二甲双胍抑制破骨细胞分化与骨吸收及其分子机制研究
摘要
背景：随着人类社会的发展和人口老龄化的进程,糖尿病(diabetes mellitus,DM)在全世界范围内呈逐年增高趋势,其急慢性并发症严重危害患者的健康,骨质疏松症(osteoporosis, OP)就是其中之一。糖尿病性骨质疏松是指糖尿病(DM)并发单位体积内骨量减少、骨组织微结构改变、骨强度减低、脆性增加等易发生骨折的一种全身性、代谢性骨病。糖尿病骨质疏松症在糖尿病的各种并发症中的发病率较高,目前发病率在24%～52%,并成为长期严重疼痛和功能障碍的主要原因。同时由于骨质疏松症易导致骨折,致残性高,使糖尿病患者的治疗和康复更加困难,严重影响患者的生活质量,给家庭和社会都带来了沉重的经济负担。二甲双胍(Metformin)是目前广泛应用的降糖药物。最近的研究发现其对骨代谢也有影响,可促进成骨细胞的分化及活性,提高eNOS和BMP-2的表达,从而促进骨形成。流行病学调查发现,口服二甲双胍的糖尿病患者较对照组相比,骨折发生率明显下降。而二甲双胍对骨质疏松的影响机制仍不清楚,目前二甲双胍对破骨细胞的作用和影响仍未有报道。因此本课题拟从分子、细胞及整体水平深入探讨二甲双胍影响骨代谢的模式、对破骨细胞的影响及其相关的分子机制,为骨质疏松的防治提供新的依据。
骨质疏松发生的根本机制在于机体骨重建的失衡,即破骨细胞(osteoclast,OC)去除旧骨(骨吸收)和成骨细胞(osteoblast, OB)形成新骨(骨形成)的失衡。而通过调节破骨细胞的活性来抑制骨吸收是治疗骨质疏松的主要的手段。OC主要来源于单核/巨噬细胞系,目前认为其分化与功能受骨髓干细胞或OB前体细胞分泌的两种关键因子所调控：巨噬细胞集落刺激因子(macrophage colony-stimulating factor, M-CSF)和核因子κB受体活化素的配体(receptor activator of nuclear factor-KB ligand, RANKL),两者与单核/巨噬细胞上受体作用使其定向发育为OC。RANKL与OC前体上的受体RANK(receptor activator of nuclear factor-KB, RANK)结合后,经TNF受体连接因子(TNF receptor associated factor, TRAF)家族蛋白激活多种信号途径,如激活转录因子NF-κB.细胞外信号调节激酶(extracellular signalregulated kinase, ERK)及c-Jun氨基末端激酶(c-Jun amino-terminal kinase, JNK),诱导转录因子AP-1家族c-fos和c-Jun等的活性,通过这些级联反应调控RANKL介导的OC分化、活化与分泌活动。同时OB前体分泌一种与RANKL竞争的可溶性分子—护骨素(osteoprotegerin, OPG),后者竞争结合RANK而抑制OC分化及其功能。OPG/RANKL/RANK轴是近年来骨代谢调节研究领域的突出成果,多种激素与细胞因子都通过上调或下调OPG与RANKL的表达参与骨代谢偶联调节。
二甲双胍是使用最广泛的降糖药物之一,能够降低肝糖输出、增加骨骼肌的葡萄糖摄取及改善脂代谢2006年,Cortizo等人通过体外实验首次提出二甲双胍拥有促成骨效应。Donghu Zhen等人报导二甲双胍能够逆转高糖对原代成骨细胞的影响,显著降低细胞内氧化应激(ROS)和细胞凋亡,同时直接促进细胞的成骨效应,而且二甲双胍的这种调节作用部分是通过增加细胞的RUNX2和IGF-1的表达。目前还没有二甲双胍调节破骨细胞增殖和分化的报道。二甲双胍的降糖效应主要通过激活腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)而实现。AMPK是细胞内调节能量代谢的蛋白激酶。当细胞内AMP/ATP比值升高到一定范围时被激活,一方面通过抑制糖原、脂肪和胆固醇的合成,减少ATP的利用；另一方面,通过促进脂肪酸氧化、葡萄糖转运等,增加ATP的产生。通过这两种作用维持细胞能量供求平衡。最近有研究发现二甲双胍还可通过激活AMPK,进而增加一氧化氮合酶(eNOS)和骨形成蛋白-2(BMP-2)的表达从而促进成骨细胞分化,增强成骨细胞活性。但二甲双胍能否通过AMPK参与对破骨细胞增殖、分化及骨吸收活性的影响?
哺乳动物雷帕霉素靶蛋白(mammalian Target Of Rapamycin, mTOR)是AMPK下游重要的信号分子,对于蛋白质翻译、细胞生长增殖、存活等具有重要的调控。有研究报道mTOR对于破骨细胞的活性有重要调控作用,细胞因子M-CSF, TNF-a和RANKL均通过mTOR信号促进破骨细胞的存活,mTOR的特异性抑制剂rapamycin能够阻断这种促存活作用,并诱导其凋亡,抑制其骨吸收的功能。那么,二甲双胍对破骨细胞的影响是否通过抑制mTOR信号而实现呢?
