肠道微生态系统是人体最大的微生态系统，栖息着大约1014数量级的细菌，其基因组的基因数约为人类基因组基因总数的150倍[1]。肠道菌群与人体相互依存，影响着人体肠道内分泌细胞、肠道神经系统、肠道通透性以及免疫系统等的功能，在促进食物消化、产生维生素等营养物质、抵御外来致病菌侵入、刺激免疫系统等方面有着重要作用[1-3]。
原发性骨质疏松症(以下简称骨质疏松症)是与社会人口老龄化关系最为密切的疾病之一，是老年人的常见病和多发病。其以骨量减少、骨组织结构退化为特征，可导致骨脆性增高及骨折危险性增加[4]。骨质疏松症的发病由20~30岁时所能达到的峰值骨量和年龄增长相关的骨丢失两方面决定，人体峰值骨量50%~85%由遗传决定，而环境因素在年龄增长相关的骨丢失中起到更加重要的作用[5]。
近年来，研究发现，肠道微生态(gut microbiome，GM)对骨骼有重要调节作用。肠道菌群缺失可导致小鼠骨量增加[6]，一些益生菌对性激素缺乏导致的骨量丢失具有保护作用[7]。一些益生菌与益生元被证明可增加动物及人类钙吸收与沉积，促进骨骼健康。本文对肠道菌群对骨骼的作用机制作一综述。
肠道菌群与骨代谢研究发现，肠道菌群的缺失导致小鼠骨量增加。肠道菌群缺乏的无菌小鼠与野生小鼠相比，胫骨近端小梁体积骨密度(volumetric bone mineral density，vBMD), 骨体积分数(bone volume fraction, BV/TV)及小梁数目均增加，骨小梁分离率减少。而将无菌小鼠定植肠道正常菌群4周后，小鼠骨小梁vBMD显著降低，接近普通小鼠vBMD水平[6]。
当无菌小鼠与普通小鼠被给予亮丙瑞林模仿卵巢切除后模型时，发现普通小鼠血清中骨吸收标志物胶原降解产物(C-terminal telopeptide, CTX)较无菌小鼠增高，皮质骨与小梁骨显著丢失。无菌小鼠可以抵抗雌激素缺乏导致的骨吸收增加作用[7]。此外，绝经后妇女肠道菌群构成的改变对骨量流失也有重大影响，因此绝经后妇女肠道菌群的紊乱会明显增加雌激素缺乏引起的骨量下降[8]。
同时，接受预防性剂量青霉素治疗3周的断奶小鼠骨矿物密度显著升高[9]。而四环素治疗能够通过抑制骨吸收预防卵巢切除后小鼠的骨量丢失，提高小鼠骨生物力学指标[10-11]。表明抗生素有调节菌群成分和骨量的作用。也间接支持了GM对骨量的调节作用。
因此，肠道菌群对骨代谢有重要调节作用，肠道菌群成分的改变将导致小鼠骨量增加，其对性激素缺乏导致的骨量丢失有保护作用。
肠道微生态对骨代谢的作用机制骨代谢与免疫
骨重建由成骨细胞与破骨细胞完成。成骨细胞来源于多能间充质细胞，破骨细胞来源于骨髓中的造血干细胞[12]。免疫系统可通过细胞因子途径调节成骨细胞与破骨细胞的活动。巨噬细胞集落刺激因子(macrophage colony-stimulating factor，M-CSF)以及核因子kB受体活化因子(receptor activator for nuclear factor-κB, RANK)的上调表达会增加破骨细胞的增生与存活[13]。由骨髓细胞产生的肿瘤坏死因子(tumor necrosis factor，TNF)与其下游调节因子白介素-1(interleakin, IL-1) 可促进破骨细胞发生[14-16]。研究表明，骨丢失与炎性反应密切相关，在一些自身免疫疾病如关节炎，破骨细胞的骨吸收由产生细胞因子的效应T细胞介导[17-18]。绝经后雌激素缺乏导致的炎性因子增加能够刺激破骨细胞的形成以及延长破骨细胞的存活[19-20]。另有研究表明，对T细胞缺乏小鼠使用抗CD4抗体、抗CD8抗体干预能够预防卵巢切除引起的骨量丢失[21]。
此外，GM作为潜在抗原，对宿主免疫系统的成熟有重要作用。无菌动物的黏膜免疫系统发育不健全，肠道相关的淋巴组织发育不完全；并且，无菌动物脾脏CD4+T细胞与生发中心明显减少[22]。
多组实验表明，肠道菌群可通过调节免疫系统影响骨代谢。其机制有以下几点。
调节免疫细胞及相关细胞因子
肠道菌群可通过减少免疫细胞数量及降低破骨细胞相关细胞因子的产生与表达来影响破骨细胞的发生，从而影响骨骼代谢。研究表明，无菌小鼠骨髓中CD4+T细胞，破骨细胞前体CD11b+/Gr1-细胞及破骨细胞减少，其骨髓中促进破骨细胞发生的免疫因子TNF-α与IL-1表达下降。卵巢切除模型的普通小鼠骨髓中TNF-α+CD4+与TNF+CD8+T细胞数量较无菌小鼠增加[23]。予以卵巢切除术后的普通小鼠罗伊氏乳杆菌6475(L.reuteri 6475)2~4周，可使其骨中TRAP5(破骨细胞的标志)及RANKL的mRNA水平降低，骨髓中CD4+T细胞及破骨细胞数量减少[24-25]。由此可推测，GM可通过减少破骨细胞前体数量及破骨细胞相关细胞因子的表达影响骨代谢。
此外，部分肠道菌群可通过提高成骨细胞活性，促进骨形成。予以普通小鼠罗伊乳杆菌6475后，小鼠肠道IL-1β、TNF基因的转录水平降低，雄性小鼠的骨小梁骨量增加，而雌性小鼠的小梁骨量无显著变化。通过检测雄性小鼠血清，发现TRAP5蛋白及其mRNA水平未发生变化，而反映成骨细胞活性的骨钙素水平显著增高。通过动态监测，发现小鼠骨形成速率显著增高。由此可说明，肠道菌群或可通过减少细胞因子IL-1β、TNF的表达，提高成骨细胞活性，促进骨形成。
调节骨髓细胞的发生
Khosravi等[26]发现，无菌小鼠骨髓中性粒细胞(Gr1hiCD115neg)和单核细胞(Gr1hiCD115hi)减少，间接反映多能造血干细胞下游粒-单核细胞前体(granulocyte-macrophage precursor，GMPs)的减少。通过细菌定植或在饮水中添加加热灭活的细菌作为微生物相关分子模式(microbe-associated molecular patterns，MAMPs)，可反转骨髓中单核细胞的减少。在Balmer等[27]的实验中也有着同样的发现，与无特定病原体小鼠(SPF小鼠)相比，无菌小鼠骨髓中CD11b+Ly6C+单核细胞与CD11b+Ly6G+粒细胞减少，通过感染SPF小鼠的血清可增加野生无菌小鼠的髓细胞生成。这说明SPF小鼠血清中的循环MAMPs促进髓细胞发生。这些实验表明，肠道正常菌群可作为MAMPs影响破骨细胞前体的发生。
影响T细胞分化
