老年膳食营养与骨质疏松关系及黄酮类化合物干预作用的实验研究
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摘要
骨质疏松(osteoporosis，OP)是一种常见的老年慢性病，它使得骨骼脆性增加，很容易骨折。早期的骨质疏松症没有任何临床症状，如果不进行预防和治疗，骨质疏松症能够渐渐地产生和发展，直到骨折发生。典型的骨折常发生在髋骨，脊柱和腕骨。骨质疏松能够影响任何部位的骨骼，特别容易发生髋骨和脊柱骨折。髋骨骨折患者需要以外科手术主要手段的住院治疗。椎骨骨折也可引起严重的不良后果，包括剧烈的背痛和脊柱畸形。使患者不能自理行走，并且可能导致终生残疾甚至死亡。
许多学者采用去势方法复制骨质疏松症动物模型，研究发现模型动物的性激素水平越低，骨矿含量越下降。Cheng SL的研究发现大豆异黄酮有类雌激素的作用，能有效减少骨丢失并增加骨形成。Arjmandi进一步明确提示大豆蛋白中的异黄酮有骨保留作用，指出长期消费大豆或其异黄酮有助于骨质持久。但是关于异黄酮对骨质疏松骨作用机制尚不清楚，有待进一步深入研究，
本课题组通过人群膳食调查、骨细胞体外培养和废用性骨质疏松(disuse osteoporosis，DOP)大鼠、去卵巢(ovariectomized，OVX)大鼠模型，探讨老年膳食营养与骨质疏松的关系及大豆异黄酮(soy isoflavone，SIFV)和鸡冠花黄酮类化合物(celosiae cristatae isoflavone，CCFV)对其干预作用。为骨质疏松症的预防、治疗提供理论依据和干预措施。本研究所报道的“鸡冠花提取物对多种大鼠模型骨代谢的影响”及“生长因子在大豆异黄酮预防骨质疏松中的作用”研究，先后经河南省科学技术情报研究所和河南省医学情报研究所查新，得出较高评价结论，认为本研究的许多报道属于国内首次。达到国内领先水平。部分研究论文已被著名的专题文献检索数据库Medline和美国化学文摘收录。
第一部分老年人营养与骨密度的关系
1．老年人膳食及头发中Zn、Cu、Ca与BMD的关系
目的探讨老年人膳食及头发中Zn、Cu、Ca水平与骨密度(bone mineral density，BMD)的关系。
方法调查并分析了470例60岁以上城市(205)和农村(256)老人，男性178名，年龄65.70±3.48y，女性292名，年龄65.90±4.02y。检测BMD与发中Zn、Cu、Fe、Ca水平，并进行三日膳食回顾调查。
结果城乡老年女性BMD显著低于老年男性，城市老年女性膳食Ca、Zn含量低于城市老年男性，农村老年女性膳食Ca、Zn，发中Zn含量低于农村老年男性(p＜0.05 or p＜0.01)。城乡比较，农村老年男女BMD、膳食Ca，发中Ca、Zn含量低于城市老年男女(p＜0.05 or p＜0.01)。BMD与膳食Ca、Zn、Cu及发中Ca、Zn含量密切相关，膳食Zn与发Ca、Zn以及膳食Ca、Cu存在显著相关(p＜0.05 or p＜0.01)。
结论膳食Ca、Zn、Cu与BMD存在一定的关系，膳食Ca、Zn等元素摄入量不足可导致BMD降低。
2．部分非循环有向图分析老年人膳食营养素对BMD影响
目的用部分非循环有向图分析老年人膳食营养素对骨密度(BMD)影响。
方法对309例60岁以上老人进行了膳食调查及健康检查，男性132名，年龄65.4±4.0y，女性177名，年龄66.7±5.8y。并作了膳食营养素对骨密度的多元回归分析，进行了因果研究，绘制出部分非循环有向图(简称p dag)作因果研究。
结果老年女性BMD显著低于男性。健康老人平均每日摄入钙、蛋白质的量男性均显著高于女性。OP患者与同性别健康老人每日矿物质摄入量相比，OP组钙、蛋白质(protein)、VitC摄入量均显著低于同性别健康老人组，BMD与钙、蛋白质、VitC均呈显著正相关，与p dag因果分析一致。
结论膳食营养素与BMD存在一定的关系，膳食钙、蛋白质、VitC等摄入量不足可导致BMD降低。
第二部分大豆异黄酮和鸡冠花黄酮对体外培养新生大鼠成骨细胞增值、分化及部分相关蛋白、基因表达的影响
1．鸡冠花黄酮类化合物对成骨细胞矿化和IGF-1的作用
目的通过研究鸡冠花黄酮类对成骨细胞体外培养矿化作用和胰岛素样生长因子-1(insulin-like growth factor，IGF-1)表达的影响，探讨鸡冠花黄酮类化合物对大鼠成骨细胞的作用机理。
方法体外培养新生大鼠颅骨成骨细胞，茜素红染色方法(ARS)研究对成骨细胞矿化的影响，SP免疫组化方法测定成骨细胞中IGF-1的表达情况，用HPIAS-2000图像分析仪对各组表达进行定量分析。
结果鸡冠花黄酮类化合物促进大鼠成骨细胞矿化结节形成及IGF-1阳性表达，鸡冠花黄酮类化合物组的IGF-1平均光密度都显著高于对照组(p＜0.05)。
结论鸡冠花黄酮类化合物可增强新生大鼠成骨细胞矿化和IGF-1的表达。
2．大豆异黄酮对成骨细胞转化生长因子-β1及其受体Ⅰ、Ⅱ表达的影响
目的探讨大豆异黄酮对成骨细胞的转化生长因子-β1(transforming growth factor，TGF-β1)信号转导系统的影响。
方法制作新生大鼠颅骨成骨细胞体外培养模型，在第三代成骨细胞培养液中添加大豆异黄酮，通过免疫组化方法观察大豆异黄酮对成骨细胞中转化生长因子-β1及其受体Ⅰ、Ⅱ表达的影响。用HPIAS图像分析仪对各组表达进行定量分析。结果免疫组织化学染色显示，大豆异黄酮培养的大鼠第三代成骨细胞中转化生长因子-β1及其受体Ⅰ、Ⅱ呈阳性表达，阳性细胞浆出现棕黄色颗粒。图像分析显示大豆异黄酮组的TGF-β_1 TGF-βRI、TGF-βR2平均光密度都显著高于阴性对照组(p＜0.05)。
结论在成骨细胞形成中，大豆异黄酮对TGF-β1及其TGF-βR1、TGF-βR2受体的表达有显著促进作用。
3．大豆异黄酮和钙对大鼠成骨细胞增殖分化和矿化的作用
目的探讨大豆异黄酮和钙对体外培养大鼠颅骨成骨细胞增殖、分化和矿化的作用。
方法体外培养新生大鼠颅骨成骨细胞，然后用噻唑盐(MTT)法测定大豆异黄酮和葡萄糖酸钙对体外培养成骨细胞的增殖作用，氨基安替比林法测成骨细胞内碱性磷酸酶(ALP)活性，茜素红染色方法(ARS)测成骨细胞的矿化作用。
结果单纯补钙对大鼠成骨细胞增殖、分化和矿化无显著促进作用，大豆异黄酮可显著地促进成骨细胞的增殖和分化，并促使成骨细胞形成矿化结节，而补充大豆异黄酮同时补钙使矿化结节团块增大。
结论大豆异黄酮可以提高成骨细胞的增殖和分化，同时补钙更有利成骨细胞的矿化。
4．大豆异黄酮对体外培养大鼠成骨细胞BMP2表达的影响
目的探讨大豆异黄酮对体外培养大鼠成骨细胞骨形态发生蛋白-2(bone morphogenetic protein 2，BMP_2)表达的影响。
方法制作新生大鼠颅骨成骨细胞体外培养模型，用不同浓度的大豆异黄酮刺激分离培养的大鼠成骨细胞，提取细胞总RNA，RT-PCR扩增BMP2基因cDNA，同时扩增β-actin cDNA作为内对照。通过免疫组化方法观察大豆异黄酮对成骨细胞BMP_2表达的影响，HPIAS图像分析仪对各组表达进行定量。扫描PCR产物电泳照片，计算BMP-2/β-actin cDNA的积分光密度比值，推算BMP2基因的相对表达水平。
结果大豆异黄酮增加成骨细胞BMP2水平，并且在基因转录水平促进大鼠成骨细胞BMP_2表达，呈剂量效应关系，(p＜0.01)。
