动物骨代谢相关信号通路研究进展

赵净颖，段小花,2，王秋婷，黄英，贾俊静，豆腾飞

动物骨代谢相关信号通路研究进展

赵净颖1，段小花1,2，王秋婷1，黄英1，贾俊静1，豆腾飞1

1. 云南农业大学动物科学技术学院，昆明 650201 2. 云南中医学院，昆明 650500

骨骼是组成脊椎动物内骨骼的坚硬器官，对机体起着运动、支撑和保护的作用。骨骼处于骨形成和骨吸收两种活动所组成的骨代谢的动态平衡状态，这种平衡对于维持骨量和矿物质稳态至关重要。在动物骨代谢过程中，存在着众多调节骨形成和骨吸收的信号通路，如BMP (bone morphogenetic protein)/SMADs、TGF-β (transforming growth factor β)、Wnt/β-catenin、OPG (osteoprotegerin)/RANKL (receptor activator of NF-κB ligand)/ RANK (receptor activator of NF-κB)、FGF (fibroblast growth factor)和Notch信号通路等。这些信号通路具有复杂的调控机制，参与骨代谢过程的调节。本文综述了在动物骨代谢过程中起关键调节作用的相关信号通路的作用机制及研究进展，以期为动物骨代谢研究奠定基础。

骨代谢；信号通路；骨形成；骨吸收

骨骼是脊椎动物机体重要的刚性组织，主要由磷酸钙矿物质和I型胶原组成，对动物机体起到支撑、保护以及运动等作用。骨骼系统的发育和维持受遗传及环境等因素的影响[1]，其中遗传因素是影响骨骼系统发育的重要因素，其对骨骼生长发育的影响在动物的整个生命过程中均有体现，包括骨相关信号通路调控机制、基因作用的发育时机、骨相关基因产物的表观遗传修饰等[2]。

骨骼发育过程包括膜内骨化和软骨内骨化两种方式[3]。在膜内骨化过程中，间充质干细胞(mesen­chymal stem cell, MSC)直接分化成成骨细胞(osteo­blast, OB)。膜内骨化主要发生在颅顶骨部位。在软骨内骨化过程中，间充质干细胞发生增殖分化形成软骨，随后被矿化骨所取代。软骨内骨化发生在颅底和颅骨后部、轴向骨和四肢骨。发育结束进入稳态维持的骨骼处于骨形成和骨吸收所组成的骨代谢动态平衡状态，其中骨形成过程是由MSC衍生的OB介导，骨吸收过程是由造血干细胞(hematopoietic stem cell, HSC)衍生的破骨细胞(osteoclast，OC)介导。骨吸收与骨形成之间的平衡对于维持骨量和维持全身矿物质稳态至关重要，因此可以保持骨骼健康。一旦这种平衡状态被破坏，骨骼便会处于病理状态，也称骨代谢性疾病，如高破骨细胞活性或低成骨细胞活性导致的低骨量(骨质减少)，以及低破骨细胞活性或高成骨细胞活性导致的高骨量(骨硬化)[4]。

在动物骨骼生长发育和骨代谢过程中，存在着生长因子、细胞因子、酶等众多调控因子，而这些调控因子通过相关的信号通路参与骨形成和骨吸收过程，如BMP (bone morphogenetic protein)/Smads、TGF-β (transforming growth factor β)、Wnt/β-catenin、OPG (osteoprotegerin)/RANKL (receptor activator of NF-κB ligand)/RANK (receptor activator of NF-κB)、FGF (fibroblast growth factor)和Notch信号通路等。其中，BMP/Smads信号通路影响成骨细胞分化及骨形成，TGF-β信号通路参与调控成骨过程中细胞的活动和代谢，Wnt/β-catenin信号通路调控骨代谢的平衡，OPG/RANKL/RANK信号通路可以维持骨骼稳态，FGF信号通路和Notch信号通路参与成骨细胞分化。本文介绍了参与动物骨代谢的信号通路相关研究进展，阐明这些信号通路在调节骨形成和骨吸收方面的作用机制，可以为临床有效防治骨异常性疾病提供理论依据。

1 影响动物骨代谢的关键信号通路

动物骨代谢的调控是一个非常复杂的过程，有众多的调节因子及信号途径参与。这些途径各自具有复杂的调控机制，单独或共同参与动物骨代谢的调控。近年来众多研究表明，动物骨代谢的平衡主要由BMP/Smads、TGF-β、Wnt/β-catenin、OPG/RANKL/ RANK和FGF等多条关键信号通路参与调控。