综上所述,我们对二甲双胍影响骨代谢的模式及其对破骨细胞的影响和相关的分子机制作出如下假设：二甲双胍通过抑制腺苷酸活化蛋白激酶(AMPK)下游的mTOR信号通路,抑制破骨细胞的增殖和分化,促进破骨细胞的凋亡,降低破骨细胞的骨吸收能力。
目的：因此本课题利用破骨细胞前体细胞株、破骨细胞体外分化细胞模型及去卵巢骨质疏松动物模型,从分子、细胞及整体层面探讨二甲双胍对破骨细胞增殖、分化及功能的影响,并探讨相关的分子机制,期望为糖尿病性骨质疏松的防治提供细胞和分子生物学依据。
方法：采用RANKL诱导鼠巨噬细胞系Raw264.7细胞及RANKL+M-CSF诱导骨髓源性巨噬细胞(Bone marrow-derived macrophages,BMMs)破骨分化模型,给予不同浓度的二甲双胍(400,800,1000μmol/L)和雷帕霉素(100μmol/L)处理后,抗酒石酸酸性磷酸酶(TRAP)染色并计数形成的TRAP阳性细胞数量,分别使用骨吸收培养板(BD BioCoatTM OsteologicTM Bone Cell Culture System)及骨片检测形成破骨细胞的骨吸收活性, RT-PCR技术检测破骨特异性基因抗酒石酸酸性磷酸酶(TRAP)、组织蛋白酶K(Cathepsin K)、降钙素(CTR)受体和金属基质蛋白酶-9(MMP-9)的表达,ELISA检测肿瘤坏死因子α(TNF-α)表达水平,Western-blot检测c-Fos蛋白以及哺乳动物雷帕霉素靶蛋白(mammalian Target OfRapamycin, mTOR)信号通路下游底物S6K1(Thr389)、S6(Ser235/236)、4EBP1(Thr37/46)的表达及磷酸化水平。构建去卵巢骨质疏松大鼠模型,同时给予二甲双胍(100mg/kg/d)、雷帕霉素(1mg/kg/d)等处理,测定模型动物体重变化、血清骨代谢生化指标(血清钙、磷、酸性磷酸酶及碱性磷酸酶)、骨组织形态学指标参数(百分骨容积、骨小梁厚度、骨小梁数量与骨小梁疏密度)及骨密度(BMD)。
结果：
1、二甲双胍抑制RANKL诱导Raw264.7和BMMs细胞向破骨细胞分化
对加入不同浓度二甲双胍的RANKL诱导的Raw264.7细胞进行TRAP染色和细胞骨架微丝荧光染色。结果显示：RANKL能有效的刺激TRAP阳性多核细胞和细胞骨架的形成。二甲双胍抑制细胞骨架的形成并且下降RANKL诱导的TRAP阳性：多核细胞的数量,二甲双胍(400、800和1000μmol/L组)与RANKL组比较,分别使形成的TRAP阳性多核细胞数量下降了17.7%、30.3%和59.3%,其抑制作用呈浓度梯度依赖性。同样的,在BMMs细胞中,我们得到了类似的结果：二甲双胍呈浓度梯度依赖性(400-1000μmol/L)的抑制!RANKL+M-CSF诱导的BMMs细胞形成TRAP阳性多核细胞的数量。细胞毒性实验结果表明:400-1000μmol/L浓度的二甲双胍对两种细胞(Raw264.7和BMMs)的生长抑制及凋亡作用并无明显统计学差异。由此表明,二甲双胍能够抑制RANKL诱导的破骨细胞分化。
2、mTORC1信号参与二甲双胍抑制破骨细胞分化的过程
RANKL能使Raw264.7细胞mTORC1信号通路下游受体S6K1(Thr389),S6(Ser235/236)和4E-BP1(Ser65)蛋白的磷酸化水平表达增加(即激活mTORC1信号),而雷帕霉素和二甲双胍均可在不影响mTORC1下游受体S6K1(Thr389),S6(Ser235/236)和4E-BP1(Ser65)总蛋白水平的基础上,抑制RANKL诱导的上述蛋白磷酸化,且随二甲双胍浓度的升高这种抑制作用越明显,具有浓度依赖性。由此说明,二甲双胍能够抑制RANKL激活的mTORC1信号。进一步研究表明,雷帕霉素也能明显的抑制RANKL诱导的TRAP阳性多核细胞的形成,加入100 nmol/L的雷帕霉素组较RANKL组比较而言,TRAP阳性多核细胞的数量下降了39.8%。这些结果表明,mTORCl信号是刺激破骨细胞分化的必要通路。当仅仅只给予800μmol/L的二甲双胍处理时(不加RANKL),也能观察到S6K1(Thr389),S6(Ser235/236)和4E-BP1(Ser65)的磷酸化受到明显的抑制。这表明mTORCl信号参与二甲双胍抑制破骨细胞分化的过程。
3、二甲双胍抑制RANKL诱导的破骨细胞特异基因的表达
RANKL组抗酒石酸酸性磷酸酶、组织蛋白酶K、降钙素受体和金属基质蛋白酶-9四种基因的表达比空白对照组明显增加。同RANKL组相比,二甲双胍+RANKL组上述基因表达量分别下降了69.6%、44.1%、69.0%和31.7%；雷帕霉素+RANKL组上述基因的表达量则分别下降了54.0%、71.3%、80.9%和78.0%。
4、二甲双胍抑制RANKL诱导的TNF-a和c-Fos蛋白表达
RANKL提高Raw264.7细胞TNF-a产物的表达,二甲双胍和雷帕霉素均能抑制RANKL诱导的TNF-a产物的表达,二甲双胍(400、800和1000μmol/L)组以及雷帕霉素(100nmol/L)组的OD值分别为1.88±0.05、1.62±0.06、1.56±0.08和1.37±0.12,比RANKL组(2.43±0.05)明显降低(F=12.257,p<0.05)。同时,二甲双胍和雷帕霉素均能抑制RANKL诱导的c-Fos蛋白的表达水平。
5、二甲双胍抑制破骨细胞骨吸收功能
在Raw264.7细胞中,与RANKL对照组相比,二甲双胍组(400、800和1000μmol/L组)明显的抑制了破骨细胞对骨的吸收功能,吸收陷窝的数量和面积百分比相对RANKL组分别下降了22.7%、55.9%和81.6%,且呈浓度梯度依赖性(p<0.05)。雷帕霉素处理组同样的显示了骨吸收陷窝在数量和面积百分比相对RANKL组下降了71.9%(p<0.05)。这些表明,二甲双胍能够抑制破骨细胞的骨吸收活性。这个结果进一步的在RANKL+M-CSF诱导的BMMs细胞上得到了验证。二甲双胍呈浓度依赖性的抑制破骨细胞骨吸收陷窝的数量和面积百分比。
6、二甲双胍与雷帕霉素在抑制破骨细胞分化和骨吸收功能方面不具有协同效用
在Raw264.7和BMMs两种细胞当中,雷帕霉素加二甲双胍(800μmol/L)组较二甲双胍(800μmol/L)组相比而言,所形成的TRAP阳性多核细胞的数量及骨吸收面积百分比都是下降的。但是,在雷帕霉素加二甲双胍(800μmol/L)组和雷帕霉素组间并无明显统计学差异。在某种程度上说明雷帕霉素(100nmol/L)较二甲双胍(800μmo1/L)具有更强的抑制破骨细胞形成和骨吸收功能的效应。鉴于此,我们分析认为二甲双胍与雷帕霉素对于抑制破骨细胞的形成和骨吸收功能并不具有明显的协同效应。
7、二甲双胍和雷帕霉素对骨质疏松模型大鼠骨丢失具有保护作用
卵巢切除手术组大鼠血清磷(P)和碱性磷酸酶(ALP)水平较假手术组分别高出42.1%和49.8%。二甲双胍组和雷帕霉素组的血清酸性磷酸酶(ACP)水平较手术组分别下降了39%和32%。各组血清钙(Ca)水平变化不具有统计学差异。雷帕霉素能增加碱性磷酸酶活性。二甲双胍及雷帕霉素均能明显下降酸性磷酸酶活性。
卵巢切除手术组大鼠在第1个月及第2个月时体重比假手术组大鼠分别要高出43%和33%。这表明雌激素对于维持体重的增长具有重要作用。然而,二甲双胍及雷帕霉素组大鼠在药物干预治疗的第1及第2个月时的体重较同时期卵巢切除组大鼠并无明显下降。
卵巢切除手术组大鼠骨密度值(0.163±0.009g/cm2)较假手术组(0.216±0.022g/cm2)明显下降,说明骨质疏松模型成功构建。二甲双胍组与雷帕霉素组大鼠的骨密度值均有不同程度的提高(分别为0.197±0.015和0.199±0.029g/cm2),较卵巢切除手术组比较具有统计学意义(p<0.05)。同时,二甲双胍及雷帕霉素处理组大鼠骨容积数值较卵巢切除手术组明显增加(p<0.05)。
卵巢切除手术组大鼠骨组织切片显示骨质破坏明显,骨小梁稀疏、变薄。二甲双胍组及雷帕霉素组则可见骨容量明显增加,无明显骨质破坏、溶解现象。而且,二甲双胍组及雷帕霉素组骨容量百分比(BV/TV)、骨小梁厚度(Tb.Th)及骨小梁数量(Tb.N)等参数指标值较卵巢切除手术组明显提高(p<0.05),而骨小梁疏密度(Tb.Sp)指标值是明显下降的(p<0.05)。结合骨密度及骨容积数据,提示二甲双胍及雷帕霉素对骨质疏松模型鼠骨丢失具有直接保护作用。结论：二甲双胍能够抑制破骨细胞分化和骨吸收活性,这一作用可能是通过抑制mTORC1信号通路而实现。二甲双胍(100 mg/kg/d)和雷帕霉素(1 mg/kg/d)对去卵巢骨质疏松模型大鼠的骨丢失具有保护作用。
小结：
在本实验研究中,从以下几个方面证实了抗糖尿病药物二甲双胍抑制RANKL诱导的Raw264.7细胞和RANKL+M-CSF诱导的BMMs细胞向破骨细胞分化的作用：(1)二甲双胍抑制RANKL诱导的Raw264.7细胞及RANKL+M-CSF诱导的BMMs细胞TRAP活性,并抑制其形成TRAP阳性破骨细胞数量；(2)二甲双胍抑制RANKL诱导的Raw264.7细胞及RANKL+M-CSF诱导的BMMs细胞形成破骨细胞的骨吸收活性；(3)二甲双胍抑制破骨特异性基因TRAP、Cathepsin K、CTR及MMP-9的表达；(4)二甲双胍抑制RANKL诱导的TNF-a蛋白的表达水平；(5)二甲双胍抑制破骨细胞特异转录因子c-Fos蛋白的表达。因此,本研究结果表明：二甲双胍不仅仅抑制破骨细胞的分化,同时也能抑制破骨细胞骨吸收活性。同时,在破骨细胞体外分化模型中,破骨细胞分化因子RANKL激活mTORC1信号通路,这表明mTORC1信号通路是破骨细胞分化的必要通路；二甲双胍与mTORC1信号通路特异性抑制剂雷帕霉素一样,均能成功抑制RANKL诱导的mTORC1信号激活。这表明mTORCl信号参与二甲双胍抑制破骨细胞分化的过程。我们进一步研究表明二甲双胍及雷帕霉素对骨质疏松模型大鼠的骨丢失具有保护作用：(1)二甲双胍(100mg/kg/d)和雷帕霉素(1mg/kg/d)治疗组较手术对照组而言,骨容量(Bone mineral content,BMC)、骨密度(Bone mineral density,BMD)、百分骨容积(Percent bone volume)、骨小梁厚度(Trabecular thickness)以及骨小梁数量(Trabecular number)等指标是明显增加的,而其骨小梁疏密度(Trabecular separation)是明显下降的；(2)去卵巢骨质疏松模型鼠血清骨代谢生化指标表明：二甲双胍(100mg/kg/d)组及雷帕霉素(1mg/kg/d)组血清酸性磷酸酶(ACP)水平较对照组(手术组)明显下降。