结论大豆异黄酮在成骨细胞形成中对BMP_2表达有显著促进作用。提示大豆异黄酮在促进成骨细胞增殖、分化的作用可能是通过增加BMP2基因的表达来实现的。
第三部分大豆异黄酮和鸡冠花黄酮对废用性骨质疏松大鼠、去卵巢大鼠骨代谢指标及相关细胞因子的影响
1．钙和鸡冠花黄酮类提取物对废用性骨质疏松大鼠的作用
目的探讨钙和鸡冠花黄酮类提取物对废用性骨质疏松(disuse osteoporosis，DOP)大鼠的作用，为老年骨质疏松的防治提供依据。
方法选用健康SD大鼠40只，雄性。随机分为5组，第1组、第2组分别为阴性对照和阳性对照，灌胃蒸馏水5ml/(kg·d)，第2～5组制造废用性骨质疏松大鼠模型，第3、4、5组为预防组分别灌胃等量鸡冠花提取物，碳酸钙，鸡冠花提取物加钙溶液。实验8周后，测骨密度，股骨X线和CT值，测血清MDA、SOD及24h尿钙、羟脯氨酸(HOP)、尿肌酐(UCR)含量等。
结果阳性组与阴性组比股骨重、灰分重、CT值、血清钙下降，尿钙、尿HOP升高(p＜0.05或0.01)，预防组与阳性组比股骨重、CT值显著增加，碳酸钙组、鸡冠花加钙组灰分重显著增加(p＜0.05)。各预防组与阳性组比股骨X线密度明显增加。与阳性组比各预防组血清钙增加，尿钙、尿HOP减少(P＜0.05或＜0.01)。鸡冠花组、鸡冠花加钙组与阳性组比股骨BMD、血清SOD升高，MDA降低，单纯补钙组仅BMD升高(P＜0.05或＜0.01)。
结论补鸡冠花提取物、补钙对废用性骨质疏松大鼠骨质代谢均有促进作用，两者合用具有更好的预防效果，能延缓或减轻DOP的发生。
2．鸡冠花黄酮对去卵巢大鼠尿无机盐、BMP_2以及对单核吞噬细胞吞噬功能的影响
目的探讨鸡冠花黄酮化合物对卵巢切除(ovariectomy，OVX)大鼠尿无机盐、骨形成蛋白(BMP_2)以及对单核吞噬细胞吞噬功能的影响。
方法雌性SD大鼠21只，随机分为假手术组(sham)，去卵巢组(OVX)，去卵巢加鸡冠花黄酮组(Celosiae cristatae isoflavone，CCFV，100mg/kg·d)，10周后测骨密度和尿液钙，钠，钾含量。用免疫组化法测骨骼中BMP_2的表达，用单核吞噬细胞体外吞噬试验和碳廓清法测定吞噬细胞的吞噬功能。
结果OVX组大鼠尿钙，钠显著增高，而尿钾及骨BMP_2表达降低。CCFV组与OVX组比大鼠尿钙，钠显著降低，骨BMP_2显著性升高。OVX组与sham组比较大鼠单核吞噬细胞体外吞噬鸡红细胞的能力有显著降低(P＜0.05或＜0.01)；CCFV有提高单核吞噬细胞吞噬功能的作用。
结论鸡冠花黄酮类化合物可调节OVX无机盐代谢表达，增加BMP_2表达，促进吞噬细胞吞噬功能，预防骨质疏松。
3．大豆异黄酮和钙对去卵巢大鼠无机盐、BMD、骨BMP_2、TGF-β_1、骨IGF-1、IGF-1mRNA表达影响
目的探讨大豆异黄酮和钙对去卵巢大鼠的骨密度、无机盐、骨形成蛋白2(BMP_2)和转移生长因子-β_1(TGF-β_1)、骨IGF-1、IGF-1mRNA表达的影响。
方法28只雌性SD大鼠随机分为假手术(sham组)、去卵巢(OVX组)、去卵巢补钙(Ca组，50mg·kg~(-1)·d~(-1))和去卵巢加大豆异黄酮(SIFV组，大豆异黄酮100mg·kg~(-1)·d~(-1))，10周后测骨密度及血清、尿液和骨骼钙、钠、钾含量。并用SP免疫组织化学检测骨BMP_2、TGF-β_1、骨IGF-1表达。用原位杂交法测IGF-1mRNA表达。用HPIAS图像分析仪对各组表达进行定量分析。
结果实验结束后，去卵巢大鼠的骨密度，骨骼钙显著下降，尿钙显著性增高(P＜0.01和P＜0.05)，骨BMP_2表达下降；与单纯去卵巢组比，大豆异黄酮组大鼠的骨密度、骨骼中钙显著升高，血清钙及尿钙显著性下降(P＜0.01和P＜0.05)，骨BMP_2表达增高(P＜0.05)，而补钙组各指标都无显著性意义。去卵巢加大豆异黄酮后骨TGF-β_1显著高于OVX组(P＜0.05)，IGF-1免疫组化和IGF-1mRNA原位杂交平均吸光度值亦显著高于OVX组(P＜0.05或P＜0.01)。
结论大豆异黄酮可能通过调节去卵巢大鼠的无机盐、骨BMP_2、TGF-β_1、IGF-1表达进而在预防骨质疏松症的发生中起到一定的作用。
4．大豆异黄酮和钙对去卵巢大鼠骨密度、矿物质及肾脏TGFβ_1表达的影响
目的探讨大豆异黄酮和钙对OVX大鼠的骨密度及微量元素的影响。
方法28只雌性SD大鼠随机分为假手术(sham组)、去卵巢(OVX组)、去卵巢补钙(Ca组，50mg·kg~(-1)·d~(-1))和去卵巢加大豆异黄酮(SIFV组，大豆异黄酮100mg·kg~(-1)·d~(-1))，10周后测骨密度和骨矿物含量及血清、尿液和骨骼钙、锌、铜、铁含量。
结果实验结束时，OVX组与sham组相比骨密度和骨矿物含量，骨骼钙、锌、铜、铁和血清锌、铁显著下降，血清钙、铜、ALP及尿钙、铜、铁显著性增高(P＜0.05)；与OVX组比，SIFV组大鼠的骨密度、骨矿物含量、血清锌、骨骼中钙、锌、铜显著升高，血清钙、铜、ALP及尿钙、铜的丢失显著性下降(P＜0.01和P＜0.05)，OVX组与sham组相比肾重/体重(％)显著增加(P＜0.01)，SIFV组与OVX组比肾重/体重(％)明显降低(P＜0.01)。对大鼠肾小球内TGFβ_1蛋白表达情况并进行计算机图像分析，显示OVX组与sham组相比肾小球TGFβ1表达显著升高(P＜0.01)，而SIFV组与OVX组比TGFβ1表达显著降低(P＜0.01)，而补钙组各指标都无显著性意义。
结论大豆异黄酮可预防OVX骨钙、锌、铜丢失，抑制肾脏TGFβ_1蛋白的过度表达，减轻肾脏损伤，预防骨质丢失。
Osteoporosis is a frequent chronic senile disease, and in which bones become fragile and more easily to break. Osteoporosis is not any clinical symptom in the initial stage. If not prevented or if left untreated, osteoporosis can produce and progress stealthily until a bone breaks. These fractures, occur typically in the hip, spine, and wrist. Any bone can be affected, but of special concern are fractures of the hip and spine. A hip bone fracture almost always requires hospitalization and major surgery. Spinal or vertebral fractures also have serious consequences, including angular curvature, severe back pain, and deformity. It can impair a person's ability to walk unassisted and may cause prolonged or permanent disability or even death.
