM (62.5, 125, 250 μg/ml)이 포함된 배지로교환하여 5일간 배양하였다.
Nanodrop(Thermo scientific, Pittsburgh, PA, USA)을 이용하여 RNA 농도를 2 μg로 정량하였으며, 그 후 superscript II reverse transcription kit를 사용하여 cDNA로 합성하였다.
그 후, RANKL (100 ng/ml)과 w-GM (62.5, 125, 250 μg/ml)이 포함된 배지로 교환하여 5일간 배양하였다.
도립현미경(Olmpus, CLX41SF, Japan)을 이용하여 붉은색의 TRAP 양성 반응을 보이는 세포와 핵이 3개이상인 세포를 파골세포로 설정하여, 그 수를 세어 정량 분석하였다.
따라서 본 연구에서는 파골세포 활성화에 관여되는 주요 인자들의단백질 및 유전자 발현에 대한 진교의 효과를 조사했다.
마지막으로 trisbuffered saline with Tween 20로 10분씩 6번씩 세척한 뒤,enhanced chemiluminescence kit (Santa Cruz, CA, USA)를 사용하여 x-ray 필름에 노출 시킨 후, 단백질 발현 양을 조사하였다.
본 연구에 사용된 primer는 Table 1에 기재하였으며, 합성된 결과물은 SYBR green(Invitrogen, CA, USA)으로 염색한 1.2% agarose gel에서 전기영동시킨 후, 발현 정도를 조사하였다.
이후 세포는 증류수로 수세하고 흡수된 면적은 도립현미경을 이용하여 관찰하였다.
RAW 264.7 세포를 5×103 cells/well의 밀도로 osteo assaysurface multiple well plate에 분주하고, 24시간 동안 안정화하였다.
RAW 264.7 세포를 파골세포로 유도하기 위해 10% FBS와 1%P/S를 첨가한 α-MEM 배지를 사용하였다.
RAW 264.7 세포에 RANKL로 유도한 파골세포에서 MAPK 및NF-κB 발현에 w-GM이 미치는 영향을 확인하였다.
w-GM의 세포 독성은 RAW 264.7 세포에 MTS assay를 실시하여 측정하였다.
w-GM의 파골세포 억제 효능을 TRAP 염색 및 활성도 그리고pit formation assay를 사용하여 확인하였다.
중국 섬서에서 채취된 소진교(Omniherb, Seoul, Korea) 300 g에 증류수 3 L을 첨가하여 2시간 동안 열수 추출하였다.
파골세포 분화 및 기능에 관여하는 유전자 발현에 w-GM이 미치는 영향을 조사하였다.
파골세포의 분화 필수 지표인 c-Fos, NFATc1의 단백질 및 유전자 발현에 w-GM이 미치는 영향을 조사하였다.
대상 데이터
Superscript II reverse transcriptase kit는 Invitrogen (Carlsbad, CA,USA)의 제품을 사용하였으며, Taq polymerase는 Kapa Biosystems (Woburn, MA, USA)에서 구매하였다.
p-extracellularsignal-regulated kinase (ERK), p-c-Jun N-terminal kinase(JNK), p-p38, T-ERK, T-JNK, T-p38은 Cell signalling Technology (Danvers, MA, USA)에서 구매하였으며, NFATc1는 BD Biosciences (San Diego, CA, USA)의 제품을 이용하였다.
그 외의 세포 배지인 αminimum essential medium (α-MEM)과 Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS) 그리고 penicillin/streptomycin (P/S)은 Gibco (Gaithersburg, MD, USA)에서 구매하였다.
Pit formation assay 실험 결과에서 w-GM은 골 흡수능을유의하게 억제하였고, 이와 연관하여 유전자 발현으로 골 흡수능과 연관된 CTK, MMP-9, CA2의 발현을 연구한 결과 w-GM은CTK의 발현을 유의하게 억제하며, MMP-9과 CA2의 발현은 억제하지 않았다.