1.1 BMP/Smads信号通路

在骨骼发育过程中，多条信号通路参与调节成骨与破骨过程的平衡，而最早发现和确认的最重要的一条通路是影响成骨细胞分化及骨形成的关键信号通路——BMP/Smads信号通路[5,6]。

骨形态发生蛋白(bone morphogenetic protein, BMPs)是转化生长因子β(transform growth factor β, TGF-β)超家族的成员，是骨骼发育的重要生长因子，具有增强骨髓干细胞向成骨细胞分化的能力，从而促进骨骼的生长发育[5]。在已发现的20余种亚型中，BMP2、4、5、6、7、9等均对成骨细胞分化有调节作用[6~8]。其中BMP2因具有增强骨形成及代谢的作用而成为研究热点。BMP2能够诱导间充质干细胞的增殖、迁移以及向成骨细胞的分化[8,9]，且能够协同其他成骨因子共同刺激成骨细胞增殖，增强成骨细胞活性，加速骨重建[10]。

BMP/Smads信号通路由BMPs及其受体、Smad蛋白和相关转录因子组成。BMP2通过自分泌或旁分泌形式释放后，其单体可通过二硫键连接形成二聚体，再结合BMPs受体。BMPs受体是丝氨酸/苏氨酸激酶受体，包括I型受体(BMPR-IA、BMPR-IB和ACVR-I)和II型受体(BMPR-II、ActR-IIA和ActR-IIB)[11]。BMP2先结合BMPR-II，发生自身磷酸化而被激活，继而磷酸化BMPR-I，使BMPR-I激活[12~14]，下游Smads 信号Smad1/5/8被BMPR-I受体激活并和受体形成短暂复合物，使其与Smad4结合，转移至核内，转录激活成骨分化基因，如上调核心结合蛋白因子2 ()[15,16]、成骨细胞特异性转录因子Osterix ()[17]和同源盒基因[18]等，促进成骨细胞分化。而Smad6和Smad7则参与了骨形态发生蛋白信号传导的负调控[19]。

Dallari等[20]和Yang等[21]在人类骨骼疾病的临床研究中发现骨质疏松性骨折患者常伴有碱性磷酸酶活性下降的现象，而导致碱性磷酸酶活性下降的重要原因是等成骨基因表达不足，并且存在抑制软骨细胞向成骨细胞的分化并伴有成骨细胞凋亡的增加现象。Zappitelli等[22]在小鼠()细胞实验中的研究显示，上调基因的表达可提高成骨细胞特定标志物的表达。Li等[19]和Zarrinkalam等[23]在山羊()的活体实验研究中发现，注射人重组的BMP2可提高骨密度，并可以治疗骨质疏松山羊腰椎骨缺损处；Cipitria等[24]研究表明注射人重组的BMP7可提高山羊胫骨的韧性和强度。Mizrahi等[25]在猪()的间充质干细胞研究中发现，人重组的BMP6比BMP2更加有效地诱导成骨分化，促进骨骼的形成。在BMP2/ Smads信号通路中，存在众多的相关调控因子。研究发现，泛素化调节因子(Smurf)与BMP2激活的Smad1、Smad5相互作用并调节成骨细胞特异性转录因子Runx2降解[26]，并且Smad6与Smurf的协同作用可下调Runx2蛋白水平，负向调控BMP/Smads信号通路[27]。而I型多发性内分泌腺瘤肿瘤抑制基因产物(Menin)可增强BMP2诱导的的转录活性。胶原三股螺旋重复蛋白1 (collagen triple helix repeat containing 1, Cthrc1)可正向调节OB骨形成，从而使骨量增加。Takeshita等[28]研究发现，缺失的小鼠在生长初期，碱性磷酸酶(alkaline phosphatase, ALP)和骨钙素(bone gamma- carboxyglutamic-acid-containing proteins, BGP)表达减少。对猪和羊的研究发现，基因是BMPs的拮抗剂，可调节BMPs对骨骼的作用，调节骨生长发育的重要细胞因子[29,30]。