效应T细胞通过刺激破骨细胞生成调节骨丢失[28]。其来源的细胞因子对破骨细胞的发生也有着重要作用。如Th1与Th2来源的IFN-γ与IL-4抑制破骨细胞生成[29-30]，由Th17细胞来源的IL17A促进破骨细胞的生成[31]，而调节性T细胞(Treg细胞)通过CTLA-4通路抑制破骨细胞生成[32-34]。最近研究发现，肠道菌群可影响辅助性T细胞与调节性T细胞分化。
Ivanov等[35-36]发现分节丝状菌，一种小鼠肠道的共生菌群，是促进Th17辅助性T细胞分化的驱动者。而另一种梭菌属则影响Treg细胞的发生。在无菌小鼠中定植该类细菌可使其结肠、肺、肝和脾中的Treg细胞恢复正常[37]。尽管这些实验未涉及骨参数，但证明了肠道菌群影响T细胞分化，而T细胞对骨细胞发挥重要作用。因此，肠道菌群或可通过影响T细胞分化而调节骨代谢[38]。
通过细菌代谢产物及酶益生元是一种通过上消化道后不被消化的纤维复合物，其可作为肠道共生细菌的底物，选择性刺激一种或少数细菌的生长与活性，而促进宿主健康。食物及益生元被肠道细菌分解后产生的代谢产物及酶类可影响骨骼代谢。
短链脂肪酸
短链脂肪酸(short-chain fatty acids, SCFA)是益生元经肠道细菌发酵后的代谢产物。包括醋酸盐、丙酸盐、丁酸盐等。SCFA可通过一系列机制增加肠道钙吸收。如降低肠腔的pH值[39]，改变钙形态及溶解度；增加钙结合蛋白D9K(位于黏膜上皮细胞基底膜转运钙的胞内载体蛋白)的表达，提高钙细胞旁路的被动转运[40-41]；增强管腔内外阳离子交换(细胞内的H+与管腔内的Ca2+)[42]，增加细胞内钙离子浓度等；乳酸盐与丁酸可为肠道细胞生长提供能量，促进肠道细胞增生，扩大矿物吸收面积[43]。
还有实验表明，SCFA能够影响调节矿物吸收或肠道细胞代谢增生的信号通路。例如在G蛋白GPR43基因沉默的条件下，SCFA能增加乳腺癌细胞MCF-7细胞的钙摄入[44]。丁酸盐可抑制组蛋白乙酰化，激活G蛋白偶联受体(G protein-coupled receptor, GPCRs)[45]。这或许对钙吸收有远在调节作用。
另一种理论是SCFA或可通过调节破骨细胞的发生影响骨代谢。丁酸盐在体外可抑制破骨细胞生成[46-47]。SCFA可调节结肠细胞内Treg细胞的数量与功能[48]，因此可能间接通过T细胞影响破骨细胞发生。此外，SCFA还可通过影响小鼠体外淋巴细胞促炎性细胞因子IL-2、干扰素-γ和免疫调节因子IL-10的表达[49]，间接调节破骨细胞发生，影响骨代谢。
血清素
肠道菌群可通过影响外周及中央血清素合成和调节血清素前体色氨酸代谢来影响骨代谢[50]。研究表明，外周血清素对小鼠骨形成有抑制作用[51-52]。而中央血清素能促进小鼠骨形成[53]。与正常小鼠相比，无菌小鼠外周血清素水平降低，近端结肠中外周血清素合成酶Tph1的mRNA表达水平降低，降解所需的血清素转运蛋白SERTmRNA表达增高。血清色氨酸(血清素前体)，海马血清素及血清素代谢升高[54]。
叶酸
实验证明，S.嗜热链球菌和双歧杆菌属能够产生叶酸[55]。而红细胞叶酸含量与绝经后妇女BMD与BMC成正相关[56]。因此，肠道细菌可通过产生叶酸影响骨代谢。
植酸酶
在谷类食物中，矿物如锌、铜、铁和钙被植酸盐络合从而影响这些矿物的吸收[57-59]。细菌所产生的植酸酶能够水解植酸的络合作用，从而提高矿物的生物利用度。单胃动物及人类缺乏内源性植酸酶，在食物中添加植物或微生物来源的植酸酶可弥补这一缺陷[60]。
植物雌激素
研究表明，双歧杆菌与乳酸杆菌在低聚果糖(fructooligosaccharide, FOS)刺激下增加肠腔细菌β-糖苷酶活性，可水解异黄酮糖苷，提高异黄酮生物利用度。从而实现植物雌激素的骨保护作用[61]。
多胺
研究表明，食物经细菌发酵后可产生多胺[62]，多胺是一种肠道黏膜生长因子[63]。有研究表明，适量剂量多胺能够保护胫骨小梁骨结构，尤其在雌激素缺乏状态下[64]。
生物活性肽
一些益生元经细菌分解后可产生一些有生物功能的生物活性肽。研究表明，瑞士乳杆菌可产生两种可显著提高矿物吸收的含脯氨酸的肽——IPP与VPP，这两种肽可使矿物由不能溶解的离子状态释放从而增加矿物吸收。此外，IPP与VPP可抑制Ang Ⅱ从Ang Ⅰ的转化[65]，在体外实验中，Ang Ⅱ被证明有促进骨吸收的作用[66]。并且可能作为血管收缩剂收缩骨血管[67]。
此外，由瑞士乳杆菌发酵的牛奶中含有干酪素-O-磷酸肽(casein phosphopeptides，CPP)，可以减少骨丢失。CPP由牛奶在肠道中消化产生，有实验表明，乳制品CPP可通过增加钙的生物利用度预防卵巢切除后小鼠骨质疏松[68-69]。
维生素及25-羟维生素D
一些细菌可参与维生素的合成[70]，而一些维生素如维生素D、C、K等可参与钙代谢，对骨形成非常必要[71-72]。此外，有研究表明，罗伊乳杆菌NCIMB 30242可增加血清25-羟维生素D，从而增加维生素D的合成，预防骨质疏松[73]。
皮质醇
肠道菌群对皮质醇的合成有重要作用。下丘脑-垂体-肾上腺轴的发生需要微生物-宿主相互作用。在应激状态下，无菌小鼠皮质醇较普通小鼠释放增加，呈现出不正常的释放状态[74-77]。皮质醇与外源性糖皮质激素对骨形成有不良反应，其可减少钙吸收，增加骨细胞及成骨细胞凋亡，抑制成骨细胞增生，延长破骨细胞存活[78]。因此，肠道菌群可通过影响皮质醇的合成与释放影响骨骼代谢。
调节钙吸收与代谢肠道微生态可通过增加钙的吸收与转运调节骨代谢。除前文所述的一些细菌的代谢产物如短链脂肪酸，维生素及生物活性肽等能够增加钙的吸收及生物利用度，肠道微生态还可通过以下机制增加钙吸收。
改善肠道健康，增强免疫防卫
研究表明，饲以果糖-菊粉混合物的小鼠小肠绒毛增多，隐窝加深，杯状细胞数目增加，结肠上皮黏液层增厚。肠道吸收功能及健康稳定性升高。而无菌小鼠肠道无改变。提示肠道黏膜的改变是由肠道细菌介导的[79]。此外，稳定的肠道菌群能够预防肠道感染及肠道细胞的氧化性损伤。实验表明某些嗜热链球菌YIT2001对实验导致的肠道脂质体的过氧化作用有很强的抑制作用[80]。这些细菌对肠道健康的促进作用均有助于钙的吸收。
开启Ca2+通道利于胞外钙向胞内扩散
非消化性低聚糖能选择性刺激结肠内双歧杆菌的生长和活性，而双歧杆菌黏附肠上皮细胞过程中，作为一种细胞外刺激因素激活细胞膜上某种受体而开启细胞的Ca2+通道，使细胞外Ca2+向胞内跨膜扩散，引起细胞内钙浓度升高[81]。
