二苯乙烯苷防治骨质疏松的作用及相关机制研究
|
摘要
骨质疏松症（osteoporosis，OP）是全身性、退行性、代谢性骨骼疾病，是由多种病因导致骨组织骨量丢失、结构改变和生物力学性能减退，容易发生骨折，严重威胁老年人的身心健康。氧化应激（oxidative stress）是机体内活性氧自由基ROS（Reactive oxygen species）产生过多，超出了机体的清除能力，导致氧化和抗氧化失衡的一种状态。高浓度的ROS可以导致核酸、蛋白质、脂质产生氧化反应，损害细胞的结构和功能，引起疾病的发生。许多衰老性疾病与氧化应激关系密切。目前，大量研究已经证实氧化应激在OP发生发展中起重要作用。过量的ROS的产生可以诱导成骨细胞凋亡和降低其分化能力，促进破骨细胞形成和活化，影响成骨细胞和破骨细胞之间的偶联，进而降低骨形成，促进骨吸收，使骨量减少。骨质疏松病人血浆氧化应激水平升高同骨密度降低具有相关性。因此，利用抗氧化剂来防治OP成为研究热点。流行病学研究发现许多营养物质及中药具有抗氧化作用，进而发挥骨保护作用。二苯乙烯苷（TSG）是从传统中药何首乌中提取的有效成分之一，具有抗氧化功效。那么，TSG能否应用于治疗OP呢？目前还未见相关报道。
本实验分为三部分，拟通过在体外研究TSG对氧化应激造成的成骨前体细胞MC3T3-E1凋亡和成骨分化抑制的逆转作用及在体内研究小鼠去卵巢骨质疏松模型中，TSG能否提高骨密度，改善骨组织微结构，进而改善OP，并对其相关机制进行初步探讨。本研究可以提供TSG治疗OP的新用途，为寻找治疗OP的中药提供理论依据。
1TSG对氧化应激导致的成骨前体细胞MC3T3-E1凋亡的预防作用及机制研究
目的研究TSG对氧化应激导致的成骨前体细胞MC3T3-E1凋亡的预防作用，并初步探讨相关机制。方法建立体外氧化应激模型，100、200、300和400μM/L的H_2O_2分别作用2h、6h、12h和24h，MTT法评价细胞存活情况，筛选合适H_2O_2作用浓度；观察不同浓度0.01、0.1、1、10、50μM/L TSG对成骨前体细胞MC3T3-E1增殖作用的影响，评价药物的毒性作用；筛选的合适浓度的TSG和H_2O_2共同作用，实验分组：①空白对照组；②单纯H_2O_2处理组；③H_2O_2+TSG0.1μM处理组；④H_2O_2+TSG1μM处理组；⑤H_2O_2+TSG10μM处理组；⑥阳性对照抗氧化剂NAC1mM处理组。通过MTT法、显微镜下大体观察、Hoechst33258染色及流式细胞仪来评价细胞凋亡情况及TSG对氧化损伤的保护作用；利用荧光酶标仪和MDA检测试剂盒来检测细胞中ROS及MDA的水平来评价细胞的氧化应激状态；Western-blot和RT-PCR分别从蛋白水平和基因水平评价Bcl-2和Bax的表达水平，初步阐述TSG对氧化应激损伤保护作用的相关机制。结果筛选出合适H_2O_2作用浓度为300μM/L，作用24h，用于后续研究；TSG在观察浓度内对MC3T3-E1无毒性作用（P>0.05）；与空白对照组相比，单纯H_2O_2处理可以导致细胞死亡，显微镜下大体观察可见许多细胞已死亡，脱壁；Hoechst33258染色可见有较多的细胞出现核固缩、核浓集现象；不同浓度的TSG预处理后，细胞凋亡现象得到改善；流式细胞仪检测结果提示：空白组、H_2O_2处理组、不同浓度TSG预处理组及NAC处理组的细胞凋亡率分别为5.8%、30.1%、22.1%、15.3%、14.3%、9.2%。可见，单独H_2O_2作用可以导致成骨前体细胞出现较大程度的凋亡，不同浓度TSG预处理后，细胞凋亡率有所降低（P<0.05）；单纯H_2O_2作用可以导致细胞水平的ROS和脂质氧化产物MDA产生增加，TSG（1-10μM）可以显著降低细胞水平ROS和MDA水平（P<0.05），改善细胞的氧化应激状态；Western-blotting和实时定量RT-PCR结果提示：TSG预处理可以减少H_2O_2处理后促凋亡蛋白Bax的表达，增加抗凋亡蛋白Bcl-2的表达。结论氧化应激可以导致成骨前体细胞MC3T3-E1凋亡增加，TSG可以通过降低氧化应激水平及改善线粒体凋亡Bcl-2/Bax信号通路来起到保护作用。
2TSG对氧化应激导致的成骨前体细胞MC3T3-E1成骨分化能力降低的预防作用及机制研究
目的探讨TSG对氧化应激导致的成骨前体细胞MC3T3-E1成骨分化能力降低的预防作用及相关机制研究，为体内实验奠定理论基础。方法将成骨前体细胞MC3T3-E1均匀接种到六孔板内，待细胞达80%铺满时，给予成骨诱导培养基培养。实验分组：①空白对照组；②单纯H_2O_2处理组；③H_2O_2+TSG0.1μM处理组；④H_2O_2+TSG1μM处理组；⑤H_2O_2+TSG10μM处理组；⑥NAC1mM处理组。诱导6、14或21天后，依组分别给予不同浓度TSG预处理24h，然后给予H_2O_2处理24h。通过ALP染色及ALP定量分析来评价成骨分化的标志物ALP的表达如何；通过茜素红染色来评价成骨细胞的矿化程度；通过实时定量RT-PCR来检测成骨分化相关基因ALP、COL-1和OCN的表达情况。为了进一步阐明相关的作用机制，我们利用荧光酶标仪检测成骨细胞内ROS的水平及MDA试剂盒检测脂质氧化水平来反应细胞内的氧化应激水平，并利用ELISA试剂盒检测骨吸收相关因子RANKL和IL-6的表达水平。结果同空白对照组相比，单纯H_2O_2处理可以明显降低成骨分化标记物ALP染色和定量结果；减少细胞外基质钙沉积量；下调成骨分化相关基因ALP、COL-1和OCN的表达水平（P<0.05）。当TSG预处理24h后，TSG (0.1-10μM)以剂量依赖方式明显改善ALP活性（P<0.05）；改善ALP染色和钙结节沉积；显著提高成骨分化基因ALP、COL-1和OCN的表达水平（P<0.05）。单纯H_2O_2处理，细胞内ROS及MDA水平显著提高，而TSG（1-10μM）预处理可以明显降低ROS及MDA水平（P<0.05）。ELISA检测也得出相同的结果，TSG（0.1-10μM）及（10μM）分别降低H_2O_2作用导致的RANKL和IL-6的升高（P<0.05）。结论氧化应激可以导致成骨前体细胞MC3T3-E1成骨分化能力抑制，TSG可能部分通过降低氧化应激水平及骨吸收因子的表达，在一定程度上逆转这种抑制作用。
3TSG对小鼠去势骨质疏松模型的预防作用
目的研究TSG对小鼠去势骨质疏松模型中骨密度、骨量及骨组织微结构改变的预防作用。方法32只9月龄BALB/c雌性小鼠，平均体重为19.73±2.011g，随机分为四组：①假手术组（SHAM）；②单纯卵巢去势组（OVX）；③卵巢去势并小剂量TSG处理组（OVX+TSG5mg/kg）；④卵巢去势并高剂量TSG处理组（OVX+TSG20mg/kg）。戊巴比妥钠（50mg/kg i.p.）麻醉成功后，通过背侧入路移除双侧卵巢，术后7天，依分组情况每天给予腹腔注射药物或双蒸水，持续3个月。心脏采血，离心后，取血清用于检测MDA和GSH表达水平，来评价血清中的氧化应激状态。取第4腰椎及左侧股骨，剔除周围软组织，4%多聚甲醛固定后，用于显微CT（Micro-CT）、HE染色、VG染色和Von kossa染色来评价TSG对骨密度、骨量及骨组织微结构的作用程度。结果与SHAM组相比，OVX组血清中MDA水平升高、GSH水平降低（P<0.05）。OVX+TSG20mg/kg可以明显改善血清中MDA水平（P<0.05），但对于GSH水平的改变无统计学差异（P>0.05）。不同剂量TSG处理组之间也无统计学差异（P>0.05）。Micro-CT结果提示：与SHAM组相比，OVX组的第四腰椎及股骨远端松质骨骨量减少，BV/TV、Conn.D、Tb.N、Tb.Th及BMD值减少(P<0.01)，SMI和Tb.Sp增加(P<0.05)。OVX+TSG20mg/kg可以明显改善去卵巢导致的这些骨组织微结构参数的改变（P<0.05）。OVX+TSG5mg/kg可以改善骨组织微结构参数值，但不具有统计学差异（P>0.05）。不同剂量TSG处理组之间也无统计学差异（P>0.05）。脱钙骨组织石蜡切片HE染色及不脱钙骨组织塑料包埋切片VG和Von kossa染色结果均提示：OVX组与SHAM组相比，骨小梁数量减少，间距增大，而经不同浓度TSG处理后，骨小梁数量及间距有不同程度改变。结论TSG可以降低小鼠去卵巢骨质疏松模型血清中的氧化应激水平，并可以改善去势导致的骨密度下降、骨量降低及骨组织微结构改变。
Osteoporosis is a systemic, degenerative and metabolic bone disease, which ischaracterized by deterioration of bone mass, microarchitecture, bone mineral density andbone biomechanics and leads to an increased risk of fracture. It is seriously threatening thephysical and mental health of the elderly. Oxidative stress is caused by excessivegeneration of reactive oxygen species (ROS) beyond the body’s scavenging capacity andresults into a state of intracellular reduction–oxidation imbalance in the body. Highconcentrations of ROS could oxidize the nucleic acids, proteins and lipids, and damage thestructure and function of the cells, leading to the development of a disease. Many degenerative diseases have been shown to be closely related to oxidative stress. In recentyears, a large number of studies have confirmed that oxidative stress plays an importantrole in the development of osteoporosis. Excessive generation of ROS can stimulateapoptosis and inhibit osteogenic differentiation of osteoblasts, stimulate formation andactivity of osteoclasts and affect the bone formation and resorption coupling. As a result,bone formation decreases and bone resorption increases, leading to bone mass loss.Meanwhile, the elevated oxidative stress level in plasma has a correlation with deceasedbone density in osteoporotic patients. Therefore, using antioxidant to prevent and treatosteoporosis has become research focus. Epidemiological studies have provided evidenceof a link between nutrient, antioxidant intake and bone health, and have led toinvestigations of the antioxidant properties of nutrients.2,3,5,4-tetrahydroxystilbene-2-o-β-D-glucoside(TSG) is a kind of extracts of Polygonum, which is a traditional Chinesemedicine, has antioxidant effect. It is unclear that whether TSG could be applied to thetreatment of OP until now.
This study was divided into three parts: firstly, we tested the protective effect of TSGon H_2O_2induced cytotoxicity in MC3T3-E1cells and clarified part of the relatedmechanism; then, we evaluated the protective effect of TSG on osteoblast dysfunctioninduced by H_2O_2; finally, we demonstrated the protective effect of TSG against bone lossin OVX mice. We confirmed a new use of TSG to the treatment of OP in this study andprovided a theoretical basis for the treatment of OP with the traditional Chinese medicine.
1. Protective effect of TSG on oxidative stress induced cytotoxicity in MC3T3-E1cells and the related mechanism
Objective: the purpose of this study was to investigate the protective effect of TSG onapoptosis of MC3T3-E1cells induced by oxidative stress and clarify the relatedmechanism. Methods: At first, we built oxidative stress model in vitro. In this study,MC3T3-E1cells were treated with different concentrations of H_2O_2(0,100,200,300and400μM/L) for2,6,12and24h and cell viability was evaluated by MTT assay. In this way,we chose the appropriate concentration of H_2O_2. Meanwhile, cells were treated with different concentrations of TSG (0,0.1,1and10μM/L) for24and72h in order toinvestigate the toxicity of TSG. The selected concentrations of H_2O_2and TSG were usedin the following studies. The experiment was divided into6groups, including the controlgroup, H_2O_2, H_2O_2+TSG(0.1μM), H_2O_2+TSG(1μM), H_2O_2+TSG(10μM) and H_2O_2+NAC(1mM). The protective effect of TSG on apoptosis of the cells induced by oxidative stresswas demonstrated by MTT assay, general observation under microscope, Hoechst33258staining and flow cytometry. The level of ROS and MDA were tested by fluorescencemicroplate reader and MDA assay kit to evaluate the cellular oxidative stress status. Inorder to further clarify the related mechanism, we examined the production of Bcl-2andBax protein using Western-blotting and the gene expression of Bcl-2and Bax by RT-PCR.Results: The suitable concentration and duration of action for H_2O_2was300μM/L for24hwhich was used in the following study. TSG alone was non-toxic to MC3T3-E1cells atthe concentrations used in this study (P>0.05). Compared with the control group, H_2O_2alone led to obvious cell death which was affirmed by observation under the microscopeand Hoechst33258staining. After being pretreated with different concentrations of TSG,the rate of apoptosis was reduced to some degree. The results of flow cytometrydemonstrated that: the rate of apoptosis in each group was5.8%、30.1%、22.1%、15.3%、14.3%、9.2%. We can deduced that treated with H_2O_2alone can lead to a great degree ofapoptosis in MC3T3-E1cells and pretreated with different concentrations of TSG coulddecrease the rate of apoptosis (P<0.05). At the same time, H_2O_2alone can increase theproduction of cellular ROS and lipid oxidation products MDA and pretreated with TSG(1-10μM) can significantly reduce the cellular ROS and MDA (P<0.05) and improve theoxidative stress status in MC3T3-E1cells. The results from western-blotting and real-timequantitative RT-PCR suggested that pretreatment with TSG can decrease the expression ofpro-apoptosis protein Bax and increase the expression of anti-apoptosis protein Bcl-2inthe level of protein and mRNA. Conclusions: Oxidative stress could lead to increasedapoptosis of MC3T3-E1cells and TSG play a protective role during this process byreducing the oxidative stress level and improving the Bcl-2/Bax signal pathway.
