李盛村，鮑捷，王靜，王國(guó)祥

骨保護(hù)素/核因子-κB受體活化因子配體/核因子-κB受體活化因子信號(hào)通路與骨質(zhì)疏松的研究進(jìn)展①

李盛村，鮑捷，王靜，王國(guó)祥

運(yùn)動(dòng)對(duì)骨質(zhì)疏松癥有積極作用，但其治療的分子生物學(xué)機(jī)制仍未清楚。骨保護(hù)素/核因子-κB受體活化因子配體/核因子-κB受體活化因子(OPG/RANKL/RANK)信號(hào)通路的發(fā)現(xiàn)，有助于骨質(zhì)疏松癥的治療。機(jī)械應(yīng)力可以調(diào)節(jié)OPG/RANKL/RANK信號(hào)通路，參與預(yù)防和治療骨質(zhì)疏松進(jìn)程。本文就應(yīng)力敏感信號(hào)通路OPG/RANKL/RANK與骨質(zhì)疏松的關(guān)系進(jìn)行綜述。

骨質(zhì)疏松癥；應(yīng)力；骨保護(hù)素；核因子-κB受體活化因子配體；核因子-κB受體活化因子；信號(hào)通路；綜述

[本文著錄格式]李盛村，鮑捷，王靜，等.骨保護(hù)素/核因子-κB受體活化因子配體/核因子-κB受體活化因子信號(hào)通路與骨質(zhì)疏松的研究進(jìn)展[J].中國(guó)康復(fù)理論與實(shí)踐,2014,20(3):250-252.

骨質(zhì)疏松癥(osteoporosis)是一種骨代謝障礙疾病，它以骨量減少、骨脆性增加和骨折風(fēng)險(xiǎn)增加為主要特征。骨質(zhì)疏松癥好發(fā)于絕經(jīng)后婦女和老年人群[1]。正常骨組織中新骨與舊骨更替而保持動(dòng)態(tài)平衡[2]。骨吸收異常增加或骨形成異常減少，均是骨質(zhì)疏松發(fā)生的生理學(xué)基礎(chǔ)。

適宜運(yùn)動(dòng)是骨質(zhì)疏松癥的有效療法，然而有關(guān)其治療的分子生物學(xué)機(jī)制尚未清楚。近年來(lái)的研究發(fā)現(xiàn)，骨保護(hù)素/核因子-κB受體活化因子配體/核因子-κB受體活化因子(OPG/ RANKL/RANK)信號(hào)通路與骨質(zhì)疏松的發(fā)生和治療有密切聯(lián)系[3]，而運(yùn)動(dòng)應(yīng)力可以調(diào)節(jié)OPG/RANKL/RANK信號(hào)通路。因此，綜合分析應(yīng)力刺激和OPG/RANKL/RANK信號(hào)通路的關(guān)系以及兩者對(duì)骨質(zhì)疏松的影響，可以為骨質(zhì)疏松的治療提供新的線索[4]。

1 OPG/RANKL/RANK信號(hào)通路

骨保護(hù)素(osteoprotegerin,OPG)是Simonet在給新生大鼠腸cDNA測(cè)序時(shí)發(fā)現(xiàn)的，包含5個(gè)外顯子，其N端D1～D4區(qū)富含半胱氨酸，結(jié)構(gòu)與腫瘤壞死因子受體2(tumor necrosis factor receptor 2,TNFR2)和CD40有高度的保守性，為發(fā)揮生理功能所必需[5]。OPG可抑制破骨細(xì)胞活性，也被稱為骨保護(hù)蛋白或護(hù)骨素[6]。OPG前體是包含401個(gè)氨基酸的糖蛋白，去除21個(gè)信號(hào)肽后，形成含380個(gè)氨基酸的成熟OPG蛋白[7]。OPG基因敲除小鼠出現(xiàn)嚴(yán)重的骨質(zhì)疏松癥，骨皮質(zhì)和骨小梁均減少；而過度表達(dá)OPG的轉(zhuǎn)基因小鼠則出現(xiàn)嚴(yán)重的骨硬化癥[8]?？梢奜PG在骨代謝中起重要作用。