The osteoporosis animal model was established by many scholars with castrated method. The sex hormone level of the model animal was lower, the bone mineral content was more descend also in the research. The research of the Cheng SL discovers that the soybean isoflavone has estrogenic function, it can valid decrease bone lose and increase the bone formation. Arjmandi definitely hints the soybean isoflavone can contain the bone reservation function, point out that consume soybean or its isoflavones to contribute the bone substance's hold out for long time, over a long period of time. But the mechanism of the isoflavone anti osteoporosis is still without thorough research. In this study we investigate the relationship of diet for aged and osteoporosis and research the intervention effect of soy isoflavone and celosiae cristatae flavone.
Part I Relationship of nutrition and bone mineral density in Elderly People
1. Relationship between BMD and Zn, Cu, Ca Levels in the Hair and Meal in Elderly People
Summary: The relationship between bone mineral density (BMD) and Zn, Cu, Ca levels in the meal and hair of urban and rural elderly people were studied. 470 subjects above 60 years old (urban 205 and rural 265), 178 males with an average age of 65.70 ± 3.48y and 292 females with an average age of 65.90±4.02y, were inquired. The BMD and Zn, Cu, Ca levels in the meal and hair were measured. The detected BMD in urban and rural female old people was significantly lower than that of the males; The Ca, Zn contents in the meal of the urban females were significantly lower than those of the urban males; The Ca, Zn in the meal and Zn in the hair of the rural females were significantly lower than those of rural males (P< 0.05 or 0.01). The BMD, Ca intakes, Ca and Zn in the hair of the rural old people were significantly lower than those of the urban old people (P< 0.05 or 0.01). There was a correlation between BMD with the Ca, Zn of the hair and dietary Ca, Zn, Cu or between dietary Zn with Ca, Zn in the hair and Ca, Cu intakes. The Zn, Cu and Ca levels in the meal nutrients were correlated with BMD to some degrees. Lack of Ca and Zn in the meal can cause the reduction of BMD. 2. P Dag Analysis of Effects of daily nutrition intake on BMD Objectives To study the effects of daily mineral intake on bone mineral density (BMD) of old people.
Data Measures The daily nutrition intake and health were investigated in 309 old people aged over 60 years. 132 male, aged 65.4±4.0y, 177 female aged 66.7±5.8y.Based on the data, the regression analysis and causal analysis are presented and thus a partial directed acyclic graph (p dag) are plotted to demonstrate a definite causal relation. Results The paper shows that BMD in female were lower than that in male. For healthy people, the daily intake of Calcium, protein, Vitamin C of male was higher than that of health female. The daily intake of Calcium, protein, Vitamin C of osteoporosis case was lower than that of health people of the same sex. BMD correlation with the daily intake of Calcium, protein, Vitamin C is significantly positive, which is consistent with directed acyclic graph (p dag) analysis.
Conclude It was suggest that there is definite relation between the daily mineral intake and BMD. The lack of daily intake of Calcium, protein, Vitamin C may decrease BMD.
Part II The effect of soy isoflavonord and flavonoid of celosia cristata
on increment, mineralization and partial related gene expression in vitro cultured
1. Effect of flavonoid of celosia cristata on mineralization and IGF-1 expression in
vitro cultured osteoblasts
Objective To study the effect of flavonoid of celosia cristata extract on mineralization and IGF-1 expression of cultured osteoblasts from rat
Methods The osteoblastic cells from cranial bone of newborn rat was culturedthe. The effect of mineralization was detected by alizarin bordeaux stain(ARS). The effect of flavonoid of celosia cristata extract on IGF-1 expression of cultured osteoblasts was detected by SP method. The expression level was monitored with HPIAS1000 photograph analysismeter. Results The IGF-1 of third cultured osteoblasts cultured used flavonoid of celosia cristata
extract was positive expression .The IGF-1 positive expression of flavonoid of celosia
cristata extract group was higher significantly than negative control group (p<0.05) .
Conclusion The flavonoid of celosia cristata extract may increase the mineralization and
IGF-1 positive expression of cultured osteoblasts from rat
2. Effect of soybean isoflavonet on TGF-β1、 TGF-βR1 and TGF-βR2 expression of cultured osteoblasts from rat
Objective To study the effect of soybean isoflavone on TGF-β1 , 、 TGF-βR1 and
TGF-βR2 expression of cultured osteoblasts from rat.
Methods The osteoblstic cells from cranial bone of newborn rat was cultured. The SP
method was used to detect the effect of soybean isoflavone on TGF-β1、 TGF-βR1 and
TGF-βR2 expression of cultured osteoblasts.The expression level was monitored with
HPIAS_1000 photograph analysismeter.
Results The TGF-β1、 TGF-βR1 and TGF-βR2 of osteoblasts cultured used soybean
isoflavone was positive expression .The TGF-β_1 、 TGF-βR1 and TGF-βR2 positive
expression of soybean isoflavone group was higher significantly than negative control
group (p<0.05) ..
Conclusion The soybean isoflavone may increase the TGF-β_1 、 TGF-βR1 and TGF-βR2
positive expression of cultured osteoblasts from rat.
3. Effects of soybean isoflavone and calcium on proliferation, differentiation and
mineralization of cultured osteoblasts in vitro
Objective To explore the effects of soybean isoflavone and calcium on proliferation , differentiation and mineralization of cultured osteoblastic cells.