2. Protective effect of TSG on osteoblast dysfunction induced by oxidative stress andthe related mechanism
Objective: The purpose of this study was to investigate the protective effect of TSG onosteoblast dysfunction induced by oxidative stress and the related mechanism, in order tolay a solid foundation for the in vivo study. Methods: Murine osteoblastic MC3T3-E1cells were divided equally into6well plates and cultured in osteogenic induction mediumat about80%confluency. The induction culture medium contains10mMβ-glycerophosphate and50μg/ml ascorbic acid to initiate differentiation. In thisexperiment, we divided into6groups according to the way of treatment, including thecontrol group, H_2O_2, H_2O_2+TSG(0.1μM), H_2O_2+TSG(1μM), H_2O_2+TSG(10μM) andH_2O_2+NAC(1mM). After induction for6,14or21days, MC3T3-E1cells werepre-incubated with different concentrations of TSG for24h before treatment with300μMH_2O_2for another24h. Alkaline phosphatase (ALP) staining and activity were used toevaluate the expression of osteogenic marker ALP; Alizarin Red S staining was used toassess calcium deposition; Real-time RT-PCR was used to test the expression level ofostegenic differentiation genes ALP, COL-I and OCN. In order to further clarify therelated mechanism, we tested the MDA and ROS levels in the cells to evaluate theoxidative stress and measured the production of bone-resorbing mediators: RANKL andIL-6by sandwich ELISA assay kit. Results: Cellular ALP staining and activity, calciumdeposition and osteogenic differentiation genes were significantly decreased in H_2O_2group when compared with the control group (P<0.05). Pre-incubated for24h, TSG(0.1-10μM) could significantly increase ALP activity in a dose-dependent manner(P<0.05), improve ALP staining and calcium deposition and significantly increase theexpression level of osteogenic differentiation genes (P<0.05). Meanwhile, the cellularROS and MDA levels were increased after H_2O_2treatment (P<0.05) and this effect wasobviously reversed by pre-incubated with TSG (1-10μM). The results from ELISA assayindicated that H_2O_2raised the production of RANKL and IL-6and these was reduced bypre-incubated with different concentrations of TSG,0.1-10μM and10μM respectively (P<0.05). Conclusions: Oxidative stress could inhibit the osteogenic differentiation andTSG could reverse this effect partially by its antioxidant ability and reducingbone-resorbing mediators.
3. Protective effect of TSG against bone loss in OVX mice
Objective: The purpose of this study was to investigate the protective effect of TSGagainst bone loss in a murine ovariectomized (OVX) osteoporosis model. Methods:Thirty-two9-week-old and weighing19.73±2.011g BALB/c female mice were randomlydivided into4groups:(1) untreated (Sham: sham-operated controls);(2) untreated (OVXcontrols);(3) OVX administered intraperitoneally with TSG (5mg/kg body weight) daily;(4) OVX administered intraperitoneally with TSG (20mg/kg body weight) daily.According to the groups, both of the ovaries were removed under anesthesia bypentobarbital sodium (50mg/kg body weight, i.p.) with dorsum approach. TSG weredissolved in distilled water. The administration of TSG started1week after the surgeryand lasted for3months. In order to evaluate the oxidative stress level in serum, we tookblood samples from the heart in anesthetized mice and prepared it by centrifugation to testthe content of MDA and GSH. The4th lumbar vertebra (L4) and the left femur from eachmouse were removed, cleaned of adherent tissue and fixed in4%paraformaldehyde for48hours. The effects of TSG on bone mineral density, bone mass and bone microarchitecturewere assessed by Micro-CT, H&E staining, Van Gieson staining and Von Kossa staining.Results: Compared with the sham group, serum MDA level was increased and GSH levelwas decreased (P<0.05) in the OVX group. TSG supplementation (20mg/kg) significantlydecreased serum MDA level (P<0.05), without significantly increased the GSH level(P>0.05). There was no difference in MDA and GSH levels in OVX mice treated withhigh or low concentration of TSG (P>0.05). The results from Micro-CT scanningindicated that ovariectomy induced deterioration of the trabecular bone microarchitecturein distal femoral metaphyses and the fourth lumber vertebrate (L4), as demonstrated by thereduction in BV/TV, Conn.D, Tb.N, Tb.Th and BMD when compared with the shamgroup (P<0.01). In contrast, SMI and Tb.Sp were significantly increased in response to OVX (P<0.05). However, supplementation of TSG with a high dose at20mg/kg couldsignificantly (P<0.05) reverse the changes induced by ovariectomy and could maintain themicroarchitecture of trabecular bone in distal femoral metaphyses and the fourth lumbervertebrate (L4). TSG at5mg/kg could improve the trabecular bone parameters, but withoutsignificant difference when compared with the OVX group (P>0.05). There was nodifference between TSG treated groups (P>0.05). Undecalcified histological examinationwith Van Gieson and Von Kossa staining demonstrated that ovariectomy could lead toreduced trabecular number and broader spaces between trabecules compared with thesham group. TSG supplementation reversed these deleterious effects, as demonstrated byan increase in trabecular number and a decrease in the trabecular space in the TSG treatedgroups. H&E staining obtained the similar result. Conclusions: TSG could reduce theserum oxidative stress level in murine ovariectomized osteoporosis model and reverse thereduced bone mineral density, bone mass loss and deleterious bone microarchitecture.
本实验分为三部分，拟通过在体外研究TSG对氧化应激造成的成骨前体细胞MC3T3-E1凋亡和成骨分化抑制的逆转作用及在体内研究小鼠去卵巢骨质疏松模型中，TSG能否提高骨密度，改善骨组织微结构，进而改善OP，并对其相关机制进行初步探讨。本研究可以提供TSG治疗OP的新用途，为寻找治疗OP的中药提供理论依据。
1TSG对氧化应激导致的成骨前体细胞MC3T3-E1凋亡的预防作用及机制研究
目的研究TSG对氧化应激导致的成骨前体细胞MC3T3-E1凋亡的预防作用，并初步探讨相关机制。方法建立体外氧化应激模型，100、200、300和400μM/L的H_2O_2分别作用2h、6h、12h和24h，MTT法评价细胞存活情况，筛选合适H_2O_2作用浓度；观察不同浓度0.01、0.1、1、10、50μM/L TSG对成骨前体细胞MC3T3-E1增殖作用的影响，评价药物的毒性作用；筛选的合适浓度的TSG和H_2O_2共同作用，实验分组：①空白对照组；②单纯H_2O_2处理组；③H_2O_2+TSG0.1μM处理组；④H_2O_2+TSG1μM处理组；⑤H_2O_2+TSG10μM处理组；⑥阳性对照抗氧化剂NAC1mM处理组。通过MTT法、显微镜下大体观察、Hoechst33258染色及流式细胞仪来评价细胞凋亡情况及TSG对氧化损伤的保护作用；利用荧光酶标仪和MDA检测试剂盒来检测细胞中ROS及MDA的水平来评价细胞的氧化应激状态；Western-blot和RT-PCR分别从蛋白水平和基因水平评价Bcl-2和Bax的表达水平，初步阐述TSG对氧化应激损伤保护作用的相关机制。结果筛选出合适H_2O_2作用浓度为300μM/L，作用24h，用于后续研究；TSG在观察浓度内对MC3T3-E1无毒性作用（P>0.05）；与空白对照组相比，单纯H_2O_2处理可以导致细胞死亡，显微镜下大体观察可见许多细胞已死亡，脱壁；Hoechst33258染色可见有较多的细胞出现核固缩、核浓集现象；不同浓度的TSG预处理后，细胞凋亡现象得到改善；流式细胞仪检测结果提示：空白组、H_2O_2处理组、不同浓度TSG预处理组及NAC处理组的细胞凋亡率分别为5.8%、30.1%、22.1%、15.3%、14.3%、9.2%。可见，单独H_2O_2作用可以导致成骨前体细胞出现较大程度的凋亡，不同浓度TSG预处理后，细胞凋亡率有所降低（P<0.05）；单纯H_2O_2作用可以导致细胞水平的ROS和脂质氧化产物MDA产生增加，TSG（1-10μM）可以显著降低细胞水平ROS和MDA水平（P<0.05），改善细胞的氧化应激状态；Western-blotting和实时定量RT-PCR结果提示：TSG预处理可以减少H_2O_2处理后促凋亡蛋白Bax的表达，增加抗凋亡蛋白Bcl-2的表达。结论氧化应激可以导致成骨前体细胞MC3T3-E1凋亡增加，TSG可以通过降低氧化应激水平及改善线粒体凋亡Bcl-2/Bax信号通路来起到保护作用。
2TSG对氧化应激导致的成骨前体细胞MC3T3-E1成骨分化能力降低的预防作用及机制研究
目的探讨TSG对氧化应激导致的成骨前体细胞MC3T3-E1成骨分化能力降低的预防作用及相关机制研究，为体内实验奠定理论基础。方法将成骨前体细胞MC3T3-E1均匀接种到六孔板内，待细胞达80%铺满时，给予成骨诱导培养基培养。实验分组：①空白对照组；②单纯H_2O_2处理组；③H_2O_2+TSG0.1μM处理组；④H_2O_2+TSG1μM处理组；⑤H_2O_2+TSG10μM处理组；⑥NAC1mM处理组。诱导6、14或21天后，依组分别给予不同浓度TSG预处理24h，然后给予H_2O_2处理24h。通过ALP染色及ALP定量分析来评价成骨分化的标志物ALP的表达如何；通过茜素红染色来评价成骨细胞的矿化程度；通过实时定量RT-PCR来检测成骨分化相关基因ALP、COL-1和OCN的表达情况。为了进一步阐明相关的作用机制，我们利用荧光酶标仪检测成骨细胞内ROS的水平及MDA试剂盒检测脂质氧化水平来反应细胞内的氧化应激水平，并利用ELISA试剂盒检测骨吸收相关因子RANKL和IL-6的表达水平。结果同空白对照组相比，单纯H_2O_2处理可以明显降低成骨分化标记物ALP染色和定量结果；减少细胞外基质钙沉积量；下调成骨分化相关基因ALP、COL-1和OCN的表达水平（P<0.05）。当TSG预处理24h后，TSG (0.1-10μM)以剂量依赖方式明显改善ALP活性（P<0.05）；改善ALP染色和钙结节沉积；显著提高成骨分化基因ALP、COL-1和OCN的表达水平（P<0.05）。单纯H_2O_2处理，细胞内ROS及MDA水平显著提高，而TSG（1-10μM）预处理可以明显降低ROS及MDA水平（P<0.05）。ELISA检测也得出相同的结果，TSG（0.1-10μM）及（10μM）分别降低H_2O_2作用导致的RANKL和IL-6的升高（P<0.05）。结论氧化应激可以导致成骨前体细胞MC3T3-E1成骨分化能力抑制，TSG可能部分通过降低氧化应激水平及骨吸收因子的表达，在一定程度上逆转这种抑制作用。
3TSG对小鼠去势骨质疏松模型的预防作用
目的研究TSG对小鼠去势骨质疏松模型中骨密度、骨量及骨组织微结构改变的预防作用。方法32只9月龄BALB/c雌性小鼠，平均体重为19.73±2.011g，随机分为四组：①假手术组（SHAM）；②单纯卵巢去势组（OVX）；③卵巢去势并小剂量TSG处理组（OVX+TSG5mg/kg）；④卵巢去势并高剂量TSG处理组（OVX+TSG20mg/kg）。戊巴比妥钠（50mg/kg i.p.）麻醉成功后，通过背侧入路移除双侧卵巢，术后7天，依分组情况每天给予腹腔注射药物或双蒸水，持续3个月。心脏采血，离心后，取血清用于检测MDA和GSH表达水平，来评价血清中的氧化应激状态。取第4腰椎及左侧股骨，剔除周围软组织，4%多聚甲醛固定后，用于显微CT（Micro-CT）、HE染色、VG染色和Von kossa染色来评价TSG对骨密度、骨量及骨组织微结构的作用程度。结果与SHAM组相比，OVX组血清中MDA水平升高、GSH水平降低（P<0.05）。OVX+TSG20mg/kg可以明显改善血清中MDA水平（P<0.05），但对于GSH水平的改变无统计学差异（P>0.05）。不同剂量TSG处理组之间也无统计学差异（P>0.05）。Micro-CT结果提示：与SHAM组相比，OVX组的第四腰椎及股骨远端松质骨骨量减少，BV/TV、Conn.D、Tb.N、Tb.Th及BMD值减少(P<0.01)，SMI和Tb.Sp增加(P<0.05)。OVX+TSG20mg/kg可以明显改善去卵巢导致的这些骨组织微结构参数的改变（P<0.05）。OVX+TSG5mg/kg可以改善骨组织微结构参数值，但不具有统计学差异（P>0.05）。不同剂量TSG处理组之间也无统计学差异（P>0.05）。脱钙骨组织石蜡切片HE染色及不脱钙骨组织塑料包埋切片VG和Von kossa染色结果均提示：OVX组与SHAM组相比，骨小梁数量减少，间距增大，而经不同浓度TSG处理后，骨小梁数量及间距有不同程度改变。结论TSG可以降低小鼠去卵巢骨质疏松模型血清中的氧化应激水平，并可以改善去势导致的骨密度下降、骨量降低及骨组织微结构改变。
Osteoporosis is a systemic, degenerative and metabolic bone disease, which ischaracterized by deterioration of bone mass, microarchitecture, bone mineral density andbone biomechanics and leads to an increased risk of fracture. It is seriously threatening thephysical and mental health of the elderly. Oxidative stress is caused by excessivegeneration of reactive oxygen species (ROS) beyond the body’s scavenging capacity andresults into a state of intracellular reduction–oxidation imbalance in the body. Highconcentrations of ROS could oxidize the nucleic acids, proteins and lipids, and damage thestructure and function of the cells, leading to the development of a disease. Many degenerative diseases have been shown to be closely related to oxidative stress. In recentyears, a large number of studies have confirmed that oxidative stress plays an importantrole in the development of osteoporosis. Excessive generation of ROS can stimulateapoptosis and inhibit osteogenic differentiation of osteoblasts, stimulate formation andactivity of osteoclasts and affect the bone formation and resorption coupling. As a result,bone formation decreases and bone resorption increases, leading to bone mass loss.Meanwhile, the elevated oxidative stress level in plasma has a correlation with deceasedbone density in osteoporotic patients. Therefore, using antioxidant to prevent and treatosteoporosis has become research focus. Epidemiological studies have provided evidenceof a link between nutrient, antioxidant intake and bone health, and have led toinvestigations of the antioxidant properties of nutrients.2,3,5,4-tetrahydroxystilbene-2-o-β-D-glucoside(TSG) is a kind of extracts of Polygonum, which is a traditional Chinesemedicine, has antioxidant effect. It is unclear that whether TSG could be applied to thetreatment of OP until now.
