【作者】 张金康；

【导师】 刘建； 杨柳； 孟国林；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【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H₂O₂分别作用2h、6h、12h和24h，MTT法评价细胞存活情况，筛选合适H₂O₂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H₂O₂共同作用，实验分组：①空白对照组；②单纯H₂O₂处理组；③H₂O₂+TSG0.1μM处理组；④H₂O₂+TSG1μM处理组；⑤H₂O₂+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H₂O₂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H₂O₂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、H₂O₂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H₂O₂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H₂O₂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H₂O₂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H₂O₂处理组；③H₂O₂+TSG0.1μM处理组；④H₂O₂+TSG1μM处理组；⑤H₂O₂+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H₂O₂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H₂O₂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H₂O₂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H₂O₂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。更多还原

【Abstract】 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 H₂O₂induced cytotoxicity in MC3T3-E1cells and clarified part of the relatedmechanism; then, we evaluated the protective effect of TSG on osteoblast dysfunctioninduced by H₂O₂; 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 mechanismObjective: 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 H₂O₂（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 H₂O₂. 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 H₂O₂and TSG were usedin the following studies. The experiment was divided into6groups, including the controlgroup, H₂O₂, H₂O₂+TSG（0.1μM）, H₂O₂+TSG（1μM）, H₂O₂+TSG（10μM） and H₂O₂+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 H₂O₂was300μM/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, H₂O₂alone 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 H₂O₂alone 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, H₂O₂alone 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 mechanismObjective: 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, H₂O₂, H₂O₂+TSG（0.1μM）, H₂O₂+TSG（1μM）, H₂O₂+TSG（10μM） andH₂O₂+NAC（1mM）. After induction for6,14or21days, MC3T3-E1cells werepre-incubated with different concentrations of TSG for24h before treatment with300μMH₂O₂for 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 H₂O₂group 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 H₂O₂treatment （P<0.05） and this effect wasobviously reversed by pre-incubated with TSG （1-10μM）. The results from ELISA assayindicated that H₂O₂raised 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 miceObjective: 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.更多还原