Methods Osteoblastic cells from newborn rat calvarial was cultured. Osteoblastic cells proliferation was masured by MTT, activity of ALP was observed with Golden's method and ARS was used to measure the mineral nodes of osteoblasts. Results The calcium group had not diferent frome the control group on stimulating the proliferation, differentiation and mineralization of rat calvarial osteoblastic cells.The soybean isoflavone group had significant effcts on stimulating the proliferation , differentiation of osteoblasts and increase the forming of the mineral nodes of osteoblasts in rat. calcium and soybean isoflavone group had more benefit for forming mineral nodes. Conclusion The soybean isoflavone stimulates the proliferation, differentiation and mineralization of rat calvarial osteoblastic cells, and increase calcium in the same time is more benefit for mineralization. 4. Effect of soybean isoflavonet on bone morphogenetic protein 2 expression of
cultured osteoblasts from rat
Objective To study the effect of soybean isoflavone on bone morphogenetic protein 2 (BMP_2)expression of cultured osteoblasts from rat.
Methods The osteoblstic cells from cranial bone of newborn rat was cultured. Cultured rat osteoblasts were treated with soybean isoflavone in difierent concentrations. Total RNA was extracted the from the cells. BMP_2 a were amplified by RT—PCR. β -actin cDNA was also amplified at the same time and used as an internal standard. The SP method was used to detect the effect of soybean isoflavone on BMP_2 expression of cultured osteoblasts. The expression level was monitored with HPIAS_2000 photograph analysismeter. The pictures of PCR production was scanned. The specific values of density of BMP_2/β -actin were calculated, and the expression levels of BMP_2 were determined.
Results Soybean isoflavone increased the level of BMP_2 in rat osteoblasts and increased the expressions of BMP_2 genes at the level of transcripaon and revealed dose-effect relation, (p<0.01) .
Conclusion The soybean isoflavone induces the proliferation and differentiated of osteoblast probably by increasing the expressions of BMP_2 genes. Part III The effect of soy isoflavonord and flavonoid of celosia cristata
on bone metabolism index in disuse osteoporosis and ovariectomized rat
1. The effect of calcium and extraction of cristata flavonoid on disuse osteoporosis in rats
Objective To research the effect of calcium and extraction of cristata flavonoid on disuse osteoporosis in rats.
Method 40 healthy , male SD rats were selected, and randomly divided into 5 groups. The group 1 (normal group) and group 2(positive group) were given with distilled water by intragastrition. Group 2-5 rats were created to the model of disuse osteoporosis. Group 3、 4 and 5 were difference given with cristata flavonoid, calcium carbonate, cristata flavonoid with calcium by intragastrition. After 8 weeks experiment, the bone mineral density(BMD), femur computer tomography (CT), serum malondialdehyde (MDA), superoxide dismutase (SOD), and urine calcium, urine hydroxyproline (HOP), urine creatinine were detected.
Results It was showed that the femur weight, ash weight , CT value in positive group were significantly lower than that of normal group and cristata flavonoid group(P<0.05 or P<0.01). Compare whit positive groups , urinary calcium and HOP were lower and serum calcium, femur BMD, serum SOD were higher in cristata flavonoid and cristata flavonoid with calcium groups(P<0.05 or P<0.01).
Conclusion It was suggested that calcium and cristata flavonoid can effect the bone matrix metabolism of disuse osteoporosis in rats, cristata flavonoid with calcium had more effect on disuse osteoporosis rats. 2. Effect of cristata L flavonoid on prevetive osteoporosis in ovariectomized rats Objective To study the effect of cristata L flavonoid on urine mineral and the expression of bone BMP_2 , and phagocytosis function of Mononuclear macrophage in ovariectomized rats.
Methods Twenty one female SD rats were randomly divided into three groups: sham, ovariectomized(OVX), ovariectomized with cristata L flavonoid (CRIST, 100mg/kg~-· d). The content of urinary calcium, sodium, potassium were measured after ten weeks. The Streptavidin-Peroxidase Histostain~(TM)-Plus Kits was used to detect the expression of BMP_2 in bone. And phagocytosis test of mononuclear macrophage in vitro, carbon clearance measurement were carried out to determine the phagocytotic functions of mononuclear macrophage.
Results Compared with sham group the calcium, sodium in urine expression of ovariectomized group were higher and the potassium of urine and the expression of BMP_2 , and the phago- percentage, and phagocytic index and the carbon clearance index were significantly decreased in OVX group(P<0.05 or <0.01). Compare with OVX group the urine calcium, sodium expression, and the phago- percentage, carbon phagocytic index were significantly in CRIST group, but the bone BMP_2 expression was significantly higher than that of OVX group (P<0.05 or<0.01 ) . Conclusion The cristat L flavonoid could decrease the urinary calcium loss, and increased the expression of BMP_2 , and increased the phagocytotic functions of mononuclear macrophage. It may be advantageous to prevent the postmenopausal Osteoporosis, and to facilitate health care in menopause woman. 3. Effect of soybean isoflavone and calcium on bone mineral density , mineral , BMP_2、 TGF-β_1、 IGF-1 and IGF-1 mRNA express in ovariectomized rats Objective To study the effect of wheat embryo extract on bone mineral density and mineral in ovariectomized rats.
Methods Twenty eight female SD rat were randomly divided into four groups:sham,ovariectomized, ovariectomized added calcium [Ca, 50 mg/kg · d] , ovariectomized added soybean isoflavone [Isof, 100 mg/kg · d], . The experiment proceed ten weeks.
Results The bone mineral density decreased, and urine calcium increased significantly in ovariectomized group; The bone BMP_2、 TGF-β_1、 IGF-1 and IGF-1 mRNA expression in ovariectomized group decreased significantly. The bone mineral density and BMP2、 TGF-β_1、 IGF-1 and IGF-1 mRNA expression in soybean isoflavone group increased significantly than ovariectomized group; The urine calcium decreased significantly. The recovery of these markers was not significantly in merely supply Calcium group than ovariectomized group.
Conclusion The soybean isoflavone may adjustment IGF-1 of growth factor expression, and prevent the lose of calcium of bone and the increase of bone mineral density induced by ovariectomy.
4. Effect of soybean isoflavone and calcium on bone mineral density and mineral
matter and kidney TGF β_1 expression in ovariectomized rats
Objective To explore the effect of soybean isoflavone and calcium on bone mineral density and trace elements in ovariectomized rats.
Methods Twenty eight female SD rats were randomly divided into four groups:sham, ovariectomized, ovariectomized added calcium (50mg.·kg~(-1)·d~(-1)), ovariectomized added soybean isoflavone (100mg.·kg~(-1)·d~(-1)).The content of bone mineral density and mineral , and serum, urine and bone calcium, zinc, copper , iron were determined after ten weeks.
Results After test , the bone mineral density and bone mineral , and bone calcium, zinc, copper , iron, and serum zinc, iron in ovariectomized group decreased significantly; The serum calcium , copper, ALP and urine calcium, copper, iron increased.The bone mineral density and bone mineral , serum zinc , bone calcium, zinc, copper in ovariectomized added soybean isoflavone group increased significantly than ovariectomized group; The serum calcium, copper, ALP and urine calcium,
copper decreased significantly ( P<0.01 or P<0.05 ) . Compare with sham group, the
OVX group kidney weight/body weight(%)increased significantly (P<0.01). Compare
with OVX group, the SIFV group kidney weight/body weight(%)decreased significantly
(P<0.01) . The glomcrulus TGFβ_1 proteinum expression increased significantly in OVX
group than sham group and SIFV group(.P<0.01).
