二仙汤及活性成分植物雌激素样作用机制研究
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摘要
一研究目的
植物雌激素(phytoestrogen, PE)是从天然植物中提取的结构与内源性雌激素相似的一类的化合物,能够与雌激素受体(estrogen receptors, ERs)结合发挥生物学效应,具有雌激素和抗雌激素双重效应,发挥哪种效应取决于内源性雌激素的水平和组织类型。流行病学研究发现植物雌激素在许多疾病的预防和治疗方面发挥有益的作用,如心血管疾病、围绝经期综合症以及与雌激素相关的肿瘤(乳腺癌、前列腺癌)等。然而,研究中也发现不一致的研究结果。因此,对其作用机制的研究自然成为研究者们关注的热点。中药复方作为中医治病用药的最主要形式之一,具有植物雌激素样作用的中药及复方在这些疾病的预防和治疗中发挥重要作用。
二仙汤具有温肾益精、滋阴泻火、调理冲任和平衡阴阳之功效,是治疗更年期综合征的经典方剂。随着研究的不断深入,动物实验和体外研究发现二仙汤具有调整生殖／内分泌,调节免疫功能和自由基代谢,在临床上有着广阔的应用前景。我们课题组在前期研究工作中发现二仙汤及方中淫羊藿具有雌激素样作用,但对于其发挥雌激素样作用靶点、作用途径和机理缺乏深入分析和探讨。因此,本实验从整体、细胞和分子水平阐明二仙汤及组方中药的植物雌激素作用以及发挥作用的物质基础和作用机制,为临床合理、科学用药提供理论指导。
二研究方法和结果
1二仙汤及其组方中药对幼年大鼠子宫作用的实验研究
采用子宫增重实验观察二仙汤及其组方中药对幼年大鼠子宫系数的影响；检测血清激素水平的变化；HE染色观察不同药物对大鼠子宫内膜厚度和上皮高度的影响；免疫组织化学染色检测ERα和ERβ在大鼠子宫中的表达情况,评价二仙汤及组方中药的雌激素样作用。
结果：已烯雌酚组、二仙汤组、淫羊藿组和仙茅组子宫系数与正常组对比增加明显,有统计学意义(P<0.05)。仅已烯雌酚组E2增高。HE染色发现已烯雌酚组子宫内膜厚度和上皮高度明显增加,并且局部增生,上皮由单层变为复层。免疫组织化学染色发现已烯雌酚组ERα和ERβ平均光密度值低于正常对照组,而二仙汤组、淫羊藿组和仙茅组的ERα和ERβ平均光密度值均高于正常对照组。
2β-谷甾醇和仙茅苷对T47D细胞增殖和细胞周期的影响
以二仙汤方剂中六味中药的活性成分β-谷甾醇、仙茅的主要活性成分仙茅苷为研究对象,通过MTT法检测它们对体外培养的ER阳性人乳腺癌细胞系T47D的细胞增殖的影响；流式细胞术检测它们对细胞周期的影响；并以雌激素受体完全拮抗剂ICI182 780对细胞增殖的干预作用,探讨β-谷甾醇和仙茅苷的植物雌激素样活性。
结果：高浓度(10-5 mol·L-1和10-6 mol·L-1)仙茅苷作用类似于雌二醇,随着浓度的降低仙茅苷对T47D细胞的促增殖作用明显降低,10-7 mol·L-1和10-8 mol·L-1仙茅苷的促增殖作用仅持续48h,其增殖作用能够被ICI182 780所抑制；高浓度(10-5 mol·L-1和10-6 mol·L-1β-谷甾醇可以抑制T47D细胞增殖,且ICI182 780不能完全拮抗其对T47D细胞的增殖抑制作用,低浓度β-谷甾醇可促进T47D细胞增殖,且具有时间与浓度依赖性,以10-7 mol·L-1和10-8 mol·L-1β-谷甾醇作用最强,ICI182,780可完全拮抗其促增殖作用。
应用流式细胞仪测定细胞周期,雌二醇使T47D细胞G。／G1期细胞的比例明显减低,而S期细胞的比例则明显升高；不同浓度的仙茅苷刺激T47D细胞,随着浓度的升高,G0／G1期细胞的比例降低,而S期细胞的比例则明显升高,细胞分裂增殖指数升高；T47D细胞在高浓度β-谷甾醇作用下,G0/G1期细胞的比例升高,S期细胞的比例下降,细胞分裂增殖指数降低,而低浓度β-谷甾醇使T47D细胞G0/G1期细胞的比例降低,S期细胞的比例升高,细胞分裂增殖指数升高。
3报告基因方法对β-谷甾醇和仙茅苷植物雌激素样作用的研究
通过荧光素酶报告基因技术方法探索二仙汤中两种活性成分β-谷甾醇和仙茅苷是否通过ERα和ERβ发挥植物雌激素样作用。
