Molecular mechanism of magnesium ions in bone metabolism: research and progress

doi:10.3969/j.issn.2095-4344.2016.48.020

Abstract

Abstract:

BACKGROUND: The magnesium ions released from the degradation of magnesium alloy are an important cation in human body, which not only participate in various metabolic activities, but also regulate bone metabolism and involved in the reconstruction and stability of bone. Therefore, the abnormal concentration of magnesium ions can lead to bone metabolism related diseases.

OBJECTIVE: To review the function and mechanisms of magnesium ions in bone metabolism and overview the related cytokines and signaling pathways.

METHODS: A computer-based research of CJFD, PubMed and WanFang databases was performed for pertinent articls published from January 2000 to February 2016 using the keywords of “magnesium, bone metabolism, PI3K/Akt signal pathway, OPG/RANKL signal pathway, TRPM, and Wnt signal pathway” in Chinese and English, respectively.

RESULTS AND CONCLUSION: The magnesium ions released from the degradation of magnesium alloy expose influence on the bone metabolism by regulating the related signaling pathways. The concentration changes of magnesium ions can induce the changes in cytokines in the signaling pathways related to bone metabolism, and further act on the bone remodeling. Whether the magnesium ions participate in bone metabolism through other pathways will be a hot spot.

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

Key words: Magnesium, Signal Transduction, Tissue Engineering

CLC Number:

R318

Shang Xiao-pan, Li Tao, Lu Yu-tong, Deng Wei, Wang Wen-ju, Yang Yong-jin. Molecular mechanism of magnesium ions in bone metabolism: research and progress[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(48): 7280-7287.