Conclusion The soybean isoflavone may prevent the lose of bone calcium, zinc ,
copper, and restraint the glomcrulus TGFβ_1 proteinum overexpression and prevent the
osteoporosis induced by ovariectomy.
许多学者采用去势方法复制骨质疏松症动物模型，研究发现模型动物的性激素水平越低，骨矿含量越下降。Cheng SL的研究发现大豆异黄酮有类雌激素的作用，能有效减少骨丢失并增加骨形成。Arjmandi进一步明确提示大豆蛋白中的异黄酮有骨保留作用，指出长期消费大豆或其异黄酮有助于骨质持久。但是关于异黄酮对骨质疏松骨作用机制尚不清楚，有待进一步深入研究，
本课题组通过人群膳食调查、骨细胞体外培养和废用性骨质疏松(disuse osteoporosis，DOP)大鼠、去卵巢(ovariectomized，OVX)大鼠模型，探讨老年膳食营养与骨质疏松的关系及大豆异黄酮(soy isoflavone，SIFV)和鸡冠花黄酮类化合物(celosiae cristatae isoflavone，CCFV)对其干预作用。为骨质疏松症的预防、治疗提供理论依据和干预措施。本研究所报道的“鸡冠花提取物对多种大鼠模型骨代谢的影响”及“生长因子在大豆异黄酮预防骨质疏松中的作用”研究，先后经河南省科学技术情报研究所和河南省医学情报研究所查新，得出较高评价结论，认为本研究的许多报道属于国内首次。达到国内领先水平。部分研究论文已被著名的专题文献检索数据库Medline和美国化学文摘收录。
第一部分老年人营养与骨密度的关系
1．老年人膳食及头发中Zn、Cu、Ca与BMD的关系
目的探讨老年人膳食及头发中Zn、Cu、Ca水平与骨密度(bone mineral density，BMD)的关系。
方法调查并分析了470例60岁以上城市(205)和农村(256)老人，男性178名，年龄65.70±3.48y，女性292名，年龄65.90±4.02y。检测BMD与发中Zn、Cu、Fe、Ca水平，并进行三日膳食回顾调查。
结果城乡老年女性BMD显著低于老年男性，城市老年女性膳食Ca、Zn含量低于城市老年男性，农村老年女性膳食Ca、Zn，发中Zn含量低于农村老年男性(p＜0.05 or p＜0.01)。城乡比较，农村老年男女BMD、膳食Ca，发中Ca、Zn含量低于城市老年男女(p＜0.05 or p＜0.01)。BMD与膳食Ca、Zn、Cu及发中Ca、Zn含量密切相关，膳食Zn与发Ca、Zn以及膳食Ca、Cu存在显著相关(p＜0.05 or p＜0.01)。
结论膳食Ca、Zn、Cu与BMD存在一定的关系，膳食Ca、Zn等元素摄入量不足可导致BMD降低。
2．部分非循环有向图分析老年人膳食营养素对BMD影响
目的用部分非循环有向图分析老年人膳食营养素对骨密度(BMD)影响。
方法对309例60岁以上老人进行了膳食调查及健康检查，男性132名，年龄65.4±4.0y，女性177名，年龄66.7±5.8y。并作了膳食营养素对骨密度的多元回归分析，进行了因果研究，绘制出部分非循环有向图(简称p dag)作因果研究。
结果老年女性BMD显著低于男性。健康老人平均每日摄入钙、蛋白质的量男性均显著高于女性。OP患者与同性别健康老人每日矿物质摄入量相比，OP组钙、蛋白质(protein)、VitC摄入量均显著低于同性别健康老人组，BMD与钙、蛋白质、VitC均呈显著正相关，与p dag因果分析一致。
结论膳食营养素与BMD存在一定的关系，膳食钙、蛋白质、VitC等摄入量不足可导致BMD降低。
第二部分大豆异黄酮和鸡冠花黄酮对体外培养新生大鼠成骨细胞增值、分化及部分相关蛋白、基因表达的影响
1．鸡冠花黄酮类化合物对成骨细胞矿化和IGF-1的作用
目的通过研究鸡冠花黄酮类对成骨细胞体外培养矿化作用和胰岛素样生长因子-1(insulin-like growth factor，IGF-1)表达的影响，探讨鸡冠花黄酮类化合物对大鼠成骨细胞的作用机理。
方法体外培养新生大鼠颅骨成骨细胞，茜素红染色方法(ARS)研究对成骨细胞矿化的影响，SP免疫组化方法测定成骨细胞中IGF-1的表达情况，用HPIAS-2000图像分析仪对各组表达进行定量分析。
结果鸡冠花黄酮类化合物促进大鼠成骨细胞矿化结节形成及IGF-1阳性表达，鸡冠花黄酮类化合物组的IGF-1平均光密度都显著高于对照组(p＜0.05)。
结论鸡冠花黄酮类化合物可增强新生大鼠成骨细胞矿化和IGF-1的表达。
2．大豆异黄酮对成骨细胞转化生长因子-β1及其受体Ⅰ、Ⅱ表达的影响
目的探讨大豆异黄酮对成骨细胞的转化生长因子-β1(transforming growth factor，TGF-β1)信号转导系统的影响。
方法制作新生大鼠颅骨成骨细胞体外培养模型，在第三代成骨细胞培养液中添加大豆异黄酮，通过免疫组化方法观察大豆异黄酮对成骨细胞中转化生长因子-β1及其受体Ⅰ、Ⅱ表达的影响。用HPIAS图像分析仪对各组表达进行定量分析。结果免疫组织化学染色显示，大豆异黄酮培养的大鼠第三代成骨细胞中转化生长因子-β1及其受体Ⅰ、Ⅱ呈阳性表达，阳性细胞浆出现棕黄色颗粒。图像分析显示大豆异黄酮组的TGF-β_1 TGF-βRI、TGF-βR2平均光密度都显著高于阴性对照组(p＜0.05)。
结论在成骨细胞形成中，大豆异黄酮对TGF-β1及其TGF-βR1、TGF-βR2受体的表达有显著促进作用。
3．大豆异黄酮和钙对大鼠成骨细胞增殖分化和矿化的作用
目的探讨大豆异黄酮和钙对体外培养大鼠颅骨成骨细胞增殖、分化和矿化的作用。
方法体外培养新生大鼠颅骨成骨细胞，然后用噻唑盐(MTT)法测定大豆异黄酮和葡萄糖酸钙对体外培养成骨细胞的增殖作用，氨基安替比林法测成骨细胞内碱性磷酸酶(ALP)活性，茜素红染色方法(ARS)测成骨细胞的矿化作用。
结果单纯补钙对大鼠成骨细胞增殖、分化和矿化无显著促进作用，大豆异黄酮可显著地促进成骨细胞的增殖和分化，并促使成骨细胞形成矿化结节，而补充大豆异黄酮同时补钙使矿化结节团块增大。
结论大豆异黄酮可以提高成骨细胞的增殖和分化，同时补钙更有利成骨细胞的矿化。
4．大豆异黄酮对体外培养大鼠成骨细胞BMP2表达的影响
目的探讨大豆异黄酮对体外培养大鼠成骨细胞骨形态发生蛋白-2(bone morphogenetic protein 2，BMP_2)表达的影响。
方法制作新生大鼠颅骨成骨细胞体外培养模型，用不同浓度的大豆异黄酮刺激分离培养的大鼠成骨细胞，提取细胞总RNA，RT-PCR扩增BMP2基因cDNA，同时扩增β-actin cDNA作为内对照。通过免疫组化方法观察大豆异黄酮对成骨细胞BMP_2表达的影响，HPIAS图像分析仪对各组表达进行定量。扫描PCR产物电泳照片，计算BMP-2/β-actin cDNA的积分光密度比值，推算BMP2基因的相对表达水平。
结果大豆异黄酮增加成骨细胞BMP2水平，并且在基因转录水平促进大鼠成骨细胞BMP_2表达，呈剂量效应关系，(p＜0.01)。
结论大豆异黄酮在成骨细胞形成中对BMP_2表达有显著促进作用。提示大豆异黄酮在促进成骨细胞增殖、分化的作用可能是通过增加BMP2基因的表达来实现的。
