臺灣博碩士論文加值系統

骨質疏鬆症普遍發生於停經後女性和老年人，當蝕骨細胞的作用大於造骨細胞的作用時，整體骨質就減少了。抑制蝕骨細胞的生成與分化，是改善骨質疏鬆症的策略之ㄧ。已知當receptor activator of nuclear factor �羠 ligand (RANKL)結合到它的接受器RANK時，活化下游的mitogen-activated protein kinases (MAPKs)訊息傳遞路徑，誘導轉錄因子nuclear factor -�羠 (NF-�羠)、activating protein 1 (AP-1)、nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)的活化，以促進蝕骨細胞的生成、tartrate-resistant acid phosphatase (TRAP)的產生，並促進骨質蝕損作用。2-Methoxystypandrone (2MS)是由虎杖(Polygonum cuspidatum)的根莖當中萃取出的naphthoquinone結構物質。本論文發現1-7.5 �嵱的2MS呈現濃度相關性地抑制RANKL誘導的RAW 264.7巨噬細胞株分化生成蝕骨細胞；結果顯示2MS對於總TRAP活性之一半有效抑制劑量(IC50)為6.8 �嵱，且無明顯細胞毒性。細胞受RANKL刺激，會增加c-Jun、c-Fos和NFATc1細胞核中表現量，這些反應會被2MS以濃度相關性抑制。RANKL也會促使inhibitory �羠 (I�羠)被降解，增加NF-�羠從細胞質轉位到細胞核的量，而這些反應也被2MS抑制，且與濃度相關。另外，RANKL誘導p38 MAP kinases (p38)、extracellular signal-regulated kinase (ERK)和c-Jun-N-terminal kinase (JNK)的磷酸化，而這些MAPKs的反應亦會被2MS以濃度相關性抑制。此外，已知matrix metalloproteinase 9 (MMP 9)參與骨質蝕損作用，dendritic cell-specific transmembrane protein (DC-STAMP)參與細胞融合過程， RT-PCR實驗分析顯示RANKL刺激可誘導MMP-9、DC-STAMP蛋白的mRNA表現增加，而此現象可被2MS抑制。因此目前的結果顯示2MS可以抑制RANKL誘導的蝕骨細胞分化，其機轉可能經由降低p38、ERK和JNK的磷酸化活化，抑制I�羠降解，以及干擾了轉錄因子(NF-κB、 c-Jun、c-Fos和NFATc1)的活化而達成。

Osteoporosis is common in menopause women and old people. When the bone resorption exceeds the bone formation, a net loss of bone occurs. Inhibition of the differentiation of osteoclasts is a potential strategy to treat the osteoporosis. Previous studies showed that the binding of the receptor activator of nuclear factor �羠 ligand (RANKL) to its receptor RANK can activate mitogen-activated protein kinases (MAPKs) and trigger activation of the transcription factors, such as NF-�羠, activating protein-1 (AP-1) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), to promote osteoclastogenesis, the generation of the tartrate-resistant acid phosphatase (TRAP) as well as bone resorption. 2-Methoxystypandrone (2MS) is a naphthoquinone compound isolated from Polygonum cuspidatum. The present results showed that 2MS (1-7.5 �嵱) concertration-dependently inhibited RANKL-induced the differentiation of RAW 264.7 macrophages into osteoclasts and the TRAP activity with an IC50 value of 6.8 �嵱 without significant cytotoxicity. Incubation the cells with RANKL significantly increased the nuclear expression of NF-�羠, c-Fos, c-Jun and NFATc1, the phosphorylation of p38, ERK and JNK, and the degradation of I�羠. All these phenomena were concentration-dependently suppressed by 2MS. Metalloproteinase-9 (MMP-9) is a digest enzyme participated in bone resorption. RT-PCR analysis revealed that RANKL-induced MMP-9 mRNA expression was inhibited by 2MS. In conclusion, the present results indicated that 2MS can inhibit RANKL-induced osteoclastogenesis probably via attenuating RANKL-induced phosphorylation of p38, ERK and JNK, the degradation of I�羠, and interfering with the activation of transcription factors (NF-κB, c-Jun, c-Fos and NFATc1).

