新橙皮苷对RANKL诱导的破骨细胞分化和去卵巢小鼠骨质疏松的影响

发布时间：2018-06-02 14:31

本文选题：骨质疏松 + 新橙皮苷　；参考：《广西医科大学》2015年博士论文

【摘要】：骨质疏松症(osteoporosis)是一种全身性骨骼疾病,它的特点为骨组织微结构破坏,骨量减少,骨强度下降,骨脆性增加。骨质疏松性骨折在骨质疏松症患者中极易发生,即在受到轻微的创伤或日常活动中即可发生骨折,容易出现严重后果,给家庭和社会造成负担。骨重建是骨生成和骨代谢最重要的过程,由成骨细胞所致的骨形成和破骨细胞所致的骨吸收两种作用之间的平衡进行调节。长期和过度的骨吸收打破这种平衡,出现骨吸收增多引起的疾病,如骨质疏松症。破骨细胞过度的骨吸收在骨质疏松症中起关键作用。因此,抑制破骨细胞的形成或活性可以作为治疗骨质疏松的一个有效途径。破骨细胞由骨髓巨噬细胞(BMM)分化而来,核因子-κB受体活化因子配体/核因子-κB受体活化因子(RANKL/RANK)信号通路与破骨细胞的分化和成熟密切相关。核因子-κB (NF-κB)属于二聚体转录因子,是细胞增殖和分化的重要调节因子,与IκB-α抑制蛋白结合并保持在无活性状态。外部刺激使IκB-α蛋白降解,激活NF-κB并进入细胞核,与相应基因的启动子和增强子区的KB序列结合启动转录。活化T细胞核因子细胞质1(NFATcl)介导由基因表达程序导致的细胞融合和破骨细胞分化,是破骨细胞分化的主转录因子。NF-κB和NFAT的活化相互影响,NF-κB和NFAT的表达量能反映RANKL/RANK信号通路激活情况。抗酒石酸酸性磷酸酶5b(TRACP5b)来源于破骨细胞,是破骨细胞和骨吸收的标记物,能将骨基质降解产物从吸收陷窝转移到功能性分泌区域,破坏有机骨基质成分。组织蛋白酶K (Cathepsin K)对Ⅰ型和Ⅱ型胶原均有切割作用,是骨生理和病理降解中主要的溶胶原蛋白酶,在破骨细胞中大量表达,对骨吸收中起到特殊作用。细胞内钙(Ca2+)振荡是兴奋和非兴奋细胞信号传导中的普遍存在的模式。RANKL可引起Ca2+振荡,导致钙调磷酸酶介导的NFATc1激活,触发破骨细胞的分化过程中持续的TNFATcl依赖的转录程序,促进破骨细胞分化。目前认为去卵巢(OVX)骨丢失小鼠模型与人类的情况相似,可以用于研究产生骨丢失的原因并进行干预,适合用于预防骨质疏松潜在治疗药物的评价。去卵巢小鼠卵巢切除后骨矿物质密度降低和骨微结构受损,非常适合作为绝经后骨质疏松症模型来进行研究。新橙皮苷是枳实的提取物,属于黄酮类化合物,是天然的抗氧化剂,具有抗炎,抗肿瘤和保护心血管系统的作用。但其在抑制骨质疏松作用方面,尚未见有相关研究。在本组实验中,我们进行体外实验,通过一系列实验分析经过新橙皮苷对RANKL诱导的破骨细胞分化的抑制作用。包括通过剂量依赖性实验观察破骨细胞分化的数量；用流式细胞仪对经过新橙皮苷处理的Raw264.7细胞进行细胞凋亡分析；通过qPCR实验对破骨细胞表达的TRACP及Cathepsin K进行测定；通过Luciferase和western blot实验对 RANKL/RANK信号通路中的NF-κB、NFAT和IκB-α进行分析；用羟基磷灰石检测破骨细胞的骨吸收作用；通过钙振荡实验对破骨细胞的分化进行分析。我们还进行了体内实验,观察新橙皮苷对去卵巢小鼠模型(OVX)骨质影响情况,结果分别用骨体积分数(BV/TV)、骨小梁厚度(Tb.Th)、骨小梁数(Tb.N)和骨小梁分离度(Tb.Sp)这四个参数进行分析。本实验包括如下两个部分第一部分新橙皮苷对RANKL诱导的破骨细胞分化的影响目的：研究新橙皮苷对体外RANKL诱导的破骨细胞的增殖分化、调节因子 NF-κB、NFAT、IκB-α、TRACP、Cathepsin K以及破骨细胞骨吸收作用和钙振荡的影响,探讨新橙皮苷对破骨细胞增殖分化以及对破骨细胞在骨代谢过程中一系列调节因子的作用机制。方法：在体外,BMM细胞用RANKL诱导,加入不同浓度的新橙皮苷进行培养,用TRAP染色,在光学显微镜下观察不同浓度新橙皮苷培养的TRAP染色阳性的破骨细胞并计数；RAW264.7细胞用RANKL诱导,并加入不同浓度的新橙皮苷培养,用流式细胞仪进行细胞凋亡分析,观察健康细胞的百分率；BMM细胞用RANKL诱导,加入10μM新橙皮苷培养,用qPCR实验对破骨细胞的TRACP及 Cathepsin K表达量进行测定；BMM细胞在含有羟基磷灰石涂层的培养基中用RANKL诱导,加入10μM新橙皮苷培养,观察羟基磷灰石涂层的吸收面积并用ImageJ软件分析；经p-NF-κB-TA-Luc转染的P3K-Luc细胞和p-NFAT-TA-Luc转染的Raw264.7细胞用RANKL诱导,加入不同浓度的新橙皮苷培养,通过Luciferase实验,用BMG Polar Star Optima荧光阅读器检测NF-κB和NFAT的表达量;BMM细胞用RANKL诱导,加入10μM的新橙皮苷培养,通过western blot实验对Iκ8-α和NFAT的表达量分别进行短时间和长时间检测,用ImageJ软件进行分析；BMM细胞用RANKL诱导,加入10μM新橙皮苷培养,通过倒置荧光显微镜对细胞钙振荡的平均峰值高度进行观察,并用Nikon Basic Research软件进行分析。结果：新橙皮苷对破骨细胞分化均有抑制作用,且浓度越大,对破骨细胞分化的抑制作用越大；在不同浓度的新橙皮苷作用下,细胞健康率均保持于90%以上的高水平,而坏死率和凋亡率均维持在低水平,不随新橙皮苷浓度的增加而增加；新橙皮苷均能抑制TRACP 及 Cathepsin K的表达；新橙皮苷能抑制破骨细胞对羟基磷灰石涂层的吸收作用；新橙皮苷能抑制NF-κB 和 NFAT的表达,且具有浓度依赖性；新橙皮苷能抑制IκB-α的降解；新橙皮苷对破骨细胞钙振荡有抑制作用。结论：新橙皮苷能从多方面抑制破骨细胞的分化,抑制破骨细胞在骨代谢中的作用,是安全有效的破骨细胞抑制剂。第二部分新橙皮苷对去卵巢小鼠骨质疏松的影响目的：研究新橙皮苷对去卵巢小鼠骨质疏松模型的骨体积分数、骨小梁厚度、骨小梁数和骨小梁分离度的影响,探讨新橙皮苷对骨质疏松的治疗作用。方法：6周龄C57BL6J雌性小鼠30只,随机分为假手术组,模型组,雌激素组,新橙皮苷低剂量组和新橙皮苷高剂量组,每组6只。每只小鼠称重,予10%水合氯醛腹腔内注射麻醉,假手术组仅切开下腹部两侧的腹部皮肤和肌层,不摘除卵巢即分层关闭腹腔。其余各组在腹腔两侧找到卵巢后,予以结扎并完整切除,分层关闭腹腔。术后各组予庆大霉素抗炎,予水和食物喂养观察1周后开始隔天按小鼠体重腹腔注射给药。假手术组和模型组予生理盐水,雌激素组予0.1mg/kg雌激素,新橙皮苷低剂量和高剂量组分别予3mg/kg 和 6mg/kg新橙皮苷。7周后,每只小鼠予乙醚麻醉,取单侧胫骨浸泡于4%FPA溶液中,20-24小时后取出,1XPBS溶液冲洗2次,用1XPBS侵泡胫骨保存于1.5m1试管中,行Micro-CT分析,对比观察各组骨体积分数、骨小梁厚度、骨小梁数和骨小梁分离度的变化。结果：新橙皮苷组增加去卵巢骨质疏松小鼠的骨体积分数,基本恢复到正常水平,效果与雌激素组相当,且有浓度依赖性,浓度越大,骨体积分数越明显；各组的骨小梁厚度无明显变化；新橙皮苷高剂量组能减少去卵巢骨质疏松小鼠的骨小梁分离度,且基本恢复正常水平,效果与雌激素相当；新橙皮苷能增加去卵巢骨质疏松小鼠的骨小梁数,有浓度依赖性,高剂量的新橙皮苷与雌激素的效果相当,使骨小梁数基本恢复正常水平。结论：新橙皮苷能抑制去卵巢小鼠骨质疏松的发展,是治疗骨质疏松症的潜在药物。