This study was divided into three parts: firstly, we tested the protective effect of TSGon H_2O_2induced cytotoxicity in MC3T3-E1cells and clarified part of the relatedmechanism; then, we evaluated the protective effect of TSG on osteoblast dysfunctioninduced by H_2O_2; finally, we demonstrated the protective effect of TSG against bone lossin OVX mice. We confirmed a new use of TSG to the treatment of OP in this study andprovided a theoretical basis for the treatment of OP with the traditional Chinese medicine.
1. Protective effect of TSG on oxidative stress induced cytotoxicity in MC3T3-E1cells and the related mechanism
Objective: the purpose of this study was to investigate the protective effect of TSG onapoptosis of MC3T3-E1cells induced by oxidative stress and clarify the relatedmechanism. Methods: At first, we built oxidative stress model in vitro. In this study,MC3T3-E1cells were treated with different concentrations of H_2O_2(0,100,200,300and400μM/L) for2,6,12and24h and cell viability was evaluated by MTT assay. In this way,we chose the appropriate concentration of H_2O_2. Meanwhile, cells were treated with different concentrations of TSG (0,0.1,1and10μM/L) for24and72h in order toinvestigate the toxicity of TSG. The selected concentrations of H_2O_2and TSG were usedin the following studies. The experiment was divided into6groups, including the controlgroup, H_2O_2, H_2O_2+TSG(0.1μM), H_2O_2+TSG(1μM), H_2O_2+TSG(10μM) and H_2O_2+NAC(1mM). The protective effect of TSG on apoptosis of the cells induced by oxidative stresswas demonstrated by MTT assay, general observation under microscope, Hoechst33258staining and flow cytometry. The level of ROS and MDA were tested by fluorescencemicroplate reader and MDA assay kit to evaluate the cellular oxidative stress status. Inorder to further clarify the related mechanism, we examined the production of Bcl-2andBax protein using Western-blotting and the gene expression of Bcl-2and Bax by RT-PCR.Results: The suitable concentration and duration of action for H_2O_2was300μM/L for24hwhich was used in the following study. TSG alone was non-toxic to MC3T3-E1cells atthe concentrations used in this study (P>0.05). Compared with the control group, H_2O_2alone led to obvious cell death which was affirmed by observation under the microscopeand Hoechst33258staining. After being pretreated with different concentrations of TSG,the rate of apoptosis was reduced to some degree. The results of flow cytometrydemonstrated that: the rate of apoptosis in each group was5.8%、30.1%、22.1%、15.3%、14.3%、9.2%. We can deduced that treated with H_2O_2alone can lead to a great degree ofapoptosis in MC3T3-E1cells and pretreated with different concentrations of TSG coulddecrease the rate of apoptosis (P<0.05). At the same time, H_2O_2alone can increase theproduction of cellular ROS and lipid oxidation products MDA and pretreated with TSG(1-10μM) can significantly reduce the cellular ROS and MDA (P<0.05) and improve theoxidative stress status in MC3T3-E1cells. The results from western-blotting and real-timequantitative RT-PCR suggested that pretreatment with TSG can decrease the expression ofpro-apoptosis protein Bax and increase the expression of anti-apoptosis protein Bcl-2inthe level of protein and mRNA. Conclusions: Oxidative stress could lead to increasedapoptosis of MC3T3-E1cells and TSG play a protective role during this process byreducing the oxidative stress level and improving the Bcl-2/Bax signal pathway.
2. Protective effect of TSG on osteoblast dysfunction induced by oxidative stress andthe related mechanism
Objective: The purpose of this study was to investigate the protective effect of TSG onosteoblast dysfunction induced by oxidative stress and the related mechanism, in order tolay a solid foundation for the in vivo study. Methods: Murine osteoblastic MC3T3-E1cells were divided equally into6well plates and cultured in osteogenic induction mediumat about80%confluency. The induction culture medium contains10mMβ-glycerophosphate and50μg/ml ascorbic acid to initiate differentiation. In thisexperiment, we divided into6groups according to the way of treatment, including thecontrol group, H_2O_2, H_2O_2+TSG(0.1μM), H_2O_2+TSG(1μM), H_2O_2+TSG(10μM) andH_2O_2+NAC(1mM). After induction for6,14or21days, MC3T3-E1cells werepre-incubated with different concentrations of TSG for24h before treatment with300μMH_2O_2for another24h. Alkaline phosphatase (ALP) staining and activity were used toevaluate the expression of osteogenic marker ALP; Alizarin Red S staining was used toassess calcium deposition; Real-time RT-PCR was used to test the expression level ofostegenic differentiation genes ALP, COL-I and OCN. In order to further clarify therelated mechanism, we tested the MDA and ROS levels in the cells to evaluate theoxidative stress and measured the production of bone-resorbing mediators: RANKL andIL-6by sandwich ELISA assay kit. Results: Cellular ALP staining and activity, calciumdeposition and osteogenic differentiation genes were significantly decreased in H_2O_2group when compared with the control group (P<0.05). Pre-incubated for24h, TSG(0.1-10μM) could significantly increase ALP activity in a dose-dependent manner(P<0.05), improve ALP staining and calcium deposition and significantly increase theexpression level of osteogenic differentiation genes (P<0.05). Meanwhile, the cellularROS and MDA levels were increased after H_2O_2treatment (P<0.05) and this effect wasobviously reversed by pre-incubated with TSG (1-10μM). The results from ELISA assayindicated that H_2O_2raised the production of RANKL and IL-6and these was reduced bypre-incubated with different concentrations of TSG,0.1-10μM and10μM respectively (P<0.05). Conclusions: Oxidative stress could inhibit the osteogenic differentiation andTSG could reverse this effect partially by its antioxidant ability and reducingbone-resorbing mediators.
3. Protective effect of TSG against bone loss in OVX mice
Objective: The purpose of this study was to investigate the protective effect of TSGagainst bone loss in a murine ovariectomized (OVX) osteoporosis model. Methods:Thirty-two9-week-old and weighing19.73±2.011g BALB/c female mice were randomlydivided into4groups:(1) untreated (Sham: sham-operated controls);(2) untreated (OVXcontrols);(3) OVX administered intraperitoneally with TSG (5mg/kg body weight) daily;(4) OVX administered intraperitoneally with TSG (20mg/kg body weight) daily.According to the groups, both of the ovaries were removed under anesthesia bypentobarbital sodium (50mg/kg body weight, i.p.) with dorsum approach. TSG weredissolved in distilled water. The administration of TSG started1week after the surgeryand lasted for3months. In order to evaluate the oxidative stress level in serum, we tookblood samples from the heart in anesthetized mice and prepared it by centrifugation to testthe content of MDA and GSH. The4th lumbar vertebra (L4) and the left femur from eachmouse were removed, cleaned of adherent tissue and fixed in4%paraformaldehyde for48hours. The effects of TSG on bone mineral density, bone mass and bone microarchitecturewere assessed by Micro-CT, H&E staining, Van Gieson staining and Von Kossa staining.Results: Compared with the sham group, serum MDA level was increased and GSH levelwas decreased (P<0.05) in the OVX group. TSG supplementation (20mg/kg) significantlydecreased serum MDA level (P<0.05), without significantly increased the GSH level(P>0.05). There was no difference in MDA and GSH levels in OVX mice treated withhigh or low concentration of TSG (P>0.05). The results from Micro-CT scanningindicated that ovariectomy induced deterioration of the trabecular bone microarchitecturein distal femoral metaphyses and the fourth lumber vertebrate (L4), as demonstrated by thereduction in BV/TV, Conn.D, Tb.N, Tb.Th and BMD when compared with the shamgroup (P<0.01). In contrast, SMI and Tb.Sp were significantly increased in response to OVX (P<0.05). However, supplementation of TSG with a high dose at20mg/kg couldsignificantly (P<0.05) reverse the changes induced by ovariectomy and could maintain themicroarchitecture of trabecular bone in distal femoral metaphyses and the fourth lumbervertebrate (L4). TSG at5mg/kg could improve the trabecular bone parameters, but withoutsignificant difference when compared with the OVX group (P>0.05). There was nodifference between TSG treated groups (P>0.05). Undecalcified histological examinationwith Van Gieson and Von Kossa staining demonstrated that ovariectomy could lead toreduced trabecular number and broader spaces between trabecules compared with thesham group. TSG supplementation reversed these deleterious effects, as demonstrated byan increase in trabecular number and a decrease in the trabecular space in the TSG treatedgroups. H&E staining obtained the similar result. Conclusions: TSG could reduce theserum oxidative stress level in murine ovariectomized osteoporosis model and reverse thereduced bone mineral density, bone mass loss and deleterious bone microarchitecture.
引文
[1] Altindag, O.; Erel, O.; Soran, N.; Celik, H.; Selek, S. Total oxidative/anti-oxidative status andrelation to bone mineral density in osteoporosis. Rheumatol Int,2008,28:317-321.
[2] Sendur, O. F.; Turan, Y.; Tastaban, E.; Serter, M. Antioxidant status in patients withosteoporosis: a controlled study. Joint Bone Spine,2009,76:514-518.
[3] Almeida, M.; Han, L.; Martin-Millan, M.; Plotkin, L. I.; Stewart, S. A.; Roberson, P. K.;Kousteni, S.; O'Brien, C. A.; Bellido, T.; Parfitt, A. M.; Weinstein, R. S.; Jilka, R. L.; Manolagas,S. C. Skeletal involution by age-associated oxidative stress and its acceleration by loss of sexsteroids. J Biol Chem,2007,282:27285-27297.
[4] Muthusami, S.; Ramachandran, I.; Muthusamy, B.; Vasudevan, G.; Prabhu, V.; Subramaniam,V.; Jagadeesan, A.; Narasimhan, S. Ovariectomy induces oxidative stress and impairs boneantioxidant system in adult rats. Clin Chim Acta,2005,360:81-86.
[5] Ozgocmen, S.; Kaya, H.; Fadillioglu, E.; Aydogan, R.; Yilmaz, Z. Role of antioxidant systems,lipid peroxidation, and nitric oxide in postmenopausal osteoporosis. Mol Cell Biochem,2007,295:45-52.
[6] Ozgocmen, S.; Kaya, H.; Fadillioglu, E.; Yilmaz, Z. Effects of calcitonin, risedronate, andraloxifene on erythrocyte antioxidant enzyme activity, lipid peroxidation, and nitric oxide inpostmenopausal osteoporosis. Arch Med Res,2007,38:196-205.
[7] Lean, J. M.; Jagger, C. J.; Kirstein, B.; Fuller, K.; Chambers, T. J. Hydrogen peroxide isessential for estrogen-deficiency bone loss and osteoclast formation. Endocrinology,2005,146:728-735.
[8] Jagger, C. J.; Lean, J. M.; Davies, J. T.; Chambers, T. J. Tumor necrosis factor-alpha mediatesosteopenia caused by depletion of antioxidants. Endocrinology,2005,146:113-118.
[9] Sanchez-Rodriguez, M. A.; Ruiz-Ramos, M.; Correa-Munoz, E.; Mendoza-Nunez, V. M.Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized byantioxidant enzymes. BMC Musculoskelet Disord,2007,8:124.
[10] Yalin, S.; Bagis, S.; Polat, G.; Dogruer, N.; Cenk, A. S.; Hatungil, R.; Erdogan, C. Is there a roleof free oxygen radicals in primary male osteoporosis?. Clin Exp Rheumatol,2005,23:689-692.
[11] Garrett, I. R.; Boyce, B. F.; Oreffo, R. O.; Bonewald, L.; Poser, J.; Mundy, G. R.Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro andin vivo. J Clin Invest,1990,85:632-639.
[12] Lee, N. K.; Choi, Y. G.; Baik, J. Y.; Han, S. Y.; Jeong, D. W.; Bae, Y. S.; Kim, N.; Lee, S. Y. Acrucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood,2005,106:852-859.
[13] Lean, J. M.; Davies, J. T.; Fuller, K.; Jagger, C. J.; Kirstein, B.; Partington, G. A.; Urry, Z. L.;Chambers, T. J. A crucial role for thiol antioxidants in estrogen-deficiency bone loss. J ClinInvest,2003,112:915-923.
[14] Koh, J. M.; Lee, Y. S.; Kim, Y. S.; Kim, D. J.; Kim, H. H.; Park, J. Y.; Lee, K. U.; Kim, G. S.Homocysteine enhances bone resorption by stimulation of osteoclast formation and activitythrough increased intracellular ROS generation. J Bone Miner Res,2006,21:1003-1011.
[15] Wittrant, Y.; Gorin, Y.; Woodruff, K.; Horn, D.; Abboud, H. E.; Mohan, S.; Abboud-Werner, S.L. High d(+)glucose concentration inhibits RANKL-induced osteoclastogenesis. Bone,2008,42:1122-1130.
[16] Hsu, H.; Lacey, D. L.; Dunstan, C. R.; Solovyev, I.; Colombero, A.; Timms, E.; Tan, H. L.;Elliott, G.; Kelley, M. J.; Sarosi, I.; Wang, L.; Xia, X. Z.; Elliott, R.; Chiu, L.; Black, T.; Scully,S.; Capparelli, C.; Morony, S.; Shimamoto, G.; Bass, M. B.; Boyle, W. J. Tumor necrosis factorreceptor family member RANK mediates osteoclast differentiation and activation induced byosteoprotegerin ligand. Proc Natl Acad Sci U S A,1999,96:3540-3545.