RANKL (100 ng/ml)을 이용하여 파골세포를 유도한뒤, 다양한 농도(62.5, 125, 250 μg/ml)의 w-GM을 처리한 결과, 대조군에 비해 w-GM 처리군에서 TRAP 양성 세포 수와 TRAP 활성도가 감소했다.
c-Fos의 유전자 발현을 확인한 결과 대조군은 정상군에비해 유의하게(p＜0.01) 증가하였지만, w-GM 250 μg/ml (p＜0.01) 처리군에서는 대조군에 비해 유의하게 억제되었다.
또한 NFATc1의 단백질 및 유전자발현 역시 w-GM 처리군에서 대조군에 비해 유의하게 감소하였음을 나타냈다.
또한 calcitonin receptor (CTR)의 발현을 조사한 결과, RANKL 유도 대조군에서 정상군에 비해 발현이 유의하게(p＜0.01) 증가하였으나, w-GM 250 μg/ml (p＜0.01) 처리군에서는대조군에 비해 유의하게 감소하였다.
또한 pit formation assay는 대조군에서는 pit형성 면적이 증가하였으나, w-GM의 농도를 증가하여 처리할수록 pit formation을 억제했다.
본 실험결과, TRAP의 발현이 w-GM 처리군에서대조군에 비해 유의하게 억제되었다.
본 실험결과, 파골세포 분화에 중요한 전사인자인 c-Fos의 단백질 및 유전자발현이w-GM 처리군에서 대조군에 비해 유의하게 억제되었다.
실험 결과, w-GM 처리군이 대조군에 비해 p-ERK, p-JNK,p-p38, NF-κB 발현을 유의하게 억제했다.
진교 물 추출물은RANKL로 유도된 파골세포에서 다핵의 TRAP-positive 세포 수, TRAP activity 그리고 pit formation을 유의하게 감소시켰고,RANKL로 유도된 파골세포의 분화와 관련한 MAPK 및 NF-κB발현을 감소시켰다.
파골세포의 골 흡수 기능에중요한 역할을 하는 cathepsin k (CTK), matrix metallopeptidase9 (MMP-9) 그리고 carbonic anhydrase 2 (CA2)의 발현을 확인한결과, 대조군에서는 정상군에 비해 유의하게(p＜0.01) 증가하였다.
하지만 연교는 CA2의 강력한 억제로 골 표면의 산성화를 억제해, CTK/MMP-9과 같은 골 흡수 enzyme이 일정이상발현되더라도 강력하게 골 흡수를 강하게 억제하는 것을 확인할수 있었다.
(A) The Gentiopicroside was identified by HPLC Chromatograms analysis on the standard. (B) HPLC chromatograms were used to identify peaks at 15 min in w-GM and Gentiopicroside was confirmed by comparison with the standard.
w-GM 내의 gentiopicroside의 동정은 표준품과 비교하여 15분대 peak로 확인하였으며, 각 peak의 PDA 스캔을 통해 표준품의 스펙트럼과 비교하여 최종적으로 gentiopicroside가 주성분임을확인하였다(Fig. 1).
후속연구
따라서 염증성 질환에서 유발된 골다공증의 치료 효과를 파악하기는 어려웠으며, 이번 연구에서는 이를 나타내지 못하였지만, 염증성 골다공증 동물 모델에서의 골다공증 억제효능과 관련 기전에 대한 추가적인 연구가 필요할 것으로 사료된다.
이상을 종합하면, 진교는 파골세포분화 관련 유전자 및 단백질의 발현 억제로 파골세포 분화 및 활성화 기전에 작용하며, 향후 염증성 골다공증 개선을 위한 치료 효능의 연구를 거쳐 임상 적용이 가능할 것으로 사료된다.