提高肠道细胞的钙摄入
研究表明发酵牛奶中瑞士乳杆菌影响绝经后妇女的钙代谢，短期内减少甲状旁腺激素，增加血清钙[82]。可推测这个效应由肠道细胞钙摄入增加导致。此外，如前文所述，细菌代谢产物SCFA能增强管腔内外H+-Ca2+离子交换，降低肠腔的pH值，改变钙形态及溶解度，增加钙结合蛋白的表达，增加细胞钙摄入。
有利于结肠中雌激素的肝肠循环
雌激素是影响钙吸收的重要因素。肠道菌群可参与雌激素的代谢。在体内循环的雌激素约60%在肝脏与葡萄糖醛酸结合并随胆汁分泌到肠道内，经过肠道中的类杆菌、链球菌、优杆菌、肠球菌等作用后(β-葡萄糖醛酸酶与硫化酶的催化脱水结合)，才能被黏膜上皮细胞重吸收进入血液系统，在肝脏激活发挥生物活性作用[80]。有报道指出，绝经妇女雌激素减少与肠道菌群失调有关。肠道菌群失调时，雌激素再吸收能力下降，粪便中的雌激素高出60倍，加重骨质疏松[83]。
综上，肠道微生态对骨骼有重要调节作用。肠道微生物可通过调节免疫系统、产生相关代谢产物及酶类及调节钙的吸收等机制影响骨代谢。但是，目前研究微生物对骨骼作用的认识仍有局限性，一些潜在的作用机制仍需探索，如微生物对宿主成骨细胞的影响，对宿主细胞信号传导及宿主基因的调节作用等。目前，益生菌与益生元已被证明可增强骨量，促进骨骼健康，并有望应用于骨质疏松的预防及治疗。肠道微生物有望成为骨折风险预测的标志物及骨质疏松防治的新靶点。
| [1] | Qin J, Li R, Raes J, et al. A human gut microbial gene catalogue established by metagenomic sequencing[J]. Nature, 2010, 464: 59–65. DOI:10.1038/nature08821 |
| [2] | Antonopoulos DA, Huse SM, Morrison HG, et al. Reproducible Community Dynamics of the Gastrointestinal Microbiota following antibiotic perturbation[J]. Infect Immun, 2009, 77: 2367–2375. DOI:10.1128/IAI.01520-08 |
| [3] | Dethlefsen L, Relman DA. Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation[J]. Proc Natl Acad Sci, 2011, 108: 4554–4561. DOI:10.1073/pnas.1000087107 |
| [4] | Sambrook P, Cooper C. Osteoporosis[J]. Endocrinol Metab Clin North Am, 2006, 367: 913–936. |
| [5] | Ralston SH, Uitterlinden AG. Genetics of osteoporosis[J]. Endocr Rev, 2010, 31: 629–662. DOI:10.1210/er.2009-0044 |
| [6] | Sjögren K, Engdahl C, Henning P, et al. The gut microbiota regulates bone mass in mice[J]. J Bone Miner Res, 2012, 27: 1357–1367. DOI:10.1002/jbmr.1588 |
| [7] | Li JY. Gut microbiota plays a pivotal role in the bone loss induced by sex steroid deficiency[J]. J Bone Miner Res, 2014, 29: 1029. |
| [8] | Claesson MJ, Cusack S, O'Sullivan O, et al. Composition, variability, and temporal stability of the intestinal microbiota of the elderly[J]. Proc Natl Acad Sci USA, 2011, 108: 4586–4591. DOI:10.1073/pnas.1000097107 |
| [9] | Cho I, Yamanishi S, Cox L, et al. Antibiotics in early life alter the murine colonic microbiome and adiposity[J]. Nature, 2012, 488: 621–626. DOI:10.1038/nature11400 |
| [10] | Williams S, Wakisaka A, Zeng QQ, et al. Minocycline prevents the decrease in bone mineral density and trabecular bone in ovariectomized aged rats[J]. Bone, 1996, 19: 637–644. DOI:10.1016/S8756-3282(96)00302-X |
| [11] | Pytlik M, Folwarczna J, Janiec W. Effects of doxycycline on mechanical properties of bones in rats with ovariectomy-induced osteopenia[J]. Calcif Tissue Int, 2004, 75: 225–230. DOI:10.1007/s00223-004-0097-x |
| [12] | Lorenzo J, Horowitz M, Choi Y. Osteoimmunology:interactions of the bone and immune system[J]. Endocr Rev, 2013, 29: 403–440. |
