王 璇，陳林珍，林瑞超，陶曉宇，于 雪，張淑靜，范琦琦，李芝奇，戴勝云，李向日*，趙崇軍*

基于斑馬魚模型的中藥抗骨質(zhì)疏松的研究進(jìn)展

王 璇1，陳林珍1，林瑞超1，陶曉宇1，于 雪2，張淑靜2，范琦琦1，李芝奇1，戴勝云3，李向日1*，趙崇軍1*

1. 北京中醫(yī)藥大學(xué) 中藥品質(zhì)評(píng)價(jià)北京市重點(diǎn)實(shí)驗(yàn)室，北京 102488 2. 北京中醫(yī)藥大學(xué)，北京 102488 3. 中國(guó)食品藥品檢定研究院，北京 102629

骨質(zhì)疏松癥（osteoporosis，OP）是一種以骨密度降低、骨骼微結(jié)構(gòu)改變和骨質(zhì)脆弱增加為特征的骨骼疾病。由于老齡化問題的逐漸加重，OP的發(fā)病率在全球范圍內(nèi)迅速增加。中藥在預(yù)防和治療OP中表現(xiàn)出極好的前景和臨床效果，然而傳統(tǒng)動(dòng)物模型具有耗時(shí)長(zhǎng)、費(fèi)用昂貴、無法準(zhǔn)確概括骨疾病復(fù)雜性等缺點(diǎn)，很大程度上限制了臨床前OP疾病的研究進(jìn)程。而斑馬魚模型可有效模擬人類骨質(zhì)減少和OP表型，通過對(duì)斑馬魚模型在治療OP研究中的優(yōu)勢(shì)和適用性及該模型在中藥抗OP的研究進(jìn)展進(jìn)行綜述，為斑馬魚模型的廣泛使用及中藥新藥研發(fā)提供參考。

斑馬魚；骨質(zhì)疏松；中藥；藥對(duì)；中藥復(fù)方；藥物篩選

骨質(zhì)疏松癥（osteoporosis，OP）是常見的骨骼疾病，也是現(xiàn)代社會(huì)長(zhǎng)期困擾患者的基礎(chǔ)疾病之一。近年來，中醫(yī)藥治療OP已顯現(xiàn)出巨大優(yōu)勢(shì)。然而，中藥化學(xué)成分復(fù)雜，不同化學(xué)成分的活性、靶點(diǎn)及作用途徑往往各不相同，因此在中藥抗OP的研究中，亟需一種新的動(dòng)物模型研究中藥調(diào)控骨合成代謝或抗再吸收化合物的發(fā)現(xiàn)和篩選。斑馬魚因其早期發(fā)育的光學(xué)透明性、與人類在生理學(xué)和遺傳學(xué)的相似性及高通量?jī)?yōu)勢(shì)，成為骨骼研究的模式生物，并隨著深入研究表現(xiàn)出愈發(fā)明顯的優(yōu)勢(shì)。本文主要對(duì)近年來斑馬魚在中藥抗OP方面的研究進(jìn)展進(jìn)行綜述，為完善基于斑馬魚模型的中藥OP評(píng)價(jià)技術(shù)與方法，進(jìn)一步促進(jìn)斑馬魚在中藥研究中的廣泛應(yīng)用提供參考。

1 OP的流行病學(xué)及治療策略

OP是老年人群中常見的骨骼代謝性疾病，其特征是骨密度低、骨結(jié)構(gòu)惡化，致使意外骨折頻率增加。作為一種高患病率疾病，隨著老齡化人口的增加，OP的流行性逐步增加，預(yù)計(jì)2040年，將有超過3.2億人處于高骨折風(fēng)險(xiǎn)狀態(tài)；到2050年，全世界男性髖部骨折的發(fā)病率將增加310%，女性將增加240%[1-2]。盡管飲食、運(yùn)動(dòng)、藥物等在內(nèi)的一系列可調(diào)節(jié)因素均可導(dǎo)致年齡相關(guān)性骨質(zhì)丟失，但原發(fā)性骨質(zhì)丟失往往由遺傳因素介導(dǎo)。由于OP相關(guān)性狀的遺傳度變化范圍較大，使得對(duì)該層面的研究存在較大難度，進(jìn)而導(dǎo)致OP的分子機(jī)制目前尚未明確。