除BMP/Smads信号通路相关调控因子以外，一些外在因素也可通过调节BMP/Smads信号通路来促进动物骨骼的生长发育。Feng等[31]对大鼠()细胞的研究显示，辛伐他汀(simvastain)可促进骨质疏松症(osteoporosi, OP)大鼠的关键成骨分化相关因子，骨保护素(osteoprotegerin,)，骨桥蛋白(osteopontin,)和的mRNA表达水平升高，可以通过BMP/Smads信号通路促进OP大鼠模型中MSCs向OB分化。Chai等[32]在去卵巢大鼠的研究中发现，传统中药配方骨疏康处理组通过上调BMP2，磷酸化Smad1和磷酸化Smad5 (p-Smad1/5)，升高Osterix和Runx2的表达来显著增强BMP/Smads信号通路，促进骨形成。Yu等[33]研究显示，大豆()苷元通过激活BMP/Smads途径促进成骨细胞增殖和分化，具体表现为上调、、基因的表达以及Runx2和Smad1蛋白的表达。可见，BMP/Smads信号通路的激活可以促进动物成骨细胞的增殖、分化及骨形成。

1.2 TGF-β信号通路

骨骼中TGF-β家族成员参与了整个成骨过程中细胞活动和代谢的调控。TGF-β家族由3种亚型组成：TGF-β1、TGF-β2和TGF-β3[34]，均在骨中表达。TGF-β与其结合蛋白(LTBP)结合形成复合物，由OB分泌，可以与细胞外基质(extracellular matrix, ECM)成分(纤维连接蛋白、纤颤蛋白1和整合素等)相互作用，增强成骨细胞分化，促进骨骼发育[35]。Dünker等[36]研究表明，TGF-β2和TGF-β3双基因敲除小鼠远端部分骨量丢失，特别是缺失的小鼠在膜内和软骨内骨化方面表现出严重的骨骼异常。

在骨基质合成终止后，成骨细胞发生凋亡或分化为骨细胞(骨衬细胞)。成熟的TGF-β通过阻止成骨细胞凋亡来调控成骨细胞的存活。骨髓巨噬细胞是破骨细胞的前体，而TGF-β直接作用于骨髓巨噬细胞，促进破骨细胞生成。Yasui等[37]研究表明，TGF-β诱导Smad2/3与泛素连接酶TRAF6 (TNF receptor associated factor 6)之间的分子相互作用，对于RANKL诱导的破骨细胞信号通路至关重要。此外，成骨细胞可产生和巨噬细胞集落刺激因子(macrophage colony stimulating factor,)。因此，TGF-β对破骨细胞的作用也来源于成骨细胞。TGF-β刺激成骨细胞不仅表达I型胶原、ALP、BGP等成骨功能蛋白，还表达、、等破骨细胞调控基因[38]。TGF-β对破骨细胞生成的影响与剂量有关，低剂量TGF-β通过增加M-CSF的表达和前列腺素的产生，以及RANKL与OPG的比值来增强破骨细胞的生成[39]，而高浓度TGF-β则通过增加的表达来抑制和的表达[40]。由于OPG是RANKL的高亲和力配体，是成骨细胞产生的RANKL的可溶性抑制剂，因此TGF-β对成骨细胞介导的破骨细胞的作用可能是骨重建的负反馈作用。

Shi等[41]研究发现，用TGF-β信号抑制剂SB431542处理的骨髓间充质干细胞(mesenchymal stem cells, MSC)，移植到猪上颌骨缺损处，可以促进成骨分化增加，成功修复小型猪严重的颌面部骨缺损。Zeng等[42]通过细胞实验发现miR-23a簇(miR-23a-24-2-27a簇)通过靶向TGF-β途径的负调节剂Prdm16来调节TGF-β信号通路，从而促进小鼠骨细胞的分化。Xu等[43]研究表明，系统或局部阻断软骨中TGF-β活性可减轻类风湿关节炎(rheumatoid arthritis, RA)关节软骨退变，表明软骨中TGF-β的异常激活与RA关节软骨退变的发生有关。可见，TGF-β对动物成骨分化起着负反馈作用。