참고문헌 (40)
World Health Organization. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. Report of a WHO Study Group. World Health Organ Tech Rep Ser. 1994 ; 843 : 1-129
Lin JT, Lane JM. Osteoporosis: a review. Clin Orthop Relat Res. 2004 ; 425 : 126-34. https://doi.org/10.3329/birdem.v5i1.28371
Majumdar SR, Lier DA, Beaupre LA, Hanley DA, Maksymowych WP, Juby AG, et al. Osteoporosis case manager for patients with hip fractures: results of a cost-effectiveness analysis conducted alongside a randomized trial. Arch Intern Med. 2009 ; 169(1) : 25-31. https://doi.org/10.1001/archinte.169.1.25
Hodgson SF, Watts NB, Bilezikian JP, Clarke BL, Gray TK, Harris DW, et al. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the prevention and treatment of postmenopausal osteoporosis: 2001 edition, with selected updates for 2003. Endocr Pract. 2003 ; 9(6) : 544-64. https://doi.org/10.4158/EP.9.6.544
Khosla S, Riggs BL. Pathophysiology of age-related bone loss and osteoporosis. Endocrinol Metab Clin North Am. 2005 ; 34(4) : 1015-30. https://doi.org/10.1016/j.ecl.2005.07.009
Larussa T, Suraci E, Nazionale I, Leone I, Montalcini T, Abenavoli L, et al. No evidence of circulating autoantibodies against osteoprotegerin in patients with celiac disease. World J Gastroenterol. 2012 ; 18(14) : 1622-27. https://doi.org/10.3748/wjg.v18.i14.1622
Cooper C, Campion G, Melton LJ 3rd. Hip fractures in the elderly: a world-wide projection. Osteoporos Int. 1992 ; 2(6) : 285-9. https://doi.org/10.1007/BF01623184
Kennel KA and Drake MT. Adverse Effects of Bisphosphonates: Implications for Osteoporosis Management. Mayo Clin Proc. 2009 ; 84(7) : 632-8. https://10.1016/S0025-6196(11)60752-0
Watts NB, Bilezikian JP, Camacho PM, Greenspan SL, Harris ST, Hodgson SF, et al. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the diagnosis and treatment of postmenopausal osteoporosis. Endocr Pract. 2010 ; 16 Suppl 3 : 1-37. https://doi.org/10.4158/ep.16.s3.1
McClung M, Harris ST, Miller PD, Bauer DC, Davison KS, Dian L, et al. Bisphosphonate therapy for osteoporosis: benefits, risks, and drug holiday. Am J Med. 2013 ; 126(1) : 13-20. https://doi.org/10.1016/j.amjmed.2012.06.023
Ruggiero SL, Dodson TB, Fantasia J, Goodday R, Aghaloo T, Mehrotra B, et al. American Association of Oral and Maxillofacial Surgeons position paper on medication-related osteonecrosis of the jaw-2014 update. J Oral Maxillofac Surg. 2014 ; 72(10) : 1938-56. https://doi.org/10.1016/j.joms.2014.04.031
Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone. 2007 ; 40(2): 251-64 https://doi.org/10.1016/j.bone.2006.09.023
Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell. 2002 ; 3(6) : 889-901. https://doi.org/10.1016/s1534-5807(02)00369-6
Shin MK, Clinical Traditional Herbology. Younglimsa 2002 ; 431-2
Yu F, Yu F, Li R, Wang R. Inhibitory effects of the gentiana macrophylla (gentianaceae) extract on rheumatoid arthritis of rats. J Ethnopharmacol. 2004 ; 95(1) : 77-81. https://doi.org/10.1016/j.jep.2004.06.025
Kwak WJ, Kim JH, Ryu KH, Cho YB, Jeon SD, Moon CK. Effects of gentianine on the production of pro-inflammatory cytokines in male Sprague-Dawley rats treated with lipopolysaccharide (LPS). Biol Pharm Bull. 2005 ; 28(4) : 750-3. https://doi.org/10.1248/bpb.28.750