中文摘要………………………………………...………………..…1
英文摘要………………………………………...……….…………2
第一章 緒論………………………………….………………..……3
1.1 骨質疏鬆症 (osteoporosis)…………………………………..4
1.2 骨骼再塑 (bone remodeling)…….……………………………..5
1.3 蝕骨細胞之生長與分化…………………………….…… …….6
1.4 RANKL誘導蝕骨細胞生成之訊息傳遞路徑……….……..… ..7
1.5 抗酒石酸酸性磷酸鹽酶 (TRAP)…………………….…....…..9
1.6 骨質蝕損…………………………………………..….…… ...10
1.7 臨床上骨質疏鬆症之治療…………………………….. …...11
1.8 虎杖(Polygonum cuspidatum)…………………… .….….13
1.9 研究目的………………………………. ……….……..…..…14
第二章 材料與方法…………………………… …….……..……21
2.1 實驗材料………………………………… ………...……….22
2.2 實驗方法……………………………… ……………...…..26
2.3 統計分析…………………………… ………………..…… .35
第三章 結果………………………………………………………….38
3.1虎杖氯仿層萃取物(PC-c)、虎杖氯仿層萃取物之次萃取物(PC-cs)及三個主要純化物對於RANKL誘導蝕骨細胞分化之影響………….39
3.2 2MS對於RANKL誘導蝕骨細胞分化之影響………….……….40
3.3 2MS對細胞存活率之影響和其溶劑(0.06 % DMSO)對蝕骨細胞生成之影響………………………………………………………….……..40
3.4 2MS對於RANKL誘導不同分化時期的蝕骨細胞影響..…..……41
3.5 RANKL誘導蝕骨細胞生成過程中，2MS對於細胞核中NFATc1表現量的影響………….…………………………………………………...42
3.6 RANKL誘導蝕骨細胞生成過程中，2MS對於細胞核中c-Fos和c-Jun表現量的影響………………………………….………….…….……43
3.7 RANKL誘導蝕骨細胞生成過程中，2MS對於細胞核中NF-�羠以及細胞中I�羠的表現量之影響………………………..……….…………43
3.8 RANKL誘導蝕骨細胞生成過程中，2MS對於細胞中p38、ERK、JNK磷酸化的影響…………………………………………………….....44
3.9 RANKL誘導蝕骨細胞生成路徑中，2MS對於MMP-9和DC-STAMP mRNA表現的影響………….…………………………………………..….45
3.10 RANKL誘導蝕骨細胞生成並進行骨質蝕損作用，2MS對於骨質蝕損的影響…………………………………………………………….....46
第四章 討論…………………………………………………….....58
4.1 篩選虎杖氯仿層萃取物之次萃取物以及純物質對於RANKL誘導的蝕骨細胞生成之影響…………………………………...59
4.2 2MS對於RANKL誘導的蝕骨細胞生成之影響……………………59
4.3 2MS對細胞存活率之影響和其溶劑 (0.06 % DMSO)對蝕骨細胞生成之影響…………………………………………………………………...60
4.4 2MS對於RANKL誘導不同分化時期的蝕骨細胞影響…………….61
4.5 RANKL誘導蝕骨細胞生成過程中，2MS對於細胞核中NFATc1表現量的影響……………………………………………………………...62 4.6 RANKL誘導蝕骨細胞生成過程中，2MS對於細胞核中c-Fos和c-Jun表現量以及p38、ERK、JNK磷酸化的影響……………………….62
4.7 RANKL誘導蝕骨細胞生成過程中，2MS對於細胞核中NF�羠與I�羠表現量的影響……………………………………………........63 4.8 RANKL誘導蝕骨細胞生成路徑中，2MS對於MMP-9 和DC-STAMP mRNA表現的影響………………………………………….………...64 4.9 RANKL誘導蝕骨細胞生成並進行骨質蝕損作用，2MS對於骨質蝕損的影響……………………………………………………………...64 4.10 未來研究方向…………………………………………… ....65 第五章 結論……………………………………………………...…..66參考文獻…………………………………………………………..….68
圖I 蝕骨細胞的起源、分化與細胞特徵……………..…………..…15圖II 蝕骨細胞生成及骨質蝕損…………………..…..…………..16圖III RANKL誘導蝕骨細胞生成之訊息傳遞路………….…………..17圖IV NFATc1調控之蝕骨細胞分化三階段….………..………………18圖V 進行骨質蝕損之蝕骨細胞…………..………………….………19圖VI 虎杖植物……...…………………………...………………….20圖VII 虎杖藥材……..…………………………………………………20圖一 2-Methoxystypandrone結構式……..…………………….…..36圖二 2MS對於不同分化時期的影響之實驗設計………………..…..37圖1 篩選中草藥之流程圖………………...…………………….….47圖2 虎杖氯仿層萃取物(PC-c)、次萃取物(PC-cs)及其純化之化合物對於RANKL誘導蝕骨細胞分化之影響……...……………………….48圖3 2MS對於RANKL誘導蝕骨細胞分化之影響……………… …….49圖4 探討溶劑對蝕骨細胞分化及評估2MS對細胞之毒性… ……..50圖5 RANKL誘導之蝕骨細胞生成，2MS對於不同分化時期的影響…..51圖6 2MS對於RANKL誘導NFATc1於細胞核中表現量的影響…… ...52圖7 2MS對於RANKL誘導c-Fos和c-Jun細胞核中表現量的影響…….53圖8 2MS對於RANKL誘導I�羠裂解及NF�羠細胞核中表現量的影響...54圖9 2MS對於RANKL誘導細胞中p38、ERK、JNK磷酸化的影響……...55圖10 2MS對於RANKL誘導MMP-9和DC-STAMP mRNA表現的影響...56 圖11 2MS對於RANKL誘導蝕骨細胞蝕損作用之影響……………...57

Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007; 40: 251–264.

Aubin JE, Bonnelye E. Osteoprotegerin and its ligand: a new paradigm for regulation of osteoclastogenesis and bone resorption. Osteoporosis International 2000; 11: 905-913.

Bharti AC, Takada Y, Aggarwal BB. Curcumin (Diferuloylmethane) Inhibits receptor activator of NF-�羠 ligand-induced NF-�羠 activation in osteoclast precursors and suppresses osteoclastogenesis. The Journal of Immunology 2004; 172: 5940–5947.

Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and
activation. Nature 2003; 423: 337-342

Hayman AR. Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy. Autoimmunity 2008; 41: 218–223.

Helena Kaija. Tartrate-resistant acid phosphatase: three-dimensional structure and structure-based functional studies. Research Center for Molecular Endocrinology, University of Oulu, 2002.

Huang H, Ryu J, Ha1 J, Chang EJ, Kim HJ, Kim HM, Kitamura T, Lee ZH, Kim HH. Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-�羠 transactivation by RANKL. Cell Death and Differentiation 2006; 13: 1879–1891.

Ishii M, Saeki Y. Osteoclast cell fusion: mechanisms and molecules. Modern Rheumatology 2008; 18: 220–227.

Kim HH, Kim JH, Kwak HB, Huang H, Han SH, Ha H, Lee SW, Woo ER, Lee ZH. Inhibition of osteoclast differentiation and bone resorption by tanshinone IIA isolated from Salvia miltiorrhiza Bunge. Biochemical Pharmacology 2004; 67: 1647–1656.

Kim K, Lee SH, Kim JH, Choi Y, Kim N. NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific ransmembrane protein (DC-STAMP). Molecular Endocrinology 2008; 22: 176–185.

Kim N, Kadono Y, Takami M, Lee J, Lee SH, Okada F, Kim JH, Kobayashi T, Odgren PR, Nakano H, Yeh WC, Lee SK, Lorenzo JA, Yongwon Choi Y. Osteoclast differentiation independent of the TRANCE–RANK–TRAF6 axis. The Journal of Experimental Medicine 2005; 202: 589-595.

Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165–176.