OP主要是由成骨細(xì)胞相關(guān)的骨形成活性和破骨細(xì)胞相關(guān)的骨吸收活性間的穩(wěn)態(tài)失衡引起的[3]。目前常用的治療OP的藥物主要包括骨吸收抑制藥物、促進(jìn)骨形成藥物及具有補(bǔ)鈣、多靶點(diǎn)的生物制劑。但大多數(shù)化學(xué)藥長(zhǎng)期服用療效有限，且易引發(fā)不良反應(yīng)，因此，在臨床使用過程中要嚴(yán)格控制用藥量和用藥時(shí)長(zhǎng)[4]。中藥在臨床上主要是從健脾補(bǔ)腎的角度出發(fā)，基于復(fù)方中多成分、多途徑、多靶點(diǎn)的特征提升骨密度，在保證用藥安全的前提下，達(dá)到較好的療效。因此，基于傳統(tǒng)中醫(yī)藥理論，開發(fā)新的具有顯著療效且安全的中藥治療，并基于現(xiàn)代技術(shù)明確其物質(zhì)基礎(chǔ)和作用機(jī)制，對(duì)于促進(jìn)中藥在臨床治療OP方面具有重要意義。目前只有少數(shù)高等脊椎動(dòng)物的體內(nèi)模型可用于骨衰老的致病和治療研究，如去卵巢嚙齒類動(dòng)物已經(jīng)成為OP研究和化合物早期測(cè)試的參考模型[5]，但高成本和低通量篩查相關(guān)問題限制了其在OP研究特定領(lǐng)域的應(yīng)用。而體外細(xì)胞模型在一定程度上無法準(zhǔn)確反映OP疾病的真實(shí)特征。基于此，尋找可替代的動(dòng)物模型用于解決OP的病理生理學(xué)機(jī)制，探討經(jīng)典抗OP藥物的作用內(nèi)涵，對(duì)于加速骨骼研究及測(cè)試、確定OP治療的新藥物尤為重要[6-7]。

2 斑馬魚研究OP的適用性

近些年，斑馬魚已成為骨骼研究的模式生物，在早期發(fā)育過程中具光學(xué)透明性，使用細(xì)胞特異性細(xì)胞可以在體內(nèi)輕松、非侵入性地監(jiān)測(cè)魚類的成骨細(xì)胞和破骨細(xì)胞，從而實(shí)現(xiàn)細(xì)胞追蹤[8-10]。此外，斑馬魚骨和軟骨特征的分析最早可以在受精后3 d開始，期間可通過觀察骨和軟骨的發(fā)育狀況進(jìn)行藥物篩選[11]。同時(shí)斑馬魚每周產(chǎn)卵量大，滿足了多元化設(shè)置實(shí)驗(yàn)條件及優(yōu)化實(shí)驗(yàn)方案的需求[12]。其他特征如體外發(fā)育快、操作簡(jiǎn)單等也增強(qiáng)了斑馬魚在骨骼研究中的受歡迎程度，是基因功能發(fā)現(xiàn)和進(jìn)行變異體功能分析的首選模型，為小鼠致死等位基因研究提供了可行的替代方案。

相似的生理、遺傳學(xué)和與人類共享的藥物反應(yīng)突出了斑馬魚作為藥物發(fā)現(xiàn)新模型的優(yōu)勢(shì)[13]。其中70%的人類基因在斑馬魚基因組中至少有一個(gè)同源基因[14]。此外，斑馬魚和哺乳動(dòng)物中幾乎所有的骨骼都是匹配的，控制骨骼發(fā)生的基因、細(xì)胞和機(jī)制及骨骼發(fā)育相關(guān)過程都是高度保守的[15]。斑馬魚模型對(duì)基因操作/編輯的適應(yīng)性，如鋅指核酸酶[16]、嗎啉反義寡核苷酸[17]等，使基于斑馬魚的OP模型快速研究變得可行。成簇的規(guī)律間隔的短回文重復(fù)序列系統(tǒng)基因組編輯技術(shù)可徹底改變斑馬魚基因功能測(cè)試的方式，能模擬人類骨骼疾病的突變系，重現(xiàn)性好、效率高，可實(shí)現(xiàn)對(duì)敲除表型的快速分析，進(jìn)而確認(rèn)相關(guān)基因的主要功能[18-19]，為OP提供了新的治療方法。斑馬魚還可以作為測(cè)試OP候選藥物的工具，并幫助評(píng)估針對(duì)OP癥狀的藥物。目前，基于斑馬魚表型藥物篩選鑒定出的多種化合物正在進(jìn)行臨床研究，如抗破骨細(xì)胞分化因子（receptor activator of nuclear factor-κB ligand，RANKL）單克隆抗體地諾單抗[20]和抗Wnt抑制劑結(jié)合硬化蛋白的單克隆抗體羅莫單抗[21]。此外，骨形態(tài)發(fā)生蛋白（bone morphogenetic protein，BMP）信號(hào)的小分子抑制劑多索嗎啡是利用斑馬魚進(jìn)行化學(xué)篩選發(fā)現(xiàn)的首批化合物之一[22]，且多索嗎啡衍生物（間變性淋巴瘤激酶2抑制劑）正在進(jìn)行治療性骨化性纖維發(fā)育不良異位骨化的臨床試驗(yàn)。