综上所述,二甲双胍能够抑制破骨细胞分化和骨吸收活性,这一作用可能是通过抑制mTORC1信号通路而实现。二甲双胍(100 mg/kg/d).和雷帕霉素(1mg/kg/d)对去卵巢骨质疏松模型大鼠的骨丢失具有保护作用。
Background:Diabetes mellitus is a serious health problem affecting millions of individuals worldwide. By the year 2050, the World Health Organization (WHO) predicts that 300 million people will have diabetes mellitus (World Health Organization/International Diabetes Federation,1999). Recently, a relationship between diabetes and osteoporotic fractures is becoming increasingly recognized. Several studies have demonstrated the deleterious effects of diabetes on bone. Diabetic patients show an increased risk of fracture compared to the non diabetic population. Patients with type 1 diabetes have low bone mass caused by reduced bone formation and impaired fracture healing, with reduced number and function of osteoblasts. There is evidence of increased risk of fracture and impaired bone healing in type 2 diabetes, which is associated with an increased bone mineral density.
Osteoclasts are terminally differentiated multinucleated cells that resorb bone. High osteoclast activity leads to bone loss associated with postmenopausal osteoporosis and Paget's disease, among others. The receptor activator of NF-kB (RANK), its ligand (RANKL), and the decoy receptor osteoprotegerin are known to be key regulators of osteoclastic bone resorption in vitro and in vivo. Interaction of RANKL with RANK on mature osteoclasts results in their activation and extended survival. The differentiation of osteoclasts is dependent on a tumor necrosis factor (TNF) family cytokine, receptor activator of nuclear factor (NF)-кB ligand(RANKL), as well as macrophage colony-stimulating factor(M-CSF). It has been reported that tumor necrosis factor-a (TNF-a), a potent bone-resorbing factor, stimulates osteoclastic bone resorbtion in vitro and in vivo and participates in inflammatory disease involving loss of bone. The fine-tuning between bone resorption by osteoclast and bone formation by osteoblast is critical for the preservation of bone mass, so regulation of osteoclast differentiation and bone-resorbing activity was proposed to be one of the mechanisms for controlling bone resorption.
Metformin has been in worldwide use for over 40 years as an anti-diabetic agent which improves glycemic control by enhancing insulin sensitivity in liver and muscle. It enhances signaling through the insulin receptors (IR), leading to reduction in insulin resistance in type 2 diabetes. The beneficial effects of metformin have been linked to activation of the AMP-activated protein kinase (AMPK) in muscle, adipose tissue, and liver. Metformin activates AMPK by increasing cellular AMP/ATP ratio through the inhibition of mitochondrial respiratory complex. Activated AMPK subsequently suppresses mammalian target of rapamycin complex 1 (mTORC1)/ ribosomal protein S6 kinase 1 (S6K1) signaling directly or indirectly. Because phosphorylation and inhibition of insulin receptor substrate (IRS) by S6K1 represents important mechanisms of insulin resistance, AMPK-mediated inactivation of mTORC1/S6K1 by metformin increases insulin sensitivity.
mTOR is a Ser/Thr protein kinase which integrates numerous extracellular and intracellular cues, and has a central role in the homeostatic control of cell growth, proliferation, differentiation and survival. There are two distinct macromolecular complexes:a rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC2. mTORC1 phosphorylates S6K1 and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) while mTORC2 controls the actin cytoskeleton and regulates activity of Akt/PKB. The mTORC1 inhibitor, rapamycin, has been implicated in the pathogenesis of post-transplant osteoporosis and affects bone growth and early fracture healing in mice. But some studies have suggested a bone-sparing effect for rapamycin. It has also been shown that rapamycin induces initiation of apoptosis in osteoclasts, ultimately reduces osteoclastic activity and bone resorption. Moreover, rapamycin affects osteoblast function by targeting osteoblast proliferation and the early stage of osteoblast differentiation. These findings indicate that mTORCl plays an important role in bone metabolism.
