大豆苷原对去势大鼠骨、子宫、乳腺组织的选择性作用及ERs/PPARγ的受体机制
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摘要
研究背景
雌激素治疗能有效减少绝经后妇女的骨丢失及骨折发生率,但同时也增加了患乳腺癌和子宫内膜癌的风险。植物雌激素以其潜在的优点,成为研究的热点。大豆苷原是一种常见的植物雌激素,具有广泛的应用前景。已有的研究提示植物雌激素对绝经后骨质疏松症有一定的防治作用,然而也有一些研究结果不尽理想,这可能与植物雌激素对骨质疏松症的作用呈剂量依赖性双相作用有关,但相关研究报道较少;植物雌激素对子宫和乳腺的作用也还没有定论。最近的研究提示雌激素受体(estrogen receptors,ERs)和过氧化物酶增殖子激活受体γ(peroxisome proliferator-activated receptors gamma,PPARγ)可能参予其调节机制,但缺乏动物研究数据,具体作用机制也不清楚。
第一部分大豆苷原防治去势大鼠骨质疏松症的量效作用及其对骨组织ERs和PPARγ表达的影响
目的
研究不同剂量大豆苷原对去势大鼠骨代谢生化指标、骨密度、生物力学、骨形态学以及对骨组织ERα、ERβ和PPARγmRNA和蛋白表达水平的影响,探讨大豆苷原防治去势大鼠骨质疏松症的量效作用及其分子机制。
方法
将4月龄大鼠随机分为6组:假手术组(Sham)、去势组(OVX)、去势+戊酸雌二醇组(E_2,戊酸雌二醇800μg/kg·d)、去势+高剂量大豆苷原组(H-Dai,大豆苷原200 mg/kg·d)、去势+中剂量大豆苷原组(M-Dai,大豆苷原50 mg/kg·d)和去势+低剂量大豆苷原组(L-Dai,大豆苷原10 mg/kg·d),除假手术组外,其余大鼠均切除卵巢,术后4周开始以相应的药物灌胃,3月后处死大鼠。取血清检测E_2、Ca、P、ALP和BGP水平;取腰椎骨L_(1-5)和左股骨检测骨密度;取腰椎骨L_3和左股骨检测生物力学;取腰椎L_4做骨形态分析;以腰椎L_6和右股骨提取总RNA,Realtime-RT-PCR方法检测ERα、ERβ和PPARγmRNA表达;免疫组化检测腰椎L_4与左股骨ERα、ERβ和PPARγ蛋白表达。
结果
1、L-Dai组大鼠腰椎BMD、最大压缩载荷、骨小梁与椎体总面积比值,以及股骨BMD和最大三点弯曲载荷均比OVX组显著增高,与E_2组无显著性差异;M-Dai组大鼠腰椎BMD、最大压缩载荷、骨小梁与椎体总面积比值以及股骨BMD比OVX组显著提高;H-Dai组大鼠只有腰椎BMD、最大压缩载荷、骨小梁与椎体总面积比值等指标显著高于OVX组。
2、M-Dai组大鼠腰椎BMD、骨小梁与椎体总面积比值以及股骨最大三点弯曲载荷等指标都显著低于L-Dai组大鼠;H-Dai组大鼠腰椎BMD、骨小梁与椎体总面积比值,以及股骨BMD和最大三点弯曲载荷均显著低于L-Dai组大鼠。
3、Dai三个剂量组大鼠腰椎和股骨ERαmRNA和蛋白表达水平均与OVX组无显著性差异。
4、L-Dai和M-Dai组大鼠腰椎和股骨ERβmRNA和蛋白表达水平均显著高于OVX组,但M-Dai组大鼠股骨ERβ蛋白表达水平却显著低于L-Dai组大鼠;H-Dai组大鼠仅腰椎ERβmRNA和蛋白表达水平显著高于OVX组,股骨则与OVX组无显著性差异,且显著低于L-Dai组大鼠。
5、Dai三个剂量组大鼠腰椎和股骨PPARγmRNA和蛋白表达水平均显著低于OVX组大鼠,H-Dai组表达水平高于另外两个剂量Dai组,但三组间差异没有显著性。
结论
1、大豆苷原对骨产生雌激素样作用,对去势大鼠骨质疏松症有防治作用,呈现剂量依赖性关系,低剂量时效果与雌二醇相当,高剂量时效果呈现降低的趋势。
2、大鼠腰椎和股骨均表达ERα和ERβ,但以ERβ为主,提示大豆苷原主要通过ERβ对骨产生保护作用。
3、大豆苷原低剂量时大鼠腰椎和股骨ERβ表达水平高,随着剂量增加,ERβ表达水平呈下降趋势,而PPARγ表达水平则呈上升趋势,这提示ERβ和PPARγ之间可能存在一种交叉对话的关系,并且参予大豆苷原防治去势大鼠骨质疏松症量效作用。
第二部分大豆苷原对去势大鼠子宫内膜的组织选择性作用
目的
研究不同剂量大豆苷原对去势大鼠子宫内膜组织形态以及ERs和PPARγmRNA和蛋白表达水平的影响,探讨大豆苷原对子宫内膜的选择性作用及其机制。
方法
本实验选用第一部分实验大鼠的子宫组织,检测子宫组织的病理形态,以Realtime-RT-PCR检测子宫内膜ERα、ERβ和PPARγmRNA表达,以免疫组化法检测其蛋白表达。
结果
1、Dai三个剂量组大鼠子宫湿重较OVX组有所增加,但与OVX组间没有显著性差异,且显著低于E_2组。
2、L-Dai和M-Dai组大鼠子宫内膜腔上皮、间质、内膜厚度和横切面面积与OVX组间无显著性差异;H-Dai组大鼠子宫内膜腔上皮、间质和内膜厚度较OVX组显著性增厚,并显著高于L-Dai组,但仍显著低于E_2组。
3、H-Dai组大鼠子宫内膜ERαmRNA和蛋白表达水平均显著高于OVX组,且显著高于L-Dai组;L-Dai和M-Dai组则与OVX组间无显著性差异。
4、子宫内膜ERβmRNA仅在Sham组和E_2组中检测出,其余组未检出;各组ERβ蛋白均未检出。
5、Dai三个剂量组大鼠子宫内膜PPARγmRNA表达水平显著低于OVX组,组间差异无显著性;各组PPARγ蛋白均未检出。
结论
1、中低剂量大豆苷原对去势大鼠子宫内膜组织没有雌激素样刺激作用,而高剂量大豆苷原可能通过刺激ERα对去势大鼠子宫内膜发挥弱雌激素样作用。
2、大鼠子宫内膜组织主要表达ERα。
3、大鼠子宫内膜PPARγ的作用还需要进一步研究以明确。
第三部分大豆苷原对去势大鼠乳腺的组织选择性作用
目的