[17] Lacey, D. L.; Timms, E.; Tan, H. L.; Kelley, M. J.; Dunstan, C. R.; Burgess, T.; Elliott, R.;Colombero, A.; Elliott, G.; Scully, S.; Hsu, H.; Sullivan, J.; Hawkins, N.; Davy, E.; Capparelli,C.; Eli, A.; Qian, Y. X.; Kaufman, S.; Sarosi, I.; Shalhoub, V.; Senaldi, G.; Guo, J.; Delaney, J.;Boyle, W. J. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation andactivation. Cell,1998,93:165-176.
[18] Bai, X. C.; Lu, D.; Liu, A. L.; Zhang, Z. M.; Li, X. M.; Zou, Z. P.; Zeng, W. S.; Cheng, B. L.;Luo, S. Q. Reactive oxygen species stimulates receptor activator of NF-kappaB ligandexpression in osteoblast. J Biol Chem,2005,280:17497-17506.
[19] Grassi, F.; Tell, G.; Robbie-Ryan, M.; Gao, Y.; Terauchi, M.; Yang, X.; Romanello, M.; Jones,D. P.; Weitzmann, M. N.; Pacifici, R. Oxidative stress causes bone loss in estrogen-deficientmice through enhanced bone marrow dendritic cell activation. Proc Natl Acad Sci U S A,2007,104:15087-15092.
[20] Chen, J. R.; Shankar, K.; Nagarajan, S.; Badger, T. M.; Ronis, M. J. Protective effects ofestradiol on ethanol-induced bone loss involve inhibition of reactive oxygen species generationin osteoblasts and downstream activation of the extracellular signal-regulated kinase/signaltransducer and activator of transcription3/receptor activator of nuclear factor-kappaB ligandsignaling cascade. J Pharmacol Exp Ther,2008,324:50-59.
[21] Pittenger, M. F.; Mackay, A. M.; Beck, S. C.; Jaiswal, R. K.; Douglas, R.; Mosca, J. D.;Moorman, M. A.; Simonetti, D. W.; Craig, S.; Marshak, D. R. Multilineage potential of adulthuman mesenchymal stem cells. Science,1999,284:143-147.
[22] Liu, A. L.; Zhang, Z. M.; Zhu, B. F.; Liao, Z. H.; Liu, Z. Metallothionein protects bone marrowstromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation. CellBiol Int,2004,28:905-911.
[23] Bai, X. C.; Lu, D.; Bai, J.; Zheng, H.; Ke, Z. Y.; Li, X. M.; Luo, S. Q. Oxidative stress inhibitsosteoblastic differentiation of bone cells by ERK and NF-kappaB. Biochem Biophys ResCommun,2004,314:197-207.
[24] Arai, M.; Shibata, Y.; Pugdee, K.; Abiko, Y.; Ogata, Y. Effects of reactive oxygen species (ROS)on antioxidant system and osteoblastic differentiation in MC3T3-E1cells. Iubmb Life,2007,59:27-33.
[25] Abraham, N. G. Molecular regulation--biological role of heme in hematopoiesis. Blood Rev,1991,5:19-28.
[26] Khosla, S.; Riggs, B. L. Pathophysiology of age-related bone loss and osteoporosis. EndocrinolMetab Clin North Am,2005,34:1015-1030.
[27] Manolagas, S. C.; Parfitt, A. M. What old means to bone. Trends Endocrinol Metab,2010,21:369-374.
[28] Strotmeyer, E. S.; Cauley, J. A.; Schwartz, A. V.; Nevitt, M. C.; Resnick, H. E.; Bauer, D. C.;Tylavsky, F. A.; de Rekeneire, N.; Harris, T. B.; Newman, A. B. Nontraumatic fracture risk withdiabetes mellitus and impaired fasting glucose in older white and black adults: the health, aging,and body composition study. Arch Intern Med,2005,165:1612-1617.
[29] Abraham, N. G.; Lutton, J. D.; Levere, R. D. Heme metabolism and erythropoiesis in abnormaliron states: role of delta-aminolevulinic acid synthase and heme oxygenase. Exp Hematol,1985,13:838-843.
[30] Vanella, L.; Kim, D. H.; Asprinio, D.; Peterson, S. J.; Barbagallo, I.; Vanella, A.; Goldstein, D.;Ikehara, S.; Kappas, A.; Abraham, N. G. HO-1expression increases mesenchymal stemcell-derived osteoblasts but decreases adipocyte lineage. Bone,2010,46:236-243.
[31] Abraham, N. G.; Kushida, T.; McClung, J.; Weiss, M.; Quan, S.; Lafaro, R.; Darzynkiewicz, Z.;Wolin, M. Heme oxygenase-1attenuates glucose-mediated cell growth arrest and apoptosis inhuman microvessel endothelial cells. Circ Res,2003,93:507-514.
[32] Pileggi, A.; Molano, R. D.; Berney, T.; Cattan, P.; Vizzardelli, C.; Oliver, R.; Fraker, C.; Ricordi,C.; Pastori, R. L.; Bach, F. H.; Inverardi, L. Heme oxygenase-1induction in islet cells results inprotection from apoptosis and improved in vivo function after transplantation. Diabetes,2001,50:1983-1991.
[33] Brouard, S.; Otterbein, L. E.; Anrather, J.; Tobiasch, E.; Bach, F. H.; Choi, A. M.; Soares, M. P.Carbon monoxide generated by heme oxygenase1suppresses endothelial cell apoptosis. J ExpMed,2000,192:1015-1026.
[34] Barbagallo, I.; Vanella, A.; Peterson, S. J.; Kim, D. H.; Tibullo, D.; Giallongo, C.; Vanella, L.;Parrinello, N.; Palumbo, G. A.; Di Raimondo, F.; Abraham, N. G.; Asprinio, D. Overexpressionof heme oxygenase-1increases human osteoblast stem cell differentiation. J Bone Miner Metab,2010,28:276-288.
[35] Bennett, C. N.; Ouyang, H.; Ma, Y. L.; Zeng, Q.; Gerin, I.; Sousa, K. M.; Lane, T. F.; Krishnan,V.; Hankenson, K. D.; MacDougald, O. A. Wnt10b increases postnatal bone formation byenhancing osteoblast differentiation. J Bone Miner Res,2007,22:1924-1932.
[36] Andrade, A. C.; Nilsson, O.; Barnes, K. M.; baron J. Wnt gene expression in the post-natalgrowth plate: regulation with chondrocyte differentiation. Bone,2007,40:1361-1369.
[37] Linares, G. R.; Xing, W.; Govoni, K. E.; Chen, S. T.; Mohan, S. Glutaredoxin5regulatesosteoblast apoptosis by protecting against oxidative stress. Bone,2009,44:795-804.
[38] Almeida, M.; Han, L.; Ambrogini, E.; Bartell, S. M.; Manolagas, S. C. Oxidative stressstimulates apoptosis and activates NF-kappaB in osteoblastic cells via a PKCbeta/p66shcsignaling cascade: counter regulation by estrogens or androgens. Mol Endocrinol,2010,24:2030-2037.
[39] Byun, C. H.; Koh, J. M.; Kim, D. K.; Park, S. I.; Lee, K. U.; Kim, G. S. Alpha-lipoic acidinhibits TNF-alpha-induced apoptosis in human bone marrow stromal cells. J Bone Miner Res,2005,20:1125-1135.
[40] Krishnan, V.; Bryant, H. U.; Macdougald, O. A. Regulation of bone mass by Wnt signaling. JClin Invest,2006,116:1202-1209.
[41] Boyden, L. M.; Mao, J.; Belsky, J.; Mitzner, L.; Farhi, A.; Mitnick, M. A.; Wu, D.; Insogna, K.;Lifton, R. P. High bone density due to a mutation in LDL-receptor-related protein5. N Engl JMed,2002,346:1513-1521.
[42] Babij, P.; Zhao, W.; Small, C.; Kharode, Y.; Yaworsky, P. J.; Bouxsein, M. L.; Reddy, P. S.;Bodine, P. V.; Robinson, J. A.; Bhat, B.; Marzolf, J.; Moran, R. A.; Bex, F. High bone mass inmice expressing a mutant LRP5gene. J Bone Miner Res,2003,18:960-974.
[43] Holmen, S. L.; Giambernardi, T. A.; Zylstra, C. R.; Buckner-Berghuis, B. D.; Resau, J. H.; Hess,J. F.; Glatt, V.; Bouxsein, M. L.; Ai, M.; Warman, M. L.; Williams, B. O. Decreased BMD andlimb deformities in mice carrying mutations in both Lrp5and Lrp6. J Bone Miner Res,2004,19:2033-2040.
[44] Day, T. F.; Guo, X.; Garrett-Beal, L.; Yang, Y. Wnt/beta-catenin signaling in mesenchymalprogenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis.Dev Cell,2005,8:739-750.
[45] Hill, T. P.; Spater, D.; Taketo, M. M.; Birchmeier, W.; Hartmann, C. CanonicalWnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell,2005,8:727-738.
[46] Bennett, C. N.; Longo, K. A.; Wright, W. S.; Suva, L. J.; Lane, T. F.; Hankenson, K. D.;MacDougald, O. A. Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc NatlAcad Sci U S A,2005,102:3324-3329.
[47] Hu, H.; Hilton, M. J.; Tu, X.; Yu, K.; Ornitz, D. M.; Long, F. Sequential roles of Hedgehog andWnt signaling in osteoblast development. Development,2005,132:49-60.
[48] Glass, D. N.; Bialek, P.; Ahn, J. D.; Starbuck, M.; Patel, M. S.; Clevers, H.; Taketo, M. M.;Long, F.; McMahon, A. P.; Lang, R. A.; Karsenty, G. Canonical Wnt signaling in differentiatedosteoblasts controls osteoclast differentiation. Dev Cell,2005,8:751-764.
[49] Kawano, Y.; Kypta, R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci,2003,116:2627-2634.
[50] Li, X.; Ominsky, M. S.; Niu, Q. T.; Sun, N.; Daugherty, B.; D'Agostin, D.; Kurahara, C.; Gao,Y.; Cao, J.; Gong, J.; Asuncion, F.; Barrero, M.; Warmington, K.; Dwyer, D.; Stolina, M.;Morony, S.; Sarosi, I.; Kostenuik, P. J.; Lacey, D. L.; Simonet, W. S.; Ke, H. Z.; Paszty, C.Targeted deletion of the sclerostin gene in mice results in increased bone formation and bonestrength. J Bone Miner Res,2008,23:860-869.
[51] Li, X.; Ominsky, M. S.; Warmington, K. S.; Morony, S.; Gong, J.; Cao, J.; Gao, Y.; Shalhoub,V.; Tipton, B.; Haldankar, R.; Chen, Q.; Winters, A.; Boone, T.; Geng, Z.; Niu, Q. T.; Ke, H. Z.;Kostenuik, P. J.; Simonet, W. S.; Lacey, D. L.; Paszty, C. Sclerostin antibody treatmentincreases bone formation, bone mass, and bone strength in a rat model of postmenopausalosteoporosis. J Bone Miner Res,2009,24:578-588.
[52] Bodine, P. V.; Zhao, W.; Kharode, Y. P.; Bex, F. J.; Lambert, A. J.; Goad, M. B.; Gaur, T.; Stein,G. S.; Lian, J. B.; Komm, B. S. The Wnt antagonist secreted frizzled-related protein-1is anegative regulator of trabecular bone formation in adult mice. Mol Endocrinol,2004,18:1222-1237.
[53] Bodine, P. V.; Stauffer, B.; Ponce-de-Leon, H.; Bhat, R. A.; Mangine, A.; Seestaller-Wehr, L.M.; Moran, R. A.; Billiard, J.; Fukayama, S.; Komm, B. S.; Pitts, K.; Krishnamurthy, G.;Gopalsamy, A.; Shi, M.; Kern, J. C.; Commons, T. J.; Woodworth, R. P.; Wilson, M. A.;Welmaker, G. S.; Trybulski, E. J.; Moore, W. J. A small molecule inhibitor of the Wntantagonist secreted frizzled-related protein-1stimulates bone formation. Bone,2009,44:1063-1068.
[54] Morvan, F.; Boulukos, K.; Clement-Lacroix, P.; Roman, R. S.; Suc-Royer, I.; Vayssiere, B.;Ammann, P.; Martin, P.; Pinho, S.; Pognonec, P.; Mollat, P.; Niehrs, C.; baron R; Rawadi, G.Deletion of a single allele of the Dkk1gene leads to an increase in bone formation and bonemass. J Bone Miner Res,2006,21:934-945.
[55] Glantschnig, H.; Hampton, R. A.; Lu, P.; Zhao, J. Z.; Vitelli, S.; Huang, L.; Haytko, P.; Cusick,T.; Ireland, C.; Jarantow, S. W.; Ernst, R.; Wei, N.; Nantermet, P.; Scott, K. R.; Fisher, J. E.;Talamo, F.; Orsatti, L.; Reszka, A. A.; Sandhu, P.; Kimmel, D.; Flores, O.; Strohl, W.; An, Z.;Wang, F. Generation and selection of novel fully human monoclonal antibodies that neutralizeDickkopf-1(DKK1) inhibitory function in vitro and increase bone mass in vivo. J Biol Chem,2010,285:40135-40147.
[56] Zanotti, S.; Canalis, E. Notch and the skeleton. Mol Cell Biol,2010,30:886-896.
[57] Nam, Y.; Sliz, P.; Song, L.; Aster, J. C.; Blacklow, S. C. Structural basis for cooperativity inrecruitment of MAML coactivators to Notch transcription complexes. Cell,2006,124:973-983.
[58] Deregowski, V.; Gazzerro, E.; Priest, L.; Rydziel, S.; Canalis, E. Notch1overexpression inhibitsosteoblastogenesis by suppressing Wnt/beta-catenin but not bone morphogenetic proteinsignaling. J Biol Chem,2006,281:6203-6210.
[59] Zanotti, S.; Smerdel-Ramoya, A.; Stadmeyer, L.; Durant, D.; Radtke, F.; Canalis, E. Notchinhibits osteoblast differentiation and causes osteopenia. Endocrinology,2008,149:3890-3899.