We have previously demonstrated that M-CSF, TNF-a and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase and Inhibition of this translational control pathway by rapamycin results in initiation of apoptosis in osteoclasts in vito and ultimately reduces bone resorption. They reported that rapamycin (30nM) could reduce the generation of mature TRAP-positive osteoclast in cocultures of murine bone marrow cells and MB 1.8 osteoblast-like cells by 75% and it could induce morphological features of apoptosis, including a loss of actin ring structure and membrane integrity as well as the formation of pyknotic nuclei in purified osteoclasts. We also learn that M-CSF rapidly induced activation of Akt by phosphorylating residues Thr308 and Ser473, while TNF-αand sRANKL had more potent induction of IкBa (Ser32/36) phosphorylation. The mTOR inhibitor, rapamycin, inhibited all stimulatory effects of M-CSF, TNF-a and sRANKL on S6K (at Thr389), S6 (Ser235/236) and 4EBP-1(Ser65) phosphorylation. These findings indicate that mTOR have an important function in osteoclast survival and bone-resorbing activity. Metformin is a widely used anti-diabetic drug inⅡdiabetes patients that activates AMPK by increasing cellular AMP/ATP ratio through the inhibition of mitochondrial respiratory complex. Further analysis has shown that metformin inhibits breast cancer cell growth in an AMPK-dependent manner, with decreased mTOR and p70S6K activity. However, it is still unclear whether or not metformin, an AMPK activator, could affect bone metabolism via the AMPK-mTOR signaling pathway in osteoclasts.
Population-based case-control study indicated that diabetes irrespective of type was associated with an increased fracture risk, while use of metformin was associated with a decreased risk of fracture. Metformin, which could not only decrease serum concentrations of leptin and insulin, but also increase E2 in ovariectomied (OVX) rats, has a direct inhibition effect on bone loss. Although several studies have indicated that metformin directly stimulated the differentiation and mineralization of osteoblastic cells, its roles in osteoclastogenesis and the underlying mechanisms are not known.
Objective:To investigate the effects of metformin on the differentiation and bone-resorbing activity of osteoclast as well as intracellular signal transduction.
Methods:The murine macrophage cell line, Raw264.7, and bone marrow-derived macrophages (BMMs) were used. RANKL and RANKL+M-CSF were used to stimulate osteoclast differentiation from Raw264.7 cells and BMMs respectively. Osteoclast differentiation was assessed by Tartrate-Resistant Acid Phosphatase (TRAP) staining and counting the TRAP-positive cells after exposure to different concentrations of metformin (0,400,800 and 1000μM) or Rapamycin (100nM) in the presence of RANKL (50 ng/ml) or RANKL (50 ng/ml)+M-CSF (25 ng/ml) for 5 days. Bone-resorbing activity was evaluated by BD BioCoatTM OsteologicTM Bone Cell Culture System and bone slices. The expression of osteoclast-specific genes like TRAP, capthesin K, CTR and MMP-9 was evaluated by RT-PCR. The expression of TNF-αand c-Fos protein was evaluated by ELISA kit and Western blot analysis respectively. Thirty-two adult Sprague-Dawley female rats weighting 180-200 g (8-10 weeks old) were anesthetized and underwent either a sham surgery or bilateral ovariectomization. Then they were randomly divided into four groups consisting of 8 rats each to receive a supplement diet via orogastric intubation:(1) Sham group:the supplement diet was distilled water; (2) OVX group:the supplement diet was distilled water; (3) OVX+metformin (100 mg/kg/day) group:the supplement diet was 100 mg/kg/day metformin (Sigma); and (4) OVX+rapamycin group:the supplement diet was rapamycin. After 2 months of therapy, all rats were scanned for bone mineral density measurement under Nembutal anesthesia before killed, and their blood and left tibiae were collected. Blood was obtained for determinations of serum calcium, phosphate, alkaline phosphatase (ALP) and acid phosphatase (ACP). Tibiae were used for bone histomorphometry. Body weight was measured at the start and at the end of 2 months study period under sedation. Results are expressed as mean±SEM. Data was statistically analyzed using one-way ANOVA and p<0.05 for F ratio was considered statistically significant. Newman-Keuls multiple comparison test was used to test the differences between groups for significance.
Results:In this study, we found that metformin dose-dependently inhibited receptor activator of NF-кB ligand (RANKL)-stimulated osteoclasts differentiation and bone resorption in Raw264.7 cell and BMMs culture, as manifested by decrease of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, down-regulation of TRAP, cathepsin K, matrix metalloproteinase (MMP-9), calcitonin receptor (CTR) and cathepsin K mRNA, reduction of tumor necrosis factor-a (TNF-a) and c-Fos protein expression, and decrease of pit erosion area. Further study revealed that RANKL activated mTOR complex 1 (mTORCl) signaling, while metformin impaired RANKL-stimulated mTORCl signaling. Rapamycin, an mTORC1-specific inhibitor and immunosuppressive macrolides could also prevent RANKL-induced osteoclast differentiation and bone resorption in vitro. Furthermore, both metformin (100 mg/kg/d) and rapamycin (1 mg/kg/d) treatment protected against ovariectomy-induced bone loss, as manifested by increase of trabecular bone connectivity and bone mineral density (BMD) in ovariectornied (OVX) rats.
Conclusion:These findings provide novel evidence that anti-diabetic drug metformin and immunosuppressive agent rapamycin inhibit osteoclastogenesis and protect against ovariectomy-induced bone loss, and that mTORC1 signaling may mediate this action of metformin.