隨著斑馬魚發(fā)育過程中透明度逐漸喪失，成年斑馬魚內(nèi)骨骼被肌肉組織和礦化鱗片覆蓋，但仍可運(yùn)用體內(nèi)成像技術(shù)檢測(cè)骨性元素（顱骨、鰭、島蓋、頜和鱗片），且其他技術(shù)（類似于在小鼠中使用的技術(shù)）在斑馬魚中同樣適用。如茜素紅染色被用于顯示礦化骨，小動(dòng)物計(jì)算機(jī)體層顯像儀可以進(jìn)行3D形態(tài)分析和精確的骨密度測(cè)量。與傳統(tǒng)嚙齒類動(dòng)物模型相比，基于斑馬魚幼魚和成魚模型進(jìn)行OP相關(guān)評(píng)價(jià)，均可以向系統(tǒng)水或胚胎培養(yǎng)基中添加化學(xué)試劑進(jìn)行暴露操作后通過骨骼表型研究來進(jìn)行檢測(cè)[23-24]，操作簡(jiǎn)單。同時(shí)，對(duì)于某些藥物，斑馬魚比小鼠模型更能重現(xiàn)特定藥物在人類臨床使用中觀察到的效果。沙利度胺不會(huì)引起出生小鼠出現(xiàn)與人類相似的出生缺陷，但是斑馬魚能夠再現(xiàn)沙利度胺對(duì)肢體的致畸性[25-26]，表明基于斑馬魚模型在骨相關(guān)遺傳疾病研究中的重要性。

3 斑馬魚OP模型的造模方法

3.1 糖皮質(zhì)激素（glucocorticoid，GC）誘導(dǎo)的OP

GC是一種有效的免疫抑制藥物，長(zhǎng)期使用會(huì)通過減少骨/血管形成及增加骨吸收影響骨代謝[27]，進(jìn)而導(dǎo)致復(fù)雜的不良反應(yīng)，包括繼發(fā)性O(shè)P。由于GC誘導(dǎo)斑馬魚幼魚模型的造模方法簡(jiǎn)便易實(shí)施，因此被作為一種常用的OP模型[28]。研究表明，潑尼松龍可通過改變參與成骨細(xì)胞和破骨細(xì)胞信號(hào)通路關(guān)鍵基因的表達(dá)水平，降低成骨細(xì)胞的增殖活性和數(shù)量，誘導(dǎo)細(xì)胞凋亡，而促進(jìn)破骨細(xì)胞的增殖和活性，進(jìn)而引起斑馬魚幼魚組織骨化減少，與人類OP存在相似之處[29]，證明潑尼松龍誘導(dǎo)的斑馬魚OP模型的準(zhǔn)確性[30]。

在OP相關(guān)研究中，常以斑馬魚鱗片作為研究部位，這種鱗片是由磷酸鈣和羥基磷灰石晶體構(gòu)成的，其結(jié)構(gòu)與人類編織骨非常相似，具有相同的細(xì)胞類型和與人體組織沉積和吸收相似的調(diào)節(jié)機(jī)制[31-32]。潑尼松龍可通過增強(qiáng)破骨細(xì)胞活性，增加基質(zhì)吸收，降低膠原編碼基因表達(dá)，誘導(dǎo)成骨細(xì)胞和破骨細(xì)胞的OP基因表達(dá)譜，使礦物質(zhì)含量減少和引起吸收陷窩，進(jìn)而誘導(dǎo)OP表型[33]。此外，潑尼松龍還可誘導(dǎo)骨標(biāo)志物的改變，如抗酒石酸酸性磷酸酶活性（tartrate-resistant acid phosphatase activity，TRAP）增加和堿性磷酸酶（alkaline phosphatase activity，ALP）活性降低[34]。Carnovali等[35]基于茜素紅S、骨基質(zhì)中的鈣和乙二胺四乙酸間的特異性結(jié)合水平量化礦物質(zhì)的含量。此外，地塞米松誘導(dǎo)的鱗片再生畸形模型[36]和斑馬魚鱗片OP模型[37]也被用于抗OP藥物的篩選和評(píng)價(jià)。為基于斑馬魚成魚鱗片進(jìn)行OP治療藥物篩選的提供理論依據(jù)。也有研究表明潑尼松龍可誘導(dǎo)老化魚鱗出現(xiàn)骨吸收，降低ALP/TRAP的值，為預(yù)防年齡相關(guān)骨改變提供新的治療策略[38]。同時(shí)，斑馬魚尾鰭也常作為研究部位探究抗礦化和促礦化化合物對(duì)骨骼元素、結(jié)構(gòu)和力學(xué)特性的影響。潑尼松龍可通過影響成骨細(xì)胞和破骨細(xì)胞的數(shù)量、活性和分化，改變關(guān)鍵生物過程相關(guān)基因的表達(dá)，惡化再生鰭的細(xì)胞運(yùn)輸，干擾骨組織恢復(fù)，從而延遲鰭條新生形成過程中的骨生長(zhǎng)和受損骨再生過程[39-40]。而阿侖膦酸鹽可拮抗?jié)娔崴升垖?duì)鰭條礦化和鈣/磷水平的影響，恢復(fù)骨彈性系數(shù)和硬度水平等生物力學(xué)性能[41]。進(jìn)一步證實(shí)了該模型的適用性。