[Abstract]:Osteoporosis (osteoporosis) is a systemic skeletal disease characterized by microstructural destruction of bone tissue, reduced bone mass, reduced bone strength, and increased bone brittleness. Osteoporotic fractures are very prone to occur in patients with osteoporosis, that is, fractures may occur in mild trauma or daily activities, and severe consequences are likely to occur. Family and society make burdens. Bone reconstruction is the most important process of bone formation and bone metabolism. The balance between the two effects of osteogenesis caused by osteoblasts and osteoclast caused by osteoclast is regulated. Long and excessive bone resorption breaks this balance and causes diseases caused by increasing bone absorption, such as osteoporosis. Osteoclast. Excessive bone resorption plays a key role in osteoporosis. Therefore, inhibition of osteoclast formation or activity can be an effective way to treat osteoporosis. Osteoclasts are differentiated from bone marrow macrophages (BMM), and nuclear factor kappa B receptor activator ligand / nuclear factor kappa B receptor activating factor (RANKL/RANK) signaling pathway It is closely related to the differentiation and maturation of osteoclasts. Nuclear factor kappa B (NF- kappa B) belongs to the two polymer transcription factor, which is an important regulator of cell proliferation and differentiation, which combines with I kappa B- alpha suppressor and remains inactive. External stimuli degrade the I kappa B- alpha protein, activate NF- kappa B and enter the nucleus, and increase the promoter and increase of the corresponding genes. The KB sequence of the hadron region combined with the activation of transcription. Activated T nuclear factor cytoplasm 1 (NFATcl) mediates the cell fusion and osteoclast differentiation caused by the gene expression program, which is the activation interaction of the main transcription factor.NF- kappa B and NFAT of the osteoclast differentiation. The expression of NF- kappa B and NFAT can reflect the activation of RANKL/RANK signaling pathway. The acid phosphatase 5b (TRACP5b) is derived from osteoclasts and is a marker of osteoclast and bone absorption. It can transfer the degradation products of bone matrix from the absorption lacunar to the functional secretory region and destroy the organic matrix components. Cathepsin K (Cathepsin K) has a cutting effect on type I and type II collagen, which is the bone physiological and pathological degradation. The main sol-gel protease, expressed in osteoclasts, plays a special role in bone resorption. Intracellular calcium (Ca2+) oscillation is a common pattern in the signal transduction of excitatory and non excitatory cells,.RANKL can cause Ca2+ oscillation, leading to calcineurin mediated NFATc1 activation, triggering a sustained TN in the differentiation of osteoclasts. The FATcl - dependent transcriptional program promotes osteoclast differentiation. It is believed that the mouse model of the ovariectomized (OVX) bone loss is similar to the human condition, and can be used to study the cause of bone loss and to intervene, suitable for the evaluation of the potential treatment drugs for osteoporosis. Bone microstructures are damaged and are very suitable for postmenopausal osteoporosis models. Neohesperidin is an extract of Fructus aurantii, which belongs to the flavonoids, is a natural antioxidant, and has the effect of anti-inflammatory, anti-tumor and protection of the cardiovascular system. However, it has not been studied in this group. In the