骨质疏松发生的根本机制在于机体骨重建的失衡,即破骨细胞(osteoclast,OC)去除旧骨(骨吸收)和成骨细胞(osteoblast, OB)形成新骨(骨形成)的失衡。而通过调节破骨细胞的活性来抑制骨吸收是治疗骨质疏松的主要的手段。OC主要来源于单核/巨噬细胞系,目前认为其分化与功能受骨髓干细胞或OB前体细胞分泌的两种关键因子所调控：巨噬细胞集落刺激因子(macrophage colony-stimulating factor, M-CSF)和核因子κB受体活化素的配体(receptor activator of nuclear factor-KB ligand, RANKL),两者与单核/巨噬细胞上受体作用使其定向发育为OC。RANKL与OC前体上的受体RANK(receptor activator of nuclear factor-KB, RANK)结合后,经TNF受体连接因子(TNF receptor associated factor, TRAF)家族蛋白激活多种信号途径,如激活转录因子NF-κB.细胞外信号调节激酶(extracellular signalregulated kinase, ERK)及c-Jun氨基末端激酶(c-Jun amino-terminal kinase, JNK),诱导转录因子AP-1家族c-fos和c-Jun等的活性,通过这些级联反应调控RANKL介导的OC分化、活化与分泌活动。同时OB前体分泌一种与RANKL竞争的可溶性分子—护骨素(osteoprotegerin, OPG),后者竞争结合RANK而抑制OC分化及其功能。OPG/RANKL/RANK轴是近年来骨代谢调节研究领域的突出成果,多种激素与细胞因子都通过上调或下调OPG与RANKL的表达参与骨代谢偶联调节。
二甲双胍是使用最广泛的降糖药物之一,能够降低肝糖输出、增加骨骼肌的葡萄糖摄取及改善脂代谢2006年,Cortizo等人通过体外实验首次提出二甲双胍拥有促成骨效应。Donghu Zhen等人报导二甲双胍能够逆转高糖对原代成骨细胞的影响,显著降低细胞内氧化应激(ROS)和细胞凋亡,同时直接促进细胞的成骨效应,而且二甲双胍的这种调节作用部分是通过增加细胞的RUNX2和IGF-1的表达。目前还没有二甲双胍调节破骨细胞增殖和分化的报道。二甲双胍的降糖效应主要通过激活腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)而实现。AMPK是细胞内调节能量代谢的蛋白激酶。当细胞内AMP/ATP比值升高到一定范围时被激活,一方面通过抑制糖原、脂肪和胆固醇的合成,减少ATP的利用；另一方面,通过促进脂肪酸氧化、葡萄糖转运等,增加ATP的产生。通过这两种作用维持细胞能量供求平衡。最近有研究发现二甲双胍还可通过激活AMPK,进而增加一氧化氮合酶(eNOS)和骨形成蛋白-2(BMP-2)的表达从而促进成骨细胞分化,增强成骨细胞活性。但二甲双胍能否通过AMPK参与对破骨细胞增殖、分化及骨吸收活性的影响?
哺乳动物雷帕霉素靶蛋白(mammalian Target Of Rapamycin, mTOR)是AMPK下游重要的信号分子,对于蛋白质翻译、细胞生长增殖、存活等具有重要的调控。有研究报道mTOR对于破骨细胞的活性有重要调控作用,细胞因子M-CSF, TNF-a和RANKL均通过mTOR信号促进破骨细胞的存活,mTOR的特异性抑制剂rapamycin能够阻断这种促存活作用,并诱导其凋亡,抑制其骨吸收的功能。那么,二甲双胍对破骨细胞的影响是否通过抑制mTOR信号而实现呢?
综上所述,我们对二甲双胍影响骨代谢的模式及其对破骨细胞的影响和相关的分子机制作出如下假设：二甲双胍通过抑制腺苷酸活化蛋白激酶(AMPK)下游的mTOR信号通路,抑制破骨细胞的增殖和分化,促进破骨细胞的凋亡,降低破骨细胞的骨吸收能力。
目的：因此本课题利用破骨细胞前体细胞株、破骨细胞体外分化细胞模型及去卵巢骨质疏松动物模型,从分子、细胞及整体层面探讨二甲双胍对破骨细胞增殖、分化及功能的影响,并探讨相关的分子机制,期望为糖尿病性骨质疏松的防治提供细胞和分子生物学依据。
方法：采用RANKL诱导鼠巨噬细胞系Raw264.7细胞及RANKL+M-CSF诱导骨髓源性巨噬细胞(Bone marrow-derived macrophages,BMMs)破骨分化模型,给予不同浓度的二甲双胍(400,800,1000μmol/L)和雷帕霉素(100μmol/L)处理后,抗酒石酸酸性磷酸酶(TRAP)染色并计数形成的TRAP阳性细胞数量,分别使用骨吸收培养板(BD BioCoatTM OsteologicTM Bone Cell Culture System)及骨片检测形成破骨细胞的骨吸收活性, RT-PCR技术检测破骨特异性基因抗酒石酸酸性磷酸酶(TRAP)、组织蛋白酶K(Cathepsin K)、降钙素(CTR)受体和金属基质蛋白酶-9(MMP-9)的表达,ELISA检测肿瘤坏死因子α(TNF-α)表达水平,Western-blot检测c-Fos蛋白以及哺乳动物雷帕霉素靶蛋白(mammalian Target OfRapamycin, mTOR)信号通路下游底物S6K1(Thr389)、S6(Ser235/236)、4EBP1(Thr37/46)的表达及磷酸化水平。构建去卵巢骨质疏松大鼠模型,同时给予二甲双胍(100mg/kg/d)、雷帕霉素(1mg/kg/d)等处理,测定模型动物体重变化、血清骨代谢生化指标(血清钙、磷、酸性磷酸酶及碱性磷酸酶)、骨组织形态学指标参数(百分骨容积、骨小梁厚度、骨小梁数量与骨小梁疏密度)及骨密度(BMD)。
结果：
1、二甲双胍抑制RANKL诱导Raw264.7和BMMs细胞向破骨细胞分化
对加入不同浓度二甲双胍的RANKL诱导的Raw264.7细胞进行TRAP染色和细胞骨架微丝荧光染色。结果显示：RANKL能有效的刺激TRAP阳性多核细胞和细胞骨架的形成。二甲双胍抑制细胞骨架的形成并且下降RANKL诱导的TRAP阳性：多核细胞的数量,二甲双胍(400、800和1000μmol/L组)与RANKL组比较,分别使形成的TRAP阳性多核细胞数量下降了17.7%、30.3%和59.3%,其抑制作用呈浓度梯度依赖性。同样的,在BMMs细胞中,我们得到了类似的结果：二甲双胍呈浓度梯度依赖性(400-1000μmol/L)的抑制!RANKL+M-CSF诱导的BMMs细胞形成TRAP阳性多核细胞的数量。细胞毒性实验结果表明:400-1000μmol/L浓度的二甲双胍对两种细胞(Raw264.7和BMMs)的生长抑制及凋亡作用并无明显统计学差异。由此表明,二甲双胍能够抑制RANKL诱导的破骨细胞分化。
2、mTORC1信号参与二甲双胍抑制破骨细胞分化的过程
RANKL能使Raw264.7细胞mTORC1信号通路下游受体S6K1(Thr389),S6(Ser235/236)和4E-BP1(Ser65)蛋白的磷酸化水平表达增加(即激活mTORC1信号),而雷帕霉素和二甲双胍均可在不影响mTORC1下游受体S6K1(Thr389),S6(Ser235/236)和4E-BP1(Ser65)总蛋白水平的基础上,抑制RANKL诱导的上述蛋白磷酸化,且随二甲双胍浓度的升高这种抑制作用越明显,具有浓度依赖性。由此说明,二甲双胍能够抑制RANKL激活的mTORC1信号。进一步研究表明,雷帕霉素也能明显的抑制RANKL诱导的TRAP阳性多核细胞的形成,加入100 nmol/L的雷帕霉素组较RANKL组比较而言,TRAP阳性多核细胞的数量下降了39.8%。这些结果表明,mTORCl信号是刺激破骨细胞分化的必要通路。当仅仅只给予800μmol/L的二甲双胍处理时(不加RANKL),也能观察到S6K1(Thr389),S6(Ser235/236)和4E-BP1(Ser65)的磷酸化受到明显的抑制。这表明mTORCl信号参与二甲双胍抑制破骨细胞分化的过程。
3、二甲双胍抑制RANKL诱导的破骨细胞特异基因的表达
RANKL组抗酒石酸酸性磷酸酶、组织蛋白酶K、降钙素受体和金属基质蛋白酶-9四种基因的表达比空白对照组明显增加。同RANKL组相比,二甲双胍+RANKL组上述基因表达量分别下降了69.6%、44.1%、69.0%和31.7%；雷帕霉素+RANKL组上述基因的表达量则分别下降了54.0%、71.3%、80.9%和78.0%。
