Studies On Estrogenic-Like Effects Of Wujiashenghua Capsule

Objective:Through this study to ovariectomized mice model and expression of estrogen receptor-positive MCF-7cell proliferation assays,to discusses the estrogen-like effects and the role of effective medication site for wujiashenghua capsule,and provide evidence for wujiashenghua capsule insteading of estrogen on treatment of perimenopausal syndrome.Methods:1. Effect of wujiashenghua capsule in mature ovariectomized mice:The experiment selected the12-week-old of kunming species of female mice only60, and divided them randomly into the sham operation group, the model group, the low-dose group (3.120g/kg), the high-dose group (4.992g/kg) and the diethylstilbestrol positive control group (0.325mg/kg), and then removed two sides ovaries after the mice were anesthized by ether(the sham operation group only remove the fat near ovarie, without removal of the ovaries),each group of12. All groups which were eating and drinking water under the same conditions gavaged once daily, but the sham and model groups were given equal volume of distilled water, continuous dosing around4weeks. We observed the estrous cycle conditions of mice in each group during experiment; and removed the eyeball within two hours after the last administration and got blood, then centrifuged and measured the E2, FSH, TC, TG in serum; quickly stripped mouse’s uterus and adrenal for weighing, meanwhile calculated the viscera index of the uterus and adrenal, with routine HE staining of uterine pathology to observe the volume of uterus, uterine cavity shape, gland number etc.2. Effect of wujiashenghua capsule65%ethanol extract on theproliferation of MCF-7cells:The65%ethanol extract of wujiashenghua capsule was separated by D-101macroporous resin column into the water, the20%ethanol, the40%ethanol, and the60%ethanol elution fraction, and prepared the elution fraction which got from above conditions as10mg/ml,lmg/ml,0.1mg/ml and0.01mg/ml four concentration with culture medium,then respectively divided them into blank control group, wujiashengh.ua group, E2positive control group, and acted on the breast cancer MCF-7cell. We seeded the MCF-7cells in96-well plates and cultured48hours after administration, for MTT color reaction in each well was measured at490nm of microplate reader (BIO-TEK)on the absorbance OD (OD is proportional to the number of live cells), and repeated the experiment three times independently. The results will be subject to the average of the three independent repeated trials.Results:1. The acted on ovariectomized mice:Compared with the sham group, after removal of the ovaries, the model group mice had been in diestrus,whose the5th consecutive estrous cycle disappeared,the uterus become shrinking, and the weight of uterus were significantly decreased, the number of glands become to less; as well as the serum E2、TG content of ovariectomized group was obviously lower than that of any other groups, On the contrary the serum FSH, TC content become increased.Comparing with the model group, wujiashenghua capsule two doses can restore the estrous cycle of ovariectomized mice, antagonize uterine atrophy and increase the weight of the uterus(p<0.01); HE staining of uterine pathology showed that the endometrium in mouse become thickened with the intercellular space enlarging, and there were an increase in the number of glands and gland cavity enlargement, which partially filled with red secreta (eosinophilic material) Although the two dose groups could make a moderate increasing to adrenal weight of ovariectomized mice, their different has no statistical significance; ELISA kits indicated that wujiashenghua capsule two dose groups could significantly increase the content of E2in serum and promote the secretion of estrogen, meanwhile could obviously reduce the levels of FSH to antagonize the FSH secretion and the high-dose group can significantly reduce the content of TC, and significantly increased the TG levels, but significantly weaker than diethylstilbestrol group, On the contrary, the low-dose group of mice compared with the model group in serum TC,TG, and no statistical difference.2. The acted on the MCF-7cell proliferation:Compared with the blank control group, when the concentration of0.01mg/ml,each drug concentration gradient had no effect on the proliferation of MCF-7cells; when the concentration of0.1mg/ml, water elution parts,20%ethanol elution parts and60%ethanol elution parts had no effect on the proliferation of MCF-7cells, while the40%ethanol elution parts can obviously promote the proliferation of MCF-7cells; When the concentration of1mg/ml, water elution parts had no effect on the proliferation of MCF-7cells, but20%,40%and60%ethanol elution parts can distinctly promote the proliferation of MCF-7cells and the40%ethanol elution parts on MCF-7cell proliferation effect is strongest, but it is still weaker then E2positive control group; when the concentration of10mg/ml, each drug concentration gradient can inhibite the proliferation of MCF-7cells, It has side effects on the cell growth. To sum up, the40%ethanol elution parts of wujiashenghua capsule for estrogen activity is more significant, it may be the main effective part for the drug to use of estrogen-like effects.Conclusion:1. Wujiashenghua capsule in vivo has obvious estrogen-like effects, it may antagonize the mouse uterine atrophy, increase the uterine weight in mice, and regulate the ovariectomized mice serum estradiol, FSH and other hormone levels, as well as the blood lipid metabolic disorder have certain improvement effect.2.40%ethanol elution positions of Wujiashenghua capsule in vitro can significantly promote the proliferation of MCF-7cells, It is an important part for Wujiashenghua capsule to play an effective role of the estrogen-like effects.