丁巧靈 王金鳳 徐小雅 周 軼 金慰芳 王洪復(fù) 高建軍

(復(fù)旦大學(xué)放射醫(yī)學(xué)研究所骨代謝研究室 上海 200032)

雌激素水平對(duì)大豆苷原(DAI)促成大鼠骨細(xì)胞分化作用的影響

丁巧靈 王金鳳 徐小雅△周 軼 金慰芳 王洪復(fù) 高建軍

(復(fù)旦大學(xué)放射醫(yī)學(xué)研究所骨代謝研究室 上海 200032)

目的 觀察基礎(chǔ)雌激素(estrogen)水平對(duì)大豆苷原(daizein,DAI)促成骨細(xì)胞分化作用的影響。方法采用酶消化法分離培養(yǎng)新生SD大鼠頭蓋骨成骨細(xì)胞,通過(guò)培養(yǎng)液中添加17β-雌二醇改變培養(yǎng)微環(huán)境(包括空白對(duì)照)雌激素水平,應(yīng)用MTT法和對(duì)硝基苯磷酸鹽(p-nitropheny-phosate,PNPP)法檢測(cè)細(xì)胞增殖率和堿性磷酸酶(alkaline phosphatase,ALP)活性,應(yīng)用real-time PCR法檢測(cè)其雌激素受體α(ERa)、雌激素受體β(ERβ)和過(guò)氧化物酶體增殖物激活受體(peroxisome proliferator-activated receptor,PPAR)γ的mRNA水平變化,研究雌激素基礎(chǔ)水平變化對(duì)DAI促成骨細(xì)胞分化作用的影響。結(jié)果 培養(yǎng)環(huán)境中雌二醇水平升高致DAI的促分化作用減弱。其中100 nmol/L DAI組ALP比活性較對(duì)照組的增幅由雌激素補(bǔ)充前的23%分別降至8%(17β-雌二醇為100 pmol/L)和-11%(17β-雌二醇為1 000 pmol/L),顯示基礎(chǔ)雌激素水平對(duì)DAI促分化的拮抗作用。為避免培養(yǎng)液中血清雌激素的可能影響,進(jìn)一步采用去激素血清培養(yǎng)(含2%去激素胎牛血清)。此時(shí)補(bǔ)充17β-雌二醇至100 pmol/L,DAI各劑量組細(xì)胞ALP比活性較對(duì)照組明顯增加(16%～21%,P<0.05)。繼續(xù)補(bǔ)充雌激素至1 000和10 000 pmol/L 時(shí),其作用反而減弱。該結(jié)果顯示,DAI促成骨細(xì)胞分化作用與雌激素基礎(chǔ)水平有關(guān),低雌激素狀態(tài)(≤100 pmol/L)可增強(qiáng)其作用。為進(jìn)一步探索其作用機(jī)制,我們初步觀察了雌激素(100 pmol/L)和DAI(100 nmol/L)單獨(dú)或聯(lián)合作用下成骨細(xì)胞ERa、ERβ和PPARγ受體表達(dá)變化。結(jié)果顯示,100 pmol/L 17β-雌二醇明顯上調(diào)ERβ表達(dá),并部分抵抗DAI下調(diào)ERβ的作用。結(jié)論 DAI的促成骨分化作用與基礎(chǔ)雌激素水平有關(guān),低雌激素(≤100 pmol/L)狀態(tài)有利于DAI的促分化作用,而雌激素水平升高可減弱其促分化作用。