Figures/Tables 1

2.1 镁合金在骨组织中的临床应用作为近年来生物金属材料研究的热点，镁合金在骨内植入材料的应用中具有巨大的优势。首先，镁合金具有良好的生物相容性、低密度、较高的强度系数等一系列生物力学特性，是一种理想的仿生骨内植入材料[6]。除此之外，镁合金生物材料的弹性模量更接近天然骨，可以减小新生骨组织的受到的机械刺激，从而影响新生骨组织的形成和改建，进而影响植入物的稳定[7-10]。其次，镁合金作为一种可降解的生物金属材料，在生理环境下可以完全降解，使其成为理想的生物降解材料[6]，近年来受到格外的关注，并被应用于硬组织植入物。对于植入体需要取出的患者，镁合金植入体能够在体内完全降解，从临床和经济学角度来讲都是有益的，避免了二次手术的费用和痛苦。更为重要的是，镁合金还具有成骨诱导作用。在动物体内可以表现出在降解过程中对周围骨组织生长的促进作用[11]。实验建立新西兰大白兔尺骨骨折模型，使用镁合金(ZK60)内固定系统进行固定，证实镁合金可作为一种理想的新型内固定材料。镁合金在体内的降解产物主要是镁离子，镁离子是人体内含量第4位的阳离子，是组成人体的重要成分，不仅可以影响多种酶的作用，还可以稳定DNA和RNA的结构[12]。镁离子除调节人体众多的新陈代谢活动外还参与调节骨代谢[13]，镁离子主要存在于骨骼中，占骨骼矿物质含量的0.5%-1%。镁离子既可通过调节骨代谢相关的激素[14]、生长因子和信号通路相关因子来影响骨矿物质和基质的代谢[15-16]，也可直接作用于骨组织本身[17]。近年来关于镁合金生物材料的研究发现，镁合金植入体周围呈现高矿化附着速率和骨量增加[18]，镁合金降解过程中释放的镁离子和周围的碱性环境对局部的骨组织生长和愈合有促进作用，显著地促进新生骨组织的形成[19-20]，有助于在植入体-骨界面形成更好的骨结合[21]，形成稳定的新骨。而多余的镁离子可以通过肾脏排泄出去，可以使血液的镁离子的浓度维持在一个安全的范围内[22-23]。因此系统的研究镁离子在骨代谢过程中分子机制对于指导镁合金在临床中的应用具有重要的意义。骨组织代谢包括骨形成和骨吸收两方面，主要涉及成骨细胞和破骨细胞，两者间的动态平衡是维持骨组织稳定的前提，而成骨细胞和破骨细胞的数量和功能受多种因素的影响，包括骨代谢过程中的相关激素、离子通道和信号通路等。近年研究发现在骨代谢过程中与镁离子相关的通路主要有PI3K/Akt信号通路、骨保护素(osteoprotegerin，OPG)/RANKL信号通路TRPM蛋白通路和Wnt信号系统等。 2.2 镁离子调节骨代谢相关通路 2.2.1 PI3K/Akt 信号通路 PI3K/Akt信号通路在细胞的多种生物学行为中扮演着重要的角色，参与到细胞的增殖、分化和凋亡的过程中。PI3K/Akt信号通路对骨代谢的调控主要是通过磷脂酰肌醇3激酶(phosphoinositide 3-kinase，PI3K)激活下游的Akt，Akt又称蛋白激酶B(protein kenase B，PKB)，从而可以进一步激活下游的靶蛋白，Akt 和其下游的靶蛋白是骨形成和骨重建的关键调控者。有实验证明敲除Akt1/Akt2 的小鼠骨化延迟[24]，而敲除Akt1的小鼠骨骼变短，并且延迟了次级骨化中心的形成[25]。说明PI3K/Akt信号通路代谢过程中Akt 和其下游的靶蛋白是骨代谢中的不可或缺的蛋白成分。成骨细胞分化过程中相关嘌呤的调节也与PI3K/Akt 信号通路相关，主要通过PI3K/Akt信号通路调控ATP和UTP来实现，ATP和UTP同成骨细胞的增殖分化密切相关。不同浓度的镁离子对成骨细胞内的PI3K/Akt信号通路的调节存在差异，研究发现6、10 mmol/L 镁离子可以使p-Akt的表达水平上调，而18 mmol/L 镁离子下调其表达水平[16]。提示镁合金对骨的促进作用可以通过其降解产物镁离子来激PI3K/Akt信号通路来实现。首先，镁离子可以通过激活PI3K/Akt信号通路可以引起一系列骨组织相关信号分子的变化，例如促进碱性磷酸酶、骨形态发生蛋白等成骨分化标志物的表达[26]，促进成骨细胞增殖和分化[27]。适当浓度的镁离子通过调节Akt 的表达而激活下游的靶蛋白，进而影响成骨细胞的增殖分化[28-31]。其次，PI3K/Akt信号通路还可以通过促进骨髓间充质干细胞向成骨细胞的分化来调节骨量，由于骨髓间充质干细胞上存在巨噬细胞生长因子受体，巨噬细胞生长因子可以通过PI3K/Akt信号通路促进骨髓间充质干细胞的增殖和分化，促进骨髓间充质干细胞向成骨细胞的分化[32]。同时，镁离子还能够作为细胞内的第二信使[33]，在成骨细胞受到外界刺激时，细胞内的游离镁离子会有所升高[34]。镁离子通过“膜-镁-有丝分裂”模式(membrane magnesium mitosis model)调节细胞的有丝分裂过程，在有丝分裂信号的刺激下，细胞内镁离子浓度会迅速升高，并且通过激活 PI3K 通路促进成骨细胞的有丝分裂[35]。 