结果：β-谷甾醇可诱导ERE报告基因的转录激活,且在10-7 mol·L-1和10-8 mol·L-1浓度下诱导作用最明显。对比β-谷甾醇通过ERα和ERβ的诱导作用发现,不同浓度β-谷甾醇通过ERβ诱导报告基因表达倍数更高,提示β-谷甾醇可能对ERβ有更高的激活效应。仙茅苷可通过ERα或ERβ诱导ERE报告基因荧光素酶的转录,且呈明显的剂量依赖效应。而低浓度仙茅苷(10'-8mol·L-1)只能通过ERβ激活报告基因荧光素酶的转录,而对过表达ERα的HEK-293细胞则没有明显的作用,提示仙茅苷对ERβ具有更高的激活效应。
4β-谷甾醇对T47D细胞ER蛋白和cyclin D1蛋白表达的影响
以ER阳性人乳腺癌细胞系T47D细胞为研究对象,应用Western blotting法观察β-谷甾醇及在雌激素受体完全拮抗剂ICI182 780干预下,对ER及下游基因cyclin D1蛋白表达的影响,探讨β-谷甾醇发挥植物雌激素样作用的分子机制。
结果：T47D细胞以ERα蛋白表达占优势,ERβ蛋白低表达,10-7 mol·L-1β-谷甾醇可诱导T47D细胞ERα、ERβ及cyclin D1蛋白表达水平升高,且可以被雌激素受体完全拮抗剂ICI182 780抑制。
5β-谷甾醇对T47D细胞ER、PS2和cyclin D1 mRNA表达的影响
采用RT-PCR技术观察β-谷甾醇对ER及其下游基因如PS2、cyclin DlmRNA表达的影响,在基因水平上进一步探讨β-谷甾醇发挥植物雌激素样作用的分子机制。
结果：β-谷甾醇可上调ERα和ERβ基因表达水平,相对表达量明显升高,且与雌二醇相比,上调ERβ表达更显著,约为溶剂对照组的7倍左右。可诱导PS2和cyclinD1mRNA表达,相对表达量明显增多,并且ICI182 780能够抑制β-谷甾醇对下游基因的诱导作用。
三研究结论
1整体动物实验显示：二仙汤及其方中温肾药(淫羊藿、仙茅)通过调节ERα和ERβ在子宫中的表达而发挥植物雌激素的功能。
2β-谷甾醇对T47D细胞增殖作用与浓度关系密切：高浓度β-谷甾醇通过细胞周期阻滞抑制细胞增殖,而低浓度β-谷甾醇通过ER介导可促进T47D细胞增殖。不同浓度仙茅苷能够通过ER介导促进T47D细胞增殖,且具有浓度依赖性。仙茅苷和低浓度β-谷甾醇具有促T47D细胞增殖的雌激素样作用。
3β-谷甾醇和仙茅苷能够通过ERα和ERβ诱导ERE报告基因的转录激活,作用强度明显低于E2,且对ERβ具有更明显的激活效应。
4β-谷甾醇通过ER介导上调下游基因cyclin D1蛋白与mRNA水平的表达,诱导下游基因PS2mRNA的表达,从而发挥植物雌激素样作用。
Phytoestrogens are a class of plant-derived compounds that exhibit structural similarity to the endogenous estrogens.They can induce biologic responses usually by binding to estrogen receptors (ERs). Phytoestrogens can therefore act as oestrogen agonists and antagonists depending on the level of the endogenous estrogens and the tissue type. Epidemiologic studies indicate that a protective effect of phytoestrogens to many diseases, such as cardiovascular diseases, climacteric syndrome and certain hormone-responsive cancers (breast and prostate cancer),and so on. However,some studies have produced inconsistent results.As a·result, interest in the possible mechanisms of phytoestrogen action has instensified and'has been the focal subject of numerous studies. Phytoestrogen-containing herbs and prescriptions may exhibit chemoprevention and treatment for these diseases.