研究不同剂量大豆苷原对去势大鼠乳腺组织形态以及ERs和PPARγmRNA和蛋白表达水平的影响,探讨大豆苷原对乳腺组织的选择性作用及其机制。
方法
本实验选用第一部分实验大鼠的乳腺组织,检测乳腺组织的病理形态,以Realtime-RT-PCR检测ERα、ERβ和PPARγmRNA表达,以免疫组化法检测其蛋白表达。
结果
1、OVX组大鼠乳腺呈明显的萎缩状态,Dai三个剂量组大鼠乳腺组织形态较之OVX组无明显改变。
2、Dai三个剂量组大鼠乳腺组织ERαmRNA表达水平与OVX组无显著性差异但显著低于E_2组。
3、乳腺组织ERβmRNA仅在Sham组和E_2组中检测出,其余组均未检出。
4、Dai三个剂量组大鼠乳腺组织PPARγmRNA表达水平显著低于OVX组,组间差异无显著性。
5、除Sham组和E_2组外,各组大鼠乳腺均未见腺泡结构,因此ERα、ERβ和PPARγ蛋白表达均未检出。
结论
1、三个剂量大豆苷原对去势大鼠乳腺组织均没有雌激素样刺激作用,可能和ERα的作用有关。
2、大鼠乳腺组织主要表达ERα。
3、大鼠乳腺PPARγ的作用还需要进一步研究以明确。
Background
Estrogen can effectively reduce the bone loss and incidence of fracture in post menopausal women.However,it may lead to an increase of breast cancer,uterine endometrial carcinoma and other adverse events.Phytoestrogen has potential benefits and becomes a hot of research.Daidzein is one of the most important phytoestrogen and show potential application prospects.It is revealed that phytoestrogens can protect bone mass to some extent,while some researches show the opposite result. These conflicting results might be due to the biphasic dose-dependent effects of phytoestrogen;however the related reports are few.It is still unknown the effects of phytoestrogens on lacteal gland and uterus.Recently,some researches have revealed that estrogen receptors and peroxisome proliferator-activated receptor gamma may mediate the dose-dependently bone-protective effect and tissue-selective effect of phytoestrogen,but whether it works in vivo is open to doubt
PartⅠThe Dose-dependent Effect of Daidzein on Osteoporosis in the Ovariectomized(OVX) Rats and its Effects on the Expression of mRNA and Protein of Bone Tissue's ERs and PPARγ
Objective
To investigate the effect of daidzein on the biochemical indexes of bone metabolism, bone mineral density,biomechanical testing,histomorphometry and the expression of ERα,ERβand PPARγmRNA and protein of bone tissue in the ovariectomized(OVX) rats,and discuss the dose-dependent effect and the molecular mechanisms of daidzein on osteoporosis in the ovariectomized rats.
Materials and methods
Sixty four-month-old Sprague-Dawley(SD) female rats were allotted to one of six groups.Four weeks after reproducing the osteoporosis model of ovariectomized SD rats,Sham group and control OVX were administrated with distilled water,whereas the other groups received valerate estriol(800μg/kg·d) and different doses of daidzein (10,50,200 mg/kg·d) by gavage respectively for three months.These parameterswere measured after the sacrifice of the rats.Using a dual energy x-ray absorptiometer,bone mineral density were measured in the lumbar spine L_(1-5) and left femur.The expressions ofmRNA of ERa,ERβand PPARγin L_6 and right femur were measured by realtime-RT-PCR,while proteins were measured by immunohistochemistry.