| [13] | Arai F, Miyamoto T, Ohneda O, et al. Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors[J]. J Exp Med, 1999, 190: 1741–1754. DOI:10.1084/jem.190.12.1741 |
| [14] | Yarilina A, Glimcher LH. TNF activates calcium-nuclear factor of activated T cells (NFAT)c1 signaling pathways in human macrophages[J]. Proc Natl Acad Sci, 2011, 108: 1573–1578. DOI:10.1073/pnas.1010030108 |
| [15] | Zwerina J, Redlich K, Polzer K, et al. TNF-induced structural joint damage is mediated by IL-1[J]. Proc Natl Acad Sci, 2007, 104: 11742–11747. DOI:10.1073/pnas.0610812104 |
| [16] | Wei S, Kitaura H, Zhou P, et al. IL-1 mediates TNF-induced osteoclastogenesis[J]. J Clin Invest, 2005, 115: 282–290. DOI:10.1172/JCI200523394 |
| [17] | Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand[J]. Nature, 1999, 402: 304–309. DOI:10.1038/46303 |
| [18] | Sato K, Suematsu A, Okamoto K, et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction[J]. J Exp Med, 2006, 203: 2673–2682. DOI:10.1084/jem.20061775 |
| [19] | Martin-Millan M, Almeida M, Ambrogini E, et al. The estrogen receptor-alpha in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone[J]. Mol Endocrinol, 2010, 24: 323–334. DOI:10.1210/me.2009-0354 |
| [20] | Nakamura T, Imai Y, Matsumoto T, et al. Estrogen prevents bone loss via estrogen receptor α and induction of Fas ligand in osteoclasts[J]. Cell, 2007, 130: 811–823. DOI:10.1016/j.cell.2007.07.025 |
| [21] | Li JY, Tawfeek H, Bedi B, et al. Ovariectomy disregulates osteoblast and osteoclast formation through the T-cell receptor CD40 ligand[J]. Proc Natl Acad Sci, 2011, 108: 768–773. DOI:10.1073/pnas.1013492108 |
| [22] | Macpherson AJ, Harris NL. Interactions between commensal intestinal bacteria and the immune system[J]. Nat Rev Immunol, 2004, 4: 478–485. DOI:10.1038/nri1373 |
| [23] | Li JY. Gut microbiota plays a pivotal role in the bone loss induced by sex steroid deficiency[J]. J Bone Miner Res, 2014, 29: 1029. |
| [24] | Britton RA, Irwin R, Quach D, et al. Probiotic L. reuteri, treatment prevents bone loss in a menopausal ovariectomized mouse model[J]. J Cell Physiol, 2014, 229: 1822–1830. DOI:10.1002/jcp.24636 |
| [25] | Mccabe LR, Irwin R, Schaefer L, et al. Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice[J]. J Cell Physiol, 2013, 228: 1793–1798. DOI:10.1002/jcp.v228.8 |
| [26] | Khosravi A, Yáñez A, Price J, et al. Gut microbiota promote hematopoiesis to control bacterial infection[J]. Cell Host Microbe, 2014, 15: 374–381. DOI:10.1016/j.chom.2014.02.006 |
| [27] | Balmer ML, Schürch CM, Saito Y, et al. Microbiota-derived compounds drive steady-state granulopoiesis via MyD88/TICAM signaling[J]. J Immunol, 2014, 193: 5273–5283. DOI:10.4049/jimmunol.1400762 |