experiment, we conducted an in vitro experiment to analyze the inhibitory effect of new hesperidin on RANKL induced osteoclast differentiation through a series of experiments, including the number of osteoclast differentiation through a dose dependent experiment, and the apoptosis analysis of Raw264.7 cells treated by new hesperidin by flow cytometry; through qPCR TRACP and Cathepsin K expressed in osteoclasts were measured, and NF- kappa B, NFAT and I kappa B- alpha in RANKL/RANK signaling pathway were analyzed by Luciferase and Western blot; the bone resorption of osteoclasts was detected by hydroxyapatite; the differentiation of osteoclasts was analyzed by calcium oscillation test. We also carried out the analysis of osteoclast differentiation. In vivo, the effect of Neo hesperidin on the ovariectomized mouse model (OVX) was observed. The results were analyzed by four parameters: bone volume fraction (BV/TV), bone small Liang Houdu (Tb.Th), bone trabecular number (Tb.N) and bone trabecular separation (Tb.Sp). The first part of the experiment included the following two parts: the first part of new hesperidin to RANKL induced osteoclast Objective: To investigate the effect of Neo hesperidin on the proliferation and differentiation of osteoclasts induced by RANKL in vitro, the effects of regulatory factor NF- kappa B, NFAT, I kappa B- a, TRACP, Cathepsin K, osteoclast absorption and calcium oscillation, and to explore the proliferation and differentiation of osteoclasts and a series of osteoclasts in bone metabolism. Methods: in vitro, in vitro, BMM cells were induced by RANKL and cultured with different concentrations of new hesperidin. TRAP staining was used to observe the TRAP staining positive osteoclasts cultured with different concentrations of new hesperidin under the optical microscope. RAW264.7 cells were induced by RANKL and added to different concentrations of new orange peel. The percentage of healthy cells was observed by flow cytometry, and the percentage of healthy cells was observed. BMM cells were induced by RANKL and cultured with 10 M new hesperidin. The expression of TRACP and Cathepsin K in osteoclasts was measured by qPCR experiment. BMM cells were induced by RANKL in the culture medium containing hydroxyapatite coating and added to 10 micron M. The absorption area of hydroxyapatite coating was observed and analyzed by ImageJ software. The P3K-Luc cells transfected by p-NF- kappa B-TA-Luc and Raw264.7 cells transfected with p-NFAT-TA-Luc were induced by RANKL and added to the culture of new hesperidin in different concentrations. The expression of T; BMM cells were induced by RANKL and added to the culture of 10 M new hesperidin. The expression of I kappa 8- A and NFAT were detected in a short and long time by Western blot experiment. The BMM cells were analyzed by ImageJ software. The BMM cells were induced by RANKL and added 10 mu new hesperidin, and the cell calcium oscillations were flat by inverted fluorescence microscope. The peak height was observed and analyzed with Nikon Basic Research software. Results: new hesperidin had inhibitory effect on osteoclast differentiation, and the greater the concentration, the greater the inhibition effect on osteoclast differentiation; the cell health rate remained at a high level above 90% under the action of different concentrations of new hesperidin, and