1.3 Wnt/β-catenin信号通路

Wnt信号通路已成为骨代谢平衡的关键调控信号通路[44]。Wnt信号控制胚胎发育和细胞增殖、分化、迁移等多个过程。β-catenin是调控Wnt信号的必要调控因子。一旦Wnt蛋白与Frizzled受体、低密度脂蛋白相关蛋白受体(Lrp5或Lrp6)结合，β-catenin将逃脱降解机制，转移到细胞核并与转录调节因子Tcf/Lef相互作用，激活Wnt靶基因的转录。在骨质疏松/高骨密度综合征中，Lrp5的功能发生缺陷[45,46]。β-catenin信号对成骨细胞发挥细胞环境相关的功能取决于分化的阶段。在早期阶段，β-catenin在前成骨细胞中失活，导致成骨细胞分化受阻，从而致使骨骼中缺乏成熟的成骨细胞[47~50]。然而，在后期阶段，β-catenin在成熟的成骨细胞和骨细胞中失活并不影响成骨细胞分化和骨形成，而是通过增加破骨细胞分化和骨吸收导致骨量降低[51~53]。在Wnt/β-catenin信号通路中，甲状旁腺激素和机械负荷等因素可下调骨细胞中基因并增强β-catenin信号[54~56]。编码硬化蛋白，是一种有效的骨形成抑制剂，通过与Lrp5/6结合来拮抗Wnt信号，而硬化蛋白表达的缺失是导致高骨量疾病范·巴克病(van Buchem)和硬化性骨化病的原因[57,58]。因此，Wnt信号的激活对动物骨代谢的调节起重要作用。

Tu等[43]研究表明，激活β-catenin的小鼠表现出四肢骨骼骨矿物质密度增加，骨小梁数量、骨松质密度、骨形成标记物显著增加，且骨膜骨形成率明显升高；骨骼中Wnt信号靶向基因、成骨细胞和骨细胞标记、原骨细胞因子和抗破骨细胞因子含量升高。因此，激活骨细胞中的Wnt/β-catenin信号可增加成骨细胞的增殖、分化，从而促进骨量增加。可见，β-catenin可激活骨细胞和成骨细胞等骨细胞的合成代谢是骨骼中Wnt/β-catenin信号促进骨形成的重要原因。Wang等[59]研究发现，脂联素(adiponectin)转基因的BMSCs中的和细胞周期蛋白D1 ()基因及其蛋白表达水平较高，且脂联素治疗组小鼠观察到更多新骨形成，表明脂联素可促进BMSCs成骨分化和成骨，而Wnt/β-catenin途径参与脂联素的成骨作用。Zhu等[60]在体外培养的BMSCs中发现，梓醇(catalpol)可显著增强成骨细胞特异性基因表达、碱性磷酸酶活性和钙沉积，可通过激活Wnt/β-catenin途径促进BMSCs的成骨分化。Mola­goda等[61]发现太平洋牡蛎()提取物可以在斑马鱼()中通过诱导Wnt/β-catenin途径来促进幼体骨矿化及尾鳍再生。因此，在动物骨骼生长发育中可通过激活Wnt/ β-catenin信号通路来促进成骨分化，促进骨量增加。

1.4 OPG/RANKL/RANK信号通路

骨转换(bone turnover)取决于成骨细胞的骨形成和破骨细胞的骨吸收之间的平衡。骨量丢失和骨质疏松症的发生是骨吸收大于骨形成导致的。OPG、细胞核因子-κB受体活化因子(RANK)和RANK配体(RANKL)是偶联成骨细胞、基质细胞和破骨细胞分化、活化及生物活性的3种主要细胞因子，OPG/ RANKL/RANK信号通路是破骨细胞生物学的基础，在骨代谢中起十分重要的作用[62]。大量研究表明，人类及动物代谢性骨病与这一系统的改变有关[63~65]。

OPG是肿瘤坏死因子(tumor necrosis factor, TNF)受体家族成员，又称破骨细胞生成抑制因子或TNF受体样分子，是一种通过抑制破骨细胞分化和活化来调节骨量的分泌蛋白。RANKL是肿瘤坏死因子超家族的一员，已被证明既能介导破骨细胞生成，又能激活成熟破骨细胞。RANK是RANKL的受体，结合RANKL发挥生物学功能。在骨组织中，RANKL由多种细胞表达，包括成骨细胞、骨细胞和免疫细胞，尤其在成骨细胞和骨细胞中的表达较高[66]。RANKL结合并激活其位于破骨细胞祖细胞和成熟破骨细胞上的受体RANK。RANK刺激导致前破骨细胞分化为活性破骨细胞，活性破骨细胞重新吸收矿化骨基质。RANKL的活化优先表达于成骨前细胞的细胞膜上，而其特异性受体RANK则表达于破骨细胞前体细胞的细胞膜上。RANKL与RANK的结合导致破骨前细胞分化、形成、融合和存活[67]。RANK- RANKL相互作用是破骨细胞形成的必要条件。