成骨細(xì)胞; 大豆苷原(DAI); 雌激素; 堿性磷酸酶(ALP); 大鼠

近年來(lái),異黃酮(isoflavones)等植物雌激素(phytoestrogens,PE)對(duì)絕經(jīng)后婦女骨質(zhì)疏松(postmenopausal,PMOP)的防治作用受到普遍關(guān)注[1-3]。異黃酮可有效延緩卵巢切除動(dòng)物的骨量丟失,但對(duì)其臨床療效仍存爭(zhēng)議[3-4]。臨床研究結(jié)果發(fā)現(xiàn),異黃酮對(duì)絕經(jīng)后婦女骨量丟失的保護(hù)作用可表現(xiàn)為有效、部分有效,甚至無(wú)效。療效差異可能與受試者絕經(jīng)時(shí)間等有關(guān)[4-5],但確切原因和機(jī)制仍未完全闡明。雌激素是絕經(jīng)后婦女骨代謝調(diào)節(jié)的關(guān)鍵激素之一[6],異黃酮的作用差異是否與絕經(jīng)后婦女體內(nèi)雌激素水平變化有關(guān)等問(wèn)題仍不明確。大豆苷原(daidzein,DAI)是一種異黃酮化合物,前期研究發(fā)現(xiàn)雌激素可影響其對(duì)成骨細(xì)胞受體的調(diào)節(jié)作用[7]。為進(jìn)一步明確雌激素在基礎(chǔ)水平對(duì)異黃酮促骨形成作用的影響,本研究以新生SD大鼠頭蓋骨成骨細(xì)胞為研究對(duì)象,觀察不同雌二醇濃度的培養(yǎng)環(huán)境對(duì)DAI促成骨細(xì)胞分化作用的影響。

材 料 和 方 法

成骨細(xì)胞分離培養(yǎng) 參考文獻(xiàn)[8],取新生SD大鼠顱蓋骨,于0.25%胰蛋白酶(美國(guó)Amersco公司)中37 ℃下預(yù)消化20 min后,將骨片剪成約1 mm2的小塊,于0.1%Ⅱ型膠原酶(美國(guó)Sigma公司)中37 ℃消化50 min,收集消化液。用新鮮膠原酶重復(fù)消化殘留骨碎片1次。將兩次收集的消化液離心(280×g,10 min),棄上清液,沉淀用MEM培養(yǎng)液重懸接種,其中含10%胎牛血清(FBS,美國(guó)Gibco公司)、100 IU/mL青霉素(華北制藥有限公司)、100 μg/mL鏈霉素(上海新先鋒藥業(yè)有限公司)。于5%CO2、37 ℃、飽和濕度條件下培養(yǎng),每2～3天換液1次。細(xì)胞匯合后,采用0.25%胰蛋白酶消化傳代。培養(yǎng)期間用倒置相差顯微鏡觀察成骨細(xì)胞的生長(zhǎng)狀況。

堿性磷酸酶染色 培養(yǎng)皿細(xì)胞用PBS洗2次,2.5%戊二醛固定10 min,蒸餾水漂洗3次,根據(jù)堿性磷酸酶(alkaline phosphatase,ALP)染色試劑盒(華美生物工程公司)說(shuō)明書的要求,按NBT∶BCIP∶AP底物液為1∶1∶100的比例配制孵育液,37 ℃下反應(yīng)30 min,蒸餾水清洗,晾干后甘油明膠封片,顯微鏡下觀察。

藥物干預(yù) 取第2繼代細(xì)胞以3 000/孔接種于96孔培養(yǎng)板(美國(guó)Costar公司),培養(yǎng)24 h后更換為含2% FBS的MEM培養(yǎng)液。給予DAI(美國(guó)Sigma公司)1、100和10 000 nmol/L和等量MEM培養(yǎng)液(對(duì)照組)分別作用3和6天,采用對(duì)硝基苯甲酸(p-nitropheny-phosate,PNPP)法和MTT法檢測(cè)細(xì)胞ALP活性和細(xì)胞增殖率。為觀察雌激素狀態(tài)的影響,通過(guò)添加17β-雌二醇(美國(guó)Sigma公司)至100、1 000和10 000 pmol/L以調(diào)整培養(yǎng)液的雌激素基礎(chǔ)水平,給予不同濃度DAI作用6天,觀察細(xì)胞ALP活性變化。為減少血清中雌激素樣物質(zhì)的影響,進(jìn)一步采用含2%去激素胎牛血清(coat-striped FBS,CSFBS)(澳大利亞Serana公司)的MEM培養(yǎng)液,觀察雌激素在基礎(chǔ)水平對(duì)DAI作用的影響。