人核因子-κB(NF-κB)受體活化因子(receptor activator of NF-κB,RANK)是Ⅰ型跨膜糖蛋白，其cDNA由616個(gè)密碼子編碼而成，屬于TNF超家族成員[9]。RANK編碼基因組成包含29個(gè)氨基酸信號(hào)肽、細(xì)胞外編碼區(qū)183個(gè)氨基酸、跨膜區(qū)21個(gè)氨基酸和細(xì)胞質(zhì)區(qū)383個(gè)氨基酸[10]。RANK蛋白和其他TNF超家族成員一樣，以三聚體形式實(shí)現(xiàn)對(duì)細(xì)胞外信號(hào)進(jìn)行接收和轉(zhuǎn)導(dǎo)[11]。

人NF-κB受體活化因子配體(receptor activator of NF-κB ligand,RANKL)是由317個(gè)氨基酸構(gòu)成的Ⅱ型跨膜蛋白，在基質(zhì)/成骨細(xì)胞、軟骨細(xì)胞和淋巴細(xì)胞中廣泛表達(dá)[12]。骨中的RANKL可結(jié)合破骨細(xì)胞表面受體RANK，激活破骨細(xì)胞?；罨钠乒羌?xì)胞分泌大量H+、Cl-等離子進(jìn)入骨陷窩，從而溶解骨質(zhì)[13]。

在骨組織中，多種因素均可以通過調(diào)節(jié)OPG/RANKL之間的比例而影響骨的生理和病理[14]。成骨/基質(zhì)細(xì)胞分泌RANKL與破骨細(xì)胞表面的RNAK結(jié)合，導(dǎo)致骨吸收增強(qiáng)[15]；成骨細(xì)胞分泌OPG可以結(jié)合RANKL，阻礙RANKL與RANK結(jié)合，從而抑制破骨細(xì)胞活性[16]。如果RANKL過度表達(dá)或OPG異常降低，骨吸收大于骨形成，最終發(fā)生以骨量和骨密度減少為特征的骨質(zhì)疏松癥[7,17]。

2 骨質(zhì)疏松與OPG/RANKL/RANK信號(hào)通路

2.1 OPG/RANKL/RANK信號(hào)通路與骨質(zhì)疏松發(fā)生

研究發(fā)現(xiàn)，可溶性RANKL過量表達(dá)的大鼠，骨骼組織中出現(xiàn)類似絕經(jīng)后骨質(zhì)疏松癥狀，包括骨吸收增加、骨密度減少和骨骼脆性增加[18]。Bashir等發(fā)現(xiàn)，骨質(zhì)疏松模型大鼠骨組織中RANKL表達(dá)升高，而采用雌激素和雷洛昔芬(raloxifene)治療12個(gè)月后，骨密度增加，同時(shí)RANKL降低[19]。由此可知，RANKL的異常增加和骨質(zhì)疏松發(fā)生密切相關(guān)，而雌激素或適宜應(yīng)力刺激均可以降低骨組織中RANKL水平，起到延緩骨質(zhì)疏松進(jìn)程的作用。

有研究發(fā)現(xiàn)，OPG基因敲除大鼠產(chǎn)生骨質(zhì)疏松癥狀[20]，這與缺乏OPG而不能阻止RANKL和RANK的結(jié)合有關(guān)。程少丹等通過非頻密繁殖法獲得OPG-/-小鼠，發(fā)現(xiàn)與同齡野生型小鼠比較，OPG基因缺失小鼠骨密度、股骨承受最大載荷、股骨剛度、腰椎椎體骨小梁數(shù)目和腰椎椎體骨小梁厚度均顯著下降[21]，證實(shí)與大鼠一樣，OPG基因缺失的小鼠也產(chǎn)生骨質(zhì)疏松初期癥狀。Bergstrm等將112名絕經(jīng)后婦女隨機(jī)分為對(duì)照組和運(yùn)動(dòng)組，分別在實(shí)驗(yàn)初期和1年運(yùn)動(dòng)訓(xùn)練結(jié)束后檢測(cè)血清OPG、RANKL水平，發(fā)現(xiàn)與對(duì)照組相比，運(yùn)動(dòng)組OPG明顯增加，但RANKL水平變化不顯著[22]。