研究表明，缺失RANKL的动物在缺乏骨基质或成骨细胞的情况下，无法产生破骨细胞，而提供外源性RANKL可刺激体外破骨细胞生成[68]。基质细胞和包括成骨细胞系在内的其他类型细胞分泌的OPG，可竞争性地结合RANKL，阻断RANK对破骨细胞的作用，进而抑制破骨细胞的活化，进而抑制破骨和骨溶解。过表达的小鼠导致严重骨丢失和成熟破骨细胞减少实验，表明OPG可作为破骨细胞生成抑制剂[69]，而RANKL缺失的小鼠表现出严重的骨丢失，并且由于不能支持破骨细胞的生成而完全缺乏破骨细胞[70]。然而，Liu等[71]研究表明敲除的小鼠骨质疏松，骨密度降低，骨折发生率高，通过静脉注射重组OPG蛋白可逆转其病理。因此，OPG的存在对维持正常骨量是绝对必要的。另外，有研究表明，作为破骨生成促进因子的和破骨生成抑制因子的表达水平的平衡决定了骨吸收的程度，RANKL上调和OPG下调都会导致骨质流失[72]，且OPG/RANKL比值的失衡可能导致骨量的丢失[73]。

Huang等[74]对家鸡()的研究表明，OPG/RANKL平衡的破坏导致骨形成改变，引起鸡胫骨结构改变、胫骨质量降低，导致胫骨软骨发育不良。血清中OPG水平、Ca2+浓度和ALP活性均显著降低，进一步证实了骨代谢受到抑制。Wu等[75]通过人乳腺癌细胞MDA-MB-231和小鼠成骨细胞MC3T3-E1共培养系统的研究结果表明，与未经处理的共培养物相比，马钱子碱(brucine)处理显著提高了共培养物中OPG/RANKL mRNA表达比率和OPG/RANKL蛋白比率，说明马钱子碱可通过调节成骨细胞中OPG和RANKL的表达和分泌来间接控制破骨细胞，从而抑制破骨细胞的分化和骨吸收功能。Hou等[76]将雌性Sprague-Dawley大鼠进行卵巢切除术，分别用10、100、1000和2000 mg/kg /d等剂量的乳铁蛋白(lactoferrin, LF)进行口服治疗，饲喂6个月后发现，LF剂量依赖性地增加了Ovx大鼠骨体积、骨小梁厚度和骨小梁数目，减少了骨小梁分离；此外，与未经处理的Ovx大鼠相比，更高剂量的LF (1000 mg/kg/d和2000 mg/kg/d)显著增加了骨密度，且总体上LF处理显著提高了Ovx大鼠mRNA水平，抑制了mRNA水平；这些结果表明，口服LF可保留骨质并改善骨骼的微结构，且LF可能通过OPG/RANKL/RANK途径的调节来增强骨形成，减少骨吸收及骨质流失。据Ma等[77]发现220 mg/kg或440 mg/kg的1,6-二磷酸果糖锶(FDP-Sr)治疗能显著提高Ovx大鼠的骨密度，改善骨微结构和骨强度，且用FDP-Sr治疗以剂量依赖性方式降低了血清中RANKL水平，增加了OPG水平，也显著下调了骨髓中的表达和上调了表达，结果表明，FDP-Sr对绝经后骨质疏松症的有效治疗，其部分作用是通过OPG-RANKL-RANK途径减少破骨细胞的生成完成。可见，OPG/RANKL的比值可调节骨吸收，OPG-RANK-RANKL信号通路在调节动物骨形成和骨吸收的过程中起着至关重要的作用。

1.5 FGF信号通路

成纤维细胞生长因子(fibroblast growth factor, FGF)信号通路已被证实在调节成骨细胞和成纤维细胞的增殖和分化、成骨以及许多其他重要的细胞过程中发挥着重要的作用，包括血管生成和伤口愈合[78]。