3.2 高糖、高脂誘導(dǎo)的OP

糖尿病是一種慢性代謝性疾病，能夠引發(fā)或加劇OP發(fā)展。當(dāng)前，糖尿病和OP是臨床常見的并發(fā)癥[42]。而斑馬魚已成為研究病理?xiàng)l件下高血糖/肥胖和骨骼相互作用的模型選擇[43]。研究表明在高血糖狀態(tài)下，糖尿病斑馬魚模型幼魚成骨和尾鰭鰭條的再生能力均受損，而維生素D類似物（帕立骨化醇）和擬鈣劑（西那卡塞）可逆轉(zhuǎn)該現(xiàn)象的發(fā)生[44]。Carnovali等[45]發(fā)現(xiàn)葡萄糖暴露可引起成年斑馬魚鱗片基質(zhì)礦化減少，誘導(dǎo)強(qiáng)烈的破骨細(xì)胞活性相關(guān)的骨吸收陷窩，改變骨調(diào)節(jié)基因的表達(dá)，且與糖尿病嚙齒類動(dòng)物骨骼情況類似[46]。此外，高脂飲食喂養(yǎng)且出現(xiàn)肥胖跡象的成年斑馬魚鱗片也發(fā)現(xiàn)類似的表型結(jié)果[47]。

3.3 鐵過載/缺鐵誘導(dǎo)的OP

鐵作為一種輔助因子，在參與關(guān)鍵代謝功能的生化反應(yīng)中具有重要作用。過量鐵會(huì)促進(jìn)破骨細(xì)胞分化并增加骨吸收[48]。在斑馬魚幼魚中，鐵過載可抑制骨形成，降低成骨細(xì)胞標(biāo)志基因的表達(dá)。膜鐵轉(zhuǎn)運(yùn)蛋白1表達(dá)缺陷的斑馬魚突變體表現(xiàn)出明顯的骨形成缺陷，包括鈣化椎骨數(shù)量減少和成骨細(xì)胞基因的異常表達(dá)[49]。高鐵應(yīng)激也被用于促進(jìn)斑馬魚OP表型，幼魚和成魚都顯示出骨礦化顯著減少和軟骨形成發(fā)育缺陷，而阿侖膦酸鹽作為靶向BMP信號(hào)通路和促進(jìn)成骨細(xì)胞分化的藥物，可顯著逆轉(zhuǎn)缺陷骨的生成[50-51]。

3.4 基因編輯技術(shù)誘導(dǎo)的OP

基因編輯技術(shù)能夠獲得部分模擬OP表型特征的突變體。這些模型是研究OP相關(guān)基因功能和發(fā)病機(jī)制的潛在工具，如ATP酶H+轉(zhuǎn)運(yùn)V1亞基H（ATPase H+transporting V1 subunit H，ATP6V1H）、G蛋白偶聯(lián)受體137b（G protein coupled receptor 137b，GPR137B）、葡萄糖神經(jīng)酰胺酶1（glucosylceramidase beta 1，GBA1）、Sp7轉(zhuǎn)錄因子（Sp7 transcription factor，SP7）突變體皆表現(xiàn)骨表型異常[52-55]。