Results
1、The BMD,biomechanical testing and histomorphometry of lumbar spine and left femur decreased in OVX group while these indexes increased in L-Dai group significantly.Except biomechanical testing of left femur,rats in M-Dai group showed all indexes higher than OVX group significantly,while only these indexes of lumbar spine of rats in H-Dai group higher significantly.
2、The BMD and histomorphometry of lumbar spine and biomechanical testing of left femur in M-Dai group,and the BMD and histomorphometry of lumbar spine and BMD and biomechanical testing of left femur in H-Dai group,were all lower than L-Dai group significantly.
3、The expressions of ERαmRNA and protein of lumbar spine and left femur in three Dai groups and OVX group showed no significant difference.
4、The expressions of ERβmRNA and protein of lumbar spine and left femur in L-Dai and M-Dai groups were significantly higher than OVX group,while the protein expression of ERβof left femur in M-Dai group was lower than L-Dai group significantly.Only the expressions of ERβmRNA and protein of lumbar spine in H-Dai group were higher than OVX group significantly,and these of left femur between two groups showed no significant difference.
5、The expressions of PPARγmRNA of bone in rats in three Dai groups decreased significantly compared with OVX group.H-Dai group was higher than other two Dai groups,but no significant difference between them.
Conclusions
1、Daidzein shows estrogen-like effect on bone,resulted into dose-dependent effect on prevention and treatment of osteoporosis in ovariectomized rats,while the low-dose of daidzein is more effective.
2、Both ERs expressed in lumbar spine and femur,however ERβwas more than ERa, which implied that the bone-protective effect of daidzein through ERβmainly.
3、When daidzein in level of low dose,the level of expression of ERβin bone was higher.And the expression of ERβshowed lower trend and PPARγshowed higher trend with the dose of daidzein increasing.Which implied that there is a cross talk between ERβand PPARγ,and it is related with the dose-dependent effect of daidzein on osteoporosis in the ovariectomized rats.