[60] Hilton, M. J.; Tu, X.; Wu, X.; Bai, S.; Zhao, H.; Kobayashi, T.; Kronenberg, H. M.; Teitelbaum,S. L.; Ross, F. P.; Kopan, R.; Long, F. Notch signaling maintains bone marrow mesenchymalprogenitors by suppressing osteoblast differentiation. Nat Med,2008,14:306-314.
[61] Engin, F.; Yao, Z.; Yang, T.; Zhou, G.; Bertin, T.; Jiang, M. M.; Chen, Y.; Wang, L.; Zheng, H.;Sutton, R. E.; Boyce, B. F.; Lee, B. Dimorphic effects of Notch signaling in bone homeostasis.Nat Med,2008,14:299-305.
[62] Zamurovic, N.; Cappellen, D.; Rohner, D.; Susa, M. Coordinated activation of notch, Wnt, andtransforming growth factor-beta signaling pathways in bone morphogenic protein2-inducedosteogenesis. Notch target gene Hey1inhibits mineralization and Runx2transcriptional activity.J Biol Chem,2004,279:37704-37715.
[63] Zanotti, S.; Smerdel-Ramoya, A.; Canalis, E. Reciprocal regulation of Notch and nuclear factorof activated T-cells (NFAT) c1transactivation in osteoblasts. J Biol Chem,2011,286:4576-4588.
[64] Isidor, B.; Lindenbaum, P.; Pichon, O.; Bezieau, S.; Dina, C.; Jacquemont, S.; Martin-Coignard,D.; Thauvin-Robinet, C.; Le, M. M.; Mandel, J. L.; David, A.; Faivre, L.; Cormier-Daire, V.;Redon, R.; Le, C. C. Truncating mutations in the last exon of NOTCH2cause a rare skeletaldisorder with osteoporosis. Nat Genet,2011,43:306-308.
[65] Simpson, M. A.; Irving, M. D.; Asilmaz, E.; Gray, M. J.; Dafou, D.; Elmslie, F. V.; Mansour, S.;Holder, S. E.; Brain, C. E.; Burton, B. K.; Kim, K. H.; Pauli, R. M.; Aftimos, S.; Stewart, H.;Kim, C. A.; Holder-Espinasse, M.; Robertson, S. P.; Drake, W. M.; Trembath, R. C. Mutationsin NOTCH2cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss. NatGenet,2011,43:303-305.
[66] Matsuo, K. Eph and ephrin interactions in bone. Adv Exp Med Biol,2010,658:95-103.
[67] Zhao, C.; Irie, N.; Takada, Y.; Shimoda, K.; Miyamoto, T.; Nishiwaki, T.; Suda, T.; Matsuo, K.Bidirectional ephrinB2-EphB4signaling controls bone homeostasis. Cell Metab,2006,4:111-121.
[68] Allan, E. H.; Hausler, K. D.; Wei, T.; Gooi, J. H.; Quinn, J. M.; Crimeen-Irwin, B.; Pompolo, S.;Sims, N. A.; Gillespie, M. T.; Onyia, J. E.; Martin, T. J. EphrinB2regulation by PTH and PTHrPrevealed by molecular profiling in differentiating osteoblasts. J Bone Miner Res,2008,23:1170-1181.
[69] Xing, W.; Kim, J.; Wergedal, J.; Chen, S. T.; Mohan, S. Ephrin B1regulates bone marrowstromal cell differentiation and bone formation by influencing TAZ transactivation via complexformation with NHERF1. Mol Cell Biol,2010,30:711-721.
[70] Irie, N.; Takada, Y.; Watanabe, Y.; Matsuzaki, Y.; Naruse, C.; Asano, M.; Iwakura, Y.; Suda, T.;Matsuo, K. Bidirectional signaling through ephrinA2-EphA2enhances osteoclastogenesis andsuppresses osteoblastogenesis. J Biol Chem,2009,284:14637-14644.
[71] Dvorak, M. M.; Siddiqua, A.; Ward, D. T.; Carter, D. H.; Dallas, S. L.; Nemeth, E. F.; Riccardi,D. Physiological changes in extracellular calcium concentration directly control osteoblastfunction in the absence of calciotropic hormones. Proc Natl Acad Sci U S A,2004,101:5140-5145.
[72] Chattopadhyay, N.; Yano, S.; Tfelt-Hansen, J.; Rooney, P.; Kanuparthi, D.; Bandyopadhyay, S.;Ren, X.; Terwilliger, E.; Brown, E. M. Mitogenic action of calcium-sensing receptor on ratcalvarial osteoblasts. Endocrinology,2004,145:3451-3462.
[73] Kameda, T.; Mano, H.; Yamada, Y.; Takai, H.; Amizuka, N.; Kobori, M.; Izumi, N.;Kawashima, H.; Ozawa, H.; Ikeda, K.; Kameda, A.; Hakeda, Y.; Kumegawa, M.Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. BiochemBiophys Res Commun,1998,245:419-422.
[74] Brown, E. M.; MacLeod, R. J. Extracellular calcium sensing and extracellular calcium signaling.Physiol Rev,2001,81:239-297.
[75] Canalis, E. Novel treatments for osteoporosis. J Clin Invest,2000,106:177-179.
[76] Marie, P. J. The calcium-sensing receptor in bone cells: a potential therapeutic target inosteoporosis. Bone,2010,46:571-576.
[77] Canalis, E.; Giustina, A.; Bilezikian, J. P. Mechanisms of anabolic therapies for osteoporosis. NEngl J Med,2007,357:905-916.
[78] Pacifici, R. T cells: critical bone regulators in health and disease. Bone,2010,47:461-471.
[79] Gao, Y.; Wu, X.; Terauchi, M.; Li, J. Y.; Grassi, F.; Galley, S.; Yang, X.; Weitzmann, M. N.;Pacifici, R. T cells potentiate PTH-induced cortical bone loss through CD40L signaling. CellMetab,2008,8:132-145.
[80] Tawfeek, H.; Bedi, B.; Li, J. Y.; Adams, J.; Kobayashi, T.; Weitzmann, M. N.; Kronenberg, H.M.; Pacifici, R. Disruption of PTH receptor1in T cells protects against PTH-induced bone loss.PLoS One,2010,5:e12290.
[81] Mann, V.; Huber, C.; Kogianni, G.; Collins, F.; Noble, B. The antioxidant effect of estrogen andSelective Estrogen Receptor Modulators in the inhibition of osteocyte apoptosis in vitro. Bone,2007,40:674-684.
[82] Valverde, P. Pharmacotherapies to manage bone loss-associated diseases: a quest for the perfectbenefit-to-risk ratio. Curr Med Chem,2008,15:284-304.
[83] Deyhim, F.; Mandadi, K.; Patil, B. S.; Faraji, B. Grapefruit pulp increases antioxidant status andimproves bone quality in orchidectomized rats. Nutrition,2008,24:1039-1044.
[84] Scalbert, A.; Manach, C.; Morand, C.; Remesy, C.; Jimenez, L. Dietary polyphenols and theprevention of diseases. Crit Rev Food Sci Nutr,2005,45:287-306.
[85] Houde, V.; Grenier, D.; Chandad, F. Protective effects of grape seed proanthocyanidins againstoxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol,2006,77:1371-1379.
[86] Ostman, B.; Michaelsson, K.; Helmersson, J.; Byberg, L.; Gedeborg, R.; Melhus, H.; Basu, S.Oxidative stress and bone mineral density in elderly men: antioxidant activity ofalpha-tocopherol. Free Radic Biol Med,2009,47:668-673.
[87]吕丽爽;汤坚;何淇傥.二苯乙烯苷特性及DPPH自由基清除活性.哈尔滨工业大学学报,2009,252-255.
[88]刘厚淳;陈万生.何首乌水溶性成分2,3,5,4’-四羟基二苯乙烯-2-O-β-D葡萄糖苷的体外抗氧化作用研究.药学实践杂志,2000.
[89]王栋;陈晓宇;刘晓利;赵欢;张腾腾;赵中原;白璐.二苯乙烯苷对沙鼠脑缺血/再灌注损伤的保护作用及部分机制.安徽医科大学学报,2012,47:379-382.
[90]袁惠琼;杨杰;曾进;郭荣静.二苯乙烯苷对老龄大鼠脑缺血再灌注后HIF-1α及EPO表达的影响.中风与神经疾病杂志,2011,28:868-871.
[91]张玲玲;陈磊;陶丽珍;李雪芬;张磊;陈良为;陈建宗.二苯乙烯苷对MPTP诱导的帕金森病小鼠模型黑质多巴胺转运体的影响.神经解剖学杂志,2012,28:33-37.
[92] Tao, L.; Li, X.; Zhang, L.; Tian, J.; Li, X.; Sun, X.; Li, X.; Jiang, L.; Zhang, X.; Chen, J.Protective effect of tetrahydroxystilbene glucoside on6-OHDA-induced apoptosis in PC12cellsthrough the ROS-NO pathway. PLoS One,2011,6:e26055.
[93] Sun, F. L.; Zhang, L.; Zhang, R. Y.; Li, L. Tetrahydroxystilbene glucoside protects humanneuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity. Eur J Pharmacol,2011,660:283-290.
[94] Qin, R.; Li, X.; Li, G.; Tao, L.; Li, Y.; Sun, J.; Kang, X.; Chen, J. Protection bytetrahydroxystilbene glucoside against neurotoxicity induced by MPP+: the involvement ofPI3K/Akt pathway activation. Toxicol Lett,2011,202:1-7.
[95] Li, X.; Li, Y.; Chen, J.; Sun, J.; Li, X.; Sun, X.; Kang, X. Tetrahydroxystilbene glucosideattenuates MPP+-induced apoptosis in PC12cells by inhibiting ROS generation and modulatingJNK activation. Neurosci Lett,2010,483:1-5.
[96] Hou, Y.; Yang, Q.; Zhou, L.; Du X; Li, M.; Yuan, M.; Zhou, Z.; Li, Z. Tetrahydroxystilbeneglucoside improves learning and (or) memory ability of aged rats and may be connected to theAPP pathway. Can J Physiol Pharmacol,2011,89:801-809.
[97]甘受益;黄俊;李宾;葛雄;李彩蓉;蔡飞.二苯乙烯苷对实验性糖尿病大鼠心脏血流动力学和氧化应激的影响.现代中西医结合杂志,2012,21:30-32.
[98]相聪坤;王蕊;袁志芳.何首乌二苯乙烯苷类提取物对高脂血症大鼠血脂代谢的影响及其抗氧化作用.中国药业,2009,18:19-20.
[99]张燕堂;张颖;郑文;杨华;王家富;商战平.二苯乙烯苷对LPC损伤血管内皮细胞的保护作用及其机制.中国病理生理杂志,2012,28:1192-1196.
[100]张彩平;杨滢;田英;乔新惠;龙石银;陈志军;田汝芳.二苯乙烯苷对H2O2诱导血管内皮细胞黏附分子表达的影响.中国药理学通报,2012,28:1088-1092.
[101] Xu, X. L.; Huang, Y. J.; Wang, Y. Q.; Chen, X. F.; Zhang, W.2,3,4',5-Tetrahydroxystilbene-2-O-beta-d-glucoside inhibits platelet-derived growth factor-induced proliferation of vascular smooth muscle cells by regulating the cell cycle. Clin ExpPharmacol Physiol,2011,38:307-313.
[102] Zhang, W.; Xu, X. L.; Wang, Y. Q.; Wang, C. H.; Zhu, W. Z. Effects of2,3,4',5-tetrahydroxystilbene2-O-beta-D-glucoside on vascular endothelial dysfunction inatherogenic-diet rats. Planta Med,2009,75:1209-1214.
[103]李军;王国荣;王燕;解砚英;牟艳玲;姚庆强.二苯乙烯苷对同型半胱氨酸诱导的人脐静脉内皮细胞MCP-1、ICAM-1和VCAM-1表达的影响.中国动脉硬化杂志,2012,20:890-894.
[104]张园;董林;许晓乐;王玉琴;张伟.二苯乙烯苷对2型糖尿病大鼠氧化应激、炎症反应及主动脉细胞凋亡相关基因的影响.中国糖尿病杂志,2012,20:775-778.
[105]李佳;贾延龙;唐婧;刘青;董又娟;胡九凡;李彩蓉;蔡飞.二苯乙烯苷对糖尿病大鼠肾脏损伤的干预作用.咸宁学院学报：医学版,2011,25:277-279.
[106] Li, C.; Cai, F.; Yang, Y.; Zhao, X.; Wang, C.; Li, J.; Jia, Y.; Tang, J.; Liu, Q.Tetrahydroxystilbene glucoside ameliorates diabetic nephropathy in rats: involvement of SIRT1and TGF-beta1pathway. Eur J Pharmacol,2010,649:382-389.
[107] Zeng, C.; Xiao, J. H.; Chang, M. J.; Wang, J. L. Beneficial effects of THSG on aceticacid-induced experimental colitis: involvement of upregulation of PPAR-gamma and inhibitionof the Nf-Kappab inflammatory pathway. Molecules,2011,16:8552-8568.
[108] Wang, X.; Zhao, L.; Han, T.; Chen, S.; Wang, J. Protective effects of2,3,5,4'-tetrahydroxystilbene-2-O-beta-d-glucoside, an active component of Polygonummultiflorum Thunb, on experimental colitis in mice. Eur J Pharmacol,2008,578:339-348.
[109]张海啸;尹智炜;李芳芳;史载祥;李恩.何首乌水提液对去卵巢大鼠骨组织的动态影响.中日友好医院学报,2006,20:217-221.
[110]朱飞鹏;王洪复;高林峰.补肾中药对肾脏1α羟化酶作用的探讨.上海中医药杂志,2003,37:42-44.
[111]胡锡琴;禚君;刘冠中;谢人明.二苯乙烯苷对大鼠骨密度与骨强度的影响.中医学报,2011,26:696-698.
[112] Denisova, N. A.; Cantuti-Castelvetri, I.; Hassan, W. N.; Paulson, K. E.; Joseph, J. A. Role ofmembrane lipids in regulation of vulnerability to oxidative stress in PC12cells: implication foraging. Free Radic Biol Med,2001,30:671-678.