4、二甲双胍抑制RANKL诱导的TNF-a和c-Fos蛋白表达
RANKL提高Raw264.7细胞TNF-a产物的表达,二甲双胍和雷帕霉素均能抑制RANKL诱导的TNF-a产物的表达,二甲双胍(400、800和1000μmol/L)组以及雷帕霉素(100nmol/L)组的OD值分别为1.88±0.05、1.62±0.06、1.56±0.08和1.37±0.12,比RANKL组(2.43±0.05)明显降低(F=12.257,p<0.05)。同时,二甲双胍和雷帕霉素均能抑制RANKL诱导的c-Fos蛋白的表达水平。
5、二甲双胍抑制破骨细胞骨吸收功能
在Raw264.7细胞中,与RANKL对照组相比,二甲双胍组(400、800和1000μmol/L组)明显的抑制了破骨细胞对骨的吸收功能,吸收陷窝的数量和面积百分比相对RANKL组分别下降了22.7%、55.9%和81.6%,且呈浓度梯度依赖性(p<0.05)。雷帕霉素处理组同样的显示了骨吸收陷窝在数量和面积百分比相对RANKL组下降了71.9%(p<0.05)。这些表明,二甲双胍能够抑制破骨细胞的骨吸收活性。这个结果进一步的在RANKL+M-CSF诱导的BMMs细胞上得到了验证。二甲双胍呈浓度依赖性的抑制破骨细胞骨吸收陷窝的数量和面积百分比。
6、二甲双胍与雷帕霉素在抑制破骨细胞分化和骨吸收功能方面不具有协同效用
在Raw264.7和BMMs两种细胞当中,雷帕霉素加二甲双胍(800μmol/L)组较二甲双胍(800μmol/L)组相比而言,所形成的TRAP阳性多核细胞的数量及骨吸收面积百分比都是下降的。但是,在雷帕霉素加二甲双胍(800μmol/L)组和雷帕霉素组间并无明显统计学差异。在某种程度上说明雷帕霉素(100nmol/L)较二甲双胍(800μmo1/L)具有更强的抑制破骨细胞形成和骨吸收功能的效应。鉴于此,我们分析认为二甲双胍与雷帕霉素对于抑制破骨细胞的形成和骨吸收功能并不具有明显的协同效应。
7、二甲双胍和雷帕霉素对骨质疏松模型大鼠骨丢失具有保护作用
卵巢切除手术组大鼠血清磷(P)和碱性磷酸酶(ALP)水平较假手术组分别高出42.1%和49.8%。二甲双胍组和雷帕霉素组的血清酸性磷酸酶(ACP)水平较手术组分别下降了39%和32%。各组血清钙(Ca)水平变化不具有统计学差异。雷帕霉素能增加碱性磷酸酶活性。二甲双胍及雷帕霉素均能明显下降酸性磷酸酶活性。
卵巢切除手术组大鼠在第1个月及第2个月时体重比假手术组大鼠分别要高出43%和33%。这表明雌激素对于维持体重的增长具有重要作用。然而,二甲双胍及雷帕霉素组大鼠在药物干预治疗的第1及第2个月时的体重较同时期卵巢切除组大鼠并无明显下降。
卵巢切除手术组大鼠骨密度值(0.163±0.009g/cm2)较假手术组(0.216±0.022g/cm2)明显下降,说明骨质疏松模型成功构建。二甲双胍组与雷帕霉素组大鼠的骨密度值均有不同程度的提高(分别为0.197±0.015和0.199±0.029g/cm2),较卵巢切除手术组比较具有统计学意义(p<0.05)。同时,二甲双胍及雷帕霉素处理组大鼠骨容积数值较卵巢切除手术组明显增加(p<0.05)。
卵巢切除手术组大鼠骨组织切片显示骨质破坏明显,骨小梁稀疏、变薄。二甲双胍组及雷帕霉素组则可见骨容量明显增加,无明显骨质破坏、溶解现象。而且,二甲双胍组及雷帕霉素组骨容量百分比(BV/TV)、骨小梁厚度(Tb.Th)及骨小梁数量(Tb.N)等参数指标值较卵巢切除手术组明显提高(p<0.05),而骨小梁疏密度(Tb.Sp)指标值是明显下降的(p<0.05)。结合骨密度及骨容积数据,提示二甲双胍及雷帕霉素对骨质疏松模型鼠骨丢失具有直接保护作用。结论：二甲双胍能够抑制破骨细胞分化和骨吸收活性,这一作用可能是通过抑制mTORC1信号通路而实现。二甲双胍(100 mg/kg/d)和雷帕霉素(1 mg/kg/d)对去卵巢骨质疏松模型大鼠的骨丢失具有保护作用。
小结：
在本实验研究中,从以下几个方面证实了抗糖尿病药物二甲双胍抑制RANKL诱导的Raw264.7细胞和RANKL+M-CSF诱导的BMMs细胞向破骨细胞分化的作用：(1)二甲双胍抑制RANKL诱导的Raw264.7细胞及RANKL+M-CSF诱导的BMMs细胞TRAP活性,并抑制其形成TRAP阳性破骨细胞数量；(2)二甲双胍抑制RANKL诱导的Raw264.7细胞及RANKL+M-CSF诱导的BMMs细胞形成破骨细胞的骨吸收活性；(3)二甲双胍抑制破骨特异性基因TRAP、Cathepsin K、CTR及MMP-9的表达；(4)二甲双胍抑制RANKL诱导的TNF-a蛋白的表达水平；(5)二甲双胍抑制破骨细胞特异转录因子c-Fos蛋白的表达。因此,本研究结果表明：二甲双胍不仅仅抑制破骨细胞的分化,同时也能抑制破骨细胞骨吸收活性。同时,在破骨细胞体外分化模型中,破骨细胞分化因子RANKL激活mTORC1信号通路,这表明mTORC1信号通路是破骨细胞分化的必要通路；二甲双胍与mTORC1信号通路特异性抑制剂雷帕霉素一样,均能成功抑制RANKL诱导的mTORC1信号激活。这表明mTORCl信号参与二甲双胍抑制破骨细胞分化的过程。我们进一步研究表明二甲双胍及雷帕霉素对骨质疏松模型大鼠的骨丢失具有保护作用：(1)二甲双胍(100mg/kg/d)和雷帕霉素(1mg/kg/d)治疗组较手术对照组而言,骨容量(Bone mineral content,BMC)、骨密度(Bone mineral density,BMD)、百分骨容积(Percent bone volume)、骨小梁厚度(Trabecular thickness)以及骨小梁数量(Trabecular number)等指标是明显增加的,而其骨小梁疏密度(Trabecular separation)是明显下降的；(2)去卵巢骨质疏松模型鼠血清骨代谢生化指标表明：二甲双胍(100mg/kg/d)组及雷帕霉素(1mg/kg/d)组血清酸性磷酸酶(ACP)水平较对照组(手术组)明显下降。
综上所述,二甲双胍能够抑制破骨细胞分化和骨吸收活性,这一作用可能是通过抑制mTORC1信号通路而实现。二甲双胍(100 mg/kg/d).和雷帕霉素(1mg/kg/d)对去卵巢骨质疏松模型大鼠的骨丢失具有保护作用。
Background:Diabetes mellitus is a serious health problem affecting millions of individuals worldwide. By the year 2050, the World Health Organization (WHO) predicts that 300 million people will have diabetes mellitus (World Health Organization/International Diabetes Federation,1999). Recently, a relationship between diabetes and osteoporotic fractures is becoming increasingly recognized. Several studies have demonstrated the deleterious effects of diabetes on bone. Diabetic patients show an increased risk of fracture compared to the non diabetic population. Patients with type 1 diabetes have low bone mass caused by reduced bone formation and impaired fracture healing, with reduced number and function of osteoblasts. There is evidence of increased risk of fracture and impaired bone healing in type 2 diabetes, which is associated with an increased bone mineral density.