PI3K/Akt信号通路除了参与调节成骨细胞代谢过程外，在破骨细胞的增殖分化中也起着重要的调控作用[36-37]。抑制PI3K/Akt信号通路活性将使破骨细胞骨吸收能力减弱[38]。实验证实，用LY294002抑制PI3K信号后，能显著的减少RANKL/CSF诱导的破骨细胞生成，提示PI3K/Akt信号通路OPG/RANKL通路在调节破骨细胞代谢中存在着交互作用[39]。PI3K/Akt 信号通路作为调控成骨细胞和破骨细胞功能的信号通路网的中心，与成骨和破骨通路紧密联系。因此适当浓度的镁离子可以激活PI3K/Akt信号通路，进而影响成骨细胞和破骨细胞存活、分化过程从而维持骨量和骨转换平衡[40]。 2.2.2 OPG/RANKL/RANK信号通路核因子κB 受体活化因子(receptor activator of nuclear factor-κB，RANK)属于Ⅰ型跨膜蛋白，是TNF家族成员之一，高度表达于许多细胞的表面。如破骨前体细胞、成熟破骨细胞、树突状细胞、哺乳动物腺体上皮细胞及某些癌细胞中等，核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand， RANKL)是一种Ⅱ型同源三聚体跨膜蛋白，RANK的配体M-CSF(巨噬细胞集落刺激因子)和RANKL对于启动破骨细胞分化时的基因转录是必不可少的，而M-CSF和RANKL是成骨细胞释放的两种关键因子，主要作用是触发破骨细胞的分化[41-42]。而骨保护素属于肿瘤坏死因子受体(Tumor Necrosis Factor Receptor，TNFR)家族成员，研究发现各种刺激骨吸收的因子均能降低骨保护素的表达[43]。骨保护素、RANK和RANKL被认为是介导各种物质诱导破骨细胞生成及功能的最终生物因子。在骨吸收过程中，RANKL与RANK结合并将骨吸收信号传导给破骨细胞前体，促进破骨细胞前体沿破骨细胞通路分化。而血清中骨保护素和RANKL的水平受镁离子的影响，镁离子可以通过调控RANK和骨保护素的水平来影响OPG/RANKL/RANK信号通路而影响破骨细胞的生物活性，维持骨代谢的平衡。骨保护素作用于破骨细胞的分化末期，能够抑制破骨细胞的分化增加骨的致密度，护骨素配体(OPGL)，也称为破骨细胞分化因子，能够在体外诱导破骨细胞的形成，体内则能抑制破骨细胞骨吸收。RANK主要在单核-巨噬细胞系表面表达，在破骨细胞前体表面高度表达，RANK的主要功能是与RANKL结合，促进破骨细胞分化成熟。镁离子缺乏时，体内骨保护素降低，RANKL水平增加，引起破骨细胞的数量增加，骨吸收增加[44]。但因骨吸收刺激因子不直接作用于破骨细胞表面，而是先作用成骨细胞，再由成骨细胞将信号传递到破骨细胞，M-CSF作用是增加破骨前体细胞池，而RANKL是通过结合到破骨前体细胞和成熟破骨细胞表面表达的RANK受体上，促进破骨细胞的分化、活化并抑制其凋亡[45]。除此之外，在骨代谢过程中成骨细胞受到刺激因子作用后除了分泌M-CSF和RANKL外还分泌骨保护素，以旁分泌的方式发挥作用。体外实验发现，RANKL与M-CSF联合应用可取代成骨细胞，而表达于成骨细胞表面的骨保护素(osteoprotegerin， OPG)与RANK竞争RANKL，从而抑制破骨细胞激活和分化[46]。由此可见，机体内镁离子浓度异常时，可以通过激活OPG/RANKL/RANK 信号通路来调节成骨细胞和破骨细胞的增殖分化，进而调节骨量的变化。 2.2.3 TRPM离子通道瞬时受体电位通道M型(melastatin-relatedtransient receptor potential， TRPM)是TRPM通道超家族的一员，包括TRPM1至TRPM8八个成员[47]，是位于哺乳动物细胞膜上一类重要的阳离子通道，TRPM蛋白组成包括六个跨膜片段、胞内氨基残端结构域和羧基残端结构域，其中氨基残端高度保守，包含大量TRPM的同源性序列。功能性TRPM为四聚体，而氨基残端结构和羧基残端结构的完整性是发挥其功能必不可少的。TRPM家族成员可以被不同因素激活，如钙离子和镁离子，并调控生物体一切的生命活动。 TRPM的8个成员中TRPM6和TRPM7参与调控细胞内的镁离子，因TRPM6与TRPM7在氨基酸排列顺序上高度(50%) 同源，均为同时包含离子通道与激酶结构域的双功能蛋白，因此在生物体内作用机制相似。但TRPM6的主要作用表现在对镁离子稳定状态的维持[48]，而 TRPM7主要参与镁离子平衡的调节[49]。研究表明，适当的细胞外镁离子浓度能够诱导成骨细胞的增殖和迁移，而成骨细胞表面表达钙镁离子的蛋白通道TRPM7(melastatin-like transientreceptor potential 7) [50]，受到胞内 Mg2 +/Mg2+-ATP 调节，外界镁离子浓度的变化，可以通过TRPM7来感知并调控细胞的相关生理活动[51]。