2.2 OPG/RANKL/RANK信號(hào)通路與骨質(zhì)疏松治療

OPG/RANKL/RANK信號(hào)通路的發(fā)現(xiàn)為闡明骨質(zhì)疏松癥的分子機(jī)制奠定了基礎(chǔ)，也可能為骨質(zhì)疏松的靶向治療提供理論依據(jù)[23]。OPG被認(rèn)為是RANKL的天然拮抗劑，在一期臨床試驗(yàn)中發(fā)現(xiàn)，OPG可以降低尿中80%的骨吸收標(biāo)志物urinary NTx[24]。West等發(fā)現(xiàn)，OPG感應(yīng)運(yùn)動(dòng)應(yīng)力刺激而抑制骨吸收；然而隨著時(shí)間的延長(zhǎng)，特異性O(shè)PG抗體也增加，從而削弱其長(zhǎng)期治療效果[25]。董潔瓊發(fā)現(xiàn)，大鼠切除卵巢后，成骨細(xì)胞和骨髓基質(zhì)細(xì)胞OPG蛋白和mRNA表達(dá)降低；運(yùn)動(dòng)3個(gè)月后，成骨細(xì)胞和骨髓基質(zhì)細(xì)胞OPG蛋白和mRNA表達(dá)升高[26]。

RANKL在骨質(zhì)疏松患者骨組織中異常升高，研究特異性RANKL抑制劑對(duì)骨質(zhì)疏松治療有重要意義。目前，正在開發(fā)的Denosumab(AMG162)單克隆抗體[27]，可通過結(jié)合RANKL，抑制破骨細(xì)胞活性[28]。Lewiecki等對(duì)絕經(jīng)后骨質(zhì)疏松婦女每6個(gè)月皮下注射Denosumab 60 mg，可以增加她們的骨密度，減少骨吸收標(biāo)志物[29]。

3 應(yīng)力調(diào)節(jié)OPG/RANKL/RANK信號(hào)通路

以往認(rèn)為，通過運(yùn)動(dòng)改善絕經(jīng)后婦女雌激素的分泌，可改善骨質(zhì)疏松患者骨密度[30-32]。值得注意的是，不同的運(yùn)動(dòng)方式改變骨密度有明顯的部位特異性，即受力越集中的骨骼，骨密度越高[33-34]。房冬梅等對(duì)105名不同專項(xiàng)女子運(yùn)動(dòng)員7個(gè)部位的骨密度進(jìn)行檢測(cè)，發(fā)現(xiàn)舉重運(yùn)動(dòng)員整體承受應(yīng)力最大，各部位骨密度絕對(duì)值都高于其他運(yùn)動(dòng)員組；自行車運(yùn)動(dòng)員前臂骨密度較高，腰椎骨密度較低[35]。此外，網(wǎng)球運(yùn)動(dòng)員持拍手的骨密度高于非持拍手，劃船運(yùn)動(dòng)員脊椎骨密度高于舞蹈和徑賽運(yùn)動(dòng)員[36]。這種應(yīng)力引起的骨密度改變不僅僅是激素的全身性效應(yīng)，也存在力對(duì)局部骨組織的直接效應(yīng)。