為研究低雌激素增強(qiáng)作用的機(jī)制,細(xì)胞分別接種于直徑35 mm的培養(yǎng)皿,匯合后更換為含2% CSFBS的MEM培養(yǎng)液。隨機(jī)分為4組,每組4皿,分別單獨(dú)或合并給予100 nmol/L DAI和100 pmol/L 17β-雌二醇(DAI、DAI-E、E組)和等量MEM培養(yǎng)液(Ctrl組)。6 h后收獲細(xì)胞,采用real-time PCR法檢測(cè)雌激素受體(estrogen receptor,ER)α、ERβ和過(guò)氧化物酶體增殖物激活受體(peroxisome proliferator-actived receptor,PPAR)γ的表達(dá)水平。

細(xì)胞ALP活性 取96孔板培養(yǎng)的細(xì)胞,經(jīng)藥物干預(yù)后,分別采用PNPP法和MTT法檢測(cè)總ALP活性和細(xì)胞增殖率,分別以D405/孔和D570/孔表示,并以D405/D570計(jì)算細(xì)胞ALP比活性。

real-time PCR分析細(xì)胞mRNA水平 以TRTzol(北京天根生化科技有限公司)一步法抽提成骨細(xì)胞總RNA,采用QuantiTect Rev.逆轉(zhuǎn)錄試劑盒(德國(guó)Qiagen公司)逆轉(zhuǎn)錄合成cDNA,采用QuantiTect SYBR Green PCR試劑盒(德國(guó)Qiagen公司)于Mx3000P實(shí)時(shí)定量PCR系統(tǒng)(品牌Stratagene,美國(guó)Agilent公司)進(jìn)行目的基因real-time RT-PCR定量分析[8]。引物序列(正義鏈/反義鏈):ERa,5′-CCG-GTCTATGGCCAGTCGAGCATC-3′/5′-GTAGAAG-GCGGGAGGGCCGGTGTC-3′(240 bp,NM012689.1);ERβ,5′-TTCCCGGCAGCACCAGTAACC-3′/5′-TCCCTCTTTGCGTTTGGACTA-3′(262 bp,NM 012754.1);PPARγ,5′-TCAGGTTTGGGCGAA-TGC-3′/5′-TTTGGTCAGCGGGAAGGA-3′(152 bp,NM013124.3);GAPDH,5′-AAACCCATCA-CCATCTTCCA-3′/5′-GTGGTTCACACCCATCACA-A-3′(198 bp,DQ 403053)。實(shí)驗(yàn)操作按試劑盒說(shuō)明,產(chǎn)物經(jīng)融解曲線單峰驗(yàn)證,獲得Ct值,計(jì)算各測(cè)試基因與內(nèi)參基因GAPDH比值作相對(duì)量分析。

結(jié) 果

DAI促進(jìn)體外培養(yǎng)成骨細(xì)胞的分化 體外培養(yǎng)大鼠成骨細(xì)胞于含2%FBS的MEM培養(yǎng)液生長(zhǎng)良好(圖1A),胞質(zhì)呈ALP染色陽(yáng)性(圖1B)。加入1、100和10 000 nmol/L DAI作用3天,細(xì)胞增殖受抑,ALP比活性較對(duì)照組略有增加(P<0.05,圖1C、D、E)。作用6天,細(xì)胞增殖受抑而ALP活性增強(qiáng)明顯,ALP比活性較對(duì)照組增加14%～22%(P<0.001),其中以100 nmol/L劑量組增幅最大(圖1F)。