此外，FGF信号通路在调节骨祖细胞膜内和软骨内骨化的信号传递过程中发挥着至关重要的作用[79]。由于FGF通路对成骨细胞分化的刺激作用以及对成骨细胞分化的抑制作用，表明FGF信号通路对成骨细胞成熟过程的影响是阶段性的[80]，且颅骨和长骨生长异常多与FGF信号通路的突变有关[81]。

在23个FGF家族成员中，一些FGF在成骨过程中起着关键作用。例如，FGF2磷酸化激活Runx2，可影响骨形成[82]。此外，已有研究表明FGFR2 (FGF receptor 2)也参与了骨生长的正向调控和成骨细胞的合成代谢功能[83]。FGFR3可通过调节成骨细胞分化来影响骨骼的骨密度和皮质骨厚度[84]。FGF9和FGF18的表达也显著影响胚胎骨形成[85]。

Kanda等[86]发现大鼠骨髓细胞在含碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)培养基中培养后，细胞数量显著增加，细胞膨胀，BMP2和骨桥蛋白表达明显增加。Furuya等[87]研究发现水凝胶bFGF治疗骨折缺损后，小鼠骨密度较高，骨矿化率较高，和基因表达上调。D’mello等[88]研究表明，转染编码FGF2蛋白(PEI-pFGF2)的聚乙烯亚胺纳米复合物(nanoplexes)的骨髓基质细胞(BMSCs)中的基因高表达。Khorsand等[89]在糖尿病兔()模型中也开发了相同的nanoplex，用于将FGF2和BMP2蛋白递送到缺陷部位。结果表明，与单纯植入胶原支架的PEI-pBMP2相比，植入胶原支架内的PEI-(pBMP2 + pFGF2)可显著改善骨再生。据Charles等[90]报道，在颅骨缺损的年老小鼠中，向BMP2中添加FGF2联合治疗后，小鼠骨缺损中心区域的骨填充增强，且骨体积增加。该研究表明，相对于单独的BMP2或FGF2，低剂量FGF2和低剂量BMP2联合应用有可能增加老年小鼠的骨愈合能力。这些结果表明BMP2和FGF2协同作用可促进骨修复。Yuan等[91]研究发现，单独的BMP4/7显著促进了BMSCs的增殖，同时，它也促进或抑制了BMSCs的成骨分化，而BMP4/7和bFGF的协同作用显著促进了BMSCs的增殖和成骨分化，协同作用的治疗取决于剂量和时间。BMP4/7和bFGF的合理组合可以促进BMSCs的增殖和成骨分化。综上所述，BMP和FGF协同作用对成骨过程的调节起重要作用。

1.6 其他骨代谢重要信号通路

1.6.1 Notch信号通路

Notch信号通路作为一种进化上高度保守的配体受体信号通路，在细胞存活、增殖、分化以及发育过程中的命运决定、稳态等方面发挥着重要的机制作用[92]。Notch信号通路在骨骼生长发育中起着对成骨细胞的直接诱导作用。而骨祖细胞中Notch信号通路受到抑制可导致骨髓源性间充质干细胞的损耗，并与各年龄段骨质流失有关[93,94]。Pan等[95]研究发现，活化的B淋巴细胞通过激活Notch信号来抑制BMSC的成骨作用，当B淋巴细胞被灭活或Notch信号被抑制时，BMSC的成骨作用将部分恢复。He等[96]在小鼠细胞实验中发现，抑制Notch1减少了BMSC的增殖并促进其成骨分化。这些结果表明Notch信号通路在BMSC分化过程中受到抑制，说明Notch信号通路对BMSC成骨分化具有抑制作用。另一方面，Fukushima等[97]研究发现，Notch 2是破骨细胞重塑刺激物，它在破骨细胞分化过程中调节活化T细胞核因子c1(NFAT-c1)的启动子并诱导破骨细胞形成。因此，Notch信号在骨骼发育中起着不可或缺的作用。

1.6.2 Hedgehog信号通路

Hedgehog(Hh)信号通路是调节骨骼发育的关键。目前在哺乳动物中已经确定了3种Hh：Sonic Hh(Shh)，Indian Hh(Ihh)和Desert Hh(Dhh)[98]。其中，Ihh是在发育中的骨骼内唯一发现的Hh，Ihh由软骨细胞分泌，调节软骨细胞的增殖和分化，对骨骼生长至关重要[99,100]。有研究发现，Hh信号通路可能通过增加和的表达来诱导MSC的成骨分化，并抑制MSC分化为脂肪细胞[101]。Zaman等[102]研究显示，在内源性抗凋亡蛋白humanin(HN)高表达小鼠、HN处理的野生型小鼠和HN处理的培养小鼠跖骨中，糖皮质激素(glucocorticoid, GC)诱导的骨生长损伤和软骨细胞凋亡被阻止，HN可通过靶向Hh途径使GC诱导的骨生长障碍受到抑制并恢复，而不会干扰GC所发挥的抗炎作用。可见，Hedgehog信号通路在调节成骨细胞分化和骨骼发育中起重要作用。