大量研究表明，OPG/RANKL/RANK信號(hào)通路是力學(xué)敏感通路[37]。Kaneuji等發(fā)現(xiàn)，對(duì)大鼠骨祖細(xì)胞施加持續(xù)3D機(jī)械應(yīng)力，骨祖細(xì)胞OPG基因表達(dá)增加[38]。Hou等應(yīng)用不同振幅機(jī)械力(40 Hz,30 min/d)刺激成骨細(xì)胞3 d以上，發(fā)現(xiàn)成骨細(xì)胞表達(dá)OPG蛋白增加，RANKL蛋白表達(dá)減少；在mRNA轉(zhuǎn)錄水平，OPG mRNA表達(dá)增強(qiáng)比RANKL表達(dá)降低幅度更加明顯[39]。Li等探索不同方式的流體剪切力對(duì)骨細(xì)胞的影響，發(fā)現(xiàn)OPG mRNA表達(dá)隨頻率加快和持續(xù)時(shí)間延長(zhǎng)及應(yīng)力刺激強(qiáng)度加大而下降[40]。Yamamoto等采用自制的靜水壓力裝置模擬牙齒咬合力，對(duì)下頜骨來(lái)源的成骨細(xì)胞施加壓力，發(fā)現(xiàn)RANKL/OPG比例上升，其中RANKL mRNA和蛋白都上調(diào)[41]。Sanchez等采用3D加力方式研究高強(qiáng)度外力(1 MPa和1.6 MPa)對(duì)成骨細(xì)胞基因表達(dá)的影響，發(fā)現(xiàn)成骨細(xì)胞受大強(qiáng)度應(yīng)力刺激后，OPG mRNA表達(dá)下降，而RANKL mRNA表達(dá)沒有發(fā)生明顯變化，RANKL/OPG比例上升[42]。

雖然上述研究結(jié)果之間還存在一定分歧，特別是應(yīng)力刺激對(duì)RANKL的影響還不一致，但大部分研究表明，適宜的中等強(qiáng)度應(yīng)力刺激引起RANKL/OPG比值下降，有利于骨骼發(fā)展；而大強(qiáng)度應(yīng)力刺激引起RANKL/OPG比值升高，不利骨骼發(fā)展。此外，有研究認(rèn)為雌激素對(duì)骨骼發(fā)揮的作用，可能也與OPG/RANKL/RANK信號(hào)通路有關(guān)[43-44]。通過適宜應(yīng)力刺激調(diào)節(jié)或直接靶向干預(yù)OPG、RANKL有望成為治療骨質(zhì)疏松的新方法。深入研究骨質(zhì)疏松和OPG/RANKL/RANK的關(guān)系，對(duì)預(yù)防和治療骨質(zhì)疏松癥有重要意義。

[1]孔祥鶴,牛銀波,李宇華,等.OPG/RANK/RANKL系統(tǒng)與骨質(zhì)疏松研究最新進(jìn)展[J].生命科學(xué)研究,2011,15(1):80-85.

[2]Wright HL,Mccarthy HS,Middleton J,et al.RANK,RANKL and osteoprotegerin in bone biology and disease[J].Curr Rev Musculoskelet Med,2009,2(1):56-64.

[3]Nakamura M,Udagawa N.Osteoporosis and RANKL signal[J].Clin Calcium,2011,21(8):1149-1155.

[4]Boyce BF,Xing L.Functions of RANKL/RANK/OPG in bone modeling and remodeling[J].Arch Biochem Biophys,2008,473(2):139-146.

[5]Yamaguchi M.RANK/RANKL/OPG during orthodontic tooth movement[J].Orthod Craniofac Res,2009,12(2):113-119.

[6]Harada S,Takahashi N.Control of bone resorption by RANKL-RANK system[J].Clin Calcium,2011,21(8):1121-1130.

[7]Narducci P,Bareggi R,Nicolin V.Receptor activator for nuclear factor kappa B ligand(RANKL)as an osteoimmune key regulator in bone physiology and pathology[J].Acta Histochemica,2011,113(2):73-81.

[8]Dempster DW,Lambing CL,Kostenuik PJ,et al.Role of RANK ligand and denosumab,a targeted RANK ligand inhibitor,in bone health and osteoporosis:a review of preclinical and clinical data[J].Clin Ther, 2012,34(3):521-536.

[9]Coluzzi F,Di Bussolo E,Mandatori I,et al.Bone metastatic disease: taking aim at new therapeutic targets[J].Curr Med Chem,2011,18 (20):3093-3115.

[10]Anastasia P,Ioannis P,Kelly M,et al.High levels of synovial fluid osteoprotegerin(OPG)and increased serum ratio of receptor activator of nuclear factor-κB ligand(RANKL)to OPG correlate with disease severity in patients with primary knee osteoarbritis[J].Clin Biochem, 2008,41(9):746-749.

[11]Sadahiro K,Masae O,Yukino C,et al.IL-27 suppresses RANKL expression in CD4+T cells in part through STAT3[J].Immunol Lett,2011, 38(1):47-53.