DAI的促分化作用與基礎(chǔ)雌激素水平有關(guān) 含2%FBS的MEM培養(yǎng)液中,通過(guò)添加17β-雌二醇調(diào)整雌激素基礎(chǔ)水平后,觀察不同濃度DAI對(duì)成骨細(xì)胞的作用。結(jié)果顯示,在17β-雌二醇為100和1000 pmol/L的基礎(chǔ)上各劑量DAI的增殖抑制作用明顯緩解(圖2B),同時(shí)其促分化作用減弱(圖2C)。100 nmol/L DAI作用下細(xì)胞ALP比活性增幅由未添加基礎(chǔ)雌激素的23%降至添加100 pmol/L17β-雌二醇時(shí)的8%和添加1 000 pmol/L雌二醇時(shí)的-11%(圖2D)。當(dāng)雌二醇基礎(chǔ)含量達(dá)10 000 pmol/L時(shí),未觀察到DAI對(duì)細(xì)胞分化的影響(圖2C、D)。

為去除血清中雌激素樣物質(zhì)的可能干擾,進(jìn)一步培養(yǎng)細(xì)胞于含2% CSFBS的MEM培養(yǎng)液中,觀察DAI對(duì)成骨細(xì)胞的作用。結(jié)果顯示,在添加100 pmol/L 17β-雌二醇的培養(yǎng)條件下,DAI各劑量組細(xì)胞的ALP比活性較對(duì)照組增加15%～21% (P<0.01),增幅較未添加基礎(chǔ)雌激素時(shí)放大2～8倍(圖3C、D)。繼續(xù)增加基礎(chǔ)雌二醇含量至1 000和10 000 pmol/L,DAI作用減弱(圖3C、D)。

圖1 DAI對(duì)體外培養(yǎng)大鼠成骨細(xì)胞分化的作用Fig 1 The effects of DAI on the differentiation of rat calvarial osteoblasts in vitro

圖2 常規(guī)培養(yǎng)下雌激素水平對(duì)DAI促成骨細(xì)胞分化作用的影響Fig 2 Influence of estrogen levels on the effects of DAI in osteoblast differentiation in normal culture

圖3 去激素血清培養(yǎng)條件下雌激素對(duì)DAI作用的影響Fig 3 Influence of estrogen levels on DAI action on osteoblasts in MEM medium containing with 2% CSFBS

圖4 成骨細(xì)胞受體ERα 、ERβ和PPARγ的mRNA水平變化Fig 4 The mRNA levels of ERα,ERβ and PPARγ in osteoblasts

低水平雌激素上調(diào)成骨細(xì)胞ERβ表達(dá) 成骨細(xì)胞ERα、ERβ和PPARγ介導(dǎo)DAI的生物學(xué)作用,其中ERβ的作用更值得重視[9]。為了解低水平雌激素增強(qiáng)DAI作用的機(jī)制,我們進(jìn)一步觀察100 pmol/L 17β-雌二醇作用下成骨細(xì)胞受體mRNA水平的變化。結(jié)果顯示,雌激素作用6 h,成骨細(xì)胞ERα mRNA水平顯著下調(diào),僅為對(duì)照組的12%;而ERβ mRNA水平上調(diào),約為對(duì)照組的2.5倍(圖4A、B)。同時(shí)雌激素有效減弱DAI對(duì)ERβ mRNA水平下調(diào)作用。DAI組ERα 和ERβ mRNA水平分別為對(duì)照組的24%和18%。DAI-雌激素組ERα mRNA水平仍維持低水平,而ERβ mRNA恢復(fù)至對(duì)照組的67%。成骨細(xì)胞PPARγ mRNA水平在各組間的差異無(wú)統(tǒng)計(jì)學(xué)意義(圖4C)。研究表明,低水平雌激素可能通過(guò)上調(diào)成骨細(xì)胞ERβ mRNA水平和抵抗DAI對(duì)其的下調(diào)作用,增強(qiáng)其結(jié)合DAI的能力。