Er-xian Decoction has the effect of warming kidney and replenishing essence, nourishing yin and dispersing fire, regulating thorough fare and conception vessels, balancing yin and yang. As a classical decoction, Er-xian Decoction was used to treat climacteric syndrome. With·the development of study, Er-xian Decoction displays a wide array of pharmacologic properties by regulating reproductive and endocrine functions,adjusting immunity and free radicals in animal and in vitro studies. Therefore, Er-xian Decoction has a wide prospect in the modern clinical aspect. Though We have found Er-xian Decoction and Herba Epimedii exert phytoestrogenic effects, the possible mechanisms,target and channel of action keep still unclear. In this study, Er-xian Decoction and its compositions were tested and discussed by means of animal experiment in vivo,cell culture in vitro and molecular level. The results will offer academic foundation for application of the clincical medicine.
Methods & Results
1 The experimental study on the effects of Er-xian Decoction and its compositions on uterus of immature Sprague-Dawley rats.
To evaluate the estrogen-like action of Er-xian Decoction and its compos it ions, the effect on the uterus coefficient was observed by uterotrophic experiment. The thickness of endometrium and the height of epithelium were measured from routine histological slides by hematoxylin-eosin staining. The expression of ERαand ERβin the uterus were examined by immunohistochemistry.
Results:
The uterus coefficient was increased significantly in diethylstilbestrol (DES) group, Er-xian Decoction group, Herba Epimedii group and Rhizoma Curculiginis group (P<0.05).The level of E2 in diethylstilbestrol group was only increased.It was found diethylstilbestrol could cause an increase in the thickness of endometrium and the height of epithelium and hypertrophy. By immunohistochemical staining, DES caused a decrease in the expression of ERαand ERβin the uterus of immature rats,but Er-xian Decoction and its compositions warming Shen(Herba Epimedii, Rhizoma Curculiginis) could induce an increase in the expression of ERαand ERβin the uterus.
2 The.influence ofβ-sitosterol and curculigoside on proliferation and cell cycle of breast cancer cell line T47D cells.
To explore the phytoestrogenic effects ofβ-sitosterol in Er-xian Decoction and curculigoside in herba Rhizoma Curcul iginis, the monotetrazolium(MTT) assay was used to evaluate the proliferation of ER-positive cell 1 ine T47D cells in vitro. Cell cycle analyses were evaluated with prop id ium iodide staining by flow cy tome try. Pure estrogen receptor antagonist, ICI182 780 was used to observe the influence on proliferation.
Results:
The influences of cell growth were different with the different concentration ofβ-sitosterol and curculigoside. The stimulatory effect of curculigoside at high concentration(10-5 mol·L-1 and 10-6 mol·L-1) is.as strong as E2. With decreased concentration of curculigoside, the stimulatory effect decreased gradually. The proliferative effect of 10-7 mol·L-1 and 10-8 mol·L-1 curculigoside only lasted 48 hours.Cell proliferation induced by cueculigoside was completely antagonized by 10-7 mol·L-1 pure estrogen receptor antagonist, ICI182 780.
