Study On Estrogen-like Effect Of Modified Shoutai Pills Based On The Model Of POI (Primary Ovarian Insufficiency)on Rats

ObjectiveUse the model of POI on rats to investigate the estrogen-effect of Modified Shoutai pills in vivo. Use the MCF-7 cell line to study the estrogen-like effect of Modified Shoutai pills in vitro, which based on the test of proliferative activity of MCF-7 cell and the test of ER luciferase reporter geneMethods:Dividing SD rats into 5 groups:Control, Model, Positive, High, Middle and Low dose of Modified Shoutai pills. Using 50mg/kg of tripterygium glycosides per day to make the model of POI. Analyzing the estrous cycle and the index of ovarian and uterus. Using radioimmunoassay to test the E2、FSH、LH and FSH/LH in serum. Using HE to test the pathmorphism of ovarian and uterus. WB was used to test the expression of ERs in ovarian, uterus, hypothalamus and hypophysis.MCF-7 cells cultured in 96 plates were divided into 5 groups:1‰DMSO, 0.1uME2,0.1mg/ml,0.01mg/ml and 0.001mg/ml extract of Modified Shoutai Pills. The reagent above were added for 48h. MTT was used to test the proliferative activity of MCF-7 cell.MCF-7 cells were cultured in 6 plates and divided into 6 groups:1‰DMSO, 0.1uME2, luMICI182,780,0. lmg/ml,0.01mg/ml and 0.001mg/ml extract of Modified Shoutai Pills. The plasmid carried ER-luciferase reporter gene was transferd into cell, The reagent above were added for 24h, After that, the activity of luciferase was tested and analyzed.Results:1. Effect of Modified Shoutai pills on model of POI(1) Estrous cycle:Compared with the group of control, the frequency of disorder of the model within D1-D30 and D21-D30 were higher, P<0.01, there was statistically significant difference. Compared with the group of model, the frequency of disorder of the positive, high, middle, and low dose of Modified Shoutai pills within D1-D30 and D21-D30 were lower, P<0.05, there was statistically significant difference.(2) Hormone in serumCompared with the group of control, FSH of the model and the low dose of Modified Shoutai pills were higher, P< 0.001, there was statistically significant difference. Compared with the group of model, FSH of the positive, the high and middle dose of Modified Shoutai pills were lower, P<0.05, there was statistically significant difference.Compared with the group of control, E2 of the model and the low dose of Modified Shoutai Pills were lower, P<0,05, there was statistically significant difference. Compared with the group of model, E2 of the positive and the high dose of Modified Shoutai pills were higher, P<0.05, there was statistically significant difference.Compared with the group of control, FSH/LH of the model and the low dose of Modified Shoutai pills were higher, P<0.05, there was statistically significant difference. Compared with the group of model, FSH/LH of the positive, the high and low dose of Modified Shoutai pills were lower, P< 0.05, there was statistically significant difference.Compared with the group of control and model, LH of the others were undifferentiated, P>0.05, the difference wasn’t statistically significant.(3) Index of ovarian and uterus:Compared with the group of control, index of ovarian of the model was lower, P< 0.001, there was statistically significant difference. Compared with the group of model, index of ovarian of the others were higher, P< 0.001, there was statistically significant difference.Compared with the group of control, index of uterus of the model was lower, P<0.001, there was statistically significant difference. Compared with the group of model, index of uterus of the other groups were higher, P<0.01, there was statistically significant difference.(4) Pathmorphism of ovarian and uterus:Follicles in ovarian of the model were less, the other group were more. There is no significant differences within all groups.(5) Expression of ERs in ovarian:Compared with the group of control, the expression of ERα in ovarian of the model was lower, P<0.001, there was statistically significant difference. Compared with the group of model, the expression of ER a in ovarian of the other groups were higher, P<0.001, there was statistically significant difference.Compared with the group of control, the expression of ERβ in ovarian of the model was lower, P< 0.001, there was statistically significant difference. Compared with the group of model, the expression of ER β in ovarian of the other groups were higher, P<0.001, there was statistically significant difference.(6) Expressio of ERs in uterus:Compared with the group of control, the expression of ERα in uterus of the model, the middle and the low dose of Modified Shoutai pills was lower, P< 0.001, there was statistically significant difference. Compared with the group of model, the expression of ERα in uterus of the other groups were higher, P< 0.001, there was statistically significant difference.Compared with the control, the expression of ERβ in uterus of the model was lower, P<0.001, there was statistically significant difference. Compared with the model, the expression of ERβ in uterus of the other groups were higher, P<0.001, there was statistically significant difference.(7) Expression of ERα in hypothalamus and hypophysis:Compared with the group of control, the expression of ER a in hypothalamus of the model was lower, P<0.01, there was statistically significant difference. Compared with the group of model, the expression of ERα in hypothalamus of the other groups were higher, P<0.01, there was statistically significant difference.Compared with the group of control, the expression of ERα in hypophysis of the model was lower, P< 0.001, there was statistically significant difference. Compared with the group of model, the expression of ERα in hypophysis of the other groups were higher, P<0.05, there was statistically significant difference.2. Infulence of Modified Shoutai pills on proliferative activity of MCF-7 cell(1) Compared with the group of control, 0.001mg/ml Modified Shoutai pills and 0.1uM E2 could improve the proliferative activity of MCF-7 cell, P<0. 05, there was statistically significant difference.(1) Compared with the group of control,0.001mg/ml Modified Shoutai pills and 0.1uM E2 could improve the expression of ERα in MCF-7 cell, P<0.05, there was statistically significant difference.3. Infulence of Modified Shoutai pills on activity of ER-luciferaseCompared with the group of control,0.1mg/ml,0. Olmg/ml extract of Modified Shoutai pills and luM ICI182,780 inhibited the activity of ER-luciferase, P<0.05, there was statistically significant difference.Compared with the group of control,0.001lmg/mlextract of Modified Shoutai pills and 0.1uME2 could improve the activity of ER-luciferase, P<0.05, there was statistically significant difference.Conclusion:1. Modified Shoutai pills could reduce the frequency of disorder in estrous cycle of POI rats, improve the level of E2, inhibit the level of FSH and FSH/LH in serum of POI rats. In addition, Modified Shoutai pills also could improve the index of ovarian and uterus of POI rats, up-regulate the expression of ERs in ovarian, uterus, hypothalamus and hypophysis of POI rats. These effects of Modified Shoutai pills on POI rats are estrogen-like.2.0.001mg/ml Modified Shoutai pills could improve the proliferative activity of MCF-7 cell by inducing the expression of ERa,this effect was estrogen-like.3.0.001mg/ml extract of Modified Shoutai pills shows estrogen-like effect in vitro, activating ER and improving the expression of ER-luciferase gene 0.1mg/ml and 0.01mg/ml extract of Modified Shoutai pills could inhibit the activity of ER and down-regulate the expression of ER-luciferase gene.