低水平的镁离子能够激活TRPM7通道[52-53]。TRPM7基因的表达增强有助于成骨细胞一系列的增殖分化，骨组织的形成依赖成骨细胞的增殖、分化、迁移和基质蛋白的分泌。TRPM7作为钙/镁的离子通道，能够调节细胞内钙/镁平衡，从而促进成骨细胞的增殖分化。事实上，成骨细胞分化过程中钙/镁水平同成骨细胞增殖分化的标志物碱性磷酸酶和骨钙素基因的表达相关。镁离子摄入不足使得血清中的碱性磷酸酶和骨钙素水平降低，影响骨组织的正常生长发育。TRPM7在成骨细胞中沉默时可抑制镁饥饿而摄入镉所导致的骨质疏松症[54]。这项研究提示镁耗竭情况下 TRPM7通过促进镉吸收从而诱导骨质疏松症。提示TRPM7在骨代谢过程中发挥着至关重要的作用[55]。以上简述可知镁离子浓度在体内的平衡是靠TRPM7通道来维持的，而TRPM7同骨细胞的增殖分化相关联，因此镁离子浓度的异常可以通过TRPM7来影响正常的骨代谢。 2.2.4 Wnt 信号通路骨代谢过程中除了以上信号通路以外，Wnt信号通路近年来也受到了人们的广泛关注，Wnt信号通路包括经典的Wnt/β-catenin信号途径和非经典的Wnt信号途径(Wnt/Ca2+途径和PCP途径)[56-57]。经典的Wnt信号通路通过β-Catenin作用来激活核内基因转录的Wnt信号通路。当成骨细胞外Wnt因子与膜受体卷曲蛋白(Frizzled)结合后，通过一系列胞膜及胞质蛋白的相互作用形成二聚体，使β-catenin在胞质内累积，然后进入细胞核内与T细胞转录因子(TCF)/淋巴增强因子(LEF)共同结合形成复合聚体，激活下游靶基因的转录，促进成骨细胞分化增殖[58]。Wnt/β-catenin 信号通路是骨细胞分化和增殖过程中重要的信号通路，该通路功能的强弱与骨量的多少有关[59]。除此之外，β-catenin还可以通过增强间充质干细胞对骨形态发生蛋白2的应答来诱导其向成骨细胞分化。这表明β-catenin在成骨前体细胞及成骨细胞增殖分化过程中提供了一种分子诱导信号，并受骨形态发生蛋白2的调节。提示骨形态发生蛋白和经典Wnt信号通路相互协作来调节成骨细胞的分化。此外，骨形态发生蛋白2也可通过作用于其受体 BMPR2，激活PI3K/AKT和β-catenin信号，从而发挥抑制成骨细胞凋亡的作[60]。而Wnt蛋白可以通过激活Src/ERK和PI3K/Akt信号通路来调控成骨细胞的增殖和存活[61]，提示骨形态发生蛋白2、PI3K/Akt信号通路同经典的Wnt信号通路在调节成骨细胞的分化和骨形成过程中也存在着一定的交互作用。而PI3K/Akt信号通路在调节骨代谢过程中受镁离子浓度影响，由此猜想Wnt信号通路在调节骨代谢过程中是否也受镁离子浓度变化的影响，但镁离子对Wnt信号通路的调节机制目前仍然没有被明确。 2.3 镁离子相关骨代谢疾病机体内镁离子的摄入和吸收是一个动态的过程，当体内镁离子浓度降低可以导致炎症因子的释放[62]，引起一系列的相关疾病，如偏头痛、Ⅱ型糖尿病、代谢综合症、高血压、动脉粥样硬化、心源性猝死甚至结肠癌等[63]。近年来研究还发现骨质疏松也与体内镁离子浓度降低相关，骨质疏松症是一种与全身骨代谢异常相关的骨疾病，主要表现为骨组织的微结构发生变化，骨小梁数量降低，骨量减少，骨质的脆性增加等症状，镁离子除了可以直接影响骨细胞的功能和羟基磷灰石晶体的生长外，还可以通过调节骨代谢过程中相关因子来影响骨组织的稳定，因此体内镁离子浓度的降低可以直接影响骨密度。然而关于镁缺乏引起骨量减少的机制存在不同的解释。目前研究提示镁离子可以通过调节骨代谢相关的因子来影响骨细胞的分裂生长，因此它的缺乏可能引起骨形成减少。除此之外，低镁还可扰乱磷酸肌醇系统和/或降低腺苷酸环化酶活性，导致骨和肾脏的抗PTH作用，而通过镁的增加，可使机体的甲状旁腺素反应恢复正常，从而影响骨的强度。除了镁离子浓度降低可以扰乱正常骨代谢外，镁离子浓度增高也同样影响骨的生理活动，体外实验发现，高镁明显会抑制成骨细胞矿物基质的沉积，并降低成骨细胞分化标志物-碱性磷酸酶的活性[16]。除此之外，高浓度的镁还可以通过对转运蛋白的竞争抑制改变细胞内包括钙离子在内的多种阳离子的浓度，进而产生对骨细胞代谢的影响。骨组织内矿物盐结晶沉积障碍时会导致成骨细胞活性降低、生长板增宽和骨骼变得短粗，从而导致骨软化样表现[64]。动物实验发现，镁过量会刺激骨吸收，但也有报道显示，高镁会抑制成骨细胞的增殖和贴壁，并且随着镁浓度的增高，细胞的矿化水平明显降低[65]。由此可见，机体内镁离子浓度异常会影响到骨组织的稳定。"

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