| [28] | Kong YY, Feige U, Sarosi I, et al. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand[J]. Nature, 1999, 402: 304–309. DOI:10.1038/46303 |
| [29] | Takayanagi H, Ogasawara K, Hida S, et al. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma[J]. Nature, 2000, 408: 600–605. DOI:10.1038/35046102 |
| [30] | Yousef AA. IL-4 abrogates osteoclastogenesis through STAT6-dependent inhibition of NF-kappaB[J]. J Clin Invest, 2001, 107: 1375–1385. DOI:10.1172/JCI10530 |
| [31] | Adamopoulos IE, Chao CC, Geissler R, et al. Interleukin-17A upregulates receptor activator of NF-κB on osteoclast precursors[J]. Arth Res her, 2010, 12: R29. |
| [32] | Zaiss MM, Axmann R, Zwerina J, et al. Treg cells suppress osteoclast formation:A new link between the immune system and bone[J]. Arth Rheumatol, 2007, 56: 4104–4112. DOI:10.1002/art.v56:12 |
| [33] | Zaiss MM, Frey B, Hess A, et al. Regulatory T cells protect from local and systemic bone destruction in arthritis[J]. J Immunol, 2010, 184: 7238–7246. DOI:10.4049/jimmunol.0903841 |
| [34] | Bozec A, Zaiss MM, Kagwiria R, et al. T cell costimulation molecules CD80/86 inhibit osteoclast differentiation by inducing the IDO/tryptophan pathway[J]. Sci Translat Med, 2014, 6: 747–751. |
| [35] | Ivanov II, Atarashi K, Manel N, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria[J]. Cell, 2009, 139: 485–498. DOI:10.1016/j.cell.2009.09.033 |
| [36] | Ivanov II, Frutos RDL, Manel N, et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine[J]. Cell Host Microbe, 2008, 4: 337–349. DOI:10.1016/j.chom.2008.09.009 |
| [37] | Atarashi K, Tanoue T, Shima T, et al. Induction of colonic regulatory T cells by indigenous clostridium species[J]. Science, 2011, 331: 337–341. DOI:10.1126/science.1198469 |
| [38] | Charles JF, Ermann J, Aliprantis AO. The intestinal microbiome and skeletal fitness:connecting bugs and bones[J]. Clin Immunol, 2015, 159: 163–169. DOI:10.1016/j.clim.2015.03.019 |
| [39] | Weaver CM, Martin BR, Story JA, et al. Novel fibers increase bone calcium content and strength beyond efficiency of large intestine fermentation[J]. J Agr Food Chem, 2010, 58: 8952–8957. DOI:10.1021/jf904086d |
| [40] | Ohta A, Motohashi Y, Sakai K, et al. Dietary fructooligosaccharides increase calcium absorption and levels of mucosal calbindin-D9k in the large intestine of gastrectomized rats[J]. Scand J Gastroenterol, 1998, 33: 1062–1068. DOI:10.1080/003655298750026769 |
| [41] | Ohta A, Sakai K, Motohashi Y, et al. Dietary short-chain fructooligosaccharides increase calbindin-D9k levels only in the large intestine in rats independent of dietary calcium deficiency or serum 1, 25 dihydroxy vitamin D levels[J]. Int J Vitamin Nutr Res, 2000, 70: 206–213. DOI:10.1024/0300-9831.70.5.206 |