[113] Fatokun, A. A.; Stone, T. W.; Smith, R. A. Hydrogen peroxide-induced oxidative stress inMC3T3-E1cells: The effects of glutamate and protection by purines. Bone,2006,39:542-551.
[114] Metrailler-Ruchonnet, I.; Pagano, A.; Carnesecchi, S.; Ody, C.; Donati, Y.; Barazzone, A. C.Bcl-2protects against hyperoxia-induced apoptosis through inhibition of themitochondria-dependent pathway. Free Radic Biol Med,2007,42:1062-1074.
[115] Tsujimoto, Y.; Croce, C. M. Analysis of the structure, transcripts, and protein products of bcl-2,the gene involved in human follicular lymphoma. Proc Natl Acad Sci U S A,1986,83:5214-5218.
[116] Hessle, L.; Johnson, K. A.; Anderson, H. C.; Narisawa, S.; Sali, A.; Goding, J. W.; Terkeltaub,R.; Millan, J. L. Tissue-nonspecific alkaline phosphatase and plasma cell membraneglycoprotein-1are central antagonistic regulators of bone mineralization. Proc Natl Acad Sci U SA,2002,99:9445-9449.
[117] Wagner, E. F.; Karsenty, G. Genetic control of skeletal development. Curr Opin Genet Dev,2001,11:527-532.
[118] Boyle, W. J.; Simonet, W. S.; Lacey, D. L. Osteoclast differentiation and activation. Nature,2003,423:337-342.
[119] Gallagher, J. C.; Sai, A. J. Molecular biology of bone remodeling: implications for newtherapeutic targets for osteoporosis. Maturitas,2010,65:301-307.
[120] Girasole, G.; Jilka, R. L.; Passeri, G.; Boswell, S.; Boder, G.; Williams, D. C.; Manolagas, S. C.17beta-estradiol inhibits interleukin-6production by bone marrow-derived stromal cells andosteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J ClinInvest,1992,89:883-891.
[121] Kurihara, N.; Bertolini, D.; Suda, T.; Akiyama, Y.; Roodman, G. D. IL-6stimulatesosteoclast-like multinucleated cell formation in long term human marrow cultures by inducingIL-1release. J Immunol,1990,144:4226-4230.
[122] Papanicolaou, D. A.; Vgontzas, A. N. Interleukin-6: the endocrine cytokine. J Clin EndocrinolMetab,2000,85:1331-1333.
[123] Baldwin, A. S. Control of oncogenesis and cancer therapy resistance by the transcription factorNF-kappaB. J Clin Invest,2001,107:241-246.
[124] Hughes, F. J.; Howells, G. L. Interleukin-6inhibits bone formation in vitro. Bone Miner,1993,21:21-28.
[125] Nielsen, F.; Mikkelsen, B. B.; Nielsen, J. B.; Andersen, H. R.; Grandjean, P. Plasmamalondialdehyde as biomarker for oxidative stress: reference interval and effects of life-stylefactors. Clin Chem,1997,43:1209-1214.
[126] Yang, P.; He, X. Q.; Peng, L.; Li, A. P.; Wang, X. R.; Zhou, J. W.; Liu, Q. Z. The role ofoxidative stress in hormesis induced by sodium arsenite in human embryo lung fibroblast (HELF)cellular proliferation model. J Toxicol Environ Health A,2007,70:976-983.
[127] Ning, J.; Grant, M. H. The role of reduced glutathione and glutathione reductase in thecytotoxicity of chromium (VI) in osteoblasts. Toxicol In Vitro,2000,14:329-335.
[128] Kinov, P.; Leithner, A.; Radl, R.; Bodo, K.; Khoschsorur, G. A.; Schauenstein, K.; Windhager,R. Role of free radicals in aseptic loosening of hip arthroplasty. J Orthop Res,2006,24:55-62.
[129] Yalin, S.; Sagir, O.; Comelekoglu, U.; Berkoz, M.; Eroglu, P. Strontium ranelate treatmentimproves oxidative damage in osteoporotic rat model. Pharmacol Rep,2012,64:396-402.
[130] Arslan, A.; Orkun, S.; Aydin, G.; Keles, I.; Tosun, A.; Arslan, M.; Caglayan, O. Effects ofovariectomy and ascorbic acid supplement on oxidative stress parameters and bone mineraldensity in rats. Libyan J Med,2011,6.
[131] Zhang, Y. B.; Zhong, Z. M.; Hou, G.; Jiang, H.; Chen, J. T. Involvement of oxidative stress inage-related bone loss. J Surg Res,2011,169:e37-e42.
[132] Makovey, J.; Macara, M.; Chen, J. S.; Hayward, C. S.; March, L.; Seibel, M. J.; Sambrook, P. N.Serum uric acid plays a protective role for bone loss in peri-and postmenopausal women: alongitudinal study. Bone,2013,52:400-406.
[2] Sendur, O. F.; Turan, Y.; Tastaban, E.; Serter, M. Antioxidant status in patients withosteoporosis: a controlled study. Joint Bone Spine,2009,76:514-518.
[3] Almeida, M.; Han, L.; Martin-Millan, M.; Plotkin, L. I.; Stewart, S. A.; Roberson, P. K.;Kousteni, S.; O'Brien, C. A.; Bellido, T.; Parfitt, A. M.; Weinstein, R. S.; Jilka, R. L.; Manolagas,S. C. Skeletal involution by age-associated oxidative stress and its acceleration by loss of sexsteroids. J Biol Chem,2007,282:27285-27297.
[4] Muthusami, S.; Ramachandran, I.; Muthusamy, B.; Vasudevan, G.; Prabhu, V.; Subramaniam,V.; Jagadeesan, A.; Narasimhan, S. Ovariectomy induces oxidative stress and impairs boneantioxidant system in adult rats. Clin Chim Acta,2005,360:81-86.
[5] Ozgocmen, S.; Kaya, H.; Fadillioglu, E.; Aydogan, R.; Yilmaz, Z. Role of antioxidant systems,lipid peroxidation, and nitric oxide in postmenopausal osteoporosis. Mol Cell Biochem,2007,295:45-52.
[6] Ozgocmen, S.; Kaya, H.; Fadillioglu, E.; Yilmaz, Z. Effects of calcitonin, risedronate, andraloxifene on erythrocyte antioxidant enzyme activity, lipid peroxidation, and nitric oxide inpostmenopausal osteoporosis. Arch Med Res,2007,38:196-205.
[7] Lean, J. M.; Jagger, C. J.; Kirstein, B.; Fuller, K.; Chambers, T. J. Hydrogen peroxide isessential for estrogen-deficiency bone loss and osteoclast formation. Endocrinology,2005,146:728-735.
[8] Jagger, C. J.; Lean, J. M.; Davies, J. T.; Chambers, T. J. Tumor necrosis factor-alpha mediatesosteopenia caused by depletion of antioxidants. Endocrinology,2005,146:113-118.
[9] Sanchez-Rodriguez, M. A.; Ruiz-Ramos, M.; Correa-Munoz, E.; Mendoza-Nunez, V. M.Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized byantioxidant enzymes. BMC Musculoskelet Disord,2007,8:124.
[10] Yalin, S.; Bagis, S.; Polat, G.; Dogruer, N.; Cenk, A. S.; Hatungil, R.; Erdogan, C. Is there a roleof free oxygen radicals in primary male osteoporosis?. Clin Exp Rheumatol,2005,23:689-692.
[11] Garrett, I. R.; Boyce, B. F.; Oreffo, R. O.; Bonewald, L.; Poser, J.; Mundy, G. R.Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro andin vivo. J Clin Invest,1990,85:632-639.
[12] Lee, N. K.; Choi, Y. G.; Baik, J. Y.; Han, S. Y.; Jeong, D. W.; Bae, Y. S.; Kim, N.; Lee, S. Y. Acrucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood,2005,106:852-859.
[13] Lean, J. M.; Davies, J. T.; Fuller, K.; Jagger, C. J.; Kirstein, B.; Partington, G. A.; Urry, Z. L.;Chambers, T. J. A crucial role for thiol antioxidants in estrogen-deficiency bone loss. J ClinInvest,2003,112:915-923.
[14] Koh, J. M.; Lee, Y. S.; Kim, Y. S.; Kim, D. J.; Kim, H. H.; Park, J. Y.; Lee, K. U.; Kim, G. S.Homocysteine enhances bone resorption by stimulation of osteoclast formation and activitythrough increased intracellular ROS generation. J Bone Miner Res,2006,21:1003-1011.
[15] Wittrant, Y.; Gorin, Y.; Woodruff, K.; Horn, D.; Abboud, H. E.; Mohan, S.; Abboud-Werner, S.L. High d(+)glucose concentration inhibits RANKL-induced osteoclastogenesis. Bone,2008,42:1122-1130.
[16] Hsu, H.; Lacey, D. L.; Dunstan, C. R.; Solovyev, I.; Colombero, A.; Timms, E.; Tan, H. L.;Elliott, G.; Kelley, M. J.; Sarosi, I.; Wang, L.; Xia, X. Z.; Elliott, R.; Chiu, L.; Black, T.; Scully,S.; Capparelli, C.; Morony, S.; Shimamoto, G.; Bass, M. B.; Boyle, W. J. Tumor necrosis factorreceptor family member RANK mediates osteoclast differentiation and activation induced byosteoprotegerin ligand. Proc Natl Acad Sci U S A,1999,96:3540-3545.
[17] Lacey, D. L.; Timms, E.; Tan, H. L.; Kelley, M. J.; Dunstan, C. R.; Burgess, T.; Elliott, R.;Colombero, A.; Elliott, G.; Scully, S.; Hsu, H.; Sullivan, J.; Hawkins, N.; Davy, E.; Capparelli,C.; Eli, A.; Qian, Y. X.; Kaufman, S.; Sarosi, I.; Shalhoub, V.; Senaldi, G.; Guo, J.; Delaney, J.;Boyle, W. J. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation andactivation. Cell,1998,93:165-176.
[18] Bai, X. C.; Lu, D.; Liu, A. L.; Zhang, Z. M.; Li, X. M.; Zou, Z. P.; Zeng, W. S.; Cheng, B. L.;Luo, S. Q. Reactive oxygen species stimulates receptor activator of NF-kappaB ligandexpression in osteoblast. J Biol Chem,2005,280:17497-17506.
[19] Grassi, F.; Tell, G.; Robbie-Ryan, M.; Gao, Y.; Terauchi, M.; Yang, X.; Romanello, M.; Jones,D. P.; Weitzmann, M. N.; Pacifici, R. Oxidative stress causes bone loss in estrogen-deficientmice through enhanced bone marrow dendritic cell activation. Proc Natl Acad Sci U S A,2007,104:15087-15092.
[20] Chen, J. R.; Shankar, K.; Nagarajan, S.; Badger, T. M.; Ronis, M. J. Protective effects ofestradiol on ethanol-induced bone loss involve inhibition of reactive oxygen species generationin osteoblasts and downstream activation of the extracellular signal-regulated kinase/signaltransducer and activator of transcription3/receptor activator of nuclear factor-kappaB ligandsignaling cascade. J Pharmacol Exp Ther,2008,324:50-59.
[21] Pittenger, M. F.; Mackay, A. M.; Beck, S. C.; Jaiswal, R. K.; Douglas, R.; Mosca, J. D.;Moorman, M. A.; Simonetti, D. W.; Craig, S.; Marshak, D. R. Multilineage potential of adulthuman mesenchymal stem cells. Science,1999,284:143-147.
[22] Liu, A. L.; Zhang, Z. M.; Zhu, B. F.; Liao, Z. H.; Liu, Z. Metallothionein protects bone marrowstromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation. CellBiol Int,2004,28:905-911.
[23] Bai, X. C.; Lu, D.; Bai, J.; Zheng, H.; Ke, Z. Y.; Li, X. M.; Luo, S. Q. Oxidative stress inhibitsosteoblastic differentiation of bone cells by ERK and NF-kappaB. Biochem Biophys ResCommun,2004,314:197-207.
[24] Arai, M.; Shibata, Y.; Pugdee, K.; Abiko, Y.; Ogata, Y. Effects of reactive oxygen species (ROS)on antioxidant system and osteoblastic differentiation in MC3T3-E1cells. Iubmb Life,2007,59:27-33.
[25] Abraham, N. G. Molecular regulation--biological role of heme in hematopoiesis. Blood Rev,1991,5:19-28.
[26] Khosla, S.; Riggs, B. L. Pathophysiology of age-related bone loss and osteoporosis. EndocrinolMetab Clin North Am,2005,34:1015-1030.
[27] Manolagas, S. C.; Parfitt, A. M. What old means to bone. Trends Endocrinol Metab,2010,21:369-374.
[28] Strotmeyer, E. S.; Cauley, J. A.; Schwartz, A. V.; Nevitt, M. C.; Resnick, H. E.; Bauer, D. C.;Tylavsky, F. A.; de Rekeneire, N.; Harris, T. B.; Newman, A. B. Nontraumatic fracture risk withdiabetes mellitus and impaired fasting glucose in older white and black adults: the health, aging,and body composition study. Arch Intern Med,2005,165:1612-1617.
[29] Abraham, N. G.; Lutton, J. D.; Levere, R. D. Heme metabolism and erythropoiesis in abnormaliron states: role of delta-aminolevulinic acid synthase and heme oxygenase. Exp Hematol,1985,13:838-843.
[30] Vanella, L.; Kim, D. H.; Asprinio, D.; Peterson, S. J.; Barbagallo, I.; Vanella, A.; Goldstein, D.;Ikehara, S.; Kappas, A.; Abraham, N. G. HO-1expression increases mesenchymal stemcell-derived osteoblasts but decreases adipocyte lineage. Bone,2010,46:236-243.
[31] Abraham, N. G.; Kushida, T.; McClung, J.; Weiss, M.; Quan, S.; Lafaro, R.; Darzynkiewicz, Z.;Wolin, M. Heme oxygenase-1attenuates glucose-mediated cell growth arrest and apoptosis inhuman microvessel endothelial cells. Circ Res,2003,93:507-514.