討 論

異黃酮類PE包括DAI、染料木黃酮(genistein)、黃豆黃素(glycitein)等,因具有雌激素樣抑制骨吸收、促進(jìn)骨形成作用而有望成為PMOP激素治療的可能替代物[1-3]。動(dòng)物實(shí)驗(yàn)研究表明異黃酮可以有效延緩卵巢切除鼠的骨量丟失[10],其中DAI的骨保護(hù)作用更明顯[11-12]。與動(dòng)物學(xué)試驗(yàn)相對(duì)一致的研究結(jié)果不同,PE延緩絕經(jīng)后婦女骨量丟失的研究結(jié)果不盡相同,出現(xiàn)有效[13-15]、部分有效[16]和無(wú)效[4,17-19]等不同甚至相反的結(jié)論。差異的產(chǎn)生可能與受試者絕經(jīng)時(shí)間、體重和基礎(chǔ)骨量等有關(guān)[5,16],也可能與受試藥物劑量(如總苷原需達(dá)到60～100 mg/d)和使用時(shí)間(如需達(dá)到2年)[2]以及藥物代謝(如牛尿酚生成量)[20-21]等有關(guān),但其確切機(jī)制尚不明確。

多位學(xué)者研究中發(fā)現(xiàn)植物雌激素的療效與受試者絕經(jīng)后時(shí)間有關(guān)[3-5]。在一項(xiàng)涉及202名60～75歲絕經(jīng)后健康婦女、為期1年的雙盲對(duì)照研究中,Kreijkamp等[4]通過(guò)以絕經(jīng)年限分層分析發(fā)現(xiàn),異黃酮(99 mg/天)干預(yù)后絕經(jīng)<14年的人群骨密度值呈增加趨勢(shì),其中粗隆間骨密度增加有統(tǒng)計(jì)學(xué)意義,而在絕經(jīng)>22年的人群骨密度值呈下降趨勢(shì)。對(duì)此作者認(rèn)為延緩骨量丟失較糾正已發(fā)生的骨量丟失更容易。Chen等[5]在一項(xiàng)針對(duì)203名48～62歲絕經(jīng)后婦女、為期1年的干預(yù)研究中發(fā)現(xiàn),異黃酮(80 mg/天)在絕經(jīng)≥4年的婦女中顯示出骨保護(hù)作用(全髖和大轉(zhuǎn)子骨礦含量增加),而在絕經(jīng)<4年的婦女中不顯示該作用。作者認(rèn)為異黃酮無(wú)法改善絕經(jīng)早期快速的骨量丟失,而隨著快速丟失期結(jié)束,異黃酮的作用開(kāi)始顯現(xiàn)?？梢?jiàn),PE對(duì)絕經(jīng)婦女骨量丟失的改善作用可能存在窗口期(絕經(jīng)后4～14年),窗口期干預(yù)多觀察到PE的骨保護(hù)作用。Marini等[13]選取的受試者絕經(jīng)時(shí)間分別為(69.5±47.4)個(gè)月(干預(yù)組,n=150)和(59.8±38.8)個(gè)月(對(duì)照組,n=154),經(jīng)過(guò)2年的干預(yù)證實(shí)染料木黃酮(54 mg/天)可改善腰椎和股骨頸骨密度[13]。當(dāng)受試者絕經(jīng)時(shí)間分別為(7±6)年(干預(yù)組)和(6±5)年(對(duì)照組)時(shí),經(jīng)過(guò)1年的干預(yù)研究也觀察到染料木黃酮改善腰椎和股骨頸骨密度等[20]。而當(dāng)受試者絕經(jīng)時(shí)間較短,如在一項(xiàng)涉及237名絕經(jīng)婦女的多中心雙盲對(duì)照研究中[平均年齡為(53±3)歲,絕經(jīng)時(shí)間為(33±15)個(gè)月],Brink等[17]未觀察到異黃酮(110 mg/天,1年)的骨保護(hù)作用。Zhu等[15]在一項(xiàng)多中心聯(lián)合研究中將受試者絕經(jīng)時(shí)間擴(kuò)展至8～25年,結(jié)果顯示仙靈骨葆膠囊可短期改善部分部位(腰椎)骨密度,療效在6月后降低,此療效的局限性是否與受試者絕經(jīng)時(shí)間的跨度大有關(guān)等問(wèn)題值得進(jìn)一步分析。這些研究提示PE對(duì)絕經(jīng)婦女骨量丟失的保護(hù)作用可能在絕經(jīng)后4～14年間比較明顯。