| [42] | Lutz T, Scharrer E. Effect of short-chain fatty acids on calcium absorption by the rat colon[J]. Exp Physiol, 1991, 76: 615–618. DOI:10.1113/expphysiol.1991.sp003530 |
| [43] | Donohoe DR, Garge N, Zhang X, et al. The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon[J]. Cell Metab, 2011, 13: 517–526. DOI:10.1016/j.cmet.2011.02.018 |
| [44] | Yonezawa T, Kobayashi Y, Obara Y. Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line[J]. Cell Sign, 2006, 19: 185–193. |
| [45] | Sharon G, Garg N, Debelius J, et al. Specialized metabolites from the microbiome in health and disease[J]. Cell Metab, 2014, 20: 719–730. DOI:10.1016/j.cmet.2014.10.016 |
| [46] | Iwami K, Moriyama T. Effects of short chain fatty acid, sodium butyrate, on osteoblastic cells and osteoclastic cells[J]. Int J Biochem, 1993, 25: 1631–1635. DOI:10.1016/0020-711X(93)90522-G |
| [47] | Morozumi A. High concentration of sodium butyrate suppresses osteoblastic differentiation and mineralized nodule formation in ROS17/2.8 cells[J]. J Oral Sci, 2011, 53: 509–516. DOI:10.2334/josnusd.53.509 |
| [48] | Smith PM, Howitt MR, Panikov N, et al. The microbial metabolites, short-chain fatty acids, regulate colonic treg cell homeostasis[J]. Science, 2013, 341: 569–573. DOI:10.1126/science.1241165 |
| [49] | Cavaglieri CR, Nishiyama A, Fernandes LC, et al. Differential effects of short-chain fatty acids on proliferation and production of pro-and anti-inflammatory cytokines by cultured lymphocytes[J]. Life Sci, 2003, 73: 1683–1690. DOI:10.1016/S0024-3205(03)00490-9 |
| [50] | Clarke G, Stilling RM, Kennedy PJ, et al. Gut microbiota:the neglected endocrine organ[J]. Mol Endocrinol, 2014, 28: 1358–1366. |
| [51] | Bliziotes M, Eshleman A, Burt-Pichat B, et al. Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells[J]. Bone, 2006, 39: 1313–1321. DOI:10.1016/j.bone.2006.06.009 |
| [52] | Yadav VK, Ryu JH, Suda N, et al. Lrp5 Controls bone formation by inhibiting serotonin synthesis in the duodenum[J]. Cell, 2008, 135: 825–837. DOI:10.1016/j.cell.2008.09.059 |
| [53] | Yadav VK, Oury F, Suda N, et al. A serotonin-dependent mechanism explains the leptin regulation of bone mass, appetite, and energy expenditure[J]. Cell, 2009, 138: 976–989. DOI:10.1016/j.cell.2009.06.051 |
| [54] | Clarke G, Grenham S, Scully P, et al. The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner[J]. Mol Psychiatr, 2013, 18: 666–673. DOI:10.1038/mp.2012.77 |
| [55] | Crittenden RG, Martinez NR, Playne MJ. Synthesis and utilisation of folate by yoghurt starter cultures and probiotic bacteria[J]. Int J Food Microbiol, 2003, 80: 217–222. DOI:10.1016/S0168-1605(02)00170-8 |