PartⅡThe Tissue-selective Effect of Daidzein on Uterine Endometrial Tissue of the Ovariectomized Rats
Objective
To investigate the effect of daidzein on the histomorphometry and the expression of mRNA and protein of ERa,ERβand PPARγin the ovariectomized(OVX) rats' uterine endometrial tissue,and discuss the tissue-selective effect and the molecular mechanisms of daidzein on uterine endometrial tissue of the ovariectomized rats.
Materials and methods
Using uterine endometrial tissues of rats in PartⅠ,we detected their histomorphometry, and the expressions of mRNA of ERa,ERβand PPARγrealtime-RT-PCR,while protein were by immunohistochemistry.
Results
1、The uterus weight of rats in three Dai groups increased,but no significant difference between them and OVX group.
2、Uterine endometrial thickness and area of transverse section of rats in L-Dai and M-Dai groups showed no significant difference with OVX group,while the uterine endometrial thickness of rats in H-Dai group higher than OVX and L-Dai groups significantly.
3、The expression of ERa mRNA and protein of uterine endometrial tissue were higher than OVX and L-Dai groups significantly.
4、The expression of ERβmRNA of uterine endometrial tissue only could be detected in Sham and E_2 group,while the protein expression could not be detected in all groups.
5、The level of PPARγof uterine endometrial tissue in three Dai groups decreased significantly,and there no significant difference between them,while the protein expression could not be detected in all groups.
Conclusions
1、L-Dai and M-Dai did not show estrogen-like effect on uterine endometrial tissue of OVX rats,however H-Dai may play weak estrogen-like effect through ERa.
2、The uterine endometrial tissue mainly expresses ERα.
3、The role of PPARγin uterine endometrial tissue of rats needs further researchs.
PartⅢThe Tissue-selective Effect of Daidzein on Lacteal Gland of the Ovariectomized Rats
Objective
To investigate the effect of daidzein on the histomorphometry and the expression of mRNA and protein of ERa,ERβand PPARγin the ovariectomized(OVX) rats' breast, and discuss the tissue-selective effect and the molecular mechanisms of daidzein on lacteal gland of the ovariectomized rats.
Materials and methods
Using breast tissues of rats in PartⅠ,we detected their histomorphometry,and the expressions of mRNA of ERα,ERβand PPARγrealtime-RT-PCR,while protein were by immunohistochemistry.
Results
1、Rats in OVX group showed atrophic breast,without obvious acinar structure,and the same in three Dai groups.
2、The expression of ERαmRNA of lacteal gland in three Dai groups were no significant difference with OVX group,but significantly lower than E_2 groups.
3、The expression of ERβof breast was only detected in Sham and E_2 groups.
4、The level of PPARγin three Dai groups decreased significantly,and there no significant difference between them.
5、The expression of ERs and PPARγprotein were not detected in all groups.
Conclusions
1、Daidzein had no estrogen-like stimulation on breast of OVX rats,which may be related with ERa.
2、The breast tissue mainly expresses ERα.
3、The role of PPARγin lacteal gland of rats needs further researchs.
Summary
1、Daidzein shows tissue-selective effect on bone,uterus and lacteal gland of the OVX Rats.
2、Daidzein shows estrogen-like effect on bone,resulted into dose-dependent effect on prevention and treatment of osteoporosis in ovafiectomized rats,while may be related with the cross talk between ERβand PPARγ.
3、L-Dai and M-Dai did not show estrogen-like effect on uterine endometrial tissue, however H-Dai may play weak estrogen-like effect through ERα.
4、Daidzein had no estrogen-like stimulation on breast.