Osteoclasts are terminally differentiated multinucleated cells that resorb bone. High osteoclast activity leads to bone loss associated with postmenopausal osteoporosis and Paget's disease, among others. The receptor activator of NF-kB (RANK), its ligand (RANKL), and the decoy receptor osteoprotegerin are known to be key regulators of osteoclastic bone resorption in vitro and in vivo. Interaction of RANKL with RANK on mature osteoclasts results in their activation and extended survival. The differentiation of osteoclasts is dependent on a tumor necrosis factor (TNF) family cytokine, receptor activator of nuclear factor (NF)-кB ligand(RANKL), as well as macrophage colony-stimulating factor(M-CSF). It has been reported that tumor necrosis factor-a (TNF-a), a potent bone-resorbing factor, stimulates osteoclastic bone resorbtion in vitro and in vivo and participates in inflammatory disease involving loss of bone. The fine-tuning between bone resorption by osteoclast and bone formation by osteoblast is critical for the preservation of bone mass, so regulation of osteoclast differentiation and bone-resorbing activity was proposed to be one of the mechanisms for controlling bone resorption.
Metformin has been in worldwide use for over 40 years as an anti-diabetic agent which improves glycemic control by enhancing insulin sensitivity in liver and muscle. It enhances signaling through the insulin receptors (IR), leading to reduction in insulin resistance in type 2 diabetes. The beneficial effects of metformin have been linked to activation of the AMP-activated protein kinase (AMPK) in muscle, adipose tissue, and liver. Metformin activates AMPK by increasing cellular AMP/ATP ratio through the inhibition of mitochondrial respiratory complex. Activated AMPK subsequently suppresses mammalian target of rapamycin complex 1 (mTORC1)/ ribosomal protein S6 kinase 1 (S6K1) signaling directly or indirectly. Because phosphorylation and inhibition of insulin receptor substrate (IRS) by S6K1 represents important mechanisms of insulin resistance, AMPK-mediated inactivation of mTORC1/S6K1 by metformin increases insulin sensitivity.
mTOR is a Ser/Thr protein kinase which integrates numerous extracellular and intracellular cues, and has a central role in the homeostatic control of cell growth, proliferation, differentiation and survival. There are two distinct macromolecular complexes:a rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC2. mTORC1 phosphorylates S6K1 and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) while mTORC2 controls the actin cytoskeleton and regulates activity of Akt/PKB. The mTORC1 inhibitor, rapamycin, has been implicated in the pathogenesis of post-transplant osteoporosis and affects bone growth and early fracture healing in mice. But some studies have suggested a bone-sparing effect for rapamycin. It has also been shown that rapamycin induces initiation of apoptosis in osteoclasts, ultimately reduces osteoclastic activity and bone resorption. Moreover, rapamycin affects osteoblast function by targeting osteoblast proliferation and the early stage of osteoblast differentiation. These findings indicate that mTORCl plays an important role in bone metabolism.
We have previously demonstrated that M-CSF, TNF-a and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase and Inhibition of this translational control pathway by rapamycin results in initiation of apoptosis in osteoclasts in vito and ultimately reduces bone resorption. They reported that rapamycin (30nM) could reduce the generation of mature TRAP-positive osteoclast in cocultures of murine bone marrow cells and MB 1.8 osteoblast-like cells by 75% and it could induce morphological features of apoptosis, including a loss of actin ring structure and membrane integrity as well as the formation of pyknotic nuclei in purified osteoclasts. We also learn that M-CSF rapidly induced activation of Akt by phosphorylating residues Thr308 and Ser473, while TNF-αand sRANKL had more potent induction of IкBa (Ser32/36) phosphorylation. The mTOR inhibitor, rapamycin, inhibited all stimulatory effects of M-CSF, TNF-a and sRANKL on S6K (at Thr389), S6 (Ser235/236) and 4EBP-1(Ser65) phosphorylation. These findings indicate that mTOR have an important function in osteoclast survival and bone-resorbing activity. Metformin is a widely used anti-diabetic drug inⅡdiabetes patients that activates AMPK by increasing cellular AMP/ATP ratio through the inhibition of mitochondrial respiratory complex. Further analysis has shown that metformin inhibits breast cancer cell growth in an AMPK-dependent manner, with decreased mTOR and p70S6K activity. However, it is still unclear whether or not metformin, an AMPK activator, could affect bone metabolism via the AMPK-mTOR signaling pathway in osteoclasts.
Population-based case-control study indicated that diabetes irrespective of type was associated with an increased fracture risk, while use of metformin was associated with a decreased risk of fracture. Metformin, which could not only decrease serum concentrations of leptin and insulin, but also increase E2 in ovariectomied (OVX) rats, has a direct inhibition effect on bone loss. Although several studies have indicated that metformin directly stimulated the differentiation and mineralization of osteoblastic cells, its roles in osteoclastogenesis and the underlying mechanisms are not known.
Objective:To investigate the effects of metformin on the differentiation and bone-resorbing activity of osteoclast as well as intracellular signal transduction.