第三部分大豆异黄酮和鸡冠花黄酮对废用性骨质疏松大鼠、去卵巢大鼠骨代谢指标及相关细胞因子的影响
1．钙和鸡冠花黄酮类提取物对废用性骨质疏松大鼠的作用
目的探讨钙和鸡冠花黄酮类提取物对废用性骨质疏松(disuse osteoporosis，DOP)大鼠的作用，为老年骨质疏松的防治提供依据。
方法选用健康SD大鼠40只，雄性。随机分为5组，第1组、第2组分别为阴性对照和阳性对照，灌胃蒸馏水5ml/(kg·d)，第2～5组制造废用性骨质疏松大鼠模型，第3、4、5组为预防组分别灌胃等量鸡冠花提取物，碳酸钙，鸡冠花提取物加钙溶液。实验8周后，测骨密度，股骨X线和CT值，测血清MDA、SOD及24h尿钙、羟脯氨酸(HOP)、尿肌酐(UCR)含量等。
结果阳性组与阴性组比股骨重、灰分重、CT值、血清钙下降，尿钙、尿HOP升高(p＜0.05或0.01)，预防组与阳性组比股骨重、CT值显著增加，碳酸钙组、鸡冠花加钙组灰分重显著增加(p＜0.05)。各预防组与阳性组比股骨X线密度明显增加。与阳性组比各预防组血清钙增加，尿钙、尿HOP减少(P＜0.05或＜0.01)。鸡冠花组、鸡冠花加钙组与阳性组比股骨BMD、血清SOD升高，MDA降低，单纯补钙组仅BMD升高(P＜0.05或＜0.01)。
结论补鸡冠花提取物、补钙对废用性骨质疏松大鼠骨质代谢均有促进作用，两者合用具有更好的预防效果，能延缓或减轻DOP的发生。
2．鸡冠花黄酮对去卵巢大鼠尿无机盐、BMP_2以及对单核吞噬细胞吞噬功能的影响
目的探讨鸡冠花黄酮化合物对卵巢切除(ovariectomy，OVX)大鼠尿无机盐、骨形成蛋白(BMP_2)以及对单核吞噬细胞吞噬功能的影响。
方法雌性SD大鼠21只，随机分为假手术组(sham)，去卵巢组(OVX)，去卵巢加鸡冠花黄酮组(Celosiae cristatae isoflavone，CCFV，100mg/kg·d)，10周后测骨密度和尿液钙，钠，钾含量。用免疫组化法测骨骼中BMP_2的表达，用单核吞噬细胞体外吞噬试验和碳廓清法测定吞噬细胞的吞噬功能。
结果OVX组大鼠尿钙，钠显著增高，而尿钾及骨BMP_2表达降低。CCFV组与OVX组比大鼠尿钙，钠显著降低，骨BMP_2显著性升高。OVX组与sham组比较大鼠单核吞噬细胞体外吞噬鸡红细胞的能力有显著降低(P＜0.05或＜0.01)；CCFV有提高单核吞噬细胞吞噬功能的作用。
结论鸡冠花黄酮类化合物可调节OVX无机盐代谢表达，增加BMP_2表达，促进吞噬细胞吞噬功能，预防骨质疏松。
3．大豆异黄酮和钙对去卵巢大鼠无机盐、BMD、骨BMP_2、TGF-β_1、骨IGF-1、IGF-1mRNA表达影响
目的探讨大豆异黄酮和钙对去卵巢大鼠的骨密度、无机盐、骨形成蛋白2(BMP_2)和转移生长因子-β_1(TGF-β_1)、骨IGF-1、IGF-1mRNA表达的影响。
方法28只雌性SD大鼠随机分为假手术(sham组)、去卵巢(OVX组)、去卵巢补钙(Ca组，50mg·kg~(-1)·d~(-1))和去卵巢加大豆异黄酮(SIFV组，大豆异黄酮100mg·kg~(-1)·d~(-1))，10周后测骨密度及血清、尿液和骨骼钙、钠、钾含量。并用SP免疫组织化学检测骨BMP_2、TGF-β_1、骨IGF-1表达。用原位杂交法测IGF-1mRNA表达。用HPIAS图像分析仪对各组表达进行定量分析。
结果实验结束后，去卵巢大鼠的骨密度，骨骼钙显著下降，尿钙显著性增高(P＜0.01和P＜0.05)，骨BMP_2表达下降；与单纯去卵巢组比，大豆异黄酮组大鼠的骨密度、骨骼中钙显著升高，血清钙及尿钙显著性下降(P＜0.01和P＜0.05)，骨BMP_2表达增高(P＜0.05)，而补钙组各指标都无显著性意义。去卵巢加大豆异黄酮后骨TGF-β_1显著高于OVX组(P＜0.05)，IGF-1免疫组化和IGF-1mRNA原位杂交平均吸光度值亦显著高于OVX组(P＜0.05或P＜0.01)。
结论大豆异黄酮可能通过调节去卵巢大鼠的无机盐、骨BMP_2、TGF-β_1、IGF-1表达进而在预防骨质疏松症的发生中起到一定的作用。
4．大豆异黄酮和钙对去卵巢大鼠骨密度、矿物质及肾脏TGFβ_1表达的影响
目的探讨大豆异黄酮和钙对OVX大鼠的骨密度及微量元素的影响。
方法28只雌性SD大鼠随机分为假手术(sham组)、去卵巢(OVX组)、去卵巢补钙(Ca组，50mg·kg~(-1)·d~(-1))和去卵巢加大豆异黄酮(SIFV组，大豆异黄酮100mg·kg~(-1)·d~(-1))，10周后测骨密度和骨矿物含量及血清、尿液和骨骼钙、锌、铜、铁含量。
结果实验结束时，OVX组与sham组相比骨密度和骨矿物含量，骨骼钙、锌、铜、铁和血清锌、铁显著下降，血清钙、铜、ALP及尿钙、铜、铁显著性增高(P＜0.05)；与OVX组比，SIFV组大鼠的骨密度、骨矿物含量、血清锌、骨骼中钙、锌、铜显著升高，血清钙、铜、ALP及尿钙、铜的丢失显著性下降(P＜0.01和P＜0.05)，OVX组与sham组相比肾重/体重(％)显著增加(P＜0.01)，SIFV组与OVX组比肾重/体重(％)明显降低(P＜0.01)。对大鼠肾小球内TGFβ_1蛋白表达情况并进行计算机图像分析，显示OVX组与sham组相比肾小球TGFβ1表达显著升高(P＜0.01)，而SIFV组与OVX组比TGFβ1表达显著降低(P＜0.01)，而补钙组各指标都无显著性意义。
结论大豆异黄酮可预防OVX骨钙、锌、铜丢失，抑制肾脏TGFβ_1蛋白的过度表达，减轻肾脏损伤，预防骨质丢失。
Osteoporosis is a frequent chronic senile disease, and in which bones become fragile and more easily to break. Osteoporosis is not any clinical symptom in the initial stage. If not prevented or if left untreated, osteoporosis can produce and progress stealthily until a bone breaks. These fractures, occur typically in the hip, spine, and wrist. Any bone can be affected, but of special concern are fractures of the hip and spine. A hip bone fracture almost always requires hospitalization and major surgery. Spinal or vertebral fractures also have serious consequences, including angular curvature, severe back pain, and deformity. It can impair a person's ability to walk unassisted and may cause prolonged or permanent disability or even death.