[32] Pileggi, A.; Molano, R. D.; Berney, T.; Cattan, P.; Vizzardelli, C.; Oliver, R.; Fraker, C.; Ricordi,C.; Pastori, R. L.; Bach, F. H.; Inverardi, L. Heme oxygenase-1induction in islet cells results inprotection from apoptosis and improved in vivo function after transplantation. Diabetes,2001,50:1983-1991.
[33] Brouard, S.; Otterbein, L. E.; Anrather, J.; Tobiasch, E.; Bach, F. H.; Choi, A. M.; Soares, M. P.Carbon monoxide generated by heme oxygenase1suppresses endothelial cell apoptosis. J ExpMed,2000,192:1015-1026.
[34] Barbagallo, I.; Vanella, A.; Peterson, S. J.; Kim, D. H.; Tibullo, D.; Giallongo, C.; Vanella, L.;Parrinello, N.; Palumbo, G. A.; Di Raimondo, F.; Abraham, N. G.; Asprinio, D. Overexpressionof heme oxygenase-1increases human osteoblast stem cell differentiation. J Bone Miner Metab,2010,28:276-288.
[35] Bennett, C. N.; Ouyang, H.; Ma, Y. L.; Zeng, Q.; Gerin, I.; Sousa, K. M.; Lane, T. F.; Krishnan,V.; Hankenson, K. D.; MacDougald, O. A. Wnt10b increases postnatal bone formation byenhancing osteoblast differentiation. J Bone Miner Res,2007,22:1924-1932.
[36] Andrade, A. C.; Nilsson, O.; Barnes, K. M.; baron J. Wnt gene expression in the post-natalgrowth plate: regulation with chondrocyte differentiation. Bone,2007,40:1361-1369.
[37] Linares, G. R.; Xing, W.; Govoni, K. E.; Chen, S. T.; Mohan, S. Glutaredoxin5regulatesosteoblast apoptosis by protecting against oxidative stress. Bone,2009,44:795-804.
[38] Almeida, M.; Han, L.; Ambrogini, E.; Bartell, S. M.; Manolagas, S. C. Oxidative stressstimulates apoptosis and activates NF-kappaB in osteoblastic cells via a PKCbeta/p66shcsignaling cascade: counter regulation by estrogens or androgens. Mol Endocrinol,2010,24:2030-2037.
[39] Byun, C. H.; Koh, J. M.; Kim, D. K.; Park, S. I.; Lee, K. U.; Kim, G. S. Alpha-lipoic acidinhibits TNF-alpha-induced apoptosis in human bone marrow stromal cells. J Bone Miner Res,2005,20:1125-1135.
[40] Krishnan, V.; Bryant, H. U.; Macdougald, O. A. Regulation of bone mass by Wnt signaling. JClin Invest,2006,116:1202-1209.
[41] Boyden, L. M.; Mao, J.; Belsky, J.; Mitzner, L.; Farhi, A.; Mitnick, M. A.; Wu, D.; Insogna, K.;Lifton, R. P. High bone density due to a mutation in LDL-receptor-related protein5. N Engl JMed,2002,346:1513-1521.
[42] Babij, P.; Zhao, W.; Small, C.; Kharode, Y.; Yaworsky, P. J.; Bouxsein, M. L.; Reddy, P. S.;Bodine, P. V.; Robinson, J. A.; Bhat, B.; Marzolf, J.; Moran, R. A.; Bex, F. High bone mass inmice expressing a mutant LRP5gene. J Bone Miner Res,2003,18:960-974.
[43] Holmen, S. L.; Giambernardi, T. A.; Zylstra, C. R.; Buckner-Berghuis, B. D.; Resau, J. H.; Hess,J. F.; Glatt, V.; Bouxsein, M. L.; Ai, M.; Warman, M. L.; Williams, B. O. Decreased BMD andlimb deformities in mice carrying mutations in both Lrp5and Lrp6. J Bone Miner Res,2004,19:2033-2040.
[44] Day, T. F.; Guo, X.; Garrett-Beal, L.; Yang, Y. Wnt/beta-catenin signaling in mesenchymalprogenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis.Dev Cell,2005,8:739-750.
[45] Hill, T. P.; Spater, D.; Taketo, M. M.; Birchmeier, W.; Hartmann, C. CanonicalWnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell,2005,8:727-738.
[46] Bennett, C. N.; Longo, K. A.; Wright, W. S.; Suva, L. J.; Lane, T. F.; Hankenson, K. D.;MacDougald, O. A. Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc NatlAcad Sci U S A,2005,102:3324-3329.
[47] Hu, H.; Hilton, M. J.; Tu, X.; Yu, K.; Ornitz, D. M.; Long, F. Sequential roles of Hedgehog andWnt signaling in osteoblast development. Development,2005,132:49-60.
[48] Glass, D. N.; Bialek, P.; Ahn, J. D.; Starbuck, M.; Patel, M. S.; Clevers, H.; Taketo, M. M.;Long, F.; McMahon, A. P.; Lang, R. A.; Karsenty, G. Canonical Wnt signaling in differentiatedosteoblasts controls osteoclast differentiation. Dev Cell,2005,8:751-764.
[49] Kawano, Y.; Kypta, R. Secreted antagonists of the Wnt signalling pathway. J Cell Sci,2003,116:2627-2634.
[50] Li, X.; Ominsky, M. S.; Niu, Q. T.; Sun, N.; Daugherty, B.; D'Agostin, D.; Kurahara, C.; Gao,Y.; Cao, J.; Gong, J.; Asuncion, F.; Barrero, M.; Warmington, K.; Dwyer, D.; Stolina, M.;Morony, S.; Sarosi, I.; Kostenuik, P. J.; Lacey, D. L.; Simonet, W. S.; Ke, H. Z.; Paszty, C.Targeted deletion of the sclerostin gene in mice results in increased bone formation and bonestrength. J Bone Miner Res,2008,23:860-869.
[51] Li, X.; Ominsky, M. S.; Warmington, K. S.; Morony, S.; Gong, J.; Cao, J.; Gao, Y.; Shalhoub,V.; Tipton, B.; Haldankar, R.; Chen, Q.; Winters, A.; Boone, T.; Geng, Z.; Niu, Q. T.; Ke, H. Z.;Kostenuik, P. J.; Simonet, W. S.; Lacey, D. L.; Paszty, C. Sclerostin antibody treatmentincreases bone formation, bone mass, and bone strength in a rat model of postmenopausalosteoporosis. J Bone Miner Res,2009,24:578-588.
[52] Bodine, P. V.; Zhao, W.; Kharode, Y. P.; Bex, F. J.; Lambert, A. J.; Goad, M. B.; Gaur, T.; Stein,G. S.; Lian, J. B.; Komm, B. S. The Wnt antagonist secreted frizzled-related protein-1is anegative regulator of trabecular bone formation in adult mice. Mol Endocrinol,2004,18:1222-1237.
[53] Bodine, P. V.; Stauffer, B.; Ponce-de-Leon, H.; Bhat, R. A.; Mangine, A.; Seestaller-Wehr, L.M.; Moran, R. A.; Billiard, J.; Fukayama, S.; Komm, B. S.; Pitts, K.; Krishnamurthy, G.;Gopalsamy, A.; Shi, M.; Kern, J. C.; Commons, T. J.; Woodworth, R. P.; Wilson, M. A.;Welmaker, G. S.; Trybulski, E. J.; Moore, W. J. A small molecule inhibitor of the Wntantagonist secreted frizzled-related protein-1stimulates bone formation. Bone,2009,44:1063-1068.
[54] Morvan, F.; Boulukos, K.; Clement-Lacroix, P.; Roman, R. S.; Suc-Royer, I.; Vayssiere, B.;Ammann, P.; Martin, P.; Pinho, S.; Pognonec, P.; Mollat, P.; Niehrs, C.; baron R; Rawadi, G.Deletion of a single allele of the Dkk1gene leads to an increase in bone formation and bonemass. J Bone Miner Res,2006,21:934-945.
[55] Glantschnig, H.; Hampton, R. A.; Lu, P.; Zhao, J. Z.; Vitelli, S.; Huang, L.; Haytko, P.; Cusick,T.; Ireland, C.; Jarantow, S. W.; Ernst, R.; Wei, N.; Nantermet, P.; Scott, K. R.; Fisher, J. E.;Talamo, F.; Orsatti, L.; Reszka, A. A.; Sandhu, P.; Kimmel, D.; Flores, O.; Strohl, W.; An, Z.;Wang, F. Generation and selection of novel fully human monoclonal antibodies that neutralizeDickkopf-1(DKK1) inhibitory function in vitro and increase bone mass in vivo. J Biol Chem,2010,285:40135-40147.
[56] Zanotti, S.; Canalis, E. Notch and the skeleton. Mol Cell Biol,2010,30:886-896.
[57] Nam, Y.; Sliz, P.; Song, L.; Aster, J. C.; Blacklow, S. C. Structural basis for cooperativity inrecruitment of MAML coactivators to Notch transcription complexes. Cell,2006,124:973-983.
[58] Deregowski, V.; Gazzerro, E.; Priest, L.; Rydziel, S.; Canalis, E. Notch1overexpression inhibitsosteoblastogenesis by suppressing Wnt/beta-catenin but not bone morphogenetic proteinsignaling. J Biol Chem,2006,281:6203-6210.
[59] Zanotti, S.; Smerdel-Ramoya, A.; Stadmeyer, L.; Durant, D.; Radtke, F.; Canalis, E. Notchinhibits osteoblast differentiation and causes osteopenia. Endocrinology,2008,149:3890-3899.
[60] Hilton, M. J.; Tu, X.; Wu, X.; Bai, S.; Zhao, H.; Kobayashi, T.; Kronenberg, H. M.; Teitelbaum,S. L.; Ross, F. P.; Kopan, R.; Long, F. Notch signaling maintains bone marrow mesenchymalprogenitors by suppressing osteoblast differentiation. Nat Med,2008,14:306-314.
[61] Engin, F.; Yao, Z.; Yang, T.; Zhou, G.; Bertin, T.; Jiang, M. M.; Chen, Y.; Wang, L.; Zheng, H.;Sutton, R. E.; Boyce, B. F.; Lee, B. Dimorphic effects of Notch signaling in bone homeostasis.Nat Med,2008,14:299-305.
[62] Zamurovic, N.; Cappellen, D.; Rohner, D.; Susa, M. Coordinated activation of notch, Wnt, andtransforming growth factor-beta signaling pathways in bone morphogenic protein2-inducedosteogenesis. Notch target gene Hey1inhibits mineralization and Runx2transcriptional activity.J Biol Chem,2004,279:37704-37715.
[63] Zanotti, S.; Smerdel-Ramoya, A.; Canalis, E. Reciprocal regulation of Notch and nuclear factorof activated T-cells (NFAT) c1transactivation in osteoblasts. J Biol Chem,2011,286:4576-4588.
[64] Isidor, B.; Lindenbaum, P.; Pichon, O.; Bezieau, S.; Dina, C.; Jacquemont, S.; Martin-Coignard,D.; Thauvin-Robinet, C.; Le, M. M.; Mandel, J. L.; David, A.; Faivre, L.; Cormier-Daire, V.;Redon, R.; Le, C. C. Truncating mutations in the last exon of NOTCH2cause a rare skeletaldisorder with osteoporosis. Nat Genet,2011,43:306-308.
[65] Simpson, M. A.; Irving, M. D.; Asilmaz, E.; Gray, M. J.; Dafou, D.; Elmslie, F. V.; Mansour, S.;Holder, S. E.; Brain, C. E.; Burton, B. K.; Kim, K. H.; Pauli, R. M.; Aftimos, S.; Stewart, H.;Kim, C. A.; Holder-Espinasse, M.; Robertson, S. P.; Drake, W. M.; Trembath, R. C. Mutationsin NOTCH2cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss. NatGenet,2011,43:303-305.
[66] Matsuo, K. Eph and ephrin interactions in bone. Adv Exp Med Biol,2010,658:95-103.
[67] Zhao, C.; Irie, N.; Takada, Y.; Shimoda, K.; Miyamoto, T.; Nishiwaki, T.; Suda, T.; Matsuo, K.Bidirectional ephrinB2-EphB4signaling controls bone homeostasis. Cell Metab,2006,4:111-121.
[68] Allan, E. H.; Hausler, K. D.; Wei, T.; Gooi, J. H.; Quinn, J. M.; Crimeen-Irwin, B.; Pompolo, S.;Sims, N. A.; Gillespie, M. T.; Onyia, J. E.; Martin, T. J. EphrinB2regulation by PTH and PTHrPrevealed by molecular profiling in differentiating osteoblasts. J Bone Miner Res,2008,23:1170-1181.
[69] Xing, W.; Kim, J.; Wergedal, J.; Chen, S. T.; Mohan, S. Ephrin B1regulates bone marrowstromal cell differentiation and bone formation by influencing TAZ transactivation via complexformation with NHERF1. Mol Cell Biol,2010,30:711-721.
[70] Irie, N.; Takada, Y.; Watanabe, Y.; Matsuzaki, Y.; Naruse, C.; Asano, M.; Iwakura, Y.; Suda, T.;Matsuo, K. Bidirectional signaling through ephrinA2-EphA2enhances osteoclastogenesis andsuppresses osteoblastogenesis. J Biol Chem,2009,284:14637-14644.
[71] Dvorak, M. M.; Siddiqua, A.; Ward, D. T.; Carter, D. H.; Dallas, S. L.; Nemeth, E. F.; Riccardi,D. Physiological changes in extracellular calcium concentration directly control osteoblastfunction in the absence of calciotropic hormones. Proc Natl Acad Sci U S A,2004,101:5140-5145.
[72] Chattopadhyay, N.; Yano, S.; Tfelt-Hansen, J.; Rooney, P.; Kanuparthi, D.; Bandyopadhyay, S.;Ren, X.; Terwilliger, E.; Brown, E. M. Mitogenic action of calcium-sensing receptor on ratcalvarial osteoblasts. Endocrinology,2004,145:3451-3462.
[73] Kameda, T.; Mano, H.; Yamada, Y.; Takai, H.; Amizuka, N.; Kobori, M.; Izumi, N.;Kawashima, H.; Ozawa, H.; Ikeda, K.; Kameda, A.; Hakeda, Y.; Kumegawa, M.Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. BiochemBiophys Res Commun,1998,245:419-422.