Chinese Library Classification:R71
雌激素治疗能有效减少绝经后妇女的骨丢失及骨折发生率,但同时也增加了患乳腺癌和子宫内膜癌的风险。植物雌激素以其潜在的优点,成为研究的热点。大豆苷原是一种常见的植物雌激素,具有广泛的应用前景。已有的研究提示植物雌激素对绝经后骨质疏松症有一定的防治作用,然而也有一些研究结果不尽理想,这可能与植物雌激素对骨质疏松症的作用呈剂量依赖性双相作用有关,但相关研究报道较少;植物雌激素对子宫和乳腺的作用也还没有定论。最近的研究提示雌激素受体(estrogen receptors,ERs)和过氧化物酶增殖子激活受体γ(peroxisome proliferator-activated receptors gamma,PPARγ)可能参予其调节机制,但缺乏动物研究数据,具体作用机制也不清楚。
第一部分大豆苷原防治去势大鼠骨质疏松症的量效作用及其对骨组织ERs和PPARγ表达的影响
目的
研究不同剂量大豆苷原对去势大鼠骨代谢生化指标、骨密度、生物力学、骨形态学以及对骨组织ERα、ERβ和PPARγmRNA和蛋白表达水平的影响,探讨大豆苷原防治去势大鼠骨质疏松症的量效作用及其分子机制。
方法
将4月龄大鼠随机分为6组:假手术组(Sham)、去势组(OVX)、去势+戊酸雌二醇组(E_2,戊酸雌二醇800μg/kg·d)、去势+高剂量大豆苷原组(H-Dai,大豆苷原200 mg/kg·d)、去势+中剂量大豆苷原组(M-Dai,大豆苷原50 mg/kg·d)和去势+低剂量大豆苷原组(L-Dai,大豆苷原10 mg/kg·d),除假手术组外,其余大鼠均切除卵巢,术后4周开始以相应的药物灌胃,3月后处死大鼠。取血清检测E_2、Ca、P、ALP和BGP水平;取腰椎骨L_(1-5)和左股骨检测骨密度;取腰椎骨L_3和左股骨检测生物力学;取腰椎L_4做骨形态分析;以腰椎L_6和右股骨提取总RNA,Realtime-RT-PCR方法检测ERα、ERβ和PPARγmRNA表达;免疫组化检测腰椎L_4与左股骨ERα、ERβ和PPARγ蛋白表达。
结果
1、L-Dai组大鼠腰椎BMD、最大压缩载荷、骨小梁与椎体总面积比值,以及股骨BMD和最大三点弯曲载荷均比OVX组显著增高,与E_2组无显著性差异;M-Dai组大鼠腰椎BMD、最大压缩载荷、骨小梁与椎体总面积比值以及股骨BMD比OVX组显著提高;H-Dai组大鼠只有腰椎BMD、最大压缩载荷、骨小梁与椎体总面积比值等指标显著高于OVX组。
2、M-Dai组大鼠腰椎BMD、骨小梁与椎体总面积比值以及股骨最大三点弯曲载荷等指标都显著低于L-Dai组大鼠;H-Dai组大鼠腰椎BMD、骨小梁与椎体总面积比值,以及股骨BMD和最大三点弯曲载荷均显著低于L-Dai组大鼠。
3、Dai三个剂量组大鼠腰椎和股骨ERαmRNA和蛋白表达水平均与OVX组无显著性差异。
4、L-Dai和M-Dai组大鼠腰椎和股骨ERβmRNA和蛋白表达水平均显著高于OVX组,但M-Dai组大鼠股骨ERβ蛋白表达水平却显著低于L-Dai组大鼠;H-Dai组大鼠仅腰椎ERβmRNA和蛋白表达水平显著高于OVX组,股骨则与OVX组无显著性差异,且显著低于L-Dai组大鼠。
5、Dai三个剂量组大鼠腰椎和股骨PPARγmRNA和蛋白表达水平均显著低于OVX组大鼠,H-Dai组表达水平高于另外两个剂量Dai组,但三组间差异没有显著性。
结论
1、大豆苷原对骨产生雌激素样作用,对去势大鼠骨质疏松症有防治作用,呈现剂量依赖性关系,低剂量时效果与雌二醇相当,高剂量时效果呈现降低的趋势。
2、大鼠腰椎和股骨均表达ERα和ERβ,但以ERβ为主,提示大豆苷原主要通过ERβ对骨产生保护作用。
3、大豆苷原低剂量时大鼠腰椎和股骨ERβ表达水平高,随着剂量增加,ERβ表达水平呈下降趋势,而PPARγ表达水平则呈上升趋势,这提示ERβ和PPARγ之间可能存在一种交叉对话的关系,并且参予大豆苷原防治去势大鼠骨质疏松症量效作用。
第二部分大豆苷原对去势大鼠子宫内膜的组织选择性作用
目的
研究不同剂量大豆苷原对去势大鼠子宫内膜组织形态以及ERs和PPARγmRNA和蛋白表达水平的影响,探讨大豆苷原对子宫内膜的选择性作用及其机制。
方法
本实验选用第一部分实验大鼠的子宫组织,检测子宫组织的病理形态,以Realtime-RT-PCR检测子宫内膜ERα、ERβ和PPARγmRNA表达,以免疫组化法检测其蛋白表达。
结果
1、Dai三个剂量组大鼠子宫湿重较OVX组有所增加,但与OVX组间没有显著性差异,且显著低于E_2组。
2、L-Dai和M-Dai组大鼠子宫内膜腔上皮、间质、内膜厚度和横切面面积与OVX组间无显著性差异;H-Dai组大鼠子宫内膜腔上皮、间质和内膜厚度较OVX组显著性增厚,并显著高于L-Dai组,但仍显著低于E_2组。
3、H-Dai组大鼠子宫内膜ERαmRNA和蛋白表达水平均显著高于OVX组,且显著高于L-Dai组;L-Dai和M-Dai组则与OVX组间无显著性差异。
4、子宫内膜ERβmRNA仅在Sham组和E_2组中检测出,其余组未检出;各组ERβ蛋白均未检出。