3.5 其他方式誘導(dǎo)的OP

骨量減少的特征是骨密度輕度降低，長(zhǎng)時(shí)間不糾正會(huì)導(dǎo)致OP，而該過程可能涉及GC的作用和神經(jīng)內(nèi)分泌軸的激活[56]。甲狀旁腺激素持續(xù)暴露可誘導(dǎo)斑馬魚幼魚骨礦化顯著減少[11]。14 d以上活動(dòng)受限可誘導(dǎo)成年斑馬魚骨量減少的穩(wěn)定模型。研究發(fā)現(xiàn)在運(yùn)動(dòng)受限和機(jī)械負(fù)荷減少的情況下，斑馬魚的椎體形狀和椎間隙骨骼參數(shù)發(fā)生顯著改變，骨體積和密度顯著降低[57]。此外，在適當(dāng)?shù)膽?yīng)激條件下（如禁食），魚類可能發(fā)展出與人類疾病相似的OP表型[58]。注射肉毒桿菌毒素后斑馬魚肌肉的短暫麻痹能夠通過減少機(jī)械負(fù)荷和破壞神經(jīng)功能，進(jìn)而損害尾鰭再生過程中的膜內(nèi)骨化[59]。

斑馬魚OP模型的造模方法見表1，基因編輯技術(shù)誘導(dǎo)斑馬魚OP模型見表2。

4 基于斑馬魚模型抗OP藥物的篩選和評(píng)價(jià)

斑馬魚OP模型已被研究者廣泛應(yīng)用于探索疾病機(jī)制，篩選新的治療靶點(diǎn)和潛在治療藥物。目前，綠色生活的理念促使人們不斷嘗試從植物中篩選提取物和化學(xué)成分作為營(yíng)養(yǎng)物質(zhì)，進(jìn)一步促進(jìn)了傳統(tǒng)中藥被大量開發(fā)。事實(shí)上，中藥在臨床治療OP方面具有顯著優(yōu)勢(shì)，其中一些天然提取物/化學(xué)成分具有抗骨吸收和骨合成代謝活性，但缺乏強(qiáng)有力的科學(xué)數(shù)據(jù)?；诖耍芯空呤褂冒唏R魚模型對(duì)經(jīng)典復(fù)方、中藥藥對(duì)及單味中藥進(jìn)行藥效學(xué)評(píng)價(jià)，篩選其主要活性物質(zhì)基礎(chǔ)，闡釋其主要活性機(jī)制，并取得了顯著的研究成果。

表1 斑馬魚OP模型的造模方法

RUNX2-成骨細(xì)胞特異性轉(zhuǎn)錄因子2 COL1a1-I型膠原蛋白α1鏈

RUNX2-Runt-related transcription factor 2 COL1a1-collagen type I α1 chain

表2 基因編輯技術(shù)誘導(dǎo)斑馬魚OP模型

LRP5-低密度脂蛋白受體相關(guān)蛋白5 EGR1-早期生長(zhǎng)反應(yīng)1 NR3C1-核受體亞家族3 C組成員1 MMP-基質(zhì)金屬蛋白酶 ACP5a-酸性磷酸酶5a

LRP5-low density lipoprotein receptor related protein 5 EGR1-early growth response 1 NR3C1-nuclear receptor subfamily 3 group C member 1 MMP-matrix metalloproteinase ACP5a-acid phosphatase 5a

4.1 在經(jīng)典復(fù)方藥效學(xué)評(píng)價(jià)中的應(yīng)用

目前斑馬魚OP模型主要被用于經(jīng)典復(fù)方、中藥藥對(duì)及單味藥的藥效學(xué)評(píng)價(jià)，如基于斑馬魚顱骨染色面積、骨礦化面積、累積光密度等不同指標(biāo)發(fā)現(xiàn)仙靈骨葆[67]、二仙湯[73]、二至丸[74]和左歸丸[75]等具有顯著的抗OP活性，且與傳統(tǒng)的經(jīng)典動(dòng)物模型具有較高的一致性，一方面揭示斑馬魚模型在復(fù)雜化學(xué)體系中藥復(fù)方抗OP藥效評(píng)價(jià)中的適用性，同時(shí)也揭示了經(jīng)典復(fù)方在對(duì)抗繼發(fā)OP方面的潛力。然而，目前中藥復(fù)方的主要物質(zhì)基礎(chǔ)和作用機(jī)制缺乏深入研究。因此，借助于斑馬魚的高通量?jī)?yōu)勢(shì)、現(xiàn)代生物信息方法的聯(lián)合優(yōu)勢(shì)，為系統(tǒng)闡釋中藥多成分、多靶點(diǎn)、多途徑的作用特點(diǎn)提供參考。Zhong等[74]結(jié)合網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)和斑馬魚的實(shí)驗(yàn)驗(yàn)證，發(fā)現(xiàn)二至丸中的槲皮素為重要代表性成分。