[12]Ryser MD,Qu Y,Komarova SV.Osteoprotegerin in bone metastases: mathematical solution to the puzzle[J].PLoS Comput Biol,2012,8 (10):e1002703.

[13]Roux S.New treatment targets in osteoporosis[J].Joint Bone Spine, 2010,77(3):222-228.

[14]Burkiewicz JS,Scarpace SL,Bruce SP.Denosumab in osteoporosis and oncology[J].Ann Pharmacother,2009,43(9):1445-1455.

[15]Huang YL,Liu YW,Huang YJ,et al.A special ingredient(VtR)containing oligostilbenes isolated from vitis thunbergii prevents bone loss in ovariectomized mice:in vitro and in vivo study[J].Evid Based ComplementAlternat Med,2013,2013:409421.

[16]Malliga DE,Wagner D,Fahrleitner PA.The role of osteoprotegerin (OPG)receptor activator for nuclear factor kappaB ligand(RANKL)in cardiovascular pathology－a review[J].Wien Med Wochenschr,2011, 161(23-24):565-570.

[17]Tat SK,Pelletier JP,Velasco CR,et al.New perspective in osteoarthritis:the OPG and RANKL system as a potential therapeutic target[J]. Keio J Med,2009,58(1):29-40.

[18]Enomoto T,Furuya Y,Tomimori Y,et al.Establishment of a new murine model of hypercalcemia with anorexia by overexpression of soluble receptor activator of NF-κB ligand using an adenovirus vector[J].J Bone Miner Metab,2011,29(4):414-412.

[19]Bashir A,Mak YT,Sankaralingam S,et al.Changes in RANKL/OPG/ RANK gene expression in peripheral mononuclear cells following treatment with estrogen or raloxifene[J].Steroids,2005,70(13):847-855.

[20]Furuya Y,Inagaki A,Khan M,et al.Stimulation of bone formation in cortical bone of mice treated with a receptor activator of nuclear factorκB ligand(RANKL)-binding peptide that possesses osteoclastogenesis inhibitory activity[J].J Biol Chem,2013,288(8):5562-5571.

[21]程少丹,王擁軍,唐德志,等.OPG基因敲除小鼠骨質(zhì)疏松情況的研究[J].中國(guó)骨質(zhì)疏松雜志,2008,14(1):16-19.

[22]Bergstrm I,Parini P,Gustafsson SA,et al.Physical training increases osteoprotegerin in postmenopausal women[J].J Bone Miner Metab, 2012,30(2):202-207.

[23]Miller PD.Anti-resorptives in the management of osteoporosis[J]. Best Pract Res Clin Endocrinol Metab,2008,22(5):849-868.

[24]Gallagher JC,Sai AJ.Molecular biology of bone remodeling:implications for new therapeutic targets for osteoporosis[J].Maturitas,2010, 65(4):301-307.

[25]West SL,Scheid JL,Souza MJ,et al.The effect of exercise and estrogen on osteoprotegerin in premenopausal women[J].Bone,2009,44 (1):137-144.

[26]董潔瓊.運(yùn)動(dòng)對(duì)去卵巢骨質(zhì)疏松大鼠OPG,RANKL表達(dá)的影響[D].上海:上海體育學(xué)院,2011.

[27]Wensel TM,Iranikhah MM,Wilborn TW,et al.Effects of denosumab on bone mineral density and bone turnover in postmenopausal women[J].Pharmacotherapy,2011,31(5):510-523.

[28]Orcel P.Advances treatment of osteoporosis:new molecules,new strategies[J].BullAcad Natl Med,2010,194(8):1516-1518.

[29]Lewiecki EM.Treatment of osteoporosis with denosumab[J].Maturitas,2010,66(2):182-186.

[30]Blahos J.Current and future options for treatment of osteoporosis[J]. Vnitr Lek,2011,57(11):888-890.

[31]Rudic JS,Poropat G,Krstic MN,et al.Hormone replacement for osteoporosis in women with primary biliary cirrhosis[J].Cochrane Database Syst Rev,2011,(12):CD009146.