1.6.3 PI3K/Akt信号通路

PI3K/Akt信号通路是一种重要的有丝分裂信号通路，在生长、存活、增殖和活性等多种细胞过程中发挥着重要作用[103]。在骨骼生长发育中，PI3K信号及其下游靶点在骨形成和骨重塑中起着重要调控功能[104]。Ke等[105]研究表明，川续断皂苷VI (asperosaponin VI, ASA VI)促进了去卵巢小鼠BMSC的增殖，增强了ALP活性，并促进了钙结节的生成；此外，ASA VI增强了和的表达，而经LY294002 (阻断PI3K/Akt信号通路的蛋白激酶抑制剂)处理则降低了以上成骨作用，并降低了ASA VI诱导的p-Akt (磷酸化Akt)水平。这些结果表明，ASA VI通过作用于PI3K/Akt信号通路来促进Ovx的BMSC的成骨分化。可见，在动物骨骼生长发育中激活PI3K/Akt信号通路可促进BMSC的成骨分化。

1.6.4 钙离子信号通路

钙离子(Ca2+)在骨骼中起着重要的结构作用，且Ca2+信号在成骨细胞分化过程中起着重要作用。在骨重塑过程中，Ca2+作为矿物相的成分，以游离离子的形式不断释放到细胞外环境中[106]。因此，Ca2+持续可作用于成骨细胞和骨祖细胞发挥生物学功能。Ca2+进入钙通道介导的细胞，Runx2起中介作用，激活磷脂酶C(PLC)和肌醇-1,4,5-三磷酸肌醇(IP3)信号[107]，进而促进细胞内存储Ca2+的释放，从而促进骨骼的生长发育。细胞外钙敏感受体(Calcium- Sensing Receptor, CaR)是一种G蛋白偶联受体，在调节细胞外钙稳态中起重要作用，Liu等[108]研究表明，高钙血症的减少在预防缺陷(CaR)小鼠的早期致死性中起关键作用，且CaR小鼠软骨内骨形成缺陷是由于血清钙浓度显著升高、血清磷浓度和骨骼甲状旁腺激素相关蛋白水平降低所致。Chang 等[109]研究表明，骨骼中的缺失会导致严重的骨骼缺陷，而软骨细胞(软骨生成细胞)中的缺失导致胚胎第13天(E13)前死亡，但在E16~E18之间诱导小鼠软骨细胞特异性缺失是可行的，但显示小鼠生长板发育延迟；这些结果显示在早期胚胎发生和骨骼发育中起着关键作用。综上所述，钙离子受体敲除小鼠实验证明了钙离子信号对骨发育非常重要。

1.6.5 Hippo信号通路

Hippo信号是调节器官大小和组织再生的主要因素，近年来的一些研究表明Hippo信号通路在调节骨形成方面发挥作用。Wang等[110]通过体外实验研究表明，小鼠BMSCs包括迁移和成骨的生物学功能与Hippo途径的关键下游效应物YAP(Yes相关蛋白)的表达增强有关。Chen等[111]发现，在成骨培养条件下，BMSCs中Hippo信号的激活抑制了成骨分化。Hippo信号通路对于骨骼发育的具体调控机制还不明确，还需开展更多的相关研究。

2 结语与展望

动物骨骼生长发育及骨代谢受BMP/Smads、TGF-β、Wnt/β-catenin、OPG/RANKL/RANK和FGF等多条关键信号通路调控，这些信号通路通过直接或间接作用于Runx2或β-catenin等关键转录因子而彼此相互联系、相互影响，从而组成了复杂的调控网络(骨代谢调控的关键信号通路图见图1)。该网络协同参与了骨代谢过程的调控，维持骨稳态。尽管目前对骨代谢相关信号通路的研究较多，也取得了一些重要的研究成果，但对于调控动物骨代谢的分子机制仍未阐明清晰。随着生命科学的不断发展，在不远的将来能够解析骨代谢调控的完整分子机制，只有理解了这些信号通路的分子作用机制，并阐明这些信号通路传导途径之间的相互作用，才能为动物病理性骨骼的治疗奠定基础。