4.2 在中藥藥對(duì)藥效學(xué)評(píng)價(jià)中的應(yīng)用

在中藥藥對(duì)抗糖尿病OP研究方面，Xu等[76]基于高通量的斑馬魚OP幼魚、成魚模型發(fā)現(xiàn)知母-黃柏（1∶1）對(duì)糖尿病-OP的治療效果最佳，可顯著抑制TRAP和促進(jìn)ALP活性，上調(diào)成骨基因、下調(diào)破骨相關(guān)基因的表達(dá)水平。Lee等[77]發(fā)現(xiàn)牛膝-杜仲（1∶1）聯(lián)合使用治療OP的效果顯著優(yōu)于單藥處理，能夠通過改善成骨、破骨蛋白、基因的表達(dá)水平緩解OP的發(fā)展。這種模式對(duì)于基于中醫(yī)藥理論和臨床使用環(huán)境來進(jìn)行中藥藥對(duì)的進(jìn)一步新藥開發(fā)具有重要的指導(dǎo)意義。

4.3 在單味中藥藥效學(xué)評(píng)價(jià)中的應(yīng)用

在單味藥藥效學(xué)評(píng)價(jià)方面，Peng等[78]基于斑馬魚模型發(fā)現(xiàn)骨碎補(bǔ)可顯著逆轉(zhuǎn)OP模型斑馬魚幼魚鈣化延遲，通過增加膠原I、骨橋蛋白（osteopontin，OPN）和骨連接蛋白的表達(dá)增加成骨細(xì)胞的活性，并通過減少M(fèi)MP9和MMP13a的表達(dá)抑制骨吸收。牡蠣通過上調(diào)成骨細(xì)胞標(biāo)志物（ALP、RUNX2和osterix），減少鈣化水平，進(jìn)而恢復(fù)成骨活性[79]。青風(fēng)藤提取物及其所含生物堿可以通過調(diào)節(jié)絲裂原活化蛋白激酶14（mitogen-activated protein kinase 14，MAPK14）、半胱氨酸天冬氨酸蛋白酶-3、CXC基序趨化因子配體8、白細(xì)胞介素-1β（nterleukin-1β，IL-1β）、IL-6等表達(dá)促進(jìn)斑馬魚骨質(zhì)形成，并能有效降低OP中TRAP含量[80]。淫羊藿中的淫羊藿素、淫羊藿苷和寶藿苷I可顯著增加斑馬魚OP模型的骨染色面積、累積光密度和Ca的含量[81]，且淫羊藿素C可通過激活磷脂酰肌醇3-激酶和蛋白激酶B信號(hào)通路，減輕地塞米松對(duì)斑馬魚幼魚細(xì)胞成骨的抑制作用[82]。生姜的正己烷提取物及其所含的10-姜酚可顯著抑制潑尼松龍誘導(dǎo)的OP再生鱗片中的破骨細(xì)胞生成以促進(jìn)正常再生，而該過程與破骨細(xì)胞特異性組織蛋白酶K（cathepsin K，CTSK）、MMP2、MMP9表達(dá)水平變化相關(guān)[83]。木槿中的多酚類化合物能夠通過抑制糖原合成酶激酶-3β（glycogen synthase kinase-3β，GSK-3β）進(jìn)而激活β-catenin，促進(jìn)成骨活性，發(fā)揮抗OP作用[84]。黃酮類化合物黃芩苷可通過調(diào)節(jié)RANK/RANKL/骨保護(hù)素（osteoprotegerin，OPG）[85]致使破骨細(xì)胞活性受損和骨吸收減少。其他酚類化合物，如丹參素[86]和丹酚酸B[65]被證明具有骨合成代謝特性。漆黃素可通過GSK-3β/β-catenin信號(hào)通路[87]、羥基紅花黃色素A[88]具有骨合成代謝和抗吸收特性。大黃酸通過腺苷酸活化蛋白激酶/Smad（small mothers against decapentaplegic）1/5/9表達(dá)[89]、環(huán)黃芪醇通過靶向端粒體酶促進(jìn)成骨細(xì)胞分化[90]。槲皮素和異槲皮素分別通過腫瘤壞死因子（tumor necrosis factor，TNF）、Kelch樣環(huán)氧氯丙烷相關(guān)蛋白-1/核因子E2相關(guān)因子2/抗氧化反應(yīng)元件信號(hào)通路緩解斑馬魚的OP癥狀[91-92]。當(dāng)歸中的藁本內(nèi)脂通過GPR30/表皮生長(zhǎng)因子受體途徑促進(jìn)骨形成[93]，吳茱萸堿可通過MMP3/OPN/MAPK促進(jìn)骨重建[61]，知母中天麻皂苷AIII可通過抑制晚期糖基化終產(chǎn)物特異性受體/ MAPK信號(hào)通路減輕糖尿病-OP[94]等。此外，菟絲子及其所含的金絲桃素、對(duì)羥基肉桂酸等[95]、甘草中甘草苷[96]、馬齒莧中多聚糖[97]、牛膝中低聚果糖[98]、白藜蘆中白藜蘆醇[62]被證實(shí)均具有顯著的抗OP活性。