| [56] | Villa ML, Marcus R, Delay RR, et al. Factors contributing to skeletal health of postmenopausal Mexican-American women[J]. J Bone Miner Res, 1995, 10: 1233–1242. |
| [57] | Torre M, Rodriguez AR, Saura-Calixto F. Effects of dietary fiber and phytic acid on mineral availability[J]. Crit Rev Food Sci Nutr, 1991, 30: 1–22. |
| [58] | Weaver CM, Heaney RP, Martin BR, et al. Human calcium absorption from whole-wheat products[J]. J Nutr, 1991, 121: 1769–1775. |
| [59] | Lopez HW, Coudray C, Levrat-Verny MA, et al. Fructooligosaccharides enhance mineral apparent absorption and counteract the deleterious effects of phytic acid on mineral homeostasis in rats[J]. J Nutr Biochem, 2000, 11: 500–508. DOI:10.1016/S0955-2863(00)00109-1 |
| [60] | Pallauf J, Rimbach G. Nutritional significance of phytic acid and phytase[J]. Arch Anim Nutr, 1997, 50: 301–319. |
| [61] | Breinholt V, Hossaini A, Svendsen GW, et al. Estrogenic activity of flavonoids in mice. The importance of estrogen receptor distribution, metabolism and bioavailability[J]. Food Chem Toxicol, 2000, 38: 555–564. DOI:10.1016/S0278-6915(00)00046-6 |
| [62] | Noack J, Kleessen B, Proll J, et al. Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats[J]. J Nutr, 1998, 128: 1385–1391. |
| [63] | Löser C, Eisel A, Harms D, et al. Dietary polyamines are essential luminal growth factors for small intestinal and colonic mucosal growth and development[J]. Gut, 1999, 44: 12–16. DOI:10.1136/gut.44.1.12 |
| [64] | Scholzahrens KE, Ade P, Marten B, et al. Prebiotics, probiotics, and synbiotics affect mineral absorption, bone mineral content, and bone structure[J]. J Nutr, 2007, 137: 838S–846S. |
| [65] | Narva M, Nevala R, Poussa T, et al. The effect of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women[J]. Eur J Nutr, 2004, 43: 61–68. DOI:10.1007/s00394-004-0441-y |
| [66] | Hagiwara H, Hiruma Y, Inoue A, et al. Deceleration by angiotensin Ⅱ of the differentiation and bone formation of rat calvarial osteoblastic cells[J]. J Endocrinol, 1998, 156: 543–550. DOI:10.1677/joe.0.1560543 |
| [67] | Ma YF, Stimpel M, Liang H, et al. Impact of antihypertensive therapy on the skeleton:effects of moexipril and hydrochlorothiazide on osteopenia in spontaneously hypertensive ovariectomized rats[J]. J Endocrinol, 1997, 154: 467–474. DOI:10.1677/joe.0.1540467 |
| [68] | Kim JG, Lee E, Kim SH, et al. Effects of a Lactobacillus casei, 393 fermented milk product on bone metabolism in ovariectomised rats[J]. Int Dairy J, 2009, 19: 690–695. DOI:10.1016/j.idairyj.2009.06.009 |