Lv J, Gu WL, Chen CX. Effect of gentiopicroside on experimental acute pancreatitis induced by retrograde injection of sodium taurocholate into the biliopancreatic duct in rats. Fitoterapia. 2015 ; 102 : 127-33. https://doi.org/10.1016/j.fitote.2015.03.002
Wang S, Xu Y, Jiang W, Zhang Y. Isolation and identification of constituents with activity of inhibiting nitric oxide production in RAW 264.7 macrophages from Gentiana triflora. Planta Med. 2013 ; 79(8) : 680-6. https://doi.org/10.1055/s-0032-1328460
Nakashima T, Hayashi M, Fukunaga T, Kurata K, Oh-Hora M, Feng JQ, et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat Med. 2011 ; 17(10) : 1231-4. https://doi.org/10.1038/nm.2452
Mansky KC, Sankar U, Han J, Ostrowski MC. Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-kappa B ligand signaling. J Biol Chem. 2002 ; 277(13) : 11077-83. https://doi.org/10.1074/jbc.M111696200
Choi J, Choi SY, Lee SY, Lee JY, Kim HS, Lee SY, et al. Caffeine enhances osteoclast differentiation and maturation through p38 MAP kinase/Mitf and DC-STAMP/CtsK and TRAP pathway. Cell Signal. 2013 ; 25(5) : 1222-27. https://doi.org/10.1016/j.cellsig.2013.02.015
Asagiri M, Sato K, Usami T, Ochi S, Nishina H, Yoshida H, et al. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J Exp Med. 2005 ; 202(9) : 1261-9. https://doi.org/10.1084/jem.20051150
Pondel M. Calcitonin and calcitonin receptors: bone and beyond. Int J Exp Pathol. 2000 ; 81(6) : 405-22. https://doi.org/10.1046/j.1365-2613.2000.00176.x
Zhuo Y, Gauthier JY, Black WC, Percival MD, Duong LT. Inhibition of bone resorption by the cathepsin K inhibitor odanacatib is fully reversible. Bone. 2014 ; 67 : 269-80. doi: https://doi.org/10.1016/j.bone.2014.07.013
Gu JH, Tong XS, Chen GH, Liu XZ, Bian JC, Yuan Y, et al. Regulation of matrix metalloproteinase-9 protein expression by $1{\alpha}$ , 25-(OH)2D3 during osteoclast differentiation. J Vet Sci. 2014 ; 15(1) : 133-40. https://doi.org/10.4142/jvs.2014.15.1.133
Lehenkari P, Hentunen TA, Laitala-Leinonen T, Tuukkanen J, Vaananen HK. Carbonic anhydrase II plays a major role in osteoclast differentiation and bone resorption by effecting the steady state intracellular pH and Ca2+. Exp Cell Res. 1998 ; 242(1) : 128-37. https://doi.org/10.1006/excr.1998.4071
Seo MS, Kim HC, Choo WG, Jeong SY, Kim SJ, Choi JU, et al. The Review on the Study of Osteoporosis in Korean Medicine Journals. The Journal of Korea CHUNA Manual Medicine for Spine & Nerves. 2013 ; 8(2) : 67-78.
Kim DH. Hwangjenaegyeong Yeongchu. Seoul : Uiseongdang. 2002 : 452.
Kim JH, Kim M, Jung HS, Sohn Y. Leonurus sibiricus L. ethanol extract promotes osteoblast differentiation and inhibits osteoclast formation. Int J Mol Med. 2019 ; 44(3) : 913-26. https://doi.org/10.3892/ijmm.2019.4269
Park WT, Jeong SH, Seo IB, Kim SJ. GCP Treatment on the Expression of NOS, C-fos, Serotonin and Substance-P in Central Nerve System of Monosodium Iodoacetate-Induced Osteoarthritic Pain Model. Korean J. Oriental Physiology & Pathology 2007 ; 21(6) : 1483-90.
Eom JW, Kim JH, Kim MS, Kim SW, Shin HJ, Jung HS, et al. Forsythiae Fructus Extract Inhibits RANKL-Induced Osteoclast Differentiation and Prevent Bone Loss in OVX-Induced Osteoporosis Rat. Korean J Acupunct. 2019 ; 36(2) : 115-26. https://doi.org/10.14406/acu.2019.008
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