Methods:The murine macrophage cell line, Raw264.7, and bone marrow-derived macrophages (BMMs) were used. RANKL and RANKL+M-CSF were used to stimulate osteoclast differentiation from Raw264.7 cells and BMMs respectively. Osteoclast differentiation was assessed by Tartrate-Resistant Acid Phosphatase (TRAP) staining and counting the TRAP-positive cells after exposure to different concentrations of metformin (0,400,800 and 1000μM) or Rapamycin (100nM) in the presence of RANKL (50 ng/ml) or RANKL (50 ng/ml)+M-CSF (25 ng/ml) for 5 days. Bone-resorbing activity was evaluated by BD BioCoatTM OsteologicTM Bone Cell Culture System and bone slices. The expression of osteoclast-specific genes like TRAP, capthesin K, CTR and MMP-9 was evaluated by RT-PCR. The expression of TNF-αand c-Fos protein was evaluated by ELISA kit and Western blot analysis respectively. Thirty-two adult Sprague-Dawley female rats weighting 180-200 g (8-10 weeks old) were anesthetized and underwent either a sham surgery or bilateral ovariectomization. Then they were randomly divided into four groups consisting of 8 rats each to receive a supplement diet via orogastric intubation:(1) Sham group:the supplement diet was distilled water; (2) OVX group:the supplement diet was distilled water; (3) OVX+metformin (100 mg/kg/day) group:the supplement diet was 100 mg/kg/day metformin (Sigma); and (4) OVX+rapamycin group:the supplement diet was rapamycin. After 2 months of therapy, all rats were scanned for bone mineral density measurement under Nembutal anesthesia before killed, and their blood and left tibiae were collected. Blood was obtained for determinations of serum calcium, phosphate, alkaline phosphatase (ALP) and acid phosphatase (ACP). Tibiae were used for bone histomorphometry. Body weight was measured at the start and at the end of 2 months study period under sedation. Results are expressed as mean±SEM. Data was statistically analyzed using one-way ANOVA and p<0.05 for F ratio was considered statistically significant. Newman-Keuls multiple comparison test was used to test the differences between groups for significance.
Results:In this study, we found that metformin dose-dependently inhibited receptor activator of NF-кB ligand (RANKL)-stimulated osteoclasts differentiation and bone resorption in Raw264.7 cell and BMMs culture, as manifested by decrease of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, down-regulation of TRAP, cathepsin K, matrix metalloproteinase (MMP-9), calcitonin receptor (CTR) and cathepsin K mRNA, reduction of tumor necrosis factor-a (TNF-a) and c-Fos protein expression, and decrease of pit erosion area. Further study revealed that RANKL activated mTOR complex 1 (mTORCl) signaling, while metformin impaired RANKL-stimulated mTORCl signaling. Rapamycin, an mTORC1-specific inhibitor and immunosuppressive macrolides could also prevent RANKL-induced osteoclast differentiation and bone resorption in vitro. Furthermore, both metformin (100 mg/kg/d) and rapamycin (1 mg/kg/d) treatment protected against ovariectomy-induced bone loss, as manifested by increase of trabecular bone connectivity and bone mineral density (BMD) in ovariectornied (OVX) rats.
Conclusion:These findings provide novel evidence that anti-diabetic drug metformin and immunosuppressive agent rapamycin inhibit osteoclastogenesis and protect against ovariectomy-induced bone loss, and that mTORC1 signaling may mediate this action of metformin.
引文
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[2]Bouillon R. Diabetic bone disease. Calcif Tissue Int 1991;49:155-60.
[3]Carnevale V, Romagnoli E, D'Erasmo E. Skeletal involvement in patients with diabetes mellitus. Diabetes Metab Res Rev 2004;20:196-204.
[4]Schwartz AV. Diabetes Mellitus:Does it Affect Bone? Calcif Tissue Int 2003;73:515-9.
[5]Vestergaard P. Discrepancies in bone mineral density and fracture risk in patients with type 1 and type 2 diabetes--a meta-analysis. Osteoporos Int 2007; 18:427-44.
[6]Schwartz AV, Sellmeyer DE, Ensrud KE, et al. Older women with diabetes have an increased risk of fracture:a prospective study. J Clin Endocrinol Metab 2001;86:32-8.
[7]Strotmeyer ES, Cauley JA, Schwartz AV, et al. Nontraumatic fracture risk with diabetes mellitus and impaired fasting glucose in older white and black adults: the health, aging, and body composition study. Arch Intern Med 2005;165:1612-7.
[8]Vestergaard P, Rejnmark L, Mosekilde L. Relative fracture risk in patients with diabetes mellitus, and the impact of insulin and oral antidiabetic medication on relative fracture risk. Diabetologia 2005;48:1292-9.
[9]Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108:1167-74.
[10]El-Mir MY, Nogueira V, Fontaine E, et al. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem 2000;275:223-8.
[11]Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 2000;348 Pt 3:607-14.
[12]Bolster DR, Crozier SJ, Kimball SR, et al. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 2002;277: 23977-80.
[13]Zakikhani M, Dowling R, Fantus IG, et al. Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. Cancer Res 2006; 66: 10269-73.
[14]Dowling RJ, Zakikhani M, Fantus IG, et al. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res 2007;67:10804-12.
[15]Zakikhani M, Blouin MJ, Piura E, et al. Metformin and rapamycin have distinct effects on the AKT pathway and proliferation in breast cancer cells. Breast Cancer Res Treat 2010; 123:271-9.
[16]Raught B, Gingras AC, Sonenberg N. The target of rapamycin (TOR) proteins. Proc Natl Acad Sci U S A 2001;98:7037-44.
[17]Guertin DA, Sabatini DM. An expanding role for mTOR in cancer. Trends Mol Med 2005;11:353-61.
[18]Martin DE, Hall MN. The expanding TOR signaling network. Curr Opin Cell Biol 2005;17:158-66.
[19]Epstein S. Post-transplantation bone disease:the role of immunosuppressive agents and the skeleton. J Bone Miner Res 1996;11:1-7.
[20]Sanchez CP, He YZ. Bone growth during rapamycin therapy in young rats. BMC Pediatr 2009;9:3.
[21]Holstein JH, Klein M, Garcia P, et al. Rapamycin affects early fracture healing in mice. Br J Pharmacol 2008; 154(5):1055-62.
[22]Romero DF, Buchinsky FJ, Rucinski B, et al. Rapamycin:a bone sparing immunosuppressant? J Bone Miner Res 1995;10:760-8.
[23]Joffe I, Katz I, Sehgal S, et al. Lack of change of cancellous bone volume with short-term use of the new immunosuppressant rapamycin in rats. Calcif Tissue Int 1993;53:45-52.
[24]Kneissel M, Luong-Nguyen NH, Baptist M, et al. Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts. Bone 2004;35:1144-56.
[25]Sugatani T, Hruska KA. Aktl/Akt2 and mammalian target of rapamycin/Bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors. J Biol Chem 2005;280:3583-9.
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