| [69] | Bennett T, Desmond A, Harrington M, et al. The effect of high intakes of casein and casein phosphopeptide on calcium absorption in the rat[J]. Br J Nutr, 2000, 83: 673–680. DOI:10.1017/S0007114500000854 |
| [70] | Hancock R, Viola R. The use of micro-organisms for L-ascorbic acid production:current status and future perspectives[J]. Appl Microbiol Biotechnol, 2001, 56: 567–576. DOI:10.1007/s002530100723 |
| [71] | Villa ML, Marcus R, Delay RR, et al. Factors contributing to skeletal health of postmenopausal Mexican-American women[J]. J Bone Miner Res, 1995, 10: 1233–1242. |
| [72] | Weber P. The role of vitamins in the prevention of osteoporosis-a brief status report[J]. Int J Vitam Nutr Res, 1999, 69: 194–197. DOI:10.1024/0300-9831.69.3.194 |
| [73] | Jones ML, Martoni CJ, Prakash S. Oral supplementation with probiotic L. reuteri NCIMB 30242 increases mean circulating 25-hydroxyvitamin D:a post hoc analysis of a randomized controlled trial[J]. J Clin Endocrinol Metab, 2013, 98: 2944–2951. DOI:10.1210/jc.2012-4262 |
| [74] | Jones ML, Martoni CJ, Prakash S. Oral supplementation with probiotic L. reuteri NCIMB 30242 increases mean circulating 25-hydroxyvitamin D:a post hoc analysis of a randomized controlled trial[J]. J Clin Endocrinol Metab, 2013, 98: 2944–2951. DOI:10.1210/jc.2012-4262 |
| [75] | Zacharowski K, Zacharowski PA, Koch A, et al. Toll-like receptor 4 pays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome[J]. Proc Natl Acad Sci, 2006, 103: 6392–6397. DOI:10.1073/pnas.0601527103 |
| [76] | Sudo N, Chida Y, Aiba Y, et al. Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice[J]. J Physiol, 2004, 558: 263–275. DOI:10.1113/jphysiol.2004.063388 |
| [77] | Clarke G, Grenham S, Scully P, et al. The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner[J]. Mol Psychiatr, 2013, 18: 666–673. DOI:10.1038/mp.2012.77 |
| [78] | Cooper MS. Sensitivity of bone to glucocorticoids[J]. Clin Sci, 2004, 107: 111–123. DOI:10.1042/CS20040070 |
| [79] | Kleessen B, Hartmann L, Blaut M. Fructans in the diet cause alterations of intestinal mucosal architecture, released mucins and mucosa-associated bifidobacteria in gnotobiotic rats[J]. Br J Nutr, 2003, 89: 597–606. DOI:10.1079/BJN2002827 |
| [80] | Ito M, Ohishi K, Yoshida Y, et al. Antioxidative effects of lactic acid bacteria on the colonic mucosa of iron-overloaded mice[J]. J Agr Food Chem, 2003, 51: 4456–4460. DOI:10.1021/jf0261957 |
| [81] | 熊德鑫. 现代微生态学[M]. 北京: 中国科技出版杜, 2000. |
| [82] | Narva M, Nevala R, Poussa T, et al. The effect of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women[J]. Eur J Nutr, 2004, 43: 61–68. DOI:10.1007/s00394-004-0441-y |
| [83] | 袁杰利. 肠道菌群与微生态制剂[M]. 大连: 大连海事出版社, 1996. |
| (收稿日期：2016-08-24) |