β-sitosterol at high concentration(10-5 mol·L-1 and 10-6mol·L-1) markedly inhibit cell proliferation that was partly antagonized by ICI182 780. However,β-sitosterol at 10-7 mol·L-1-10-10 mol·L-1 concentration enhanced cell proliferation. The concentration at 10-7 mol·L-1 and 10-8 mol·L-1 was the largest. The effect was completely inhibited by ICI182 780.
Cell cycle distribution analysis revealed that percentage of T47D cells treated with E2 at G0/G1 phase decreased and percentage at S phase increased significant ly. With increased concentration of curculigoside, percentage at G0/G1 phase decreased and per centage at S phase increased markedly. So,proliferation index obviously increased in a concentration-dependent manner. These phenomena were observed withβ-sitosterol at 10-7 mol·L-1-10-10 mol·L-1 concentration, too. Butβ-sitosterol at 10-5 mol·L-1-10-6 mol·L-1 concentration arrested cells at G0/G1 phase,and the amount of cell in S phase and proliferation index decreased.
3 The study ofβ-sitosterol and curculigoside on phytoestrogenic effects by reporter gene assays.
To investigate the selective activation effect ofβ-sitosterol and curculigoside on-different estrogen receptor subtype, the luciferase reporter gene assay was used.
Results:
β-si tosterol could induce luciferase expression, and under the concentration of 10-7 mol·L-1 was much higher. Compared to E2, the effect was slight. The expression of reporter gene induced byβ-sitosterol was larger through ERβ.
Luciferase act ivi ties showed dose-responed relationship with curculigoside. ERβwas activated by curculigoside at a lowest concentration of 10-8 mol·L-1,while ERαwas not activated under this concentration.
4 The influence ofβ-sitosterol on ER and cyclin D1 proteins expression in breast cancer cell line T47D cells.
To explore the possible mechanisms of action ofβ-sitosterol, the expression ofβ-sitosterol-induced ERα, ERβand cyclin D1 proteins was assessed by using P-sitosterol and ICI182 780 by western-blotting analyses.
Results:
We found that the predominant.ER protein in T47D cells is ER a,while the expression of ERβprotein in these cells is slight.β-sitosterol increased the level of T47D cells ERα,ERβand cyclin D1 proteins expression, respectively. The effect was completely antagonized by pure estrogen receptor antagonist, ICI182 780.
5 The effect ofβ-sitosterol on ER,cyclin D1 and PS2 gene expression in T47D cells.
To detect the possible mechanisms of action ofβ-sitosterol, the expression ofβ-sitosterol-induced ERα, ERβ,cyclin D1 and PS2 gene expression was assessed by real-time PCR analyses.
Results:
β-sitosterol up-regulated ER mRNA expression and cyclin D1,PS2 gene are induced in response toβ-si tosterol treatment. The effect on cyclin D1 and PS2 gene expression was inhibited by ICI182 780.
Conclusions
1 Er-xian Decoction and its compositions warming Shen(Herba Epimedii, Rhizoma Curculiginis) could possess phytoestrogenic effects by upregulating the expression of ERαand ERβin the uterus of immature rats.
2 (3-sitosterol at high concentration inhibited proliferation of T47D cells in vitro through arresting cell at G0/G1.However,β-sitosterol at low concen-tration and different concentration curculigoside possessed estrogen-like activity of enhancing proliferation in T47D cells.The effect was mediated by ER.
3β-sitosterol and curculigoside as an agonist of ER, promoted luciferase expression involving activation of ER. The effect on ERβwas greater than ER a.
4β-sitosterol up-regulated cyclin D1 protein and gene expression and PS2 gene expression through ER.β-sitosterol possessed phytoestrogenic effect by activating ER.