Lee SW, Han SI, Kim HH, Lee ZH. TAK1-dependent activation of AP-1 and c-Jun N-terminal kinase by receptor activator of NF-�羠. Journal of Biochemistry and Molecular Biology 2002; 35: 371-376.

Metzler I, Krebbel H, Hecht M, Manz RA, Fleissner C, Mieth M, Kaiser M, Jakob C, Sterz J, Kleeberg L, Heider U, Sezer O. Bortezomib inhibits human osteoclastogenesis. Leukemia 2007; 21: 2025–2034.

Mizukami J, Takaesu G, Akatsuka H, Sakurai H, Tsuji JN, Matsumoto K, Sakurai N. Receptor activator of NF-�羠 ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Molecular and Cellular Biology 2002; 22: 992–1000.

Oddie GW, Schenk G, Angel NZ, Walsh N, Guddat LW, Jersey JD, Cassady AI, Hamilton SE, Hume DA. Structure, function, and regulation of tartrate-resistant acid phosphatase. Bone 2000; 27: 575–584.

Park CK, Kim HJ, Kwak HB, Lee TH, Bang MH, Kim CM, Lee Y, Chung DK, Baek NI, Kim J, Lee ZH, Kim HH. Inhibitory effects of stewartia koreana on osteoclast differentiation and bone resorption. International Immunopharmacology 2007; 7: 1507–1516.

Rahman MM, Bhattacharya A, Fernandes G. Docosahexaenoic acid is more potent inhibitor of osteoclast differentiation in RAW 264.7 cells than eicosapentaenoic acid. Journal of Cellular Physiology 2008; 214: 201–209.

Riggs BL, Melton LJ. Osteoporosis: Etiology, Diagnosis, and Management, 2nd ed. Philadelphia, Lippincott-Raven Publishers, 1995.

Romas E, Gillespie MT, Martin TJ. Involvement of receptor activator of NF�羠 ligand and tumor necrosis factor-�� in bone destruction in rheumatoid arthritis. Bone 2002; 30: 340–346.

Singh SB, Graham PL, Reamer RA, Cordingley MG. Discovery, total synthesis, HRV 3C-protease inhibitory activity, and structure–activity relationships of 2-Methoxystypandrone and its analogues. Bioorganic & Medicinal Chemistry Letters 2001; 11: 3143–3146.

Souza LF, Jardim FR, Sauter IP, Souza MM, Barreto F, Margis R, Bernard EA. Lipoteichoic acid from Staphylococcus aureus increases matrix metalloproteinase 9 expression in RAW264.7 macrophages: Modulation by A2A and A2B adenosine receptors. Molecular Immunology 2009; 46: 937–942.

Soysa NS, Alles N. NF-jB functions in osteoclasts. Biochemical and Biophysical Research Communications 2009; 378: 1–5.

Takayanagi H. Mechanistic insight into osteoclast differentiation in osteoimmunology. Journal of Molecular Medicine 2005; 83: 170-179.

Takayanagi H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nature Reviews Immunology 2007; 7: 292-304.

Takayanagi H. The role of NFAT in osteoclast formation. Annals of the New York Academy of Sciences 2007; 1116: 227–237.

Teitelbaum SL. Bone resorption by osteoclasts. Science 2000; 289: 1504-1508.

Teitelbaum SL. RANKing c-Jun in osteoclast development. The Journal of Clinical Investigation 2004; 114: 463-465.

Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nature 2003; 4: 638-649.

Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T, Koga T, Martin TJ, Suda T. Origin of osteoclasts: Mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Cell Biology 1990; 87: 7260-7264.

Vaananen HK, Leinonen TL. Osteoclast lineage and function. Archives of Biochemistry and Biophysics 2008; 473: 132–138.

Zaidi M. Skeletal remodeling in health and disease. Nature Medicine 2007; 13: 791-801.

Micromedex® Healthcare Series

黃永彥. 骨質疏鬆症-基礎與臨床. 合記圖書出版社, 台灣, 1997; 1: 3-4; 17: 93-126.

蕭培根、連文琰、胡廷松主編. 中國本草圖錄. 商務印書館, 香港, 1989;第一卷: 33.

行政院衛生署 民國94年國民健康訪問調查, 1995.