5、Dai三个剂量组大鼠子宫内膜PPARγmRNA表达水平显著低于OVX组,组间差异无显著性;各组PPARγ蛋白均未检出。
结论
1、中低剂量大豆苷原对去势大鼠子宫内膜组织没有雌激素样刺激作用,而高剂量大豆苷原可能通过刺激ERα对去势大鼠子宫内膜发挥弱雌激素样作用。
2、大鼠子宫内膜组织主要表达ERα。
3、大鼠子宫内膜PPARγ的作用还需要进一步研究以明确。
第三部分大豆苷原对去势大鼠乳腺的组织选择性作用
目的
研究不同剂量大豆苷原对去势大鼠乳腺组织形态以及ERs和PPARγmRNA和蛋白表达水平的影响,探讨大豆苷原对乳腺组织的选择性作用及其机制。
方法
本实验选用第一部分实验大鼠的乳腺组织,检测乳腺组织的病理形态,以Realtime-RT-PCR检测ERα、ERβ和PPARγmRNA表达,以免疫组化法检测其蛋白表达。
结果
1、OVX组大鼠乳腺呈明显的萎缩状态,Dai三个剂量组大鼠乳腺组织形态较之OVX组无明显改变。
2、Dai三个剂量组大鼠乳腺组织ERαmRNA表达水平与OVX组无显著性差异但显著低于E_2组。
3、乳腺组织ERβmRNA仅在Sham组和E_2组中检测出,其余组均未检出。
4、Dai三个剂量组大鼠乳腺组织PPARγmRNA表达水平显著低于OVX组,组间差异无显著性。
5、除Sham组和E_2组外,各组大鼠乳腺均未见腺泡结构,因此ERα、ERβ和PPARγ蛋白表达均未检出。
结论
1、三个剂量大豆苷原对去势大鼠乳腺组织均没有雌激素样刺激作用,可能和ERα的作用有关。
2、大鼠乳腺组织主要表达ERα。
3、大鼠乳腺PPARγ的作用还需要进一步研究以明确。
Background
Estrogen can effectively reduce the bone loss and incidence of fracture in post menopausal women.However,it may lead to an increase of breast cancer,uterine endometrial carcinoma and other adverse events.Phytoestrogen has potential benefits and becomes a hot of research.Daidzein is one of the most important phytoestrogen and show potential application prospects.It is revealed that phytoestrogens can protect bone mass to some extent,while some researches show the opposite result. These conflicting results might be due to the biphasic dose-dependent effects of phytoestrogen;however the related reports are few.It is still unknown the effects of phytoestrogens on lacteal gland and uterus.Recently,some researches have revealed that estrogen receptors and peroxisome proliferator-activated receptor gamma may mediate the dose-dependently bone-protective effect and tissue-selective effect of phytoestrogen,but whether it works in vivo is open to doubt
PartⅠThe Dose-dependent Effect of Daidzein on Osteoporosis in the Ovariectomized(OVX) Rats and its Effects on the Expression of mRNA and Protein of Bone Tissue's ERs and PPARγ
Objective
To investigate the effect of daidzein on the biochemical indexes of bone metabolism, bone mineral density,biomechanical testing,histomorphometry and the expression of ERα,ERβand PPARγmRNA and protein of bone tissue in the ovariectomized(OVX) rats,and discuss the dose-dependent effect and the molecular mechanisms of daidzein on osteoporosis in the ovariectomized rats.