the necrosis rate and the rate of necrosis were observed. The apoptosis rate remained at a low level and did not increase with the increase of the concentration of new hesperidin, and Neo hesperidin could inhibit the expression of TRACP and Cathepsin K; neo hesperidin could inhibit the absorption of osteoclast to hydroxyapatite coating; neo hesperidin could inhibit the expression of NF- kappa B and NFAT, and was dependent on the concentration of new hesperidin. The degradation of I kappa B- alpha and the inhibitory effect of Neo hesperidin on osteoclast calcium oscillation. Conclusion: Neo hesperidin can inhibit the differentiation of osteoclast from many aspects and inhibit the role of osteoclast in bone metabolism. It is a safe and effective osteoclast inhibitor. The aim of the study is to study the effect of the second part of new hesperidin on osteoporosis in ovariectomized mice. The effect of Neo hesperidin on bone volume fraction, bone trabecular thickness, bone trabecular number and bone trabecular separation in ovariectomized mice was studied. Methods: 30 C57BL6J female mice of 6 weeks old were randomly divided into sham operation group, model group, estrogen group, new hesperidin low dose group and new orange. A high dose of peridoside, each group of 6. Each mouse was weighed and injected with 10% chloral chloral intraperitoneal injection. The sham operation group only cut the abdominal skin and muscle layer on both sides of the lower abdomen, and did not remove the ovaries and closed the abdominal cavity. The other groups were ligated and completely removed and closed the abdominal cavity after the ovaries were found on both sides of the abdominal cavity. Mycophentin was injected with water and food for 1 weeks after 1 weeks. The sham operation group and the model group were given physiological saline, estrogen group was given estrogen, the low dose and high dose group of new hesperidin were given 3mg/kg and 6mg/kg new hesperidin.7 weeks respectively, each mouse was given eether anesthesia for unilateral tibia soaking. In 4%FPA solution, 20-24 hours after removal, 1XPBS solution flushed 2 times, 1XPBS invaded tibia in 1.5m1 test tube, Micro-CT analysis, compare the volume fraction of bone, bone trabecular thickness, bone trabecular number and bone trabecular separation. Results: new hesperidin group increased bone volume fraction of ovariectomized osteoporosis mice, basic Restore to the normal level, the effect is equivalent to the estrogen group, and the concentration dependence, the greater the concentration, the more obvious bone volume, the thickness of bone trabecula in each group has no obvious change; the high dose group of new hesperidin can reduce the bone trabecular separation degree of ovariectomized mice, and basically restore normal level, the effect is equivalent to the estrogens; new orange Peridoside can increase the number of bone trabeculae in ovariectomized mice and have a concentration dependence. The high dose of new hesperidin has the same effect as estrogen, making the number of bone trabeculae basically restored to normal level. Conclusion: Neo hesperidin can inhibit the development of osteoporosis in ovariectomized mice and be a potential drug for the treatment of osteoporosis.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R580

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