表3 中藥抗斑馬魚OP模型的作用機(jī)制

續(xù)表3

NFATC-1-活化T細(xì)胞核因子1 TGF-β-轉(zhuǎn)化生長(zhǎng)因子-β Bcl-2-B淋巴細(xì)胞瘤-2

NFATC-1-nuclear factor of activated T cells 1 TGF-β-transforming growth factor-β Bcl-2-B-cell lymphoma-2

綜上，天然提取物富含很有前景的骨活性化合物，有望開發(fā)為治療OP的下一代藥物。而斑馬魚具有基因高度保守、生長(zhǎng)周期短和高通量等特征，為繁瑣的藥效物質(zhì)篩選提供了明顯的技術(shù)優(yōu)勢(shì)，當(dāng)前斑馬魚OP模型已被首選作為對(duì)中藥潛在抗OP活性成分進(jìn)行篩選的一線模型。中藥抗OP具體作用機(jī)制見表3。

5 結(jié)語與展望

5.1 斑馬魚模型逐步成為OP的替代模型

斑馬魚作為OP研究的主要替代模型，具光學(xué)透明性、體積小、發(fā)育快等優(yōu)勢(shì)，同時(shí)因其與人類骨骼結(jié)構(gòu)、功能和調(diào)控機(jī)制等方面的高度同源性、光學(xué)透明性、體積小、發(fā)育快和轉(zhuǎn)基因技術(shù)的不斷發(fā)展促使斑馬魚被廣泛用于骨生理病理過程研究、人類骨骼疾病模型的模擬和構(gòu)建、高通量生物活性化合物篩選等。另一方面，成年斑馬魚的一些功能也為成人骨骼疾病如骨轉(zhuǎn)換、修復(fù)、退行性變的治療研究提供了一種新型的強(qiáng)大資源?；诖?，斑馬魚模型被廣泛應(yīng)用于經(jīng)典抗OP復(fù)方、補(bǔ)肝腎中藥的藥效學(xué)評(píng)價(jià)[112-113]，且大多數(shù)研究采用斑馬魚、傳統(tǒng)動(dòng)物聯(lián)合評(píng)價(jià)，進(jìn)一步證實(shí)了斑馬魚模型的可靠性。目前，斑馬魚模型還被廣泛用于天然化合物庫(kù)活性成分[114]、動(dòng)物（海洋生物）活性大分子的篩選[115-116]及功能食品的制備[117-118]、老藥新用的發(fā)現(xiàn)等。

5.2 斑馬魚OP模型的參照標(biāo)準(zhǔn)亟需建立

目前，研究者常用GC誘導(dǎo)的斑馬魚OP模型進(jìn)行中藥藥效評(píng)價(jià)及活性成分篩選，但是在不同研究者構(gòu)建模型的方法（包括暴露濃度、暴露時(shí)間、魚齡的選擇）及藥效評(píng)價(jià)的基準(zhǔn)不同。因此，筆者建議相關(guān)研究領(lǐng)域應(yīng)盡快完善斑馬魚OP模型的標(biāo)準(zhǔn)參數(shù)，為治療OP篩選候選藥物和開展新藥研發(fā)奠定基礎(chǔ)。

5.3 斑馬魚OP模型應(yīng)綜合考慮機(jī)體其他并發(fā)癥對(duì)研究結(jié)果的影響

OP是一種代謝性疾病，具有明顯的年齡相關(guān)性，往往伴隨著激素降低，糖尿病、高血壓和老年癡呆等[119]。目前建立的斑馬魚OP相關(guān)模型及基于該模型進(jìn)行的藥物篩選過程大多數(shù)只是針對(duì)機(jī)體單一的骨骼生理病理過程進(jìn)行的篩選和評(píng)價(jià)，不能完全反映大多數(shù)OP患者臨床狀態(tài)。因此，基于臨床疾病狀態(tài)，建議重點(diǎn)關(guān)注和構(gòu)建以年齡為核心、不同并發(fā)癥同時(shí)存在的OP模型，準(zhǔn)確反映藥物的臨床治療效果。同時(shí)，在研究中要關(guān)注遺傳性疾病，如成骨不全、糖尿病和肥胖等遺傳性疾病對(duì)模型和藥效的影響。