[74] Brown, E. M.; MacLeod, R. J. Extracellular calcium sensing and extracellular calcium signaling.Physiol Rev,2001,81:239-297.
[75] Canalis, E. Novel treatments for osteoporosis. J Clin Invest,2000,106:177-179.
[76] Marie, P. J. The calcium-sensing receptor in bone cells: a potential therapeutic target inosteoporosis. Bone,2010,46:571-576.
[77] Canalis, E.; Giustina, A.; Bilezikian, J. P. Mechanisms of anabolic therapies for osteoporosis. NEngl J Med,2007,357:905-916.
[78] Pacifici, R. T cells: critical bone regulators in health and disease. Bone,2010,47:461-471.
[79] Gao, Y.; Wu, X.; Terauchi, M.; Li, J. Y.; Grassi, F.; Galley, S.; Yang, X.; Weitzmann, M. N.;Pacifici, R. T cells potentiate PTH-induced cortical bone loss through CD40L signaling. CellMetab,2008,8:132-145.
[80] Tawfeek, H.; Bedi, B.; Li, J. Y.; Adams, J.; Kobayashi, T.; Weitzmann, M. N.; Kronenberg, H.M.; Pacifici, R. Disruption of PTH receptor1in T cells protects against PTH-induced bone loss.PLoS One,2010,5:e12290.
[81] Mann, V.; Huber, C.; Kogianni, G.; Collins, F.; Noble, B. The antioxidant effect of estrogen andSelective Estrogen Receptor Modulators in the inhibition of osteocyte apoptosis in vitro. Bone,2007,40:674-684.
[82] Valverde, P. Pharmacotherapies to manage bone loss-associated diseases: a quest for the perfectbenefit-to-risk ratio. Curr Med Chem,2008,15:284-304.
[83] Deyhim, F.; Mandadi, K.; Patil, B. S.; Faraji, B. Grapefruit pulp increases antioxidant status andimproves bone quality in orchidectomized rats. Nutrition,2008,24:1039-1044.
[84] Scalbert, A.; Manach, C.; Morand, C.; Remesy, C.; Jimenez, L. Dietary polyphenols and theprevention of diseases. Crit Rev Food Sci Nutr,2005,45:287-306.
[85] Houde, V.; Grenier, D.; Chandad, F. Protective effects of grape seed proanthocyanidins againstoxidative stress induced by lipopolysaccharides of periodontopathogens. J Periodontol,2006,77:1371-1379.
[86] Ostman, B.; Michaelsson, K.; Helmersson, J.; Byberg, L.; Gedeborg, R.; Melhus, H.; Basu, S.Oxidative stress and bone mineral density in elderly men: antioxidant activity ofalpha-tocopherol. Free Radic Biol Med,2009,47:668-673.
[87]吕丽爽;汤坚;何淇傥.二苯乙烯苷特性及DPPH自由基清除活性.哈尔滨工业大学学报,2009,252-255.
[88]刘厚淳;陈万生.何首乌水溶性成分2,3,5,4’-四羟基二苯乙烯-2-O-β-D葡萄糖苷的体外抗氧化作用研究.药学实践杂志,2000.
[89]王栋;陈晓宇;刘晓利;赵欢;张腾腾;赵中原;白璐.二苯乙烯苷对沙鼠脑缺血/再灌注损伤的保护作用及部分机制.安徽医科大学学报,2012,47:379-382.
[90]袁惠琼;杨杰;曾进;郭荣静.二苯乙烯苷对老龄大鼠脑缺血再灌注后HIF-1α及EPO表达的影响.中风与神经疾病杂志,2011,28:868-871.
[91]张玲玲;陈磊;陶丽珍;李雪芬;张磊;陈良为;陈建宗.二苯乙烯苷对MPTP诱导的帕金森病小鼠模型黑质多巴胺转运体的影响.神经解剖学杂志,2012,28:33-37.
[92] Tao, L.; Li, X.; Zhang, L.; Tian, J.; Li, X.; Sun, X.; Li, X.; Jiang, L.; Zhang, X.; Chen, J.Protective effect of tetrahydroxystilbene glucoside on6-OHDA-induced apoptosis in PC12cellsthrough the ROS-NO pathway. PLoS One,2011,6:e26055.
[93] Sun, F. L.; Zhang, L.; Zhang, R. Y.; Li, L. Tetrahydroxystilbene glucoside protects humanneuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity. Eur J Pharmacol,2011,660:283-290.
[94] Qin, R.; Li, X.; Li, G.; Tao, L.; Li, Y.; Sun, J.; Kang, X.; Chen, J. Protection bytetrahydroxystilbene glucoside against neurotoxicity induced by MPP+: the involvement ofPI3K/Akt pathway activation. Toxicol Lett,2011,202:1-7.
[95] Li, X.; Li, Y.; Chen, J.; Sun, J.; Li, X.; Sun, X.; Kang, X. Tetrahydroxystilbene glucosideattenuates MPP+-induced apoptosis in PC12cells by inhibiting ROS generation and modulatingJNK activation. Neurosci Lett,2010,483:1-5.
[96] Hou, Y.; Yang, Q.; Zhou, L.; Du X; Li, M.; Yuan, M.; Zhou, Z.; Li, Z. Tetrahydroxystilbeneglucoside improves learning and (or) memory ability of aged rats and may be connected to theAPP pathway. Can J Physiol Pharmacol,2011,89:801-809.
[97]甘受益;黄俊;李宾;葛雄;李彩蓉;蔡飞.二苯乙烯苷对实验性糖尿病大鼠心脏血流动力学和氧化应激的影响.现代中西医结合杂志,2012,21:30-32.
[98]相聪坤;王蕊;袁志芳.何首乌二苯乙烯苷类提取物对高脂血症大鼠血脂代谢的影响及其抗氧化作用.中国药业,2009,18:19-20.
[99]张燕堂;张颖;郑文;杨华;王家富;商战平.二苯乙烯苷对LPC损伤血管内皮细胞的保护作用及其机制.中国病理生理杂志,2012,28:1192-1196.
[100]张彩平;杨滢;田英;乔新惠;龙石银;陈志军;田汝芳.二苯乙烯苷对H2O2诱导血管内皮细胞黏附分子表达的影响.中国药理学通报,2012,28:1088-1092.
[101] Xu, X. L.; Huang, Y. J.; Wang, Y. Q.; Chen, X. F.; Zhang, W.2,3,4',5-Tetrahydroxystilbene-2-O-beta-d-glucoside inhibits platelet-derived growth factor-induced proliferation of vascular smooth muscle cells by regulating the cell cycle. Clin ExpPharmacol Physiol,2011,38:307-313.
[102] Zhang, W.; Xu, X. L.; Wang, Y. Q.; Wang, C. H.; Zhu, W. Z. Effects of2,3,4',5-tetrahydroxystilbene2-O-beta-D-glucoside on vascular endothelial dysfunction inatherogenic-diet rats. Planta Med,2009,75:1209-1214.
[103]李军;王国荣;王燕;解砚英;牟艳玲;姚庆强.二苯乙烯苷对同型半胱氨酸诱导的人脐静脉内皮细胞MCP-1、ICAM-1和VCAM-1表达的影响.中国动脉硬化杂志,2012,20:890-894.
[104]张园;董林;许晓乐;王玉琴;张伟.二苯乙烯苷对2型糖尿病大鼠氧化应激、炎症反应及主动脉细胞凋亡相关基因的影响.中国糖尿病杂志,2012,20:775-778.
[105]李佳;贾延龙;唐婧;刘青;董又娟;胡九凡;李彩蓉;蔡飞.二苯乙烯苷对糖尿病大鼠肾脏损伤的干预作用.咸宁学院学报：医学版,2011,25:277-279.
[106] Li, C.; Cai, F.; Yang, Y.; Zhao, X.; Wang, C.; Li, J.; Jia, Y.; Tang, J.; Liu, Q.Tetrahydroxystilbene glucoside ameliorates diabetic nephropathy in rats: involvement of SIRT1and TGF-beta1pathway. Eur J Pharmacol,2010,649:382-389.
[107] Zeng, C.; Xiao, J. H.; Chang, M. J.; Wang, J. L. Beneficial effects of THSG on aceticacid-induced experimental colitis: involvement of upregulation of PPAR-gamma and inhibitionof the Nf-Kappab inflammatory pathway. Molecules,2011,16:8552-8568.
[108] Wang, X.; Zhao, L.; Han, T.; Chen, S.; Wang, J. Protective effects of2,3,5,4'-tetrahydroxystilbene-2-O-beta-d-glucoside, an active component of Polygonummultiflorum Thunb, on experimental colitis in mice. Eur J Pharmacol,2008,578:339-348.
[109]张海啸;尹智炜;李芳芳;史载祥;李恩.何首乌水提液对去卵巢大鼠骨组织的动态影响.中日友好医院学报,2006,20:217-221.
[110]朱飞鹏;王洪复;高林峰.补肾中药对肾脏1α羟化酶作用的探讨.上海中医药杂志,2003,37:42-44.
[111]胡锡琴;禚君;刘冠中;谢人明.二苯乙烯苷对大鼠骨密度与骨强度的影响.中医学报,2011,26:696-698.
[112] Denisova, N. A.; Cantuti-Castelvetri, I.; Hassan, W. N.; Paulson, K. E.; Joseph, J. A. Role ofmembrane lipids in regulation of vulnerability to oxidative stress in PC12cells: implication foraging. Free Radic Biol Med,2001,30:671-678.
[113] Fatokun, A. A.; Stone, T. W.; Smith, R. A. Hydrogen peroxide-induced oxidative stress inMC3T3-E1cells: The effects of glutamate and protection by purines. Bone,2006,39:542-551.
[114] Metrailler-Ruchonnet, I.; Pagano, A.; Carnesecchi, S.; Ody, C.; Donati, Y.; Barazzone, A. C.Bcl-2protects against hyperoxia-induced apoptosis through inhibition of themitochondria-dependent pathway. Free Radic Biol Med,2007,42:1062-1074.
[115] Tsujimoto, Y.; Croce, C. M. Analysis of the structure, transcripts, and protein products of bcl-2,the gene involved in human follicular lymphoma. Proc Natl Acad Sci U S A,1986,83:5214-5218.
[116] Hessle, L.; Johnson, K. A.; Anderson, H. C.; Narisawa, S.; Sali, A.; Goding, J. W.; Terkeltaub,R.; Millan, J. L. Tissue-nonspecific alkaline phosphatase and plasma cell membraneglycoprotein-1are central antagonistic regulators of bone mineralization. Proc Natl Acad Sci U SA,2002,99:9445-9449.
[117] Wagner, E. F.; Karsenty, G. Genetic control of skeletal development. Curr Opin Genet Dev,2001,11:527-532.
[118] Boyle, W. J.; Simonet, W. S.; Lacey, D. L. Osteoclast differentiation and activation. Nature,2003,423:337-342.
[119] Gallagher, J. C.; Sai, A. J. Molecular biology of bone remodeling: implications for newtherapeutic targets for osteoporosis. Maturitas,2010,65:301-307.
[120] Girasole, G.; Jilka, R. L.; Passeri, G.; Boswell, S.; Boder, G.; Williams, D. C.; Manolagas, S. C.17beta-estradiol inhibits interleukin-6production by bone marrow-derived stromal cells andosteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J ClinInvest,1992,89:883-891.
[121] Kurihara, N.; Bertolini, D.; Suda, T.; Akiyama, Y.; Roodman, G. D. IL-6stimulatesosteoclast-like multinucleated cell formation in long term human marrow cultures by inducingIL-1release. J Immunol,1990,144:4226-4230.
[122] Papanicolaou, D. A.; Vgontzas, A. N. Interleukin-6: the endocrine cytokine. J Clin EndocrinolMetab,2000,85:1331-1333.
[123] Baldwin, A. S. Control of oncogenesis and cancer therapy resistance by the transcription factorNF-kappaB. J Clin Invest,2001,107:241-246.
[124] Hughes, F. J.; Howells, G. L. Interleukin-6inhibits bone formation in vitro. Bone Miner,1993,21:21-28.
[125] Nielsen, F.; Mikkelsen, B. B.; Nielsen, J. B.; Andersen, H. R.; Grandjean, P. Plasmamalondialdehyde as biomarker for oxidative stress: reference interval and effects of life-stylefactors. Clin Chem,1997,43:1209-1214.
[126] Yang, P.; He, X. Q.; Peng, L.; Li, A. P.; Wang, X. R.; Zhou, J. W.; Liu, Q. Z. The role ofoxidative stress in hormesis induced by sodium arsenite in human embryo lung fibroblast (HELF)cellular proliferation model. J Toxicol Environ Health A,2007,70:976-983.
[127] Ning, J.; Grant, M. H. The role of reduced glutathione and glutathione reductase in thecytotoxicity of chromium (VI) in osteoblasts. Toxicol In Vitro,2000,14:329-335.
[128] Kinov, P.; Leithner, A.; Radl, R.; Bodo, K.; Khoschsorur, G. A.; Schauenstein, K.; Windhager,R. Role of free radicals in aseptic loosening of hip arthroplasty. J Orthop Res,2006,24:55-62.
[129] Yalin, S.; Sagir, O.; Comelekoglu, U.; Berkoz, M.; Eroglu, P. Strontium ranelate treatmentimproves oxidative damage in osteoporotic rat model. Pharmacol Rep,2012,64:396-402.
[130] Arslan, A.; Orkun, S.; Aydin, G.; Keles, I.; Tosun, A.; Arslan, M.; Caglayan, O. Effects ofovariectomy and ascorbic acid supplement on oxidative stress parameters and bone mineraldensity in rats. Libyan J Med,2011,6.
[131] Zhang, Y. B.; Zhong, Z. M.; Hou, G.; Jiang, H.; Chen, J. T. Involvement of oxidative stress inage-related bone loss. J Surg Res,2011,169:e37-e42.
[132] Makovey, J.; Macara, M.; Chen, J. S.; Hayward, C. S.; March, L.; Seibel, M. J.; Sambrook, P. N.Serum uric acid plays a protective role for bone loss in peri-and postmenopausal women: alongitudinal study. Bone,2013,52:400-406.