The osteoporosis animal model was established by many scholars with castrated method. The sex hormone level of the model animal was lower, the bone mineral content was more descend also in the research. The research of the Cheng SL discovers that the soybean isoflavone has estrogenic function, it can valid decrease bone lose and increase the bone formation. Arjmandi definitely hints the soybean isoflavone can contain the bone reservation function, point out that consume soybean or its isoflavones to contribute the bone substance's hold out for long time, over a long period of time. But the mechanism of the isoflavone anti osteoporosis is still without thorough research. In this study we investigate the relationship of diet for aged and osteoporosis and research the intervention effect of soy isoflavone and celosiae cristatae flavone.
Part I Relationship of nutrition and bone mineral density in Elderly People
1. Relationship between BMD and Zn, Cu, Ca Levels in the Hair and Meal in Elderly People
Summary: The relationship between bone mineral density (BMD) and Zn, Cu, Ca levels in the meal and hair of urban and rural elderly people were studied. 470 subjects above 60 years old (urban 205 and rural 265), 178 males with an average age of 65.70 ± 3.48y and 292 females with an average age of 65.90±4.02y, were inquired. The BMD and Zn, Cu, Ca levels in the meal and hair were measured. The detected BMD in urban and rural female old people was significantly lower than that of the males; The Ca, Zn contents in the meal of the urban females were significantly lower than those of the urban males; The Ca, Zn in the meal and Zn in the hair of the rural females were significantly lower than those of rural males (P< 0.05 or 0.01). The BMD, Ca intakes, Ca and Zn in the hair of the rural old people were significantly lower than those of the urban old people (P< 0.05 or 0.01). There was a correlation between BMD with the Ca, Zn of the hair and dietary Ca, Zn, Cu or between dietary Zn with Ca, Zn in the hair and Ca, Cu intakes. The Zn, Cu and Ca levels in the meal nutrients were correlated with BMD to some degrees. Lack of Ca and Zn in the meal can cause the reduction of BMD. 2. P Dag Analysis of Effects of daily nutrition intake on BMD Objectives To study the effects of daily mineral intake on bone mineral density (BMD) of old people.
Data Measures The daily nutrition intake and health were investigated in 309 old people aged over 60 years. 132 male, aged 65.4±4.0y, 177 female aged 66.7±5.8y.Based on the data, the regression analysis and causal analysis are presented and thus a partial directed acyclic graph (p dag) are plotted to demonstrate a definite causal relation. Results The paper shows that BMD in female were lower than that in male. For healthy people, the daily intake of Calcium, protein, Vitamin C of male was higher than that of health female. The daily intake of Calcium, protein, Vitamin C of osteoporosis case was lower than that of health people of the same sex. BMD correlation with the daily intake of Calcium, protein, Vitamin C is significantly positive, which is consistent with directed acyclic graph (p dag) analysis.
Conclude It was suggest that there is definite relation between the daily mineral intake and BMD. The lack of daily intake of Calcium, protein, Vitamin C may decrease BMD.
Part II The effect of soy isoflavonord and flavonoid of celosia cristata
on increment, mineralization and partial related gene expression in vitro cultured
1. Effect of flavonoid of celosia cristata on mineralization and IGF-1 expression in
vitro cultured osteoblasts
Objective To study the effect of flavonoid of celosia cristata extract on mineralization and IGF-1 expression of cultured osteoblasts from rat
Methods The osteoblastic cells from cranial bone of newborn rat was culturedthe. The effect of mineralization was detected by alizarin bordeaux stain(ARS). The effect of flavonoid of celosia cristata extract on IGF-1 expression of cultured osteoblasts was detected by SP method. The expression level was monitored with HPIAS1000 photograph analysismeter. Results The IGF-1 of third cultured osteoblasts cultured used flavonoid of celosia cristata
extract was positive expression .The IGF-1 positive expression of flavonoid of celosia
cristata extract group was higher significantly than negative control group (p<0.05) .
Conclusion The flavonoid of celosia cristata extract may increase the mineralization and
IGF-1 positive expression of cultured osteoblasts from rat
2. Effect of soybean isoflavonet on TGF-β1、 TGF-βR1 and TGF-βR2 expression of cultured osteoblasts from rat
Objective To study the effect of soybean isoflavone on TGF-β1 , 、 TGF-βR1 and
TGF-βR2 expression of cultured osteoblasts from rat.
Methods The osteoblstic cells from cranial bone of newborn rat was cultured. The SP
method was used to detect the effect of soybean isoflavone on TGF-β1、 TGF-βR1 and
TGF-βR2 expression of cultured osteoblasts.The expression level was monitored with
HPIAS_1000 photograph analysismeter.
Results The TGF-β1、 TGF-βR1 and TGF-βR2 of osteoblasts cultured used soybean
isoflavone was positive expression .The TGF-β_1 、 TGF-βR1 and TGF-βR2 positive
expression of soybean isoflavone group was higher significantly than negative control
group (p<0.05) ..
Conclusion The soybean isoflavone may increase the TGF-β_1 、 TGF-βR1 and TGF-βR2
positive expression of cultured osteoblasts from rat.
3. Effects of soybean isoflavone and calcium on proliferation, differentiation and
mineralization of cultured osteoblasts in vitro
Objective To explore the effects of soybean isoflavone and calcium on proliferation , differentiation and mineralization of cultured osteoblastic cells.
Methods Osteoblastic cells from newborn rat calvarial was cultured. Osteoblastic cells proliferation was masured by MTT, activity of ALP was observed with Golden's method and ARS was used to measure the mineral nodes of osteoblasts. Results The calcium group had not diferent frome the control group on stimulating the proliferation, differentiation and mineralization of rat calvarial osteoblastic cells.The soybean isoflavone group had significant effcts on stimulating the proliferation , differentiation of osteoblasts and increase the forming of the mineral nodes of osteoblasts in rat. calcium and soybean isoflavone group had more benefit for forming mineral nodes. Conclusion The soybean isoflavone stimulates the proliferation, differentiation and mineralization of rat calvarial osteoblastic cells, and increase calcium in the same time is more benefit for mineralization. 4. Effect of soybean isoflavonet on bone morphogenetic protein 2 expression of
cultured osteoblasts from rat
Objective To study the effect of soybean isoflavone on bone morphogenetic protein 2 (BMP_2)expression of cultured osteoblasts from rat.