植物雌激素(phytoestrogen, PE)是从天然植物中提取的结构与内源性雌激素相似的一类的化合物,能够与雌激素受体(estrogen receptors, ERs)结合发挥生物学效应,具有雌激素和抗雌激素双重效应,发挥哪种效应取决于内源性雌激素的水平和组织类型。流行病学研究发现植物雌激素在许多疾病的预防和治疗方面发挥有益的作用,如心血管疾病、围绝经期综合症以及与雌激素相关的肿瘤(乳腺癌、前列腺癌)等。然而,研究中也发现不一致的研究结果。因此,对其作用机制的研究自然成为研究者们关注的热点。中药复方作为中医治病用药的最主要形式之一,具有植物雌激素样作用的中药及复方在这些疾病的预防和治疗中发挥重要作用。
二仙汤具有温肾益精、滋阴泻火、调理冲任和平衡阴阳之功效,是治疗更年期综合征的经典方剂。随着研究的不断深入,动物实验和体外研究发现二仙汤具有调整生殖／内分泌,调节免疫功能和自由基代谢,在临床上有着广阔的应用前景。我们课题组在前期研究工作中发现二仙汤及方中淫羊藿具有雌激素样作用,但对于其发挥雌激素样作用靶点、作用途径和机理缺乏深入分析和探讨。因此,本实验从整体、细胞和分子水平阐明二仙汤及组方中药的植物雌激素作用以及发挥作用的物质基础和作用机制,为临床合理、科学用药提供理论指导。
二研究方法和结果
1二仙汤及其组方中药对幼年大鼠子宫作用的实验研究
采用子宫增重实验观察二仙汤及其组方中药对幼年大鼠子宫系数的影响；检测血清激素水平的变化；HE染色观察不同药物对大鼠子宫内膜厚度和上皮高度的影响；免疫组织化学染色检测ERα和ERβ在大鼠子宫中的表达情况,评价二仙汤及组方中药的雌激素样作用。
结果：已烯雌酚组、二仙汤组、淫羊藿组和仙茅组子宫系数与正常组对比增加明显,有统计学意义(P<0.05)。仅已烯雌酚组E2增高。HE染色发现已烯雌酚组子宫内膜厚度和上皮高度明显增加,并且局部增生,上皮由单层变为复层。免疫组织化学染色发现已烯雌酚组ERα和ERβ平均光密度值低于正常对照组,而二仙汤组、淫羊藿组和仙茅组的ERα和ERβ平均光密度值均高于正常对照组。
2β-谷甾醇和仙茅苷对T47D细胞增殖和细胞周期的影响
以二仙汤方剂中六味中药的活性成分β-谷甾醇、仙茅的主要活性成分仙茅苷为研究对象,通过MTT法检测它们对体外培养的ER阳性人乳腺癌细胞系T47D的细胞增殖的影响；流式细胞术检测它们对细胞周期的影响；并以雌激素受体完全拮抗剂ICI182 780对细胞增殖的干预作用,探讨β-谷甾醇和仙茅苷的植物雌激素样活性。
结果：高浓度(10-5 mol·L-1和10-6 mol·L-1)仙茅苷作用类似于雌二醇,随着浓度的降低仙茅苷对T47D细胞的促增殖作用明显降低,10-7 mol·L-1和10-8 mol·L-1仙茅苷的促增殖作用仅持续48h,其增殖作用能够被ICI182 780所抑制；高浓度(10-5 mol·L-1和10-6 mol·L-1β-谷甾醇可以抑制T47D细胞增殖,且ICI182 780不能完全拮抗其对T47D细胞的增殖抑制作用,低浓度β-谷甾醇可促进T47D细胞增殖,且具有时间与浓度依赖性,以10-7 mol·L-1和10-8 mol·L-1β-谷甾醇作用最强,ICI182,780可完全拮抗其促增殖作用。
应用流式细胞仪测定细胞周期,雌二醇使T47D细胞G。／G1期细胞的比例明显减低,而S期细胞的比例则明显升高；不同浓度的仙茅苷刺激T47D细胞,随着浓度的升高,G0／G1期细胞的比例降低,而S期细胞的比例则明显升高,细胞分裂增殖指数升高；T47D细胞在高浓度β-谷甾醇作用下,G0/G1期细胞的比例升高,S期细胞的比例下降,细胞分裂增殖指数降低,而低浓度β-谷甾醇使T47D细胞G0/G1期细胞的比例降低,S期细胞的比例升高,细胞分裂增殖指数升高。
3报告基因方法对β-谷甾醇和仙茅苷植物雌激素样作用的研究
通过荧光素酶报告基因技术方法探索二仙汤中两种活性成分β-谷甾醇和仙茅苷是否通过ERα和ERβ发挥植物雌激素样作用。
结果：β-谷甾醇可诱导ERE报告基因的转录激活,且在10-7 mol·L-1和10-8 mol·L-1浓度下诱导作用最明显。对比β-谷甾醇通过ERα和ERβ的诱导作用发现,不同浓度β-谷甾醇通过ERβ诱导报告基因表达倍数更高,提示β-谷甾醇可能对ERβ有更高的激活效应。仙茅苷可通过ERα或ERβ诱导ERE报告基因荧光素酶的转录,且呈明显的剂量依赖效应。而低浓度仙茅苷(10'-8mol·L-1)只能通过ERβ激活报告基因荧光素酶的转录,而对过表达ERα的HEK-293细胞则没有明显的作用,提示仙茅苷对ERβ具有更高的激活效应。
