| 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 & Results1 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.Conclusions1 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.
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