[32]Duggan ST,McKeage K.Bazedoxifene:a review of its use in the treatment of postmenopausal osteoporosis[J].Drugs,2011,71(16):2193-2212.

[33]李世昌,季瀏,劉體偉,等.不同方式運(yùn)動(dòng)對(duì)去卵巢大鼠骨密度、骨生物力學(xué)及代謝指標(biāo)的影響[J].體育學(xué)刊,2012,19(2):132-137.

[34]Iwamoto J.Effect of exercise on developing bone mass and cortical bone geometry[J].Clin Calcium,2011,21(9):1323-1328.

[35]房冬悔.生長(zhǎng)期骨量與不同沖擊力運(yùn)動(dòng)的關(guān)系[J].中國(guó)組織工程研究與臨床康復(fù),2007,11(36):7248-7251.

[36]樓紅侃,成羿,黃良夫.運(yùn)動(dòng)對(duì)骨質(zhì)疏松的影響和機(jī)制[J].浙江臨床醫(yī)學(xué),2008,10(8):1009-1011.

[37]Zhang Y,Paul EM,Sathyendra V,et al.Enhanced osteoclastic resorption and responsiveness to mechanical load in gap junction deficient bone[J].PLoS One,2011,6(8):e23516.

[38]Kaneuji T,Ariyoshi W,Okinaga T,et al.Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts[J].Biochem Biophys Res Commun,2011,408(1):103-109.

[39]Hou WW,Zhu ZL,Zhou Y,et al.Involvement of Wnt activation in the micromechanical vibration-enhanced osteogenic response of osteoblasts[J].J Orthop Sci,2011,16(5):598-605.

[40]Li J,Rose E,Frances D,et al.Effect of oscillating fluid flow stimulation on osteocyte mRNA expression[J].J Biomech,2012,45(2):247-251.

[41]Yamamoto K,Yamamoto T,Ichioka H,et al.Effects of mechanical stress on cytokine production in mandible-derived osteoblasts[J].Oral Dis,2011,17(7):712-719.

[42]Sanchez C,Gabay O,Salvat C,et al.Mechanical loading highly increase IL-6 production and decrease OPG expression by osteoblasts[J]. Osteoarthritis Cartilage,2009,17(4):473-481.

[43]Jules J,Ashley JW,Feng X.Selective targeting of RANK signaling pathways as new therapeutic strategies for osteoporosis[J].Expert Opin Ther Targets,2010,14(9):923-934.

[44]Frenkel B,Hong A,Baniwal SK,et al.Regulation of adult bone turnover by sex steroids[J].J Cell Physiol,2010,224(2):305-310.

Stress Sensitive Signal Pathway Osteoprotegerin/ReceptorActivator of Nuclear Factor-κB ligand/ReceptorActivator of Nuclear Factor-κB and Osteoporosis(review)

LI Sheng-cun,BAO Jie,WANG Jing,et al.School of Physical Education of Soochow University,Suzhou 215021,Jiangsu,China

Exercise is benefic for osteoporosis,without clear molecular biology mechanism.The osteoprotegerin/receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factor-κB(OPG/RANKL/RANK)signal pathway contributes to osteoporosis,which can be mediated by mechanical force.Research progress on osteoporosis and the stress sensitive signal pathway OPG/RANKL/RANK were reviewed in this paper.

osteoporosis;stress;osteoprotegerin;receptor activator of nuclear factor-κB ligand;receptor activator of nuclear factor-κB; signal pathway;review

R681

A

1006-9771(2014)03-0250-03

2013-09-02

2013-11-01)

江蘇省2012年度普通高校研究生科研創(chuàng)新計(jì)劃項(xiàng)目(No.CXZZ12_0787)。

蘇州大學(xué)體育學(xué)院，江蘇蘇州市215021。作者簡(jiǎn)介：李盛村(1985-)，男，漢族，浙江溫州市人，博士研究生，主要研究方向：運(yùn)動(dòng)醫(yī)學(xué)。通訊作者：王國(guó)祥，男，教授，博士生導(dǎo)師。

10.3969/j.issn.1006-9771.2014.03.013