4β-谷甾醇对T47D细胞ER蛋白和cyclin D1蛋白表达的影响
以ER阳性人乳腺癌细胞系T47D细胞为研究对象,应用Western blotting法观察β-谷甾醇及在雌激素受体完全拮抗剂ICI182 780干预下,对ER及下游基因cyclin D1蛋白表达的影响,探讨β-谷甾醇发挥植物雌激素样作用的分子机制。
结果：T47D细胞以ERα蛋白表达占优势,ERβ蛋白低表达,10-7 mol·L-1β-谷甾醇可诱导T47D细胞ERα、ERβ及cyclin D1蛋白表达水平升高,且可以被雌激素受体完全拮抗剂ICI182 780抑制。
5β-谷甾醇对T47D细胞ER、PS2和cyclin D1 mRNA表达的影响
采用RT-PCR技术观察β-谷甾醇对ER及其下游基因如PS2、cyclin DlmRNA表达的影响,在基因水平上进一步探讨β-谷甾醇发挥植物雌激素样作用的分子机制。
结果：β-谷甾醇可上调ERα和ERβ基因表达水平,相对表达量明显升高,且与雌二醇相比,上调ERβ表达更显著,约为溶剂对照组的7倍左右。可诱导PS2和cyclinD1mRNA表达,相对表达量明显增多,并且ICI182 780能够抑制β-谷甾醇对下游基因的诱导作用。
三研究结论
1整体动物实验显示：二仙汤及其方中温肾药(淫羊藿、仙茅)通过调节ERα和ERβ在子宫中的表达而发挥植物雌激素的功能。
2β-谷甾醇对T47D细胞增殖作用与浓度关系密切：高浓度β-谷甾醇通过细胞周期阻滞抑制细胞增殖,而低浓度β-谷甾醇通过ER介导可促进T47D细胞增殖。不同浓度仙茅苷能够通过ER介导促进T47D细胞增殖,且具有浓度依赖性。仙茅苷和低浓度β-谷甾醇具有促T47D细胞增殖的雌激素样作用。
3β-谷甾醇和仙茅苷能够通过ERα和ERβ诱导ERE报告基因的转录激活,作用强度明显低于E2,且对ERβ具有更明显的激活效应。
4β-谷甾醇通过ER介导上调下游基因cyclin D1蛋白与mRNA水平的表达,诱导下游基因PS2mRNA的表达,从而发挥植物雌激素样作用。
Phytoestrogens are a class of plant-derived compounds that exhibit structural similarity to the endogenous estrogens.They can induce biologic responses usually by binding to estrogen receptors (ERs). Phytoestrogens can therefore act as oestrogen agonists and antagonists depending on the level of the endogenous estrogens and the tissue type. Epidemiologic studies indicate that a protective effect of phytoestrogens to many diseases, such as cardiovascular diseases, climacteric syndrome and certain hormone-responsive cancers (breast and prostate cancer),and so on. However,some studies have produced inconsistent results.As a·result, interest in the possible mechanisms of phytoestrogen action has instensified and'has been the focal subject of numerous studies. Phytoestrogen-containing herbs and prescriptions may exhibit chemoprevention and treatment for these diseases.