5.4 斑馬魚OP模型結(jié)果向臨床研究結(jié)果轉(zhuǎn)化存在的問題

基于斑馬魚模型進(jìn)行OP相關(guān)研究，須考慮斑馬魚模型與哺乳動(dòng)物、人類在骨骼組成和功能方面的差異性。如雖然斑馬魚與哺乳動(dòng)物細(xì)胞的重吸收活性和分子途徑高度保守，但是不同時(shí)期斑馬魚成骨細(xì)胞、破骨細(xì)胞的存在狀態(tài)和比例與哺乳動(dòng)物存在差異，如斑馬魚破骨細(xì)胞通常是單核細(xì)胞，而哺乳動(dòng)物大多為多核細(xì)胞。因此，欲將斑馬魚系統(tǒng)評(píng)價(jià)結(jié)果擴(kuò)展到臨床研究結(jié)果或應(yīng)用時(shí)應(yīng)謹(jǐn)慎。未來仍需系統(tǒng)評(píng)價(jià)基于斑馬魚不同細(xì)胞類型、細(xì)胞生物過程及特定靶點(diǎn)的評(píng)價(jià)結(jié)果與對(duì)應(yīng)傳統(tǒng)哺乳動(dòng)物評(píng)價(jià)模型結(jié)果間的一致性，進(jìn)一步確認(rèn)斑馬魚模型在OP疾病機(jī)制研究、藥物評(píng)價(jià)中的地位和作用。

利益沖突 所有作者均聲明不存在利益沖突

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Research progress on anti-osteoporosis of traditional Chinese medicine based on zebrafish model

WANG Xuan1, CHEN Lin-zhen1, LIN Rui-chao1, TAO Xiao-yu1, YU Xue2, ZHANG Shu-jing2, FAN Qi-qi1, LI Zhi-qi1, DAI Sheng-yun3, LI Xiang-ri1, ZHAO Chong-jun1

1. Beijing Key Laboratory for Quality Evaluation of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China 2. Beijing University of Chinese Medicine, Beijing 102488, China 3. National Institutes for Food and Drug Control, Beijing 102629, China

Osteoporosis is a bone disease characterized by decreased bone density, changes in bone microstructure, and increased bone fragility. Due to the increasing aging problem, the incidence of osteoporosis is increasing rapidly around the world. Traditional Chinese medicine has shown excellent prospects and clinical effects in the prevention and treatment of osteoporosis. However, traditional animal models have disadvantages of time-consuming, high cost and inability to accurately summarize the complexity of bone diseases, which greatly limits the research process of preclinical osteoporosis diseases. The zebrafish model can effectively simulate the human osteopenia and osteoporosis phenotype. The advantages and applicability of zebrafish model in the treatment of osteoporosis and the research progress of this model in the anti-osteoporosis of Chinese medicine were reviewed, with view to providing references for the widespread use of zebrafish model and the development of new Chinese medicine drugs.

zebrafish; osteoporosis; traditional Chinese medicine; herb pair; traditional Chinese medicine compound; drug screening

R285

A

0253 - 2670(2023)15 - 5088 - 13

10.7501/j.issn.0253-2670.2023.15.032

2023-02-09

國(guó)家自然科學(xué)基金資助項(xiàng)目（82204753）；國(guó)家科技重大專項(xiàng)（2018ZX09735005）；國(guó)家中醫(yī)藥管理局公益性中醫(yī)藥行業(yè)科研專項(xiàng)（201507004）

王 璇（2000—），女，碩士研究生，研究方向?yàn)橹兴幇踩栽u(píng)價(jià)及主要活性/毒性物質(zhì)基礎(chǔ)篩選。E-mail: wangxuan9962@163.com

通信作者：李向日（1972—），女，教授，博士生導(dǎo)師，從事中藥炮制、質(zhì)量控制及藥效物質(zhì)基礎(chǔ)研究。E-mail: lixiangri@sina.com

趙崇軍（1988—），男，助理研究員，從事中藥安全性評(píng)價(jià)及主要活性/毒性物質(zhì)基礎(chǔ)篩選。E-mail: 1014256537@qq.com

[責(zé)任編輯 趙慧亮]