絕經(jīng)后骨量經(jīng)過(guò)3～5年快速丟失后逐漸進(jìn)入年平均丟失1%的緩慢丟失階段,盡管對(duì)該階段骨量丟失機(jī)制仍有不同看法,雌激素?zé)o疑仍發(fā)揮重要調(diào)節(jié)作用[6]。絕經(jīng)后雌激素水平下降。研究顯示,當(dāng)血清雌二醇水平低于40 pg/mL(約108 pmol/L)時(shí)就可能發(fā)生骨量丟失[21],故常以E2<100 pmol/L作為判斷絕經(jīng)與否或受試者入選條件之一。隨著絕經(jīng)時(shí)間延長(zhǎng),雌激素水平仍持續(xù)減低,如女性絕經(jīng)時(shí)間為15～16年則血清E2僅為16～18 pmol/L[15]。絕經(jīng)后基礎(chǔ)雌激素水平的變動(dòng)是否會(huì)影響PE的作用尚不清楚。

本課題組前期研究中觀察到,DAI通過(guò)調(diào)節(jié)ER和PPARγ等受體基因表達(dá)可間接影響成骨細(xì)胞的藥物反應(yīng),并且其受體調(diào)節(jié)作用受雌激素的影響[7]。為進(jìn)一步觀察雌激素狀態(tài)的作用,我們通過(guò)添加17β-雌二醇以改變培養(yǎng)液的雌激素基礎(chǔ)水平,觀察DAI體外促大鼠成骨細(xì)胞分化的作用變化。結(jié)果顯示,低雌激素狀態(tài)有利于DAI促分化的作用。隨著培養(yǎng)液中雌激素濃度增加(100～10 000 pmol/L),DAI的作用減弱。而當(dāng)處于無(wú)激素或低激素狀態(tài)下(采用含2%CSFBS培養(yǎng)液以去除血清中雌激素樣物質(zhì)),DAI促分化作用反而微弱,此時(shí)補(bǔ)充100 pmol/L 17β-雌二醇后,其作用明顯增強(qiáng)。各劑量組細(xì)胞ALP比活性較對(duì)照組增加16%～21%,增幅較基礎(chǔ)雌激素補(bǔ)充前放大3.6～7.5倍,顯示一定量雌激素(～100 pmol/L)環(huán)境有利于DAI發(fā)揮作用。Sacco等[24]也觀察到低劑量雌激素(17β-雌二醇,13 μg,90天緩釋)增強(qiáng)亞麻籽延緩卵巢切除大鼠腰椎骨丟失的作用。繼續(xù)補(bǔ)充雌激素至1 000和10 000 pmol/L 時(shí),DAI的作用減弱并呈低劑量促進(jìn)/高劑量抑制的雙相變化趨勢(shì)。

成骨細(xì)胞ER和PPARγ是PE的主要靶受體,分別介導(dǎo)促進(jìn)和抑制細(xì)胞成骨作用[9]。由于與PE的高親和力,ERβ介導(dǎo)的作用更突出[25-27]。為了解低雌激素(100 pmol/L)增強(qiáng)DAI作用的機(jī)制,我們初步觀察了成骨細(xì)胞受體ERα、ERβ和PPARγ表達(dá)變化。結(jié)果顯示,100 pmol/L 17β-雌二醇作用下ERα表達(dá)顯著下調(diào),而ERβ轉(zhuǎn)錄明顯上調(diào)(約為對(duì)照組的2.5倍)。當(dāng)與DAI共同作用下(DAI-E組),ERα表達(dá)維持低水平,而ERβ上升至對(duì)照組的67%(P>0.05),反映低濃度雌激素對(duì)成骨細(xì)胞ERβ表達(dá)的提升作用及對(duì)DAI下調(diào)作用的抵抗。上調(diào)的ERβ可能通過(guò)增強(qiáng)其結(jié)合DAI的能力[23-24]或抑制PPARγ信號(hào)傳遞[25]來(lái)促進(jìn)成骨細(xì)胞分化。