Er-xian Decoction has the effect of warming kidney and replenishing essence, nourishing yin and dispersing fire, regulating thorough fare and conception vessels, balancing yin and yang. As a classical decoction, Er-xian Decoction was used to treat climacteric syndrome. With·the development of study, Er-xian Decoction displays a wide array of pharmacologic properties by regulating reproductive and endocrine functions,adjusting immunity and free radicals in animal and in vitro studies. Therefore, Er-xian Decoction has a wide prospect in the modern clinical aspect. Though We have found Er-xian Decoction and Herba Epimedii exert phytoestrogenic effects, the possible mechanisms,target and channel of action keep still unclear. In this study, Er-xian Decoction and its compositions were tested and discussed by means of animal experiment in vivo,cell culture in vitro and molecular level. The results will offer academic foundation for application of the clincical medicine.
Methods & Results
1 The experimental study on the effects of Er-xian Decoction and its compositions on uterus of immature Sprague-Dawley rats.
To evaluate the estrogen-like action of Er-xian Decoction and its compos it ions, the effect on the uterus coefficient was observed by uterotrophic experiment. The thickness of endometrium and the height of epithelium were measured from routine histological slides by hematoxylin-eosin staining. The expression of ERαand ERβin the uterus were examined by immunohistochemistry.
Results:
The uterus coefficient was increased significantly in diethylstilbestrol (DES) group, Er-xian Decoction group, Herba Epimedii group and Rhizoma Curculiginis group (P<0.05).The level of E2 in diethylstilbestrol group was only increased.It was found diethylstilbestrol could cause an increase in the thickness of endometrium and the height of epithelium and hypertrophy. By immunohistochemical staining, DES caused a decrease in the expression of ERαand ERβin the uterus of immature rats,but Er-xian Decoction and its compositions warming Shen(Herba Epimedii, Rhizoma Curculiginis) could induce an increase in the expression of ERαand ERβin the uterus.
2 The.influence ofβ-sitosterol and curculigoside on proliferation and cell cycle of breast cancer cell line T47D cells.
To explore the phytoestrogenic effects ofβ-sitosterol in Er-xian Decoction and curculigoside in herba Rhizoma Curcul iginis, the monotetrazolium(MTT) assay was used to evaluate the proliferation of ER-positive cell 1 ine T47D cells in vitro. Cell cycle analyses were evaluated with prop id ium iodide staining by flow cy tome try. Pure estrogen receptor antagonist, ICI182 780 was used to observe the influence on proliferation.
Results:
The influences of cell growth were different with the different concentration ofβ-sitosterol and curculigoside. The stimulatory effect of curculigoside at high concentration(10-5 mol·L-1 and 10-6 mol·L-1) is.as strong as E2. With decreased concentration of curculigoside, the stimulatory effect decreased gradually. The proliferative effect of 10-7 mol·L-1 and 10-8 mol·L-1 curculigoside only lasted 48 hours.Cell proliferation induced by cueculigoside was completely antagonized by 10-7 mol·L-1 pure estrogen receptor antagonist, ICI182 780.