Materials and methods
Sixty four-month-old Sprague-Dawley(SD) female rats were allotted to one of six groups.Four weeks after reproducing the osteoporosis model of ovariectomized SD rats,Sham group and control OVX were administrated with distilled water,whereas the other groups received valerate estriol(800μg/kg·d) and different doses of daidzein (10,50,200 mg/kg·d) by gavage respectively for three months.These parameterswere measured after the sacrifice of the rats.Using a dual energy x-ray absorptiometer,bone mineral density were measured in the lumbar spine L_(1-5) and left femur.The expressions ofmRNA of ERa,ERβand PPARγin L_6 and right femur were measured by realtime-RT-PCR,while proteins were measured by immunohistochemistry.
Results
1、The BMD,biomechanical testing and histomorphometry of lumbar spine and left femur decreased in OVX group while these indexes increased in L-Dai group significantly.Except biomechanical testing of left femur,rats in M-Dai group showed all indexes higher than OVX group significantly,while only these indexes of lumbar spine of rats in H-Dai group higher significantly.
2、The BMD and histomorphometry of lumbar spine and biomechanical testing of left femur in M-Dai group,and the BMD and histomorphometry of lumbar spine and BMD and biomechanical testing of left femur in H-Dai group,were all lower than L-Dai group significantly.
3、The expressions of ERαmRNA and protein of lumbar spine and left femur in three Dai groups and OVX group showed no significant difference.
4、The expressions of ERβmRNA and protein of lumbar spine and left femur in L-Dai and M-Dai groups were significantly higher than OVX group,while the protein expression of ERβof left femur in M-Dai group was lower than L-Dai group significantly.Only the expressions of ERβmRNA and protein of lumbar spine in H-Dai group were higher than OVX group significantly,and these of left femur between two groups showed no significant difference.
5、The expressions of PPARγmRNA of bone in rats in three Dai groups decreased significantly compared with OVX group.H-Dai group was higher than other two Dai groups,but no significant difference between them.
Conclusions
1、Daidzein shows estrogen-like effect on bone,resulted into dose-dependent effect on prevention and treatment of osteoporosis in ovariectomized rats,while the low-dose of daidzein is more effective.
2、Both ERs expressed in lumbar spine and femur,however ERβwas more than ERa, which implied that the bone-protective effect of daidzein through ERβmainly.
3、When daidzein in level of low dose,the level of expression of ERβin bone was higher.And the expression of ERβshowed lower trend and PPARγshowed higher trend with the dose of daidzein increasing.Which implied that there is a cross talk between ERβand PPARγ,and it is related with the dose-dependent effect of daidzein on osteoporosis in the ovariectomized rats.
PartⅡThe Tissue-selective Effect of Daidzein on Uterine Endometrial Tissue of the Ovariectomized Rats
Objective
To investigate the effect of daidzein on the histomorphometry and the expression of mRNA and protein of ERa,ERβand PPARγin the ovariectomized(OVX) rats' uterine endometrial tissue,and discuss the tissue-selective effect and the molecular mechanisms of daidzein on uterine endometrial tissue of the ovariectomized rats.
Materials and methods
Using uterine endometrial tissues of rats in PartⅠ,we detected their histomorphometry, and the expressions of mRNA of ERa,ERβand PPARγrealtime-RT-PCR,while protein were by immunohistochemistry.
Results
1、The uterus weight of rats in three Dai groups increased,but no significant difference between them and OVX group.
2、Uterine endometrial thickness and area of transverse section of rats in L-Dai and M-Dai groups showed no significant difference with OVX group,while the uterine endometrial thickness of rats in H-Dai group higher than OVX and L-Dai groups significantly.