本研究表明,DAI的促成骨分化作用與微環(huán)境中雌激素基礎(chǔ)水平有關(guān),低雌激素(≤100 pmol/L)環(huán)境有利于DAI發(fā)揮作用,而雌激素水平升高則減弱其促分化作用。盡管體外實(shí)驗(yàn)結(jié)果有一定的局限性,并且未涉及更低雌激素水平(<100 pmol/L)的影響,但本研究仍從細(xì)胞水平觀察到雌激素狀態(tài)對(duì)DAI促成骨作用的影響,提示雌激素基礎(chǔ)水平可能影響PE骨改善作用的發(fā)揮,并為PE防治骨質(zhì)疏松癥的臨床研究提供實(shí)驗(yàn)依據(jù)。

致謝 復(fù)旦大學(xué)放射醫(yī)學(xué)研究所邵春林教授在基因分析過(guò)程中提供了幫助。

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Influence of estrogen levels in medium on actions of daidzein (DAI) in osteoblast differentiation of ratsinvitro

DING Qiao-ling, WANG Jin-feng, XU Xiao-ya△, ZHOU Yi, JIN Wei-fang, WANG Hong-fu, GAO Jian-jun

(DepartmentofBoneMetabolism,InstituteofRadiationMedicine,FudanUniversity,Shanghai200032,China)

Objective To investigate the contribution of circulating estrogen to the action of daidzein (DAI) on osteoblast differentiationinvitro. Methods Osteoblasts were prepared from calvaria of neonatal SD rats by sequential collagenase digestion and treated with DAI in MEM medium supplied with 2% fetal bovine serum (FBS) or 2% coat-striped fetal bovine serum (CSFBS) respectively.17β-estradiol was added to all medium to modify the estrogen levels as required.The cell proliferation and activity of alkaline phosphatase (ALP) were measured by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and p-nitropheny-phosate (PNPP) assays respectively.The expression of ERa、ERβ and peroxisome proliferator-activated receptor (PPAR) γ were determined by real-time RT-PCR. Results Daidzein promoted osteoblasts differentiationinvitro,which was modified by estrogen levels in medium.Compared to the control,the cell ALP activity (D405/D570) was increased to 23% (P<0.001) by 100nmol/L of daidzein in medium containing with 2% FBS.The increase was fell down to 8% in presence of 100 pmol/L 17β-estradiol and -11% in presence of 1 000 pmol/L.When cultured in MEM medium containing with 2% CSFBS,the cell ALP activities,which were slightly increased by 1-100 nmol/L of daidzein,were obviously increased by 16%-21% (P<0.05) in presence of 100 pmol/L 17β-estradiol.The promotions was weaken by the continued increase of estrogen in medium.The expressions of ERα were dramatically down regulated by 100 nmol/L of daidzein alone or the combination with 100 pmol/L of estradiol.The transcriptional levels of ERβ were markedly increased (up to 2.5 times) by estradiol,but decreased to 18% (P<0.05) by daidzein,while back to 67% (P>0.05) by the combination.The expressions of PPARγ were not altered significantly in transcriptional levels. Conclusions The results indicated that effects of daidzein in osteogenesis was modified by the levels of estrogen in medium and the optimal lower level of estrogen (≤100 pmol/L) was in favour of daidzein in promoting osteoblast differentiation.

osteoblast; daidzein (DAI); estrogen; alkaline phosphatase (ALP); rat

R 336, R 589.5

A

10.3969/j.issn.1672-8467.2015.02.008

2013-10-19;編輯:段佳)

△Corresponding author E-mail:mulei80@sina.com