Methods The osteoblstic cells from cranial bone of newborn rat was cultured. Cultured rat osteoblasts were treated with soybean isoflavone in difierent concentrations. Total RNA was extracted the from the cells. BMP_2 a were amplified by RT—PCR. β -actin cDNA was also amplified at the same time and used as an internal standard. The SP method was used to detect the effect of soybean isoflavone on BMP_2 expression of cultured osteoblasts. The expression level was monitored with HPIAS_2000 photograph analysismeter. The pictures of PCR production was scanned. The specific values of density of BMP_2/β -actin were calculated, and the expression levels of BMP_2 were determined.
Results Soybean isoflavone increased the level of BMP_2 in rat osteoblasts and increased the expressions of BMP_2 genes at the level of transcripaon and revealed dose-effect relation, (p<0.01) .
Conclusion The soybean isoflavone induces the proliferation and differentiated of osteoblast probably by increasing the expressions of BMP_2 genes. Part III The effect of soy isoflavonord and flavonoid of celosia cristata
on bone metabolism index in disuse osteoporosis and ovariectomized rat
1. The effect of calcium and extraction of cristata flavonoid on disuse osteoporosis in rats
Objective To research the effect of calcium and extraction of cristata flavonoid on disuse osteoporosis in rats.
Method 40 healthy , male SD rats were selected, and randomly divided into 5 groups. The group 1 (normal group) and group 2(positive group) were given with distilled water by intragastrition. Group 2-5 rats were created to the model of disuse osteoporosis. Group 3、 4 and 5 were difference given with cristata flavonoid, calcium carbonate, cristata flavonoid with calcium by intragastrition. After 8 weeks experiment, the bone mineral density(BMD), femur computer tomography (CT), serum malondialdehyde (MDA), superoxide dismutase (SOD), and urine calcium, urine hydroxyproline (HOP), urine creatinine were detected.
Results It was showed that the femur weight, ash weight , CT value in positive group were significantly lower than that of normal group and cristata flavonoid group(P<0.05 or P<0.01). Compare whit positive groups , urinary calcium and HOP were lower and serum calcium, femur BMD, serum SOD were higher in cristata flavonoid and cristata flavonoid with calcium groups(P<0.05 or P<0.01).
Conclusion It was suggested that calcium and cristata flavonoid can effect the bone matrix metabolism of disuse osteoporosis in rats, cristata flavonoid with calcium had more effect on disuse osteoporosis rats. 2. Effect of cristata L flavonoid on prevetive osteoporosis in ovariectomized rats Objective To study the effect of cristata L flavonoid on urine mineral and the expression of bone BMP_2 , and phagocytosis function of Mononuclear macrophage in ovariectomized rats.
Methods Twenty one female SD rats were randomly divided into three groups: sham, ovariectomized(OVX), ovariectomized with cristata L flavonoid (CRIST, 100mg/kg~-· d). The content of urinary calcium, sodium, potassium were measured after ten weeks. The Streptavidin-Peroxidase Histostain~(TM)-Plus Kits was used to detect the expression of BMP_2 in bone. And phagocytosis test of mononuclear macrophage in vitro, carbon clearance measurement were carried out to determine the phagocytotic functions of mononuclear macrophage.
Results Compared with sham group the calcium, sodium in urine expression of ovariectomized group were higher and the potassium of urine and the expression of BMP_2 , and the phago- percentage, and phagocytic index and the carbon clearance index were significantly decreased in OVX group(P<0.05 or <0.01). Compare with OVX group the urine calcium, sodium expression, and the phago- percentage, carbon phagocytic index were significantly in CRIST group, but the bone BMP_2 expression was significantly higher than that of OVX group (P<0.05 or<0.01 ) . Conclusion The cristat L flavonoid could decrease the urinary calcium loss, and increased the expression of BMP_2 , and increased the phagocytotic functions of mononuclear macrophage. It may be advantageous to prevent the postmenopausal Osteoporosis, and to facilitate health care in menopause woman. 3. Effect of soybean isoflavone and calcium on bone mineral density , mineral , BMP_2、 TGF-β_1、 IGF-1 and IGF-1 mRNA express in ovariectomized rats Objective To study the effect of wheat embryo extract on bone mineral density and mineral in ovariectomized rats.
Methods Twenty eight female SD rat were randomly divided into four groups:sham,ovariectomized, ovariectomized added calcium [Ca, 50 mg/kg · d] , ovariectomized added soybean isoflavone [Isof, 100 mg/kg · d], . The experiment proceed ten weeks.
Results The bone mineral density decreased, and urine calcium increased significantly in ovariectomized group; The bone BMP_2、 TGF-β_1、 IGF-1 and IGF-1 mRNA expression in ovariectomized group decreased significantly. The bone mineral density and BMP2、 TGF-β_1、 IGF-1 and IGF-1 mRNA expression in soybean isoflavone group increased significantly than ovariectomized group; The urine calcium decreased significantly. The recovery of these markers was not significantly in merely supply Calcium group than ovariectomized group.
Conclusion The soybean isoflavone may adjustment IGF-1 of growth factor expression, and prevent the lose of calcium of bone and the increase of bone mineral density induced by ovariectomy.
4. Effect of soybean isoflavone and calcium on bone mineral density and mineral
matter and kidney TGF β_1 expression in ovariectomized rats
Objective To explore the effect of soybean isoflavone and calcium on bone mineral density and trace elements in ovariectomized rats.
Methods Twenty eight female SD rats were randomly divided into four groups:sham, ovariectomized, ovariectomized added calcium (50mg.·kg~(-1)·d~(-1)), ovariectomized added soybean isoflavone (100mg.·kg~(-1)·d~(-1)).The content of bone mineral density and mineral , and serum, urine and bone calcium, zinc, copper , iron were determined after ten weeks.
Results After test , the bone mineral density and bone mineral , and bone calcium, zinc, copper , iron, and serum zinc, iron in ovariectomized group decreased significantly; The serum calcium , copper, ALP and urine calcium, copper, iron increased.The bone mineral density and bone mineral , serum zinc , bone calcium, zinc, copper in ovariectomized added soybean isoflavone group increased significantly than ovariectomized group; The serum calcium, copper, ALP and urine calcium,
copper decreased significantly ( P<0.01 or P<0.05 ) . Compare with sham group, the
OVX group kidney weight/body weight(%)increased significantly (P<0.01). Compare
with OVX group, the SIFV group kidney weight/body weight(%)decreased significantly
(P<0.01) . The glomcrulus TGFβ_1 proteinum expression increased significantly in OVX
group than sham group and SIFV group(.P<0.01).
Conclusion The soybean isoflavone may prevent the lose of bone calcium, zinc ,
copper, and restraint the glomcrulus TGFβ_1 proteinum overexpression and prevent the
osteoporosis induced by ovariectomy.
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