3、The expression of ERa mRNA and protein of uterine endometrial tissue were higher than OVX and L-Dai groups significantly.
4、The expression of ERβmRNA of uterine endometrial tissue only could be detected in Sham and E_2 group,while the protein expression could not be detected in all groups.
5、The level of PPARγof uterine endometrial tissue in three Dai groups decreased significantly,and there no significant difference between them,while the protein expression could not be detected in all groups.
Conclusions
1、L-Dai and M-Dai did not show estrogen-like effect on uterine endometrial tissue of OVX rats,however H-Dai may play weak estrogen-like effect through ERa.
2、The uterine endometrial tissue mainly expresses ERα.
3、The role of PPARγin uterine endometrial tissue of rats needs further researchs.
PartⅢThe Tissue-selective Effect of Daidzein on Lacteal Gland of the Ovariectomized Rats
Objective
To investigate the effect of daidzein on the histomorphometry and the expression of mRNA and protein of ERa,ERβand PPARγin the ovariectomized(OVX) rats' breast, and discuss the tissue-selective effect and the molecular mechanisms of daidzein on lacteal gland of the ovariectomized rats.
Materials and methods
Using breast tissues of rats in PartⅠ,we detected their histomorphometry,and the expressions of mRNA of ERα,ERβand PPARγrealtime-RT-PCR,while protein were by immunohistochemistry.
Results
1、Rats in OVX group showed atrophic breast,without obvious acinar structure,and the same in three Dai groups.
2、The expression of ERαmRNA of lacteal gland in three Dai groups were no significant difference with OVX group,but significantly lower than E_2 groups.
3、The expression of ERβof breast was only detected in Sham and E_2 groups.
4、The level of PPARγin three Dai groups decreased significantly,and there no significant difference between them.
5、The expression of ERs and PPARγprotein were not detected in all groups.
Conclusions
1、Daidzein had no estrogen-like stimulation on breast of OVX rats,which may be related with ERa.
2、The breast tissue mainly expresses ERα.
3、The role of PPARγin lacteal gland of rats needs further researchs.
Summary
1、Daidzein shows tissue-selective effect on bone,uterus and lacteal gland of the OVX Rats.
2、Daidzein shows estrogen-like effect on bone,resulted into dose-dependent effect on prevention and treatment of osteoporosis in ovafiectomized rats,while may be related with the cross talk between ERβand PPARγ.
3、L-Dai and M-Dai did not show estrogen-like effect on uterine endometrial tissue, however H-Dai may play weak estrogen-like effect through ERα.
4、Daidzein had no estrogen-like stimulation on breast.
Chinese Library Classification:R71
引文
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1. Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocrine Reviews,2000,21:115-137.
2. Manolagas SC and Kousteni S. Perspective: Nonreproductive Sites of Action of Reproductive Hormonesl. Endocrinology,2001,142(6):2200～2204.
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5. Bellido T, Plotkin LI, Han L, et al. Estrogen in hibit apoptosis of osteoblast and osteocytes through rapid (nongenomic) activation of extra cellular signal regulated kinases (ERKs) . J Bone Miner Res, 1999,23:SA135.
6. Lorenzo J. A new hypothesis for how sex steroid hormones regulate bone mass. J Clin Invest, 2003, 111:1641 — 1643.
7. Kousteni S, Bellido T, Plotkin LI, et al. Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell, 2001,104:719-730.
8. Piva R, Penolazzi L, Lambertini E, et al. Induction of apoptosis of human primary osteoclasts treated with a transcription factor decoy mimicking a promoter region of estrogen receptor alpha. Apoptosis, 2005,10(5):1079—1094.
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15. Zecchi-Orlandini S, Formigli L, Tani A, et al. 17beta-estradiol induces apoptosis in the preosteoclastic FLG 29.1 cell line. Biochemical & Biophysical Research Communications. 1999,255(3):680～685.
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17. Gangji V, Hauzeur JP, Scboutens A, et al. Abnormalities in the replicative capacity of osteoblastic cells in the proximal femur of patients with osteonecrosis of the femoral head. J Rhenmatol,2003,30(2):348～351.
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