β-sitosterol at high concentration(10-5 mol·L-1 and 10-6mol·L-1) markedly inhibit cell proliferation that was partly antagonized by ICI182 780. However,β-sitosterol at 10-7 mol·L-1-10-10 mol·L-1 concentration enhanced cell proliferation. The concentration at 10-7 mol·L-1 and 10-8 mol·L-1 was the largest. The effect was completely inhibited by ICI182 780.
Cell cycle distribution analysis revealed that percentage of T47D cells treated with E2 at G0/G1 phase decreased and percentage at S phase increased significant ly. With increased concentration of curculigoside, percentage at G0/G1 phase decreased and per centage at S phase increased markedly. So,proliferation index obviously increased in a concentration-dependent manner. These phenomena were observed withβ-sitosterol at 10-7 mol·L-1-10-10 mol·L-1 concentration, too. Butβ-sitosterol at 10-5 mol·L-1-10-6 mol·L-1 concentration arrested cells at G0/G1 phase,and the amount of cell in S phase and proliferation index decreased.
3 The study ofβ-sitosterol and curculigoside on phytoestrogenic effects by reporter gene assays.
To investigate the selective activation effect ofβ-sitosterol and curculigoside on-different estrogen receptor subtype, the luciferase reporter gene assay was used.
Results:
β-si tosterol could induce luciferase expression, and under the concentration of 10-7 mol·L-1 was much higher. Compared to E2, the effect was slight. The expression of reporter gene induced byβ-sitosterol was larger through ERβ.
Luciferase act ivi ties showed dose-responed relationship with curculigoside. ERβwas activated by curculigoside at a lowest concentration of 10-8 mol·L-1,while ERαwas not activated under this concentration.
4 The influence ofβ-sitosterol on ER and cyclin D1 proteins expression in breast cancer cell line T47D cells.
To explore the possible mechanisms of action ofβ-sitosterol, the expression ofβ-sitosterol-induced ERα, ERβand cyclin D1 proteins was assessed by using P-sitosterol and ICI182 780 by western-blotting analyses.
Results:
We found that the predominant.ER protein in T47D cells is ER a,while the expression of ERβprotein in these cells is slight.β-sitosterol increased the level of T47D cells ERα,ERβand cyclin D1 proteins expression, respectively. The effect was completely antagonized by pure estrogen receptor antagonist, ICI182 780.
5 The effect ofβ-sitosterol on ER,cyclin D1 and PS2 gene expression in T47D cells.
To detect the possible mechanisms of action ofβ-sitosterol, the expression ofβ-sitosterol-induced ERα, ERβ,cyclin D1 and PS2 gene expression was assessed by real-time PCR analyses.
Results:
β-sitosterol up-regulated ER mRNA expression and cyclin D1,PS2 gene are induced in response toβ-si tosterol treatment. The effect on cyclin D1 and PS2 gene expression was inhibited by ICI182 780.
Conclusions
1 Er-xian Decoction and its compositions warming Shen(Herba Epimedii, Rhizoma Curculiginis) could possess phytoestrogenic effects by upregulating the expression of ERαand ERβin the uterus of immature rats.
2 (3-sitosterol at high concentration inhibited proliferation of T47D cells in vitro through arresting cell at G0/G1.However,β-sitosterol at low concen-tration and different concentration curculigoside possessed estrogen-like activity of enhancing proliferation in T47D cells.The effect was mediated by ER.
3β-sitosterol and curculigoside as an agonist of ER, promoted luciferase expression involving activation of ER. The effect on ERβwas greater than ER a.
4β-sitosterol up-regulated cyclin D1 protein and gene expression and PS2 gene expression through ER.β-sitosterol possessed phytoestrogenic effect by activating ER.
引文
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