Experimental Research On The Phytoestrogenic Effects And Its Mechanisms Of HSYA

Purposes:Estrogen is one of the most important regulating hormone in human body. Its biological effect is realized via estrogen receptor.Estrogen plays much more important roles in female. Postmenopausal woman usually tops the list of ovarian cancer, endometrial cancer, osteoporosis, Alzheimer’s disease and atherosclerosis.This is because estrogen level significantly reduced after menopause.Although estrogen replacement therapy can alleviate the symptoms,the side effect is obvious.So the extracts from natrual plants which can play estrogenic roles with no obvious side effects attract much attention and they can be called phytoestrogens (PE).Many kinds of PE come from the TCM such as sichuan safflower, achyranthes and scurfpea fruit which are frequently applied in gynecological diseases treating.The chemical structure of phytoestrogen is similar to endogenous estrogen which can combine with estrogen receptor(ER) and display estrogenic or anti-estrogenic effects.On one hand, when the level of estrogen inside is low, PE combined with ER and played estrogenic effect. On the other hand,when the level of estrogen inside is high,PE can inhibit endogenous estrogen competitively play a role of anti-estrogenic effect.Hydroxy safflor yellow A(HSYA) is the strongest bioactive constituents of safflower. It can play estrogenic role inside the human body.But the mechanism and pathway of its estrogenic effects has not yet been reported.Our study focused on the phytocstrogenic effects of HSYA and its possible mechanism at cell and molecular level.We expect to provide theoretical basis for clinical application of safflower and its proprietary Chinese medicine preparations.Methods and results:1. The influence of HSYA on proliferation and cell cycle distribution of T47D and SK-BR-3cell.1.1MTT test was used to evaluate cell proliferation.The flow cytometry was used to detect the of cell cycle distribution change induced by T47D.1.1.1The effects of different concentration of HSYA on proliferation of T47D cell.Results:HSYA showed a significant effect on promoting cell proliferation rate at24h, the effect of HSYA is the strongest at48h,and the effect is weeken at72h. 1.1.2The effect of HSYA on T47D cell proliferation with ER antagonist, ERa antagonist, ERβ antagonist, ERa agonist and ERβ agonist.Results:The promoting effect of HSYA on T47D cell proliferation can be antagonisted by ER antagonist.ERa antagonist MPP and ERβ antagonist PHTPP showed an obvious antagonistic action on cell proliferation promoted by estradiol and HSYA.The antagonistic effect of PHTPP is more obvious than the same dose of MPP.The effect of ERa agonist PPT and ERP agonist DPN on cell proliferation of HSYA are not obvious. But at72h,the effect of PPT is more significant than10-7mol·L-1HSYA.1.1.3The effects of different concentration of HSYA on cell cycle distribution of T47D cellResults:We selected the48h group to perform the cell cycle test.10-6mol-L-1and10-7mol·L-1HSYA increased cell proportion in both S phase and G2/M phase. It resulted in increasing cell proliferation index.1.2SK-BR-3(ER-) cell was applied in MTT assay to evaluate the effect of HSYA on ER(-) cell proliferation.Results:10-5mol·L-1-10-7mol·L-11HSYA has inhibitory effect on SK-BR-3cell, and the effect lasts48h.10"9mol·L-11HSYA slightly stimulate cell proliferation. All the results show no significant difference compared with the normal control group at48h.2. The effect of HSYA on ERmRNA expression of T47D. Real-time fluorescence quantitative RT-PCR technique was used to test the effect of HSYA on ERmRNA expression.Results:HSYA significantly promote the expression of ERa, ERβ mRNA. The ratio of ERβ mRNA/ERa mRNA in HSYA group is significantly higher than the normal control group.3. The effect of HSYA on on ER and ER related protein expression in T47D and SK-BR-3cell.3.1T47D was used in Western blot analysis to evaluate the expression of six kinds of ER and ER related protein including ERa,ERP,ERK1/2,p-ERK1/2,P27and PR. Results:10-7mol·L-1HSYA promote the expression of ERa significantly.10-7mol·L-1and10-8mol·L-1HSYA promote the expression of ERβ obviously. The ratio of ERα and ERβ could be changed by HSYA and it reached the highest level with10-7mol·L-1and10-8mol·L-1HSYA treating while cell proliferation is also at its highest level.10-7mol·L-1and10-8mol·L-1HSYA significantly promote the expression of ERK1/2.10-7mol·L-1HSYA significantly promote the expression of p-ERK1/2.The expression of P27protein was significantly decreased by HSYA treating.10-7mol·L-1-10-8mol·L-1HSYA significantly promote expression of PR while10-9mol·L-1HSYA inhibit expression of PR. 3.2Western blot analysis of ERK1/2and p-ERK1/2expression affected by HSYA in SK-BR-3cell.Results:10-7mol·L-1-10-9mol·L-1HSYA significantly inhibit ERK1/2expression.But the effect of HSYA on p-ERK1/2expression is not obvious.4. Establishment of the specific ER subtype positive cell model by ER plasmid transfection. ERα and ERβ plasmid were.transfected into HEK293cells by liposome.Results:Western blot were applied to test plasmid transfection and ER expression in transfected HEK293cells.The result showed the transfection was successful.