此外，本文介绍了中草药及其提取物可通过这些信号通路调节骨形成或骨吸收过程，从而维持骨代谢的平衡。目前已有较多研究表明，淫羊藿()、补骨脂(.)、当归()、山药()、杜仲()等单体及其提取物，或是由多味单剂组成的方剂如更年春、密骨胶囊等均对动物骨骼的生长发育起作用，说明中草药在防治动物病理性骨骼方面具有广阔的前景。但动物骨骼疾病的分子机制复杂，中草药对于骨代谢相关信号通路的调控研究还处于初始阶段，还需进一步深入研究。将中草药应用与动物骨骼发育和骨代谢的分子机制研究相结合，不仅可以深入揭示中草药防治骨骼疾病的机制，还能为动物骨骼疾病的防治提供依据，使中草药的应用前景更广阔。

图1 骨代谢调控的关键信号通路图

TGF-β信号通路通过Smad2/3路径调节BMSCs的增殖、分化及其成骨细胞分化；FGF信号通路中FGF和FGFR可调节BMSCs的增殖和成骨分化；BMP/Smads信号通路通过激活Smads1/5/8，结合Smad4，再与Runx2和OSX等相互作用，调节成骨细胞分化与骨重建；Wnt/β-catenin信号通路通过Wnt蛋白与Frizzled 和Lrp5/6结合，激活β-catenin并将其转移到细胞核与TCF/LEF等相互作用，激活Wnt靶基因的转录，从而调控成骨细胞的增殖、分化及骨形成；OPG/RANKL/RANK信号通路中OPG和RANKL竞争性结合，阻止RANKL和RANK之间的结合，通过调节OPG/RANKL比值来调控骨吸收过程。该5条关键信号通路共同参与调节动物骨骼的生长发育与骨代谢平衡。

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Progress on signal pathways related to bone metabolism in animals

Jingying Zhao1, Xiaohua Duan1,2, Qiuting Wang1, Ying Huang1, Junjing Jia1, Tengfei Dou1

Bone is a hard organ that makes up vertebrate endoskeleton, which plays a role in movement, support and protection for the body. The normal growth and development of bone is in the dynamic balance of bone metabolism, which is composed of bone formation and bone absorption. This balance is very important for maintaining bone mass and mineral homeostasis. In the process of bone growth and metabolism, there are many signaling pathways regulating bone formation and absorption, such as BMP (bone morphogenetic protein)/SMADs, TGF-β (transforming growth factor β), Wnt/β-catenin, OPG (osteoprotegerin)/RANKL (receptor activator of NF-κB ligand)/RANK (receptor activator of NF-κB), FGF (fibroblast growth factor) and Notch signaling pathway. These signaling pathways have complex regulatory mechanisms and are involved in the regulation of bone metabolism. In this review, we summarize the mechanism and research progress of signal pathways that play key regulatory roles in the process of animal bone metabolism, thereby laying a foundation for research in animal bone metabolism.

bone metabolism; signaling pathway; bone formation; bone resorption

2020-03-11;

2020-06-14

国家自然科学基金项目(编号：U1702232)，云南省创新团队项目(编号：2019HC011)，云南省科技厅南希·莱恩院士工作站项目(编号：2017IC048)，云岭产业技术领军人才培养和云南省博士研究生学术新人奖资助[Supported by National Natural Science Foundation of China (No. U1702232), Yunnan Innovation Team Project (No. 2019HC011), Nancy Lane Academician Workstation Project of Yunnan Science and Technology Department (No. 2017IC048), Yunling Industrial Technology Leader Training, and New Academic Award for Doctoral Candidates in Yunnan Province.]

赵净颖，在读硕士研究生，专业方向：动物营养与饲料科学。E-mail: 1849680658@qq.com

豆腾飞，讲师，研究方向：动物遗传育种与繁殖。E-mail: tengfeidou@sina.com

10.16288/j.yczz.20-066

2020/7/30 9:38:52

URI: https://kns.cnki.net/kcms/detail/11.1913.R.20200728.1634.001.html

(责任编委: 张雷)